Discovering disease-causing pathogens in resource-scarce Southeast Asia using a global metagenomic pathogen monitoring system.

SignificanceMetagenomic pathogen sequencing offers an unbiased approach to characterizing febrile illness. In resource-scarce settings with high biodiversity, it is critical to identify disease-causing pathogens in order to understand burden and to prioritize efforts for control. Here, metagenomic next-generation sequencing (mNGS) characterization of the pathogen landscape in Cambodia revealed diverse vector-borne and zoonotic pathogens irrespective of age and gender as risk factors. Identification of key pathogens led to changes in national program surveillance. This study is a "real world" example of the use of mNGS surveillance of febrile individuals, executed in-country, to identify outbreaks of vector-borne, zoonotic, and other emerging pathogens in a resource-scarce setting.

High Prevalence of Echinostoma mekongi Infection in Schoolchildren and Adults, Kandal Province, Cambodia.

A high prevalence of Echinostoma mekongi infection (13.9%; 260/1,876) was found among schoolchildren and adults in Kandal Province, Cambodia, by fecal examination, worm expulsion, and molecular analysis of cox1 and nd1 genes. The source of infection was consumption of Pila sp. snails, a finding confirmed morphologically and molecularly.

National dengue surveillance, Cambodia 2002-2020.

Global dengue incidence has increased dramatically over the past few decades from approximately 500 000 reported cases in 2000 to over 5 million in 2019. This trend has been attributed to population growth in endemic areas, rapid unplanned urbanization, increasing global connectivity, and climate change expanding the geographic range of the Aedes spp. mosquito, among other factors. Reporting dengue surveillance data is key to understanding the scale of the problem, identifying important changes in the landscape of disease, and developing policies for clinical management, vector control and vaccine rollout. However, surveillance practices are not standardized, and data may be difficult to interpret particularly in low- and middle-income countries with fragmented health-care systems. The latest national dengue surveillance data for Cambodia was published in 2010. Since its publication, the country experienced marked changes in health policies, population demographics, climate and urbanization. How these changes affected dengue control remains unknown. In this article, we summarize two decades of policy changes, published literature, country statistics, and dengue case data collected by the Cambodia National Dengue Control Programme to: (i) identify important changes in the disease landscape; and (ii) derive lessons to inform future surveillance and disease control strategies. We report that while dengue case morbidity and mortality rates in Cambodia fell between 2002 and 2020, dengue incidence doubled and age at infection increased. Future national surveillance, disease prevention and treatment, and vector control policies will have to account for these changes to optimize disease control.

Le taux d'incidence de la dengue dans le monde a considérablement augmenté au cours des dernières décennies, passant d'environ 500 000 cas notifiés en 2000 à plus de 5 millions en 2019. Cette tendance est attribuée à la croissance démographique dans les zones d'endémie, à l'urbanisation rapide non planifiée, au développement de la connectivité à l'échelle internationale, ainsi qu'au changement climatique, qui agrandit le territoire géographique du moustique Aedes spp., entre autres. La communication des données de surveillance de la dengue est essentielle pour comprendre l'étendue du problème, identifier les principales variations de contexte entourant la maladie et mettre au point des politiques pour la prise en charge clinique, la lutte contre les vecteurs et le déploiement des vaccins. Les pratiques en matière de surveillance ne sont toutefois pas standardisées et les données peuvent être difficiles à interpréter, surtout dans les pays à revenu faible et intermédiaire où les systèmes de soins de santé sont fragmentés. Les données de surveillance les plus récentes concernant la dengue au Cambodge ont été publiées en 2010. Depuis leur publication, le pays a subi de profondes mutations au niveau des politiques de santé, de l'évolution démographique, du climat et de l'urbanisation. L'impact de ces mutations sur la lutte contre la dengue reste à établir. Dans le présent article, nous résumons deux décennies d'amendements politiques, de documentation, de statistiques nationales et d'informations collectées sur les cas par le programme cambodgien de lutte contre la dengue afin de: (i) définir les changements importants survenus dans le contexte entourant la maladie; mais aussi (ii) tirer des leçons en vue d'élaborer, à l'avenir, des stratégies de surveillance et de lutte contre la maladie. Nous signalons qu'en dépit d'une baisse des taux de morbidité et de mortalité liés aux cas de dengue entre 2002 et 2020 au Cambodge, son incidence a doublé et l'âge des patients au moment de l'infection a augmenté. Les futures politiques nationales de surveillance, de prévention et de traitement de la dengue, mais aussi de lutte contre ses vecteurs, devront tenir compte de ces changements de façon à mieux maîtriser la maladie.

