Comparison of Two Real-Time PCR Assays Targeting Ribosomal Sequences for the Identification of Cystoisospora belli in Human Stool Samples

Hahn²,

et al. 2021

Pathogens

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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: Methodsmentioning

confidence: 99%

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

Hahn²,

et al. 2021

Pathogens

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“…The study was conducted as a comparative head-to-head assessment of different real-time-PCR assays with historical residual sample materials without microscopic characterization. For the comparison of the screening PCR assays targeting G. dudodenalis , residual volumes of nucleic acid extractions from stool samples of 905 Ghanaian HIV patients from previous epidemiological and technical assessments [ 38 , 39 , 40 , 41 , 42 , 43 ] were used, so an acceptable pre-test probability due to a high prevalence of giardiasis in Ghana could be assumed [ 44 , 45 ]. All available residual sample materials with sufficient volumes were included.…”

Section: Methodsmentioning

confidence: 99%

“…After testing, samples showing PCR inhibition in the inhibition control PCR as detailed below were excluded from the assessments. For the subsequent comparison of the assemblage-specific duplex real-time PCR assays, 55 residual volumes of nucleic acid extractions from samples with positive screening PCR results for G. duodenalis from previous epidemiological studies [ 38 , 39 , 40 , 41 , 42 , 43 , 46 ] were applied. Inclusion criterion was that at least two out of three G. duodenalis -specific screening PCR assays provided a positive signal, making abundance of G. duodenalis DNA likely.…”

Section: Methodsmentioning

confidence: 99%

Comparative Evaluation of Real-Time Screening PCR Assays for Giardia duodenalis and of Assays Discriminating the Assemblages A and B

Hahn²,

et al. 2022

Microorganisms

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Due to superior sensitivity compared to traditional microscopy, real-time PCR has been well established for the diagnosis of Giardia duodenalis in human stool samples. In this study, screening real-time PCRs for different target genes of G. duodenalis, i.e., the 18S rRNA gene, the gdh (glutamate dehydrogenase) gene and the bg (beta-giardin) gene, were comparatively assessed next to various real-time PCR assays for the discrimination of the assemblages A and B of G. duodenalis targeting the bg gene with and without locked nucleic acid–containing probes as well as the tpi (triose phosphate isomerase) gene. The screening PCRs were assessed by including 872 non-preselected samples with a high pre-test probability for G. duodenalis in the statistical analysis, while 53 G. duodenalis-positive samples as indicated by at least two screening PCRs were finally included in the assessment of the assemblage-specific PCRs. For the screening PCRs, sensitivity estimated with latent class analysis (LCA) ranged from 17.5% to 100%, specificity from 92.3% to 100% with an accuracy-adjusted prevalence of 7.2% for G. duodenalis within the non-preselected sample collection. In detail, sensitivity and specificity were 100% and 100% for the 18S rRNA gene-specific assay, 17.5% and 92.3% for the gdh gene-specific assay, and 31.7% and 100% for the bg gene-specific assay, respectively. Agreement kappa was slight with only 15.5%. For the assemblage-specific PCRs, estimated sensitivity ranged from 82.1% to 100%, specificity from 84.0% to 100% with nearly perfect agreement kappa of 90.1% for assemblage A and yet substantial agreement of 74.8% for assemblage B. In detail for assemblage A, sensitivity and specificity were 100% and 100% for the bg gene-specific assay without locked nucleic acids (LNA) as well as 100% and 97.8% for both the bg gene-specific assay with LNA and the tri gene-specific assay, respectively. For assemblage B, sensitivity and specificity were 100% and 100% for the bg gene-specific assay without LNA, 96.4% and 84.0% for the bg gene-specific assay with LNA, and 82.1% and 100% for the tri gene-specific assay, respectively. Within the assessed sample collection, the observed proportion comprised 15.1% G. duodenalis assemblage A, 52.8% G. duodenalis assemblage B and 32.1% non-resolved assemblages. Only little differences were observed regarding the cycle threshold (Ct) values when comparing the assays. In conclusion, best diagnostic accuracy was shown for an 18S rRNA gene-specific screening assay for G. duodenalis and for a differentiation assay discriminating the G. duodenalis assemblages A and B by targeting the bg gene with probes not containing locked nucleic acids. By adding additional highly specific competitor assays for confirmation testing, diagnostic specificity can be further increased on the cost of sensitivity if optimized specificity is desired.

“…A total of 905 nucleic acid extractions from stool samples collected from Ghanaian HIV patients (n = 905) [35,36] were used for this study. The samples had already been successfully applied for comparative evaluations of real-time PCR assays targeting either microsporidia [37] or other coccidian parasites [38,39], so a high likelihood of a relevant proportion of samples positive for various parasites could be assumed for the assessment. Assessments for C. cayetanensis had not been performed so far with these samples prior to this study.…”

Section: Residual Sample Collection and Nucleic Acid Extractionmentioning

confidence: 99%

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

Hahn²,

et al. 2022

Pathogens

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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.