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

Abstract

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.

Diagnostic Techniques for Soil-Transmitted Helminths – Recent Advances

“… 35 In addition, pooling of samples has been tested as a strategy to reduce cost and time and found to be cost-effective, time and labour saving without loss of sensitivity or accuracy. 71 …”

“…35 In addition, pooling of samples has been tested as a strategy to reduce cost and time and found to be costeffective, time and labour saving without loss of sensitivity or accuracy. 71 A newer molecular tool, Loop-mediated amplification assay (LAMP) is a single-step method amplifying target DNA sequences isothermally. [72][73][74][75][76] It only requires a heat block or incubator/water bath and is more tolerant of biological inhibition.…”

What does soil-transmitted helminth elimination look like? Results from a targeted molecular detection survey in Japan

“…One sample from a preschool child was excluded due to age-related exclusion criteria. A single sample per participant was obtained [50][51][52] and, in total, 682 samples were analyzed in this study. This sample size provides the design effect of 1.09.…”

Assessment of sample pooling for clinical SARS-CoV-2 testing

“…When used as an epidemiological surveillance tool, acceptable parameters and limits may be quite different to those when the same system is applied in a clinical testing setting (6,(13)(14)(15)(16). In the latter, the issue of detection sensitivity for every individual specimen becomes critical.…”