Evaluation of loop mediated isothermal amplification, quantitative real-time PCR, conventional PCR methods for identifying Ascaris lumbricoides in human stool samples

Abstract

Ascariasis, caused by Ascaris lumbricoides, is a widespread parasitic infection. Traditional diagnostic methods, such as microscopy, can miss infections with low worm burdens, leading to false negatives. This study compares four diagnostic methods—microscopy, conventional PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP)—for detecting A. lumbricoides in 400 stool samples from children aged 2-16. Microscopy methods (direct wet mount, Kato-Katz, and concentration) detected 17, 23, and 21 positive samples, respectively. Molecular techniques identified 23 positive samples by conventional PCR, 29 by real-time PCR, and 25 by LAMP. Notably, real-time PCR detected two samples missed by microscopy, while conventional PCR failed to detect three samples positive by real-time PCR and LAMP. In limit-of-detection assays, conventional PCR detected A. lumbricoides DNA down to 150 pg, while qPCR and LAMP could detect as low as 15 fg. For egg number analysis, conventional PCR detected DNA from 100 eggs, while qPCR and LAMP identified DNA from just 10 eggs. The methods specifically targeted A. lumbricoides, without cross-reacting with other co-occurring parasites. Sensitivity and specificity analysis revealed that microscopy had sensitivities of 81.3 %, while conventional PCR, qPCR, and LAMP had sensitivities of 81.1 %, 99.2 %, and 88.1 %, respectively. Microscopy and conventional PCR had 100 % specificity, while qPCR and LAMP had 99.2 % and 99.9 % specificity. While Kato-Katz is advantageous for detecting active infections, molecular techniques, particularly LAMP's field applicability in resource-limited settings makes it a promising tool for surveillance and control of low-intensity infections.