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

Abstract

There is growing interest in local elimination of soil-transmitted helminth (STH) infection 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 2,799 stool samples from children aged 2-12 years in a setting in rural Bangladesh with predominantly low STH infection intensity. We estimated the sensitivity and specificity of each diagnostic using Bayesian latent class analysis. Compared to double-slide 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 double-slide Kato-Katz has few false positives, our results indicate otherwise and highlight inherent limitations of the Kato-Katz technique. qPCR had higher sensitivity than Kato-Katz in this low intensity infection setting.

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

“…This suggests that qPCR results can be used to group infections into low, medium and high intensity. Similar results were found by Benjamin-Chung et al [38] using C t values instead of DNA copy numbers; the caveats this entails have been previously reported.…”

“…There was evidence for a linear relationship between DNA copy number and EPG for A. lumbricoides (Kendall Tau-b = 0.68) and weaker evidence for T. trichiura (Kendall Tau-b = 0.56) and hookworm (Kendall Tau-b = 0.50). This magnitude of association is lower than found by Easton et al in Kenya (Spearman's rank correlation coefficients between DNA (ng/µl) and EPG of 0.83 for A. lumbricoides and 0.55 for T. trichiura) but higher than reported by Benjamin-Chung et al in Bangladesh (Tau-b value between qPCR C t and EPG was − 0.44 for A. lumbricoides and − 0.25 for T. trichiura) [38]. It is important that there is evidence for association between the two intensity measures, as it infers that the higher amount of DNA released by a higher number of eggs is being detected by qPCR.…”

“…As discussed above, there remains the possibility of false-positives, supported by evidence from the Easton et al [16] worm expulsion study. Surprisingly, the study in Bangladesh by Benjamin-Chung et al [38] found that the detection rate for A. lumbricoides was 14% lower by qPCR than KK. Although the authors equate this finding to misclassification of Ascaris ova during microscopy, the possibility of false positive results by qPCR was not ruled out [38].…”

“…MDA should continue at its current frequency (twice a year) [35]. Studies in Kenya [16], Timor-Leste [25], Ecuador [20] and Bangladesh [38] have all found qPCR to have greater sensitivity for detecting STH infections than KK. Unfortunately, due to time and expertise constraints, in this study we were only carrying out one KK slide per sample instead of the recommended two.…”

Detection of Ascaris lumbricoides infection by ABA-1 coproantigen ELISA

“…Copromicroscopy for detection of Ascaris eggs in fecal samples, as well as qPCR-based detection of worm DNA in fecal samples, are the only currently applied techniques to diagnose this gastrointestinal infection [ 7 , 25 ]. Microscopy lacks accuracy and is operator dependent, while qPCR requires specific laboratory infrastructure and currently lacks standardization [ 25 , 28 – 30 ]. Detection of specific serum antibodies to A .…”