Optimus Cambodia
Eri Pfaff
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.
Copyright: 2013 Khieu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was financed by the UBS Optimus Foundation (www.ubs.com/global/en/wealth_management/optimusfoundation.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
In Cambodia, information on S. stercoralis infection is scarce. Earlier studies indicate prevalence rates of up to 20% in school children. However, they were assessed using a diagnostic approach with low sensitivity [16], [17], [35].
In semi-rural Cambodia, we determined infection rates and risk factors, compared two methods (KAP culture and Baermann technique) for diagnosing S. stercoralis infection using a multiple stool examination approach, and assessed efficacy of ivermectin treatment. We performed a cross-sectional study on S. stercoralis infection in four primary schools in semi-urban villages in Kandal province, examining three stool samples per child.
The study was approved by the National Ethics Committee for Health Research (NECHR; number 033, dated 20 March 2009), Ministry of Health, Cambodia and by the Ethics Committees of the Cantons of Basel-Stadt and Baselland (EKBB; number 21/09, dated 29 January 2009), Switzerland. All relevant authorities (village chiefs, school teachers and headmasters) were informed about the purpose and procedures of the study. Written informed consent was obtained from the parent or the legal guardian of the child or appropriate literate substitutes, prior to study onset.
The study was carried out in four semi-rural villages (Ang, Roka, Koh Khel and Damrey Chhlang villages), located in the Saang District (11.22N and 105.01E longitude), Kandal province, 45 kilometers south of Phnom Penh. Rice subsistence farming is the main economic activity in the villages. Pigs, poultry and cattle are the most common domestic animals. The villages were selected because hookworm infections were previously reported (used as a proxy for likely S. stercoralis transmission), and the villages were accessible by car to ensure rapid transfer of stool samples to the Parasitological Laboratory of the National Center for Parasitology, Entomology and Malaria Control (CNM) in Phnom Penh.
First, parents or legal guardian of the children were interviewed at home, using a pre-tested household questionnaire, to obtain the demographic data (age, sex, education level, profession), personal risk-perception (knowledge about helminth infections, health seeking behavior), living conditions (type of house, sanitation infrastructure, domestic animals) and personal hygiene.
Second, at school, a pre-tested child questionnaire was administered to the schoolchildren to obtain demographic data (age, sex, school grade), personal risk-perception (knowledge about helminth infection) and behavior data (wearing shoes, food consumption and personal hygiene) from the child. After the interview, each child received a pre-labeled plastic container (ID code, name, sex, age and date) for stool sample collection. Each morning, after collecting the filled container, another empty pre-labeled one was provided for the following day. This procedure was repeated until three stool samples were obtained per child or over a period of five days.
Within 90 minutes after collection, the stool samples arrived at the laboratory at ambient temperature. Upon arrival, experienced laboratory technicians from the Parasitological Laboratory of CNM immediately examined the specimens, as explained below.
First, KAP culture [27] was used for identifying S. stercoralis and possibly hookworm larvae. For this purpose, agar plates were prepared once per week and stored at 4C in humid conditions. A hazelnut-sized stool sample was placed in the middle of the plate and the closed Petri dish was incubated in a humid chamber for 48 hours at 28C. Afterwards, the plates were rinsed with sodium acetate-acetic acid-formalin (SAF) solution. The eluent was centrifuged and the sediment microscopically examined for the presence of S. stercoralis and hookworm larvae. The two species were distinguished by the characteristic morphology of the larvae (i.e., size of buccal cavity, presence of genital primordium (L1), presence of forked tail-end (L3)).
Finally, the Baermann technique [30] was performed. A walnut-sized stool sample was placed on gauze inserted into a glass funnel, and covered with water. The apparatus was exposed for two hours to artificial light directed from below. After centrifuging of the collected liquid, the sediment was examined under a microscope for presence of S. stercoralis larvae. If insufficient stool was submitted Baermann technique was dropped first.
For quality control, the technicians were specifically trained for three days on morphological criteria distinguishing hookworm and S. stercoralis larvae. During the whole study period, beside the permanent and rigorous supervision by a qualified microscopist from the Swiss Tropical and Public Health Institute (Swiss TPH), Basel, Switzerland, any unclear diagnosis was immediately discussed and solved with the qualified microscopist and study supervisor. Additionally, ten percent of the slides were re-examined by the same qualified technician from Swiss TPH. Slides yielding discrepant results were re-read by involved reader. A definitive infection(s) was found to be such by consensus.
