Comparison of individual and pooled stool samples for the assessment of soil-transmitted helminth infection intensity and drug efficacy

Background: In veterinary parasitology samples are often pooled for a rapid assessment of infection intensity and drug efficacy. Currently, studies evaluating this strategy in large-scale drug administration programs to control human soil-transmitted helminths (STHs; Ascaris lumbricoides,Trichuris trichiura, and hookworm), are absent. Therefore, we developed and evaluated a pooling strategy to assess intensity of STH infections and drug efficacy. Methods/Principal Findings: Stool samples from 840 children attending 14 primary schools in Jimma, Ethiopia were pooled (pool sizes of 10, 20, and 60) to evaluate the infection intensity of STHs. In addition, the efficacy of a single dose of mebendazole (500 mg) in terms of fecal egg count reduction (FECR; synonym of egg reduction rate) was evaluated in 600 children from two of these schools. Individual and pooled samples were examined with the McMaster egg counting method. For each of the three STHs, we found a significant positive correlation between mean fecal egg counts (FECs) of individual stool samples and FEC of pooled stool samples, ranging from 0.62 to 0.98. Only for A. lumbricoides was any significant difference in mean FEC of the individual and pooled samples found. For this STH species, pools of 60 samples resulted in significantly higher FECs. FECR for the different number of samples pooled was comparable in all pool sizes,except for hookworm. For this parasite, pools of 10 and 60 samples provided significantly higher FECR results. Conclusion/Significance: This study highlights that pooling stool samples holds promise as a strategy for rapidly assessing infection intensity and efficacy of administered drugs in programs to control human STHs. However, further research is required to determine when and how pooling of stool samples can be cost-effectively applied along a control program, and to verify whether this approach is also applicable to other NTDs

Pooling stool samples : a cost-effective strategy to assess infection intensity of soil-transmitted helminths and to monitor drug efficacy?

Up to date cost-effective strategies to guide health care decision makers on how to optimize control of soil-transmitted helminths (STH) and on how to detect the development of anthelmintic resistance are scarce. In the present study, we developed and evaluated a novel pooling strategy to assess intensity of STH infections and to monitor drug efficacy

Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool

Background : A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. Methodology and principal findings : In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNA later and were stored at 4 degrees C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. Conclusions : The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs

Evaluation of copromicroscopy and serology to measure the exposure to Ascaris infections across age groups and to assess the impact of 3 years of biannual mass drug administration in Jimma Town, Ethiopia

Background: The scientific community has recently summarized the desired characteristics for diagnostic tools across the different phases of a soil-transmitted helminth (STH) mass drug administration (MDA) program. Although serology meets some of the desired criteria, there is a scarcity of data on baseline serological profiles in human populations, both prior to and during MDA programs. Methods: In this study, we compared the copromicroscopic and the serological infection profiles in 600 school-aged children (SAC) and 600 adults at the advent of the MDA program in Jimma Town, Ethiopia. The serological profiles were examined by two ELISAs that measure IgG4 responses to the Ascaris suum haemoglobin antigen (AsHb) and a somatic extract of lung stage larvae (AsLungL3). Three years into the MDA program, we sampled another group of 600 SAC from the same schools to assess the reduction in prevalence and intensity of Ascaris infections measured by copromicroscopy and serology. Principal findings: Prior to the start of MDA, copromicroscopy revealed an Ascaris prevalence of 31.0% and a mean fecal egg count of 2,919 eggs per gram (EPG) in SAC. Following three years of biannual treatment, the prevalence reduced to 13.2% (57.8% reduction) and the mean fecal egg count to 1,513 EPG (48.1% reduction). This reduction was also reflected in the serological results. The seroprevalence reduced with 40.9% and 27.4% and the mean optical density ratio reduced with 44.2% and 38.2% as measured by the AsHb or AsLungL3 ELISA respectively. We also showed that, despite a decreasing coproprevalence, seroprevalence to Ascaris increased with age. Conclusions: This study is the first to provide IgG4 response profiles of an endemic population to two different A. suum antigens. The results suggest that exposure to the infectious stages of Ascaris reaches beyond SAC alone. Furthermore, it highlights the possible use of serological assays to monitor changes in STH exposure during MDA programs

