Publications 2016

Leang R, Khu NH, Mukaka M, Debackere M, Tripura R, Kheang ST, Chy S, Kak N, Buchy P, Tarantola A et al. 2016. Erratum to: An optimised age-based dosing regimen for single low-dose primaquine for blocking malaria transmission in Cambodia. BMC Med, 14 (1), pp. 213. | Show Abstract | Read more

© 2016 The Author(s).After publication of the original article [1], it came to the authors' attention that there was an error in the PQ pharmacokinetics sub-section of the Background section. The following sentence is affected: "There is no PK interaction between PQ and either artesunate-pyronaridine [76] or mefloquine [69, 77]; no PK interaction data exist for PQ and artemetherlumefantrine (AL)." This sentence should have read as follows: "AS pyronaridine increased PQ exposure by 15% without affecting significantly cPQ exposure [76]. There is no PK interaction between PQ and mefloquine [69, 77]; no PK interaction data exist for PQ and artemetherlumefantrine (AL).".

Wuthiekanun V, White NJ, Amornchai P, Day NP, Limmathurotsakul D. 2016. Quality controls for antimicrobial disk diffusion testing on Leptospira Vanaporn Wuthiekanun agar. Trans R Soc Trop Med Hyg, 110 (11), pp. 673-675. | Show Abstract | Read more

BACKGROUND: Disk diffusion susceptibility testing for Leptospira spp. on Leptospira Vanaporn Wuthiekanun (LVW) solid agar was reported recently. However, it was unclear whether the zone sizes obtained on LVW agar were comparable with those of other bacteria on Mueller-Hinton agar. METHODS: Here, we evaluate the disk diffusion assay on LVW agar using the standard quality control (QC) bacterial strains for 22 antimicrobials. RESULTS: All antimicrobials provided zone sizes within the standard range for each QC bacterial strain, except for fosfomycin. CONCLUSIONS: In conclusion, the simple disk diffusion assay can be used to assess antimicrobial activity against Leptospira on LVW agar using standard bacterial strains for QC with the standard breakpoints (except for fosfomycin).

Newton PN, Timmermann B. 2016. Fake penicillin, The Third Man, and Operation Claptrap. BMJ, 355 pp. i6494. | Read more

Ya-Umphan P, Cerqueira D, Parker DM, Cottrell G, Poinsignon A, Remoue F, Brengues C, Chareonviriyaphap T, Nosten F, Corbel V. 2016. Anopheles Salivary Biomarker to Assess Malaria Transmission Risk Along the Thailand-Myanmar Border. J Infect Dis, | Show Abstract | Read more

BACKGROUND: The modalities of malaria transmission along the Thailand-Myanmar border are poorly understood. Here we address the relevance of using a specific Anopheles salivary biomarker to measure the risk of human exposure to anopheles bites. METHODS: Serologic surveys were conducted from May 2013 to December 2014 in 4 sentinel villages. Dried blood spots (n>9,400) were collected in filter papers among all inhabitants at baseline and then every three months up to 18 months. The relationship between the intensity of human antibody response and entomological indicators of transmission (Human biting rates and Entomological Inoculation rates, EIR) was studied using a multivariate three-level mixed model analysis. "Heatmaps" for human IgG responses for each village and survey time point were created using QGIS 2.4. RESULTS: The levels of IgG response of participants significantly varied according to the village, season and age (P<0.001) and were positively associated with the abundance of total Anopheles spp. and primary malaria vectors and EIR (P<0.001). Spatial clusters of high IgG responders were identified across space and time within study villages. CONCLUSIONS: The gSG6-P1 biomarker has great potential to address the risk of transmission along the Thailand-Myanmar border and represents a promising tool to guide malaria interventions.

Dance D. 2016. Melioidosis parotitis in children. J Venom Anim Toxins Incl Trop Dis, 22 (1), pp. 33. | Citations: 1 (Scopus) | Show Abstract | Read more

A recent paper published in JVATiTD reporting a child in Hainan with parotitis caused by Burkholderia pseudomallei misleadingly described parotitis as a rare manifestation of melioidosis. In fact, it is one of the commonest forms of paediatric melioidosis seen in other parts of Southeast Asia, although interestingly not in Australia.

White NJ. 2016. Why Do Some Primate Malarias Relapse? Trends Parasitol, 32 (12), pp. 918-920. | Show Abstract | Read more

Relapse may have evolved in malaria as a mechanism to avoid suppression by more virulent species in mixed infections, thereby increasing transmission opportunities. Later evolution of long latency in Plasmodium vivax was a necessary adaptation as early hominins moved to colder areas with shorter mosquito breeding seasons. Genetic diversity was maintained through heterologous hypnozoite activation.

Rongkard P, Hantrakun V, Dittrich S, Srilohasin P, Amornchai P, Langla S, Lim C, Day NP, AuCoin D, Wuthiekanun V, Limmathurotsakul D. 2016. Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples. PLoS Negl Trop Dis, 10 (12), pp. e0005204. | Show Abstract | Read more

BACKGROUND: Culture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples. METHODOLOGY/PRINCIPAL FINDINGS: First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei. CONCLUSIONS/SIGNIFICANCE: The LFI can be used to detect B. pseudomallei in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis in resource-limited settings.

Parker DM, Landier J, von Seidlein L, Dondorp A, White L, Hanboonkunupakarn B, Maude RJ, Nosten FH. 2016. Limitations of malaria reactive case detection in an area of low and unstable transmission on the Myanmar-Thailand border. Malar J, 15 (1), pp. 571. | Show Abstract | Read more

BACKGROUND: Reactive case detection is an approach that has been proposed as a tool for malaria elimination in low-transmission settings. It is an intuitively justified approach based on the concept of space-time clustering of malaria cases. When an index malaria clinical case is detected, it triggers reactive screening and treatment in the index house and neighbouring houses. However, the efficacy of this approach at varying screening radii and malaria prevalence remains ill defined. METHODS: Data were obtained from a detailed demographic and geographic surveillance study in four villages on the Myanmar-Thailand border. Clinical cases were recorded at village malaria clinics and were linked back to patients' residencies. These data were used to simulate the efficacy of reactive case detection for clinical cases using rapid diagnostic tests (RDT). Simulations took clinical cases in a given month and tabulated the number of cases that would have been detected in the following month at varying screening radii around the index houses. Simulations were run independently for both falciparum and vivax malaria. Each simulation of a reactive case detection effort was run in comparison with a strategy using random selection of houses for screening. RESULTS: In approximately half of the screenings for falciparum and 10% for vivax it would have been impossible to detect any malaria cases regardless of the screening strategy because the screening would have occurred during times when there were no cases. When geographically linked cases were present in the simulation, reactive case detection would have only been successful at detecting most malaria cases using larger screening radii (150-m radius and above). At this screening radius and above, reactive case detection does not perform better than random screening of an equal number of houses in the village. Screening within very small radii detects only a very small proportion of cases, but despite this low performance is better than random screening with the same sample size. CONCLUSIONS: The results of these simulations indicate that reactive case detection for clinical cases using RDTs has limited ability in halting transmission in regions of low and unstable transmission. This is linked to high spatial heterogeneity of cases, acquisition of malaria infections outside the village, as well missing asymptomatic infections. When cases are few and sporadic, reactive case detection would result in major time and budgetary losses.

Wuthiekanun V, White NJ, Amornchai P, Day NP, Limmathurotsakul D. 2016. Quality controls for antimicrobial disk diffusion testing on Leptospira Vanaporn Wuthiekanun agar. Trans R Soc Trop Med Hyg, 110 (11), pp. 673-675. | Read more

Turner P, Kloprogge S, Miliya T, Soeng S, Tan P, Sar P, Yos P, Moore CE, Wuthiekanun V, Limmathurotsakul D et al. 2016. A retrospective analysis of melioidosis in Cambodian children, 2009-2013. BMC Infect Dis, 16 (1), pp. 688. | Show Abstract | Read more

BACKGROUND: Melioidiosis, infection by Burkholderia pseudomallei, is an important but frequently under-recognised cause of morbidity and mortality in Southeast Asia and elsewhere in the tropics. Data on the epidemiology of paediatric melioidosis in Cambodia are extremely limited. METHODS: Culture-positive melioidosis cases presenting to Angkor Hospital for Children, a non-governmental paediatric hospital located in Siem Reap, Northern Cambodia, between 1(st) January 2009 and 31(st) December 2013 were identified by searches of hospital and laboratory databases and logbooks. RESULTS: One hundred seventy-three evaluable cases were identified, presenting from eight provinces. For Siem Reap province, the median commune level incidence was estimated to be 28-35 cases per 100,000 children <15 years per year. Most cases presented during the wet season, May to October. The median age at presentation was 5.7 years (range 8 days-15.9 years). Apart from undernutrition, co-morbidities were rare. Three quarters (131/173) of the children had localised infection, most commonly skin/soft tissue infection (60 cases) or suppurative parotitis (51 cases). There were 39 children with B. pseudomallei bacteraemia: 29 (74.4%) of these had clinical and/or radiological evidence of pneumonia. Overall mortality was 16.8% (29/173) with mortality in bacteraemic cases of 71.8% (28/39). At least seven children did not receive an antimicrobial with activity against B. pseudomallei prior to death. CONCLUSIONS: This retrospective study demonstrated a considerable burden of melioidosis in Cambodian children. Given the high mortality associated with bacteraemic infection, there is an urgent need for greater awareness amongst healthcare professionals in Cambodia and other countries where melioidosis is known or suspected to be endemic. Empiric treatment guidelines should ensure suspected cases are treated early with appropriate antimicrobials.

Chaguza C, Cornick JE, Harris SR, Andam CP, Bricio-Moreno L, Yang M, Yalcin F, Ousmane S, Govindpersad S, Senghore M et al. 2016. Understanding pneumococcal serotype 1 biology through population genomic analysis. BMC Infect Dis, 16 (1), pp. 649. | Show Abstract | Read more

BACKGROUND: Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries. METHODS: We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes. RESULTS: Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat pC194), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1's rarity in carriage and consequently its lower recombination rates. CONCLUSIONS: The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates.

Srimuang K, Miotto O, Lim P, Fairhurst RM, Kwiatkowski DP, Woodrow CJ, Imwong M, Tracking Resistance to Artemisinin Collaboration. 2016. Analysis of anti-malarial resistance markers in pfmdr1 and pfcrt across Southeast Asia in the Tracking Resistance to Artemisinin Collaboration. Malar J, 15 (1), pp. 541. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Declining anti-malarial efficacy of artemisinin-based combination therapy, and reduced Plasmodium falciparum susceptibility to individual anti-malarials are being documented across an expanding area of Southeast Asia (SEA). Genotypic markers complement phenotypic studies in assessing the efficacy of individual anti-malarials. METHODS: The markers pfmdr1 and pfcrt were genotyped in parasite samples obtained in 2011-2014 at 14 TRAC (Tracking Resistance to Artemisinin Collaboration) sites in mainland Southeast Asia using a combination of PCR and next-generation sequencing methods. RESULTS: Pfmdr1 amplification, a marker of mefloquine and lumefantrine resistance, was highly prevalent at Mae Sot on the Thailand-Myanmar border (59.8% of isolates) and common (more than 10%) at sites in central Myanmar, eastern Thailand and western Cambodia; however, its prevalence was lower than previously documented in Pailin, western Cambodia. The pfmdr1 Y184F mutation was common, particularly in and around Cambodia, and the F1226Y mutation was found in about half of samples in Mae Sot. The functional significance of these two mutations remains unclear. Other previously documented pfmdr1 mutations were absent or very rare in the region. The pfcrt mutation K76T associated with chloroquine resistance was found in 98.2% of isolates. The CVIET haplotype made up 95% or more of isolates in western SEA while the CVIDT haplotype was common (30-40% of isolates) in north and northeastern Cambodia, southern Laos, and southern Vietnam. CONCLUSIONS: These findings generate cause for concern regarding the mid-term efficacy of artemether-lumefantrine in Myanmar, while the absence of resistance-conferring pfmdr1 mutations and SVMNT pfcrt haplotypes suggests that amodiaquine could be an efficacious component of anti-malarial regimens in SEA.

Dondorp AM, Hoang MN, Mer M, Sepsis in Resource-Limited Settings-Expert Consensus Recommendations Group of the European Society of Intensive Care Medicine (ESICM) and the Mahidol-Oxford Research Unit (MORU) in Bangkok, Thailand. 2016. Recommendations for the management of severe malaria and severe dengue in resource-limited settings. Intensive Care Med, pp. 1-3. | Read more

Stirnemann J, Villar J, Salomon LJ, Ohuma E, Ruyan P, Altman DG, Nosten F, Craik R, Munim S, Cheikh Ismail L et al. 2016. International estimated fetal weight standards of the INTERGROWTH-21(st) Project. Ultrasound Obstet Gynecol, | Show Abstract | Read more

OBJECTIVE: Estimated fetal weight (EFW) and fetal biometry are complementary measures used to screen for fetal growth disturbances. Our aim was to provide international EFW standards to complement the INTERGROWTH-21(st) Fetal Growth Standards that are available for use worldwide. METHODS: Women with an accurate gestational-age assessment, who were enrolled in the prospective, international, multicenter, population-based Fetal Growth Longitudinal Study (FGLS) and INTERBIO-21(st) Fetal Study (FS), two components of the INTERGROWTH-21(st) Project, had ultrasound scans every 5 weeks from 9-14 weeks' until 40 weeks' gestation. At each visit, measurements of fetal head circumference (HC), biparietal diameter, occipitofrontal diameter, abdominal circumference (AC) and femur length (FL) were obtained blindly by dedicated research sonographers using standardized methods and identical ultrasound machines. Birth weight was measured within 12 h of delivery by dedicated research anthropometrists using standardized methods and identical electronic scales. Live babies without any congenital abnormality, who were born within 14 days of the last ultrasound scan, were selected for inclusion. As most births occurred at around 40 weeks' gestation, we constructed a bootstrap model selection and estimation procedure based on resampling of the complete dataset under an approximately uniform distribution of birth weight, thus enriching the sample size at extremes of fetal sizes, to achieve consistent estimates across the full range of fetal weight. We constructed reference centiles using second-degree fractional polynomial models. RESULTS: Of the overall population, 2404 babies were born within 14 days of the last ultrasound scan. Mean time between the last scan and birth was 7.7 (range, 0-14) days and was uniformly distributed. Birth weight was best estimated as a function of AC and HC (without FL) as log(EFW) = 5.084820 - 54.06633 × (AC/100)(3)  - 95.80076 × (AC/100)(3)  × log(AC/100) + 3.136370 × (HC/100), where EFW is in g and AC and HC are in cm. All other measures, gestational age, symphysis-fundus height, amniotic fluid indices and interactions between biometric measures and gestational age, were not retained in the selection process because they did not improve the prediction of EFW. Applying the formula to FGLS biometric data (n = 4231) enabled gestational age-specific EFW tables to be constructed. At term, the EFW centiles matched those of the INTERGROWTH-21(st) Newborn Size Standards but, at < 37 weeks' gestation, the EFW centiles were, as expected, higher than those of babies born preterm. Comparing EFW cross-sectional values with the INTERGROWTH-21(st) Preterm Postnatal Growth Standards confirmed that preterm postnatal growth is a different biological process from intrauterine growth. CONCLUSIONS: We provide an assessment of EFW, as an adjunct to routine ultrasound biometry, from 22 to 40 weeks' gestation. However, we strongly encourage clinicians to evaluate fetal growth using separate biometric measures such as HC and AC, as well as EFW, to avoid the minimalist approach of focusing on a single value. © 2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Adhikari B, James N, Newby G, von Seidlein L, White NJ, Day NP, Dondorp AM, Pell C, Cheah PY. 2016. Community engagement and population coverage in mass anti-malarial administrations: a systematic literature review. Malar J, 15 (1), pp. 523. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Mass anti-malarial administration has been proposed as a key component of the malaria elimination strategy in South East Asia. The success of this approach depends on the local malaria epidemiology, nature of the anti-malarial regimen and population coverage. Community engagement is used to promote population coverage but little research has systematically analysed its impact. This systematic review examines population coverage and community engagement in programmes of mass anti-malarial drug administration. METHODS: This review builds on a previous review that identified 3049 articles describing mass anti-malarial administrations published between 1913 and 2011. Further search and application of a set of criteria conducted in the current review resulted in 51 articles that were retained for analysis. These 51 papers described the population coverage and/or community engagement in mass anti-malarial administrations. Population coverage was quantitatively assessed and a thematic analysis was conducted on the community engagement activities. RESULTS: The studies were conducted in 26 countries: in diverse healthcare and social contexts where various anti-malarial regimens under varied study designs were administered. Twenty-eight articles reported only population coverage; 12 described only community engagement activities; and 11 community engagement and population coverage. Average population coverage was 83% but methods of calculating coverage were frequently unclear or inconsistent. Community engagement activities included providing health education and incentives, using community structures (e.g. existing hierarchies or health infrastructure), mobilizing human resources, and collaborating with government at some level (e.g. ministries of health). Community engagement was often a process involving various activities throughout the duration of the intervention. CONCLUSION: The mean population coverage was over 80% but incomplete reporting of calculation methods limits conclusions and comparisons between studies. Various community engagement activities and approaches were described, but many articles contained limited or no details. Other factors relevant to population coverage, such as the social, cultural and study context were scarcely reported. Further research is needed to understand the factors that influence population coverage and adherence in mass anti-malarial administrations and the role community engagement activities and approaches play in satisfactory participation.

Hanboonkunupakarn B, White NJ. 2016. The threat of artemisinin resistant malaria in Southeast Asia. Travel Med Infect Dis, 14 (6), pp. 548-550. | Citations: 1 (Scopus) | Read more

Leang R, Khu NH, Mukaka M, Debackere M, Tripura R, Kheang ST, Chy S, Kak N, Buchy P, Tarantola A et al. 2016. An optimised age-based dosing regimen for single low-dose primaquine for blocking malaria transmission in Cambodia. BMC Med, 14 (1), pp. 171. | Citations: 2 (Scopus) | Show Abstract | Read more

BACKGROUND: In 2012, the World Health Organization recommended the addition of single low-dose primaquine (SLDPQ, 0.25 mg base/kg body weight) to artemisinin combination therapies to block the transmission of Plasmodium falciparum without testing for glucose-6-phosphate dehydrogenase deficiency. The targeted group was non-pregnant patients aged ≥ 1 year (later changed to ≥ 6 months) with acute uncomplicated falciparum malaria, primarily in countries with artemisinin-resistant P. falciparum (ARPf). No dosing regimen was suggested, leaving malaria control programmes and clinicians in limbo. Therefore, we designed a user-friendly, age-based SLDPQ regimen for Cambodia, the country most affected by ARPf. METHODS: By reviewing primaquine's pharmacology, we defined a therapeutic dose range of 0.15-0.38 mg base/kg (9-22.5 mg in a 60-kg adult) for a therapeutic index of 2.5. Primaquine doses (1-20 mg) were tested using a modelled, anthropometric database of 28,138 Cambodian individuals (22,772 healthy, 4119 with malaria and 1247 with other infections); age distributions were: 0.5-4 years (20.0 %, n = 5640), 5-12 years (9.1 %, n = 2559), 13-17 years (9.1 %, n = 2550), and ≥ 18 years (61.8 %, n = 17,389). Optimal age-dosing groups were selected according to calculated mg base/kg doses and proportions of individuals receiving a therapeutic dose. RESULTS: Four age-dosing bands were defined: (1) 0.5-4 years, (2) 5-9 years, (3) 10-14 years, and (4) ≥15 years to receive 2.5, 5, 7.5, and 15 mg of primaquine base, resulting in therapeutic doses in 97.4 % (5494/5640), 90.5 % (1511/1669), 97.7 % (1473/1508), and 95.7 % (18,489/19,321) of individuals, respectively. Corresponding median (1st-99th centiles) mg base/kg doses of primaquine were (1) 0.23 (0.15-0.38), (2) 0.29 (0.18-0.45), (3) 0.27 (0.15-0.39), and (4) 0.29 (0.20-0.42). CONCLUSIONS: This age-based SLDPQ regimen could contribute substantially to malaria elimination and requires urgent evaluation in Cambodia and other countries with similar anthropometric characteristics. It guides primaquine manufacturers on suitable tablet strengths and doses for paediatric-friendly formulations. Development of similar age-based dosing recommendations for Africa is needed.

Grist EP, Flegg JA, Humphreys G, Mas IS, Anderson TJ, Ashley EA, Day NP, Dhorda M, Dondorp AM, Faiz MA et al. 2016. Optimal health and disease management using spatial uncertainty: a geographic characterization of emergent artemisinin-resistant Plasmodium falciparum distributions in Southeast Asia. Int J Health Geogr, 15 (1), pp. 37. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Artemisinin-resistant Plasmodium falciparum malaria parasites are now present across much of mainland Southeast Asia, where ongoing surveys are measuring and mapping their spatial distribution. These efforts require substantial resources. Here we propose a generic 'smart surveillance' methodology to identify optimal candidate sites for future sampling and thus map the distribution of artemisinin resistance most efficiently. METHODS: The approach uses the 'uncertainty' map generated iteratively by a geostatistical model to determine optimal locations for subsequent sampling. RESULTS: The methodology is illustrated using recent data on the prevalence of the K13-propeller polymorphism (a genetic marker of artemisinin resistance) in the Greater Mekong Subregion. CONCLUSION: This methodology, which has broader application to geostatistical mapping in general, could improve the quality and efficiency of drug resistance mapping and thereby guide practical operations to eliminate malaria in affected areas.

Bang ND, Caws M, Truc TT, Duong TN, Dung NH, Ha DT, Thwaites GE, Heemskerk D, Tarning J, Merson L et al. 2016. Clinical presentations, diagnosis, mortality and prognostic markers of tuberculous meningitis in Vietnamese children: a prospective descriptive study. BMC Infect Dis, 16 (1), pp. 573. | Show Abstract | Read more

BACKGROUND: Tuberculous meningitis in adults is well characterized in Vietnam, but there are no data on the disease in children. We present a prospective descriptive study of Vietnamese children with TBM to define the presentation, course and characteristics associated with poor outcome. METHODS: A prospective descriptive study of 100 consecutively admitted children with TBM at Pham Ngoc Thach Hospital, Ho Chi Minh City. Cox and logistic regression were used to identify factors associated with risk of death and a combined endpoint of death or disability at treatment completion. RESULTS: The study enrolled from October 2009 to March 2011. Median age was 32.5 months; sex distribution was equal. Median duration of symptoms was 18.5 days and time from admission to treatment initiation was 11 days. Fifteen of 100 children died, 4 were lost to follow-up, and 27/81 (33 %) of survivors had intermediate or severe disability upon treatment completion. Microbiological confirmation of disease was made in 6 %. Baseline characteristics associated with death included convulsions (HR 3.46, 95CI 1.19-10.13, p = 0.02), decreased consciousness (HR 22.9, 95CI 3.01-174.3, p < 0.001), focal neurological deficits (HR 15.7, 95CI 1.67-2075, p = 0.01), Blantyre Coma Score (HR 3.75, 95CI 0.99-14.2, p < 0.001) and CSF protein, lactate and glucose levels. Neck stiffness, MRC grade (children aged >5 years) and hydrocephalus were also associated with the combined endpoint of death or disability. CONCLUSIONS: Tuberculous meningitis in Vietnamese children has significant mortality and morbidity. There is significant delay in diagnosis; interventions that increase the speed of diagnosis and treatment initiation are likely to improve outcomes.

Maude RR, Ghose A, Samad R, de Jong HK, Fukushima M, Wijedoru L, Hassan MU, Hossain MA, Karim MR, Sayeed AA et al. 2016. A prospective study of the importance of enteric fever as a cause of non-malarial febrile illness in patients admitted to Chittagong Medical College Hospital, Bangladesh. BMC Infect Dis, 16 (1), pp. 567. | Show Abstract | Read more

BACKGROUND: Fever is a common cause of hospital admission in Bangladesh but causative agents, other than malaria, are not routinely investigated. Enteric fever is thought to be common. METHODS: Adults and children admitted to Chittagong Medical College Hospital with a temperature of ≥38.0 °C were investigated using a blood smear for malaria, a blood culture, real-time PCR to detect Salmonella Typhi, S. Paratyphi A and other pathogens in blood and CSF and an NS1 antigen dengue ELISA. RESULTS: We enrolled 300 febrile patients with a negative malaria smear between January and June 2012: 156 children (aged ≤15 years) and 144 adults with a median (interquartile range) age of 13 (5-31) years and median (IQR) illness duration before admission of five (2-8) days. Clinical enteric fever was diagnosed in 52 patients (17.3 %), lower respiratory tract infection in 48 (16.0 %), non-specific febrile illness in 48 (16.0 %), a CNS infection in 37 patients (12.3 %), urinary sepsis in 23 patients (7.7 %), an upper respiratory tract infection in 21 patients (7.0 %), and diarrhea or dysentery in 21 patients (7.0 %). Malaria was still suspected in seven patients despite a negative microscopy test. S. Typhi was detected in blood by culture or PCR in 34 (11.3 %) of patients. Of note Rickettsia typhi and Orientia tsutsugamushi were detected by PCR in two and one patient respectively. Twenty-nine (9 %) patients died during their hospital admission (15/160 (9.4 %) of children and 14/144 (9.7 %) adults). Two of 52 (3.8 %) patients with enteric fever, 5/48 (10.4 %) patients with lower respiratory tract infections, and 12/37 (32.4 %) patients with CNS infection died. CONCLUSION: Enteric fever was confirmed in 11.3 % of patients admitted to this hospital in Bangladesh with non-malaria fever. Lower respiratory tract and CNS infections were also common. CNS infections in this location merit more detailed study due to the high mortality.

Thwaites CL, Lundeg G, Dondorp AM, sepsis in resource-limited settings–expert consensus recommendations group* of the European Society of Intensive Care Medicine (ESICM) and the Mahidol-Oxford Research Unit (MORU) in Bangkok, Thailand. 2016. Infection management in patients with sepsis and septic shock in resource-limited settings. Intensive Care Med, 42 (12), pp. 2117-2118. | Read more

Baird JK, Valecha N, Duparc S, White NJ, Price RN. 2016. Diagnosis and Treatment of Plasmodium vivax Malaria. Am J Trop Med Hyg, 95 (6 Suppl), pp. 35-51. | Show Abstract | Read more

The diagnosis and treatment of Plasmodium vivax malaria differs from that of Plasmodium falciparum malaria in fundamentally important ways. This article reviews the guiding principles, practices, and evidence underpinning the diagnosis and treatment of P. vivax malaria.

Wong VK, Baker S, Connor TR, Pickard D, Page AJ, Dave J, Murphy N, Holliman R, Sefton A, Millar M et al. 2016. An extended genotyping framework for Salmonella enterica serovar Typhi, the cause of human typhoid. Nat Commun, 7 pp. 12827. | Citations: 1 (Scopus) | Show Abstract | Read more

The population of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, exhibits limited DNA sequence variation, which complicates efforts to rationally discriminate individual isolates. Here we utilize data from whole-genome sequences (WGS) of nearly 2,000 isolates sourced from over 60 countries to generate a robust genotyping scheme that is phylogenetically informative and compatible with a range of assays. These data show that, with the exception of the rapidly disseminating H58 subclade (now designated genotype 4.3.1), the global S. Typhi population is highly structured and includes dozens of subclades that display geographical restriction. The genotyping approach presented here can be used to interrogate local S. Typhi populations and help identify recent introductions of S. Typhi into new or previously endemic locations, providing information on their likely geographical source. This approach can be used to classify clinical isolates and provides a universal framework for further experimental investigations.

Cheah PY, Newton PN, Mayxay M. 2016. The first Science Café in Laos. Lancet, 388 (10052), pp. 1376. | Read more

Hantrakun V, Rongkard P, Oyuchua M, Amornchai P, Lim C, Wuthiekanun V, Day NP, Peacock SJ, Limmathurotsakul D. 2016. Soil Nutrient Depletion Is Associated with the Presence of Burkholderia pseudomallei. Appl Environ Microbiol, 82 (24), pp. 7086-7092. | Show Abstract | Read more

Burkholderia pseudomallei is a soil-dwelling bacterium and the cause of melioidosis, which kills an estimated 89,000 people per year worldwide. Agricultural workers are at high risk of infection due to repeated exposure to the bacterium. Little is known about the soil physicochemical properties associated with the presence or absence of the organism. Here, we evaluated the soil physicochemical properties and presence of B. pseudomallei in 6,100 soil samples collected from 61 rice fields in Thailand. The presence of B. pseudomallei was negatively associated with the proportion of clay, proportion of moisture, level of salinity, percentage of organic matter, presence of cadmium, and nutrient levels (phosphorus, potassium, calcium, magnesium, and iron). The presence of B. pseudomallei was not associated with the level of soil acidity (P = 0.54). In a multivariable logistic regression model, the presence of B. pseudomallei was negatively associated with the percentage of organic matter (odds ratio [OR], 0.06; 95% confidence interval [CI], 0.01 to 0.47; P = 0.007), level of salinity (OR, 0.06; 95% CI, 0.01 to 0.74; P = 0.03), and percentage of soil moisture (OR, 0.81; 95% CI, 0.66 to 1.00; P = 0.05). Our study suggests that B. pseudomallei thrives in rice fields that are nutrient depleted. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected areas. IMPORTANCE: Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis. Humans acquire the disease following skin inoculation, inhalation, or ingestion of the bacterium in the environment. The presence of B. pseudomallei in soil defines geographic regions where humans and livestock are at risk of melioidosis, yet little is known about the soil properties associated with the presence of the organism. We evaluated the soil properties and presence of B. pseudomallei in 61 rice fields in East, Central, and Northeast Thailand. We demonstrated that the organism was more commonly found in soils with lower levels of organic matter and nutrients, including phosphorus, potassium, calcium, magnesium, and iron. We also demonstrated that crop residue burning after harvest, which can reduce soil nutrients, was not uncommon. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected areas.