Questionnaire and laboratory data were double-entered in EpiData version 3.1 (EpiData Association; Odense, Denmark) and validated. Statistical analyses were performed with STATA version 10.1 (StataCorp.; College Station, TX, USA). Only schoolchildren with a complete record (three stool samples examined with all three methods and complete questionnaire information) were retained for analyses.
Univariate logistic regression was used to associate infection status with demographic variables, hygienic status, and knowledge of the child's guardian and the recent medical history of the child. Population attributable fraction was calculated for significantly associated risks. P-values under or equal to 0.05 were considered as significant.
The observed and estimated prevalence of eight different intestinal parasite species found from different stool samples and diagnostic methods are detailed in Figure 2. In total, half of the schoolchildren (49.3%) were infected with hookworm, and one quarter of them (24.4%) were diagnosed with S. stercoralis infection. T. trichiura was found in 17.3%, while 7.9% harbored small trematode eggs. Both, A. lumbricoides and E. vermicularis, were observed in 2.0% of participants, whereas H. nana and Taenia sp. were found in 3.7% and 0.4% of children, respectively.
The effect of the sampling effort for multiple stool samples examination on infection prevalence and on the sensitivity of the different diagnostic methods are presented in Table 2 for S. stercoralis and in Table 3 for hookworms. The number of S. stercoralis and hookworm infections detected by either method increased considerably by analyzing three stool samples. For S. stercoralis the prevalence rose from 15.9% to 21.6% and from 12.0% to 18.3%, as detected by KAP culture and Baermann method, respectively. The combined results of both methods showed an increase from 18.6% to 24.4% when three stool samples were examined instead of one.
The cure rates of treatments on the other intestinal helminth infections are presented in Table 4. Overall, 97 (82.9%) cases were co-infected with hookworm. Three weeks after mebendazole treatment (500 mg single dose), 65 (55.5%) participants were found hookworm positive in the follow-up examination. The cure rate of mebendazole was 33.0%.
Of 112 S. stercoralis cases, 108 (96.4%) were diagnosed in schoolchildren under 16 years, 42.0% were girls. As shown in Table 5, gender and age were not statistically different between infected and non-infected children. However, reported personal hygiene characteristics were significantly associated with S. stercoralis infection. The children who reported having shoes, and defecating in a toilet were half as likely to be infected with S. stercoralis than those who did not report shoes and latrine use (p
Population attributable risk analysis showed that the number of strongyloidiasis cases would be reduced by 72% and 40% if all children had a toilet at home and used it for defecation, respectively. Bivariate analysis of population attributable risk showed that when the children in this population had toilet at home and defecated in it, strongyloidiasis cases could be reduced by 74%.
An in-depth parasitological investigation of S. stercoralis in Cambodia, including the performance of different diagnostic methods and the efficacy of treatment has not been carried out before. Our study confirms the validity of the KAP culture and Baermann method for detecting S. stercoralis larvae with high sensitivity and the high efficacy of oral ivermectin treatment (100 g/kg/day for two days) in curing S. stercoralis infection. A cumulative prevalence of 24.4% was found among 458 schoolchildren in four semi-rural villages in Kandal province, south of Phnom Penh, by applying two methods on three stool samples collected over five days. This prevalence is substantially higher than those stated in three previous reports from Cambodia [16], [17], , and in reports from neighboring Laos and Thailand [14], [19]. This is most likely due to the fact that we used a much more rigorous diagnostic approach (number of stool samples, multiple diagnostic methods) than did the other studies, where it was common to examine a single stool sample with a single method. Nevertheless, a prevalence (20.2%) similar to the one found in our study was observed in 2006, among school-aged children living in villages bordering Tonl Sap Lake, northern Cambodia [35] and using only the Baermann technique to analyze a single stool sample. This observation indicates that in villages close to Tonl Sap Lake the true prevalence was actually considerably higher. It further underlines the reason why S. stercoralis is so often underdiagnosed.
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