La incidencia del dengue a nivel mundial ha aumentado considerablemente en las últimas décadas, desde aproximadamente 500 000 casos notificados en el año 2000 a más de 5 millones en 2019. Esta tendencia se ha atribuido al crecimiento de la población en zonas endémicas, a una urbanización rápida y no planificada, al aumento de la conectividad a nivel mundial y al cambio climático, que está permitiendo una distribución geográfica más amplia del mosquito Aedes spp., entre otros factores. Para comprender la magnitud del problema resulta clave la notificación de datos sobre vigilancia del dengue, la identificación de cambios importantes dentro del escenario de la enfermedad, la creación de políticas enfocadas a la gestión clínica, así como el control de vectores y la implantación de la vacuna. Sin embargo, las prácticas sobre vigilancia no están estandarizadas y es posible que sea difícil interpretar los datos, especialmente en países con ingresos medios y bajos, que cuentan con sistemas fragmentados de atención sanitaria. Los datos nacionales más recientes sobre vigilancia del dengue en Camboya se publicaron en 2010. Desde su publicación, el país experimentó cambios significativos en las políticas sanitarias, la demografía de la población, el clima y la urbanización. Aún no se sabe cómo afectaron dichos cambios al control del dengue. En el presente artículo, resumimos dos décadas de cambios políticos, de bibliografía publicada, de datos estadísticos a nivel nacional y datos sobre casos de dengue recopilados por el programa nacional de control de dengue en Camboya, con el fin de: (i) identificar cambios importantes en el escenario de la enfermedad; y (ii) extraer conclusiones para orientar futuras estrategias sobre vigilancia y control de la enfermedad. Informamos de que, aunque las tasas de morbilidad y mortalidad de los casos de dengue en Camboya descendieron entre 2002 y 2020, la incidencia del dengue se duplicó y la edad de infección aumentó. Las futuras políticas nacionales sobre vigilancia, prevención y tratamiento de la enfermedad y control de vectores deberán tener en cuenta estos cambios para optimizar el control de la enfermedad.

Development of Inapparent Dengue Associated With Increased Antibody Levels to Aedes aegypti Salivary Proteins: A Longitudinal Dengue Cohort in Cambodia.

BACKGROUND: We established the first prospective cohort to understand how infection with dengue virus is influenced by vector-specific determinants such as humoral immunity to Aedes aegypti salivary proteins. METHODS: Children aged 2-9 years were enrolled in the PAGODAS (Pediatric Assessment Group of Dengue and Aedes Saliva) cohort with informed consent by their guardians. Children were followed semi-annually for antibodies to dengue and to proteins in Ae. aegypti salivary gland homogenate using enzyme-linked immunosorbent assays and dengue-specific neutralization titers. Children presented with fever at any time for dengue testing. RESULTS: From 13 July to 30 August 2018, we enrolled 771 children. At baseline, 22% (173/770) had evidence of neutralizing antibodies to 1 or more dengue serotypes. By April 2020, 51 children had symptomatic dengue while 148 dengue-naive children had inapparent dengue defined by neutralization assays. In a multivariate model, individuals with higher antibodies to Ae. aegypti salivary proteins were 1.5 times more likely to have dengue infection (hazard ratio [HR], 1.47 [95% confidence interval {CI}, 1.05-2.06]; P = .02), particularly individuals with inapparent dengue (HR, 1.64 [95% CI, 1.12-2.41]; P = .01). CONCLUSIONS: High levels of seropositivity to Ae. aegypti salivary proteins are associated with future development of dengue infection, primarily inapparent, in dengue-naive Cambodian children. CLINICAL TRIALS REGISTRATION: NCT03534245.

