scholarly journals 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

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1131
Author(s):  
Felix Weinreich ◽  
Andreas Hahn ◽  
Kirsten Alexandra Eberhardt ◽  
Torsten Feldt ◽  
Fred Stephen Sarfo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Protein Gene ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Ssu Rrna Gene ◽  
Cryptosporidium Spp ◽  
Stool Samples ◽  
Pcr Assays

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.

scholarly journals A critical assessment of two real-time PCR assays targeting the (SSU) rRNA and gdh genes for the molecular identification of Giardia intestinalis in a clinical laboratory

2014 ◽  
Vol 67 (9) ◽  
pp. 811-816 ◽  
Author(s):  
Samuel Boadi ◽  
Spencer D Polley ◽  
Sally Kilburn ◽  
Graham A Mills ◽  
Peter L Chiodini
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Laboratory ◽  
Protozoan Parasite ◽  
Molecular Techniques ◽  
Giardia Intestinalis ◽  
Diagnostic Sensitivity ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Pcr Assays

IntroductionGiardiasis is an intestinal diarrhoeal illness caused by the flagellate protozoan parasite Giardia intestinalis. Molecular techniques for the identification of G. intestinalis have generally been shown to offer a better detection rate of the parasite than the traditional faecal concentration and microscopy techniques.AimThe aim of this study was to critically assess the performance of a commercial and a published real-time PCR assay for their potential use as frontline tests for the diagnosis of giardiasis.MethodsA composite reference standard of enzyme immunoassay and rapid membrane test was used in a diagnostic accuracy study to assess the performance of Primerdesign's, and Verweij et alG. intestinalis real-time PCR assays, comparing them with the traditional ova, cysts and parasite microscopy test (OCP-M).ResultsThe Verweij real-time PCR used primers for the (SSU) rRNA gene, and produced a diagnostic sensitivity of 93.4% (95% CI 88.30% to 98.50%) and an efficiency of 100%. Primerdesign's real-time PCR used primers for the glutamate dehydrogenase gene and produced a diagnostic sensitivity of 61.5% (95% CI 51.50% to 71.50%) and an efficiency of 203%. The OCP-M sensitivity was 83.5% (95% CI 75.87% to 91.13%).ConclusionsThe Verweij real-time PCR was robust and the most sensitive assay suited for use as a first-line diagnostic test for giardiasis.

Improving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay

2019 ◽  
Vol 72 (7) ◽  
pp. 487-492 ◽  
Author(s):  
Samson S Y Wong ◽  
Rosana W S Poon ◽  
Kelvin K W To ◽  
Jasper F W Chan ◽  
Gang Lu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
28S Rrna ◽  
Pcr Assay ◽  
Single Tube ◽  
Histological Sections ◽  
Pcr Products ◽  
Stool Samples

AimsHelminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay.MethodsWe designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested.ResultsThe PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples.ConclusionsThe highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 656
Author(s):  
Konstantin Tanida ◽  
Andreas Hahn ◽  
Kirsten Alexandra Eberhardt ◽  
Egbert Tannich ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Real Time ◽  
Indigenous People ◽  
Real Time Pcr ◽  
Latent Class ◽  
Enterocytozoon Bieneusi ◽  
Enteric Disease ◽  
Immunosuppressed Patients ◽  
Study Population ◽  
Stool Samples ◽  
Pcr Assays

Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen’s kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied.

scholarly journals Cryptosporidium spp. during chemotherapy: a cross-sectional study of 94 patients with malignant solid tumor

2021 ◽  
Vol 41 (5) ◽  
pp. 293-298
Author(s):  
Mehmet Karabey ◽  
Hüseyin Can ◽  
Tülay Öncü Öner ◽  
Mert Döşkaya ◽  
Sedef Erkunt Alak ◽  
...  
Keyword(s):  
Sample Size ◽  
Real Time ◽  
Solid Tumors ◽  
Real Time Pcr ◽  
Tertiary Care ◽  
Diagnostic Methods ◽  
Cross Sectional ◽  
Cryptosporidium Spp ◽  
Stool Samples ◽  
Pcr Targeting

BACKGROUND: Cryptosporidium spp . is a protozoan parasite that infects many vertebrate animals, including humans. Since Cryptosporidium spp . can cause chronic life-threatening diarrhea and severe malabsorption in immunocompromised patients, we investigated the prevalence of this parasite among patients undergoing chemotherapy for malignant solid tumors. OBJECTIVE: Investigate the prevalence of Cryptosporidium spp . in stool samples. DESIGN: Cross-sectional. SETTING: Tertiary care. PATIENTS AND METHODS: Stool samples were collected from adult patients with malignant solid tumors receiving chemotherapy and diarrhea. Cryptosporidium spp . prevalence was determined using Ziehl–Neelsen staining, ELISA, and real-time PCR targeting of the COWP gene. MAIN OUTCOME MEASURE: The prevalence of Cryptosporidium spp . in patients undergoing chemotherapy for malignant solid tumors. SAMPLE SIZE: 94 RESULTS: The prevalence was 2.1% (2/94), 5.3% (5/94), and 5.3% (5/94) as detected by Ziehl–Neelsen staining, real-time PCR and ELISA, respectively. The prevalence reached 8.5% (8/94) using all results obtained from the three methods. Among eight positive stool samples, four were positive by at least two different methods (Ziehl–Neelsen staining-ELISA or ELISA-real-time PCR) whereas the remaining four were positive by either ELISA or real-time PCR. CONCLUSION: These findings show the risk of cryptosporidiosis in cancer patients and the necessity to use at least two diagnostic methods during the diagnosis of cryptosporidiosis to reach more accurate and trustworthy results. LIMITATIONS: Further studies with a larger sample size are recommended. CONFLICT OF INTEREST: None.

