Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the approximately 90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict approximately 11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during approximately 350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.
We developed and evaluated real-time polymerase chain reaction (PCR) assays for detecting Wuchereria bancrofti DNA in human blood and in mosquitoes. An assay based on detection of the W. bancrofti "LDR" repeat DNA sequence was more sensitive than an assay for Wolbachia 16S rDNA. The LDR-based assay was sensitive for detecting microfilarial DNA on dried membrane filters or on filter paper. We also compared real-time PCR with conventional PCR (C-PCR) for detecting W. bancrofti DNA in mosquito samples collected in endemic areas in Egypt and Papua New Guinea. Although the two methods had comparable sensitivity for detecting filarial DNA in reference samples, real-time PCR was more sensitive than C-PCR in practice with field samples. Other advantages of real-time PCR include its high-throughput capacity and decreased risk of cross-contamination between test samples. We believe that real-time PCR has great potential as a tool for monitoring progress in large-scale filariasis elimination programs.
To identify Wuchereria bancrofti DNA sequences that could be used as the basis for a simple and rapid parasite detection assay, a genomic library of W. bancrofti was constructed and screened for highly repeated DNA. The repeat found with the highest copy number was 195 basepairs (bps) long, 77% AT, and 300 copies per haploid genome. This sequence was designated the Ssp I repeat because it has a unique recognition site for that restriction endonuclease in all or most of the repeat copies. The Ssp I repeat DNA family is dispersed, genus-specific, and exists in all of the different geographic isolates of W. bancrofti tested. Based on DNA sequence analysis of this repeat, we have developed an assay to detect very small quantities of W. bancrofti DNA using the polymerase chain reaction (PCR). With this PCR assay, the Ssp I repeat was detected in as little as 1 pg of W. bancrofti genomic DNA (about 1% of the DNA in one microfilaria) added to 100 pA of human blood. The PCR assay also amplified Ssp I repeat DNA from geographic isolates of W. bancrofti from around the world but not from other species of filariae or from human or mosquito DNA. Microfilaria-positive human blood samples collected in Mauke, Cook Islands were shown to be Ssp I PCR-positive, while microfilaria-negative samples were PCR-negative. The specificity and sensitivity of the Ssp I PCR assay indicates that this approach has significant potential for improved screening of large human populations for active W. bancrofti infection.
Background Helminth and protozoan infections affect more than 1 billion children globally. Improving water quality, sanitation, handwashing, and nutrition could be more sustainable control strategies for parasite infections than mass drug administration, while providing other quality of life benefits. Methods and findings We enrolled geographic clusters of pregnant women in rural western Kenya into a cluster-randomized controlled trial (ClinicalTrials.gov NCT01704105) that tested 6 interventions: water treatment, improved sanitation, handwashing with soap, combined water treatment, sanitation, and handwashing (WSH), improved nutrition, and combined WSH and nutrition (WSHN). We assessed intervention effects on parasite infections by measuring Ascaris lumbricoides , Trichuris trichiura , hookworm, and Giardia duodenalis among children born to the enrolled pregnant women (index children) and their older siblings. After 2 years of intervention exposure, we collected stool specimens from 9,077 total children aged 2 to 15 years in 622 clusters, including 2,346 children in an active control group (received household visits but no interventions), 1,117 in the water treatment arm, 1,160 in the sanitation arm, 1,141 in the handwashing arm, 1,064 in the WSH arm, 1,072 in the nutrition arm, and 1,177 in the WSHN arm. In the control group, 23% of children were infected with A . lumbricoides , 1% with T . trichiura , 2% with hookworm, and 39% with G . duodenalis . The analysis included 4,928 index children (median age in years: 2) and 4,149 older siblings (median age in years: 5); study households had an average of 5 people, <10% had electricity access, and >90% had dirt floors. Compared to the control group, Ascaris infection prevalence was lower in the water treatment arm (prevalence ratio [PR]: 0.82 [95% CI 0.67, 1.00], p = 0.056), the WSH arm (PR: 0.78 [95% CI 0.63, 0.96], p = 0.021), and the WSHN arm (PR: 0.78 [95% CI 0.64, 0.96], p = 0.017). We did not observe differences in Ascaris infection prevalence between the control group and the arms with the individual interventions sanitation (PR: 0.89 [95% CI 0.73, 1.08], p = 0.228), handwashing (PR: 0.89 [95% CI 0.73, 1.09], p = 0.277), or nutrition (PR: 86 [95% CI 0.71, 1.05], p = 0.148). Integrating nutrition with WSH did not provide additional benefit. Trichuris and hookworm were rarely detected, resulting in imprecise effect estimates. No intervention reduced Giardia . Reanalysis of stool samples by quantitative polymera...
