Biology subjects

Pilotte, N.

Publications and source records attributed to Pilotte, N..

5 recordsLinked to original sources

bioRxiv2026

Determination of diagnostic cycle threshold (Ct) cut-offs for qPCR-based prevalence surveys of soil-transmitted helminth infections

WHO guidelines for control of soil-transmitted helminths (STH) rely on coproscopic methods to assess population prevalence. In low-prevalence and light-intensity STH settings, quantitative PCR (qPCR) has higher sensitivity and specificity for detection. For qPCR to accurately identify transmissible infections of public health significance, it is essential to interpret the qPCR cycle threshold (Ct) results. As part of the DeWorm3 community-based cluster randomized trial on interrupting STH transmission, we conducted population-based surveys using high-throughput qPCR and aimed to establish appropriate Ct cut-offs to detect transmissible infections. Experimental approaches including egg and genome-equivalent spiking experiments were hindered by inefficient fecal DNA extraction despite optimization efforts. The Ct results for 29,980 samples (pre-intervention, cross-sectional surveys) revealed a bimodal distribution for two of the four species tested, N. americanus and A. lumbricoides. The first peak was assumed to represent transmissible infections, and the second peak to represent indeterminate or non-transmissible infections. Using a finite mixture model, we defined true qPCR positivity as any Ct result with a [≥]5% chance of belonging to the first peak. This approach yielded Ct cut-offs of 34.4398 for N. americanus and 28.57587 for A. lumbricoides. For hookworms, sensitivity of qPCR was 96.7%, compared to 73.2% for Kato-Katz and moderate- to heavy-intensity infections (median Ct: 19.1, interquartile range [IQR]: 17.9-19.8) were differentiated from light-intensity infections and Kato-Katz negative samples (25.3, IQR: 22.5-27.9). Our findings demonstrate the feasibility and utility of evidence-based Ct cut-offs to identify transmissible STH infections in large scale surveys, and to categorize infection intensity as programmatically relevant. Author SummaryQuantitative PCR (qPCR) has been used for the detection of soil-transmitted helminths but with limited emphasis on determining cycle threshold (Ct) cut-offs to accurately identify transmissible infections which are of public health significance. When qPCR is used to assess interventions, or, in the future, to potentially make programmatic decisions, it will be crucial to validate positivity criteria to avoid underestimation (false negatives) or overestimation (false positives) of results. As part of the DeWorm3 trial, a community-based cluster randomized trial on interrupting transmission of STH, we developed and applied a validated STH qPCR to test 29,980 samples collected pre-intervention and explored experimental approaches to establish assay-specific Ct cut-offs. The Ct values from these samples showed a bimodal distribution for N. americanus and A. lumbricoides, suggesting the presence of two distinct groups. For this reason, a statistical approach with a finite mixture model was employed to determine Ct cut-offs that differentiated epidemiologically relevant, transmissible, egg-positive STH infections from those that are likely to represent detection of non-transmissible DNA or indeterminate results. While our data were applied in three different country settings, India, Benin and Malawi, it is important to note that no single Ct cut-off may be applicable across all epidemiological scenarios. Our findings demonstrate the feasibility of developing evidence-based Ct cut-offs with high sensitivity to accurately detect transmissible STH infections in large scale surveys.

microbiology↗

bioRxiv2024

Environmental surveillance of soil-transmitted helminths and other enteric pathogens in settings without networked wastewater infrastructure

Soil-transmitted helminths (STH) are one of the most prevalent enteric infections world-wide. To control STH-related morbidity, the World Health Organization recommends targeted deworming and improvements in water, sanitation and hygiene. Current surveillance strategies for STH focus on identifying and quantifying eggs in stool samples via microscopy, which exhibits poor specificity and sensitivity, especially in settings with low-intensity infections. Wastewater-based epidemiology is a surveillance tool used to monitor pathogen circulation and could replace stool- based approaches for STH detection. However, sampling strategies for settings lacking networked sanitation outside large urban settlements are not well developed. Here, we report evaluation of sampling strategies for soil and wastewater STH surveillance in rural and peri-urban settings without networked sanitation. We used multi-parallel qPCR assays to detect STH DNA in soil collected from high foot-traffic locations and three types of wastewater samples (passive Moore swabs, grab samples, and sediment from drainage ditches) in Come, Benin and Timiri and Jawadhu Hills in Tamil Nadu, India. We detected STH in soil (India = 32/95, Benin = 39/121) and wastewater (India = 24/60, Benin = 8/64) with a detection frequency across all sample types of 36% in India and 25% in Benin. We evaluated which sample locations and types allowed for more sensitive detection of STH DNA and determined that STH prevalence varied by sample site but did not vary significantly within a given sample site location (e.g., samples collected from multiple locations within one market). Further, we determined that wastewater sediment samples outperformed grab and Moore swab sample types for STH detection. Finally, we expanded our methods to include detection of other enteric pathogens using multiplexed qPCR for wastewater samples. Our results establish sampling strategies for environmental and wastewater surveillance of a wide range of enteric pathogens in settings without networked sanitation.