Parasitological versus molecular diagnosis of strongyloidiasis in serial stool samples: how many?

2017 ◽  
Vol 92 (1) ◽  
pp. 12-16 ◽  
Author(s):  
E. Dacal ◽  
J.M. Saugar ◽  
T. Soler ◽  
J.M. Azcárate ◽  
M.S. Jiménez ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Correct Diagnosis ◽  
Molecular Techniques ◽  
Maximum Sensitivity ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Techniques ◽  
Molecular Technique ◽  
Asymptomatic Patients ◽  
Stool Samples

AbstractStrongyloidiasis is usually an asymptomatic disease in immunocompetent patients, caused by Strongyloides stercoralis. However, in immunocompromised patients it can produce a severe clinical profile. Therefore, a correct diagnosis is necessary in these cases and in those chronic asymptomatic patients. The low sensitivity of classical parasitological techniques requires the analysis of multiple serial stool samples. Molecular diagnostic techniques represent an improvement in the detection of the parasite. The objective of this study was to evaluate the minimum number of samples necessary to achieve maximum sensitivity by real-time polymerase chain reaction (PCR). A total of 116 stool samples from 39 patients were analysed by direct microscopic observation, agar culture, Harada–Mori and real-time PCR, in one, two, three and four or more consecutive samples. After two serial samples, 6 out of 39 patients were positive by parasitological and molecular techniques, while 16 of them were real-time PCR positive, and all the patients detected by parasitology were also detected by the molecular technique, reaching 100.00% sensitivity versus 83.00% when analysing a single sample. These data also reflect apparently low specificity (51.52%) and positive predictive value (PPV) (27.27 %) values, due to the high number of cases detected by real-time PCR and not by parasitological techniques. These cases were confirmed as true positives when analysing three, four or more samples from the same patient. In conclusion, the application of molecular techniques decreases the number of serial stool samples necessary to give a diagnosis with the maximum sensitivity.

scholarly journals Prevalence and molecular characterization of Cryptosporidium spp. in Père David’s deer (Elaphurus davidianus) in Jiangsu, China

2020 ◽  
Vol 29 (2) ◽  
Author(s):  
Si-Yang Huang ◽  
Yi-Min Fan ◽  
Yi Yang ◽  
Yi-Jun Ren ◽  
Jing-Zhi Gong ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Small Subunit ◽  
Basic Knowledge ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Ssu Rrna Gene ◽  
Cryptosporidium Spp ◽  
Père David’S Deer ◽  
Rrna Gene Sequencing

Abstract Cryptosporidium is a zoonotic parasite that causes diarrhea in a broad range of animals, including deer. Little is known about the prevalence and genotype of Cryptosporidium spp. in Père David’s deer. In this study, 137 fecal samples from Père David’s deer were collected between July 2017 and August 2018 in the Dafeng Reserve and analyzed for Cryptosporidium spp. by nested-PCR based on the small subunit ribosomal RNA (SSU rRNA) gene, followed by sequence analyses to determine the species. The 60 kDa glycoprotein (gp60) gene was used to characterize Cryptosporidium spp. Among 137 samples, 2 (1.46%) were positive for Cryptosporidium spp. according to SSU rRNA gene sequencing results. Both samples belonged to the Cryptosporidium deer genotype, with two nucleotide deletions and one nucleotide substitution. The prevalence data and molecular characterization of this study provide basic knowledge for controlling and preventing Cryptosporidium infections in Père David’s deer in this area.

scholarly journals Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays

2004 ◽  
Vol 42 (12) ◽  
pp. 5636-5643 ◽  
Author(s):  
M. Rougemont ◽  
M. Van Saanen ◽  
R. Sahli ◽  
H. P. Hinrikson ◽  
J. Bille ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Plasmodium Species ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Pcr Assays ◽  
Species Specific

Design and testing of real-time PCR primers for the quantification ofMethanoculleus,Methanosarcina,Methanothermobacter, and a group of uncultured methanogens

2009 ◽  
Vol 55 (5) ◽  
pp. 611-616 ◽  
Author(s):  
Ingrid H. Franke-Whittle ◽  
Marta Goberna ◽  
Heribert Insam
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Biogas Production ◽  
Pcr Primers ◽  
Sybr Green ◽  
Rrna Gene ◽  
Pcr Assays ◽  
Design And Testing ◽  
Uncultured Archaea ◽  
Detection And Quantification

In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta , Methanosarcina , and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter , and a group of yet uncultured archaea from anaerobic habitats.

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