A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.

Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

Baseline data of parasite clearance in patients with falciparum malaria treated with an artemisinin derivative: an individual patient data meta-analysis.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum manifests as slow parasite clearance but this measure is also influenced by host immunity, initial parasite biomass and partner drug efficacy. This study collated data from clinical trials of artemisinin derivatives in falciparum malaria with frequent parasite counts to provide reference parasite clearance estimates stratified by location, treatment and time, to examine host factors affecting parasite clearance, and to assess the relationships between parasite clearance and risk of recrudescence during follow-up. METHODS: Data from 24 studies, conducted from 1996 to 2013, with frequent parasite counts were pooled. Parasite clearance half-life (PC1/2) was estimated using the WWARN Parasite Clearance Estimator. Random effects regression models accounting for study and site heterogeneity were used to explore factors affecting PC1/2 and risk of recrudescence within areas with reported delayed parasite clearance (western Cambodia, western Thailand after 2000, southern Vietnam, southern Myanmar) and in all other areas where parasite populations are artemisinin sensitive. RESULTS: PC1/2 was estimated in 6975 patients, 3288 of whom also had treatment outcomes evaluate d during 28-63 days follow-up, with 93 (2.8 %) PCR-confirmed recrudescences. In areas with artemisinin-sensitive parasites, the median PC1/2 following three-day artesunate treatment (4 mg/kg/day) ranged from 1.8 to 3.0 h and the proportion of patients with PC1/2 >5 h from 0 to 10 %. Artesunate doses of 4 mg/kg/day decreased PC1/2 by 8.1 % (95 % CI 3.2-12.6) compared to 2 mg/kg/day, except in populations with delayed parasite clearance. PC1/2 was longer in children and in patients with fever or anaemia at enrolment. Long PC1/2 (HR = 2.91, 95 % CI 1.95-4.34 for twofold increase, p < 0.001) and high initial parasitaemia (HR = 2.23, 95 % CI 1.44-3.45 for tenfold increase, p < 0.001) were associated independently with an increased risk of recrudescence. In western Cambodia, the region with the highest prevalence of artemisinin resistance, there was no evidence for increasing PC1/2 since 2007. CONCLUSIONS: Several factors affect PC1/2. As substantial heterogeneity in parasite clearance exists between locations, early detection of artemisinin resistance requires reference PC1/2 data. Studies with frequent parasite count measurements to characterize PC1/2 should be encouraged. In western Cambodia, where PC1/2 values are longest, there is no evidence for recent emergence of higher levels of artemisinin resistance.

Efficacy of dihydroartemisinin-piperaquine for treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax in Cambodia, 2008 to 2010.

We describe here the results of antimalarial therapeutic efficacy studies conducted in Cambodia from 2008 to 2010. A total of 15 studies in four sentinel sites were conducted using dihydroartemisinin-piperaquine (DP) for the treatment of Plasmodium falciparum infection and chloroquine (CQ) and DP for the treatment of P. vivax infection. All studies were performed according to the standard World Health Organization protocol for the assessment of antimalarial treatment efficacy. Among the studies of DP for the treatment of P. falciparum, an increase in treatment failure was observed in the western provinces. In 2010, the PCR-corrected treatment failure rates for DP on day 42 were 25% (95% confidence interval [CI] = 10 to 51%) in Pailin and 10.7% (95% CI = 4 to 23%) in Pursat, while the therapeutic efficacy of DP remained high (100%) in Ratanakiri and Preah Vihear provinces, located in northern and eastern Cambodia. For the studies of P. vivax, the day 28 uncorrected treatment failure rate among patients treated with CQ ranged from 4.4 to 17.4%; DP remained 100% effective in all sites. Further study is required to investigate suspected P. falciparum resistance to piperaquine in western Cambodia; the results of in vitro and molecular studies were not found to support the therapeutic efficacy findings. The emergence of artemisinin resistance in this region has likely put additional pressure on piperaquine. Although DP appears to be an appropriate new first-line treatment for P. vivax in Cambodia, alternative treatments are urgently needed for P. falciparum-infected patients in western Cambodia.

Effect of single nucleotide polymorphisms in cytochrome P450 isoenzyme and N-acetyltransferase 2 genes on the metabolism of artemisinin-based combination therapies in malaria patients from Cambodia and Tanzania.

