Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction with Parasitological Methods for Detection of Strongyloides stercoralis in Human Fecal Samples

Abstract

Abstract. This study was performed to evaluate nested polymerase chain reaction (PCR) and real-time PCR methods for detection of Strongyloides stercoralis in fecal samples compared with parasitological methods. A total of 466 stool samples were examined by conventional parasitological methods (formalin ether concentration [FEC] and agar plate culture [APC]). DNA was extracted using an in-house method, and mitochondrial cytochrome c oxidase subunit 1 and 18S ribosomal genes were amplified by nested PCR and real-time PCR, respectively. Among 466 samples, 12.7% and 18.2% were found infected with S. stercoralis by FEC and APC, respectively. DNA of S. stercoralis was detected in 18.9% and 25.1% of samples by real-time PCR and nested PCR, respectively. Considering parasitological methods as the diagnostic gold standard, the sensitivity and specificity of nested PCR were 100% and 91.6%, respectively, and that of real-time PCR were 84.7% and 95.8%, respectively. However, considering sequence analyzes of the selected nested PCR products, the specificity of nested PCR is increased. In general, molecular methods were superior to parasitological methods. They were more sensitive and more reliable in detection of S. stercoralis in comparison with parasitological methods. Between the two molecular methods, the sensitivity of nested PCR was higher than real-time PCR.

Molecular Diagnostics for Soil-Transmitted Helminths

“…However, this test also can give a positive result in the setting of hookworm infection and can contribute to perceived false negatives and an overall low sensitivity seen in some studies using qPCR 26,61,62. However, in many epidemiologic surveys, qPCR has typically increased overall diagnostic yield by 2- to 8-fold when compared with a variety of microscopic techniques 12,19,27,45,51,63,73. Unlike the other STH, however, the quantification of larvae in the stool has not been shown to predict the adult worm burden, and so qPCR would not be used as a reflection of adult worm burden in a clinical setting.…”

The laboratory diagnosis of Strongyloides stercoralis

“…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.…”

“…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. 24,25,27,29 . Sitta et al found a number of false positives, using published genus and species-specific primers, based on non-target sized bands on gel electrophoresis 19,25 .…”

“…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…”

Strongyloides stercoralis: current perspectives

“…61 Similar approach was adopted to detect S. stercoralis in human feces using real-time PCR with species-specific target genes, such as cytochrome c oxidase, 18S rRNA, and 5.8S rRNA. [62][63][64][65][66] The real-time PCR was 100% specific but failed to enhance the sensitivity to detect strongyloidiasis compared to parasite concentration methods. 67 The major disadvantage of DNAbased detection is that the amount of parasite-specific DNA in the feces is variable because of sporadic larval release, and in asymptomatic patients with very low levels of larval output, the test is unlikely to achieve a higher sensitivity unless repeated stool samples are tested.…”