Accelerating malaria elimination in Cambodia: an intensified approach for targeting at-risk populations.

BACKGROUND: Malaria in Cambodia has decreased by 90.8% between 2010 and 2020, driven by the commitment of the National Center for Parasitology, Entomology and Malaria (CNM) and the achievements of the roll-out of a village malaria worker programme. However, in the first seven months of 2018, CNM identified a 207% increase (11,969 to 36,778) in confirmed malaria cases compared to the same months in the previous year. To address this increase, CNM developed the "Intensification Plan" (IP), implemented between October 2018 and December 2020. METHODS: The structure of the IP was summarized, including the selection of sites, the interventions implemented in the selected health facility catchment areas (HFCAs) and the monitoring and evaluation process. Data on IP interventions were collected by CNM and civil society organisations. Data on malaria cases and tests from all HFCAs in Cambodia from January 2018 to December 2020 were sourced from the Cambodia Malaria Information System (MIS) and WHO Malaria Elimination Database. Malaria data from IP HFCAs and non-IP HFCAs was analysed and compared to present the changes in malaria testing and confirmed cases before and during implementation of the IP. RESULTS: Between October 2018 and December 2020, through the IP 16,902 forest packs and 293,090 long-lasting insecticide treated nets were distributed. In the 45 HFCAs included in the IP, 431,143 malaria tests were performed and 29,819 malaria cases were diagnosed, 5364 (18%) of which were Plasmodium falciparum/mixed cases. During the intervention period, over all HFCAs included in IP, P. falciparum/mixed cases declined from 1029 to 39, a 96.2% decrease, and from 25.4 P. falciparum/mixed cases per HFCA to 0.9. HFCAs not included in IP declined from 468 to 43 cases, a 90.8% decrease, showing that routine malaria activities in Cambodia were also playing an important contribution to malaria control. CONCLUSIONS: Over the course of IP implementation there was a substantial increase in malaria testing and both overall malaria cases and P. falciparum/mixed cases decreased month on month. The initiative yields lessons learned for Cambodia to reach the final stage of elimination as well as for other countries aiming to accelerate their malaria control programmes.

Echinostoma mekongi: Discovery of Its Metacercarial Stage in Snails, Filopaludina martensi cambodjensis, in Pursat Province, Cambodia.

Echinostoma mekongi was reported as a new species in 2020 based on specimens collected from humans in Kratie and Takeo Province, Cambodia. In the present study, its metacercarial stage has been discovered in Filopaludina martensi cambodjensis snails purchased from a local market nearby the Tonle Sap Lake, Pursat Province, Cambodia. The metacercariae were fed orally to an experimental hamster, and adult flukes were recovered at day 20 post-infection. They were morphologically examined using light and scanning electron microscopes and molecularly analyzed by sequencing of their mitochondrial cox1 and nad1 genes. A total of 115 metacercariae (1-8 per snail) were detected in 60 (60.0%) out of 100 Filopaludina snails examined. The metacercariae were round, 174 µm in average diameter (163-190 µm in range), having a thin cyst wall, a head collar armed with 37 collar spines, and characteristic excretory granules. The adult flukes were elongated, ventrally curved, 7.3 (6.4-8.2)×1.4 (1.1-1.7) mm in size, and equipped with 37 collar spines on the head collar (dorsal spines in 2 alternating rows), being consistent with E. mekongi. In phylogenetic analyses, the adult flukes showed 99.0-100% homology based on cox1 sequences and 98.9-99.7% homology based on nad1 sequences with E. mekongi. The results evidenced that F. martensi cambodjensis snails act as the second intermediate host of E. mekongi, and hamsters can be used as a suitable experimental definitive host. As local people favor to eat undercooked snails, these snails seem to be an important source of human infection with E. mekongi in Cambodia.