The pharmacogenetics of antimalarial agents are poorly known, although the application of pharmacogenetics might be critical in optimizing treatment. This population pharmacokinetic-pharmacogenetic study aimed at assessing the effects of single nucleotide polymorphisms (SNPs) in cytochrome P450 isoenzyme genes (CYP, namely, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) and the N-acetyltransferase 2 gene (NAT2) on the pharmacokinetics of artemisinin-based combination therapies in 150 Tanzanian patients treated with artemether-lumefantrine, 64 Cambodian patients treated with artesunate-mefloquine, and 61 Cambodian patients treated with dihydroartemisinin-piperaquine. The frequency of SNPs varied with the enzyme and the population. Higher frequencies of mutant alleles were found in Cambodians than Tanzanians for CYP2C9*3, CYP2D6*10 (100C → T), CYP3A5*3, NAT2*6, and NAT2*7. In contrast, higher frequencies of mutant alleles were found in Tanzanians for CYP2D6*17 (1023C → T and 2850C → T), CYP3A4*1B, NAT2*5, and NAT2*14. For 8 SNPs, no significant differences in frequencies were observed. In the genetic-based population pharmacokinetic analyses, none of the SNPs improved model fit. This suggests that pharmacogenetic data need not be included in appropriate first-line treatments with the current artemisinin derivatives and quinolines for uncomplicated malaria in specific populations. However, it cannot be ruled out that our results represent isolated findings, and therefore more studies in different populations, ideally with the same artemisinin-based combination therapies, are needed to evaluate the influence of pharmacogenetic factors on the clearance of antimalarials.

Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia.

The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.

Therapeutic efficacy of fixed dose artesunate-mefloquine for the treatment of acute, uncomplicated Plasmodium falciparum malaria in Kampong Speu, Cambodia.

BACKGROUND: Cambodia stopped using co-blistered, non-fixed, artesunate-mefloquine (ASMQ) in 2008 when treatment failure rates approximated 20%. Fixed dose combination (FDC) ASMQ is efficacious against acute uncomplicated, drug resistant Plasmodium falciparum malaria in Southeast Asia but has not been tested in Cambodia. METHODS: A 42-day WHO therapeutic efficacy study (TES) was conducted in 2010 in Oral, Kampong Speu province, south-west Cambodia, in patients with acute uncomplicated P. falciparum. Daily administered FDC ASMQ for three days was dosed by age. Genotyping of isolates at day 0 and day of recrudescence by polymerase chain reaction (PCR) classified post-treatment recurrent falciparum parasitaemia. Ex vivo drug sensitivity testing ([3H] hypoxanthine method) was performed on baseline parasites and reported as the drug concentration inhibiting 50% parasite growth vs no drug (IC50). RESULTS: Recruited patients numbered 45; five aged <15 years. On day 3, five of 45 [11.1 (3.7-24.05)] % patients were still parasite-positive; one of whom later failed treatment on day 21. There were 5/45 (11.1%) late treatment failures on day 21, 28 and 35; all were PCR diagnosed recrudescent infections. The day 0 MQ IC50s ranged from 11.5-238.9 (median 58.6) nM. CONCLUSIONS: This TES demonstrated reasonable efficacy in an area of possible reduced artemisinin sensitivity and high MQ IC50s. Efficacy testing of FDC ASMQ should continue in Cambodia and be considered for reintroduction if efficacy returns.

Population pharmacokinetics of mefloquine, piperaquine and artemether-lumefantrine in Cambodian and Tanzanian malaria patients.

BACKGROUND: Inter-individual variability in plasma concentration-time profiles might contribute to differences in anti-malarial treatment response. This study investigated the pharmacokinetics of three different forms of artemisinin combination therapy (ACT) in Tanzania and Cambodia to quantify and identify potential sources of variability. METHODS: Drug concentrations were measured in 143 patients in Tanzania (artemether, dihydroartemisinin, lumefantrine and desbutyl-lumefantrine), and in 63 (artesunate, dihydroartemisinin and mefloquine) and 60 (dihydroartemisinin and piperaquine) patients in Cambodia. Inter- and intra-individual variabilities in the pharmacokinetic parameters were assessed and the contribution of demographic and other covariates was quantified using a nonlinear mixed-effects modelling approach (NONMEM®). RESULTS: A one-compartment model with first-order absorption from the gastrointestinal tract fitted the data for all drugs except piperaquine (two-compartment). Inter-individual variability in concentration exposure was about 40% and 12% for mefloquine. From all the covariates tested, only body weight (for all antimalarials) and concomitant treatment (for artemether only) showed a significant influence on these drugs' pharmacokinetic profiles. Artesunate and dihydroartemisinin could not be studied in the Cambodian patients due to insufficient data-points. Modeled lumefantrine kinetics showed that the target day 7 concentrations may not be achieved in a substantial proportion of patients. CONCLUSION: The marked variability in the disposition of different forms of ACT remained largely unexplained by the available covariates. Dosing on body weight appears justified. The concomitance of unregulated drug use (residual levels found on admission) and sub-optimal exposure (variability) could generate low plasma levels that contribute to selecting for drug-resistant parasites.

