# Diagnostic accuracy of nucleic acid amplification tests for human intestinal nematode infections: A systematic review and meta-analysis

## TL;DR

This study compares molecular tests with traditional microscopy for diagnosing intestinal worm infections and finds molecular tests are more accurate, especially for low-intensity infections.

## Contribution

The study provides the first comprehensive meta-analysis comparing nucleic acid amplification tests with conventional methods for diagnosing human intestinal nematode infections.

## Key findings

- NAATs showed significantly higher sensitivity than Kato-Katz and flotation methods for detecting Ascaris lumbricoides and hookworm.
- For Strongyloides stercoralis, NAATs had 93% sensitivity against composite reference standards compared to 59% for the Baermann technique.
- Combining microscopy with targeted NAAT use can improve diagnostic accuracy and support WHO 2030 deworming goals.

## Abstract

The use of diverse diagnostic methods in the absence of a definitive gold standard makes it challenging to determine the most appropriate test for diagnosing human intestinal nematode infections (HINIs), particularly across various clinical settings with varying endemicity. The ideal diagnostic method should be feasible, cost-effective, and accurate. This review evaluates the diagnostic accuracy of nucleic acid amplification tests (NAATs), comparing them to the Kato-Katz (KK) and flotation methods for the detection of ascariasis, trichuriasis, and hookworm infection, the Baermann technique (BT) for strongyloidiasis, the Scotch tape test for enterobiasis, and a composite reference standard (CRS).

We systematically searched PubMed, CINAHL, Scopus, Trip, Web of Science, Cochrane Library, and the academic search engine Google Scholar for studies published within the 12 years preceding September 2024. After the title, abstract and full-text screening, the selected studies were assessed for their methodological quality using Quality Assessment of Diagnostic Accuracy Studies - Version 2 (QUADAS-2). Data were extracted into 2x2 contingency tables, and sensitivity and specificity were pooled using the Reitsma bivariate random-effects model. Forest plots and summary ROC curves were used to explore heterogeneity.

Of the 3,239 articles screened, 35 met the inclusion criteria. Overall, NAATs showed higher pooled sensitivity for HINIs. For Ascaris lumbricoides, NAATs showed markedly higher sensitivities of 96–98% against the CRSs, compared with KK and flotation methods (57–67%). For Trichuris trichiura, NAAT sensitivity ranged from 74 to 87% across CRSs, whereas KK and flotation exhibited slightly lower but comparable sensitivities (70–83%). For hookworm, NAATs achieved sensitivities of 88–95% against CRS, substantially exceeding those of KK (43%) and flotation (59%) against CRS, with specificities above 87%. In detecting Strongyloides stercoralis, NAATs showed 80% sensitivity versus the BT, increasing to 93% against CRS, while the BT showed a sensitivity of 59%. When all soil-transmitted helminths were analysed collectively, pooled sensitivities of NAATs (75–84%) exceeded those of KK (64%), with consistently high specificity across all diagnostic methods. For hookworm, NAATs detected approximately two to threefold more infections than KK and flotation methods, when evaluated against a CRS, highlighting the substantial under-detection by conventional microscopy.

NAATs provide markedly higher sensitivity than copro-microscopy, especially for low-intensity or post-MDA infections. Combining routine microscopy with targeted NAAT deployment and emerging low-cost molecular approaches can optimise diagnostic accuracy and surveillance feasibility, strengthening control programmes and accelerating progress toward the WHO 2030 deworming and elimination goals.

Human intestinal nematode infections continue to affect millions of people, particularly in low- and middle-income countries. Accurate diagnosis is critical for treatment, control, and elimination programs. Traditionally, infections are detected through microscopic examination of stool samples using methods like wet smear, sedimentation methods, flotation methods, Kato-Katz, and the Baermann technique. However, these methods may miss infections, particularly when the infection intensity is low. In recent years, molecular tests such as nucleic acid amplification tests (NAATs) have emerged as a promising alternative, offering higher sensitivity. In this systematic review, we compared NAATs with conventional microscopy for detecting Ascaris lumbricoides, Trichuris trichiura, hookworm, and Strongyloides stercoralis. NAATs consistently showed higher sensitivity, particularly against composite reference standards and for low-intensity infections. Evidence for Enterobius vermicularis remained limited, with no studies comparing NAATs to the Scotch tape test. Our findings support integrating NAATs into surveillance, especially in settings approaching elimination, to improve case detection and strengthen control efforts. Using microscopy for routine diagnosis, together with targeted use of NAATs with emerging low-cost molecular tools, provides a feasible approach to strengthen diagnostic capacity and support progress toward the WHO 2030 goals for soil-transmitted helminthiases control programmes.