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.

Felix Weinreich,Fred Stephen Sarfo,Ulrike Loderstädt,Torsten Feldt,Andreas Hahn,Hagen Frickmann,Veronica Di Cristanziano,Kirsten Alexandra Eberhardt

doi:10.3390/pathogens11020165

Abstract

Diagnostic real-time PCR for the detection of Cyclospora cayetanensis in human stool samples has been applied for two decades. However, recent comparative assessments between in-house and commercial assays suggested room for improvement regarding the agreement of positive signals of the applied real-time PCRs. In order to assess the effect of the choice of the target sequence, 3 inhouse real time PCR assays targeting the 18S rRNA gene (n = 2, one of them later referred to as SSU rRNA gene assay to avoid confusion) and the hsp70 gene of C. cayetanensis were compared in a head-to-head comparison with 905 samples with high pretest probability for C. cayetanensis infections from Ghanaian HIV patients in a test comparison without a reference standard. Only slight agreement kappa of 0.095 was observed. In the assays targeting the SSU rRNA gene, the 18S rRNA gene, and hsp70, positive signals were recorded in 63, 45, and 0 instances, respectively, with latent class analysis-based estimation of sensitivity of 32.2%, 23.3%, 0% as well as of specificity of 99.7%, 99.9% and 100%, respectively. High cycle threshold values with an average of about 35 indicated low quantities of target DNA in the samples with similar Ct values in concordantly and discordantly positive samples. In conclusion, the study suggested target-gene-specific differences in the diagnostic accuracy of real-time PCR-based diagnosis of C. cayetanensis as well as an ongoing need for further standardization of this diagnostic approach.

Highlights

Introduction distributed under the terms andCyclospora cayetanensis are coccidian parasites causing enteric disease in human patients following fecal-oral transmission [1,2] with an obligatory environmental sporulation step in water or soil [1]
While the diagnosis of C. cayetanensis has traditionally been based on microscopy [2], usually following acid-fast staining or alternative approaches to increase visibility within stool specimens as well as enrichment steps to increase sensitivity [2,18,19,20,21], both in-house and commercial molecular diagnostic assays have been described in the meantime [5,22,23,24,25,26]
As the applied oligonucleotides are usually not published for commercial molecular diagnostic assays, it could not be checked whether or not different target sequences of the real-time PCR assays could be the reason for the observed discrepancy [25,26]

Summary

Introduction

Introduction distributed under the terms andCyclospora cayetanensis are coccidian parasites causing enteric disease in human patients following fecal-oral transmission [1,2] with an obligatory environmental sporulation step in water or soil [1]. In recent comparisons of commercial and in-house real-time PCR assays, there was a surprisingly low agreement between different molecular C. cayetanensis-specific assays [25,26]. This finding was accompanied by a generally higher sensitivity of real-time PCR compared to microscopy [25,27]. As the applied oligonucleotides are usually not published for commercial molecular diagnostic assays, it could not be checked whether or not different target sequences of the real-time PCR assays could be the reason for the observed discrepancy [25,26]. As the choice of the target sequence is of critical importance for the reliability of a diagnostic real-time PCR assay [28,29], it is likely that different target sequences as previously reported for C. cayetanensis [25,26,30,31,32,33] may have played a role

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Conclusion