Molecular diagnosis of Strongyloides stercoralis in faecal samples using real-time PCR

Abstract

A real-time PCR method targeting the small subunit of the rRNA gene was developed for the detection of Strongyloides stercoralis DNA in faecal samples, including an internal control to detect inhibition of the amplification process. The assay was performed on a range of well-defined control samples (n=145), known positive faecal samples (n=38) and faecal samples from a region in northern Ghana where S. stercoralis infections are highly endemic (n=212), and achieved 100% specificity and high sensitivity. The use of this assay could facilitate monitoring the prevalence and intensity of S. stercoralis infections during helminth intervention programs. Moreover, the use of this assay in diagnostic laboratories could make the introduction of molecular diagnostics feasible in the routine diagnosis of S. stercoralis infections, with a two-fold increase in the detection rate as compared with the commonly used Baermann sedimentation method.

The laboratory diagnosis of Strongyloides stercoralis

“…Nucleic acid tests complement non-molecular methodologies for the diagnosis of S. stercoralis, and allow the use of refrigerated, frozen, or preserved specimens 11,19,20 . This simplifies specimen transportation, particularly where collection occurs some distance from the testing laboratory, and there is no risk of laboratoryacquired strongyloidiasis 21 .…”

“…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 .…”

“…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.…”

Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats

“…Interestingly, PCR has been associated with higher sensitivity than classical parasitological methods during infections with lower intensity (Wongratanacheewin et al 2001). Verweij et al (2009) evaluated the molecular diagnosis of S. stercoralis in faecal samples using real-time PCR. The authors demonstrated that this methodology could be a useful alternative to the commonly used Baermann method, offering a two-fold increase in the detection rate.…”