World Malaria Day 2016 in the Kingdom of Cambodia: high-level governmental support embodies the WHO call for "political will to end malaria".

On World Malaria Day 2016, The Kingdom of Cambodia's National celebrations served as a prime of example of how political will is currently being exercised in Cambodia through high-level governmental support for malaria elimination. The main country event was well-planned and coordinated by the National Programme for Parasitology, Entomology and Malaria Control (CNM), and included key contributions from high-ranking political figures, such as His Excellency (H.E) Mam Bun Heng (Minister of Health), and H.E. Keut Sothea (Governor of Pailin Province). There were more than 1000 attendees, ranging from Village Malaria Workers and high school students to CNM's director and other officials in Pailin Province, Western Cambodia. A strong inter-sectoral participation included attendances from the Ministry of Education and high-level representatives of the Cambodian Armed Forces, as well as Malaria Partners like the World Health Organization.

High prevalence of Ancylostoma ceylanicum hookworm infections in humans, Cambodia, 2012.

Ancylostoma ceylanicum, a hookworm of canids and felids in Asia, is becoming the second most common hookworm infecting humans. In 2012, we investigated the prevalence and infection dynamics of and risk factors for hookworm infections in humans and dogs in a rural Cambodian village. Over 57% of the population was infected with hookworms; of those, 52% harbored A. ceylanicum hookworms. The greatest intensities of A. ceylanicum eggs were in persons 21-30 years of age. Over 90% of dogs also harbored A. ceylanicum hookworms. Characterization of the cytochrome oxidase-1 gene divided isolates of A. ceylanicum hookworms into 2 groups, 1 containing isolates from humans only and the other a mix of isolates from humans and animals. We hypothesize that preventative chemotherapy in the absence of concurrent hygiene and animal health programs may be a factor leading to emergence of A. ceylanicum infections; thus, we advocate for a One Health approach to control this zoonosis.

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.

Individual Plasmodium vivax msp1 variants within polyclonal P. vivax infections display different propensities for relapse.

Using a newly developed Plasmodium vivax merozoite surface protein 1 gene (Pvmsp1) heteroduplex tracking assay, we genotyped 107 P. vivax infections in individuals from Cambodia, 45 of whom developed recurrent parasitemia within 42 days. The majority of isolates were polyclonal, but recurrent parasitemias displayed fewer variants compared to initial parasitemias. Two Pvmsp1 gene variants occurred more frequently in the initial genotypes of those who developed recurrent parasitemia, representing the first time P. vivax variants associated with a higher risk of relapse have been described.

Origin and evolution of sulfadoxine resistant Plasmodium falciparum.

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.

Multiple genetic backgrounds of the amplified Plasmodium falciparum multidrug resistance (pfmdr1) gene and selective sweep of 184F mutation in Cambodia.

BACKGROUND: The emergence of artesunate-mefloquine (AS+MQ)-resistant Plasmodium falciparum in the Thailand-Cambodia region is a major concern for malaria control. Studies indicate that copy number increase and key alleles in the pfmdr1 gene are associated with AS+MQ resistance. In the present study, we investigated evidence for a selective sweep around pfmdr1 because of the spread of adaptive mutation and/or multiple copies of this gene in the P. falciparum population in Cambodia. METHODS: We characterized 13 microsatellite loci flanking (+/-99 kb) pfmdr1 in 93 single-clone P. falciparum infections, of which 31 had multiple copies and 62 had a single copy of the pfmdr1 gene. RESULTS: Genetic analysis revealed no difference in the mean (+/- standard deviation) expected heterozygosity (H(e)) at loci around single (0.75+/-0.03) and multiple (0.76+/-0.04) copies of pfmdr1. Evidence of genetic hitchhiking with the selective sweep of certain haplotypes was seen around mutant (184F) pfmdr1 allele, irrespective of the copy number. There was an overall reduction of 28% in mean H(e) (+/-SD) around mutant allele (0.56+/-0.05), compared with wild-type allele (0.84+/-0.02). Significant linkage disequilibrium was also observed between the loci flanking mutant pfmdr1 allele. CONCLUSION: The 184F mutant allele is under selection, whereas amplification of pfmdr1 gene in this population occurs on multiple genetic backgrounds.

Risk profiling and efficacy of albendazole against the hookworms Necator americanus and Ancylostoma ceylanicum in Cambodia to support control programs in Southeast Asia and the Western Pacific.

