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.

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.

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.

Ivermectin Treatment and Sanitation Effectively Reduce Strongyloides stercoralis Infection Risk in Rural Communities in Cambodia.

BACKGROUND: Strongyloides stercoralis is the only soil-transmitted helminth with the ability to replicate within its host, leading to long-lasting and potentially fatal infections. It is ubiquitous and its worldwide prevalence has recently been estimated to be at least half that of hookworm. Information on the epidemiology of S. stercoralis remains scarce and modalities for its large-scale control are yet to be determined. METHODOLOGY/PRINCIPAL FINDINGS: A community-based two-year cohort study was conducted among the general population in a rural province in North Cambodia. At each survey, participants infected with S. stercoralis were treated with a single oral dose of ivermectin (200µg/kg BW). Diagnosis was performed using a combination of the Baermann method and Koga agar plate culture on two stool samples. The cohort included participants from eight villages who were either positive or negative for S. stercoralis at baseline. Mixed logistic regression models were employed to assess risk factors for S. stercoralis infection at baseline and re-infection at follow-up. A total of 3,096 participants were examined at baseline, revealing a S. stercoralis prevalence of 33.1%. Of these participants, 1,269 were followed-up over two years. Re-infection and infection rates among positive and negative participants at baseline were 14.4% and 9.6% at the first and 11.0% and 11.5% at the second follow-up, respectively. At follow-up, all age groups were at similar risk of acquiring an infection, while infection risk significantly decreased with increasing village sanitation coverage. CONCLUSIONS/SIGNIFICANCE: Chemotherapy-based control of S. stercoralis is feasible and highly beneficial, particularly in combination with improved sanitation. The impact of community-based ivermectin treatment on S. stercoralis was high, with over 85% of villagers remaining negative one year after treatment. The integration of S. stercoralis into existing STH control programs should be considered without further delay.

Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment.

Western Cambodia is recognized as the epicenter of Plasmodium falciparum multidrug resistance. Recent reports of the efficacy of dihydroartemisinin (DHA)-piperaquine (PP), the latest of the artemisinin-based combination therapies (ACTs) recommended by the WHO, have prompted further investigations. The clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 plasma piperaquine concentrations were measured and day 0 isolates tested for in vitro susceptibilities to piperaquine and mefloquine, polymorphisms in the K13 gene, and the copy number of the Pfmdr-1 gene. A total of 425 patients were recruited in 2011 to 2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) than in eastern (2.5%) Cambodia (P <10(-3)). Day 7 plasma PP concentrations and median 50% inhibitory concentrations (IC50) of PP were independent of treatment outcomes, in contrast to median mefloquine IC50, which were found to be lower for isolates from patients with recrudescent infections (18.7 versus 39.7 nM; P = 0.005). The most significant risk factor associated with DHA-PP treatment failure was infection by parasites carrying the K13 mutant allele (odds ratio [OR], 17.5; 95% confidence interval [CI], 1 to 308; P = 0.04). Our data show evidence of P. falciparum resistance to PP in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies, such as the use of triple ACTs, are urgently needed and must be tested. (This study has been registered at the Australian New Zealand Clinical Trials Registry under registration no. ACTRN12614000344695.).

Malaria PCR detection in Cambodian low-transmission settings: dried blood spots versus venous blood samples.

In the context of malaria elimination, novel strategies for detecting very low malaria parasite densities in asymptomatic individuals are needed. One of the major limitations of the malaria parasite detection methods is the volume of blood samples being analyzed. The objective of the study was to compare the diagnostic accuracy of a malaria polymerase chain reaction assay, from dried blood spots (DBS, 5 µL) and different volumes of venous blood (50 µL, 200 µL, and 1 mL). The limit of detection of the polymerase chain reaction assay, using calibrated Plasmodium falciparum blood dilutions, showed that venous blood samples (50 µL, 200 µL, 1 mL) combined with Qiagen extraction methods gave a similar threshold of 100 parasites/mL, ∼100-fold lower than 5 µL DBS/Instagene method. On a set of 521 field samples, collected in two different transmission areas in northern Cambodia, no significant difference in the proportion of parasite carriers, regardless of the methods used was found. The 5 µL DBS method missed 27% of the samples detected by the 1 mL venous blood method, but most of the missed parasites carriers were infected by Plasmodium vivax (84%). The remaining missed P. falciparum parasite carriers (N = 3) were only detected in high-transmission areas.