Diagnostic sensitivity of direct wet mount microscopy for soil-transmitted helminth infections in Jimma Town, Ethiopia

Introduction: Soil-transmitted helminthiasis (STH) remains a major public health problem in school children in Ethiopia. Although direct wet mount microscopy (DWMM) is the means to diagnose parasitic diseases in health care facilities in Ethiopia, it remains unclear what its diagnostic performance is for STH. Methodology: A cross-sectional study was performed in Jimma Town (Ethiopia) and included 600 children from 10 primary schools. The diagnostic sensitivity of DWMM was compared to a composite reference standard (CRS) consisting of Kato-Katz, McMaster and Mini-FLOTAC. We also explored the impact of intensity of infection (the highest faecal egg counts (FECs; expressed as eggs per gram of stool (EPG)) across the CRS) on the diagnostic sensitivity of DWMM. Results: Based on the CRS, there were 210 Ascaris (35.0%), 312 Trichuris (52.0%) and 102 hookworm cases (17.0%). The median intensity of infections equalled 2,057 EPG for Ascaris, 200 EPG for Trichuris and 110 EPG for hookworms. The sensitivity of DWMM was 73.8% for Ascaris, but was around 17% for both Trichuris and hookworms. The sensitivity significantly increased with intensity of STH. For Ascaris, the odds for detecting an infection intensity of 1,000 EPG was 6.2 times higher than detecting an infection of 100 EPG. For Trichuris and hookworms, these odds ratios were 7.1 and 14. Conclusions: The diagnostic sensitivity of DWMM is low for STH, but it is able to detect those subjects that are in the highest need of treatment, and hence contributes to the global goal to eliminate STH as a public health problem

Quantitative PCR in soil-transmitted helminth control programs : four potential applications

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Comprehensive evaluation of stool-based diagnostic methods and benzimidazole resistance markers to assess drug efficacy and detect the emergence of anthelmintic resistance : a Starworms study protocol

Background : To work towards reaching the WHO goal of eliminating soil-transmitted helminth (STH) infections as a public health problem, the total number of children receiving anthelmintic drugs has strongly increased over the past few years. However, as drug pressure levels rise, the development of anthelmintic drug resistance (AR) is more and more likely to appear. Currently, any global surveillance system to monitor drug efficacy and the emergence of possible AR is lacking. Consequently, it remains unclear to what extent the efficacy of drugs may have dropped and whether AR is already present. The overall aim of this study is to recommend the best diagnostic methods to monitor drug efficacy and molecular markers to assess the emergence of AR in STH control programs. Methods : A series of drug efficacy trials will be performed in four STH endemic countries with varying drug pressure (Ethiopia and Brazil: low drug pressure, Lao PDR: moderate drug pressure and Tanzania: high drug pressure). These trials are designed to assess the efficacy of a single oral dose of 400 mg albendazole (ALB) against STH infections in school-aged children (SAC) by microscopic (duplicate Kato-Katz thick smear, Mini-FLOTAC and FECPAK(G2)) and molecular stool-based diagnostic methods (quantitative PCR (qPCR)). Data will be collected on the cost of the materials used, as well as the time required to prepare and examine stool samples for the different diagnostic methods. Following qPCR, DNA samples will also be submitted for pyrosequencing to assess the presence and prevalence of single nucleotide polymorphisms (SNPs) in the beta-tubulin gene. These SNPs are known to be linked to AR in animal STHs. Discussion : The results obtained by these trials will provide robust evidence regarding the cost-efficiency and diagnostic performance of the different stool-based diagnostic methods for the assessment of drug efficacy in control programs. The assessment of associations between the frequency of SNPs in the beta-tubulin gene and the history of drug pressure and drug efficacy will allow the validation of these SNPs as a marker for AR in human STHs