Bernier MC, Li F, Musselman B, Newton PN, Fernández FM. 2016. Fingerprinting of falsified artemisinin combination therapies via direct analysis in real time coupled to a compact single quadrupole mass spectrometer Anal. Methods, 8 (36), pp. 6616-6624. | Show Abstract | Read more

© The Royal Society of Chemistry 2016.Falsified anti-malarial treatments continue to constitute a major health crisis, especially in malarious Africa. Even after detection of poor quality pharmaceuticals, it is critical that they be fully analyzed to determine their components, in order to assess their health effects and ultimately allow forensic tracing of their sources of production and distribution. Timely assessment requires robust and complete field-testing, or at the very least timely analysis after seizure or purchase. Ideally, low-cost and simple analytical equipment such as portable mass spectrometry (MS) is the best approach for achieving this quick and informative analysis. To date, Direct Analysis in Real Time (DART) MS has been successfully implemented to rapidly analyze falsified artemisinin-based combination therapies (ACTs) in laboratory settings, but this approach typically translates into high-cost and the need for high-resolution instrumentation. Here, we examine the use of DART ionization coupled with a portable low-resolution single-quadrupole instrument, and compare its success in fingerprinting anti-malarial tablets with higher resolution instrumentation. Using single quadrupole DART-MS, the same sample components were detected as with the high-resolution instrument, while needing significantly less consumables and power, and the additional advantages of increased portability and ease of use. Using Principal Component Analysis (PCA) of DART data, specific classes of falsified ACTs were identified, providing a more straightforward method for sourcing counterfeits and assessing their similarities.

Ravinetto R, De Weggheleire A, Dorlo TP, Francque S, Sokkab A, Pouget C, Meessen B, Tabernero P, Newton PN, Lynen L. 2016. Predictable threats to public health through delaying universal access to innovative medicines for hepatitis C: a pharmaceutical standpoint. Trop Med Int Health, 21 (12), pp. 1490-1495. | Citations: 1 (Scopus) | Read more

Poovorawan K, Pan-Ngum W, White LJ, Soonthornworasiri N, Wilairatana P, Wasitthankasem R, Tangkijvanich P, Poovorawan Y. 2016. Estimating the Impact of Expanding Treatment Coverage and Allocation Strategies for Chronic Hepatitis C in a Direct Antiviral Agent Era. PLoS One, 11 (9), pp. e0163095. | Show Abstract | Read more

Hepatitis C virus (HCV) infection is an important worldwide public health problem, and most of the global HCV burden is in low- to middle-income countries. This study aimed to estimate the future burden of chronic hepatitis C (CHC) and the impact of public health policies using novel antiviral agents in Thailand. A mathematical model of CHC transmission dynamics was constructed to examine the disease burden over the next 20 years using different treatment strategies. We compared and evaluated the current treatment (PEGylated interferon and ribavirin) with new treatments using novel direct-acting antiviral agents among various treatment policies. Thailand's CHC prevalence was estimated to decrease 1.09%-0.19% in 2015-2035. Expanding treatment coverage (i.e., a five-fold increment in treatment accessibility) was estimated to decrease cumulative deaths (33,007 deaths avoided, 25.5% reduction) from CHC-related decompensated cirrhosis and hepatocellular carcinoma (HCC). The yearly incidence of HCC-associated HCV was estimated to decrease from 2,305 to 1,877 cases yearly with expanding treatment coverage. A generalized treatment scenario (i.e., an equal proportional distribution of available treatment to individuals at all disease stages according to the number of cases at each stage) was predicted to further reduce death from HCC (9,170 deaths avoided, 11.3% reduction) and the annual incidence of HCC (i.e., a further decrease from 1,877 to 1,168 cases yearly, 37.7% reduction), but cumulative deaths were predicted to increase (by 3,626 deaths, 3.7% increase). Based on the extensive coverage scenario and the generalized treatment scenario, we estimated near-zero death from decompensated cirrhosis in 2031. In conclusion, CHC-related morbidity and mortality in Thailand are estimated to decrease dramatically over the next 20 years. Treatment coverage and allocation strategies are important factors that affect the future burden of CHC in resource-limited countries like Thailand.

Brown A, Halliday JS, Swadling L, Madden RG, Bendall R, Hunter JG, Maggs J, Simmonds P, Smith DB, Vine L et al. 2016. Characterization of the Specificity, Functionality, and Durability of Host T-Cell Responses Against the Full-Length Hepatitis E Virus. Hepatology, 64 (6), pp. 1934-1950. | Show Abstract | Read more

The interplay between host antiviral immunity and immunopathology during hepatitis E virus (HEV) infection determines important clinical outcomes. We characterized the specificity, functionality, and durability of host T-cell responses against the full-length HEV virus and assessed a novel "Quantiferon" assay for the rapid diagnosis of HEV infection. Eighty-nine volunteers were recruited from Oxford, Truro (UK), and Toulouse (France), including 44 immune-competent patients with acute HEV infection, 18 HEV-exposed immunosuppressed organ-transplant recipients (8 with chronic HEV), and 27 healthy volunteers. A genotype 3a peptide library (616 overlapping peptides spanning open reading frames [ORFs] 1-3) was used in interferon-gamma (IFN-γ) T-cell ELISpot assays. CD4(+) /CD8(+) T-cell subsets and polyfunctionality were defined using ICCS and SPICE analysis. Quantification of IFN-γ used whole-blood stimulation with recombinant HEV-capsid protein in the QuantiFERON kit. HEV-specific T-cell responses were detected in 41/44 immune-competent HEV exposed volunteers (median magnitude: 397 spot-forming units/10(6) peripheral blood mononuclear cells), most frequently targeting ORF2. High-magnitude, polyfunctional CD4 and CD8(+) T cells were detected during acute disease and maintained to 12 years, but these declined over time, with CD8(+) responses becoming more monofunctional. Low-level responses were detectable in immunosuppressed patients. Twenty-three novel HEV CD4(+) and CD8(+) T-cell targets were mapped predominantly to conserved genomic regions. QuantiFERON testing demonstrated an inverse correlation between IFN-γ production and the time from clinical presentation, providing 100% specificity, and 71% sensitivity (area under the receiver operator characteristic curve of 0.86) for HEV exposure at 0.3 IU/mL. CONCLUSION: Robust HEV-specific T-cell responses generated during acute disease predominantly target ORF2, but decline in magnitude and polyfunctionality over time. Defining HEV T-cell targets will be important for the investigation of HEV-associated autoimmune disease. (Hepatology 2016;64:1934-1950).

Cheah PY, Parker M, Dondorp AM. 2016. Development of drugs for severe malaria in children. Int Health, 8 (5), pp. 313-316. | Citations: 1 (Scopus) | Show Abstract | Read more

Over 90% of deaths attributable to malaria are in African children under 5 years old. Yet, new treatments are often tested primarily in adult patients and extrapolations have proven to be sometimes invalid, especially in dosing regimens. For studies in severe malaria an additional complication is that the decline in severe malaria in adult patients precludes sufficiently powered trials in adults, before the intervention can be tested in the ultimate target group, paediatric severe malaria. In this paper we propose an alternative pathway to the development of drugs for use in paediatric severe malaria. We argue that following the classical phase I and II studies, small safety and efficacy studies using well-chosen surrogate endpoints in adult severe malaria be conducted, instead of larger mortality endpoint trials. If the drug appears safe and promising small pilot studies in paediatric severe malaria using the same endpoints can follow. Finally, with carefully observed safeguards in place to ensure high ethical standards, promising candidate interventions can be taken forward into mortality endpoint, well-powered, large paediatric studies in African children with severe malaria. Given the available research capacity, limited numbers of prudently selected interventions can be studied in phase III trials, and adaptive designs should be considered.

Weitzel T, Dittrich S, López J, Phuklia W, Martinez-Valdebenito C, Velásquez K, Blacksell SD, Paris DH, Abarca K. 2016. Endemic Scrub Typhus in South America. N Engl J Med, 375 (10), pp. 954-961. | Citations: 1 (Scopus) | Show Abstract | Read more

Scrub typhus is a life-threatening zoonosis caused by Orientia tsutsugamushi organisms that are transmitted by the larvae of trombiculid mites. Endemic scrub typhus was originally thought to be confined to the so called "tsutsugamushi triangle" within the Asia-Pacific region. In 2006, however, two individual cases were detected in the Middle East and South America, which suggested that the pathogen was present farther afield. Here, we report three autochthonous cases of scrub typhus caused by O. tsutsugamushi acquired on Chiloé Island in southern Chile, which suggests the existence of an endemic focus in South America. (Funded by the Chilean Comisión Nacional de Investigación Científica y Tecnológica and the Wellcome Trust.).

Lim C, Takahashi E, Hongsuwan M, Wuthiekanun V, Thamlikitkul V, Hinjoy S, Day NP, Peacock SJ, Limmathurotsakul D. 2016. Epidemiology and burden of multidrug-resistant bacterial infection in a developing country. Elife, 5 (September), | Show Abstract | Read more

Little is known about the excess mortality caused by multidrug-resistant (MDR) bacterial infection in low- and middle-income countries (LMICs). We retrospectively obtained microbiology laboratory and hospital databases of nine public hospitals in northeast Thailand from 2004 to 2010, and linked these with the national death registry to obtain the 30-day mortality outcome. The 30-day mortality in those with MDR community-acquired bacteraemia, healthcare-associated bacteraemia, and hospital-acquired bacteraemia were 35% (549/1555), 49% (247/500), and 53% (640/1198), respectively. We estimate that 19,122 of 45,209 (43%) deaths in patients with hospital-acquired infection due to MDR bacteria in Thailand in 2010 represented excess mortality caused by MDR. We demonstrate that national statistics on the epidemiology and burden of MDR in LMICs could be improved by integrating information from readily available databases. The prevalence and mortality attributable to MDR in Thailand are high. This is likely to reflect the situation in other LMICs.

Phyo AP, Ashley EA, Anderson TJ, Bozdech Z, Carrara VI, Sriprawat K, Nair S, White MM, Dziekan J, Ling C et al. 2016. Declining Efficacy of Artemisinin Combination Therapy Against P. Falciparum Malaria on the Thai-Myanmar Border (2003-2013): The Role of Parasite Genetic Factors. Clin Infect Dis, 63 (6), pp. 784-791. | Citations: 4 (Scopus) | Show Abstract | Read more

BACKGROUND: Deployment of mefloquine-artesunate (MAS3) on the Thailand-Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial. METHODS: Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance. RESULTS: Polymerase chain reaction (PCR)-adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8% (95% confidence interval [CI], 45.4, 68.3) compared with 97.8% (95% CI, 93.3, 99.3) in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P = .009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%. CONCLUSIONS: The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand-Myanmar border.

White NJ, Duong TT, Uthaisin C, Nosten F, Phyo AP, Hanboonkunupakarn B, Pukrittayakamee S, Jittamala P, Chuthasmit K, Cheung MS et al. 2016. Antimalarial Activity of KAF156 in Falciparum and Vivax Malaria. N Engl J Med, 375 (12), pp. 1152-1160. | Citations: 2 (Scopus) | Show Abstract | Read more

Background KAF156 belongs to a new class of antimalarial agents (imidazolopiperazines), with activity against asexual and sexual blood stages and the preerythrocytic liver stages of malarial parasites. Methods We conducted a phase 2, open-label, two-part study at five centers in Thailand and Vietnam to assess the antimalarial efficacy, safety, and pharmacokinetic profile of KAF156 in adults with acute Plasmodium vivax or P. falciparum malaria. Assessment of parasite clearance rates in cohorts of patients with vivax or falciparum malaria who were treated with multiple doses (400 mg once daily for 3 days) was followed by assessment of the cure rate at 28 days in a separate cohort of patients with falciparum malaria who received a single dose (800 mg). Results Median parasite clearance times were 45 hours (interquartile range, 42 to 48) in 10 patients with falciparum malaria and 24 hours (interquartile range, 20 to 30) in 10 patients with vivax malaria after treatment with the multiple-dose regimen and 49 hours (interquartile range, 42 to 54) in 21 patients with falciparum malaria after treatment with the single dose. Among the 21 patients who received the single dose and were followed for 28 days, 1 had reinfection and 7 had recrudescent infections (cure rate, 67%; 95% credible interval, 46 to 84). The mean (±SD) KAF156 terminal elimination half-life was 44.1±8.9 hours. There were no serious adverse events in this small study. The most common adverse events included sinus bradycardia, thrombocytopenia, hypokalemia, anemia, and hyperbilirubinemia. Vomiting of grade 2 or higher occurred in 2 patients, 1 of whom discontinued treatment because of repeated vomiting after receiving the single 800-mg dose. More adverse events were reported in the single-dose cohort, which had longer follow-up, than in the multiple-dose cohorts. Conclusions KAF156 showed antimalarial activity without evident safety concerns in a small number of adults with uncomplicated P. vivax or P. falciparum malaria. (Funded by Novartis and others; number, NCT01753323 .).

Taylor AJ, Vongphayloth K, Vongsouvath M, Grandadam M, Brey PT, Newton PN, Sutherland IW, Dittrich S. 2016. Large-scale survey for tickborne bacteria, Khammouan province, Laos Emerging Infectious Diseases, 22 (9), pp. 1635-1639. | Show Abstract | Read more

© 2016, Centers for Disease Control and Prevention (CDC). All rights reserved.We screened 768 tick pools containing 6,962 ticks from Khammouan Province, Laos, by using quantitative realtime PCR and identified Rickettsia spp., Ehrlichia spp., and Borrelia spp. Sequencing of Rickettsia spp.–positive and Borrelia spp.–positive pools provided evidence for distinct genotypes. Our results identified bacteria with human disease potential in ticks in Laos.

International Typhoid Consortium, Wong VK, Holt KE, Okoro C, Baker S, Pickard DJ, Marks F, Page AJ, Olanipekun G, Munir H et al. 2016. Molecular Surveillance Identifies Multiple Transmissions of Typhoid in West Africa. PLoS Negl Trop Dis, 10 (9), pp. e0004781. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: The burden of typhoid in sub-Saharan African (SSA) countries has been difficult to estimate, in part, due to suboptimal laboratory diagnostics. However, surveillance blood cultures at two sites in Nigeria have identified typhoid associated with Salmonella enterica serovar Typhi (S. Typhi) as an important cause of bacteremia in children. METHODS: A total of 128 S. Typhi isolates from these studies in Nigeria were whole-genome sequenced, and the resulting data was used to place these Nigerian isolates into a worldwide context based on their phylogeny and carriage of molecular determinants of antibiotic resistance. RESULTS: Several distinct S. Typhi genotypes were identified in Nigeria that were related to other clusters of S. Typhi isolates from north, west and central regions of Africa. The rapidly expanding S. Typhi clade 4.3.1 (H58) previously associated with multiple antimicrobial resistances in Asia and in east, central and southern Africa, was not detected in this study. However, antimicrobial resistance was common amongst the Nigerian isolates and was associated with several plasmids, including the IncHI1 plasmid commonly associated with S. Typhi. CONCLUSIONS: These data indicate that typhoid in Nigeria was established through multiple independent introductions into the country, with evidence of regional spread. MDR typhoid appears to be evolving independently of the haplotype H58 found in other typhoid endemic countries. This study highlights an urgent need for routine surveillance to monitor the epidemiology of typhoid and evolution of antimicrobial resistance within the bacterial population as a means to facilitate public health interventions to reduce the substantial morbidity and mortality of typhoid.

Whistler T, Kaewpan A, Blacksell SD. 2016. A Biological Safety Cabinet Certification Program: Experiences in Southeast Asia. Appl Biosaf, 21 (3), pp. 121-127. | Show Abstract | Read more

Biological safety cabinets (BSCs) are the primary means of containment used in laboratories worldwide for the safe handling of infectious microorganisms. They provide protection to the laboratory worker and the surrounding environment from pathogens. To ensure the correct functioning of BSCs, they need to be properly maintained beyond the daily care routines of the laboratory. This involves annual maintenance and certification by a qualified technician in accordance to the NSF/American National Standards Institute 49-2014 Biosafety Cabinetry: Design, Construction, Performance, and Field Certification. Service programs can be direct from the manufacturer or through third-party service companies, but in many instances, technicians are not accredited by international bodies, and these services are expensive. This means that a large number of BSCs may not be operating in a safe manner. In this article, we discuss our approach to addressing the lack of trained and qualified personnel in Thailand who can install, maintain, and certify BSCs in a cost-effective and practical manner. We initiated a program to create both local and regional capacity for repair, maintenance, and certification of BSCs and share our experiences with the reader.

Atwal S, Giengkam S, VanNieuwenhze M, Salje J. 2016. Live imaging of the genetically intractable obligate intracellular bacteria Orientia tsutsugamushi using a panel of fluorescent dyes. J Microbiol Methods, 130 pp. 169-176. | Show Abstract | Read more

Our understanding of the molecular mechanisms of bacterial infection and pathogenesis are disproportionally derived from a small number of well-characterised species and strains. One reason for this is the enormous time and resources required to develop a new organism into experimental system that can be interrogated at the molecular level, in particular with regards to the development of genetic tools. Live cell imaging by fluorescence microscopy is a powerful technique to study biological processes such as bacterial motility, host cell invasion, and bacterial growth and division. In the absence of genetic tools that enable exogenous expression of fluorescent proteins, fluorescent chemical probes can be used to label and track living cells. A large number of fluorescent chemical probes are commercially available, but these have overwhelmingly been applied to the study of eukaryotic cell systems. Here, we present a methodical analysis of four different classes of probes, which can be used to delineate the cytoplasm, nucleic acids, cell membrane or peptidoglycan of living bacterial cells. We have tested these in the context of the important but neglected human pathogen Orientia tsutsugamushi but expect that the methodology would be broadly applicable to other bacterial species.

Phyo AP, Ashley EA, Anderson TJ, Carrara VI, Woodrow CJ, White NJ, Nosten F. 2016. Reply to Meshnick and Hastings et al. Clin Infect Dis, 63 (11), pp. 1528-1529. | Read more

Salisbury P, Hall L, Kulkus S, Paw MK, Tun NW, Min AM, Chotivanich K, Srikanok S, Ontuwong P, Sirinonthachai S et al. 2016. Family planning knowledge, attitudes and practices in refugee and migrant pregnant and post-partum women on the Thailand-Myanmar border - a mixed methods study. Reprod Health, 13 (1), pp. 94. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Lack of data in marginalized populations on knowledge, attitudes and practices (KAP) hampers efforts to improve modern contraceptive practice. A mixed methods study to better understand family planning KAP amongst refugee and migrant women on the Thailand-Myanmar border was conducted as part of an ongoing effort to improve reproductive health, particularly maternal mortality, through Shoklo Malaria Research Unit (SMRU) antenatal and birthing services. METHODS: Cross-sectional surveys and focus group discussions (FGDs) in currently pregnant women; and in-depth interviews (IDIs) in selected post-partum women with three children or more; were conducted. Quantitative data were described with medians and proportions and compared using standard statistical tests. Risk factors associated with high parity (>3) were identified using logistic regression analysis. Qualitative data were coded and grouped and discussed using identified themes. RESULTS: In January-March 2015, 978 women participated in cross-sectional studies, 120 in FGD and 21 in IDI. Major positive findings were: > 90 % of women knew about contraceptives for birth spacing, >60 % of women in the FGD and IDI reported use of family planning (FP) in the past and nearly all women knew where they could obtain FP supplies. Major gaps identified included: low uptake of long acting contraception (LAC), lack of awareness of emergency contraception (>90 % of women), unreliable estimates of when child bearing years end, and misconceptions surrounding female sterilization. Three was identified as the ideal number of children in the cross-sectional survey but less than half of the women with this parity or higher in the IDI actually adopted LAC leaving them at risk for unintended pregnancy. Discussing basic female anatomy using a simple diagram was well received in FGD and IDIs. LAC uptake has increased particularly the IUD from 2013-2015. CONCLUSION: Definitive contextual issues were identified during this study and a significant range of action points have been implemented in FP services at SMRU as a result, particularly in regard to the IUD. The importance of the role and attitudes of husbands were acknowledged by women and studies to investigate male perspectives in future may enhance FP practice in this area.

Chu CS, White NJ. 2016. Management of relapsing Plasmodium vivax malaria. Expert Rev Anti Infect Ther, 14 (10), pp. 885-900. | Citations: 1 (Scopus) | Show Abstract | Read more

INTRODUCTION: Relapses are important contributors to illness and morbidity in Plasmodium vivax and P. ovale infections. Relapse prevention (radical cure) with primaquine is required for optimal management, control and ultimately elimination of Plasmodium vivax malaria. A review was conducted with publications in English, French, Portuguese and Spanish using the search terms 'P. vivax' and 'relapse'. AREAS COVERED: Hypnozoites causing relapses may be activated weeks or months after initial infection. Incidence and temporal patterns of relapse varies geographically. Relapses derive from parasites either genetically similar or different from the primary infection indicating that some derive from previous infections. Malaria illness itself may activate relapse. Primaquine is the only widely available treatment for radical cure. However, it is often not given because of uncertainty over the risks of primaquine induced haemolysis when G6PD deficiency testing is unavailable. Recommended dosing of primaquine for radical cure in East Asia and Oceania is 0.5 mg base/kg/day and elsewhere is 0.25 mg base/kg/day. Alternative treatments are under investigation. Expert commentary: Geographic heterogeneity in relapse patterns and chloroquine susceptibility of P. vivax, and G6PD deficiency epidemiology mean that radical treatment should be given much more than it is today. G6PD testing should be made widely available so primaquine can be given more safely.

Zhang R, Lee WC, Lau YL, Albrecht L, Lopes SC, Costa FT, Suwanarusk R, Nosten F, Cooke BM, Rénia L, Russell B. 2016. Rheopathologic Consequence of Plasmodium vivax Rosette Formation. PLoS Negl Trop Dis, 10 (8), pp. e0004912. | Show Abstract | Read more

Malaria parasites dramatically alter the rheological properties of infected red blood cells. In the case of Plasmodium vivax, the parasite rapidly decreases the shear elastic modulus of the invaded RBC, enabling it to avoid splenic clearance. This study highlights correlation between rosette formation and altered membrane deformability of P. vivax-infected erythrocytes, where the rosette-forming infected erythrocytes are significantly more rigid than their non-rosetting counterparts. The adhesion of normocytes to the PvIRBC is strong (mean binding force of 440pN) resulting in stable rosette formation even under high physiological shear flow stress. Rosetting may contribute to the sequestration of PvIRBC schizonts in the host microvasculature or spleen.

Do NT, Ta NT, Tran NT, Than HM, Vu BT, Hoang LB, van Doorn HR, Vu DT, Cals JW, Chandna A et al. 2016. Point-of-care C-reactive protein testing to reduce inappropriate use of antibiotics for non-severe acute respiratory infections in Vietnamese primary health care: a randomised controlled trial. Lancet Glob Health, 4 (9), pp. e633-e641. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Inappropriate antibiotic use for acute respiratory tract infections is common in primary health care, but distinguishing serious from self-limiting infections is difficult, particularly in low-resource settings. We assessed whether C-reactive protein point-of-care testing can safely reduce antibiotic use in patients with non-severe acute respiratory tract infections in Vietnam. METHOD: We did a multicentre open-label randomised controlled trial in ten primary health-care centres in northern Vietnam. Patients aged 1-65 years with at least one focal and one systemic symptom of acute respiratory tract infection were assigned 1:1 to receive either C-reactive protein point-of-care testing or routine care, following which antibiotic prescribing decisions were made. Patients with severe acute respiratory tract infection were excluded. Enrolled patients were reassessed on day 3, 4, or 5, and on day 14 a structured telephone interview was done blind to the intervention. Randomised assignments were concealed from prescribers and patients but not masked as the test result was used to assist treatment decisions. The primary outcome was antibiotic use within 14 days of follow-up. All analyses were prespecified in the protocol and the statistical analysis plan. All analyses were done on the intention-to-treat population and the analysis of the primary endpoint was repeated in the per-protocol population. This trial is registered under number NCT01918579. FINDINGS: Between March 17, 2014, and July 3, 2015, 2037 patients (1028 children and 1009 adults) were enrolled and randomised. One adult patient withdrew immediately after randomisation. 1017 patients were assigned to receive C-reactive protein point-of-care testing, and 1019 patients were assigned to receive routine care. 115 patients in the C-reactive protein point-of-care group and 72 patients in the routine care group were excluded in the intention-to-treat analysis due to missing primary endpoint. The number of patients who used antibiotics within 14 days was 581 (64%) of 902 patients in the C-reactive protein group versus 738 (78%) of 947 patients in the control group (odds ratio [OR] 0·49, 95% CI 0·40-0·61; p<0·0001). Highly significant differences were seen in both children and adults, with substantial heterogeneity of the intervention effect across the 10 sites (I(2)=84%, 95% CI 66-96). 140 patients in the C-reactive protein group and 137 patients in the routine care group missed the urine test on day 3, 4, or 5. Antibiotic activity in urine on day 3, 4, or 5 was found in 267 (30%) of 877 patients in the C-reactive protein group versus 314 (36%) of 882 patients in the routine treatment group (OR 0·78, 95% CI 0·63-0·95; p=0·015). Time to resolution of symptoms was similar in both groups. Adverse events were rare, with no deaths and a total of 14 hospital admissions (six in the C-reactive protein group and eight in the control group). INTERPRETATION: C-reactive protein point-of-care testing reduced antibiotic use for non-severe acute respiratory tract infection without compromising patients' recovery in primary health care in Vietnam. Health-care providers might have become familiar with the clinical picture of low C-reactive protein, leading to reduction in antibiotic prescribing in both groups, but this would have led to a reduction in observed effect, rather than overestimation. Qualitative analysis is needed to address differences in context in order to implement this strategy to improve rational antibiotic use for patients with acute respiratory infection in low-income and middle-income countries. FUNDING: Wellcome Trust, UK, and Global Antibiotic Resistance Partnership, USA.

Boudhar A, Ng XW, Loh CY, Chia WN, Tan ZM, Nosten F, Dymock BW, Tan KS. 2016. Overcoming chloroquine resistance in malaria: Design, synthesis and structure-activity relationships of novel chemoreversal agents. Eur J Med Chem, 119 pp. 231-249. | Citations: 2 (Scopus) | Show Abstract | Read more

Malaria remains a significant infectious disease with even artemisinin-based therapies now facing resistance in the field. Development of new therapies is urgently needed, either by finding new compounds with unique modes of action, or by reversing resistance towards known drugs with 'chemosensitizers' or 'chemoreversal' agents (CRA). Concerning the latter, we have focused on the resistance mechanisms developed against chloroquine (CQ). We have synthesized a series of compounds related to previously identified CRAs, and found promising novel compounds. These compounds show encouraging results in a coumarin labeled chloroquine uptake assay, exhibiting a dose response in resensitising parasites to the antimalarial effects of chloroquine. Selected compounds show consistent potency across a panel of chloroquine and artemisinin sensitive and resistant parasites, and a wide therapeutic window. This data supports further study of CRAs in the treatment of malaria and, ultimately, their use in chloroquine-based combination therapies.

Boullé M, Witkowski B, Duru V, Sriprawat K, Nair SK, McDew-White M, Anderson TJ, Phyo AP, Menard D, Nosten F. 2016. Artemisinin-Resistant Plasmodium falciparum K13 Mutant Alleles, Thailand-Myanmar Border. Emerg Infect Dis, 22 (8), pp. 1503-1505. | Citations: 1 (Scopus) | Read more

Dittrich S, Card E, Phuklia W, Rudgard WE, Silousok J, Phoumin P, Bouthasavong L, Azarian S, Davong V, Dance DAB et al. 2016. Survival and growth of Orientia tsutsugamushi in conventional hemocultures Emerging Infectious Diseases, 22 (8), pp. 1460-1463. | Show Abstract | Read more

© 2016, Centers for Disease Control and Prevention (CDC). All rights reserved.Orientia tsutsugamushi, which requires specialized facilities for culture, is a substantial cause of disease in Asia. We demonstrate that O. tsutsugamushi numbers increased for up to 5 days in conventional hemocultures. Performing such a culture step before molecular testing could increase the sensitivity of O. tsutsugamushi molecular diagnosis.

Chung The H, Rabaa MA, Pham Thanh D, De Lappe N, Cormican M, Valcanis M, Howden BP, Wangchuk S, Bodhidatta L, Mason CJ et al. 2016. South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study. PLoS Med, 13 (8), pp. e1002055. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Antimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei. METHODS AND FINDINGS: We performed whole-genome sequencing on a collection of 60 contemporaneous ciprofloxacin-resistant S. sonnei isolated in four countries within Asia (Vietnam, n = 11; Bhutan, n = 12; Thailand, n = 1; Cambodia, n = 1) and two outside of Asia (Australia, n = 19; Ireland, n = 16). We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny, we found that all ciprofloxacin-resistant S. sonnei formed a single clade within a Central Asian expansion of lineage III. Furthermore, our data show that resistance to ciprofloxacin within S. sonnei may be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I, and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the United States and Europe. Our analysis was limited by the number of S. sonnei sequences available from diverse geographical areas and time periods, and we cannot discount the potential existence of other unsampled reservoir populations of antimicrobial-resistant S. sonnei. CONCLUSIONS: This study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.

Auburn S, Serre D, Pearson RD, Amato R, Sriprawat K, To S, Handayuni I, Suwanarusk R, Russell B, Drury E et al. 2016. Genomic Analysis Reveals a Common Breakpoint in Amplifications of the Plasmodium vivax Multidrug Resistance 1 Locus in Thailand. J Infect Dis, 214 (8), pp. 1235-1242. | Show Abstract | Read more

In regions of coendemicity for Plasmodium falciparum and Plasmodium vivax where mefloquine is used to treat P. falciparum infection, drug pressure mediated by increased copy numbers of the multidrug resistance 1 gene (pvmdr1) may select for mefloquine-resistant P. vivax Surveillance is not undertaken routinely owing in part to methodological challenges in detection of gene amplification. Using genomic data on 88 P. vivax samples from western Thailand, we identified pvmdr1 amplification in 17 isolates, all exhibiting tandem copies of a 37.6-kilobase pair region with identical breakpoints. A novel breakpoint-specific polymerase chain reaction assay was designed to detect the amplification. The assay demonstrated high sensitivity, identifying amplifications in 13 additional, polyclonal infections. Application to 132 further samples identified the common breakpoint in all years tested (2003-2015), with a decline in prevalence after 2012 corresponding to local discontinuation of mefloquine regimens. Assessment of the structure of pvmdr1 amplification in other geographic regions will yield information about the population-specificity of the breakpoints and underlying amplification mechanisms.