Background: Hookworm disease is endemic throughout many parts of the Asia Pacific, despite targeted control programs of at-risk populations. The success of these programs has been hindered by the limited efficacy of widely-used mebendazole, rapid re-infection rates linked to persistent reservoirs of untreated people and dogs, and the low sensitivity of conventional coprodiagnostic techniques employed. Methods: Here, we used standard faecal flotation (SFF) and a multiplex qPCR (mqPCR) assay to calculate and compare species-specific cure and egg reduction rates of single dose albendazole (400 mg) against hookworm infections at community level. Data from a cross-sectional survey in 1,232 people from Cambodia were used to inform a generalised linear mixed model to identify risk factors linked to hookworm infection(s) at baseline. Furthermore, we calculated risk factors associated to the probability of being cured after albendazole administration. Findings: Overall, 13·5% of all 1,232 people tested by SFF were positive for hookworm infection(s). Most (80·1%) infected people were >12 years of age, hence above the age targeted by the WHO control program. We estimate that as age increases, the odds of being infected increases at a faster rate for females than for males. We revealed a substantial difference in cure rate of hookworm infection(s) following albendazole treatment using the SFF (81·5%) and mqPCR (46·4%) assays, and provide the first data on the efficacy of this drug against the zoonotic hookworm Ancylostoma ceylanicum. We estimated that as age increases by one year, the odds of being cured decreases by 0·4%-3·7%. Similarly, the odds of being cured for people who boiled drinking water was estimated to be between 1·02 and 6·82. Interpretation: These findings show that the adoption of refined diagnostic techniques is central to monitoring hookworm infection(s) and the success of control strategies, which can ultimately aid in reducing associated morbidity in human populations. The approach taken is likely to be directly applicable to other parts of Southeast Asia and the Western Pacific, where specific epidemiological conditions might hamper the success of targeted treatment programs. Funding: Faculty of Veterinary and Agricultural Sciences Strategic Research Funds, The University of Melbourne.

Failure of artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria in southern Cambodia.

BACKGROUND: Resistance to anti-malarial drugs hampers control efforts and increases the risk of morbidity and mortality from malaria. The efficacy of standard therapies for uncomplicated Plasmodium falciparum and Plasmodium vivax malaria was assessed in Chumkiri, Kampot Province, Cambodia. METHODS: One hundred fifty-one subjects with uncomplicated falciparum malaria received directly observed therapy with 12 mg/kg artesunate (over three days) and 25 mg/kg mefloquine, up to a maximum dose of 600 mg artesunate/1,000 mg mefloquine. One hundred nine subjects with uncomplicated vivax malaria received a total of 25 mg/kg chloroquine, up to a maximum dose of 1,500 mg, over three days. Subjects were followed for 42 days or until recurrent parasitaemia was observed. For P. falciparum infected subjects, PCR genotyping of msp1, msp2, and glurp was used to distinguish treatment failures from new infections. Treatment failure rates at days 28 and 42 were analyzed using both per protocol and Kaplan-Meier survival analysis. Real Time PCR was used to measure the copy number of the pfmdr1 gene and standard 48-hour isotopic hypoxanthine incorporation assays were used to measure IC50 for anti-malarial drugs. RESULTS: Among P. falciparum infected subjects, 47.0% were still parasitemic on day 2 and 11.3% on day 3. The PCR corrected treatment failure rates determined by survival analysis at 28 and 42 days were 13.1% and 18.8%, respectively. Treatment failure was associated with increased pfmdr1 copy number, higher initial parasitaemia, higher mefloquine IC50, and longer time to parasite clearance. One P. falciparum isolate, from a treatment failure, had markedly elevated IC50 for both mefloquine (130 nM) and artesunate (6.7 nM). Among P. vivax infected subjects, 42.1% suffered recurrent P. vivax parasitaemia. None acquired new P. falciparum infection. CONCLUSION: The results suggest that artesunate-mefloquine combination therapy is beginning to fail in southern Cambodia and that resistance is not confined to the provinces at the Thai-Cambodian border. It is unclear whether the treatment failures are due solely to mefloquine resistance or to artesunate resistance as well. The findings of delayed clearance times and elevated artesunate IC50 suggest that artesunate resistance may be emerging on a background of mefloquine resistance.