Occurrence of a Hybrid Between Taenia saginata and Taenia asiatica Tapeworms in Cambodia.

Human infection with Taenia asiatica or a hybrid between Taenia saginata and T. asiatica has not been reported in Cambodia. We detected for the first time a hybrid form between T. saginata and T. asiatica in Preah Vihear Province, Cambodia. An adult tapeworm specimen, i.e., 75 cm long strobila without scolex, was expelled from a 27-year-old man after praziquantel medication and purging. It was morphologically indistinguishable between T. saginata and T. asiatica. Several proglottids were molecularly analyzed to confirm the tapeworm species. The mitochondrial gene encoding cytochrome c oxidase subunit 1 (cox1) and nuclear genes encoding elongation factor-1α (ef1) and ezrin-radixin-moesin (ERM)-like protein (elp) were sequenced, and a single-allele analysis was performed to confirm the haploid genotype. The results revealed that our sample showed a discrepancy between the mitochondrial and 2 nuclear genes. It possessed homozygous sequences typical of T. saginata at cox1 and ef1 loci. However, it was heterozygous at the elp locus, with 1 allele in T. asiatica (elpA) and 1 in T. saginata (elpC), which indicates that it is a hybrid between T. saginata and T. asiatica. The present results confirmed the presence of a hybrid between T. saginata and T. asiatica in Cambodia and strongly suggest the existence of also 'pure' T. asiatica in Cambodia.

Morphological and Molecular Diagnosis of Necator americanus and Ancylostoma ceylanicum Recovered from Villagers in Northern Cambodia.

Human hookworm infections caused by adult Ancylostoma spp. and Necator americanus are one of the most important tropical diseases. We performed a survey of intestinal helminths using the Kato-Katz fecal examination technique targeting 1,156 villagers residing in 2 northern provinces (Preah Vihear and Stung Treng) of Cambodia in 2018. The results revealed a high overall egg positive rate of intestinal helminths (61.9%), and the egg positive rate of hookworms was 11.6%. Nine of the hookworm egg positive cases in Preah Vihear Province were treated with 5-10 mg/kg pyrantel pamoate followed by purging with magnesium salts, and a total of 65 adult hookworms were expelled in diarrheic stools. The adult hookworms were analyzed morphologically and molecularly to confirm the species. The morphologies of the buccal cavity and dorsal rays on the costa were observed with a light microscope, and the nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. The majority of the hookworm adults (90.7%) were N. americanus, whereas the remaining 9.3% were Ancylostoma ceylanicum, a rare hookworm species infecting humans. The results revealed a high prevalence of hookworm infections among people in a northern part of Cambodia, suggesting the necessity of a sustained survey combined with control measures against hookworm infections.

Molecular Diagnosis of Taenia saginata Tapeworms from Two Residents of Northern Cambodia.

Taenia saginata infection has seldom been reported in Cambodia. In this study, we performed a survey of intestinal parasites in 1,156 residents of Preah Vihear and Stung Treng Provinces in 2018. The results revealed that 26 (2.4%) cases were positive for Taenia spp. eggs. In order to obtain the strobilae of the tapeworms, 2 patients in Preah Vihear were treated with praziquantel and purged with magnesium salts. The proglottids expelled after the medication were morphologically and molecularly analyzed to determine the species. The main uterine lateral braches in gravid proglottids were >15 in number suggesting that they are either T. saginata or Taenia asiatica. The sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and 2 nuclear loci, elongation factor-1 alpha (ef1) and ezrin-radixin-moesin-like protein (elp), were identical to the sequences of T. saginata available in GenBank but distant from Taenia solium, T. asiatica, and T. saginata-T. asiatica hybrid. This is the first report of the presence of T. saginata in the northern part of Cambodia bordering Lao PDR based on a molecular confirmation.

Predicting Dengue Outbreaks in Cambodia.

In Cambodia, dengue outbreaks occur each rainy season (May-October) but vary in magnitude. Using national surveillance data, we designed a tool that can predict 90% of the variance in peak magnitude by April, when typically <10% of dengue cases have been reported. This prediction may help hospitals anticipate excess patients.