Moore CE, Giess A, Soeng S, Sar P, Kumar V, Nhoung P, Bousfield R, Turner P, Stoesser N, Day NP, Parry CM. 2016. Characterisation of Invasive Streptococcus pneumoniae Isolated from Cambodian Children between 2007 - 2012. PLoS One, 11 (7), pp. e0159358. | Show Abstract | Read more

BACKGROUND: The 13-valent pneumococcal vaccine (PCV13) was introduced in Cambodia in January 2015. There are limited data concerning the common serotypes causing invasive pneumococcal disease (IPD). Knowledge of the circulating pneumococcal serotypes is important to monitor epidemiological changes before and after vaccine implementation. METHODS: All episodes of IPD defined by the isolation of Streptococcus pneumoniae from blood, cerebrospinal fluid or other sterile site in Cambodian children admitted to the Angkor Hospital for Children in Siem Reap, Northwestern Cambodia, between 1st January 2007 and 1st July 2012 were retrospectively studied. Streptococcus pneumoniae isolates that could be retrieved underwent phenotypic typing and whole genome sequencing. RESULTS: There were 90 Cambodian children hospitalized with IPD with a median (IQR) age of 2.3 years (0.9-6.2). The case fatality was 15.6% (95% CI 8-23). Of 50 Streptococcus pneumoniae isolates available for further testing, 46% were penicillin non-susceptible and 8% were ceftriaxone non-susceptible, 78% were cotrimoxazole resistant, 30% were erythromycin resistant and 30% chloramphenicol resistant. There were no significant changes in resistance levels over the five-year period. The most common serotypes were 1 (11/50; 22%), 23F (8/50; 16%), 14 (6/50; 12%), 5 (5/50; 10%) and 19A (3/50; 6%). Coverage by PCV7, PCV10 and PCV13 was 44%, 76% and 92% respectively. We identified novel multilocus sequence types and resistotypes using whole genome sequencing. CONCLUSIONS: This study suggests IPD is an important disease in Cambodian children and can have a significant mortality. PCV13 coverage of the serotypes determined in studied strains was high and consistent with another recent study. The phenotypic resistance patterns observed were similar to other regional studies. The use of whole genome sequencing in the present study provides additional typing and resistance information together with the description of novel sequence types and resistotypes.

Douangngeun B, Theppangna W, Soukvilay V, Senaphanh C, Phithacthep K, Phomhaksa S, Yingst S, Lombardini E, Hansson E, Selleck PW, Blacksell SD. 2016. Seroprevalence of Q Fever, Brucellosis, and Bluetongue in Selected Provinces in Lao People's Democratic Republic. Am J Trop Med Hyg, 95 (3), pp. 558-561. | Show Abstract | Read more

This study has determined the proportional seropositivity of two zoonotic diseases, Q fever and brucellosis, and bluetongue virus (BTV) which is nonzoonotic, in five provinces of Lao People's Democratic Republic (PDR) (Loungphabang, Luangnumtha, Xayaboury, Xiengkhouang, and Champasak, and Vientiane Province and Vientiane capital). A total of 1,089 samples from buffalo, cattle, pigs, and goats were tested, with seropositivity of BTV (96.7%), Q fever (1.2%), and brucellosis (0.3%). The results of this survey indicated that Q fever seropositivity is not widely distributed in Lao PDR; however, Xayaboury Province had a cluster of seropositive cattle in seven villages in four districts (Botan, Kenthao, Paklaiy, and Phiang) that share a border with Thailand. Further studies are required to determine if Xayaboury Province is indeed an epidemiological hot spot of Q fever activity. There is an urgent need to determine the levels of economic loss and human health-related issues caused by Q fever, brucellosis, and BTV in Lao PDR.

Vanobberghen F, Penny MA, Duthaler U, Odermatt P, Sayasone S, Keiser J, Tarning J. 2016. Population Pharmacokinetic Modeling of Tribendimidine Metabolites in Opisthorchis viverrini-Infected Adults. Antimicrob Agents Chemother, 60 (10), pp. 5695-5704. | Citations: 1 (Scopus) | Show Abstract | Read more

There is a pressing need for alternative treatments against the liver fluke Opisthorchis viverrini Oral tribendimidine is a promising candidate, but its population pharmacokinetic properties are unknown. Two phase IIa trials were conducted in Laos in O. viverrini-infected adults receiving single oral doses of 25 to 600 mg tribendimidine administered as different formulations in each study (study 1 used 200-mg tablets, and study 2 used 50-mg tablets). Venous whole blood, plasma, and capillary dried blood spots were sampled frequently from 68 adults, and concentrations of the tribendimidine metabolites dADT (deacetylated amidantel) and adADT (acetylated dADT) were measured. Population pharmacokinetics were assessed by using nonlinear mixed-effects modeling. The relationship between drug exposure and cure (assessed at 21 days posttreatment) was evaluated by using univariable logistic regression. A six-transit compartment absorption model with a one-disposition compartment for each metabolite described the data well. Compared to the 50-mg formulation (study 2), the 200-mg formulation (study 1) had a 40.1% higher mean transit absorption time, a 113% higher dADT volume of distribution, and a 364% higher adADT volume of distribution. Each 10-year increase in age was associated with a 12.7% lower dADT clearance and a 21.2% lower adADT clearance. The highest cure rates (≥55%) were observed with doses of ≥100 mg. Higher dADT, but not adADT, peak concentrations and exposures were associated with cure (P = 0.004 and 0.003, respectively). For the first time, population pharmacokinetics of tribendimidine have been described. Known differences in the 200-mg versus 50-mg formulations were captured by covariate modeling. Further studies are needed to validate the structural model and confirm covariate relationships. (This study has been registered with the ISRCTN Registry under no. ISRCTN96948551.).

Paris DH, Dumler JS. 2016. State of the art of diagnosis of rickettsial diseases: the use of blood specimens for diagnosis of scrub typhus, spotted fever group rickettsiosis, and murine typhus. Curr Opin Infect Dis, 29 (5), pp. 433-439. | Citations: 1 (Scopus) | Show Abstract | Read more

PURPOSE OF REVIEW: With improved malaria control, acute undifferentiated febrile illness studies in tropical regions reveal a startling proportion of rickettsial illnesses, especially scrub typhus, murine typhus, and spotted fever group rickettsioses. Laboratory diagnosis of these infections evolved little over the past 40 years, but combinations of technologies like PCR and loop-mediated isothermal amplification, with refined rapid diagnostic tests and/or ELISA, are promising for guidance for early antirickettsial treatment. RECENT FINDINGS: The long-term reliance on serological tests - useful only late in rickettsial infections - has led to underdiagnosis, inappropriate therapies, and undocumented morbidity and mortality. Recent approaches integrate nucleic acid amplification and recombinant protein-based serological tests for diagnosing scrub typhus. Optimized using Bayesian latent class analyses, this strategy increases diagnostic confidence and enables early accurate diagnosis and treatment - a model to follow for lagging progress in murine typhus and spotted fever. SUMMARY: A laboratory diagnostic paradigm shift in rickettsial infections is evolving, with replacement of indirect immunofluorescence assay by the more objective ELISA coupled with nucleic acid amplification assays to expand the diagnostic window toward early infection intervals. This approach supports targeted antirickettsial therapy, reduces morbidity and mortality, and provides a robust evidence base for further development of diagnostics and vaccines.

Moore CE, Elwin K, Phot N, Seng C, Mao S, Suy K, Kumar V, Nader J, Bousfield R, Perera S et al. 2016. Molecular Characterization of Cryptosporidium Species and Giardia duodenalis from Symptomatic Cambodian Children. PLoS Negl Trop Dis, 10 (7), pp. e0004822. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: In a prospective study, 498 single faecal samples from children aged under 16 years attending an outpatient clinic in the Angkor Hospital for Children, northwest Cambodia, were examined for Cryptosporidium oocysts and Giardia cysts using microscopy and molecular assays. METHODOLOGY/PRINCIPAL FINDINGS: Cryptosporidium oocysts were detected in 2.2% (11/498) of samples using microscopy and in 7.7% (38/498) with molecular tests. Giardia duodenalis cysts were detected in 18.9% (94/498) by microscopy and 27.7% (138/498) by molecular tests; 82% of the positive samples (by either method) were from children aged 1-10 years. Cryptosporidium hominis was the most common species of Cryptosporidium, detected in 13 (34.2%) samples, followed by Cryptosporidium meleagridis in 9 (23.7%), Cryptosporidium parvum in 8 (21.1%), Cryptosporidium canis in 5 (13.2%), and Cryptosporidium suis and Cryptosporidium ubiquitum in one sample each. Cryptosporidium hominis and C. parvum positive samples were subtyped by sequencing the GP60 gene: C. hominis IaA16R6 and C. parvum IIeA7G1 were the most abundant subtypes. Giardia duodenalis was typed using a multiplex real-time PCR targeting assemblages A and B. Assemblage B (106; 76.8% of all Giardia positive samples) was most common followed by A (12.3%) and mixed infections (5.1%). Risk factors associated with Cryptosporidium were malnutrition (AOR 9.63, 95% CI 1.67-55.46), chronic medical diagnoses (AOR 4.51, 95% CI 1.79-11.34) and the presence of birds in the household (AOR 2.99, 95% CI 1.16-7.73); specifically C. hominis (p = 0.03) and C. meleagridis (p<0.001) were associated with the presence of birds. The use of soap was protective against Giardia infection (OR 0.74, 95% CI 0.58-0.95). CONCLUSIONS/SIGNIFICANCE: This is the first report to describe the different Cryptosporidium species and subtypes and Giardia duodenalis assemblages in Cambodian children. The variety of Cryptosporidium species detected indicates both anthroponotic and zoonotic transmission in this population. Interventions to improve sanitation, increase hand washing after defecation and before preparing food and promote drinking boiled water may reduce the burden of these two parasites.

Pholwat S, Sakai F, Turner P, Vidal JE, Houpt ER. 2016. Development of a TaqMan Array Card for Pneumococcal Serotyping on Isolates and Nasopharyngeal Samples. J Clin Microbiol, 54 (7), pp. 1842-1850. | Show Abstract | Read more

Streptococcus pneumoniae is both a commensal and a major pathogen that causes invasive disease in people of all ages. The introduction of serotype-specific pneumococcal vaccines has reduced the burden of disease but has also led to replacement with new strains; thus, serotyping remains important for vaccine-related disease surveillance. Conventional serotyping methods are laborious and expensive. We developed an easy-to-perform genotypic TaqMan array card (TAC) to identify S. pneumoniae strains, including lytA-based sequences, and 53 sequence-specific PCRs to identify 74 serotypes/serogroups covering all current vaccine types as well as prevalent nonvaccine types. The TAC method was evaluated on 146 clinical S. pneumoniae isolates and 13 nonpneumococcal species that naturally inhabit the upper respiratory tract and yielded 97% (142/146) sensitivity and 100% (13/13) specificity versus results of standard Quellung serotyping. The calculated limit of detection was 20 to 200 fg (∼8 to 84 genome equivalents) per reaction. On 23 blinded nasopharyngeal specimens that were pneumococcus culture positive, the TAC pan-pneumococcus lytA assay was positive in 21 (91% sensitivity versus culture). On TAC lytA-positive specimens, a serotype result was obtained on 86%, and the result was 95% accurate versus the subsequent culture's Quellung result. TAC also detected mixed serotypes in two specimens where Quellung detected only the predominant serotype. This TAC method yields fast and comprehensive serotyping compared to the standard method and may be useful on direct specimens.

White NJ. 2016. Can new treatment developments combat resistance in malaria? Expert Opin Pharmacother, 17 (10), pp. 1303-1307. | Citations: 3 (Scopus) | Read more

Kohler C, Dunachie SJ, Müller E, Kohler A, Jenjaroen K, Teparrukkul P, Baier V, Ehricht R, Steinmetz I. 2016. Rapid and Sensitive Multiplex Detection of Burkholderia pseudomallei-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach. PLoS Negl Trop Dis, 10 (7), pp. e0004847. | Show Abstract | Read more

BACKGROUND: The environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking. METHODS AND PRINCIPAL FINDINGS: In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response. CONCLUSIONS: Our protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-B. pseudomallei antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.

Cheah PY, White NJ. 2016. Antimalarial mass drug administration: ethical considerations. Int Health, 8 (4), pp. 235-238. | Citations: 1 (Scopus) | Show Abstract | Read more

Falciparum malaria is a major cause of death and illness in tropical countries, particularly in childhood. In endemic countries, a significant proportion of the community is infected with malaria asymptomatically. One promising way to eliminate malaria is to give the entire population malaria treatment. This is called mass drug administration (MDA) and it raises a number of ethical issues, as possible long-term benefits are uncertain. The effectiveness of MDA is critically dependent on level of participation, so the promised benefits to the community can be annulled by non-participation of a small number of individuals. These potential benefits range a wide spectrum, from the permanent elimination of malaria (success) to a transient reduction in the prevalence of infection and the incidence of illness (failure). The drawbacks of MDA are: inconvenience, potential toxicity, loss of confidence in the elimination campaign, possible drug resistance (though highly unlikely), and the potential for a rebound of malaria illness (if immunity is lost and malaria is reintroduced later). Other ethical issues are related to balancing individual and public health interests, and potentially limiting individual autonomy by making MDA compulsory.

Pearson RD, Amato R, Auburn S, Miotto O, Almagro-Garcia J, Amaratunga C, Suon S, Mao S, Noviyanti R, Trimarsanto H et al. 2016. Genomic analysis of local variation and recent evolution in Plasmodium vivax. Nat Genet, 48 (8), pp. 959-964. | Citations: 16 (Scopus) | Show Abstract | Read more

The widespread distribution and relapsing nature of Plasmodium vivax infection present major challenges for the elimination of malaria. To characterize the genetic diversity of this parasite in individual infections and across the population, we performed deep genome sequencing of >200 clinical samples collected across the Asia-Pacific region and analyzed data on >300,000 SNPs and nine regions of the genome with large copy number variations. Individual infections showed complex patterns of genetic structure, with variation not only in the number of dominant clones but also in their level of relatedness and inbreeding. At the population level, we observed strong signals of recent evolutionary selection both in known drug resistance genes and at new loci, and these varied markedly between geographical locations. These findings demonstrate a dynamic landscape of local evolutionary adaptation in the parasite population and provide a foundation for genomic surveillance to guide effective strategies for control and elimination of P. vivax.

Pham Thanh D, Thompson CN, Rabaa MA, Sona S, Sopheary S, Kumar V, Moore C, Tran Vu Thieu N, Wijedoru L, Holt KE et al. 2016. The Molecular and Spatial Epidemiology of Typhoid Fever in Rural Cambodia. PLoS Negl Trop Dis, 10 (6), pp. e0004785. | Citations: 1 (Scopus) | Show Abstract | Read more

Typhoid fever, caused by the bacterium Salmonella Typhi, is an endemic cause of febrile disease in Cambodia. The aim of this study was to better understand the epidemiology of pediatric typhoid fever in Cambodia. We accessed routine blood culture data from Angkor Hospital for Children (AHC) in Siem Reap province between 2007 and 2014, and performed whole genome sequencing (WGS) on the isolated bacteria to characterize the S. Typhi population. The resulting phylogenetic information was combined with conventional epidemiological approaches to investigate the spatiotemporal distribution of S. Typhi and population-level risk factors for reported disease. During the study period, there were 262 cases of typhoid within a 100 km radius of AHC, with a median patient age of 8.2 years (IQR: 5.1-11.5 years). The majority of infections occurred during the rainy season, and commune incidences as high as 11.36/1,000 in children aged <15 years were observed over the study period. A population-based risk factor analysis found that access to water within households and increasing distance from Tonle Sap Lake were protective. Spatial mapping and WGS provided additional resolution for these findings, and confirmed that proximity to the lake was associated with discrete spatiotemporal disease clusters. We confirmed the dominance of MDR H58 S. Typhi in this population, and found substantial evidence of diversification (at least seven sublineages) within this single lineage. We conclude that there is a substantial burden of pediatric typhoid fever in rural communes in Cambodia. Our data provide a platform for additional population-based typhoid fever studies in this location, and suggest that this would be a suitable setting in which to introduce a school-based vaccination programme with Vi conjugate vaccines.

Thwaites CL, Lundeg G, Dondorp AM, sepsis in resource-limited settings–expert consensus recommendations group of the European Society of Intensive Care Medicine (ESICM) and the Mahidol-Oxford Research Unit (MORU) in Bangkok, Thailand. 2016. Recommendations for infection management in patients with sepsis and septic shock in resource-limited settings. Intensive Care Med, 42 (12), pp. 2040-2042. | Citations: 3 (Scopus) | Read more

Busby GBJ, Band G, Le QS, Jallow M, Bougama E, Mangano VD, Amenga-Etego LN, Enimil A, Apinjoh T, Ndila CM et al. 2016. Admixture into and within sub-Saharan Africa eLife, 5 (JUN2016), | Citations: 1 (Scopus) | Show Abstract | Read more

© Busby et al.Similarity between two individuals in the combination of genetic markers along their chromosomes indicates shared ancestry and can be used to identify historical connections between different population groups due to admixture. We use a genome-wide, haplotype-based, analysis to characterise the structure of genetic diversity and gene-flow in a collection of 48 sub-Saharan African groups. We show that coastal populations experienced an influx of Eurasian haplotypes over the last 7000 years, and that Eastern and Southern Niger-Congo speaking groups share ancestry with Central West Africans as a result of recent population expansions. In fact, most sub-Saharan populations share ancestry with groups from outside of their current geographic region as a result of gene-flow within the last 4000 years. Our in-depth analysis provides insight into haplotype sharing across different ethno-linguistic groups and the recent movement of alleles into new environments, both of which are relevant to studies of genetic epidemiology.

Tabernero P, Parker M, Ravinetto R, Phanouvong S, Yeung S, Kitutu FE, Cheah PY, Mayxay M, Guerin PJ, Newton PN. 2016. Ethical challenges in designing and conducting medicine quality surveys. Trop Med Int Health, 21 (6), pp. 799-806. | Show Abstract | Read more

OBJECTIVES: In this paper we discuss the main ethical challenges related to the conduct of medicine quality surveys and make suggestions on how to address them. METHOD: Most evidence-based information regarding medicine quality derives from surveys. However, existing research ethical guidelines do not provide specific guidance for medicine quality surveys. Hence, those conducting surveys are often left wondering how to judge what counts as best practice. A list of the main ethical challenges in the design and conduct of surveys is presented. RESULTS AND CONCLUSIONS: It is vital that the design and conduct of medicine quality surveys uphold moral and ethical obligations and analyse the ethical implications and consequences of such work. These aspects include the impact on the local availability of and access to medicines; the confidentiality and privacy of the surveyors and the surveyed; questions as to whether outlet staff personnel should be told they are part of a survey; the need of ethical and regulatory approvals; and how the findings should be disseminated. Medicine quality surveys should ideally be conducted in partnership with the relevant national Medicine Regulatory Authorities. An international, but contextually sensitive, model of good ethical practice for such surveys is needed.

Boonyuen U, Chamchoy K, Swangsri T, Saralamba N, Day NP, Imwong M. 2016. Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PDViangchan and G6PDViangchan+Mahidol: Decreased stability and catalytic efficiency contribute to the clinical phenotype. Mol Genet Metab, 118 (2), pp. 84-91. | Citations: 1 (Scopus) | Show Abstract | Read more

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is an X-linked hereditary genetic defect that is the most common polymorphism and enzymopathy in humans. To investigate functional properties of two clinical variants, G6PDViangchan and G6PDViangchan+Mahidol, these two mutants were created by overlap-extension PCR, expressed in Escherichia coli and purified to homogeneity. We describe an overexpression and purification method to obtain substantial amounts of functionally active protein. The KM for G6P of the two variants was comparable to the KM of the native enzyme, whereas the KM for NADP(+) was increased 5-fold for G6PDViangchan and 8-fold for G6PDViangchan+Mahidol when compared with the native enzyme. Additionally, kcat of the mutant enzymes was markedly reduced, resulting in a 10- and 18-fold reduction in catalytic efficiency for NADP(+) catalysis for G6PDViangchan and G6PDViangchan+Mahidol, respectively. Furthermore, the two variants demonstrated significant reduction in thermostability, but similar susceptibility to trypsin digestion, when compared with the wild-type enzyme. The presence of NADP(+) is shown to improve the stability of G6PD enzymes. This is the first report indicating that protein instability and reduced catalytic efficiency are responsible for the reduced catalytic activity of G6PDViangchan and G6PDViangchan+Mahidol and, as a consequence, contribute to the clinical phenotypes of these two clinical variants.

James SL, Blacksell SD, Nawtaisong P, Tanganuchitcharnchai A, Smith DJ, Day NP, Paris DH. 2016. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand. PLoS Negl Trop Dis, 10 (6), pp. e0004723. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. CONCLUSIONS/SIGNIFICANCE: Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes.

Abdulla S, Achan J, Yeka A, D'Alessandro U, Adam I, Alemayehu BH, Allan R, Temu EA, Allen EN, Barnes KI et al. 2016. Gametocyte carriage in uncomplicated Plasmodium falciparum malaria following treatment with artemisinin combination therapy: a systematic review and meta-analysis of individual patient data BMC Medicine, 14 (1), | Citations: 3 (Scopus) | Show Abstract | Read more

© 2016 WWARN Gametocyte Study Group.Background: Gametocytes are responsible for transmission of malaria from human to mosquito. Artemisinin combination therapy (ACT) reduces post-treatment gametocyte carriage, dependent upon host, parasite and pharmacodynamic factors. The gametocytocidal properties of antimalarial drugs are important for malaria elimination efforts. An individual patient clinical data meta-analysis was undertaken to identify the determinants of gametocyte carriage and the comparative effects of four ACTs: artemether-lumefantrine (AL), artesunate/amodiaquine (AS-AQ), artesunate/mefloquine (AS-MQ), and dihydroartemisinin-piperaquine (DP). Methods: Factors associated with gametocytaemia prior to, and following, ACT treatment were identified in multivariable logistic or Cox regression analysis with random effects. All relevant studies were identified through a systematic review of PubMed. Risk of bias was evaluated based on study design, methodology, and missing data. Results: The systematic review identified 169 published and 9 unpublished studies, 126 of which were shared with the WorldWide Antimalarial Resistance Network (WWARN) and 121 trials including 48,840 patients were included in the analysis. Prevalence of gametocytaemia by microscopy at enrolment was 12.1 % (5887/48,589), and increased with decreasing age, decreasing asexual parasite density and decreasing haemoglobin concentration, and was higher in patients without fever at presentation. After ACT treatment, gametocytaemia appeared in 1.9 % (95 % CI, 1.7-2.1) of patients. The appearance of gametocytaemia was lowest after AS-MQ and AL and significantly higher after DP (adjusted hazard ratio (AHR), 2.03; 95 % CI, 1.24-3.12; P = 0.005 compared to AL) and AS-AQ fixed dose combination (FDC) (AHR, 4.01; 95 % CI, 2.40-6.72; P < 0.001 compared to AL). Among individuals who had gametocytaemia before treatment, gametocytaemia clearance was significantly faster with AS-MQ (AHR, 1.26; 95 % CI, 1.00-1.60; P = 0.054) and slower with DP (AHR, 0.74; 95 % CI, 0.63-0.88; P = 0.001) compared to AL. Both recrudescent (adjusted odds ratio (AOR), 9.05; 95 % CI, 3.74-21.90; P < 0.001) and new (AOR, 3.03; 95 % CI, 1.66-5.54; P < 0.001) infections with asexual-stage parasites were strongly associated with development of gametocytaemia after day 7. Conclusions: AS-MQ and AL are more effective than DP and AS-AQ FDC in preventing gametocytaemia shortly after treatment, suggesting that the non-artemisinin partner drug or the timing of artemisinin dosing are important determinants of post-treatment gametocyte dynamics.

Weehuizen TA, Lankelma JM, De Jong HK, De Boer OJ, Roelofs JJ, Day NP, Gram H, De Vos AF, Wiersinga WJ. 2016. Therapeutic Administration of a Monoclonal Anti-Il-1β Antibody Protects Against Experimental Melioidosis. Shock, 46 (5), pp. 566-574. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Melioidosis, caused by the gram-negative bacterium Burkholderia pseudomallei, is a common cause of community-acquired sepsis in Southeast Asia and Northern Australia. The NLRP3 inflammasome and its downstream product interleukin-1 beta (IL-1β) have been proposed to play crucial roles in melioidosis. In this study, we characterized the role of IL-1β more closely and we assessed its therapeutic potential. METHODS: mRNA expression of inflammasome components was determined in isolated leukocytes of 32 healthy controls and 34 patients with sepsis caused by B pseudomallei.Wild-type (WT), NLRP3-deficient (Nlrp3), and Asc mice were infected with B pseudomallei. In additional experiments, infected WT mice were treated with an anti-IL-1β antibody. After 24, 48, and 72 hours (h) mice were sacrificed and organs were harvested. Furthermore, survival studies were performed. RESULTS: Patients with melioidosis exhibited lower mRNA levels of caspase-1, NLRP3, and ASC. Bacterial dissemination and organ damage were increased in B pseudomallei-infected Nlrp3 and Asc mice, together with a reduced pulmonary cell influx. Anti-IL-1β treatment of B pseudomallei challenged mice resulted in strongly reduced bacterial counts, organ damage, and pulmonary granulocyte influx together with reduced mortality. Postponement of anti-IL-1β treatment for 24 h postinfection still protected mice during melioidosis. CONCLUSION: Expression of caspase-1, NLRP3, and ASC is altered in melioidosis patients. In mice, both NLRP3 and ASC contribute to the host defense against melioidosis. Anti-IL-1β treatment protects mice against B pseudomallei infection and might be a novel treatment strategy in melioidosis.

Newton PN, Caillet C, Guerin PJ. 2016. A link between poor quality antimalarials and malaria drug resistance? Expert Rev Anti Infect Ther, 14 (6), pp. 531-533. | Read more

Vongsouvath M, Phommasone K, Sengvilaipaseuth O, Kosoltanapiwat N, Chantratita N, Blacksell SD, Lee SJ, de Lamballerie X, Mayxay M, Keomany S et al. 2016. Using Rapid Diagnostic Tests as a Source of Viral RNA for Dengue Serotyping by RT-PCR - A Novel Epidemiological Tool. PLoS Negl Trop Dis, 10 (5), pp. e0004704. | Show Abstract | Read more

BACKGROUND: Dengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype. CONCLUSIONS/SIGNIFICANCE: Therefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions.

Govindasamy G, Barber BE, Ghani SA, William T, Grigg MJ, Borooah S, Dhillon B, Dondorp AM, Yeo TW, Anstey NM, Maude RJ. 2016. Retinal Changes in Uncomplicated and Severe Plasmodium knowlesi Malaria. J Infect Dis, 213 (9), pp. 1476-1482. | Show Abstract | Read more

BACKGROUND: Plasmodium knowlesi causes severe malaria, but its pathogenesis is poorly understood. Retinal changes provide insights into falciparum malaria pathogenesis but have not been studied in knowlesi malaria. METHODS: An observational study was conducted in Malaysian adults hospitalized with severe (n = 20) and nonsevere (n = 24) knowlesi malaria using indirect ophthalmoscopy (n = 44) and fundus photography (n = 29). RESULTS: The patients' median age was 44 years (range, 18-74 years). No coma or deaths occurred. Photography detected retinal changes in 11 of 12 patients (92%) with severe and 14 of 17 (82%) with nonsevere knowlesi malaria. Nonspecific retinal whitening occurred in 3 (35%) and 5 (29%) patients with severe and nonsevere disease, respectively; hemorrhages in 2 (17%) and 3 (18%); loss of retinal pigment epithelium in 1 (8%) and 4 (24%); and drusen in 9 (71%) and 12 (75%). All changes were mild, with no significant differences between severe and nonsevere disease. Patients with retinal hemorrhages had lower platelet counts than those without (median, 22 vs 43 × 10(9)/L; P= .04). CONCLUSIONS: The paucity of specific retinal findings associated with disease severity in knowlesi malaria contrasts with the retinopathy of severe adult falciparum malaria with and without coma, suggesting that falciparum-like microvascular sequestration in the brain is not a major component in severe knowlesi malaria pathogenesis.

Phetsouvanh R, Sonthayanon P, Pukrittayakamee S, Paris DH, Newton PN, Feil EJ, Day NP. 2016. Correction: The Diversity and Geographical Structure of Orientia tsutsugamushi Strains from Scrub Typhus Patients in Laos. PLoS Negl Trop Dis, 10 (5), pp. e0004742. | Show Abstract | Read more

[This corrects the article DOI: 10.1371/journal.pntd.0004024.].