microbiology↗

bioRxiv2019

Field evaluation of DNA amplification of human filarial and malaria parasites using mosquito excreta/feces

BackgroundWe recently developed a superhydrophobic cone-based method for the collection of mosquito excreta/feces (E/F) for the molecular xenomonitoring of vector-borne parasites showing higher throughput compared to the traditional approach. To test its field applicability, we used this platform to detect the presence of filarial and malaria parasites in two villages of Ghana and compared results to those for detection in mosquito carcasses and human blood. Methodology and principal findingsWe compared the molecular detection of three parasites (Wuchereria bancrofti, Plasmodium falciparum and Mansonella perstans) in mosquito E/F, mosquito carcasses and human blood collected from the same households in two villages in the Savannah Region of the country. We successfully detected the parasite DNA in mosquito E/F from indoor resting mosquitoes, including W. bancrofti which had a very low community prevalence (2.5-3.8%). Detection in the E/F samples was concordant with detection in insect whole carcasses and people, and laboratory tests showed that the risk of mosquito carcass cross-contamination with positive excreta when insects are held together in the device is minimal. ConclusionsOur approach to collect and test mosquito E/F successfully detected a variety of parasites at varying prevalence in the human population under field conditions, including a pathogen (M. perstans) which is not transmitted by mosquitoes. The method shows promise for further development and applicability for the early detection and surveillance of a variety of pathogens carried in human blood. Author summaryMolecular xenomonitoring of parasites or viruses using mosquitoes as "flying syringes" is a promising and non-invasive tool for early detection and surveillance of various pathogens, particularly in low prevalence settings, but there is a need for cost-effective and higher throughput alternatives. We recently developed a novel approach based on the collection of mosquito excreta/feces (E/F) using a superhydrophobic cone that directs the sample into a tube at the bottom of the collection cup. We tested this methods ability to detect the presence of lymphatic filariasis, malaria, and mansonellosis from households in two endemic rural communities of Ghana and compared it to the molecular detection from blood and mosquito carcass samples from corresponding households. The detection of parasite DNA in mosquito E/F was successful for all three pathogens, and it showed good concordance with the detection in the insects whole carcasses. Given the successful detection of Mansonella perstans, which is not transmitted by mosquitoes, our tool shows promise for use as an alternative method for the early and non-invasive detection and surveillance of various pathogens in human blood, including those not strictly mosquito-borne, in endemic settings.

microbiology↗

bioRxiv2019

Comparison of multi-parallel qPCR and Kato-Katz for detection of soil-transmitted helminth infection among children in rural Bangladesh

