scholarly journals Comparison of real-time PCR and the Kato-Katz method for the diagnosis of soil-transmitted helminthiasis and assessment of cure in a randomized controlled trial

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Beatrice Barda ◽  
Christian Schindler ◽  
Rahel Wampfler ◽  
Shaali Ame ◽  
Said M. Ali ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Controlled Trial ◽  
Bead Beating ◽  
Novel Treatments ◽  
Two Samples ◽  
Stool Samples ◽  
Pcr Method

Abstract Background Diagnosis of soil-transmitted helminths (STHs) in developing countries is commonly based on microscopic detection of eggs in stool samples, using the Kato-Katz (KK) method, which has a poor sensitivity for detecting light intensity infections. We compared the performance of the KK method and real-time PCR in the framework of a randomized trial, which evaluated four novel treatments against Trichuris trichiura and concomitant STH infections. Results Two stool samples obtained from 320 participants were examined at baseline and follow-up with quadruplicate KK and PCR analyses of one of the two samples using “bead-beating” for DNA extraction. At follow-up, 80 samples were negative according to both PCR and KK and 173 were positive with both methods for any of the STHs. Relative to PCR, the calculated sensitivity of KK at follow-up was 83.6%, 43.0% and 53.8% for T. trichiura, for hookworm and for Ascaris lumbricoides, respectively. The sensitivity of PCR compared with KK at this time point was 89.1% for T. trichiura, 72.7% for hookworm and 87.5% for A. lumbricoides. Cure rates (CRs) for T. trichiura and A. lumbricoides were slightly lower with the PCR method. For hookworm CRs with KK were mostly significantly lower, namely 36.7%, 91.1%, 72.2% and 77.8% for moxidectin, moxidectin in combination with tribendimidine, moxidectin in combination with albendazole and albendazole in combination with oxantel pamoate, respectively, whereas with PCR the CRs were 8.3%, 82.6%, 37.1% and 57.1%, respectively. Conclusions In conclusion, a single real-time PCR is as sensitive as quadruplicate KK for T. trichiura and A. lumbricoides detection but more sensitive for hookworm, which has an influence on the estimated treatment efficacy. PCR method with DNA extraction using the “bead-beating protocol” should be further promoted in endemic areas and laboratories that can afford the needed equipment. The study is registered at ISRCTN (no. 20398469).

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 Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

2015 ◽  
Vol 35 (3) ◽  
pp. 306-313 ◽  
Author(s):  
Abdullah Kilic ◽  
Mohammad J. Alam ◽  
Naradah L. Tisdel ◽  
Dhara N. Shah ◽  
Mehmet Yapar ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Real Time ◽  
Real Time Pcr ◽  
Stool Samples ◽  
Pcr Method ◽  
Simultaneous Identification

scholarly journals ORAL HIV-ASSOCIATED KAPOSI SARCOMA: A COMPARISON BETWEEN IMMUNOHISTOCHEMISTRY AND qPCR TECHNIQUES FOR DETECTION OF HHV8

2015 ◽  
Vol 21 (1) ◽  
pp. 29
Author(s):  
Priscila Lie Tobouti ◽  
Juliana Seo ◽  
Michella Bezerra Lima ◽  
Bruno Tavares Sedassari ◽  
Norberto Nobuo Sugaya ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Kaposi Sarcoma ◽  
Positive Staining ◽  
Extraction Protocol ◽  
Simple Phenol ◽  
Two Samples ◽  
Hematoxylin And Eosin ◽  
The Mean

<p><strong>Objective: </strong>To compare the diagnostic accuracy of immunohistochemistry compared to real-time PCR (using a simple phenol-chloroform DNA extraction protocol) in the detection of HHV8 in small oral biopsies of Kaposi sarcoma. Also to validate the use of this DNA extraction protocol to extract HHV8 DNA.</p><p><strong>Material and methods:</strong> Seventeen cases of oral KS were examined. Data including gender, age, and anatomic location were obtained from patient´s records and histological sections stained with hematoxylin and eosin (H&amp;E) were reviewed. Immunohistochemistry was used to detect HHV8 in lesions of interest, as well as real-time PCR.</p><p><strong>Results: </strong>All the patients were HIV positive, the mean age was 35.5 years old, and the affected oral sites were palate (47%), gingiva (29.4%), tongue (11.8%), lip (5.9%), and oral floor (5.9%). Fifteen samples showed positive staining for HHV8 and only two samples were negative. The same results were observed using real-time PCR HHV8-DNA detection.</p><p><strong>Relevance: </strong>Our findings suggest that immunohistochemistry is faster and cheaper to perform than real-time PCR and was shown to have similar levels of sensitivity and accuracy for detection of HHV8 even in small biopsies. Additionally DNA extraction using a non-commercial kit, as done in this study can further reduce the costs to a pathology service.</p>

Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

2007 ◽  
Vol 70 (6) ◽  
pp. 1373-1378 ◽  
Author(s):  
ANNA-CLARA RÖNNER ◽  
HANS LINDMARK
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Dna Extraction ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Close Correlation ◽  
Quantitative Detection ◽  
Quantitative Real Time Pcr ◽  
Direct Plating ◽  
Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

Detection of Rendered Meat and Bone Meals by PCR Is Dependent on Animal Species of Origin and DNA Extraction Method

2010 ◽  
Vol 73 (6) ◽  
pp. 1090-1096 ◽  
Author(s):  
MICHAEL J. MYERS ◽  
DOROTHY E. FARRELL ◽  
CHRISTINE M. DEAVER ◽  
JACQULINE MASON ◽  
HEIDI L. SWAIM ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Animal Species ◽  
Animal Feed ◽  
Functional Assay ◽  
Threshold Values ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Pcr Method

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed.

scholarly journals Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR

2006 ◽  
Vol 55 (9) ◽  
pp. 1187-1191 ◽  
Author(s):  
Lisa J. Griffiths ◽  
Martin Anyim ◽  
Sarah R. Doffman ◽  
Mark Wilks ◽  
Michael R. Millar ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Diagnostic Technique ◽  
Extraction Methods ◽  
Simple Method ◽  
Bead Beating ◽  
Fungal Dna ◽  
Dna Extraction Methods ◽  
High Level

Newer methods such as PCR are being investigated in order to improve the diagnosis of invasive aspergillosis. One of the major obstacles to using PCR to diagnose aspergillosis is a reliable, simple method for extraction of the fungal DNA. The presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of the DNA by conventional methods employed for bacteria. Numerous fungal DNA extraction protocols have been described in the literature. However, these methods are time-consuming, require a high level of skill and may not be suitable for use as a routine diagnostic technique. Here, a number of extraction methods were compared: a freeze–thaw method, a freeze–boil method, enzyme extraction and a bead-beating method using Mini-BeadBeater-8. The quality and quantity of the DNA extracted was compared using real-time PCR. It was found that the use of a bead-beating method followed by extraction with AL buffer (Qiagen) was the most successful extraction technique, giving the greatest yield of DNA, and was also the least time-consuming method assessed.

scholarly journals Multicenter Comparative Study of Six Cryptosporidium parvum DNA Extraction Protocols Including Mechanical Pretreatment from Stool Samples

2020 ◽  
Vol 8 (9) ◽  
pp. 1450
Author(s):  
Nicolas Valeix ◽  
Damien Costa ◽  
Louise Basmaciyan ◽  
Stéphane Valot ◽  
Anne Vincent ◽  
...  
Keyword(s):  
Comparative Study ◽  
Real Time ◽  
Dna Extraction ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Sample Pretreatment ◽  
Dna Amplification ◽  
Extraction Methods ◽  
Mechanical Pretreatment ◽  
Stool Samples

Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Methods: A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Results: The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of C. parvum oocysts (i.e., 0–94.4% and 33.3–100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep® manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24® with Lysing Matrix E®. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods.

Long-Term Monitoring of Cancer-Free Subjects Carrying Non-Lymphoma Associated bcl2/IgH Rearrangements (NLABR): Prolonged Persistence of Clonal Populations Potentially Related to Follicular Lymphoma (FL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1373-1373
Author(s):  
Marco Ladetto ◽  
Barbara Mantoan ◽  
Federica De Marco ◽  
Berardino Pollio ◽  
Daniela Drandi ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
Real Time ◽  
Median Time ◽  
Real Time Pcr ◽  
Direct Sequencing ◽  
Two Samples ◽  
Long Term Monitoring ◽  
Term Monitoring