Fellmeth G, Paw MK, Wiladphaingern J, Charunwatthana P, Nosten FH, McGready R. 2016. Maternal suicide risk among refugees and migrants. Int J Gynaecol Obstet, 134 (2), pp. 223-224. | Citations: 1 (Scopus) | Read more

Turner P, Pol S, Soeng S, Sar P, Neou L, Chea P, Day NP, Cooper BS, Turner C. 2016. High Prevalence of Antimicrobial-resistant Gram-negative Colonization in Hospitalized Cambodian Infants. Pediatr Infect Dis J, 35 (8), pp. 856-861. | Citations: 2 (Scopus) | Show Abstract | Read more

BACKGROUND: Antimicrobial-resistant Gram-negative infections are a significant cause of mortality in young infants. We aimed to determine characteristics of, and risk factors for, colonization and invasive infection caused by 3rd generation cephalosporin (3GC) or carbapenem-resistant organisms in outborn infants admitted to a neonatal unit (NU) in Cambodia. METHODS: During the first year of operation, patients admitted to the Angkor Hospital for Children NU, Siem Reap, Cambodia, underwent rectal swabbing on admission and twice weekly until discharge. Swabs were taken also from 7 environmental sites. Swabs were cultured to identify 3GC or carbapenem-resistant Acinetobacter sp., Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. RESULTS: The study included 333 infants with a median age at NU admission of 10 days (range, 0-43). Colonization by ≥1 3GC-resistant organism was detected in 85.9% (286/333). Admission swabs were collected in 289 infants: 61.9% were colonized by a 3GC-resistant organism at the time of admission, and a further 23.2% were colonized during hospitalization, at a median of 4 days [95% confidence interval: 3-5]. Probiotic treatment (hazard ratio: 0.58; 95% confidence interval: 0.35-0.98) was associated with delayed colonization. Colonization by a carbapenem-resistant organism occurred in 25 (7.5%) infants. Six infants had NU-associated K. pneumoniae bacteremia; phenotypically identical colonizing strains were found in 3 infants. Environmental colonization occurred early. CONCLUSIONS: Colonization by antimicrobial-resistant Gram-negative organisms occurred early in hospitalized Cambodian infants and was associated with subsequent invasive infection. Trials of potential interventions such as probiotics are needed.

Nakeesathit S, Saralamba N, Pukrittayakamee S, Dondorp A, Nosten F, White NJ, Imwong M. 2016. Limited Polymorphism of the Kelch Propeller Domain in Plasmodium malariae and P. ovale Isolates from Thailand. Antimicrob Agents Chemother, 60 (7), pp. 4055-4062. | Show Abstract | Read more

Artemisinin resistance in Plasmodium falciparum, the agent of severe malaria, is currently a major obstacle to malaria control in Southeast Asia. A gene named "kelch13" has been associated with artemisinin resistance in P. falciparum The orthologue of the kelch gene in P. vivax was identified and a small number of mutations were found in previous studies. The kelch orthologues in the other two human malaria parasites, P. malariae and P. ovale, have not yet been studied. Therefore, in this study, the orthologous kelch genes of P. malariae, P. ovale wallikeri, and P. ovale curtisi were isolated and analyzed for the first time. The homologies of the kelch genes of P. malariae and P. ovale were 84.8% and 82.7%, respectively, compared to the gene in P. falciparum kelch polymorphisms were studied in 13 P. malariae and 5 P. ovale isolates from Thailand. There were 2 nonsynonymous mutations found in these samples. One mutation was P533L, which was found in 1 of 13 P. malariae isolates, and the other was K137R, found in 1 isolate of P. ovale wallikeri (n = 4). This result needs to be considered in the context of widespread artemisinin used within the region; their functional consequences for artemisinin sensitivity in P. malariae and P. ovale will need to be elucidated.

Holt HR, Inthavong P, Khamlome B, Blaszak K, Keokamphe C, Somoulay V, Phongmany A, Durr PA, Graham K, Allen J et al. 2016. Endemicity of Zoonotic Diseases in Pigs and Humans in Lowland and Upland Lao PDR: Identification of Socio-cultural Risk Factors. PLoS Negl Trop Dis, 10 (4), pp. e0003913. | Citations: 3 (Scopus) | Show Abstract | Read more

In Lao People's Democratic Republic pigs are kept in close contact with families. Human risk of infection with pig zoonoses arises from direct contact and consumption of unsafe pig products. This cross-sectional study was conducted in Luang Prabang (north) and Savannakhet (central-south) Provinces. A total of 59 villages, 895 humans and 647 pigs were sampled and serologically tested for zoonotic pathogens including: hepatitis E virus (HEV), Japanese encephalitis virus (JEV) and Trichinella spiralis; In addition, human sera were tested for Taenia spp. and cysticercosis. Seroprevalence of zoonotic pathogens in humans was high for HEV (Luang Prabang: 48.6%, Savannakhet: 77.7%) and T. spiralis (Luang Prabang: 59.0%, Savannakhet: 40.5%), and lower for JEV (around 5%), Taenia spp. (around 3%) and cysticercosis (Luang Prabang: 6.1, Savannakhet 1.5%). Multiple correspondence analysis and hierarchical clustering of principal components was performed on descriptive data of human hygiene practices, contact with pigs and consumption of pork products. Three clusters were identified: Cluster 1 had low pig contact and good hygiene practices, but had higher risk of T. spiralis. Most people in cluster 2 were involved in pig slaughter (83.7%), handled raw meat or offal (99.4%) and consumed raw pigs' blood (76.4%). Compared to cluster 1, cluster 2 had increased odds of testing seropositive for HEV and JEV. Cluster 3 had the lowest sanitation access and had the highest risk of HEV, cysticercosis and Taenia spp. Farmers which kept their pigs tethered (as opposed to penned) and disposed of manure in water sources had 0.85 (95% CI: 0.18 to 0.91) and 2.39 (95% CI: 1.07 to 5.34) times the odds of having pigs test seropositive for HEV, respectively. The results have been used to identify entry-points for intervention and management strategies to reduce disease exposure in humans and pigs, informing control activities in a cysticercosis hyper-endemic village.

Kulsantiwong P, Pudla M, Boondit J, Wikraiphat C, Dunachie SJ, Chantratita N, Utaisincharoen P. 2016. Erratum to: Burkholderia pseudomallei induces IL-23 production in primary human monocytes. Med Microbiol Immunol, 205 (3), pp. 261. | Read more

Imwong M, Stepniewska K, Tripura R, Peto TJ, Lwin KM, Vihokhern B, Wongsaen K, von Seidlein L, Dhorda M, Snounou G et al. 2016. Numerical Distributions of Parasite Densities During Asymptomatic Malaria. J Infect Dis, 213 (8), pp. 1322-1329. | Citations: 13 (Scopus) | Show Abstract | Read more

BACKGROUND: Asymptomatic parasitemia is common even in areas of low seasonal malaria transmission, but the true proportion of the population infected has not been estimated previously because of the limited sensitivity of available detection methods. METHODS: Cross-sectional malaria surveys were conducted in areas of low seasonal transmission along the border between eastern Myanmar and northwestern Thailand and in western Cambodia. DNA was quantitated by an ultrasensitive polymerase chain reaction (uPCR) assay (limit of accurate detection, 22 parasites/mL) to characterize parasite density distributions for Plasmodium falciparum and Plasmodium vivax, and the proportions of undetected infections were imputed. RESULTS: The prevalence of asymptomatic malaria as determined by uPCR was 27.5% (1303 of 4740 people tested). Both P. vivax and P. falciparum density distributions were unimodal and log normal, with modal values well within the quantifiable range. The estimated proportions of all parasitemic individuals identified by uPCR were >70% among individuals infected with P. falciparum and >85% among those infected with P. vivax. Overall, 83% of infections were predicted to be P. vivax infections, 13% were predicted to be P. falciparum infections, and 4% were predicted to be mixed infections. Geometric mean parasite densities were similar; 5601 P. vivax parasites/mL and 5158 P. falciparum parasites/mL. CONCLUSIONS: This uPCR method identified most infected individuals in malaria-endemic areas. Malaria parasitemia persists in humans at levels that optimize the probability of generating transmissible gametocyte densities without causing illness.

Beane A, Stephens T, Silva AP, Welch J, Sigera C, De Alwis S, Athapattu P, Dharmagunawardene D, Peiris L, Siriwardana S et al. 2016. A sustainable approach to training nurses in acute care skills in a resource limited setting (Network for Intensive Care Skills Training, NICST). Resuscitation, 101 pp. e1-e2. | Read more

Chansamouth V, Thammasack S, Phetsouvanh R, Keoluangkot V, Moore CE, Blacksell SD, Castonguay-Vanier J, Dubot-Pérès A, Tangkhabuanbutra J, Tongyoo N et al. 2016. The Aetiologies and Impact of Fever in Pregnant Inpatients in Vientiane, Laos. PLoS Negl Trop Dis, 10 (4), pp. e0004577. | Show Abstract | Read more

INTRODUCTION: Laos has the highest maternal mortality ratio in mainland Southeast Asia and a high incidence of infectious diseases. Globally, malaria has been the pathogen most intensively investigated in relation to impact on pregnancy, but there has been relatively little research on the aetiology and impact of other diseases. We therefore aimed to determine the causes and impact of fever in pregnant women admitted to two central hospitals in Vientiane City, Lao PDR (Laos). MATERIALS AND METHODS: This hospital-based prospective study was conducted in Mahosot Hospital and the Mother and Child Hospital, Vientiane, between 2006 and 2010, with the aim to recruit 250 consenting pregnant women admitted with tympanic temperature ≥37.5°C. Primary outcome was the cause of fever and secondary outcomes were pregnancy outcomes. Specific investigations (culture, antigen, molecular and serological tests) were performed to investigate causes of fever. After discharge, all pregnant women were asked to return for review and convalescence serum on day 10-14 and were monitored until delivery. PRINCIPLE FINDINGS: 250 pregnant women were recruited to this study between February 2006 and November 2010. Fifty percent were pregnant for the first time. Their median (range) gestational age on admission was 24 (4-43) weeks. The median (range) tympanic admission temperature was 38.5°C (37.5-40.5°C). Fifteen percent of patients stated that they had taken antibiotics before admission. Headache, myalgia, back pain and arthralgia were described by >60% of patients and 149 (60%) were given a laboratory diagnosis. Of those with confirmed diagnoses, 132 (53%) had a single disease and 17 (7%) had apparent mixed diseases. Among those who had a single disease, dengue fever was the most common diagnosis, followed by pyelonephritis, scrub typhus, murine typhus and typhoid. Patients were also diagnosed with tuberculosis, appendicitis, Staphylococcus aureus septicemia, leptospirosis, Japanese encephalitis virus infection and Plasmodium falciparum malaria. Severe consequences, including maternal death, miscarriage, stillbirth, low birth weight and preterm birth, were found among 28 (78%) mothers with dengue fever, rickettsioses and typhoid. CONCLUSION: Fevers other than malaria, such as dengue, pyelonephritis, rickettsioses and typhoid are common causes of fever during pregnancy in the Asian tropics. Further investigations of their impact in the community on maternal death, fetal loss, vertical transmission, low birth weight and preterm birth are needed.

Thuan PD, Ca NTN, Toi PV, Nhien NTT, Thanh NV, Anh ND, Phu NH, Thai CQ, Thai LH, Hoa NT et al. 2016. A Randomized Comparison of Chloroquine versus Dihydroartemisinin-Piperaquine for the Treatment of Plasmodium vivax Infection in Vietnam AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 94 (4), pp. 879-885. | Read more

Caillet C, Sichanh C, Assemat G, Malet-Martino M, Sommet A, Bagheri H, Mayxay M, Syhakhang L, Lapeyre-Mestre M, Newton PN, Roussin A. 2016. Adverse drug reaction-related hospitalizations of adults in the Lao People's Democratic Republic: health risks of unknown medicines FUNDAMENTAL & CLINICAL PHARMACOLOGY, 30 pp. 10-10.

Tun KM, Jeeyapant A, Imwong M, Thein M, Aung SS, Hlaing TM, Yuentrakul P, Promnarate C, Dhorda M, Woodrow CJ et al. 2016. Parasite clearance rates in Upper Myanmar indicate a distinctive artemisinin resistance phenotype: a therapeutic efficacy study. Malar J, 15 (1), pp. 185. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance in Plasmodium falciparum extends across Southeast Asia where it is associated with worsening partner drug resistance and a decline in the efficacy of frontline artemisinin-based combination therapy. Dihydroartemisinin-piperaquine (DP) is an essential component of preventive and curative treatment in the region, but its therapeutic efficacy has fallen in Cambodia. METHODS: A prospective clinical and parasitological evaluation of DP was conducted at two sites in Upper Myanmar between August 2013 and December 2014, enrolling 116 patients with acute uncomplicated falciparum malaria. Patients received DP orally for 3 days together with primaquine 0.25 mg/kg on admission. Parasite clearance half-lives based on 6 hourly blood smears, and day 42 therapeutic responses were assessed as well as parasite K13 genotypes. RESULTS: Median parasite clearance half-life was prolonged, and clearance half-life was greater than 5 h in 21% of patients. Delayed parasite clearance was significantly associated with mutations in the propeller region of the parasite k13 gene. The k13 F446I mutation was found in 25.4% of infections and was associated with a median clearance half-life of 4.7 h compared with 2.7 h for infections without k13 mutations (p < 0.001). There were no failures after 42 days of follow-up, although 18% of patients had persistent parasitaemia on day 3. CONCLUSION: The dominant k13 mutation observed in Upper Myanmar, F446I, appears to be associated with an intermediate rate of parasite clearance compared to other common mutations described elsewhere in the Greater Mekong Subregion. Discerning this phenotype requires relatively detailed clearance measurements, highlighting the importance of methodology in assessing artemisinin resistance.

Worby CJ, O'Neill PD, Kypraios T, Robotham JV, De Angelis D, Cartwright EJ, Peacock SJ, Cooper BS. 2016. Reconstructing transmission trees for communicable diseases using densely sampled genetic data. Ann Appl Stat, 10 (1), pp. 395-417. | Show Abstract | Read more

Whole genome sequencing of pathogens from multiple hosts in an epidemic offers the potential to investigate who infected whom with unparalleled resolution, potentially yielding important insights into disease dynamics and the impact of control measures. We considered disease outbreaks in a setting with dense genomic sampling, and formulated stochastic epidemic models to investigate person-to-person transmission, based on observed genomic and epidemiological data. We constructed models in which the genetic distance between sampled genotypes depends on the epidemiological relationship between the hosts. A data augmented Markov chain Monte Carlo algorithm was used to sample over the transmission trees, providing a posterior probability for any given transmission route. We investigated the predictive performance of our methodology using simulated data, demonstrating high sensitivity and specificity, particularly for rapidly mutating pathogens with low transmissibility. We then analyzed data collected during an outbreak of methicillin-resistant Staphylococcus aureus in a hospital, identifying probable transmission routes and estimating epidemiological parameters. Our approach overcomes limitations of previous methods, providing a framework with the flexibility to allow for unobserved infection times, multiple independent introductions of the pathogen, and within-host genetic diversity, as well as allowing forward simulation.

Blacksell SD, Lim C, Tanganuchitcharnchai A, Jintaworn S, Kantipong P, Richards AL, Paris DH, Limmathurotsakul D, Day NP. 2016. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay. J Clin Microbiol, 54 (6), pp. 1472-1478. | Citations: 3 (Scopus) | Show Abstract | Read more

The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus.

Rachlin A, Dittrich S, Phommasone K, Douangnouvong A, Phetsouvanh R, Newton PN, Dance DA. 2016. Investigation of Recurrent Melioidosis in Lao People's Democratic Republic by Multilocus Sequence Typing. Am J Trop Med Hyg, 94 (6), pp. 1208-1211. | Citations: 1 (Scopus) | Show Abstract | Read more

Melioidosis is an infectious disease caused by the saprophytic bacterium Burkholderia pseudomallei In northeast Thailand and northern Australia, where the disease is highly endemic, a range of molecular tools have been used to study its epidemiology and pathogenesis. In the Lao People's Democratic Republic (Laos) where melioidosis has been recognized as endemic since 1999, no such studies have been undertaken. We used a multilocus sequence typing scheme specific for B. pseudomallei to investigate nine cases of culture-positive recurrence occurring in 514 patients with melioidosis between 2010 and 2015: four were suspected to be relapses while the other five represented reinfections. In addition, two novel sequence types of the bacterium were identified. The low overall recurrence rates (2.4%) and proportions of relapse and reinfection in the Laos are consistent with those described in the recent literature, reflecting the effective use of appropriate antimicrobial therapy.

Wei Y, Kypraios T, O'Neill PD, Huang SS, Rifas-Shiman SL, Cooper BS. 2016. Evaluating hospital infection control measures for antimicrobial-resistant pathogens using stochastic transmission models: Application to vancomycin-resistant enterococci in intensive care units. Stat Methods Med Res, | Show Abstract | Read more

Nosocomial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) are the cause of significant morbidity and mortality among hospital patients. It is important to be able to assess the efficacy of control measures using data on patient outcomes. In this paper, we describe methods for analysing such data using patient-level stochastic models which seek to describe the underlying unobserved process of transmission. The methods are applied to detailed longitudinal patient-level data on vancomycin-resistant Enterococci from a study in a US hospital with eight intensive care units (ICUs). The data comprise admission and discharge dates, dates and results of screening tests, and dates during which precautionary measures were in place for each patient during the study period. Results include estimates of the efficacy of the control measures, the proportion of unobserved patients colonized with vancomycin-resistant Enterococci, and the proportion of patients colonized on admission.

Tarning J. 2016. Treatment of Malaria in Pregnancy. N Engl J Med, 374 (10), pp. 981-982. | Citations: 1 (Scopus) | Read more

Sheppard AE, Vaughan A, Jones N, Turner P, Turner C, Efstratiou A, Patel D, Modernising Medical Microbiology Informatics Group, Walker AS, Berkley JA et al. 2016. Capsular Typing Method for Streptococcus agalactiae Using Whole-Genome Sequence Data. J Clin Microbiol, 54 (5), pp. 1388-1390. | Citations: 5 (Scopus) | Show Abstract | Read more

Group B streptococcus (GBS) capsular serotypes are major determinants of virulence and affect potential vaccine coverage. Here we report a whole-genome-sequencing-based method for GBS serotype assignment. This method shows strong agreement (kappa of 0.92) with conventional methods and increased serotype assignment (100%) to all 10 capsular types.

Boudhar A, Ng XW, Loh CY, Chia WN, Tan ZM, Nosten F, Dymock BW, Tan KS. 2016. Overcoming Chloroquine Resistance in Malaria: Design, Synthesis, and Structure-Activity Relationships of Novel Hybrid Compounds. Antimicrob Agents Chemother, 60 (5), pp. 3076-3089. | Citations: 1 (Scopus) | Show Abstract | Read more

Resistance to antimalarial therapies, including artemisinin, has emerged as a significant challenge. Reversal of acquired resistance can be achieved using agents that resensitize resistant parasites to a previously efficacious therapy. Building on our initial work describing novel chemoreversal agents (CRAs) that resensitize resistant parasites to chloroquine (CQ), we herein report new hybrid single agents as an innovative strategy in the battle against resistant malaria. Synthetically linking a CRA scaffold to chloroquine produces hybrid compounds with restored potency toward a range of resistant malaria parasites. A preferred compound, compound 35, showed broad activity and good potency against seven strains resistant to chloroquine and artemisinin. Assessment of aqueous solubility, membrane permeability, and in vitro toxicity in a hepatocyte line and a cardiomyocyte line indicates that compound 35 has a good therapeutic window and favorable drug-like properties. This study provides initial support for CQ-CRA hybrid compounds as a potential treatment for resistant malaria.

Ishioka H, Ghose A, Charunwatthana P, Maude R, Plewes K, Kingston H, Intharabut B, Woodrow C, Chotivanich K, Sayeed AA et al. 2016. Sequestration and Red Cell Deformability as Determinants of Hyperlactatemia in Falciparum Malaria. J Infect Dis, 213 (5), pp. 788-793. | Show Abstract | Read more

BACKGROUND: Hyperlactatemia is a strong predictor of mortality in severe falciparum malaria. Sequestered parasitized erythrocytes and reduced uninfected red blood cell deformability (RCD) compromise microcirculatory flow, leading to anaerobic glycolysis. METHODS: In a cohort of patients with falciparum malaria hospitalized in Chittagong, Bangladesh, bulk RCD was measured using a laser diffraction technique, and parasite biomass was estimated from plasma concentrations of Plasmodium falciparum histidine-rich protein 2 (PfHRP2). A multiple linear regression model was constructed to examine their associations with plasma lactate concentrations. RESULTS: A total of 286 patients with falciparum malaria were studied, of whom 224 had severe malaria, and 70 died. Hyperlactatemia (lactate level, ≥ 4 mmol/L) was present in 111 cases. RCD at shear stresses of 1.7 Pa and 30 Pa was reduced significantly in patients who died, compared with survivors, individuals with uncomplicated malaria, or healthy individuals (P < .05, for all comparisons). Multiple linear regression analysis showed that the plasma PfHRP2 level, parasitemia level, total bilirubin level, and RCD at a shear stress of 1.7 Pa were each independently correlated with plasma lactate concentrations (n = 278; R(2) = 0.35). CONCLUSIONS: Sequestration of parasitized red blood cells and reduced RCD both contribute to decreased microcirculatory flow in severe disease.

Croucher NJ, Mostowy R, Wymant C, Turner P, Bentley SD, Fraser C. 2016. Horizontal DNA Transfer Mechanisms of Bacteria as Weapons of Intragenomic Conflict. PLoS Biol, 14 (3), pp. e1002394. | Citations: 8 (Scopus) | Show Abstract | Read more

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated.

Amaratunga C, Lim P, Suon S, Sreng S, Mao S, Sopha C, Sam B, Dek D, Try V, Amato R et al. 2016. Dihydroartemisinin-piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study. Lancet Infect Dis, 16 (3), pp. 357-365. | Citations: 41 (Scopus) | Show Abstract | Read more

BACKGROUND: Artemisinin resistance in Plasmodium falciparum threatens to reduce the efficacy of artemisinin combination therapies (ACTs), thus compromising global efforts to eliminate malaria. Recent treatment failures with dihydroartemisinin-piperaquine, the current first-line ACT in Cambodia, suggest that piperaquine resistance may be emerging in this country. We explored the relation between artemisinin resistance and dihydroartemisinin-piperaquine failures, and sought to confirm the presence of piperaquine-resistant P falciparum infections in Cambodia. METHODS: In this prospective cohort study, we enrolled patients aged 2-65 years with uncomplicated P falciparum malaria in three Cambodian provinces: Pursat, Preah Vihear, and Ratanakiri. Participants were given standard 3-day courses of dihydroartemisinin-piperaquine. Peripheral blood parasite densities were measured until parasites cleared and then weekly to 63 days. The primary outcome was recrudescent P falciparum parasitaemia within 63 days. We measured piperaquine plasma concentrations at baseline, 7 days, and day of recrudescence. We assessed phenotypic and genotypic markers of drug resistance in parasite isolates. The study is registered with, number NCT01736319. FINDINGS: Between Sept 4, 2012, and Dec 31, 2013, we enrolled 241 participants. In Pursat, where artemisinin resistance is entrenched, 37 (46%) of 81 patients had parasite recrudescence. In Preah Vihear, where artemisinin resistance is emerging, ten (16%) of 63 patients had recrudescence and in Ratanakiri, where artemisinin resistance is rare, one (2%) of 60 patients did. Patients with recrudescent P falciparum infections were more likely to have detectable piperaquine plasma concentrations at baseline compared with non-recrudescent patients, but did not differ significantly in age, initial parasite density, or piperaquine plasma concentrations at 7 days. Recrudescent parasites had a higher prevalence of kelch13 mutations, higher piperaquine 50% inhibitory concentration (IC50) values, and lower mefloquine IC50 values; none had multiple pfmdr1 copies, a genetic marker of mefloquine resistance. INTERPRETATION: Dihydroartemisinin-piperaquine failures are caused by both artemisinin and piperaquine resistance, and commonly occur in places where dihydroartemisinin-piperaquine has been used in the private sector. In Cambodia, artesunate plus mefloquine may be a viable option to treat dihydroartemisinin-piperaquine failures, and a more effective first-line ACT in areas where dihydroartemisinin-piperaquine failures are common. The use of single low-dose primaquine to eliminate circulating gametocytes is needed in areas where artemisinin and ACT resistance is prevalent. FUNDING: National Institute of Allergy and Infectious Diseases.

Bharucha T, Chanthongthip A, Phuangpanom S, Phonemixay O, Sengvilaipaseuth O, Vongsouvath M, Lee S, Newton PN, Dubot-Pérès A. 2016. Pre-cut Filter Paper for Detecting Anti-Japanese Encephalitis Virus IgM from Dried Cerebrospinal Fluid Spots. PLoS Negl Trop Dis, 10 (3), pp. e0004516. | Show Abstract | Read more

BACKGROUND: The use of filter paper as a simple, inexpensive tool for storage and transportation of blood, 'Dried Blood Spots' or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA). METHODOLOGY AND PRINCIPAL FINDINGS: Sample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200 μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009-15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25-30 °C showed 81.6% (65.7-92.3 95%CI) positive agreement, 96.8% (91.0-99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70-0.92 95%CI). CONCLUSIONS/SIGNIFICANCE: The novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids.

Boni MF, White NJ, Baird JK. 2016. The Community As the Patient in Malaria-Endemic Areas: Preempting Drug Resistance with Multiple First-Line Therapies. PLoS Med, 13 (3), pp. e1001984. | Show Abstract | Read more

Maciej F. Boni and colleagues propose deploying multiple first-line combination therapies against malaria within a community to delay drug-resistance evolution.

Leang R, Canavati SE, Khim N, Vestergaard LS, Borghini Fuhrer I, Kim S, Denis MB, Heng P, Tol B, Huy R et al. 2016. Efficacy and Safety of Pyronaridine-Artesunate for Treatment of Uncomplicated Plasmodium falciparum Malaria in Western Cambodia. Antimicrob Agents Chemother, 60 (7), pp. 3884-3890. | Citations: 2 (Scopus) | Show Abstract | Read more

Pyronaridine-artesunate efficacy for the treatment of uncomplicated Plasmodium falciparum malaria was assessed in an area of artemisinin resistance in western Cambodia. This nonrandomized, single-arm, observational study was conducted between 2014 and 2015. Eligible patients were adults or children with microscopically confirmed P. falciparum infection and fever. Patients received pyronaridine-artesunate once daily for 3 days, dosed according to body weight. The primary outcome was an adequate clinical and parasitological response (ACPR) on day 42, estimated by using Kaplan-Meier analysis, PCR adjusted to exclude reinfection. One hundred twenty-three patients were enrolled. Day 42 PCR-crude ACPRs were 87.2% (95% confidence interval [CI], 79.7 to 92.6%) for the overall study, 89.8% (95% CI, 78.8 to 95.3%) for Pursat, and 82.1% (95% CI, 68.4 to 90.2%) for Pailin. Day 42 PCR-adjusted ACPRs were 87.9% (95% CI, 80.6 to 93.2%) for the overall study, 89.8% (95% CI, 78.8 to 95.3%) for Pursat, and 84.0% (95% CI, 70.6 to 91.7%) for Pailin (P = 0.353 by a log rank test). Day 28 PCR-crude and -adjusted ACPRs were 93.2% (95% CI, 82.9 to 97.4%) and 88.1% (95% CI, 75.3 to 94.5%) for Pursat and Pailin, respectively. A significantly lower proportion of patients achieved day 3 parasite clearance in Pailin (56.4% [95% CI, 43.9 to 69.6%]) than in Pursat (86.7% [95% CI, 76.8 to 93.8%]; P = 0.0019). Fever clearance was also extended at Pailin versus Pursat (P < 0.0001). Most patients (95.9% [116/121]) harbored P. falciparum kelch13 C580Y mutant parasites. Pyronaridine-artesunate was well tolerated; mild increases in hepatic transaminase levels were consistent with data from previous reports. Pyronaridine-artesunate efficacy was below the World Health Organization-recommended threshold at day 42 for medicines with a long half-life (90%) for first-line treatment of P. falciparum malaria in western Cambodia despite high efficacy elsewhere in Asia and Africa. (This study has been registered at under registration number NCT02389439.).

Suttisunhakul V, Wuthiekanun V, Brett PJ, Khusmith S, Day NP, Burtnick MN, Limmathurotsakul D, Chantratita N. 2016. Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei. J Clin Microbiol, 54 (5), pp. 1259-1268. | Citations: 3 (Scopus) | Show Abstract | Read more

Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic.

Dondorp AM, Iyer SS, Schultz MJ. 2016. Critical Care in Resource-Restricted Settings. JAMA, 315 (8), pp. 753-754. | Citations: 4 (Scopus) | Read more

Deeny SR, Worby CJ, Tosas Auguet O, Cooper BS, Edgeworth J, Cookson B, Robotham JV. 2016. More Research Is Needed to Quantify Risks, Benefits, and Cost-Effectiveness of Universal Mupirocin Usage. Clin Infect Dis, 62 (9), pp. 1193-1194. | Citations: 1 (Scopus) | Read more

Sigera PC, Tunpattu TM, Jayashantha TP, De Silva AP, Athapattu PL, Dondorp A, Haniffa R. 2016. National Profile of Physical Therapists in Critical Care Units of Sri Lanka: Lower Middle-Income Country. Phys Ther, 96 (7), pp. 933-939. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: The availability and role of physical therapists in critical care is variable in resource-poor settings, including lower middle-income countries. OBJECTIVE: The aim of this study was to determine: (1) the availability of critical care physical therapist services, (2) the equipment and techniques used and needed, and (3) the training and continuous professional development of physical therapists. METHODS: All physical therapists working in critical care units (CCUs) of state hospitals in Sri Lanka were contacted. The study tool used was an interviewer-administered telephone questionnaire. RESULTS: The response rate was 100% (N=213). Sixty-one percent of the physical therapists were men. Ninety-four percent of the respondents were at least diploma holders in physical therapy, and 6% had non-physical therapy degrees. Most (n=145, 68%) had engaged in some continuous professional development in the past year. The majority (n=119, 56%) attended to patients after referral from medical staff. Seventy-seven percent, 98%, and 96% worked at nights, on weekends, and on public holidays, respectively. Physical therapists commonly perform manual hyperinflation, breathing exercises, manual airway clearance techniques, limb exercises, mobilization, positioning, and postural drainage in the CCUs. Lack of specialist training, lack of adequate physical therapy staff numbers, a heavy workload, and perceived lack of infection control in CCUs were the main difficulties they identified. LIMITATIONS: Details on the proportions of time spent by the physical therapists in the CCUs, wards, or medical departments were not collected. CONCLUSIONS: The availability of physical therapist services in CCUs in Sri Lanka, a lower middle-income country, was comparable to that in high-income countries, as per available literature, in terms of service availability and staffing, although the density of physical therapists remained very low, critical care training was limited, and resource limitations to physical therapy practices were evident.

