Comparison of Multi-Parallel Quantitative Real-Time PCRs Targeting Different DNA Regions and Detecting Soil-Transmitted Helminths in Stool

Authors

Marina Papaiakovou
Rubén O Cimino
Nils Pilotte
Julia Dunn
D Timothy J Littlewood
Steven A Williams
Alejandro J Krolewiecki
Rojelio Mejia

Publication Date

9-13-2024

Journal

Parasites & Vectors

DOI

10.1186/s13071-024-06464-6

PMID

39272159

PMCID

PMC11397029

PubMedCentral® Posted Date

9-13-2024

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Feces, Animals, Real-Time Polymerase Chain Reaction, Humans, DNA, Helminth, Soil, Helminthiasis, Helminths, Parasite Egg Count, Sensitivity and Specificity, Trichuris

Abstract

BACKGROUND: Soil-transmitted helminths infect an estimated 18% of the world's population, causing a significant health burden. Microscopy has been the primary tool for diagnosing eggs from fecal samples, but its sensitivity drops in low-prevalence settings. Quantitative real-time polymerase chain reaction (qPCR) is slowly increasing in research and clinical settings. However, there is still no consensus on preferred qPCR targets.

METHODS: We aimed to compare soil-transmitted helminth (STH) DNA detection methods by testing naïve stool samples spiked with known quantities of STH eggs and larvae. DNA extracts from spiked samples were tested using independent quantitative realtime PCR (qPCR) assays targeting ribosomal or putative non-protein coding satellite sequences.

RESULTS: For Trichuris trichiura, there was a strong correlation between egg/larvae counts and qPCR results using either qPCR method (0.86 and 0.87, respectively). Strong correlations also existed for A. lumbricoides (0.60 and 0.63, respectively), but weaker correlations were found for Ancylostoma duodenale (0.41 for both assays) and Strongyloides stercoralis (0.48 and 0.65, respectively). No correlation for Necator americanus was observed when testing with either qPCR assay. Both assays had fair-to-moderate agreement across targets when using field-collected stool samples (0.28-0.45, for all STHs), except for S. stercoralis (0.12) with slight agreement.

CONCLUSIONS: There is a strong correlation between qPCR results and egg/larvae counts. Our study confirms that qPCR is an effective diagnostic tool, even with low-intensity infections, regardless of the DNA-based diagnostic marker used. However, the moderate agreement between the two different qPCR assays when testing field samples highlights the need to understand the role of these targets in the genome so that the parasite burden can be quantified more accurately and consistently by qPCR.

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