Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths

Abstract

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin–ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

Evaluation of the Allplex™ GI-Helminth(I) Assay, the first marketed multiplex PCR for helminth diagnosis

“…For each parasite targeted, we compared our results to those in the literature ( Table 3 ) [ 3 , 5 , 10 , 13 , 15 , 17 , 18 , 20 , 25 ]. Overall, the performances of the Allplex™ GIPH assay were similar to other PCR techniques, except for hookworm and T. trichiura detection, the latter being improved when applying bead-beating pretreatment.…”

“… Targets Micro + Micro + Proportion of false-negative from PCR Reference PCR + PCR − Ascaris spp. 7 1 13% This study 154 7 4% [ 17 ] 34 a 2 a 6% a [ 13 ] 23 5 18% [ 20 ] 8 0 0% This study (+bb) 35 a 1 a 3% a [ 13 ] (+bb) 192 27 12% [ 15 ] (+bb) E. verm...…”

Clinical value of serology for the diagnosis of strongyloidiasis in travelers and migrants: A 4-year retrospective study using the Bordier IVD®Strongyloides rattiELISA assay

“…Recently, PCR assays have been developed for S. stercoralis detection in stools, often yielding better performances than microscopy [ 3 , 5 , 26 ]. However, these assays showed heterogeneous performances, greatly depending on the patient population and the reference method used for clinical evaluation [ 2 , 9 ].…”

Whip-LAMP: a novel LAMP assay for the detection of Trichuris muris-derived DNA in stool and urine samples in a murine experimental infection model

“…Over the past several years, efforts have been made to use molecular diagnostic methods, particularly qPCR and multiplex-qPCR, to detect soil-transmitted helminths [ 20 , 46 – 48 ]. Traditionally, most of these techniques have been restricted to the research setting since they are not easily accessible for low-income countries that are the endemic areas of STH infections.…”