Dittrich S, Rudgard WE, Woods KL, Silisouk J, Phuklia W, Davong V, Vongsouvath M, Phommasone K, Rattanavong S, Knappik M et al. 2016. The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira: A Prospective Evaluation. Am J Trop Med Hyg, 94 (4), pp. 736-740. | Citations: 2 (Scopus) | Show Abstract | Read more

Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N= 109) and negative (N= 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55-76) and 59% (95% CI: 49-68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N= 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used.

Hearn P, Turner C, Suy K, Soeng S, Day NP, Turner P. 2016. Lack of Utility of Nasopharyngeal Swabs for Diagnosis of Burkholderia pseudomallei Pneumonia in Paediatric Patients. J Trop Pediatr, 62 (4), pp. 328-330. | Show Abstract | Read more

Diagnosis of Burkholderia pseudomallei pneumonia in children is challenging. We investigated the utility of nasopharyngeal swabs taken from 194 paediatric patients on admission with radiologically proven pneumonia. Melioidosis was proven in 0.5% of samples tested and only in a third of those known to be bacteraemic with B. pseudomallei. It appears unlikely that culture of nasopharyngeal secretions is helpful to confirm B. pseudomallei pneumonia in paediatric patients.

Beardsley J, Wolbers M, Kibengo FM, Ggayi AB, Kamali A, Cuc NT, Binh TQ, Chau NV, Farrar J, Merson L et al. 2016. Adjunctive Dexamethasone in HIV-Associated Cryptococcal Meningitis. N Engl J Med, 374 (6), pp. 542-554. | Citations: 17 (Scopus) | Show Abstract | Read more

BACKGROUND: Cryptococcal meningitis associated with human immunodeficiency virus (HIV) infection causes more than 600,000 deaths each year worldwide. Treatment has changed little in 20 years, and there are no imminent new anticryptococcal agents. The use of adjuvant glucocorticoids reduces mortality among patients with other forms of meningitis in some populations, but their use is untested in patients with cryptococcal meningitis. METHODS: In this double-blind, randomized, placebo-controlled trial, we recruited adult patients with HIV-associated cryptococcal meningitis in Vietnam, Thailand, Indonesia, Laos, Uganda, and Malawi. All the patients received either dexamethasone or placebo for 6 weeks, along with combination antifungal therapy with amphotericin B and fluconazole. RESULTS: The trial was stopped for safety reasons after the enrollment of 451 patients. Mortality was 47% in the dexamethasone group and 41% in the placebo group by 10 weeks (hazard ratio in the dexamethasone group, 1.11; 95% confidence interval [CI], 0.84 to 1.47; P=0.45) and 57% and 49%, respectively, by 6 months (hazard ratio, 1.18; 95% CI, 0.91 to 1.53; P=0.20). The percentage of patients with disability at 10 weeks was higher in the dexamethasone group than in the placebo group, with 13% versus 25% having a prespecified good outcome (odds ratio, 0.42; 95% CI, 0.25 to 0.69; P<0.001). Clinical adverse events were more common in the dexamethasone group than in the placebo group (667 vs. 494 events, P=0.01), with more patients in the dexamethasone group having grade 3 or 4 infection (48 vs. 25 patients, P=0.003), renal events (22 vs. 7, P=0.004), and cardiac events (8 vs. 0, P=0.004). Fungal clearance in cerebrospinal fluid was slower in the dexamethasone group. Results were consistent across Asian and African sites. CONCLUSIONS: Dexamethasone did not reduce mortality among patients with HIV-associated cryptococcal meningitis and was associated with more adverse events and disability than was placebo. (Funded by the United Kingdom Department for International Development and others through the Joint Global Health Trials program; Current Controlled Trials number, ISRCTN59144167.).

Charnaud SC, McGready R, Herten-Crabb A, Powell R, Guy A, Langer C, Richards JS, Gilson PR, Chotivanich K, Tsuboi T et al. 2016. Maternal-foetal transfer of Plasmodium falciparum and Plasmodium vivax antibodies in a low transmission setting. Sci Rep, 6 pp. 20859. | Citations: 2 (Scopus) | Show Abstract | Read more

During pregnancy immunoglobulin G (IgG) antibodies are transferred from mother to neonate across the placenta. Studies in high transmission areas have shown transfer of P. falciparum-specific IgG, but the extent and factors influencing maternal-foetal transfer in low transmission areas co-endemic for both P. falciparum and P. vivax are unknown. Pregnant women were screened weekly for Plasmodium infection. Mother-neonate paired serum samples at delivery were tested for IgG to antigens from P. falciparum, P. vivax and other infectious diseases. Antibodies to malarial and non-malarial antigens were highly correlated between maternal and neonatal samples (median [range] spearman ρ = 0.78 [0.57-0.93]), although Plasmodium spp. antibodies tended to be lower in neonates than mothers. Estimated gestational age at last P. falciparum infection, but not P. vivax infection, was positively associated with antibody levels in the neonate (P. falciparum merozoite, spearman ρ median [range] 0.42 [0.33-0.66], PfVAR2CSA 0.69; P. vivax ρ = 0.19 [0.09-0.3]). Maternal-foetal transfer of anti-malarial IgG to Plasmodium spp. antigens occurs in low transmission settings. P. vivax IgG acquisition is not associated with recent exposure unlike P. falciparum IgG, suggesting a difference in acquisition of antibodies. IgG transfer is greatest in the final weeks of pregnancy which has implications for the timing of future malaria vaccination strategies in pregnant women.

Thuan PD, Ca NT, Van Toi P, Nhien NT, Thanh NV, Anh ND, Phu NH, Thai CQ, Thai LEH, Hoa NT et al. 2016. A Randomized Comparison of Chloroquine Versus Dihydroartemisinin-Piperaquine for the Treatment of Plasmodium vivax Infection in Vietnam. Am J Trop Med Hyg, 94 (4), pp. 879-885. | Citations: 1 (Scopus) | Show Abstract | Read more

A total of 128 Vietnamese patients with symptomatic Plasmodium vivax mono-infections were enrolled in a prospective, open-label, randomized trial to receive either chloroquine or dihydroartemisinin-piperaquine (DHA-PPQ). The proportions of patients with adequate clinical and parasitological responses were 47% in the chloroquine arm (31 of 65 patients) and 66% in the DHA-PPQ arm (42 of 63 patients) in the Kaplan-Meier intention-to-treat analysis (absolute difference 19%, 95% confidence interval = 0-37%), thus establishing non-inferiority of DHA-PPQ. Fever clearance time (median 24 versus 12 hours,P= 0.02), parasite clearance time (median 36 versus 18 hours,P< 0.001), and parasite clearance half-life (mean 3.98 versus 1.80 hours,P< 0.001) were all significantly shorter in the DHA-PPQ arm. All cases of recurrent parasitemia in the chloroquine arm occurred from day 33 onward, with corresponding whole blood chloroquine concentration lower than 100 ng/mL in all patients. Chloroquine thus remains efficacious for the treatment of P. vivax malaria in southern Vietnam, but DHA-PPQ provides more rapid symptomatic and parasitological recovery.

Drake TL, Devine A, Yeung S, Day NP, White LJ, Lubell Y. 2016. Dynamic Transmission Economic Evaluation of Infectious Disease Interventions in Low- and Middle-Income Countries: A Systematic Literature Review. Health Econ, 25 Suppl 1 pp. 124-139. | Citations: 1 (Scopus) | Show Abstract | Read more

Economic evaluation using dynamic transmission models is important for capturing the indirect effects of infectious disease interventions. We examine the use of these methods in low- and middle-income countries, where infectious diseases constitute a major burden. This review is comprised of two parts: (1) a summary of dynamic transmission economic evaluations across all disease areas published between 2011 and mid-2014 and (2) an in-depth review of mosquito-borne disease studies focusing on health economic methods and reporting. Studies were identified through a systematic search of the MEDLINE database and supplemented by reference list screening. Fifty-seven studies were eligible for inclusion in the all-disease review. The most common subject disease was HIV/AIDS, followed by malaria. A diverse range of modelling methods, outcome metrics and sensitivity analyses were used, indicating little standardisation. Seventeen studies were included in the mosquito-borne disease review. With notable exceptions, most studies did not employ economic evaluation methods beyond calculating a cost-effectiveness ratio or net benefit. Many did not adhere to health care economic evaluations reporting guidelines, particularly with respect to full model reporting and uncertainty analysis. We present a summary of the state-of-the-art and offer recommendations for improved implementation and reporting of health economic methods in this crossover discipline.

Stuetz W, Carrara VI, Mc Gready R, Lee SJ, Sriprawat K, Po B, Hanboonkunupakarn B, Grune T, Biesalski HK, Nosten FH. 2016. Impact of Food Rations and Supplements on Micronutrient Status by Trimester of Pregnancy: Cross-Sectional Studies in the Maela Refugee Camp in Thailand. Nutrients, 8 (2), pp. 66. | Citations: 1 (Scopus) | Show Abstract | Read more

Micronutrient fortified flour (MFF), supplementary food rations and micronutrient (MN) supplements may prevent deficiencies among pregnant women. Objectives of cross-sectional surveys in 2004 (n = 533) and 2006 (n = 515) were to assess the impact of new food rations (flour, oil) and supplements on MN status by trimester of pregnancy in the Maela refugee camp. Hemoglobin, iron status, zinc, retinol, β-carotene and tryptophan decreased, while α-/γ-tocopherol and 5-methyltetrahydrofolate (5-MTHF) increased from first to third trimester. In 2006, mean zinc and α-tocopherol for each trimester was significantly higher than in 2004. The weeks of supplemented thiamine and folic acid were positively correlated with thiamine diphosphate (TDP) and 5-MTHF, but not for ferrous sulfate as iron deficiency was observed in 38.5% of third-trimester women. Frequent consumption of fish paste and owning a garden or animal were associated with significantly higher iron status, retinol, β-carotene, and 5-MTHF. In conclusion, MFF and supplementary oil were most likely to explain improved zinc and α-tocopherol status, while thiamine and folate supplements ensured high TDP and 5-MTHF in late pregnancy. MN supplements, MN-rich staple food, small gardens, and programs to improve iron compliance are promising strategies to prevent MN deficiencies during pregnancy in vulnerable populations.

Carter MJ, Emary KR, Moore CE, Parry CM, Sona S, Putchhat H, Reaksmey S, Chanpheaktra N, Stoesser N, Dobson AD et al. 2016. Correction: Rapid Diagnostic Tests for Dengue Virus Infection in Febrile Cambodian Children: Diagnostic Accuracy and Incorporation into Diagnostic Algorithms. PLoS Negl Trop Dis, 10 (2), pp. e0004453. | Read more

Sadique Z, Lopman B, Cooper BS, Edmunds WJ. 2016. Cost-effectiveness of Ward Closure to Control Outbreaks of Norovirus Infection in United Kingdom National Health Service Hospitals. J Infect Dis, 213 Suppl 1 (suppl 1), pp. S19-S26. | Citations: 2 (Web of Science Lite) | Show Abstract | Read more

BACKGROUND: Norovirus is the most common cause of outbreaks of acute gastroenteritis in National Health Service hospitals in the United Kingdom. Wards (units) are often closed to new admissions to stop the spread of the virus, but there is limited evidence describing the cost-effectiveness of ward closure. METHODS: An economic analysis based on the results from a large, prospective, active-surveillance study of gastroenteritis outbreaks in hospitals and from an epidemic simulation study compared alternative ward closure options evaluated at different time points since first infection, assuming different efficacies of ward closure. RESULTS: A total of 232 gastroenteritis outbreaks occurring in 14 hospitals over a 1-year period were analyzed. The risk of a new outbreak in a hospital is significantly associated with the number of admission, general medical, and long-stay wards that are concurrently affected but is less affected by the level of community transmission. Ward closure leads to higher costs but reduces the number of new outbreaks by 6%-56% and the number of clinical cases by 1%-55%, depending on the efficacy of the intervention. The incremental cost per outbreak averted varies from £10 000 ($14 000) to £306 000 ($428 000), and the cost per case averted varies from £500 ($700) to £61 000 ($85 000). The cost-effectiveness of ward closure decreases as the efficacy of the intervention increases, and the cost-effectiveness increases with the timing of the intervention. The efficacy of ward closure is critical from a cost-effectiveness perspective. CONCLUSIONS: Ward closure may be cost-effective, particularly if targeted to high-throughput units.

Moore KA, Simpson JA, Paw MK, Pimanpanarak M, Wiladphaingern J, Rijken MJ, Jittamala P, White NJ, Fowkes FJ, Nosten F, McGready R. 2016. Safety of artemisinins in first trimester of prospectively followed pregnancies: an observational study. Lancet Infect Dis, 16 (5), pp. 576-583. | Citations: 6 (Scopus) | Show Abstract | Read more

BACKGROUND: Artemisinins, the most effective antimalarials available, are not recommended for falciparum malaria during the first trimester of pregnancy because of safety concerns. Therefore, quinine is used despite its poor effectiveness. Assessing artemisinin safety requires weighing the risks of malaria and its treatment. We aimed to assess the effect of first-trimester malaria and artemisinin treatment on miscarriage and major congenital malformations. METHODS: In this observational study, we assessed data from antenatal clinics on the Thai-Myanmar border between Jan 1, 1994, and Dec 31, 2013. We included women who presented to antenatal clinics during their first trimester with a viable fetus. Women were screened for malaria, and data on malaria, antimalarial treatment, and birth outcomes were collected. The relationship between artemisinin treatments (artesunate, dihydroartemisinin, or artemether) and miscarriage or malformation was assessed using Cox regression with left-truncation and time-varying exposures. FINDINGS: Of 55 636 pregnancies registered between 1994 and 2013, 25 485 pregnancies were analysed for first-trimester malaria and miscarriage, in which 2558 (10%) had first-trimester malaria. The hazard of miscarriage increased 1·61-fold after an initial first-trimester falciparum episode (95% CI 1·32-1·97; p<0·0001), 3·24-fold following falciparum recurrence (2·24-4·68; p<0·0001), and 2·44-fold (1·01-5·88; p=0·0473) following recurrent symptomatic vivax malaria. No difference was noted in miscarriage in first-line falciparum treatments with artemisinin (n=183) versus quinine (n=842; HR 0·78 [95% CI 0·45-1·34]; p=0·3645) or in risk of major congenital malformations (two [2%] of 109 [95% CI 0·22-6·47] versus eight (1%) of 641 [0·54-2·44], respectively). INTERPRETATION: First-trimester falciparum and vivax malaria both increase the risk of miscarriage. We noted no evidence of an increased risk of miscarriage or of major congenital malformations associated with first-line treatment with an artemisinin derivative compared with quinine. In view of the low efficacy of quinine and wide availability of highly effective artemisinin-based combination therapies, it is time to reconsider first-trimester antimalarial treatment recommendations. FUNDING: The Wellcome Trust and The Bill & Melinda Gates Foundation.

Tosas Auguet O, Betley JR, Stabler RA, Patel A, Ioannou A, Marbach H, Hearn P, Aryee A, Goldenberg SD, Otter JA et al. 2016. Evidence for Community Transmission of Community-Associated but Not Health-Care-Associated Methicillin-Resistant Staphylococcus Aureus Strains Linked to Social and Material Deprivation: Spatial Analysis of Cross-sectional Data. PLoS Med, 13 (1), pp. e1001944. | Show Abstract | Read more

BACKGROUND: Identifying and tackling the social determinants of infectious diseases has become a public health priority following the recognition that individuals with lower socioeconomic status are disproportionately affected by infectious diseases. In many parts of the world, epidemiologically and genotypically defined community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) strains have emerged to become frequent causes of hospital infection. The aim of this study was to use spatial models with adjustment for area-level hospital attendance to determine the transmission niche of genotypically defined CA- and health-care-associated (HA)-MRSA strains across a diverse region of South East London and to explore a potential link between MRSA carriage and markers of social and material deprivation. METHODS AND FINDINGS: This study involved spatial analysis of cross-sectional data linked with all MRSA isolates identified by three National Health Service (NHS) microbiology laboratories between 1 November 2011 and 29 February 2012. The cohort of hospital-based NHS microbiology diagnostic services serves 867,254 usual residents in the Lambeth, Southwark, and Lewisham boroughs in South East London, United Kingdom (UK). Isolates were classified as HA- or CA-MRSA based on whole genome sequencing. All MRSA cases identified over 4 mo within the three-borough catchment area (n = 471) were mapped to small geographies and linked to area-level aggregated socioeconomic and demographic data. Disease mapping and ecological regression models were used to infer the most likely transmission niches for each MRSA genetic classification and to describe the spatial epidemiology of MRSA in relation to social determinants. Specifically, we aimed to identify demographic and socioeconomic population traits that explain cross-area extra variation in HA- and CA-MRSA relative risks following adjustment for hospital attendance data. We explored the potential for associations with the English Indices of Deprivation 2010 (including the Index of Multiple Deprivation and several deprivation domains and subdomains) and the 2011 England and Wales census demographic and socioeconomic indicators (including numbers of households by deprivation dimension) and indicators of population health. Both CA-and HA-MRSA were associated with household deprivation (CA-MRSA relative risk [RR]: 1.72 [1.03-2.94]; HA-MRSA RR: 1.57 [1.06-2.33]), which was correlated with hospital attendance (Pearson correlation coefficient [PCC] = 0.76). HA-MRSA was also associated with poor health (RR: 1.10 [1.01-1.19]) and residence in communal care homes (RR: 1.24 [1.12-1.37]), whereas CA-MRSA was linked with household overcrowding (RR: 1.58 [1.04-2.41]) and wider barriers, which represent a combined score for household overcrowding, low income, and homelessness (RR: 1.76 [1.16-2.70]). CA-MRSA was also associated with recent immigration to the UK (RR: 1.77 [1.19-2.66]). For the area-level variation in RR for CA-MRSA, 28.67% was attributable to the spatial arrangement of target geographies, compared with only 0.09% for HA-MRSA. An advantage to our study is that it provided a representative sample of usual residents receiving care in the catchment areas. A limitation is that relationships apparent in aggregated data analyses cannot be assumed to operate at the individual level. CONCLUSIONS: There was no evidence of community transmission of HA-MRSA strains, implying that HA-MRSA cases identified in the community originate from the hospital reservoir and are maintained by frequent attendance at health care facilities. In contrast, there was a high risk of CA-MRSA in deprived areas linked with overcrowding, homelessness, low income, and recent immigration to the UK, which was not explainable by health care exposure. Furthermore, areas adjacent to these deprived areas were themselves at greater risk of CA-MRSA, indicating community transmission of CA-MRSA. This ongoing community transmission could lead to CA-MRSA becoming the dominant strain types carried by patients admitted to hospital, particularly if successful hospital-based MRSA infection control programmes are maintained. These results suggest that community infection control programmes targeting transmission of CA-MRSA will be required to control MRSA in both the community and hospital. These epidemiological changes will also have implications for effectiveness of risk-factor-based hospital admission MRSA screening programmes.

Chairat K, Jittamala P, Hanpithakpong W, Day NP, White NJ, Pukrittayakamee S, Tarning J. 2016. Population pharmacokinetics of oseltamivir and oseltamivir carboxylate in obese and non-obese volunteers. Br J Clin Pharmacol, 81 (6), pp. 1103-1112. | Citations: 2 (Scopus) | Show Abstract | Read more

AIMS: The aims of the present study were to compare the pharmacokinetics of oseltamivir and its active antiviral metabolite oseltamivir carboxylate in obese and non-obese individuals and to determine the effect of obesity on the pharmacokinetic properties of oseltamivir and oseltamivir carboxylate. METHODS: The population pharmacokinetic properties of oseltamivir and oseltamivir carboxylate were evaluated in 12 obese [body mass index (BMI) ≥30 kg m(-2) ) and 12 non-obese (BMI <30 kg m(-2) ) Thai adult volunteers receiving a standard dose of 75 mg and a double dose of 150 mg in a randomized sequence. Concentration-time data were collected and analysed using nonlinear mixed-effects modelling. RESULTS: The pharmacokinetics of oseltamivir and oseltamivir carboxylate were described simultaneously by first-order absorption, with a one-compartment disposition model for oseltamivir, followed by a metabolism compartment and a one-compartment disposition model for oseltamivir carboxylate. Creatinine clearance was a significant predictor of oseltamivir carboxylate clearance {3.84% increase for each 10 ml min(-1) increase in creatinine clearance [95% confidence interval (CI) 0.178%, 8.02%]}. Obese individuals had an approximately 25% (95% CI 24%, 28%) higher oseltamivir clearance, 20% higher oseltamivir volume of distribution (95% CI 19%, 23%) and 10% higher oseltamivir carboxylate clearance (95% CI 9%, 11%) compared with non-obese individuals. However, these altered pharmacokinetic properties were small and did not change the overall exposure to oseltamivir carboxylate. CONCLUSIONS: The results confirmed that a dose adjustment for oseltamivir in obese individuals is not necessary on the basis of its pharmacokinetics.

Ribolzi O, Rochelle-Newall E, Dittrich S, Auda Y, Newton PN, Rattanavong S, Knappik M, Soulileuth B, Sengtaheuanghoung O, Dance DA, Pierret A. 2016. Land use and soil type determine the presence of the pathogen Burkholderia pseudomallei in tropical rivers. Environ Sci Pollut Res Int, 23 (8), pp. 7828-7839. | Citations: 4 (Scopus) | Show Abstract | Read more

Burkholderia pseudomallei is the bacterium that causes melioidosis in humans. While B. pseudomallei is known to be endemic in South East Asia (SEA), the occurrence of the disease in other parts of the tropics points towards a potentially large global distribution. We investigated the environmental factors that influence the presence (and absence) of B. pseudomallei in a tropical watershed in SEA. Our main objective was to determine whether there is a link between the presence of the organism in the hydrographic network and the upstream soil and land-use type. The presence of B. pseudomallei was determined using a specific quantitative real-time PCR assay following enrichment culture. Land use, soil, geomorphology, and environmental data were then analyzed using partial least squares discriminant analysis (PLSDA) to compare the B. pseudomallei positive and negative sites. Soil type in the surrounding catchment and turbidity had a strong positive influence on the presence (acrisols and luvisols) or absence (ferralsols) of B. pseudomallei. Given the strong apparent links between soil characteristics, water turbidity, and the presence/absence of B. pseudomallei, actions to raise public awareness about factors increasing the risk of exposure should be undertaken in order to reduce the incidence of melioidosis in regions of endemicity.

Bousfield R, Thyl M, Samol O, Rithea L, Sona S, Chhat HP, Poda S, Moore CE, Chheng K, Kumar V et al. 2016. A retrospective study of factors which determine a negative blood culture in Cambodian children diagnosed with enteric fever. Paediatr Int Child Health, pp. 1-7. | Show Abstract | Read more

BACKGROUND: Blood cultures are used to confirm a diagnosis of enteric fever but reported sensitivities can be as low as 40%. AIMS: To determine the factors associated with a negative blood culture in Cambodian children with suspected enteric fever. METHODS: In a retrospective study of hospitalised Cambodian children given a discharge diagnosis of enteric fever, the following factors associated with a negative blood culture were analysed: age, blood culture volume, prior antibiotic therapy, duration of illness and disease severity. RESULTS: In 227 hospitalised Cambodian children with a discharge diagnosis of enteric fever, it was confirmed in 70% by a positive blood culture. There was no association between a negative blood culture and younger age, lower blood volumes for culture, prior antibiotic therapy, a late presentation or milder disease. CONCLUSIONS: Although blood culture sensitivity was higher than expected, alternative simple, rapid and sensitive tests are needed for diagnosing enteric fever.

Moore CE, Sona S, Poda S, Putchhat H, Kumar V, Sopheary S, Stoesser N, Bousfield R, Day N, Parry CM. 2016. Antimicrobial susceptibility of uropathogens isolated from Cambodian children. Paediatr Int Child Health, pp. 1-5. | Show Abstract | Read more

BACKGROUND: Bacterial resistance to commonly used antimicrobials is an increasing problem in Asia but information concerning the antimicrobial susceptibility of bacteria causing urinary tract infections (UTIs) in children is limited. METHODS: This was a 5-year retrospective study of children with suspected UTI attending a paediatric hospital in north-west Cambodia. Urines with a positive culture containing a single organism with a count of >10(5) colony-forming units (CFU)/ml were considered diagnostic of infection. The organism was identified and the resistance pattern (using CLSI guidelines) and presence of an extended-spectrum β-lactamase (ESBL) phenotype was determined. RESULTS: In total, there were 217 episodes of infection, 210 (97%) with Gram-negative bacteria. Escherichia coli was the most common infecting isolate with high levels of resistance to most oral antibiotics, except nitrofurantoin. Nearly half of the E. coli (44%) were extended-spectrum cephalosporin (ESC)-resistant with the proportion increasing significantly over the 5-year period. ESC-resistant E. coli were more likely to be multi-drug-resistant and 91% demonstrated an ESBL phenotype. CONCLUSION: The data highlight the importance of microbiological surveillance of UTIs in children, particularly in areas where there are known to be multiply resistant organisms.

Limmathurotsakul D, Golding N, Dance DA, Messina JP, Pigott DM, Moyes CL, Rolim DB, Bertherat E, Day NP, Peacock SJ, Hay SI. 2016. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat Microbiol, 1 (1), pp. 15008-15008. | Show Abstract | Read more

Burkholderia pseudomallei, a highly pathogenic bacterium that causes melioidosis, is commonly found in soil in Southeast Asia and Northern Australia(1,2). Melioidosis can be difficult to diagnose due to its diverse clinical manifestations and the inadequacy of conventional bacterial identification methods(3). The bacterium is intrinsically resistant to a wide range of antimicrobials, and treatment with ineffective antimicrobials may result in case fatality rates (CFRs) exceeding 70%(4,5). The importation of infected animals has, in the past, spread melioidosis to non-endemic areas(6,7). The global distribution of B. pseudomallei and burden of melioidosis, however, remain poorly understood. Here, we map documented human and animal cases, and the presence of environmental B. pseudomallei, and combine this in a formal modelling framework(8-10) to estimate the global burden of melioidosis. We estimate there to be 165,000 (95% credible interval 68,000-412,000) human melioidosis cases per year worldwide, of which 89,000 (36,000-227,000) die. Our estimates suggest that melioidosis is severely underreported in the 45 countries in which it is known to be endemic and that melioidosis is likely endemic in a further 34 countries which have never reported the disease. The large numbers of estimated cases and fatalities emphasise that the disease warrants renewed attention from public health officials and policy makers.

Limmathurotsakul D, Golding N, Dance DA, Messina JP, Pigott DM, Moyes CL, Rolim DB, Bertherat E, Day NP, Peacock SJ, Hay SI. 2016. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat Microbiol, 1 pp. 15008. | Show Abstract | Read more

Burkholderia pseudomallei, a highly pathogenic bacterium that causes melioidosis, is commonly found in soil in Southeast Asia and Northern Australia(1,2). Melioidosis can be difficult to diagnose due to its diverse clinical manifestations and the inadequacy of conventional bacterial identification methods(3). The bacterium is intrinsically resistant to a wide range of antimicrobials, and treatment with ineffective antimicrobials may result in case fatality rates (CFRs) exceeding 70%(4,5). The importation of infected animals has, in the past, spread melioidosis to non-endemic areas(6,7). The global distribution of B. pseudomallei and the burden of melioidosis, however, remain poorly understood. Here, we map documented human and animal cases and the presence of environmental B. pseudomallei and combine this in a formal modelling framework(8-10) to estimate the global burden of melioidosis. We estimate there to be 165,000 (95% credible interval 68,000-412,000) human melioidosis cases per year worldwide, from which 89,000 (36,000-227,000) people die. Our estimates suggest that melioidosis is severely underreported in the 45 countries in which it is known to be endemic and that melioidosis is probably endemic in a further 34 countries that have never reported the disease. The large numbers of estimated cases and fatalities emphasize that the disease warrants renewed attention from public health officials and policy makers.

Canavati SE, Lawford HL, Fatunmbi BS, Lek D, Leang R, Top Samphor N, Dondorp AM, Huy R, Kazadi WM. 2016. The Cambodia Research Consortium: expediting research for malaria elimination with the emergency response to artemisinin resistance framework. Malar J, 15 (1), pp. 5. | Citations: 2 (Scopus) | Show Abstract | Read more

This commentary offers insight into how to best address barriers that may hinder the translation of malaria research findings into policy. It also proposes viable methods of implementing these policies in Cambodia. Currently, a wide range of malaria research is being conducted by in-country stakeholders, including Cambodia's National Programme for Parasitology, Entomology and Malaria Control's (CNM), non-governmental organizations, and academic institutions. Coordinating research amongst these partners, as well as within the Ministry of Health, is a challenge. Results are rarely disseminated widely and seldom inform programme and policy decisions. CNM and its research partners have severely limited access to each other's databases. This lack of accessibility, timeliness, engagement and cooperation between CNM and its partners greatly impacts overall research efficiency in this field, and is stifling innovation both within and beyond CNM. Cambodia has set a goal to eradicate all forms of malaria by 2030. As countries approach the elimination phase, there is a greater need for sharing research-generated evidence amongst partners, in order to ensure that appropriate and impactful activities are conducted. The Cambodian Research Consortium was established to serve as a framework for partners, stakeholders and researchers to share research projects, information and results, and to promote the goals of CNM. The sharing of malaria research results will help to inform prevention, control and elimination activities in the country. It will also determine and address the country's operational research needs, and could potentially become a framework model to be used in other countries aiming to transition from malaria control to elimination.

