Development of a Quantitative Real-Time PCR Assay for Detection of <i>Cryptosporidium</i> spp. Infection and Threatening Caused by <i>Cryptosporidium parvum</i> Subtype IIdA19G1 in Diarrhea Calves from Northeastern China

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.

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

Weinreich

Hahn²,

Eberhardt

et al. 2021

Pathogens

“…The 18ssu rRNA gene is highly abundant in terms of genomic copies and it has high transcripts that can increase the sensitivity of the assay, while the COWP is available only in transcriptionally active live cryptosporidial oocysts. (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(28)(29)(30)(31) Hence, in the current study we report an mRNA based TaqMan® probe duplex real time PCR that can able to detect the metabolically active Cryptosporidium oocysts from dead oocysts in passively infected animals…”

mentioning

confidence: 86%

“…Microscopy is an excellent tool and can be highly specific but requires expertise and experience in identification of the weak acid fast round oocysts from other artefacts similar to them. Besides conventional microscopy and staining, molecular diagnostic approaches like PCR are also gaining momentum Molecular diagnostic assays are usually regarded as more sensitive compared to microscopy and serological diagnosis in human and animals for the detection of Cryptosporidium in several studies (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). The molecular assays are determined by the factors like stage of life cycle/infection as well as method of nucleic acid extraction for targeting the Cryptosporidium spp.…”

mentioning

confidence: 99%

Development of duplex real time PCR for quick detection of Cryptosporidia in goats

Kumaresan

et al. 2022

Preprint

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and the leading cause of mortality from foodborne and waterborne gastrointestinal disease. In neonates of domestic animals it is associated with consistent diarrhoea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy, antigen trap ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA based duplex TaqMan® probe PCR (dRT-qPCR) was developed to target the Cryptosporidium oocyst wall protein (COWP) gene and 18ssu rRNA gene in a single tube that can detect metabolically active Cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and it will help decipher the active stages of its lifecycle in host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection. For COWP and 188ssu rRNA genes the limit of detection was 7.08x1004 and 5.95x1005 respectively. During active infections the oocyst wall protein will be active and so its COWP gene transcripts will act as marker for active infection. While transcripts for 18SSU rRNA are constitutively expressing in Cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among the healthy animals.

Development of duplex real‐time PCR for quick detection of cryptosporidiosis in goats

Kumaresan

Cell Biochemistry & Function

et al. 2022

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and a leading cause of mortality from foodborne and waterborne gastrointestinal diseases. In neonates of domestic animals, it is associated with consistent diarrhea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy and antigen trap-ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA-based duplex TaqMan ® probe PCR was developed to target the Cryptosporidium oocyst wall protein gene and 18SSU rRNA gene in a single tube that can detect metabolically active cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and they will help decipher the active stages of its lifecycle in a host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection (LOD). For COWP and 18SSU rRNA genes, the LOD was 7.08 × 10 04 and 5.95 × 10 05 , respectively. During active infections, the oocyst wall protein will be active and so its COWP gene transcripts will act as a marker for active infection. While transcripts for 18SSU rRNA are constitutively expressed in cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess the active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among healthy animals.dead and live oocysts, duplex real-time PCR assay, oocyst wall protein | INTRODUCTIONCryptosporidiosis disease is caused by the intracellular, extracytoplasmic protozoan parasite, which belongs to the phylum Apicomplexa. Cryptosporidiosis is the primary cause of chronic diarrhea among immunocompromised patients, especially in children suffering from HIV, AIDS-defining illness because of its high association with mortality. [1][2][3][4] Cryptosporidium is accountable for 8%-19% of diarrhea in less developed countries, 5 and till the year 2010, it already caused about 100,000 deaths globally. 6 More than 15% of the world's population is drinking contaminated water and suffering from waterborne parasitic protozoan diseases globally, 7 it is responsible for four billion cases of mild to severe diarrhea and 1.6 million deaths annually. 8,9 Cryptosporidium oocysts are