Asymptomatic Dengue Virus Infections, Cambodia, 2012-2013.

We investigated dengue virus (DENV) and asymptomatic DENV infections in rural villages of Kampong Cham Province, Cambodia, during 2012 and 2013. We conducted perifocal investigations in and around households for 149 DENV index cases identified through hospital and village surveillance. We tested participants 0.5-30 years of age by using nonstructural 1 rapid tests and confirmed DENV infections using quantitative reverse transcription PCR or nonstructural 1-capture ELISA. We used multivariable Poisson regressions to explore links between participants' DENV infection status and household characteristics. Of 7,960 study participants, 346 (4.4%) were infected with DENV, among whom 302 (87.3%) were <15 years of age and 225 (65.0%) were <9 years of age. We identified 26 (7.5%) participants with strictly asymptomatic DENV infection at diagnosis and during follow-up. We linked symptomatic DENV infection status to familial relationships with index cases. During the 2-year study, we saw fewer asymptomatic DENV infections than expected based on the literature.

Efficacy and Safety of Pyronaridine-Artesunate plus Single-Dose Primaquine for the Treatment of Malaria in Western Cambodia.

This single-arm trial (n = 104) in western Cambodia showed high efficacy for 3-day treatment with pyronaridine-artesunate plus single-dose primaquine in Plasmodium falciparum malaria. Day 42 PCR-adjusted adequate clinical and parasitological response (ACPR) was 98.3% (58/59) (95% confidence interval [CI], 90.9 to 100.0) in Trapeng Chau in Kampong Speu and 100% (41/41) (95% CI, 91.4 to 100) in Veal Veng in Pursat; 80.6% (83/103) of the patients had P. falciparum with drug resistance molecular markers. For Plasmodium vivax malaria, pyronaridine-artesunate day 28 ACPR was 98.3% (59/60) (95% CI, 91.1 to 100) and 100% (60/60) (95% CI, 94.0 to 100), respectively. (This study is registered in the Australian New Zealand Clinical Trials Registry [ANZCTR] under reference no. ACTRN12618001999224.).

Efficacy and Safety of Pyronaridine-Artesunate plus Single-Dose Primaquine for Treatment of Uncomplicated Plasmodium falciparum Malaria in Eastern Cambodia.

In Cambodia, multidrug-resistant Plasmodium falciparum undermines the treatment of uncomplicated malaria, and new therapeutic options are needed. Pyronaridine-artesunate has not previously been evaluated in eastern Cambodia. We conducted a single-arm, open-label, prospective study between July and December 2017 at the Koh Gnek (Mondulkiri) and Veun Sai (Rattanakiri) health centers in eastern Cambodia. Eligible patients were aged ≥7 years (females, ages 12 to 18 years, were excluded), weighing ≥20 kg, with microscopically confirmed P. falciparum monoinfection and fever. Oral pyronaridine-artesunate was administered once daily for 3 days, dosed according to body weight, plus a single dose of primaquine on day 0. Sixty patients were recruited to Koh Gnek, and 61 patients were recruited to Veun Sai. The primary outcomes, i.e., the day 42 PCR-adjusted adequate clinical and parasitological responses (ACPRs), were 98.3% (95% confidence interval [CI], 88.4 to 99.8) in Koh Gnek and 96.7% (95% CI, 87.3 to 99.2) in Veun Sai (Kaplan-Meier). In a per-protocol analysis, the proportions of patients with day 42 PCR-adjusted ACPRs were 98.3% (57/58; 95% CI, 90.8 to 100.0) at Koh Gnek and 96.7% (58/60; 95% CI, 88.5 to 99.6) at Veun Sai. The Kelch13 (C580Y) mutation was present in 70.0% (77/110) of isolates. The copy numbers were increased in 61.3% (73/119) of isolates for Pfpm2 and in 1.7% (2/119) for Pfmdr1 There was no relationship between outcome and the 50% inhibitory concentration of pyronaridine. Adverse events were consistent with malaria, and there were no serious adverse events. Pyronaridine-artesunate has high efficacy in eastern Cambodia and could be used to increase the diversity of antimalarial therapy in the region. (This study is registered in the Australian New Zealand Clinical Trials Registry [ANZCTR] under no. ACTRN12618001300268.).