Kyaw SS, Drake T, Thi A, Kyaw MP, Hlaing T, Smithuis FM, White LJ, Lubell Y. 2016. Malaria community health workers in Myanmar: a cost analysis. Malar J, 15 (1), pp. 41. | Citations: 3 (Scopus) | Show Abstract | Read more

BACKGROUND: Myanmar has the highest malaria incidence and attributed mortality in South East Asia with limited healthcare infrastructure to manage this burden. Establishing malaria Community Health Worker (CHW) programmes is one possible strategy to improve access to malaria diagnosis and treatment, particularly in remote areas. Despite considerable donor support for implementing CHW programmes in Myanmar, the cost implications are not well understood. METHODS: An ingredients based micro-costing approach was used to develop a model of the annual implementation cost of malaria CHWs in Myanmar. A cost model was constructed based on activity centres comprising of training, patient malaria services, monitoring and supervision, programme management, overheads and incentives. The model takes a provider perspective. Financial data on CHWs programmes were obtained from the 2013 financial reports of the Three Millennium Development Goal fund implementing partners that have been working on malaria control and elimination in Myanmar. Sensitivity and scenario analyses were undertaken to outline parameter uncertainty and explore changes to programme cost for key assumptions. RESULTS: The range of total annual costs for the support of one CHW was US$ 966-2486. The largest driver of CHW cost was monitoring and supervision (31-60% of annual CHW cost). Other important determinants of cost included programme management (15-28% of annual CHW cost) and patient services (6-12% of annual CHW cost). Within patient services, malaria rapid diagnostic tests are the major contributor to cost (64% of patient service costs). CONCLUSION: The annual cost of a malaria CHW in Myanmar varies considerably depending on the context and the design of the programme, in particular remoteness and the approach to monitoring and evaluation. The estimates provide information to policy makers and CHW programme planners in Myanmar as well as supporting economic evaluations of their cost-effectiveness.

Zhu L, Mok S, Imwong M, Jaidee A, Russell B, Nosten F, Day NP, White NJ, Preiser PR, Bozdech Z. 2016. New insights into the Plasmodium vivax transcriptome using RNA-Seq. Sci Rep, 6 pp. 20498. | Citations: 4 (Scopus) | Show Abstract | Read more

Historically seen as a benign disease, it is now becoming clear that Plasmodium vivax can cause significant morbidity. Effective control strategies targeting P. vivax malaria is hindered by our limited understanding of vivax biology. Here we established the P. vivax transcriptome of the Intraerythrocytic Developmental Cycle (IDC) of two clinical isolates in high resolution by Illumina HiSeq platform. The detailed map of transcriptome generates new insights into regulatory mechanisms of individual genes and reveals their intimate relationship with specific biological functions. A transcriptional hotspot of vir genes observed on chromosome 2 suggests a potential active site modulating immune evasion of the Plasmodium parasite across patients. Compared to other eukaryotes, P. vivax genes tend to have unusually long 5' untranslated regions and also present multiple transcription start sites. In contrast, alternative splicing is rare in P. vivax but its association with the late schizont stage suggests some of its significance for gene function. The newly identified transcripts, including up to 179 vir like genes and 3018 noncoding RNAs suggest an important role of these gene/transcript classes in strain specific transcriptional regulation.

Phommasone K, Althaus T, Souvanthong P, Phakhounthong K, Soyvienvong L, Malapheth P, Mayxay M, Pavlicek RL, Paris DH, Dance D et al. 2016. Accuracy of commercially available c-reactive protein rapid tests in the context of undifferentiated fevers in rural Laos. BMC Infect Dis, 16 (1), pp. 61. | Citations: 2 (Scopus) | Show Abstract | Read more

BACKGROUND: C-Reactive Protein (CRP) has been shown to be an accurate biomarker for discriminating bacterial from viral infections in febrile patients in Southeast Asia. Here we investigate the accuracy of existing rapid qualitative and semi-quantitative tests as compared with a quantitative reference test to assess their potential for use in remote tropical settings. METHODS: Blood samples were obtained from consecutive patients recruited to a prospective fever study at three sites in rural Laos. At each site, one of three rapid qualitative or semi-quantitative tests was performed, as well as a corresponding quantitative NycoCard Reader II as a reference test. We estimate the sensitivity and specificity of the three tests against a threshold of 10 mg/L and kappa values for the agreement of the two semi-quantitative tests with the results of the reference test. RESULTS: All three tests showed high sensitivity, specificity and kappa values as compared with the NycoCard Reader II. With a threshold of 10 mg/L the sensitivity of the tests ranged from 87-98 % and the specificity from 91-98 %. The weighted kappa values for the semi-quantitative tests were 0.7 and 0.8. CONCLUSION: The use of CRP rapid tests could offer an inexpensive and effective approach to improve the targeting of antibiotics in remote settings where health facilities are basic and laboratories are absent. This study demonstrates that accurate CRP rapid tests are commercially available; evaluations of their clinical impact and cost-effectiveness at point of care is warranted.

Ponsuwanna P, Kochakarn T, Bunditvorapoom D, Kümpornsin K, Otto TD, Ridenour C, Chotivanich K, Wilairat P, White NJ, Miotto O, Chookajorn T. 2016. Comparative genome-wide analysis and evolutionary history of haemoglobin-processing and haem detoxification enzymes in malarial parasites. Malar J, 15 (1), pp. 51. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Malaria parasites have evolved a series of intricate mechanisms to survive and propagate within host red blood cells. Intra-erythrocytic parasitism requires these organisms to digest haemoglobin and detoxify iron-bound haem. These tasks are executed by haemoglobin-specific proteases and haem biocrystallization factors that are components of a large multi-subunit complex. Since haemoglobin processing machineries are functionally and genetically linked to the modes of action and resistance mechanisms of several anti-malarial drugs, an understanding of their evolutionary history is important for drug development and drug resistance prevention. METHODS: Maximum likelihood trees of genetic repertoires encoding haemoglobin processing machineries within Plasmodium species, and with the representatives of Apicomplexan species with various host tropisms, were created. Genetic variants were mapped onto existing three-dimensional structures. Genome-wide single nucleotide polymorphism data were used to analyse the selective pressure and the effect of these mutations at the structural level. RESULTS: Recent expansions in the falcipain and plasmepsin repertoires are unique to human malaria parasites especially in the Plasmodium falciparum and P. reichenowi lineage. Expansion of haemoglobin-specific plasmepsins occurred after the separation event of Plasmodium species, but the other members of the plasmepsin family were evolutionarily conserved with one copy for each sub-group in every Apicomplexan species. Haemoglobin-specific falcipains are separated from invasion-related falcipain, and their expansions within one specific locus arose independently in both P. falciparum and P. vivax lineages. Gene conversion between P. falciparum falcipain 2A and 2B was observed in artemisinin-resistant strains. Comparison between the numbers of non-synonymous and synonymous mutations suggests a strong selective pressure at falcipain and plasmepsin genes. The locations of amino acid changes from non-synonymous mutations mapped onto protein structures revealed clusters of amino acid residues in close proximity or near the active sites of proteases. CONCLUSION: A high degree of polymorphism at the haemoglobin processing genes implicates an imposition of selective pressure. The identification in recent years of functional redundancy of haemoglobin-specific proteases makes them less appealing as potential drug targets, but their expansions, especially in the human malaria parasite lineages, unequivocally point toward their functional significance during the independent and repetitive adaptation events in malaria parasite evolutionary history.

Birgersson S, Van Toi P, Truong NT, Dung NT, Ashton M, Hien TT, Abelö A, Tarning J. 2016. Population pharmacokinetic properties of artemisinin in healthy male Vietnamese volunteers. Malar J, 15 (1), pp. 90. | Show Abstract | Read more

BACKGROUND: Artemisinin-based combination therapy is recommended as first-line anti-malarial treatment worldwide. A combination of artemisinin with the long acting drug piperaquine has shown high efficacy and tolerability in patients with uncomplicated Plasmodium falciparum infections. The aim of this study was to characterize the population pharmacokinetic properties of artemisinin in healthy male Vietnamese volunteers after two different dose sizes, formulations and in a combination with piperaquine. A secondary aim was to compare two different methods for the evaluation of bioequivalence of the formulations. METHODS: Fifteen subjects received four different dose regimens of a single dose of artemisinin as a conventional formulation (160 and 500 mg) and as a micronized test formulation (160 mg alone and in combination with piperaquine phosphate, 360 mg) with a washout period of 3 weeks between each period (i.e. four-way cross-over). Venous plasma samples were collected frequently up to 12 h after dose in each period. Artemisinin was quantified in plasma using liquid chromatography coupled with tandem mass spectrometry. A nonlinear mixed-effects modelling approach was utilized to evaluate the population pharmacokinetic properties of the drug and to investigate the clinical impact of different formulations. RESULTS: The plasma concentration-time profiles for artemisinin were adequately described by a transit-absorption model with a one-compartment disposition, in all four sequences simultaneously. The mean oral clearance, volume of distribution and terminal elimination half-life was 417 L/h, 1210 L and 1.93 h, respectively. Influence of formulation, dose and possible interaction of piperaquine was evaluated as categorical covariates in full covariate approaches. No clinically significant differences between formulations were shown which was in accordance with the previous results using a non-compartmental bioequivalence approach. CONCLUSIONS: This is the first population pharmacokinetic characterization of artemisinin in healthy volunteers. Increasing the dose resulted in a significant increase in the mean transit-time but the micronized formulation or concomitant piperaquine administration did not affect the pharmacokinetic properties of artemisinin. The results from the traditional bioequivalence evaluation were comparable with results obtained from mixed-effects modelling.

Dicko A, Brown JM, Diawara H, Baber I, Mahamar A, Soumare HM, Sanogo K, Koita F, Keita S, Traore SF et al. 2016. Primaquine to reduce transmission of Plasmodium falciparum malaria in Mali: a single-blind, dose-ranging, adaptive randomised phase 2 trial. Lancet Infect Dis, 16 (6), pp. 674-684. | Citations: 8 (Scopus) | Show Abstract | Read more

BACKGROUND: Single low doses of primaquine, when added to artemisinin-based combination therapy, might prevent transmission of Plasmodium falciparum malaria to mosquitoes. We aimed to establish the activity and safety of four low doses of primaquine combined with dihydroartemisinin-piperaquine in male patients in Mali. METHODS: In this phase 2, single-blind, dose-ranging, adaptive randomised trial, we enrolled boys and men with uncomplicated P falciparum malaria at the Malaria Research and Training Centre (MRTC) field site in Ouelessebougou, Mali. All participants were confirmed positive carriers of gametocytes through microscopy and had normal function of glucose-6-phosphate dehydrogenase (G6PD) on colorimetric quantification. In the first phase, participants were randomly assigned (1:1:1) to one of three primaquine doses: 0 mg/kg (control), 0·125 mg/kg, and 0·5 mg/kg. Randomisation was done with a computer-generated randomisation list (in block sizes of six) and concealed with sealed, opaque envelopes. In the second phase, different participants were sequentially assigned (1:1) to 0·25 mg/kg primaquine or 0·0625 mg/kg primaquine. Primaquine tablets were dissolved into a solution and administered orally in a single dose. Participants were also given a 3 day course of dihydroartemisinin-piperaquine, administered by weight (320 mg dihydroartemisinin and 40 mg piperaquine per tablet). Outcome assessors were masked to treatment allocation, but participants were permitted to find out group assignment. Infectivity was assessed through membrane-feeding assays, which were optimised through the beginning part of phase one. The primary efficacy endpoint was the mean within-person percentage change in mosquito infectivity 2 days after primaquine treatment in participants who completed the study after optimisation of the infectivity assay, had both a pre-treatment infectivity measurement and at least one follow-up infectivity measurement, and who were given the correct primaquine dose. The safety endpoint was the mean within-person change in haemoglobin concentration during 28 days of study follow-up in participants with at least one follow-up visit. This study is registered with, number NCT01743820. FINDINGS: Between Jan 2, 2013, and Nov 27, 2014, we enrolled 81 participants. In the primary analysis sample (n=71), participants in the 0·25 mg/kg primaquine dose group (n=15) and 0·5 mg/kg primaquine dose group (n=14) had significantly lower mean within-person reductions in infectivity at day 2-92·6% (95% CI 78·3-100; p=0·0014) for the 0·25 mg/kg group; and 75·0% (45·7-100; p=0·014) for the 0·5 mg/kg primaquine group-compared with those in the control group (n=14; 11·3% [-27·4 to 50·0]). Reductions were not significantly different from control for participants assigned to the 0·0625 mg/kg dose group (n=16; 41·9% [1·4-82·5]; p=0·16) and the 0·125 mg/kg dose group (n=12; 54·9% [13·4-96·3]; p=0·096). No clinically meaningful or statistically significant drops in haemoglobin were recorded in any individual in the haemoglobin analysis (n=70) during follow-up. No serious adverse events were reported and adverse events did not differ between treatment groups. INTERPRETATION: A single dose of 0·25 mg/kg primaquine, given alongside dihydroartemisinin-piperaquine, was safe and efficacious for the prevention of P falciparum malaria transmission in boys and men who are not deficient in G6PD. Future studies should assess the safety of single-dose primaquine in G6PD-deficient individuals to define the therapeutic range of primaquine to enable the safe roll-out of community interventions with primaquine. FUNDING: Bill & Melinda Gates Foundation.

Awab GR, Imwong M, Pukrittayakamee S, Alim F, Hanpithakpong W, Tarning J, Dondorp AM, Day NP, White NJ, Woodrow CJ. 2016. Clinical trials of artesunate plus sulfadoxine-pyrimethamine for Plasmodium falciparum malaria in Afghanistan: maintained efficacy a decade after introduction. Malar J, 15 (1), pp. 121. | Citations: 2 (Scopus) | Show Abstract | Read more

BACKGROUND: Combination therapy with artesunate plus sulfadoxine-pyrimethamine (SP) was adopted as recommended treatment for Plasmodium falciparum infection in Afghanistan in 2003. METHODS: A series of prospective clinical studies examining the efficacy of artesunate plus sulfadoxine-pyrimethamine (AS + SP) against P. falciparum were undertaken in sentinel sites in Afghanistan from 2007 to 2014, accompanied by relevant molecular studies. The first study was a randomized trial of AS + SP versus dihydroartemisinin-piperaquine, while two subsequent studies were standard therapeutic efficacy studies of AS + SP. RESULTS: Three hundred and three patients were enrolled across four provinces in the north and east of the country. Curative efficacy was high in all the trials, with an adequate clinical and parasitological response (ACPR) of more than 95 % in all groups and trial stages. Genotyping for drug-resistance alleles at dhfr indicated fixation of the S108 N mutation and a prevalence of the C59R mutation of approximately 95 % across all sites. Other mutations in dhfr and dhps remained rare or absent entirely, although five isolates from the first trial carried the dhps triple mutant SGEGA haplotype. In the last study undertaken in 2012-2014 the K13 artemisinin resistance marker was examined; only two of 60 successfully sequenced samples carried a K13-propeller mutation. CONCLUSIONS: These data confirm maintained efficacy 10 years after introduction of artesunate plus SP as combination treatment of P. falciparum in Afghanistan. The molecular data indicate that despite a substantial fall in incidence, resistance has not developed to artemisinins, or intensified to the ACT partner drug components. Trial Registration NCT00682578, NCT01115439 and NCT01707199.

Grigg MJ, Barber BE, Marfurt J, Imwong M, William T, Bird E, Piera KA, Aziz A, Boonyuen U, Drakeley CJ et al. 2016. Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia. PLoS One, 11 (3), pp. e0149519. | Citations: 2 (Scopus) | Show Abstract | Read more

BACKGROUND: Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. METHODS: The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. RESULTS: Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. CONCLUSION: Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.

Canavati SE, Lawford HL, Fatunmbi BS, Lek D, Top-Samphor N, Leang R, Dondorp AM, Huy R, Kazadi WM. 2016. Establishing research priorities for malaria elimination in the context of the emergency response to artemisinin resistance framework-the Cambodian approach. Malar J, 15 (1), pp. 120. | Citations: 3 (Scopus) | Show Abstract | Read more

BACKGROUND: Countries of the greater Mekong subregion have made a transition from malaria control to an aim for falciparum and vivax malaria elimination. The elimination of falciparum malaria will have to be achieved against a background of increasing artemisinin and multi-drug resistance. This ambitious goal requires an operational research (OR) agenda that addresses the dynamic challenges encountered on the path to elimination, which will need to be flexible and developed in close relation with the cambodian national programme for parasitology, entomology and malaria control (CNM). In Cambodia, a number of meetings with stakeholders were convened by the CNM and emergency response to artemisinin resistance (ERAR) hub, producing an initial list of priority OR topics. The process and outcome of these meetings are described, which could serve as a template for other countries in the region. METHODS: A landscaping exercise was conducted to gather all past, on-going and planned malaria focussed OR activities conducted by the cambodian research consortium in Cambodia and categorized according to research theme. The six themes included (1) malaria epidemiology, surveillance and response, (2) malaria case management, (3) malaria vector control, (4) malaria behavioural issues, (5) malaria clinical studies, and (6) other vector-borne diseases (dengue, neglected tropical diseases, soil-transmitted helminths). The different themes were discussed in small focus groups, which made an initial prioritization list which was then presented to a plenary group for further discussion. This produced a list of research questions ranked according to priority. RESULTS: OR priorities produced by the thematic groups were discussed in the plenary meeting and given a priority score by group voting. A list of 17 OR questions were developed, finalized and listed, which included questions on surveillance, active case detection and treatment efficacy. CONCLUSION: This paper describes ERAR's work on supporting Cambodia's transition to malaria elimination by identifying national operational research priorities. ERAR has initiated and currently plays a critical role in the development of country specific research agendas for malaria elimination. The first example of this has been the described exercise in Cambodia, which could serve a template for setting OR priorities in the wider region.

MalariaGEN Plasmodium falciparum Community Project. 2016. Genomic epidemiology of artemisinin resistant malaria. Elife, 5 (MARCH2016), | Citations: 14 (Scopus) | Show Abstract | Read more

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.

Lubell Y, Althaus T, Blacksell SD, Paris DH, Mayxay M, Pan-Ngum W, White LJ, Day NP, Newton PN. 2016. Modelling the Impact and Cost-Effectiveness of Biomarker Tests as Compared with Pathogen-Specific Diagnostics in the Management of Undifferentiated Fever in Remote Tropical Settings. PLoS One, 11 (3), pp. e0152420. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Malaria accounts for a small fraction of febrile cases in increasingly large areas of the malaria endemic world. Point-of-care tests to improve the management of non-malarial fevers appropriate for primary care are few, consisting of either diagnostic tests for specific pathogens or testing for biomarkers of host response that indicate whether antibiotics might be required. The impact and cost-effectiveness of these approaches are relatively unexplored and methods to do so are not well-developed. METHODS: We model the ability of dengue and scrub typhus rapid tests to inform antibiotic treatment, as compared with testing for elevated C-Reactive Protein (CRP), a biomarker of host-inflammation. Using data on causes of fever in rural Laos, we estimate the proportion of outpatients that would be correctly classified as requiring an antibiotic and the likely cost-effectiveness of the approaches. RESULTS: Use of either pathogen-specific test slightly increased the proportion of patients correctly classified as requiring antibiotics. CRP testing was consistently superior to the pathogen-specific tests, despite heterogeneity in causes of fever. All testing strategies are likely to result in higher average costs, but only the scrub typhus and CRP tests are likely to be cost-effective when considering direct health benefits, with median cost per disability adjusted life year averted of approximately $48 USD and $94 USD, respectively. CONCLUSIONS: Testing for viral infections is unlikely to be cost-effective when considering only direct health benefits to patients. Testing for prevalent bacterial pathogens can be cost-effective, having the benefit of informing not only whether treatment is required, but also as to the most appropriate antibiotic; this advantage, however, varies widely in response to heterogeneity in causes of fever. Testing for biomarkers of host inflammation is likely to be consistently cost-effective despite high heterogeneity, and can also offer substantial reductions in over-use of antimicrobials in viral infections.

Stoesser N, Sheppard AE, Pankhurst L, De Maio N, Moore CE, Sebra R, Turner P, Anson LW, Kasarskis A, Batty EM et al. 2016. Evolutionary History of the Global Emergence of the Escherichia coli Epidemic Clone ST131. MBio, 7 (2), pp. e02162. | Citations: 9 (Scopus) | Show Abstract | Read more

UNLABELLED: Escherichia colisequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n= 215) of sequenced ST131E. coliisolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of ablaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration ofblaCTX-Mwithin subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, theblaCTX-M-14/14-likegroup. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages ofE. coli These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages. IMPORTANCE: Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specificE. colilineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements.

Bancone G, Chowwiwat N, Somsakchaicharoen R, Poodpanya L, Moo PK, Gornsawun G, Kajeechiwa L, Thwin MM, Rakthinthong S, Nosten S et al. 2016. Single Low Dose Primaquine (0.25 mg/kg) Does Not Cause Clinically Significant Haemolysis in G6PD Deficient Subjects. PLoS One, 11 (3), pp. e0151898. | Citations: 6 (Scopus) | Show Abstract | Read more

BACKGROUND: Primaquine is the only drug consistently effective against mature gametocytes of Plasmodium falciparum. The transmission blocking dose of primaquine previously recommended was 0.75 mg/kg (adult dose 45 mg) but its deployment was limited because of concerns over haemolytic effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is an inherited X-linked enzymatic defect that affects an estimated 400 million people around the world with high frequencies (15-20%) in populations living in malarious areas. To reduce transmission in low transmission settings and facilitate elimination of P. falciparum, the World Health Organization now recommends adding a single dose of 0.25 mg/kg (adult dose 15 mg) to Artemisinin-based Combination Therapies (ACTs) without G6PD testing. Direct evidence of the safety of this low dose is lacking. Adverse events and haemoglobin variations after this treatment were assessed in both G6PD normal and deficient subjects in the context of targeted malaria elimination in a malaria endemic area on the North-Western Myanmar-Thailand border where prevalence of G6PD deficiency (Mahidol variant) approximates 15%. METHODS AND FINDINGS: The tolerability and safety of primaquine (single dose 0.25 mg base/kg) combined with dihydroartemisinin-piperaquine (DHA-PPQ) given three times at monthly intervals was assessed in 819 subjects. Haemoglobin concentrations were estimated over the six months preceding the ACT + primaquine rounds of mass drug administration. G6PD deficiency was assessed with a phenotypic test and genotyping was performed in male subjects with deficient phenotypes and in all females. Fractional haemoglobin changes in relation to G6PD phenotype and genotype and primaquine round were assessed using linear mixed-effects models. No adverse events related to primaquine were reported during the trial. Mean fractional haemoglobin changes after each primaquine treatment in G6PD deficient subjects (-5.0%, -4.2% and -4.7%) were greater than in G6PD normal subjects (0.3%, -0.8 and -1.7%) but were clinically insignificant. Fractional drops in haemoglobin concentration larger than 25% following single dose primaquine were observed in 1.8% of the population but were asymptomatic. CONCLUSIONS: The single low dose (0.25mg/kg) of primaquine is clinically well tolerated and can be used safely without prior G6PD testing in populations with high prevalence of G6PD deficiency. The present evidence supports a broader use of low dose primaquine without G6PD testing for the treatment and elimination of falciparum malaria. TRIAL REGISTRATION: NCT01872702.

Wattanakul T, Teerapong P, Plewes K, Newton PN, Chierakul W, Silamut K, Chotivanich K, Ruengweerayut R, White NJ, Dondorp AM, Tarning J. 2016. Pharmacokinetic properties of intramuscular versus oral syrup paracetamol in Plasmodium falciparum malaria. Malar J, 15 (1), pp. 244. | Show Abstract | Read more

BACKGROUND: Fever is an inherent symptom of malaria in both adults and children. Paracetamol (acetaminophen) is the recommended antipyretic as it is inexpensive, widely available and has a good safety profile, but patients may not be able to take the oral drug reliably. A comparison between the pharmacokinetics of oral syrup and intramuscular paracetamol given to patients with acute falciparum malaria and high body temperature was performed. METHODS: A randomized, open-label, two-treatment, crossover, pharmacokinetic study of paracetamol dosed orally and intramuscularly was conducted. Twenty-one adult patients with uncomplicated falciparum malaria were randomized to receive a single 600 mg dose of paracetamol either as syrup or intramuscular injection on day 0 followed by a single dose administered by the alternative route on day 1. Paracetamol plasma concentrations were quantified frequently and modelled simultaneously using nonlinear mixed-effects modelling. The final population pharmacokinetic model was used for dose optimization simulations. Relationships between paracetamol concentrations with temperature and parasite half-life were investigated using linear and non-linear regression analyses. RESULTS: The population pharmacokinetic properties of paracetamol were best described by a two-compartment disposition model, with zero-order and first-order absorption for intramuscular and oral syrup administration, respectively. The relative bioavailability of oral syrup was 84.4 % (95 % CI 68.2-95.1 %) compared to intramuscular administration. Dosing simulations showed that 1000 mg of intramuscular or oral syrup administered six-hourly reached therapeutic steady state concentrations for antipyresis, but more favourable concentration-time profiles were achieved with a loading dose of 1500 mg, followed by a 1000 mg maintenance dose. This ensured that maximum therapeutic concentrations were reached rapidly during the first 6 h. No significant relationships between paracetamol concentrations and temperature or parasite half-life were found. CONCLUSIONS: Paracetamol plasma concentrations after oral syrup and intramuscular administration in patients with acute falciparum malaria were described successfully by a two-compartment disposition model. Relative oral bioavailability compared to intramuscular dosing was estimated as 84.4 % (95 % CI 68.2-95.1 %). Dosing simulations showed that a loading dose followed by six-hourly dosing intervals reduced the time delay to reach therapeutic drug levels after both routes of administration. The safety and efficacy of loading dose paracetamol antipyretic regimens now needs to be established in larger studies.

Pouplin T, Bang ND, Toi PV, Phuong PN, Dung NH, Duong TN, Caws M, Thwaites GE, Tarning J, Day JN. 2016. Naïve-pooled pharmacokinetic analysis of pyrazinamide, isoniazid and rifampicin in plasma and cerebrospinal fluid of Vietnamese children with tuberculous meningitis. BMC Infect Dis, 16 (1), pp. 144. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Among the various forms of TB, tuberculous meningitis (TBM) is the most severe, with about 30% mortality and 50% of survivors left with neurological sequelae. Children suffer more frequently from TBM than adults and outcomes are often poor due to difficulties in making the diagnosis and uncertainty regarding the best anti-tuberculosis drug regimen. The aim of this prospective study was to describe the pharmacokinetics of pyrazinamide, isoniazid and rifampicin in plasma and cerebrospinal fluid of children with tuberculous meningitis treated with the standard TBM regimen. METHODS: We performed a prospective observational study of 100 consecutively treated children (≤ 15 years of age) with tuberculous meningitis in Ho Chi Minh City, Vietnam. Children were treated according to the 2006 WHO recommended pediatric treatment regimen consisting of isoniazid (5 mg/kg), rifampicin (10 mg/kg) and ethambutol (15 mg/kg) for 8 months, with the addition of pyrazinamide (25 mg/kg) for the first 3 months and streptomycin (15 mg/kg) for the first 2 months. Pyrazinamide, isoniazid and rifampicin concentrations were measured in plasma at day 14 and in cerebrospinal fluid (CSF) at 1 month by HPLC-UV. A naïve-pooled non-compartmental data analysis was used to describe the pharmacokinetic properties of drugs in the two-age groups of children ≤ 4 years or > 4 years of age. RESULTS: Younger children, when compared to older children, presented a higher body weight-normalized clearance and volume of distribution, and lower median total plasma exposures for the three studied drugs with -14%, -22% and -16% for Pyrazinamide, Isoniazid and Rifampicin, respectively. In CSF, individual concentrations of isoniazid and pyrazinamide were comparable to that in plasma in both age groups; but rifampicin concentrations were lower than the minimum inhibitory concentration of susceptible bacteria in all but two children. CONCLUSIONS: There is an age-dependent variation in the plasma and cerebrospinal fluid pharmacokinetics of rifampicin, isoniazid and pyrazinamide. The safety and efficacy of higher doses of rifampicin should be investigated for the treatment of childhood tuberculous meningitis.

Tripura R, Peto TJ, Chalk J, Lee SJ, Sirithiranont P, Nguon C, Dhorda M, von Seidlein L, Maude RJ, Day NP et al. 2016. Persistent Plasmodium falciparum and Plasmodium vivax infections in a western Cambodian population: implications for prevention, treatment and elimination strategies. Malar J, 15 (1), pp. 181. | Citations: 3 (Scopus) | Show Abstract | Read more

BACKGROUND: Subclinical Plasmodium parasitaemia is an important reservoir for the transmission and persistence of malaria, particularly in low transmission areas. METHODS: Using ultrasensitive quantitative PCR (uPCR) for the detection of parasitaemia, the entire population of three Cambodian villages in Pailin province were followed for 1 year at three-monthly intervals. A cohort of adult participants found initially to have asymptomatic malaria parasitaemia was followed monthly over the same period. RESULTS: The initial cross sectional survey in June 2013 (M0) of 1447 asymptomatic residents found that 32 (2.2%) had Plasmodium falciparum, 48 (3.3%) had P. vivax, 4 (0.3%) had mixed infections and in 142/1447 (9.8%) malaria was detected but there was insufficient DNA to identify the species (Plasmodium. species). Polymorphisms in the 'K13-propeller' associated with reduced susceptibility to artemisinin derivatives (C580Y) were found in 17/32 (51%) P. falciparum strains. Monthly follow-up without treatment of 24 adult participants with asymptomatic mono or mixed P. falciparum infections found that 3/24 (13%) remained parasitaemic for 2-4 months, whereas the remaining 21/24 (87%) participants had cleared their parasitaemia after 1 month. In contrast, 12/34 (35%) adult participants with P. vivax mono-infection at M0 had malaria parasites (P. vivax or P. sp.) during four or more of the following 11 monthly surveys. CONCLUSIONS: This longitudinal survey in a low transmission setting shows limited duration of P. falciparum carriage, but prolonged carriage of P. vivax infections. Radical treatment of P. vivax infections by 8-aminoquinoline regimens may be required to eliminate all malaria from Cambodia. Trial registration NCT01872702.