G6PD deficiency in Plasmodium falciparum and Plasmodium vivax malaria-infected Cambodian patients.

BACKGROUND: Glucose-6-phosphate-dehydrogenase deficiency (G6PDd) rates are unknown in malaria-infected Cambodian patients. These data are key to a rational drug policy for malaria elimination of Plasmodium falciparum and Plasmodium vivax. METHODS: From September 2010-2012, a two-year survey of G6PDd and haemoglobinopathies assessed by quantitative enzyme activity assay and haemoglobin electrophoresis, respectively, was conducted in malaria-infected patients presenting to 19 health centres throughout Cambodia. RESULTS: A total of 2,408 confirmed malaria patients of mean age 26.7 (range 2-81) years were recruited from mostly western Cambodia (n = 1,732, 71.9%); males outnumbered females by 3.9:1. Plasmodium falciparum was present in 1,443 (59.9%) and P. vivax in 965 (40.1%) patients. Mean G6PD activity was 11.6 (CI 95%: 11.4-11.8) U/g Hb, G6PDd was present in 13.9% of all patients (335/2,408) and severe G6PDd (including WHO Class I and II variants) was more common in western (158/1,732, 9.1%) versus eastern (21/414, 5.1%) Cambodia (P = 0.01). Of 997/2,408 (41.4%) had a haemoglobinopathy. Mean haemoglobin concentrations were inversely related to age: 8.1 g/dL < five years, 8.7 g/dL five to 14 years, and 10.4 g/dL >15 years (P <0.001). CONCLUSIONS: G6PDd prevalence, anaemia and haemoglobinopathies were common in malaria-infected patients. The deployment of primaquine in Cambodia should be preceded by primaquine safety studies paralleled with evaluations of easy to use tests to detect G6PDd.

Higher microsatellite diversity in Plasmodium vivax than in sympatric Plasmodium falciparum populations in Pursat, Western Cambodia.

Previous microsatellite analyses of sympatric populations of Plasmodium vivax and Plasmodium falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n=87) and P. falciparum (n=164) parasites from Pursat province, Western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P=0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P=0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax.

Plasmodium knowlesi infection in humans, Cambodia, 2007-2010.

Two cases of Plasmodium knowlesi infection in humans were identified in Cambodia by 3 molecular detection assays and sequencing. This finding confirms the widespread distribution of P. knowlesi malaria in humans in Southeast Asia. Further wide-scale studies are required to assess the public health relevance of this zoonotic malaria parasite.

Increasing access to early malaria diagnosis and prompted treatment in remote cambodian villages.

Malaria poses a significant public health burden in the remote areas of western Cambodia, where access to health services and information is limited. Recognizing the potential of village malaria workers to reach these communities, the US Agency for International Development-funded Malaria Control in Cambodia project used a multipronged approach to strengthen the village malaria workers network. As a result, the proportion of confirmed malaria cases treated by village malaria workers has doubled during the past 2 years, significantly increasing the numbers being properly diagnosed and treated. Key to the program's success has been the integration of village malaria workers with public health facilities, improved patient access to prompt diagnosis and treatment, and resolution of systemic barriers such as logistics for rapid diagnostic tests.

Residual antimalarial concentrations before treatment in patients with malaria from Cambodia: indication of drug pressure.

BACKGROUND: The Thai-Cambodian border has been known as the origin of antimalarial drug resistance for the past 30 years. There is a highly diverse market for antimalarials in this area, and improved knowledge of drug pressure would be useful to target interventions aimed at reducing inappropriate drug use. METHODS: Baseline samples from 125 patients with falciparum malaria recruited for 2 in vivo studies (in Preah Vihear and Pursat provinces) were analyzed for the presence of 14 antimalarials in a single run, by means of a liquid chromatography-tandem mass spectrometry assay. RESULTS: Half of the patients had residual drug concentrations above the lower limit of calibration for at least 1 antimalarial at admission. Among the drugs detected were the currently used first-line drugs mefloquine (25% and 35% of patients) and piperaquine (15% of patients); the first-line drug against vivax malaria, chloroquine (25% and 41% of patients); and the former first-line drug, quinine (5% and 34% patients). CONCLUSIONS: The findings demonstrate that there is high drug pressure and that many people still seek treatment in the private and informal sector, where appropriate treatment is not guaranteed. Promotion of comprehensive behavioral change, communication, community-based mobilization, and advocacy are vital to contain the emergence and spread of parasite resistance against new antimalarials.