Is Opisthorchis viverrini Emerging in Cambodia?

Opisthorchis viverrini infection is widely prevalent in Southeast Asia. In Cambodia information on this helminth infection is scare. Recent reports suggest that O. viverrini is an emerging public health problem. We aimed to synthesize all information in relation to the infection, epidemiology, and morbidity of O. viverrini in Cambodia; from published as well as thus far unpublished sources. First reports on O. viverrini date back to 1995. In 2006 an O. viverrini initiative was launched by the national helminth control program. Since then O. viverrini has been reported in all - except two - provinces. Villages with high prevalences (>20%) were found in provinces from Preah Vihear to Takeo. The infection has a highly focal distribution. In many villages no infections were detected. O. viverrini infection was also reported in cats, dogs and intermediate hosts. No report on morbidity associated with O. viverrini was found. The current evidence suggests that O. viverrini infection remains underreported in Cambodia. It is likely that the transmission will further increase in the future with potentially serious consequences for human health.

Strongyloides stercoralis: Spatial distribution of a highly prevalent and ubiquitous soil-transmitted helminth in Cambodia.

BACKGROUND: Strongyloides stercoralis is a neglected soil-transmitted helminth that occurs worldwide, though it is particularly endemic in tropical and subtropical areas. It can cause long-lasting and potentially fatal infections due to its ability to replicate within its host. S. stercoralis causes gastrointestinal and dermatological morbidity. The objective of this study was to assess the S. stercoralis infection risk and, using geostatistical models, to predict its geographical distribution in Cambodia. METHODOLOGY / PRINCIPAL FINDINGS: A nation-wide, community-based parasitological survey was conducted among the Cambodian population, aged 6 years and older. S. stercoralis was diagnosed using a serological diagnostic test that detects IgG antibodies in urine. Data on demography, hygiene and knowledge about helminth infection were collected. S. stercoralis prevalence among 7,246 participants with a complete data record was 30.5%, ranging from 10.9% to 48.2% across provinces. The parasite was ubiquitous in Cambodia; only five south-eastern provinces had prevalence rates below 20%. Infection risk increased with age for both men and women, although girls under the age of 13 and women aged 50 years and over had lower odds of infection than their male counterparts. Open defecation was associated with higher odds of infection, while having some knowledge of the health problems caused by worms was a protective factor. Infection risk was positively associated with nighttime maximum temperature, minimum rainfall, and distance to water; it was negatively associated with land occupied by rice fields. CONCLUSIONS / SIGNIFICANCE: S. stercoralis infection is rampant in Cambodia. Control programs delivering ivermectin are needed to manage the parasite. However, the high cost of this drug in Cambodia currently precludes the implementation of control initiatives. Donations, subsidies or affordable generics are needed so that S. stercoralis, which infects almost a third of the Cambodian population, can be addressed through an adequate control program.

Elimination of Schistosomiasis Mekongi from Endemic Areas in Cambodia and the Lao People's Democratic Republic: Current Status and Plans.

The areas endemic for schistosomiasis in the Lao People's Democratic Republic and in Cambodia were first reported 50 and 60 years ago, respectively. However, the causative parasite Schistosoma mekongi was not recognized as a separate species until 1978. The infection is distributed along a limited part of the Mekong River, regulated by the focal distribution of the intermediate snail host Neotricula aperta. Although more sensitive diagnostics imply a higher figure, the current use of stool examinations suggests that only about 1500 people are presently infected. This well-characterized setting should offer an exemplary potential for the elimination of the disease from its endemic areas; yet, the local topography, reservoir animals, and a dearth of safe water sources make transmission control a challenge. Control activities based on mass drug administration resulted in strong advances, and prevalence was reduced to less than 5% according to stool microscopy. Even so, transmission continues unabated, and the true number of infected people could be as much as 10 times higher than reported. On-going control activities are discussed together with plans for the future.

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.

Strongyloides stercoralis and hookworm co-infection: spatial distribution and determinants in Preah Vihear Province, Cambodia.