The Global Programme to Eliminate Lymphatic Filariasis (GPELF) was launched in 2000. To understand why some national programs have been more successful than others, a panel of individuals with expertise in LF elimination efforts met to assess available data from programs in 8 countries. The goal was to identify: 1) the factors determining success for national LF elimination programs (defined as the rapid, sustained reduction in microfilaremia/antigenemia after repeated mass drug administration [MDA]); 2) the priorities for operational research to enhance LF elimination efforts. Of more than 40 factors identified, the most prominent were 1) initial level of LF endemicity; 2) effectiveness of vector mosquitoes; 3) MDA drug regimen; 4) population compliance. Research important for facilitating program success was identified as either biologic (i.e., [1] quantifying differences in vectorial capacity; [2] identifying seasonal variations affecting LF transmission) or programmatic (i.e., [1] identifying quantitative thresholds, especially the population compliance levels necessary for success, and the antigenemia or microfilaremia prevalence at which MDA programs can stop with minimal risk of resumption of transmission; [2] defining optimal drug distribution strategies and timing; [3] identifying those individuals who are "persistently non-compliant" during MDAs, the reasons for this non-compliance and approaches to overcoming it). While addressing these challenges is important, many key determinants of program success are already clearly understood; operationalizing these as soon as possible will greatly increase the potential for national program success.
Background With the expansion of soil transmitted helminth (STH) intervention efforts and the corresponding decline in infection prevalence, there is an increased need for sensitive and specific STH diagnostic assays. Previously, through next generation sequencing (NGS)-based identification and targeting of non-coding, high copy-number repetitive DNA sequences, we described the development of a panel of improved quantitative real-time PCR (qPCR)-based assays for the detection of Necator americanus , Ancylostoma duodenale , Ancylostoma ceylanicum , Trichuris trichiura , and Strongyloides stercoralis . However, due to the phenomenon of chromosome diminution, a similar assay based on high copy-number repetitive DNA was not developed for the detection of Ascaris lumbricoides . Recently, the publication of a reference-level germline genome sequence for A . lumbricoides has facilitated our development of an improved assay for this human pathogen of vast global importance. Methodology/Principal findings Repurposing raw DNA sequence reads from a previously published Illumina-generated, NGS-based A . lumbricoides germline genome sequencing project, we performed a cluster-based repeat analysis utilizing RepeatExplorer2 software. This analysis identified the most prevalent repetitive DNA element of the A . lumbricoides germline genome (AGR, Ascaris germline repeat), which was then used to develop an improved qPCR assay. During experimental validation, this assay demonstrated a fold increase in sensitivity of ~3,100, as determined by relative Cq values, when compared with an assay utilizing a previously published, frequently employed, ribosomal internal transcribed spacer (ITS) DNA target. A comparative analysis of 2,784 field-collected samples was then performed, successfully verifying this improved sensitivity. Conclusions/Significance Through analysis of the germline genome sequence of A . lumbricoides , a vastly improved qPCR assay has been developed. This assay, utilizing a high copy-number repeat target found in eggs and embryos (the AGR repeat), will improve prevalence estimates that are fundamental to the programmatic decision-making process, while simultaneously strengthening mathematical models used to examine STH infection rates. Furthermore, through the identification of an optimal target for PCR, future assay development efforts will also benefit, as the identity of the optimized repeat DNA target is likely to remain unchanged despite continued improvement in PCR-based diagnostic technologies.
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