Oo NN, Bancone G, Maw LZ, Chowwiwat N, Bansil P, Domingo GJ, Htun MM, Thant KZ, Htut Y, Nosten F. 2016. Validation of G6PD Point-of-Care Tests among Healthy Volunteers in Yangon, Myanmar. PLoS One, 11 (4), pp. e0152304. | Citations: 1 (Scopus) | Show Abstract | Read more

Primaquine and other 8-amnoquinoline based anti-malarials can cause haemolysis in subjects with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Correct diagnosis of G6PD status in patients is crucial for safe treatment of both relapsing stages of Plasmodium vivax and transmitting forms of Plasmodium falciparum. Lack of suitable point-of-care tests has hampered a much needed wide use of primaquine for malaria elimination. In this study we have assessed the performances of two qualitative tests, the fluorescent spot test (FST) and the G6PD CareStart test (CST), against the gold standard quantitative spectrophotometric assay in a population of 1000 random adult healthy volunteers living in Yangon, Myanmar. The prevalence of G6PD deficiency in the Bamar, Karen and in the whole sample set was 6.6% (10.1% in males), 9.2% (21.0% in males) and 6.8% (11.1% in males) respectively. The FST and CST showed comparable performances with sensitivity over 95% and specificity over 90%, however for cases with severe G6PD activity the FTS had improved performance. If used with a conservative interpretation of the signal, the CareStart test has the potential to be used in the field and, by allowing a wider use of primaquine, to help malaria elimination.

Numminen E, Chewapreecha C, Turner C, Goldblatt D, Nosten F, Bentley SD, Turner P, Corander J. 2016. Corrigendum: Climate induces seasonality in pneumococcal transmission. Sci Rep, 6 pp. 23307. | Read more

Herdman MT, Maude RJ, Chowdhury MS, Kingston HW, Jeeyapant A, Samad R, Karim R, Dondorp AM, Hossain MA. 2016. The Relationship between Poverty and Healthcare Seeking among Patients Hospitalized with Acute Febrile Illnesses in Chittagong, Bangladesh. PLoS One, 11 (4), pp. e0152965. | Show Abstract | Read more

Delays in seeking appropriate healthcare can increase the case fatality of acute febrile illnesses, and circuitous routes of care-seeking can have a catastrophic financial impact upon patients in low-income settings. To investigate the relationship between poverty and pre-hospital delays for patients with acute febrile illnesses, we recruited a cross-sectional, convenience sample of 527 acutely ill adults and children aged over 6 months, with a documented fever ≥38.0 °C and symptoms of up to 14 days' duration, presenting to a tertiary referral hospital in Chittagong, Bangladesh, over the course of one year from September 2011 to September 2012. Participants were classified according to the socioeconomic status of their households, defined by the Oxford Poverty and Human Development Initiative's multidimensional poverty index (MPI). 51% of participants were classified as multidimensionally poor (MPI>0.33). Median time from onset of any symptoms to arrival at hospital was 22 hours longer for MPI poor adults compared to non-poor adults (123 vs. 101 hours) rising to a difference of 26 hours with adjustment in a multivariate regression model (95% confidence interval 7 to 46 hours; P = 0.009). There was no difference in delays for children from poor and non-poor households (97 vs. 119 hours; P = 0.394). Case fatality was 5.9% vs. 0.8% in poor and non-poor individuals respectively (P = 0.001)-5.1% vs. 0.0% for poor and non-poor adults (P = 0.010) and 6.4% vs. 1.8% for poor and non-poor children (P = 0.083). Deaths were attributed to central nervous system infection (11), malaria (3), urinary tract infection (2), gastrointestinal infection (1) and undifferentiated sepsis (1). Both poor and non-poor households relied predominantly upon the (often informal) private sector for medical advice before reaching the referral hospital, but MPI poor participants were less likely to have consulted a qualified doctor. Poor participants were more likely to attribute delays in decision-making and travel to a lack of money (P<0.001), and more likely to face catastrophic expenditure of more than 25% of monthly household income (P<0.001). We conclude that multidimensional poverty is associated with greater pre-hospital delays and expenditure in this setting. Closer links between health and development agendas could address these consequences of poverty and streamline access to adequate healthcare.

Peto TJ, Kloprogge SE, Tripura R, Nguon C, Sanann N, Yok S, Heng C, Promnarate C, Chalk J, Song N et al. 2016. History of malaria treatment as a predictor of subsequent subclinical parasitaemia: a cross-sectional survey and malaria case records from three villages in Pailin, western Cambodia. Malar J, 15 (1), pp. 240. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: Treatment of the sub-clinical reservoir of malaria, which may maintain transmission, could be an important component of elimination strategies. The reliable detection of asymptomatic infections with low levels of parasitaemia requires high-volume quantitative polymerase chain reaction (uPCR), which is impractical to conduct on a large scale. It is unknown to what extent sub-clinical parasitaemias originate from recent or older clinical episodes. This study explored the association between clinical history of malaria and subsequent sub-clinical parasitaemia. METHODS: In June 2013 a cross-sectional survey was conducted in three villages in Pailin, western Cambodia. Demographic and epidemiological data and blood samples were collected. Blood was tested for malaria by high-volume qPCR. Positive samples were analysed by nested PCR to determine the Plasmodium species. To identify previous episodes of malaria, case records were collected from village malaria workers and local health facilities and linked to study participants. RESULTS: Among 1343 participants, 40/122 (32.8 %) with a history of clinical malaria were parasitaemic during the cross-sectional survey, compared to 172/1221 (14.1 %) without this history (p < 0.001). Among the 212 parasitaemic participants in the survey, 40 (18.9 %) had a history of clinical malaria, compared to 87 out of 1131 (7.7 %) parasite-negative participants; p < 0.001, adjusted OR 3.3 (95 % CI; 2.1-5.1). A history of Plasmodium vivax was associated with sub-clinical P. vivax parasitaemia in the survey (p < 0.001), but this association was not seen with Plasmodium falciparum (p = 0.253); only three participants had both P. falciparum parasites in the survey and a clinical history of P. falciparum. CONCLUSIONS: A clinical episode of vivax malaria was associated with subsequent sub-clinical parasitaemia. Treatment of P. vivax with artemisinin-based combination therapy without primaquine often resulted in recurrent episodes. Targeting individuals with a history of clinical malaria will be insufficient to eliminate the sub-clinical reservoir as they constitute a minority of parasitaemias.

Banks T, Kang J, Watts I, Tyrosvoutis ME, Min AM, Tun NW, Keereecharoen L, Simmawong W, Wanyatip S, Hanboonkunupakarn B et al. 2016. High hepatitis B seroprevalence and risk factors for infection in pregnant women on the Thailand-Myanmar Border. J Infect Dev Ctries, 10 (4), pp. 384-388. | Show Abstract | Read more

INTRODUCTION: Infection from Hepatitis B primarily results from peri-partum vertical transmission and the risk increases in the presence of hepatitis B e antigen. We aimed to evaluate a new screening program for hepatitis B in pregnant women as a component of antenatal services in a marginalized population. METHODOLOGY: Counseling and screening for hepatitis B screening was offered to all women at the first visit, at Shoklo Malaria Research Unit (SMRU) antenatal clinics on the Thai-Myanmar border.  Point-of-care rapid diagnostic tests (RDT) were used throughout the period of evaluation. A certified Thai Public Health laboratory at Mae Sot Hospital verified RDT positive cases using enzyme-linked immunosorbent assay (ELISA) for HBsAb and HBeAg. Risk factors for hepatitis B were identified by data linkage to antenatal care records. RESULTS: There were 523 (8.5%) RDT positive for HBsAg among 6158 women tested (Aug-2012 to April-2014). Of these 373 (96.9%) of 385 sent for confirmation were positive by ELISA i.e. RDT false positive rate of 3.1% (95% CI 1.7- 5.4). The overall confirmed HbsAg prevalence was 8.3% (511/6158) (95% CI 7.6-9.0). HBeAg prevalence was 32.7% (114/350) (95% CI 27.9-37.7) of cases tested. Risk factors for HBsAg positivity included age >25 years (OR 1.24, CI 1.03-1.49, p 0.021) and Karen heritage (OR 1.73, CI 1.39-2.15, p < 0.01). CONCLUSIONS: High hepatitis B seroprevalence amongst migrants and refugees accessing SMRU antenatal services likely reflects that of Kayin State, Myanmar, and perinatal prevention programs are required. False positive cases with HBsAg RDT complicate what is theoretically a straightforward screening.

Phommasone K, Adhikari B, Henriques G, Pongvongsa T, Phongmany P, von Seidlein L, White NJ, Day NP, M Dondorp A, Newton PN et al. 2016. Asymptomatic Plasmodium infections in 18 villages of southern Savannakhet Province, Lao PDR (Laos). Malar J, 15 (1), pp. 296. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: A large fraction of Plasmodium infections do not cause clinical signs and symptoms of disease and persist at densities in blood that are not detectable by microscopy or rapid diagnostic tests. These infections may be critical as a transmission reservoir in areas of low malaria endemicity. Understanding the epidemiology of these infections would be helpful for malaria elimination. METHODS: A cross-sectional survey was conducted in Thapangthong and Nong Districts of Savannakhet Province, Lao PDR, to determine the prevalence of parasitaemia. A total of 888 blood samples were collected from afebrile volunteers aged ≥15 years in 18 villages during March and July 2015. Plasmodium infections were diagnosed by rapid diagnostic tests (RDT) and high volume, ultra-sensitive quantitative polymerase chain reaction (uPCR). RESULTS: uPCR detected Plasmodium infections in 175 of 888 samples (20 %). The species distribution was Plasmodium falciparum 3.6 % (32/888), Plasmodium vivax 11.1 % (99/888), mixed infections with P. falciparum and P. vivax 1.6 % (14/888) and Plasmodium of undetermined species 3.4 % (30/888). RDT identified only 2 % (18/888) positive cases. Using uPCR as reference, the sensitivity and specificity of RDTs were 28 and 100 %, respectively, in detecting P. falciparum infections, and 3 and 99 % in detecting asymptomatic P. vivax infections. The K13 kelch propeller domain C580Y mutation, associated with reduced susceptibility to artemisinin derivatives, was found in 75 % (12/18) of P. falciparum isolates from Thapangthong and in 7 % (2/28) from Nong (p < 0.001). In a multivariate analysis, males were more likely to have P. vivax infections [adjusted odds ratio (aOR) 4.76 (95 % CI 2.84-8.00)] while older villagers were at lower risk for parasitaemia [aOR for increasing age 0.98 (95 % CI 0.96-0.99)]. CONCLUSION: There is a high prevalence of asymptomatic Plasmodium infections in southern Savannakhet. Artemisinin-resistant P. falciparum strains form an increasing proportion of the parasite population in Thapangthong District and are already present in the more remote Nong District. This worrying trend has wider implications for Laos and could reverse the gains achieved by the successful control of malaria in Laos and the Greater Mekong Sub-region (GMS). Rapid elimination of P. falciparum has to be a top priority in Laos as well as in the wider GMS.

Carrara VI, Darakomon MC, Thin NW, Paw NT, Wah N, Wah HG, Helen N, Keereecharoen S, Paw NT, Jittamala P et al. 2016. Evaluation and Acceptability of a Simplified Test of Visual Function at Birth in a Limited-Resource Setting. PLoS One, 11 (6), pp. e0157087. | Show Abstract | Read more

Neurological examination, including visual fixation and tracking of a target, is routinely performed in the Shoklo Malaria Research Unit postnatal care units on the Thailand-Myanmar border. We aimed to evaluate a simple visual newborn test developed in Italy and performed by non-specialized personnel working in neonatal care units. An intensive training of local health staff in Thailand was conducted prior to performing assessments at 24, 48 and 72 hours of life in healthy, low-risk term singletons. The 48 and 72 hours results were then compared to values obtained to those from Italy. Parents and staff administering the test reported on acceptability. One hundred and seventy nine newborns, between June 2011 and October 2012, participated in the study. The test was rapidly completed if the infant remained in an optimal behavioral stage (7 ± 2 minutes) but the test duration increased significantly (12 ± 4 minutes, p < 0.001) if its behavior changed. Infants were able to fix a target and to discriminate a colored face at 24 hours of life. Horizontal tracking of a target was achieved by 96% (152/159) of the infants at 48 hours. Circular tracking, stripe discrimination and attention to distance significantly improved between each 24-hour test period. The test was easily performed by non-specialized local staff and well accepted by the parents. Healthy term singletons in this limited-resource setting have a visual response similar to that obtained to gestational age matched newborns in Italy. It is possible to use these results as a reference set of values for the visual assessment in Karen and Burmese infants in the first 72 hours of life. The utility of the 24 hours test should be pursued.

Tschirhart N, Nosten F, Foster AM. 2016. Access to free or low-cost tuberculosis treatment for migrants and refugees along the Thailand-Myanmar border in Tak province, Thailand. Int J Equity Health, 15 (1), pp. 100. | Citations: 1 (Scopus) | Show Abstract | Read more

BACKGROUND: In Tak province, Thailand migrants and refugees from Myanmar navigate a pluralistic healthcare system to seek Tuberculosis (TB) care from a variety of government and non-governmental providers. This multi-methods qualitative study examined access to TB, TB/HIV and multidrug-resistant tuberculosis (MDR-TB) treatment with an emphasis on barriers to care and enabling factors. METHODS: In the summer and fall of 2014, we conducted 12 key informant interviews with public health officials and TB treatment providers. We also conducted 11 focus group discussions with migrants and refugees who were receiving TB, TB/HIV and MDR-TB treatment in Tak province as well as non-TB patients. We analyzed these data through thematic analysis using both predetermined and emergent codes. As a second step in the qualitative analysis, we explored the barriers and enabling factors separately for migrants and refugees. RESULTS: We found that refugees face fewer barriers to accessing TB treatment than migrants. For both migrants and refugees, legal status plays an important intermediary role in influencing the population's ability to access care and eligibility for treatment. Our results suggest that there is a large geographical catchment area for migrants who seek TB treatment in Tak province that extends beyond provincial boundaries. Migrant participants described their ability to seek care as linked to the financial and non-financial resources required to travel and undergo treatment. Patients identified language of health services, availability of free or low cost services, and psychosocial support as important health system characteristics that affect accessibility. CONCLUSION: Access to TB treatment for migrants and refugees occurs at the interface of health system accessibility, population ability and legal status. In Tak province, migrant patients draw upon their social networks and financial resources to navigate a pathway to treatment. We revised a conceptual framework for access to healthcare to incorporate legal status and the cyclical pathways through which migrants access TB treatment in this region. We recommend that organizations continue to collaborate to provide supportive services that help migrants to access and continue TB treatment.

Landier J, Parker DM, Thu AM, Carrara VI, Lwin KM, Bonnington CA, Pukrittayakamee S, Delmas G, Nosten FH. 2016. The role of early detection and treatment in malaria elimination. Malar J, 15 (1), pp. 363. | Citations: 2 (Scopus) | Show Abstract | Read more

Falciparum malaria persists in hard-to-reach areas or demographic groups that are missed by conventional healthcare systems but could be reached by trained community members in a malaria post (MP). The main focus of a MP is to provide uninterrupted and rapid access to rapid diagnostic tests (RDTs) and artemisinin-based combination therapy (ACT) too all inhabitants of a village. RDTs allow trained community members to perform malaria diagnosis accurately and prescribe appropriate treatment, reducing as much as possible any delay between the onset of fever and treatment. Early treatment with ACT and with a low-dose of primaquine prevents further transmission from human to mosquito. A functioning MP represents an essential component of any malaria elimination strategy. Implementing large-scale, high-coverage, community-based early diagnosis and treatment through MPs requires few technological innovations but relies on a very well structured organization able to train, supervise and supply MPs, to monitor activity and to perform strict malaria surveillance.

Chaumeau V, Andolina C, Fustec B, Tuikue Ndam N, Brengues C, Herder S, Cerqueira D, Chareonviriyaphap T, Nosten F, Corbel V. 2016. Comparison of the Performances of Five Primer Sets for the Detection and Quantification of Plasmodium in Anopheline Vectors by Real-Time PCR. PLoS One, 11 (7), pp. e0159160. | Citations: 1 (Scopus) | Show Abstract | Read more

Quantitative real-time polymerase chain reaction (qrtPCR) has made a significant improvement for the detection of Plasmodium in anopheline vectors. A wide variety of primers has been used in different assays, mostly adapted from molecular diagnosis of malaria in human. However, such an adaptation can impact the sensitivity of the PCR. Therefore we compared the sensitivity of five primer sets with different molecular targets on blood stages, sporozoites and oocysts standards of Plasmodium falciparum (Pf) and P. vivax (Pv). Dilution series of standard DNA were used to discriminate between methods at low concentrations of parasite and to generate standard curves suitable for the absolute quantification of Plasmodium sporozoites. Our results showed that the best primers to detect blood stages were not necessarily the best ones to detect sporozoites. Absolute detection threshold of our qrtPCR assay varied between 3.6 and 360 Pv sporozoites and between 6 and 600 Pf sporozoites per mosquito according to the primer set used in the reaction mix. In this paper, we discuss the general performance of each primer set and highlight the need to use efficient detection methods for transmission studies.

Goh YS, Peng K, Chia WN, Siau A, Chotivanich K, Gruner AC, Preiser P, Mayxay M, Pukrittayakamee S, Sriprawat K et al. 2016. Neutralizing Antibodies against Plasmodium falciparum Associated with Successful Cure after Drug Therapy. PLoS One, 11 (7), pp. e0159347. | Citations: 1 (Scopus) | Show Abstract | Read more

An effective antibody response can assist drug treatment to contribute to better parasite clearance in malaria patients. To examine this, sera were obtained from two groups of adult patients with acute falciparum malaria, prior to drug treatment: patients who (1) have subsequent recrudescent infection, or (2) were cured by Day 28 following treatment. Using a Plasmodium falciparum antigen library, we examined the antibody specificities in these sera. While the antibody repertoire of both sera groups was extremely broad and varied, there was a differential antibody profile between the two groups of sera. The proportion of cured patients with antibodies against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 was higher than the proportion of patients with recrudescent infection. The presence of these antibodies was associated with higher odds of treatment cure. Sera containing all six antibodies impaired the invasion of P. falciparum clinical isolates into erythrocytes. These results suggest that antibodies specific against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 in P. falciparum infections could assist anti-malarial drug treatment and contribute to the resolution of the malarial infection.

Peto TJ, Tripura R, Lee SJ, Althaus T, Dunachie S, Nguon C, Dhorda M, Promnarate C, Chalk J, Imwong M et al. 2016. Association between Subclinical Malaria Infection and Inflammatory Host Response in a Pre-Elimination Setting. PLoS One, 11 (7), pp. e0158656. | Show Abstract | Read more

BACKGROUND: Subclinical infections in endemic areas of Southeast Asia sustain malaria transmission. These asymptomatic infections might sustain immunity against clinical malaria and have been considered benign for the host, but if they are associated with chronic low-grade inflammation this could be harmful. We conducted a case-control study to explore the association between subclinical malaria and C-reactive protein (CRP), an established biomarker of inflammation. METHODS: Blood samples from asymptomatic villagers in Pailin, Western Cambodia were tested for malaria by high-volume ultra-sensitive polymerase chain reaction (uPCR) to determine the Plasmodium species. Plasma CRP concentration was measured in 328 individuals with parasitaemia (cases) and compared with: i) the same individual's value at the first time point when they had no detectable parasites (n = 282); and ii) age- sex- and village-matched controls (n = 328) free of Plasmodium infection. Plasma CRP concentrations were compared against thresholds of 3mg/L and 10mg/L. Subgroup analysis was carried out for cases with P vivax and P falciparum mono-infections. RESULTS: Median plasma CRP level for all samples was 0.59mg/L (interquartile range: 0.24-1.64mg/L). CRP concentrations were higher in parasitaemic individuals compared with same-person-controls (p = 0.050); and matched-controls (p = 0.025). 4.9% of samples had CRP concentrations above 10mg/L and 14.6% were above 3mg/L. Cases were more likely to have plasma CRP concentrations above these thresholds than age/sex matched controls, odds ratio 3.5 (95%CI 1.5-9.8) and 1.8 (95%CI 1.1-2.9), respectively. Amongst cases, parasite density and CRP were positively correlated (p<0.001), an association that remained significant when controlling for age and fever. Individuals with P.vivax mono-infections had the highest plasma CRP concentrations with the greatest association with parasitaemia. DISCUSSION: In this setting persistent malaria infections in asymptomatic individuals were associated with moderately elevated plasma CRP concentrations; chiefly evident in cases with P.vivax mono-infections. As well as interrupting malaria transmission within the community, treatment of asymptomatic malaria infections, in particular radical cure of vivax malaria, may benefit the health of infected individuals.

Tschirhart N, Sein T, Nosten F, Foster AM. 2016. Migrant and Refugee Patient Perspectives on Travel and Tuberculosis along the Thailand-Myanmar Border: A Qualitative Study. PLoS One, 11 (8), pp. e0160222. | Citations: 2 (Scopus) | Show Abstract | Read more

BACKGROUND: The Thailand-Myanmar border separates two very different health systems. The healthcare system in eastern Myanmar remains underdeveloped as a result of decades of instability. Comparatively, Tak province, Thailand has more healthcare resources. In this Thai border province government hospitals and non-governmental organizations provide tuberculosis (TB) treatment to migrants and refugees. OBJECTIVES: Our overall study aimed to explore accessibility of TB treatment, TB surveillance and health system responsiveness specific to migrant and refugee populations in Tak province. In this paper, we focus on the perspectives of migrant and refugee TB patients with respect to travel and treatment in Tak province. METHODS: In 2014 we conducted focus group discussions with 61 TB, Tuberculosis and Human Immunodeficiency Virus co-infection, and multidrug-resistant TB patients in Tak province. We analyzed the data for content and themes and documented individual travel trajectories. RESULTS AND DISCUSSION: Migrants are travelling with active TB within the country and between Thailand and Myanmar. Migrants primarily travelled to obtain treatment but two participants reported travelling home to seek family care in Myanmar before returning to Thailand for treatment. Travel, while expensive and arduous, is an adaptive strategy that migrants use to access healthcare. CONCLUSIONS: Migrant's need for travel points to larger difficulties associated with healthcare access in the border region. Long distance travel with an infectious disease can be seen as an indicator that local healthcare is not available or affordable. These findings suggest that public health officials from both sides of the border should discuss the factors that contribute to travel with active TB and explore potential solutions to mitigate disease transmission in migrant populations.

McLean AR, Boel ME, McGready R, Ataide R, Drew D, Tsuboi T, Beeson JG, Nosten F, Simpson JA, Fowkes FJ. 2016. Antibody responses to Plasmodium falciparum and Plasmodium vivax blood-stage and sporozoite antigens in the postpartum period. Sci Rep, 6 pp. 32159. | Citations: 1 (Scopus) | Show Abstract | Read more

During pregnancy a variety of immunological changes occur to accommodate the fetus. It is unknown whether these changes continue to affect humoral immunity postpartum or how quickly they resolve. IgG levels were measured to P. falciparum and P. vivax antigens in 201 postpartum and 201 controls over 12 weeks. Linear mixed-effects models assessed antibody maintenance over time and the effect of microscopically confirmed Plasmodium spp. infection on antibody levels, and whether this was different in postpartum women compared with control women. Postpartum women had reduced Plasmodium spp. antibody levels compared to controls at baseline. Over 12 weeks, mean antibody levels in postpartum women increased to levels observed in control women. Microscopically confirmed P. falciparum and P. vivax infections during follow-up were associated with an increase in species-specific antibodies with similar magnitudes of boosting observed in postpartum and control women. Antibodies specific for pregnancy-associated, VAR2CSA-expressing parasites did not rapidly decline postpartum and did not boost in response to infection in either postpartum or control women. After pregnancy, levels of malaria-specific antibodies were reduced, but recovered to levels seen in control women. There was no evidence of an impaired ability to mount a boosting response in postpartum women.

Weerasuriya CK, Tan SO, Alexakis LC, Set AK, Rijken MJ, Martyn P, Nosten F, McGready R. 2016. Erratum to: Evaluation of a surgical service in the chronic phase of a refugee camp: an example from the Thai-Myanmar border. Confl Health, 10 (1), pp. 24. | Show Abstract | Read more

[This corrects the article DOI: 10.1186/1752-1505-6-5.].

Dittrich S, Tadesse BT, Moussy F, Chua A, Zorzet A, Tängdén T, Dolinger DL, Page AL, Crump JA, D'Acremont V et al. 2016. Target Product Profile for a Diagnostic Assay to Differentiate between Bacterial and Non-Bacterial Infections and Reduce Antimicrobial Overuse in Resource-Limited Settings: An Expert Consensus. PLoS One, 11 (8), pp. e0161721. | Show Abstract | Read more

Acute fever is one of the most common presenting symptoms globally. In order to reduce the empiric use of antimicrobial drugs and improve outcomes, it is essential to improve diagnostic capabilities. In the absence of microbiology facilities in low-income settings, an assay to distinguish bacterial from non-bacterial causes would be a critical first step. To ensure that patient and market needs are met, the requirements of such a test should be specified in a target product profile (TPP). To identify minimal/optimal characteristics for a bacterial vs. non-bacterial fever test, experts from academia and international organizations with expertise in infectious diseases, diagnostic test development, laboratory medicine, global health, and health economics were convened. Proposed TPPs were reviewed by this working group, and consensus characteristics were defined. The working group defined non-severely ill, non-malaria infected children as the target population for the desired assay. To provide access to the most patients, the test should be deployable to community health centers and informal health settings, and staff should require <2 days of training to perform the assay. Further, given that the aim is to reduce inappropriate antimicrobial use as well as to deliver appropriate treatment for patients with bacterial infections, the group agreed on minimal diagnostic performance requirements of >90% and >80% for sensitivity and specificity, respectively. Other key characteristics, to account for the challenging environment at which the test is targeted, included: i) time-to-result <10 min (but maximally <2 hrs); ii) storage conditions at 0-40°C, ≤90% non-condensing humidity with a minimal shelf life of 12 months; iii) operational conditions of 5-40°C, ≤90% non-condensing humidity; and iv) minimal sample collection needs (50-100μL, capillary blood). This expert approach to define assay requirements for a bacterial vs. non-bacterial assay should guide product development, and enable targeted and timely efforts by industry partners and academic institutions.

Chanthavilay P, Reinharz D, Mayxay M, Phongsavan K, Marsden DE, Moore L, White LJ. 2016. The economic evaluation of human papillomavirus vaccination strategies against cervical cancer in women in Lao PDR: a mathematical modelling approach. BMC Health Serv Res, 16 (1), pp. 418. | Show Abstract | Read more

BACKGROUND: Cervical cancer, a preventable disease, is the third leading cause of cancer morbidity and mortality in the Lao People's Democratic Republic (Lao PDR). Since many cervical cancers are linked to human papilloma virus (HPV) infection, vaccination against this virus may lead to a reduction in these types of cancer. The study described here is the first to compare the cost-effectiveness of different HPV vaccination options in Lao PDR. METHODS: A dynamic compartment model was created. The model included routine screening activities already in place, as well as theoretical interventions that included a 10-year old girl-only vaccination programme combined with/without a 10-year old boy vaccination programme and/or a catch-up component. The simulation was run over 100 years. In base case analyses, we assumed 70 % vaccination coverage with lifelong protection and 100 % efficacy against HPV types 16/18. The outcomes of interest were the incremental cost per Disability-Adjusted Life Year (DALY) averted. RESULTS: In base case analyses, according to the WHO definition of cost-effectiveness thresholds, vaccinating 10-year-old girls was very cost-effective. Adding a catch-up vaccination element for females aged 11-25 years was also very cost-effective, costing 1559 international dollars (I$) per DALY averted. Increasing the age limit of the catch-up vaccination component to 75 years old showed that this remained a cost-effective option (I$ 5840 per DALY averted). Adding a vaccination programme for 10-year-old boys was not found to be cost-effective unless a short time simulation (30 years or less) was considered, along with a catch-up vaccination component for both males and females. CONCLUSIONS: Adding a catch-up female vaccination component is more attractive than adding a 10-year-old boy vaccination component.

Briand V, Le Hesran JY, Mayxay M, Newton PN, Bertin G, Houzé S, Keomany S, Inthavong Y, Vannavong N, Chindavongsa K et al. 2016. Prevalence of malaria in pregnancy in southern Laos: a cross-sectional survey. Malar J, 15 (1), pp. 436. | Show Abstract | Read more

BACKGROUND: There are no data on the burden of malaria in pregnancy (MiP) in Laos, where malaria still remains prevalent in the south. METHODS: Two cross-sectional surveys were conducted in 2014 to assess the prevalence of MiP in Vapi District, Salavan Province, southern Laos: the first consisted of screening 204 pregnant women during pregnancies [mean (95 % CI) gestational age: 23 (22-25) weeks] living in 30 randomly selected villages in Vapi District; the second was conducted among 331 pregnant women, who delivered during the study period in Vapi and Toumlane District Hospitals and in Salavan Provincial Hospital. Peripheral and placental malaria was detected using rapid diagnostic tests (RDT), thick blood smears (TBS) and real-time quantitative polymerase chain reactions (RT-qPCR). Factors associated with low birth weight (LBW) and maternal anaemia were assessed. RESULTS: In the villages, 12/204 women (5.9 %; 95 % CI 3.1-10.0) were infected with malaria as determined by RT-qPCR: 11 were Plasmodium vivax infections and 1 was mixed Plasmodium vivax/Plasmodium falciparum infection, among which 9 were sub-microscopic (as not detected by TBS). History of malaria during current pregnancy tended to be associated with a higher risk of MiP (aIRR 3.05; 95 % CI 0.94-9.88). At delivery, two Plasmodium falciparum sub-microscopic infections (one peripheral and one placental) were detected (4.5 %; 0.6-15.5) in Vapi District. In both surveys, all infected women stated they had slept under a bed net the night before the survey, and 86 % went to the forest for food-finding 1 week before the survey in median. The majority of infections (94 %) were asymptomatic and half of them were associated with anaemia. Overall, 24 % of women had LBW newborns. Factors associated with a higher risk of LBW were tobacco use (aIRR 2.43; 95 % CI 1.64-3.60) and pre-term delivery (aIRR 3.17; 95 % CI 2.19-4.57). Factors associated with a higher risk of maternal anaemia were no iron supplementation during pregnancy, Lao Theung ethnicity and place of living. CONCLUSIONS: The prevalence of MiP in this population was noticeable. Most infections were asymptomatic and sub-microscopic vivax malaria, which raises the question of reliability of recommended national strategies for the screening and prevention of MiP in Laos.

