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 <i>Cryptosporidium</i> spp. in Stool Samples

• Main Authors: Felix Weinreich, Andreas Hahn, Kirsten Alexandra Eberhardt, Torsten Feldt, Fred Stephen Sarfo, Veronica Di Cristanziano, Hagen Frickmann, Ulrike Loderstädt
• Format: Article
• Language: English
• Published: MDPI AG 2021-09-01
• Series: Pathogens
• Subjects: <i>Cryptosporidium</i> spp.; real-time PCR; test comparison; latent class analysis; sensitivity; specificity
• Online Access: https://www.mdpi.com/2076-0817/10/9/1131

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 <i>Cryptosporidium</i> 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 <i>Cryptosporidium</i> spp. by real-time PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene, the <i>Cryptosporidium</i> 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 <i>Cryptosporidium</i> 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 <i>Cryptosporidium</i> spp. in stool samples even in low prevalence settings.