BACKGROUND: Strongyloides stercoralis and hookworm are two soil-transmitted helminths (STH) that are highly prevalent in Cambodia. Strongyloides stercoralis causes long-lasting infections and significant morbidity but is largely neglected, while hookworm causes the highest public health burden among STH. The two parasites have the same infection route, i.e. skin penetration. The extent of co-distribution, which could result in potential high co-morbidities, is unknown in highly endemic settings like Cambodia. The aim of this study was to predict the spatial distribution of S. stercoralis-hookworm co-infection risk and to investigate determinants of co-infection in Preah Vihear Province, North Cambodia. METHODS: A cross-sectional survey was conducted in 2010 in 60 villages of Preah Vihear Province. Diagnosis was performed on two stool samples, using combined Baermann technique and Koga agar culture plate for S. stercoralis and Kato-Katz technique for hookworm. Bayesian multinomial geostatistical models were used to assess demographic, socioeconomic, and behavioural determinants of S. stercoralis-hookworm co-infection and to predict co-infection risk at non-surveyed locations. RESULTS: Of the 2576 participants included in the study, 48.6% and 49.0% were infected with S. stercoralis and hookworm, respectively; 43.8% of the cases were co-infections. Females, preschool aged children, adults aged 19-49 years, and participants who reported regularly defecating in toilets, systematically boiling drinking water and having been treated with anthelmintic drugs had lower odds of co-infection. While S. stercoralis infection risk did not appear to be spatially structured, hookworm mono-infection and co-infection exhibited spatial correlation at about 20 km. Co-infection risk was positively associated with longer walking distances to a health centre and exhibited a small clustering tendency. The association was only partly explained by climatic variables, suggesting a role for underlying factors, such as living conditions and remoteness. CONCLUSIONS: Both parasites were ubiquitous in the province, with co-infections accounting for almost half of all cases. The high prevalence of S. stercoralis calls for control measures. Despite several years of school-based de-worming programmes, hookworm infection levels remain high. Mebendazole efficacy, as well as coverage of and compliance to STH control programmes should be investigated.

How elimination of lymphatic filariasis as a public health problem in the Kingdom of Cambodia was achieved.

BACKGROUND: Endemicity of lymphatic filariasis (LF) in Cambodia was proven in 1956 when microfilariae were detected in mosquitos in the Kratié province. In 2001, an extensive study confirmed the presence of both Brugia malayi and Wuchereria bancrofti microfilariae. In 2003, the Ministry of Health established a national task force to develop policies and strategies for controlling and eliminating neglected tropical diseases (NTDs), with the goal of eliminating LF by 2015. This article summarizes the work accomplished to eliminate LF as a public health problem in Cambodia. METHODS: The National Program to Eliminate Lymphatic Filariasis made excellent progress in the goal towards elimination due to strong collaboration between ministries, intensive supervision by national staff, and advocacy for mobilization of internal and external resources. Mass drug administration (MDA) with diethylcarbamazine citrate and albendazole was conducted in six implementation units, achieving > 70% epidemiological coverage for five consecutive rounds, from 2005 to 2009. In 2006, in 14 provinces, healthcare workers developed a line list of lymphedema and hydrocele patients, many of whom were > 40 years old and had been affected by LF for many years. The national program also trained healthcare workers and provincial and district staff in morbidity management and disability prevention, and designated health centers to provide care for lymphedema and acute attack. Two reference hospitals were designated to administer hydrocele surgery. RESULTS: Effectiveness of MDA was proven with transmission assessment surveys. These found that less than 1% of school children had antigenemia in 2010, which fell to 0% in both 2013 and 2015. A separate survey in one province in 2015 using Brugia Rapid tests to test for LF antibody found one child positive among 1677 children. The list of chronic LF patients was most recently updated and confirmed in 2011-2012, with 32 lymphoedema patients and 17 hydrocele patients listed. All lymphedema patients had been trained on self-management and all hydrocele patients had been offered free surgery. CONCLUSIONS: Due to the success of the MDA and the development of health center capacity for patient care, along with benefits gained from socioeconomic improvements and other interventions against vector-borne diseases and NTDs, Cambodia was validated by the World Health Organization as achieving LF elimination as a public health problem in 2016.

A review of malaria diagnostic tools: microscopy and rapid diagnostic test (RDT).

