Comparison of Three Real-Time PCR Assays Targeting the SSU rRNA Gene, the COWP Gene and the DnaJ-Like Protein Gene for the Diagnosis of Cryptosporidium spp. in Stool Samples

Abstract

As qualified microscopy of enteric parasitoses as defined by high diagnostic accuracy is difficult to maintain in non-endemic areas due to scarce opportunities for practicing with positive sample materials, molecular diagnostic options provide less investigator-dependent alternatives. Here, we compared three molecular targets for the real-time PCR-based detection of Cryptosporidium spp. From a population of 1000 individuals comprising both Ghanaian HIV (human immunodeficiency virus) patients and military returnees after deployment in the tropics, stool samples were assessed for Cryptosporidium spp. by real-time PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene, the Cryptosporidium oocyst wall (COWP) gene, and the DnaJ-like protein gene (DnaJ), respectively. In declining order, sensitivity of 100% for the SSU rRNA gene PCR, 90.0% for the COWP PCR and 88.8% for the DnaJ PCR, respectively, as well as specificity of 99.6% for the COWP PCR and 96.9% for both the SSU rRNA gene PCR and the DnaJ PCR, respectively, were recorded. Substantial agreement (kappa value 0.663) between the three assays was observed. Further, an accuracy-adjusted Cryptosporidium spp. prevalence of 6.0% was calculated for the study population. In conclusion, none of the assessed real-time PCR assays were associated with perfect test accuracy. However, a combination of highly sensitive SSU rRNA gene PCR for screening purposes and more specific COWP PCR for confirmatory testing should allow reliable diagnosis of Cryptosporidium spp. in stool samples even in low prevalence settings.

Comparison of Three Real-Time PCR Assays for the Detection of Cyclospora cayetanensis in Stool Samples Targeting the 18S rRNA Gene and the hsp70 Gene

“…This is surprisingly high, compared to 6.0% for Cryptosporidium spp. and 3.2% for Cystoisospora belli as previously reported for this population [38,39]. While an association of increased prevalence of the coccidian parasites Cryptosporidium spp.…”

“…A total of 905 nucleic acid extractions from stool samples collected from Ghanaian HIV patients (n = 905) [35,36] were used for this study. The samples had already been successfully applied for comparative evaluations of real-time PCR assays targeting either microsporidia [37] or other coccidian parasites [38,39], so a high likelihood of a relevant proportion of samples positive for various parasites could be assumed for the assessment. Assessments for C. cayetanensis had not been performed so far with these samples prior to this study.…”

Comparative Evaluation of Real-Time Screening PCR Assays for Giardia duodenalis and of Assays Discriminating the Assemblages A and B

“…The study was conducted as a comparative head-to-head assessment of different real-time-PCR assays with historical residual sample materials without microscopic characterization. For the comparison of the screening PCR assays targeting G. dudodenalis , residual volumes of nucleic acid extractions from stool samples of 905 Ghanaian HIV patients from previous epidemiological and technical assessments [ 38 , 39 , 40 , 41 , 42 , 43 ] were used, so an acceptable pre-test probability due to a high prevalence of giardiasis in Ghana could be assumed [ 44 , 45 ]. All available residual sample materials with sufficient volumes were included.…”

“…After testing, samples showing PCR inhibition in the inhibition control PCR as detailed below were excluded from the assessments. For the subsequent comparison of the assemblage-specific duplex real-time PCR assays, 55 residual volumes of nucleic acid extractions from samples with positive screening PCR results for G. duodenalis from previous epidemiological studies [ 38 , 39 , 40 , 41 , 42 , 43 , 46 ] were applied. Inclusion criterion was that at least two out of three G. duodenalis -specific screening PCR assays provided a positive signal, making abundance of G. duodenalis DNA likely.…”

Diagnostic Methods of Common Intestinal Protozoa: Current and Future Immunological and Molecular Methods

“…They also suggested the use of the combination of primers from both genes for optimum accuracy of Cryptosporidium spp. detection in stool samples, particularly in low-prevalence settings [ 44 ].…”