Decreased in vitro susceptibility of Plasmodium falciparum isolates to artesunate, mefloquine, chloroquine, and quinine in Cambodia from 2001 to 2007.

This study describes the results of in vitro antimalarial susceptibility assays and molecular polymorphisms of Plasmodium falciparum isolates from Cambodia. The samples were collected from patients enrolled in therapeutic efficacy studies (TES) conducted by the Cambodian National Malaria Control Program for the routine efficacy monitoring of artemisinin-based combination therapy (ACT) (artesunate-mefloquine and artemether-lumefantrine combinations). The isolates (n = 2,041) were obtained from nine sentinel sites during the years 2001 to 2007. Among these, 1,588 were examined for their in vitro susceptibilities to four antimalarials (artesunate, mefloquine, chloroquine, and quinine), and 851 isolates were genotyped for single nucleotide polymorphisms (SNPs). The geometric means of the 50% inhibitory concentrations (GMIC(50)s) of the four drugs tested were significantly higher for isolates from western Cambodia than for those from eastern Cambodia. GMIC(50)s for isolates from participants who failed artesunate-mefloquine therapy were significantly higher than those for patients who were cured (P, <0.001). In vitro correlation of artesunate with the other drugs was observed. The distributions of the SNPs differed between eastern and western Cambodia, suggesting different genetic backgrounds of the parasite populations in these two parts of the country. The GMIC(50)s of the four drugs tested increased significantly in eastern Cambodia during 2006 to 2007. These results are worrisome, because they may signal deterioration of the efficacy of artesunate-mefloquine beyond the Cambodian-Thai border.

Cost of dengue and other febrile illnesses to households in rural Cambodia: a prospective community-based case-control study.

BACKGROUND: The average annual reported dengue incidence in Cambodia is 3.3/1,000 among children < 15 years of age (2002-2007). To estimate the economic burden of dengue, accurate cost-of-illness data are essential. We conducted a prospective, community-based, matched case-control study to assess the cost and impact of an episode of dengue fever and other febrile illness on households in rural Cambodia. METHODS: In 2006, active fever surveillance was conducted among a cohort of 6,694 children aged < or = 15 years in 16 villages in Kampong Cham province, Cambodia. Subsequently, a case-control study was performed by individually assigning one non-dengue febrile control from the cohort to each laboratory-confirmed dengue case. Parents of cases and controls were interviewed using a standardized questionnaire to determine household-level, illness-related expenditures for medical and non-medical costs, and estimated income loss (see Additional file 1). The household socio-economic status was determined and its possible association with health seeking behaviour and the ability to pay for the costs of a febrile illness. RESULTS: Between September and November 2006, a total of 60 household heads were interviewed: 30 with dengue-positive and 30 with dengue-negative febrile children. Mean total dengue-related costs did not differ from those of other febrile illnesses (31.5 vs. 27.2 US dollars, p = 0.44). Hospitalization almost tripled the costs of dengue (from 14.3 to 40.1 US dollars) and doubled the costs of other febrile illnesses (from 17.0 to 36.2 US dollars). To finance the cost of a febrile illness, 67% of households incurred an average debt of 23.5 US dollars and higher debt was associated with hospitalization compared to outpatient treatment (23.1 US dollars vs. 4.5 US dollars, p < 0.001). These costs compared to an average one-week expenditure on food of 9.5 US dollars per household (range 2.5-21.3). In multivariate analysis, higher socio-economic status (odds ratio [OR] 4.4; 95% confidence interval [CI] 1.4-13.2), duration of fever (OR 2.1; 95%CI 1.3-3.5), and age (OR 0.8; 95%CI 0.7-0.9) were independently associated with hospitalization. CONCLUSION: In Cambodia, dengue and other febrile illnesses pose a financial burden to households. A possible reason for a lower rate of hospitalization among children from poor households could be the burden of higher illness-related costs and debts.

Molecular surveillance for multidrug-resistant Plasmodium falciparum, Cambodia.