The absolute necessity for rational therapy in the face of rampant drug resistance places increasing importance on the accuracy of malaria diagnosis. Giemsa microscopy and rapid diagnostic tests (RDTs) represent the two diagnostics most likely to have the largest impact on malaria control today. These two methods, each with characteristic strengths and limitations, together represent the best hope for accurate diagnosis as a key component of successful malaria control. This review addresses the quality issues with current malaria diagnostics and presents data from recent rapid diagnostic test trials. Reduction of malaria morbidity and drug resistance intensity plus the associated economic loss of these two factors require urgent scaling up of the quality of parasite-based diagnostic methods. An investment in anti-malarial drug development or malaria vaccine development should be accompanied by a parallel commitment to improve diagnostic tools and their availability to people living in malarious areas.

First report of human intestinal sarcocystosis in Cambodia.

Human intestinal sarcocystosis (HIS), caused by Sarcocystis species, is acquired by eating undercooked meat from sarcocyst-containing cattle (S. hominis, S. heydorni) and pigs (S. suihominis). We report on the detection of human intestinal Sarcocystis infections in a cross-sectional survey of Strongyloides stercoralis in early 2014, in Rovieng District, Preah Vihear Province, northern Cambodia. Among 1081 participants, 108 (10.0%) were diagnosed with Sarcocystis spp. oocysts in stool samples. Males had a significantly higher risk of infection than females (OR: 1.9, 95% CI: 1.3-2.9, p=0.001). None of the reported symptoms (abdominal discomfort, diarrhea, muscle pain and itching skin) occurring in the two weeks preceding the examinations were associated with a Sarcocystis infection. Many Sarcocystis cases were found among those who had participated in a wedding celebration and Chinese New Year festivities, where they had consumed raw or insufficiently cooked beef (83.3%) and pork (38.9%) based dishes. This report documents the first HIS cases in Cambodia.

Different but overlapping populations of Strongyloides stercoralis in dogs and humans-Dogs as a possible source for zoonotic strongyloidiasis.

Strongyloidiasis is a much-neglected soil born helminthiasis caused by the nematode Strongyloides stercoralis. Human derived S. stercoralis can be maintained in dogs in the laboratory and this parasite has been reported to also occur in dogs in the wild. Some authors have considered strongyloidiasis a zoonotic disease while others have argued that the two hosts carry host specialized populations of S. stercoralis and that dogs play a minor role, if any, as a reservoir for zoonotic S. stercoralis infections of humans. We isolated S. stercoralis from humans and their dogs in rural villages in northern Cambodia, a region with a high incidence of strongyloidiasis, and compared the worms derived from these two host species using nuclear and mitochondrial DNA sequence polymorphisms. We found that in dogs there exist two populations of S. stercoralis, which are clearly separated from each other genetically based on the nuclear 18S rDNA, the mitochondrial cox1 locus and whole genome sequence. One population, to which the majority of the worms belong, appears to be restricted to dogs. The other population is indistinguishable from the population of S. stercoralis isolated from humans. Consistent with earlier studies, we found multiple sequence variants of the hypervariable region I of the 18 S rDNA in S. stercoralis from humans. However, comparison of mitochondrial sequences and whole genome analysis suggest that these different 18S variants do not represent multiple genetically isolated subpopulations among the worms isolated from humans. We also investigated the mode of reproduction of the free-living generations of laboratory and wild isolates of S. stercoralis. Contrary to earlier literature on S. stercoralis but similar to other species of Strongyloides, we found clear evidence of sexual reproduction. Overall, our results show that dogs carry two populations, possibly different species of Strongyloides. One population appears to be dog specific but the other one is shared with humans. This argues for the strong potential of dogs as reservoirs for zoonotic transmission of S. stercoralis to humans and suggests that in order to reduce the exposure of humans to infective S. stercoralis larvae, dogs should be treated for the infection along with their owners.

Strongyloides stercoralis is associated with significant morbidity in rural Cambodia, including stunting in children.