Abstract Introduction: NLABRs are frequently observed in cancer free-subjects. We recently observed that NLABR-positive clones can persist up to 60 days (Ladetto et al, J Clin Oncol 2003). However the long-term kinetics and potential pre-neoplastic role of NLABR-carrying cells are unknown. To define the natural history of NLABR-positive clones, long term monitoring of cancer-free subjects carrying these lesions has been performed. Methods: 118 subjects undergoing periodical blood examinations for warfarin therapy were screened for the bcl-2/IgH translocation. PCR-positive subjects underwent subsequent monitoring at least once every three months. NLABR-positive clones were monitored using both nested and real time-PCR according to previously published approaches (Ladetto et al Exp Hematol 2001). Sequence homology of NLABRs has always been confirmed by direct sequencing of nested PCR products. Results: 15 NLABR-positive subjects were identified out of 118 (12.7%) subjects. NLABR-positive subjects were monitored for a median time of 13 months (mos) (range 3–30 mos) for a total number of 60 timepoints. In eight subjects (53%), NLABRs detected at study initiation were not detected again in follow-up samples. These eight subjects have been monitored for median period of 12 mos (range 3–28 mos). Follow-up samples in this group were usually PCR-negative, although transient PCR-positivity due to unrelated NLABRs were noticed in two samples. In seven subjects (47%), the same NLABR observed at study initiation was detected one or more times at follow-up. In four subjects, NLABRs detected at diagnosis were amplified in every available follow-up sample (three to seven samples were available for each subject). In three, NLABRs detected at diagnosis were amplified only in a fraction of follow-up samples while the remaining were PCR-negative. Overall, persistent NLABRs were followed on these subjects for a median time of 15 months (range 3–30). The median burden of persistent NLABRs assessed by real-time PCR was 33 rearrangements (rg)/106 diploid genomes (dg) (range <10–760), while the median burden of short-lived NLABRs was <10rg/106 dg (range <10–330). The number of NLABR-positive cells appeared to be rather stable in subjects with persistent NLABR-positive clones. In none of these subjects we could detect differences greater than 1 log among available follow-up samples. Subjects having mixed PCR-positive and PCR-negative results had a smaller tumor burden compared to those constantly PCR-positive. This is consistent with the presence of a small though persistent clonal population. Studies on selected populations showed that NLABR-positive cells were CD19-positive. Discussion: NLABR-positive clones are long-lived cell populations in approximately 50% of cases. Based on this finding it is reasonable to hypothesize the existence of a follicular lymphoma (FL)-related lymphoproliferation of undetermined significance. Since NLABRs occurs in more than than 10% of healthy subjects, this condition is expected to be highly prevalent in the general population (as observed in MGUS and CLUS) and of potential relevance for the pathogenesis of FL.

scholarly journals Comparability of CMV DNA Extraction Methods and Validation of Viral Load

2022 ◽  
Vol 5 (1) ◽  
pp. 6
Author(s):  
Théophile Uwiringiyeyezu ◽  
Bouchra El Khalfi ◽  
Rachid Saile ◽  
Jamal Belhachmi ◽  
Abdelaziz Soukri
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Human Cytomegalovirus ◽  
Real Time Pcr ◽  
Biological Fluids ◽  
Extraction Methods ◽  
Acid Extraction ◽  
Viral Loads ◽  
Transplant Patients

Human cytomegalovirus is a herpesvirus that has a worldwide seroprevalence of more than 60% of adults in developed countries and 90% in developing countries. Severe disabilities in newborns are characteristic of the human cytomegalovirus congenital infection, and this virus is implicated in graft rejection in transplant patients. To treat and follow-up the infection, the CMVPCR viral loads are required, and the DNA extraction step remains very important; however, the quantity, quality, and purity of extracted DNA from different biological fluids influence the results of PCR amplification, that is why for reliable results, the choice of nucleic acid extraction methods requires careful attention. Materials and methods: In this study, we compare 4 protocols, I (EZ1 DSP Virus kit), II (EZ1 Virus mini kit), III (QIAamp DSP virus kit), and IV (heating); the extractions are made from plasma collected on EDTA tubes, and the concentration of extracted DNA was measured on NanoDrop Lite followed by real-time CMVPCR using an Artus CMV QS-RGQ kit. All protocols are performed following the manufacturer’s instructions. Results: This study is conducted on the samples of 135 transplant patients whose follow-up medical tests related to human cytomegalovirus infection; since most of the CMVPCR results are negative, we have chosen the 10 CMVPCR positive samples and 2 negative samples as controls to conduct this comparison study. By using NanoDrop Lite to evaluate the DNA concentration, the yield of extracted DNA is higher in our heating protocol than other protocols, the EZ1 DSP virus kit and EZ1 Virus mini kit show homogeneous quantities, and the QIAamp DSP virus kit shows very low DNA yields. Comparing cycle threshold and viral loads by real-time PCR, all these protocols identified negative samples (100%), and the previously positive samples used were as follows: protocol IV (90%), protocol II (60%), and protocol I (40%). QIAamp DSP virus kit results were not real-time PCR applicable and were non-conclusive because of the low DNA yields. Conclusion: Our developed heating method (protocol IV) is very effective, reliable, simple, fast, and cheap compared to the other protocols in our study.

Sign in / Sign up

Export Citation Format

Share Document