We conducted surveillance for multidrug-resistant Plasmodium falciparum in Cambodia during 2004-2006 by assessing molecular changes in pfmdr1. The high prevalence of isolates with multiple pfmdr1 copies found in western Cambodia near the Thai border, where artesunate-mefloquine therapy failures occur, contrasts with isolates from eastern Cambodia, where this combination therapy remains highly effective.

Production and validation of durable, high quality standardized malaria microscopy slides for teaching, testing and quality assurance during an era of declining diagnostic proficiency.

BACKGROUND: Sets of Giemsa-stained, blood smear slides with systematically verified composite diagnoses would contribute substantially to development of externally validated quality assurance systems for the microscopic diagnosis of malaria. METHODS: whole blood from Plasmodium-positive donors in Cambodia and Indonesia and individuals with no history of risk for malaria was collected. Using standard operating procedures, technicians prepared Giemsa-stained thick and thin smears from each donor. One slide from each of the first 35 donations was distributed to each of 28 individuals acknowledged by reputation as having expertise in the microscopic diagnosis of malaria. These reference readers recorded presence or absence of Plasmodium species and parasite density. A composite diagnosis for each donation was determined based on microscopic findings and species-specific small subunit ribosomal RNA (ssrRNA) DNA polymerase chain reaction (PCR) amplification. RESULTS: More than 12,000 slides were generated from 124 donations. Reference readers correctly identified presence of parasites on 85% of slides with densities <100 parasites/microl, which improved to 100% for densities >350 parasites/microl. Percentages of agreement with composite diagnoses were highest for Plasmodium falciparum (99%), followed by Plasmodium vivax (86%). CONCLUSION: Herein, a standardized method for producing large numbers of consistently high quality, durable Giemsa-stained blood smears and validating composite diagnoses for the purpose of creating a malaria slide repository in support of initiatives to improve training and competency assessment amidst a background of variability in diagnosis is described.

High prevalence and spatial distribution of Strongyloides stercoralis in rural Cambodia.

BACKGROUND: The threadworm, Strongyloides stercoralis, endemic in tropical and temperate climates, is a neglected tropical disease. Its diagnosis requires specific methods, and accurate information on its geographic distribution and global burden are lacking. We predicted prevalence, using Bayesian geostatistical modeling, and determined risk factors in northern Cambodia. METHODS: From February to June 2010, we performed a cross-sectional study among 2,396 participants from 60 villages in Preah Vihear Province, northern Cambodia. Two stool specimens per participant were examined using Koga agar plate culture and the Baermann method for detecting S. stercoralis infection. Environmental data was linked to parasitological and questionnaire data by location. Bayesian mixed logistic models were used to explore the spatial correlation of S. stercoralis infection risk. Bayesian Kriging was employed to predict risk at non-surveyed locations. PRINCIPAL FINDINGS: Of the 2,396 participants, 44.7% were infected with S. stercoralis. Of 1,071 strongyloidiasis cases, 339 (31.6%) were among schoolchildren and 425 (39.7%) were found in individuals under 16 years. The incidence of S. stercoralis infection statistically increased with age. Infection among male participants was significantly higher than among females (OR: 1.7; 95% CI: 1.4-2.0; P<0.001). Participants who defecated in latrines were infected significantly less than those who did not (OR: 0.6; 95% CI: 0.4-0.8; P=0.001). Strongyloidiasis cases would be reduced by 39% if all participants defecated in latrines. Incidence of S. stercoralis infections did not show a strong tendency toward spatial clustering in this province. The risk of infection significantly decreased with increasing rainfall and soil organic carbon content, and increased in areas with rice fields. CONCLUSIONS/SIGNIFICANCE: Prevalence of S. stercoralis in rural Cambodia is very high and school-aged children and adults over 45 years were the most at risk for infection. Lack of access to adequate treatment for chronic uncomplicated strongyloidiasis is an urgent issue in Cambodia. We would expect to see similar prevalence rates elsewhere in Southeast Asia and other tropical resource poor countries.

Diagnosis, treatment and risk factors of Strongyloides stercoralis in schoolchildren in Cambodia.