Wolkewitz M, Cooper BS, Palomar-Martinez M, Alvarez-Lerma F, Olaechea-Astigarraga P, Barnett AG, Schumacher M. 2016. Multiple time scales in modeling the incidence of infections acquired in intensive care units. BMC Med Res Methodol, 16 (1), pp. 116. | Show Abstract | Read more

BACKGROUND: When patients are admitted to an intensive care unit (ICU) their risk of getting an infection will be highly depend on the length of stay at-risk in the ICU. In addition, risk of infection is likely to vary over calendar time as a result of fluctuations in the prevalence of the pathogen on the ward. Hence risk of infection is expected to depend on two time scales (time in ICU and calendar time) as well as competing events (discharge or death) and their spatial location. The purpose of this paper is to develop and apply appropriate statistical models for the risk of ICU-acquired infection accounting for multiple time scales, competing risks and the spatial clustering of the data. METHODS: A multi-center data base from a Spanish surveillance network was used to study the occurrence of an infection due to Methicillin-resistant Staphylococcus aureus (MRSA). The analysis included 84,843 patient admissions between January 2006 and December 2011 from 81 ICUs. Stratified Cox models were used to study multiple time scales while accounting for spatial clustering of the data (patients within ICUs) and for death or discharge as competing events for MRSA infection. RESULTS: Both time scales, time in ICU and calendar time, are highly associated with the MRSA hazard rate and cumulative risk. When using only one basic time scale, the interpretation and magnitude of several patient-individual risk factors differed. Risk factors concerning the severity of illness were more pronounced when using only calendar time. These differences disappeared when using both time scales simultaneously. CONCLUSIONS: The time-dependent dynamics of infections is complex and should be studied with models allowing for multiple time scales. For patient individual risk-factors we recommend stratified Cox regression models for competing events with ICU time as the basic time scale and calendar time as a covariate. The inclusion of calendar time and stratification by ICU allow to indirectly account for ICU-level effects such as local outbreaks or prevention interventions.

Falq G, Van Den Bergh R, De Smet M, Etienne W, Nguon C, Rekol H, Imwong M, Dondorp A, Kindermans JM. 2016. Assessing the asymptomatic reservoir and dihydroartemisinin-piperaquine effectiveness in a low transmission setting threatened by artemisinin resistant Plasmodium falciparum. Malar J, 15 (1), pp. 446. | Show Abstract | Read more

BACKGROUND: In Cambodia, elimination of artemisinin resistance through direct elimination of the Plasmodium falciparum parasite may be the only strategy. Prevalence and incidence at district and village levels were assessed in Chey Saen district, Preah Vihear province, North of Cambodia. Molecular and clinical indicators for artemisinin resistance were documented. METHODS: A cross sectional prevalence survey was conducted at village level in the district of Chey Saen from September to October 2014. Plasmodium spp. was assessed with high volume quantitative real-time polymerase chain reaction (qPCR). Plasmodium falciparum-positive samples were screened for mutations in the k13-propeller domain gene. Treatment effectiveness was established after 28 days (D28) using the same qPCR technique. Data from the provincial surveillance system targeting symptomatic cases, supported by Médecins Sans Frontières (MSF), were used to assess incidence. RESULTS: District P. falciparum prevalence was of 0.74 % [0.41; 1.21]; village prevalence ranged from 0 to 4.6 % [1.4; 10.5]. The annual incidence of P. falciparum was 16.8 cases per 1000 inhabitants in the district; village incidence ranged from 1.3 to 54.9 for 1000 inhabitants. Two geographical clusters with high number of cases were identified by both approaches. The marker for artemisinin resistance was found in six samples out of the 11 tested (55 %). 34.9 % of qPCR blood analysis of symptomatic patients were still positive at D28. CONCLUSIONS: The overall low prevalence of P. falciparum was confirmed in Chey Saen district in Cambodia, while there were important variations between villages. Symptomatic cases had a different pattern and were likely acquired outside the villages. It illustrates the importance of prevalence surveys in targeting interventions for elimination. Mutations in the k13-propeller domain gene (C580Y), conferring artemisinin resistance, were highly prevalent in both symptomatic and asymptomatic cases (realizing the absolute figures remain low). Asymptomatic individuals could be an additional reservoir for artemisinin resistance. The low effectiveness of dihydroartemisinin-piperaquine (DHA-PPQ) for symptomatic cases indicates that PPQ is no longer able to complement the reduced potency of DHA to treat falciparum malaria and highlights the need for an alternative first-line treatment.

Chanthavilay P, Reinharz D, Mayxay M, Phongsavan K, Marsden DE, Moore L, White LJ. 2016. Economic Evaluation of Screening Strategies Combined with HPV Vaccination of Preadolescent Girls for the Prevention of Cervical Cancer in Vientiane, Lao PDR. PLoS One, 11 (9), pp. e0162915. | Show Abstract | Read more

BACKGROUND: Several approaches to reduce the incidence of invasive cervical cancers exist. The approach adopted should take into account contextual factors that influence the cost-effectiveness of the available options. OBJECTIVE: To determine the cost-effectiveness of screening strategies combined with a vaccination program for 10-year old girls for cervical cancer prevention in Vientiane, Lao PDR. METHODS: A population-based dynamic compartment model was constructed. The interventions consisted of a 10-year old girl vaccination program only, or this program combined with screening strategies, i.e., visual inspection with acetic acid (VIA), cytology-based screening, rapid human papillomavirus (HPV) DNA testing, or combined VIA and cytology testing. Simulations were run over 100 years. In base-case scenario analyses, we assumed a 70% vaccination coverage with lifelong protection and a 50% screening coverage. The outcome of interest was the incremental cost per Disability-Adjusted Life Year (DALY) averted. RESULTS: In base-case scenarios, compared to the next best strategy, the model predicted that VIA screening of women aged 30-65 years old every three years, combined with vaccination, was the most attractive option, costing 2 544 international dollars (I$) per DALY averted. Meanwhile, rapid HPV DNA testing was predicted to be more attractive than cytology-based screening or its combination with VIA. Among cytology-based screening options, combined VIA with conventional cytology testing was predicted to be the most attractive option. Multi-way sensitivity analyses did not change the results. Compared to rapid HPV DNA testing, VIA had a probability of cost-effectiveness of 73%. Compared to the vaccination only option, the probability that a program consisting of screening women every five years would be cost-effective was around 60% and 80% if the willingness-to-pay threshold is fixed at one and three GDP per capita, respectively. CONCLUSIONS: A VIA screening program in addition to a girl vaccination program was predicted to be the most attractive option in the health care context of Lao PDR. When compared with other screening methods, VIA was the primary recommended method for combination with vaccination in Lao PDR.

Saralamba N, Nakeesathit S, Mayxay M, Newton PN, Osorio L, Kim JR, White NJ, Day NP, Dondorp AM, Imwong M. 2016. Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia. Malar J, 15 (1), pp. 484. | Show Abstract | Read more

BACKGROUND: Non-synonymous mutations in dhfr and dhps genes in Plasmodium vivax are associated with sulfadoxine-pyrimethamine (SP) resistance. The present study aimed to assess the prevalence of point mutations in P. vivax dhfr (pvdhfr) and P. vivax dhps (pvdhps) genes in three countries: Lao PDR, India and Colombia. METHODS: Samples from 203 microscopically diagnosed vivax malaria were collected from the three countries. Five codons at positions 13, 57, 58, 61, and 117 of pvdhfr and two codons at positions 383 and 553 of pvdhps were examined by polymerase chain reaction-restriction fragment length polymorphism methodology. RESULTS: The largest number of 58R/117 N double mutations in pvdhfr was observed in Colombia (94.3 %), while the corresponding wild-type amino acids were found at high frequencies in Lao PDR during 2001-2004 (57.8 %). Size polymorphism analysis of the tandem repeats within pvdhfr revealed that 74.3 % of all the isolates carried the type B variant. Eighty-nine per cent of all the isolates examined carried wild-type pvdhps A383 and A553. CONCLUSIONS: Although SP is not generally used to treat P. vivax infections, mutations in dhfr and dhps that confer antifolate resistance in P. vivax are common. The data strongly suggest that, when used primarily to treat falciparum malaria, SP can exert a substantial selective pressure on P. vivax populations, and this can lead to point mutations in dhfr and dhps. Accurate data on the global geographic distribution of dhfr and dhps genotypes should help to inform anti-malarial drug-use policies.

Kajeechiwa L, Thwin MM, Shee PW, Yee NL, Elvina E, Peapah P, Kyawt K, Oo PT, PoWah W, Min JR et al. 2016. The acceptability of mass administrations of anti-malarial drugs as part of targeted malaria elimination in villages along the Thai-Myanmar border. Malar J, 15 (1), pp. 494. | Citations: 2 (Scopus) | Show Abstract | Read more

BACKGROUND: A targeted malaria elimination project, including mass drug administrations (MDA) of dihydroartemisinin/piperaquine plus a single low dose primaquine is underway in villages along the Thailand Myanmar border. The intervention has multiple components but the success of the project will depend on the participation of the entire communities. Quantitative surveys were conducted to study reasons for participation or non-participation in the campaign with the aim to identify factors associated with the acceptance and participation in the mass drug administrations. METHODS: The household heads in four study villages in which MDAs had taken place previously were interviewed between January 2014 and July 2015. RESULTS: 174/378 respondents (46 %) completed three rounds of three drug doses each, 313/378 (83 %) took at least three consecutive doses and 56/378 (15 %) did not participate at all in the MDA. The respondents from the two villages (KNH and TPN) were much more likely to participate in the MDA than respondents from the other two villages (HKT and TOT). The more compliant villages KNH and TPN had both an appearance of cohesive communities with similar demographic and ethnic backgrounds. By contrast the villages with low participation were unique. One village was fragmented following years of armed conflict and many respondents gave little inclination to cooperate with outsiders. The other village with low MDA coverage was characterised by a high percentage of short-term residents with little interest in community interventions. A universal reason for non-participation in the MDA applicable to all villages was an inadequate understanding of the intervention. CONCLUSIONS: It is unlikely that community engagement can unite fragmented communities in participating in an intervention, which benefits the community. Understanding the purpose and the reasons underlying the intervention is an important pre-condition for participation. In the absence of direct benefits and a complete understanding of the indirect benefits trust in the investigators is critical for participation.

Fairhurst RM, Dondorp AM. 2016. Artemisinin-resistant Plasmodium falciparum malaria Microbiology Spectrum, 4 (3), | Show Abstract | Read more

© 2016 American Society for Microbiology.For more than five decades, Southeast Asia (SEA) has been fertile ground for the emergence of drug-resistant Plasmodium falciparum malaria. After generating parasites resistant to chloroquine, sulfadoxine, pyrimethamine, quinine, and mefloquine, this region has now spawned parasites resistant to artemisinins, the world's most potent antimalarial drugs. In areas where artemisinin resistance is prevalent, artemisinin combination therapies (ACTs)-the first-line treatments for malaria-are failing fast. This worrisome development threatens to make malaria practically untreatable in SEA, and threatens to compromise global endeavors to eliminate this disease. A recent series of clinical, in vitro, genomics, and transcriptomics studies in SEA have defined in vivo and in vitro phenotypes of artemisinin resistance, identified its causal genetic determinant, explored its molecular mechanism, and assessed its clinical impact. Specifically, these studies have established that artemisinin resistance manifests as slow parasite clearance in patients and increased survival of early-ring-stage parasites in vitro; is caused by single nucleotide polymorphisms in the parasite's K13 gene, is associated with an upregulated "unfolded protein response" pathway that may antagonize the pro-oxidant activity of artemisinins, and selects for partner drug resistance that rapidly leads to ACT failures. In SEA, clinical studies are urgently needed to monitor ACT efficacy where K13 mutations are prevalent, test whether new combinations of currently available drugs cure ACT failures, and advance new antimalarial compounds through preclinical pipelines and into clinical trials. Intensifying these efforts should help to forestall the spread of artemisinin and partner drug resistance from SEA to sub-Saharan Africa, where the world's malaria transmission, morbidity, and mortality rates are highest.

Wihokhoen B, Dondorp AM, Turner P, Woodrow CJ, Imwong M. 2016. Use of Blood Smears and Dried Blood Spots for Polymerase Chain Reaction-Based Detection and Quantification of Bacterial Infection and Plasmodium falciparum in Severely Ill Febrile African Children. Am J Trop Med Hyg, 94 (2), pp. 322-326. | Show Abstract | Read more

Molecular approaches offer a means of testing archived samples stored as dried blood spots in settings where standard blood cultures are not possible. Peripheral blood films are one suggested source of material, although the sensitivity of this approach has not been well defined. Thin blood smears and dried blood spots from a severe pediatric malaria study were assessed using specific polymerase chain reaction (PCR) primers to detect non-typhoidal Salmonella (NTS; MisL gene), Streptococcus pneumoniae (lytA), and Plasmodium falciparum (18S rRNA). Of 16 cases of NTS and S. pneumoniae confirmed on blood culture, none were positive by PCR using DNA extracts from blood films or dried blood spots. In contrast, four of 36 dried blood spots and two of 178 plasma samples were PCR positive for S. pneumoniae, despite negative bacterial blood cultures, suggesting false positives. Quantitative assessment revealed that the effective concentration of P. falciparum DNA in blood films was three log orders of magnitude lower than for dried blood spots. The P. falciparum kelch13 gene could not be amplified from blood films. These findings question the value of blood PCR-based approaches for detection of NTS and S. pneumoniae, and show that stored blood films are an inefficient method of studying P. falciparum.

Cross R, Ling C, Day NP, McGready R, Paris DH. 2016. Revisiting doxycycline in pregnancy and early childhood--time to rebuild its reputation? Expert Opin Drug Saf, 15 (3), pp. 367-382. | Citations: 2 (Scopus) | Show Abstract | Read more

INTRODUCTION: Doxycycline is highly effective, inexpensive with a broad therapeutic spectrum and exceptional bioavailability. However these benefits have been overshadowed by its classification alongside the tetracyclines - class D drugs, contraindicated in pregnancy and in children under 8 years of age. Doxycycline-treatable diseases are emerging as leading causes of undifferentiated febrile illness in Southeast Asia. For example scrub typhus and murine typhus have an unusually severe impact on pregnancy outcomes, and current mortality rates for scrub typhus reach 12-13% in India and Thailand. The emerging evidence for these important doxycycline-treatable diseases prompted us to revisit doxycycline usage in pregnancy and childhood. AREAS COVERED: A systematic review of the available literature on doxycycline use in pregnant women and children revealed a safety profile of doxycycline that differed significantly from that of tetracycline; no correlation between the use of doxycycline and teratogenic effects during pregnancy or dental staining in children was found. EXPERT OPINION: The change of the US FDA pregnancy classification scheme to an evidence-based approach will enable adequate evaluation of doxycycline in common tropical illnesses and in vulnerable populations in clinical treatment trials, dosage-optimization pharmacokinetic studies and for the empirical treatment of undifferentiated febrile illnesses, especially in pregnant women and children.

Rattanavong S, Dance DA, Davong V, Baker C, Frost H, Phetsouvanh R, Vongsouvath M, Newton PN, Steer AC, Smeesters PR. 2016. Group A streptococcal strains isolated in Lao People's Democratic Republic from 2004 to 2013. Epidemiol Infect, 144 (8), pp. 1770-1773. | Show Abstract | Read more

Epidemiological data regarding group A streptococcal (GAS) infections in South East Asia are scarce with no information from Laos. We characterized emm types, emm clusters and the antibiotic resistance profile of 124 GAS isolates recovered in Laos during 2004-2013. Most strains were recovered from skin and invasive infections (76% and 19%, respectively). Thirty-four emm types were identified as belonging to 12 emm clusters and no novel emm types were identified. No significant differences were observed in the distribution of emm types or emm clusters according to age or site of recovery (skin or invasive infections). There was moderate strain diversity in this country but considerable differences in emm-type distribution between Laos, Thailand and Cambodia. Vaccine coverage was high for the J8 vaccine candidate. The theoretical coverage for the 30-valent vaccine candidate needs further investigation. Antibiotic resistance was moderate to erythromycin and chloramphenicol (8% and 7%, respectively) and low to ofloxacin (<1%).

Cheeseman IH, Miller B, Tan JC, Tan A, Nair S, Nkhoma SC, De Donato M, Rodulfo H, Dondorp A, Branch OH et al. 2016. Population Structure Shapes Copy Number Variation in Malaria Parasites. Mol Biol Evol, 33 (3), pp. 603-620. | Citations: 3 (Scopus) | Show Abstract | Read more

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.

Kulsantiwong P, Pudla M, Boondit J, Wikraiphat C, Dunachie SJ, Chantratita N, Utaisincharoen P. 2016. Burkholderia pseudomallei induces IL-23 production in primary human monocytes. Med Microbiol Immunol, 205 (3), pp. 255-260. | Citations: 1 (Scopus) | Show Abstract | Read more

Burkholderia pseudomallei, a gram-negative intracellular bacterium, is a causative agent of melioidosis. The bacterium has been shown to induce the innate immune response, particularly pro-inflammatory cytokine production in several of both mouse and human cell types. In the present study, we investigate host immune response in B. pseudomallei-infected primary human monocytes. We discover that wild-type B. pseudomallei is able to survive and multiply inside the primary human monocytes. In contrast, B. pseudomallei LPS mutant, a less virulent strain, is susceptible to host killing during bacterial infection. Moreover, microarray result showed that wild-type B. pseudomallei but not B. pseudomallei LPS mutant is able to activate gene expression of IL-23 as demonstrated by the up-regulation of p19 and p40 subunit expression. Consistent with gene expression analysis, the secretion of IL-23 analyzed by ELISA also showed that wild-type B. pseudomallei induces a significantly higher level of IL-23 secretion than that of B. pseudomallei LPS mutant. These results implied that IL-23 may be an important cytokine for the innate immune response during B. pseudomallei infection. The regulation of IL-23 production may drive the different host innate immune responses between patients and may relate to the severity of melioidosis.

Kaji A, Parker DM, Chu CS, Thayatkawin W, Suelaor J, Charatrueangrongkun R, Salathibuppha K, Nosten FH, McGready R. 2016. Immunization Coverage in Migrant School Children Along the Thailand-Myanmar Border. J Immigr Minor Health, 18 (5), pp. 1038-1045. | Show Abstract | Read more

The objective of this project was to document and increase vaccine coverage in migrant school children on the Thailand-Myanmar border. Migrant school children (n = 12,277) were enrolled in a school-based immunization program in four Thai border districts. The children were evaluated for vaccination completion and timing, for six different vaccines: Bacille Calmette-Guerin (BCG); Oral Polio vaccine (OPV); Hepatitis B vaccine (HepB); Diphtheria, Pertussis and Tetanus vaccine (DTP); Measles Containing Vaccine or Measles, Mumps and Rubella vaccine (MMR); Tetanus and Diphtheria containing vaccine (Td). Vaccine coverage proportions for BCG, OPV3, DTP3, HepB3 and measles containing vaccine were 92.3, 85.3, 63.8, 72.2, and 90.9 % respectively. Most children were able to receive vaccines in a time appropriate manner. School-based immunization programs offer a suitable vaccine delivery mechanism for hard-to-reach populations. However, these data suggest overall low vaccine coverage in migrant populations. Further efforts toward improving appropriate vaccine coverage and methods of retaining documentation of vaccination in mobile migrant populations are necessary for improved health.

Hetem DJ, Derde LPG, Empel J, Mroczkowska A, Orczykowska-Kotyna M, Kozińska A, Hryniewicz W, Goossens H, Bonten MJM. 2016. Molecular epidemiology of MRSA in 13 ICUs from eight European countries Journal of Antimicrobial Chemotherapy, 71 (1), pp. 45-52. | Read more

Zhang R, Suwanarusk R, Malleret B, Cooke BM, Nosten F, Lau YL, Dao M, Lim CT, Renia L, Tan KS, Russell B. 2016. A Basis for Rapid Clearance of Circulating Ring-Stage Malaria Parasites by the Spiroindolone KAE609. J Infect Dis, 213 (1), pp. 100-104. | Citations: 2 (Scopus) | Show Abstract | Read more

Recent clinical trials revealed a surprisingly rapid clearance of red blood cells (RBCs) infected with malaria parasites by the spiroindolone KAE609. Here, we show that ring-stage parasite-infected RBCs exposed to KAE609 become spherical and rigid, probably through osmotic dysregulation consequent to the disruption of the parasite's sodium efflux pump (adenosine triphosphate 4). We also show that this peculiar drug effect is likely to cause accelerated splenic clearance of the rheologically impaired Plasmodium vivax- and Plasmodium falciparum-infected RBCs.

Cho JS, Russell B, Kosasaivee V, Zhang R, Colin Y, Bertrand O, Chandramohanadas R, Chu CS, Nosten F, Renia L, Malleret B. 2016. Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry. Int J Parasitol, 46 (1), pp. 31-39. | Citations: 1 (Scopus) | Show Abstract | Read more

The invasion of CD71+ reticulocytes by Plasmodium vivax is a crucial yet poorly characterised event. The application of flow cytometry to ex vivo invasion assays promises to facilitate the quantitative analysis of P. vivax reticulocyte invasion. However, current protocols suffer from a low level of sensitivity due to the absence of a particular design for P. vivax cell tropism. Importantly, merozoite invasion into contaminating red blood cells from the schizont inoculum (auto-invasion) may confound the analysis. Here we present a stable two-color flow cytometry assay for the accurate quantification of P. vivax merozoite invasion into intracellularly labelled CD71+ reticulocytes. Various enzymatic treatments, antibodies and invasion inhibitory molecules were used to successfully demonstrate the utility of this method. Fluorescent labelling of red blood cells did not affect the invasion and early intra-erythrocytic development of P. vivax. Importantly, this portable field assay allows for the economic usage of limited biological material (parasites and reticulocytes) and the intracellular labeling of the target cells reduces the need for highly purified schizont inoculums. This assay will facilitate the study of P. vivax merozoite biology and the testing of vaccine candidates against vivax malaria.

Bousfield R, Thyl M, Samol O, Rithea L, Sona S, Chhat HP, Poda S, Moore CE, Chheng K, Kumar V et al. 2016. A retrospective study of factors which determine a negative blood culture in Cambodian children diagnosed with enteric fever. Paediatr Int Child Health, 36 (2), pp. 118-121. | Show Abstract | Read more

BACKGROUND: Blood cultures are used to confirm a diagnosis of enteric fever but reported sensitivities can be as low as 40%. AIMS: To determine the factors associated with a negative blood culture in Cambodian children with suspected enteric fever. METHODS: In a retrospective study of hospitalised Cambodian children given a discharge diagnosis of enteric fever, the following factors associated with a negative blood culture were analysed: age, blood culture volume, prior antibiotic therapy, duration of illness and disease severity. RESULTS: In 227 hospitalised Cambodian children with a discharge diagnosis of enteric fever, it was confirmed in 70% by a positive blood culture. There was no association between a negative blood culture and younger age, lower blood volumes for culture, prior antibiotic therapy, a late presentation or milder disease. CONCLUSIONS: Although blood culture sensitivity was higher than expected, alternative simple, rapid and sensitive tests are needed for diagnosing enteric fever.

Moore CE, Sona S, Poda S, Putchhat H, Kumar V, Sopheary S, Stoesser N, Bousfield R, Day N, Parry CM. 2016. Antimicrobial susceptibility of uropathogens isolated from Cambodian children. Paediatr Int Child Health, 36 (2), pp. 113-117. | Show Abstract | Read more

BACKGROUND: Bacterial resistance to commonly used antimicrobials is an increasing problem in Asia but information concerning the antimicrobial susceptibility of bacteria causing urinary tract infections (UTIs) in children is limited. METHODS: This was a 5-year retrospective study of children with suspected UTI attending a paediatric hospital in north-west Cambodia. Urines with a positive culture containing a single organism with a count of >10(5) colony-forming units (CFU)/ml were considered diagnostic of infection. The organism was identified and the resistance pattern (using CLSI guidelines) and presence of an extended-spectrum β-lactamase (ESBL) phenotype was determined. RESULTS: In total, there were 217 episodes of infection, 210 (97%) with Gram-negative bacteria. Escherichia coli was the most common infecting isolate with high levels of resistance to most oral antibiotics, except nitrofurantoin. Nearly half of the E. coli (44%) were extended-spectrum cephalosporin (ESC)-resistant with the proportion increasing significantly over the 5-year period. ESC-resistant E. coli were more likely to be multi-drug-resistant and 91% demonstrated an ESBL phenotype. CONCLUSION: The data highlight the importance of microbiological surveillance of UTIs in children, particularly in areas where there are known to be multiply resistant organisms.

Phyo AP, Jittamala P, Nosten FH, Pukrittayakamee S, Imwong M, White NJ, Duparc S, Macintyre F, Baker M, Möhrle JJ. 2016. Antimalarial activity of artefenomel (OZ439), a novel synthetic antimalarial endoperoxide, in patients with Plasmodium falciparum and Plasmodium vivax malaria: an open-label phase 2 trial. Lancet Infect Dis, 16 (1), pp. 61-69. | Citations: 1 (Web of Science Lite) | Show Abstract | Read more

BACKGROUND: Artefenomel (OZ439) is a novel synthetic trioxolane with improved pharmacokinetic properties compared with other antimalarial drugs with the artemisinin pharmacophore. Artefenomel has been generally well tolerated in volunteers at doses up to 1600 mg and is being developed as a partner drug in an antimalarial combination treatment. We investigated the efficacy, tolerability, and pharmacokinetics of artefenomel at different doses in patients with Plasmodium falciparum or Plasmodium vivax malaria. METHODS: This phase 2a exploratory, open-label trial was done at the Hospital for Tropical Diseases, Bangkok, and the Shoklo Malaria Research Unit in Thailand. Adult patients with acute, uncomplicated P falciparum or P vivax malaria received artefenomel in a single oral dose (200 mg, 400 mg, 800 mg, or 1200 mg). The first cohort received 800 mg. Testing of a new dose of artefenomel in a patient cohort was decided on after safety and efficacy assessment of the preceding cohort. The primary endpoint was the natural log parasite reduction per 24 h. Definitive oral treatment was given at 36 h. This trial is registered with, number NCT01213966. FINDINGS: Between Oct 24, 2010, and May 25, 2012, 82 patients were enrolled (20 in each of the 200 mg, 400 mg, and 800 mg cohorts, and 21 in the 1200 mg cohort). One patient withdrew consent (before the administration of artefenomel) but there were no further dropouts. The parasite reduction rates per 24 h ranged from 0·90 to 1·88 for P falciparum, and 2·09 to 2·53 for P vivax. All doses were equally effective in both P falciparum and P vivax malaria, with median parasite clearance half-lives of 4·1 h (range 1·3-6·7) to 5·6 h (2·0-8·5) for P falciparum and 2·3 h (1·2-3·9) to 3·2 h (0·9-15·0) for P vivax. Maximum plasma concentrations, dose-proportional to 800 mg, occurred at 4 h (median). The estimated elimination half-life was 46-62 h. No serious drug-related adverse effects were reported; other adverse effects were generally mild and reversible, with the highest number in the 1200 mg cohort (17 [81%] patients with at least one adverse event). The most frequently reported adverse effect was an asymptomatic increase in plasma creatine phosphokinase concentration (200 mg, n=5; 400 mg, n=3; 800 mg, n=1; 1200 mg, n=3). INTERPRETATION: Artefenomel is a new synthetic antimalarial peroxide with a good safety profile that clears parasitaemia rapidly in both P falciparum and P vivax malaria. Its long half-life suggests a possible use in a single-dose treatment in combination with other drugs. FUNDING: Bill & Melinda Gates Foundation, Wellcome Trust, and UK Department for International Development.

Stoesser N, Mathers AJ, Moore CE, Day NP, Crook DW. 2016. Colistin resistance gene mcr-1 and pHNSHP45 plasmid in human isolates of Escherichia coli and Klebsiella pneumoniae. Lancet Infect Dis, 16 (3), pp. 285-286. | Citations: 30 (Scopus) | Read more

Auburn S, Böhme U, Steinbiss S, Trimarsanto H, Hostetler J, Sanders M, Gao Q, Nosten F, Newbold CI, Berriman M et al. 2016. A new Plasmodium vivax reference sequence with improved assembly of the subtelomeres reveals an abundance of pir genes. Wellcome Open Res, 1 pp. 4. | Show Abstract | Read more

Plasmodium vivax is now the predominant cause of malaria in the Asia-Pacific, South America and Horn of Africa. Laboratory studies of this species are constrained by the inability to maintain the parasite in continuous ex vivo culture, but genomic approaches provide an alternative and complementary avenue to investigate the parasite's biology and epidemiology. To date, molecular studies of P. vivax have relied on the Salvador-I reference genome sequence, derived from a monkey-adapted strain from South America. However, the Salvador-I reference remains highly fragmented with over 2500 unassembled scaffolds.  Using high-depth Illumina sequence data, we assembled and annotated a new reference sequence, PvP01, sourced directly from a patient from Papua Indonesia. Draft assemblies of isolates from China (PvC01) and Thailand (PvT01) were also prepared for comparative purposes. The quality of the PvP01 assembly is improved greatly over Salvador-I, with fragmentation reduced to 226 scaffolds. Detailed manual curation has ensured highly comprehensive annotation, with functions attributed to 58% core genes in PvP01 versus 38% in Salvador-I. The assemblies of PvP01, PvC01 and PvT01 are larger than that of Salvador-I (28-30 versus 27 Mb), owing to improved assembly of the subtelomeres.  An extensive repertoire of over 1200 Plasmodium interspersed repeat (pir) genes were identified in PvP01 compared to 346 in Salvador-I, suggesting a vital role in parasite survival or development. The manually curated PvP01 reference and PvC01 and PvT01 draft assemblies are important new resources to study vivax malaria. PvP01 is maintained at GeneDB and ongoing curation will ensure continual improvements in assembly and annotation quality.

Total citations for publications on this page: 301