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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.

SUBMITTER: Weinreich F

PROVIDER: S-EPMC8472038 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The protozoan parasite Cryptosporidium is an important cause of diarrheal disease (cryptosporidiosis) in humans and animals, with significant morbidity and mortality especially in severely immunocompromised people and in young children in low-resource settings. Due to the sexual life cycle of the parasite, transmission is complex. There are no restrictions on sexual recombination between sub-populations, meaning that large-scale genetic recombination may occur within a host, potentially confounding epidemiological analysis. To clarify the relationships between infections in different hosts, it is first necessary to correctly identify species and genotypes, but these differentiations are not made by standard diagnostic tests and more sophisticated molecular methods have been developed. For instance, multilocus genotyping has been utilized to differentiate isolates within the major human pathogens, Cryptosporidium parvum and Cryptosporidium hominis. This has allowed mixed populations with multiple alleles to be identified: recombination events are considered to be the driving force of increased variation and the emergence of new subtypes. As yet, whole genome sequencing (WGS) is having limited impact on public health investigations, due in part to insufficient numbers of oocysts and purity of DNA derived from clinical samples. Moreover, because public health agencies have not prioritized parasites, validation has not been performed on user-friendly data analysis pipelines suitable for public health practitioners. Nonetheless, since the first whole genome assembly in 2004 there are now numerous genomes of human and animal-derived cryptosporidia publically available, spanning nine species. It has also been demonstrated that WGS from very low numbers of oocysts is possible, through the use of amplification procedures. These data and approaches are providing new insights into host-adapted infectivity, the presence and frequency of multiple sub-populations of Cryptosporidium spp. within single clinical samples, and transmission of infection. Analyses show that although whole genome sequences do indeed contain many alleles, they are invariably dominated by a single highly abundant allele. These insights are helping to better understand population structures within hosts, which will be important to develop novel prevention strategies in the fight against cryptosporidiosis.

Dataset Information

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.

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