An active area of research investigates whether soil-transmitted helminths (STH) can be locally eliminated in endemic settings. In such settings, highly sensitive diagnostics are needed to detect STH infection. We compared double-slide Kato-Katz, the most commonly used copromicroscopic detection method, to multi-parallel quantitative polymerase chain reaction (qPCR) in 2,800 stool samples from children 2-12 years in rural Bangladesh. We estimated the sensitivity and specificity of each diagnostic using Bayesian latent class analysis. Compared to Kato-Katz, STH prevalence using qPCR was almost 3-fold higher for hookworm species and nearly 2-fold higher for Trichuris trichiura. Ascaris lumbricoides prevalence was lower using qPCR, and 26% of samples classified as A. lumbricoides positive by Kato-Katz were negative by qPCR. Amplicon sequencing of the 18S rDNA from 10 samples confirmed that A. lumbricoides was absent in samples classified as positive by Kato-Katz and negative by qPCR. The sensitivity of Kato-Katz was 49% for A. lumbricoides, 32% for hookworm, and 52% for T. trichiura; the sensitivity of qPCR was 79% for A. lumbricoides, 93% for hookworm, and 90% for T. trichiura. Specificity was [≥] 97% for both tests for all STH except for Kato-Katz for A. lumbricoides (specificity = 68%). There were moderate negative, monotonic correlations between qPCR cycle quantification values and eggs per gram quantified by Kato-Katz. While it is widely assumed that Kato-Katz has few false positives, our results indicate otherwise. Our findings suggest that qPCR is more appropriate than Kato-Katz in low intensity infection settings because of its higher sensitivity and specificity.\n\nAuthor summarySoil-transmitted helminth infections (STH) (e.g., Ascaris, hookworm, Trichuris) contribute to a large burden of disease among children in low- and middle-income countries. There is increasing interest in implementing large-scale deworming programs to eliminate STH in certain settings. Efforts to monitor whether local elimination has occurred require sensitive diagnostic tests that will not miss positive cases. Kato-Katz, a microscopy-based diagnostic test, has commonly been used to identify STH eggs in stool, but in settings where infection intensity is low, this method frequently misses positive samples because it requires visual identification of small numbers of eggs, and eggs may degrade prior to visualization. Quantitative polymerase chain reaction (qPCR) is a molecular diagnostic method that may miss fewer infections because it identifies STH DNA in stool, which can be detected in very small quantities and is less likely to degrade. This study compared the performance of Kato-Katz and qPCR using 2,800 stool samples from children aged 2-12 years in rural Bangladesh. qPCR detected substantially more hookworm and Trichuris infections than Kato-Katz. 26% of samples were classified as Ascaris positive by Kato-Katz and negative by qPCR. We conclude that qPCR is a more appropriate diagnostic method than Kato-Katz in low infection intensity settings.

epidemiology↗

bioRxiv2018

Integrating water, sanitation, handwashing, and nutrition interventions to reduce child soil-transmitted helminth and Giardia infections: a cluster-randomized controlled trial in rural Kenya

Background.Helminth and protozoan infections affect >1 billion children globally. Improved water, sanitation, handwashing, and nutrition could be more sustainable control strategies for parasite infections than mass drug administration (MDA), while providing other quality of life benefits.\n\nMethods and Findings.We enrolled geographic clusters of pregnant women into a cluster-randomized controlled trial that tested six interventions: disinfecting drinking water(W), improved sanitation(S), handwashing with soap(H), combined WSH, improved nutrition(N), and combined WSHN. We assessed intervention effects on parasite infections by measuring Ascaris lumbricoides, Trichuris trichiura, hookworm, and Giardia duodenalis among individual children born to enrolled mothers and their older siblings (ClinicalTrials.gov NCT01704105). We collected stool specimens from 9077 total children in 622 clusters, including 2346 children in control, 1117 in water, 1160 in sanitation, 1141 in handwashing, 1064 in WSH, 1072 in nutrition, and 1177 in WSHN. In the control group, 23% of children were infected with Ascaris lumbricoides, 1% with Trichuris trichuria, 2% with hookworm and 39% with Giardia duodenalis. After two years of intervention exposure, Ascaris infection prevalence was 18% lower in the water treatment arm (95% confidence interval (CI) 0%, 33%), 22% lower in the WSH arm (CI 4%, 37%), and 22% lower in the WSHN arm (CI 4%, 36%) compared to control. Individual sanitation, handwashing, and nutrition did not significantly reduce Ascaris infection on their own, and 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 polymerase chain reaction (qPCR) confirmed the reductions in Ascaris infections measured by microscopy in the WSH and WSHN groups. Lab technicians and data analysts were blinded to treatment assignment, but participants and sample collectors were not blinded. The trial was funded by the Bill & Melinda Gates Foundation and USAID.\n\nConclusions.Our results suggest integration of improved water quality, sanitation, and handwashing could contribute to sustainable control strategies for Ascaris infections, particularly in similar settings with recent or ongoing deworming programs. Water treatment alone was similarly effective to integrated WSH, providing new evidence that drinking water should be given increased attention as a transmission pathway for Ascaris.

clinical trials↗