BACKGROUND: Strongyloides stercoralis is a soil-transmitted nematode that can replicate within its host, leading to long-lasting and potentially fatal infections. It is ubiquitous and highly prevalent in Cambodia. The extent of morbidity associated with S. stercoralis infection is difficult to assess due to the broad spectrum of symptoms and, thus, remains uncertain. METHODOLOGY/PRINCIPAL FINDINGS: Clinical signs were compared among S. stercoralis infected vs. non-infected participants in a cross-sectional survey conducted in 2012 in eight villages of Northern Cambodia, and before and after treatment with a single oral dose of ivermectin (200µg/kg BW) among participants harboring S. stercoralis. Growth retardation among schoolchildren and adolescents was assessed using height-for-age and thinness using body mass index-for-age. S. stercoralis prevalence was 31.1% among 2,744 participants. Urticaria (55% vs. 47%, OR: 1.4, 95% CI: 1.1-1.6) and itching (52% vs. 48%, OR: 1.2, 95% CI: 1.0-1.4) were more frequently reported by infected participants. Gastrointestinal, dermatological, and respiratory symptoms were less prevalent in 103 mono-infected participants after treatment. Urticaria (66% vs. 11%, OR: 0.03, 95% CI: 0.01-0.1) and abdominal pain (81 vs. 27%, OR: 0.07, 95% CI: 0.02-0.2) mostly resolved by treatment. S. stercoralis infection was associated with stunting, with 2.5-fold higher odds in case of heavy infection. CONCLUSIONS/SIGNIFICANCE: The morbidity associated with S. stercoralis confirmed the importance of gastrointestinal and dermatological symptoms unrelated to parasite load, and long-term chronic effects when associated with malnutrition. The combination of high prevalence and morbidity calls for the integration of S. stercoralis into ongoing STH control measures in Cambodia.

Comparison of novel and standard diagnostic tools for the detection of Schistosoma mekongi infection in Lao People's Democratic Republic and Cambodia.

BACKGROUND: Given the restricted distribution of Schistosoma mekongi in one province in Lao People's Democratic Republic (Lao PDR) and two provinces in Cambodia, together with progress of the national control programmes aimed at reducing morbidity and infection prevalence, the elimination of schistosomiasis mekongi seems feasible. However, sensitive diagnostic tools will be required to determine whether elimination has been achieved. We compared several standard and novel diagnostic tools in S. mekongi-endemic areas. METHODS: The prevalence and infection intensity of S. mekongi were evaluated in 377 study participants from four villages in the endemic areas in Lao PDR and Cambodia using Kato-Katz stool examination, antibody detection based on an enzyme-linked immunosorbent assay (ELISA) and schistosome circulating antigen detection by lateral-flow tests. Two highly sensitive test systems for the detection of cathodic and anodic circulating antigens (CCA, CAA) in urine and serum were utilized. RESULTS: Stool microscopy revealed an overall prevalence of S. mekongi of 6.4% (one case in Cambodia and 23 cases in Lao PDR), while that of Opisthorchis viverrini, hookworm, Trichuris trichiura, Ascaris lumbricoides and Taenia spp. were 50.4%, 28.1%, 3.5%, 0.3% and 1.9%, respectively. In the urine samples, the tests for CCA and CAA detected S. mekongi infections in 21.0% and 38.7% of the study participants, respectively. In the serum samples, the CAA assay revealed a prevalence of 32.4%, while a combination of the CAA assay in serum and in urine revealed a prevalence of 43.2%. There was a difference between the two study locations with a higher prevalence reached in the samples from Lao PDR. CONCLUSIONS: The CCA, CAA and ELISA results showed substantially higher prevalence estimates for S. mekongi compared to Kato-Katz thick smears. Active schistosomiasis mekongi in Lao PDR and Cambodia might thus have been considerably underestimated previously. Hence, sustained control efforts are still needed to break transmission of S. mekongi. The pivotal role of highly sensitive diagnostic assays in areas targeting elimination cannot be overemphasised.

Reader technique as a source of variability in determining malaria parasite density by microscopy.

BACKGROUND: Accurate identification and quantification of malaria parasites are critical for measuring clinical trial outcomes. Positive and negative diagnosis is usually sufficient for the assessment of therapeutic outcome, but vaccine or prophylactic drug trials require measuring density of infection as a primary endpoint. Microscopy is the most established and widely-used technique for quantifying parasite densities in the blood. METHODS: Results obtained by 24-27 expert malaria microscopists, who had independently read 895 slides from 35 donors, were analysed to understand how reader technique contributes to discrepancy in measurements of parasite density over a wide range of densities. RESULTS: Among these 35 donations, standard deviations ranged from 30% to 250% of the mean parasite density and the percent discrepancy was inversely correlated with the mean parasite density. The number of white blood cells indexed and whether parasites were counted in the thick film or thin film were shown to significantly contribute to discrepancy amongst microscopists. CONCLUSION: Errors in microscopy measurements are not widely appreciated or addressed but have serious consequences for efficacy trials, including possibly abandoning promising vaccine candidates.