BACKGROUND: Worldwide, an estimated 30 to 100 million people are infected with Strongyloides stercoralis, a soil-transmitted helminth. Information on the parasite is scarce in most settings. In semi-rural Cambodia, we determined infection rates and risk factors; compared two diagnostic methods (Koga agar plate [KAP] culture and Baermann technique) for detecting S. stercoralis infections, using a multiple stool examination approach; and assessed efficacy of ivermectin treatment. METHODS/PRINCIPAL FINDINGS: We performed a cross-sectional study in 458 children from four primary schools in semi-rural villages in Kandal province, using three diagnostic procedures (Kato-Katz, KAP culture and Baermann technique) on three stool samples. Infected children were treated with ivermectin (100 µg/kg/day for two days) and re-examined three weeks after treatment. Hookworm, S. stercoralis, Trichuris trichiura, and small trematode eggs were most prevalent, with 24.4% of children being infected with S. stercoralis. The sensitivity of KAP culture and Baermann technique was 88.4% and 75.0%, respectively and their negative predictive values were 96.4% and 92.5%, respectively. The cumulative prevalence of S. stercoralis increased from 18.6% to 24.4%, after analyzing three stool samples, which was close to the modeled 'true' prevalence of 24.8%. Children who reported defecating in latrines were significantly less infected with S. stercoralis than those who did not use latrines (p<0.001). Itchy skin and diarrhea were significantly associated with S. stercoralis infection. The cure rate of ivermectin was 98.3%. CONCLUSIONS/SIGNIFICANCE: S. stercoralis infection is highly prevalent among semi-rural Cambodian schoolchildren. The sensitivity of KAP culture is higher than that of the Baermann technique. In the absence of a "gold standard", analysis of multiple stool samples by different diagnostic methods is required to achieve a satisfactory level of sensitivity. Almost three-quarters of the infections could have been avoided by proper sanitation. Ivermectin is highly efficacious against S. stercoralis but prohibitive costs render the drug inaccessible to most Cambodians.

Evaluation of real-time PCR for Strongyloides stercoralis and hookworm as diagnostic tool in asymptomatic schoolchildren in Cambodia.

Diagnosis of soil-transmitted helminths such as Strongyloides stercoralis and hookworms (Ancylostoma duodenale and Necator americanus) is challenging due to irregular larval and egg output in infected individuals and insensitive conventional diagnostic procedures. Sensitive novel real-time PCR assays have been developed. Our study aimed to evaluate the real-time PCR assays as a diagnostic tool for detection of Strongyloides spp. and hookworms in a random stool sample of 218 asymptomatic schoolchildren in Cambodia. Overall prevalence of 17.4% (38/218) and 34.9% (76/218) were determined by real-time PCR for S. stercoralis and hookworms, respectively. Sensitivity and specificity of S. stercoralis specific real-time PCR as compared to the combination of Baermann/Koga Agar as gold standard were 88.9% and 92.7%, respectively. For hookworm specific real-time PCR a sensitivity of 78.9% and specificity of 78.9% were calculated. Co-infections were detectable by PCR in 12.8% (28/218) of individuals. S. stercoralis real-time PCR applied in asymptomatic cases showed a lower sensitivity compared to studies undertaken with symptomatic patients with the same molecular tool, yet it proved to be a valid supplement in the diagnosis of STH infection in Cambodia.

Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin monotherapy or artemisinin-based combination therapy. Absence of in-vitro and ex-vivo correlates of artemisinin resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate the stage-dependent susceptibility of parasites to dihydroartemisinin in the in-vitro RSA, and to assess whether an ex-vivo RSA can identify artemisinin-resistant P falciparum infections. METHODS: We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study done in Pursat, Cambodia, in 2010 (registered with ClinicalTrials.gov, number NCT00341003) and used novel in-vitro survival assays to explore the stage-dependent susceptibility of slow-clearing and fast-clearing parasites to dihydroartemisinin. In 2012, we implemented the RSA in prospective parasite clearance studies in Pursat, Preah Vihear, and Ratanakiri, Cambodia (NCT01736319), to measure the ex-vivo responses of parasites from patients with malaria. Continuous variables were compared with the Mann-Whitney U test. Correlations were analysed with the Spearman correlation test. FINDINGS: In-vitro survival rates of culture-adapted parasites from 13 slow-clearing and 13 fast-clearing infections differed significantly when assays were done on 0-3 h ring-stage parasites (10·88% vs 0·23%; p=0·007). Ex-vivo survival rates significantly correlated with in-vivo parasite clearance half-lives (n=30, r=0·74, 95% CI 0·50-0·87; p<0·0001). INTERPRETATION: The in-vitro RSA of 0-3 h ring-stage parasites provides a platform for the molecular characterisation of artemisinin resistance. The ex-vivo RSA can be easily implemented where surveillance for artemisinin resistance is needed. FUNDING: Institut Pasteur du Cambodge and the Intramural Research Program, NIAID, NIH.