A Controlled Trial of Mass Drug Administration to Interrupt Transmission of Multidrug-Resistant Falciparum Malaria in Cambodian Villages.

Background: The increase in multidrug-resistant Plasmodium falciparum in Southeast Asia suggests a need for acceleration of malaria elimination. We evaluated the effectiveness and safety of mass drug administration (MDA) to interrupt malaria transmission. Methods: Four malaria-endemic villages in western Cambodia were randomized to 3 rounds of MDA (a 3-day course of dihydroartemisinin with piperaquine-phosphate), administered either early in or at the end of the study period. Comprehensive malaria treatment records were collected during 2014-2017. Subclinical parasite prevalence was estimated by ultrasensitive quantitative polymerase chain reaction quarterly over 12 months. Results: MDA coverage with at least 1 complete round was 88% (1999/2268), ≥2 rounds 73% (1645/2268), and all 3 rounds 58% (1310/2268). Plasmodium falciparum incidence in intervention and control villages was similar over the 12 months prior to the study: 39 per 1000 person-years (PY) vs 45 per 1000 PY (P = .50). The primary outcome, P. falciparum incidence in the 12 months after MDA, was lower in intervention villages (1.5/1000 PY vs 37.1/1000 PY; incidence rate ratio, 24.5 [95% confidence interval], 3.4-177; P = .002). Following MDA in 2016, there were no clinical falciparum malaria cases over 12 months (0/2044 PY) in all 4 villages. Plasmodium vivax prevalence decreased markedly in intervention villages following MDA but returned to approximately half the baseline prevalence by 12 months. No severe adverse events were attributed to treatment. Conclusions: Mass drug administrations achieved high coverage, were safe, and associated with the absence of clinical P. falciparum cases for at least 1 year. Clinical Trials Registration: NCT01872702.

Asymptomatic humans transmit dengue virus to mosquitoes.

Three-quarters of the estimated 390 million dengue virus (DENV) infections each year are clinically inapparent. People with inapparent dengue virus infections are generally considered dead-end hosts for transmission because they do not reach sufficiently high viremia levels to infect mosquitoes. Here, we show that, despite their lower average level of viremia, asymptomatic people can be infectious to mosquitoes. Moreover, at a given level of viremia, DENV-infected people with no detectable symptoms or before the onset of symptoms are significantly more infectious to mosquitoes than people with symptomatic infections. Because DENV viremic people without clinical symptoms may be exposed to more mosquitoes through their undisrupted daily routines than sick people and represent the bulk of DENV infections, our data indicate that they have the potential to contribute significantly more to virus transmission to mosquitoes than previously recognized.

Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity.

Background: Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine. Methods: Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset. Results: Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity. Conclusions: Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.

Low Circulation of Zika Virus, Cambodia, 2007-2016.

We describe a retrospective study on circulation of Zika virus in Cambodia during 2007-2016 among patients with dengue-like symptoms and Aedes aegypti mosquitoes. Our findings suggest that Zika virus in Cambodia belongs to the Asia genotype, is endemic, has low prevalence, and has had low-level impact on public health.

Local constraints to access appropriate malaria treatment in the context of parasite resistance in Cambodia: a qualitative study.

