Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis

Abstract

Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People's Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.

Infección por Strongyloides stercoralis: metanálisis sobre evaluación de métodos diagnósticos convencionales (1980-2013)

“…Además se pueden producir reacciones cruzadas con otros helmintos y no permiten diferenciar entre las DIAGNÓSTICO DE LA INFECCIÓN POR STRONGYLOIDES STERCORALIS: METANÁLISIS SOBRE EVALUACIÓN DE MÉTODOS ... infecciones actuales y las pasadas 2 . Igualmente, la reacción en cadena de la polimerasa (PCR) también se ha utilizado como método diagnóstico, presentando diferencias en la sensibilidad obtenida en diferentes ensayos [27][28][29] .…”

The laboratory diagnosis of Strongyloides stercoralis

“…the 18S rRNA small subunit (SSU); the internal transcribed spacer region 1 (ITS-1); the 28S rRNA gene; or the cyclooxygenase gene (cox1) 19,[23][24][25][26][27][28][29][30][32][33][34][35][36][37] . Published sensitivities and specificities for…”

“…The majority of Strongyloides PCR publications have used a real-time method with primers and probe published by Verweij et al 19 . This has also allowed for the development of multiplexed PCR 10,30,34,38 . Some studies evaluating the diagnostic accuracy of these PCR methods have used both morphological diagnosis and detection of PCR products as their reference standards, and are not reviewed here.…”

“…No PCR studies have reported false positive results when analytical specificity has been tested using DNA extracted from bacteria, viruses, fungi, protozoa, and other helminths 19,23,24,27,29,30 . Studies have also assessed the specificity of the PCR products by sequence analysis, with all finding 100% sequence homology with the target sequence of S. stercoralis.…”

Molecular Detection of <i>Strongyloides ratti</i> in Faecal Samples from Wild Rats in Serdang, Malaysia

“…Approximately 30 million people are infected in 70 countries [1,2]. However, strongyloidiasis is a neglected parasitic disease the prevalence of which might be underestimated in many countries.…”