The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration

Background The most commonly used diagnostic tool for soil-transmitted helminths (STH) is the Kato-Katz (KK) thick smear technique. However, numerous studies have suggested that the sensitivity of KK can be problematic, especially in low prevalence and low intensity settings. An emerging alternative is quantitative polymerase chain reaction (qPCR). Methods In this study, both KK and qPCR were conducted on stool samples from 648 participants in an STH epidemiology study conducted in the delta region of Myanmar in June 2016. Results Prevalence of any STH was 20.68% by KK and 45.06% by qPCR. Prevalence of each individual STH was also higher by qPCR than KK, the biggest difference was for hookworm with an approximately 4-fold increase between the two diagnostic techniques. Prevalence of Ancylostoma ceylanicum, a parasite predominately found in dogs, was 4.63%, indicating that there is the possibility of zoonotic transmission in the study setting. In individuals with moderate to high intensity infections there is evidence for a linear relationship between eggs per gram (EPG) of faeces, derived from KK, and DNA copy number, derived from qPCR which is particularly strong for Ascaris lumbricoides. Conclusions The use of qPCR in low prevalence settings is important to accurately assess the epidemiological situation and plan control strategies for the ‘end game’. However, more work is required to accurately assess STH intensity from qPCR results and to reduce the cost of qPCR so that is widely accessible in STH endemic countries.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data

Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility

Background The strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low- and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy. Results The sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a ‘pooling approach’ can yield a low frequency of ‘missed’ infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools. Conclusions Our results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in ‘pools-of-five’. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method.Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

Pooling as a Strategy for the Timely Diagnosis of Soil-Transmitted Helminths in Stool: Value and Reproducibility

Background: The strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low- and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy. Results: The sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a \u27pooling approach\u27 can yield a low frequency of \u27missed\u27 infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools. Conclusions: Our results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in \u27pools-of-five\u27. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method