Strongyloides stercoralis infection and re-infection in a cohort of children in Cambodia.

Information on Strongyloides stercoralis re-infection after ivermectin treatment is scarce in S. stercoralis endemic countries. In semi-rural Cambodia, we determined S. stercoralis infection and re-infection rates among schoolchildren, two years after ivermectin treatment (2×100 µg/kg PO, 24 h apart). The study was conducted among 484 children from four primary schools in semi-rural villages in Kandal province from 2009 to 2011, using Koga agar plate culture and the Baermann method on two stool samples per child. Complete data were available for 302 participants. We observed infections in 24.2% and 22.5% of the children at baseline and at follow-up, respectively. At baseline, 73 children were treated for S. stercoralis infection. At follow-up, one-third of those treated for S. stercoralis infection had been reinfected, while 19.6% of the 229 healthy children (at baseline) had been newly infected with S. stercoralis. Possession of shoes and defecation in toilet were negatively associated with S. stercoralis infection at follow-up. Infection and re-infection rates of S. stercoralis among schoolchildren are considerably high. However, 68.5% of infected children remained free of infection for at least two years. A large-scale cohort study is required to understand age-specific infection and re-infection dynamics in endemic countries.

Prevalence and risk factors of Strongyloides stercoralis in Takeo Province, Cambodia.

BACKGROUND: The threadworm Strongyloides stercoralis, the most neglected helminth, affects an estimated 30-100 million people worldwide. Information on S. stercoralis infection is scarce in tropical and sub-tropical resource poor countries, including Cambodia. We determined S. stercoralis infection prevalence and risk factors for infection in the general population in Southern Cambodia. METHODS: A cross-sectional study was carried out between January and April 2011 among 2,861 participants living in 60 villages of Takeo province, using Koga-agar plate culture, the Baermann technique and the Kato-Katz technique on a single stool sample. RESULTS: Eight intestinal helminth species were diagnosed. Hookworm (31.4%) and S. stercoralis (21.0%) occurred most frequently. Prevalence of S. stercoralis infection increased with age. In all age groups a higher prevalence was found among males than among females (OR: 1.7; 95% CI: 1.4 - 2.0; P < 0.001). Participants who had a latrine at home were significantly less frequently infected with S. stercoralis than those who did not (OR: 0.7; 95% CI: 0.4 - 0.8; P = 0.003). Muscle pain (OR: 1.3; 95% CI: 1.0 - 1.6; P = 0.028) and urticaria (OR: 1.4; 95% CI: 1.1 - 1.8; P = 0.001) were significantly associated with S. stercoralis infection. CONCLUSIONS: S. stercoralis is highly prevalent among the general Cambodian population and should no longer be neglected. Access to adequate diagnosis and treatment is urgently needed.

Development of a capillary electrophoresis-based heteroduplex tracking assay to measure in-host genetic diversity of initial and recurrent Plasmodium vivax infections in Cambodia.

A heteroduplex tracking assay used to genotype Plasmodium vivax merozoite surface protein 1 was adapted to a capillary electrophoresis format, obviating the need for radiolabeled probes and allowing its use in settings where malaria is endemic. This new assay achieved good allelic discrimination and detected high multiplicities of infection in 63 P. vivax infections in Cambodia. More than half of the recurrent parasitemias sampled displayed identical or highly related genotypes compared to the initial genotype, suggesting that they represented relapses.

Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in western, northern, and eastern Cambodia, 2011-2012: association with molecular markers.

In 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC50s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC50s (GMIC50s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P ≤ 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC50s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC50 and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance.

Reemergence of Chikungunya virus in Cambodia.

Chikungunya virus (CHIKV), probably Asian genotype, was first detected in Cambodia in 1961. Despite no evidence of acute or recent CHIKV infections since 2000, real-time reverse transcription PCR of serum collected in 2011 detected CHIKV, East Central South African genotype. Spatiotemporal patterns and phylogenetic clustering indicate that the virus probably originated in Thailand.