BACKGROUND: Despite emerging drug resistance in Cambodia, artemisinin-based combination therapy (ACT) is still the most efficacious therapy. ACT is available free of charge in the Cambodian public sector and at a subsidized rate in the private sector. However, un- and mistreated cases in combination with population movements may lead to the further spread of resistant parasites, stressing the importance of understanding how the perceived aetiology of malaria and associated health-seeking behaviour may delay access to appropriate treatment. A qualitative study explored these factors after an epidemiological survey confirmed parasite resistance in Preah Vihear province. RESULTS: In Cambodian cosmology, illnesses can be inflicted by supernatural beings or originate from 'natural' causes because of disorder in the social, domestic or outdoor environment. Initial treatment options consist of cheap and accessible home-based care (manual therapy, herbs and biomedical medication) targeting single symptoms. If there is no steady recovery or if the condition quickly aggravates, care will be sought from 'village doctors', public health facilities, private pharmacies or, in case of suspicion of a supernatural cause, from a specialized indigenous healer. The choice of provider is mostly based on the family's financial situation, access to and trust in the provider, and the congruence between the suspected aetiology of the illness and the treatment offered by the provider. Different treatment options are often combined during the same illness episode through a serial process of trial and error guided by the observable improvements in the patient's condition. CONCLUSIONS: Cambodian perceptions of illness that focus on single symptoms and their perceived severity may lead to the identification of one or multiple illnesses at the same time, rarely suspecting malaria from the start and implying different patterns of health seeking behaviour and treatment choice. However, decisions to self-diagnose and treat at home are also pragmatic and must be understood in the context of poverty, a major barrier to seeking prompt and appropriate care for malaria in an area characterized by parasite resistance.

Measuring ex vivo drug susceptibility in Plasmodium vivax isolates from Cambodia.

BACKGROUND: While intensive Plasmodium falciparum multidrug resistance surveillance continues in Cambodia, relatively little is known about Plasmodium vivax drug resistance in Cambodia or elsewhere. To investigate P. vivax anti-malarial susceptibility in Cambodia, 76 fresh P. vivax isolates collected from Oddar Meanchey (northern Cambodia) in 2013-2015 were assessed for ex vivo drug susceptibility using the microscopy-based schizont maturation test (SMT) and a Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. P. vivax multidrug resistance gene 1 (pvmdr1) mutations, and copy number were analysed in a subset of isolates. RESULTS: Ex vivo testing was interpretable in 80% of isolates using the pLDH-ELISA, but only 25% with the SMT. Plasmodium vivax drug susceptibility by pLDH-ELISA was directly compared with 58 P. falciparum isolates collected from the same locations in 2013-4, tested by histidine-rich protein-2 ELISA. Median pLDH-ELISA IC50 of P. vivax isolates was significantly lower for dihydroartemisinin (3.4 vs 6.3 nM), artesunate (3.2 vs 5.7 nM), and chloroquine (22.1 vs 103.8 nM) than P. falciparum but higher for mefloquine (92 vs 66 nM). There were not significant differences for lumefantrine or doxycycline. Both P. vivax and P. falciparum had comparable median piperaquine IC50 (106.5 vs 123.8 nM), but some P. falciparum isolates were able to grow in much higher concentrations above the normal standard range used, attaining up to 100-fold greater IC50s than P. vivax. A high percentage of P. vivax isolates had pvmdr1 Y976F (78%) and F1076L (83%) mutations but none had pvmdr1 amplification. CONCLUSION: The findings of high P. vivax IC50 to mefloquine and piperaquine, but not chloroquine, suggest significant drug pressure from drugs used to treat multidrug resistant P. falciparum in Cambodia. Plasmodium vivax isolates are frequently exposed to mefloquine and piperaquine due to mixed infections and the long elimination half-life of these drugs. Difficulty distinguishing infection due to relapsing hypnozoites versus blood-stage recrudescence complicates clinical detection of P. vivax resistance, while well-validated molecular markers of chloroquine resistance remain elusive. The pLDH assay may be a useful adjunctive tool for monitoring for emerging drug resistance, though more thorough validation is needed. Given high grade clinical chloroquine resistance observed recently in neighbouring countries, low chloroquine IC50 values seen here should not be interpreted as susceptibility in the absence of clinical data. Incorporating pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax will help to further validate this field-expedient method.

Efficacy and Safety of Pyronaridine-Artesunate for Treatment of Uncomplicated Plasmodium falciparum Malaria in Western Cambodia.

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 ClinicalTrials.gov under registration number NCT02389439.).