Dataset Information

Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples.

ABSTRACT: The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples.

SUBMITTER: Santana BN

PROVIDER: S-EPMC9901886 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples.

Santana Bruna Nicoleti BN Ferrari Elis Domingos ED Nakamura Alex Akira AA Silva Giane Serafim da GSD Meireles Marcelo Vasconcelos MV

Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria 20220321 1

The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventiona ...[more]

PMID: 35320826

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Project description:Early diagnosis of Pneumocystis carinii pneumonia, a life-threatening complication in immunosuppressed patients, may lower morbidity and mortality. We have developed a one-tube nested PCR assay for the detection of P. carinii in respiratory specimens. Four primers were selected from the sequence of the small-subunit rRNA gene of P. carinii to amplify a 265-bp fragment, and their specificities for P. carinii were confirmed by both theoretical evaluations (by computer-assisted comparison with the sequences in GenBank) and empirical evaluations (with DNA from medically important fungi and diagnostic samples). The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (digestion with proteinase K directly in PCR buffer at room temperature in the presence of 10% Chelex 100 and no further purification steps). Bovine serum albumin (1 mg/ml) and glycerol (10%) in the amplification buffer reduced the number of samples inhibitory to the PCR, as assessed by control reactions containing a size-modified target. A total of 749 clinical specimens (312 bronchoalveolar lavage, 403 sputum or induced sputum, and 34 other specimens) from 507 patients (295 human immunodeficiency virus [HIV]-infected and 164 non-HIV-infected patients and 48 patients whose HIV status was unknown) were tested by PCR, and the results were compared with those of an indirect immunofluorescence assay (IFA). Concordant results were obtained for 732 samples (646 negative and 86 positive). There were 17 discrepant results: 12 were PCR positive and IFA negative, and 5 were PCR negative and IFA positive. After resolution of the discrepant results by review of the patients' clinical data, the sensitivity and specificity were 94.8 and 99.1%, respectively, for PCR and 93.8 and 100%, respectively, for IFA. In conclusion, the short procedure time and the technical ease of this PCR assay render it suitable for implementation in routine diagnostic laboratories.

Project description:The enteric protozoan parasites Giardia duodenalis and Cryptosporidium spp. are common cause of diarrhea in pet dogs and cats, affecting primarily young animals. This comparative study evaluates the diagnostic performance of conventional and molecular methods for the detection of G. duodenalis and Cryptosporidium spp. infection in dogs and cats.The compared diagnostic assays included merthiolate-iodine-formalin (MIF) method, lateral flow immunochromatography rapid test (ICT) and real-time PCR; using direct immunofluorescence assay (DFA) as golden standard. The study included the analysis of 328 fecal samples from different dog (n = 225) and cat (n = 103) populations.According to DFA, the overall prevalence of G. duodenalis was 24.4% (80/328, 95% CI: 19.8-29.4), varying from 11.6% (12/103, 95% CI: 6.2-19.5) in cats to 30.2% (68/225, 95% CI: 24.3-36.7) in dogs. The overall prevalence of Cryptosporidium spp. was 4.0% (13/328, 95% CI: 2.1-6.7), varying from 2.9% (3/103, 95% CI: 0.6-8.3) in cats to 4.4% (10/225, 95% CI: 2.1-8.0) in dogs. MIF was only used for the detection of G. duodenalis, which was identified by this method in 22.7% of dogs and 7.8% of cats, respectively. DFA was the most sensitive technique for detecting G. duodenalis in samples from dogs and cats (p-value: < 0.001), followed by real-time PCR. Identification of Cryptosporidium infections was most effectively accomplished by the combination of DFA and PCR technique (p-value: < 0.001). In addition, epidemiological (sex, age, origin) and clinical (fecal consistency) variables were collected to assess their potential associations with an increased likelihood of infection by G. duodenalis and/or Cryptosporidium spp. Breeder dogs were more likely to harbor G. duodenalis infection (p-value: 0.004), whereas female cats were significantly more infected with Cryptosporidium (p-value: 0.003).In conclusion, DFA (alone or in combination with PCR) has been identified as the most accurate and cost-effective method for detecting G. duodenalis and Cryptosporidium spp. in fecal samples from pet dogs and cats. This highlights their importance in both veterinary and clinical settings for enabling prompt treatment and preventing potential transmission to humans.

Dataset Information

Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples.

Publications

Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples.

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