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

Abstract

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.

Author summary

Soil-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.