Accuracy of real-time polymerase chain reaction to detect Schistosoma mansoni – infected individuals from an endemic area with low parasite loads

Parasitology ◽  
2020 ◽  
Vol 147 (10) ◽  
pp. 1140-1148
Author(s):  
Fernanda do Carmo Magalhães ◽  
Samira Diniz Resende ◽  
Carolina Senra ◽  
Carlos Graeff-Teixeira ◽  
Martin Johannes Enk ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Schistosoma Mansoni ◽  
Real Time ◽  
Diagnostic Methods ◽  
Control Measures ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Cross Sectional ◽  
Intestinal Schistosomiasis ◽  
Polymerase Chain

AbstractDue to the efforts to control schistosomiasis transmission in tropical countries, a large proportion of individuals from endemic areas present low parasite loads, which hinders diagnosis of intestinal schistosomiasis by the Kato-Katz (KK) method. Therefore, the development of more sensitive diagnostic methods is essential for efficient control measures. The aim was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) to detect Schistosoma mansoni DNA in fecal samples of individuals with low parasite loads. A cross-sectional population-based study was conducted in a rural community (n = 257) in Brazil. POC-CCA® was performed in urine and feces were used for RT-PCR. In addition, fecal exams were completed by 18 KK slides, saline gradient and Helmintex techniques. The combined results of the three parasitological tests detected schistosome eggs in 118 participants (45.9%) and composed the consolidated reference standard (CRS). By RT-PCR, 117 out of 215 tested samples were positive, showing 91.4% sensitivity, 80.2% specificity and good concordance with the CRS (kappa = 0.71). RT-PCR identified 86.9% of the individuals eliminating less than 12 eggs/g of feces, demonstrating much better performance than POC-CCA® (50.8%). Our results showed that RT-PCR is a valuable alternative for the diagnosis of intestinal schistosomiasis in individuals with very low parasite loads.

scholarly journals Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus

2012 ◽  
Vol 24 (5) ◽  
pp. 959-963 ◽  
Author(s):  
Dolores Buitrago ◽  
Ana Rocha ◽  
Cristina Tena-Tomás ◽  
Marta Vigo ◽  
Montserrat Agüero ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Diagnostic Methods ◽  
Nucleotide Sequencing ◽  
Alectoris Rufa ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Field Samples ◽  
Polymerase Chain

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5’-Taq nuclease-3’ minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses ( Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak ( n = 11), as well as samples collected from partridges during surveillance programs ( n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

scholarly journals Usefulness of TrueNat: A Chip-based Real-time PCR Test for COVID-19

2021 ◽  
Author(s):  
Swarnim Swarn ◽  
Indu Prasad ◽  
Amit Kumar Anand ◽  
Binod Shankar Singh
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Reaction System ◽  
Kappa Coefficient ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Cross Sectional ◽  
Percent Agreement ◽  
Polymerase Chain

Introduction: For the containment of growing Coronavirus Disease (COVID-19) pandemic, rapid diagnostic facilities are need of today. Indigenously developed TrueNat assay is a point-of-care assay developed for early diagnosis of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). It is a portable, fully automated, chip-based, real-time quantitative polymerase chain reaction system with a turnaround time of 1.5-2 hours. Aim: To assess the practical utility and diagnostic accuracy of TrueNat testing for COVID-19 in a pandemic situation. Materials and Methods: A cohort selection cross-sectional study was conducted from July to September 2020 at Department of Biochemistry, Vardhaman Institute of Medical Sciences, Pawapuri, Bihar, India, after obtaining Institutional Ethics Committee (IEC) approval. A total of 296 cases with symptoms of COVID-19 were selected for the study. Assuming real-time Reverse Transcription- Polymerase Chain Reaction (rRT-PCR) to be the gold standard, we collected oropharyngeal swabs from symptomatic COVID-19 suspected cases and tested by both TrueNat and standard RT-PCR. Agreement between both the assays were assessed by overall, Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) and Cohen’s kappa coefficient using Epitools (Ausvet 2020). Results: Out of 296 oropharyngeal swabs taken from suspected COVID-19 patients, 19 were read as “invalid” and discarded; hence only 277 samples were tested by TrueNat and RT-PCR both. Assuming RT-PCR as standard, TrueNat assay demonstrated an overall percent agreement of 99.64%, PPA of 95.65%, NPA 99.81%. The kappa coefficient was 0.9546. Conclusion: TrueNat assay offers a rapid, accurate and affordable technique for COVID-19. It may be deployed for mass screening and confirmation of COVID-19 cases in hospitals and remote areas.

scholarly journals Real-time PCR assay for the diagnosis of pleural tuberculosis

2017 ◽  
pp. 47-52
Author(s):  
Martha Alejandra Casallas-Rivera ◽  
Ana María Cardenas Bernal ◽  
Luis Fernando Giraldo-Cadavid ◽  
Enrique Prieto Diago ◽  
Paola Santander
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reference Method ◽  
Operating Characteristics ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Cross Sectional ◽  
Pleural Tuberculosis ◽  
Hybridization Probes ◽  
Polymerase Chain

Introduction: The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak.Objective: To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis.Method: The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen or Mycobacterium tuberculosis culture in fluid or pleural tissue, or pleural biopsy with granulomas. Pleural tuberculosis was ruled out (in 31 patients) if all three tests were negative. The operating characteristics of the RT-PCR, using the Mid-P Exact Test, were determined using the OpenEpi 2.3 Software (2009).Results: The RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia.Conclusions: The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis.

scholarly journals Prevalence of COVID-19 Positive Cases Diagnosed By Real Time Polymerase Chain Reaction and Mortality from SARS-Cov-2 among Suspected Population

2021 ◽  
Vol 01 (10) ◽  
Author(s):  
Aklima Akter ◽  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Disease Transmission ◽  
Virus Disease ◽  
Age Group ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Cross Sectional ◽  
Total Recovery ◽  
Polymerase Chain

SARS-CoV-2, a newly emergent virus is the responsible agent for causing Corona Virus Disease 2019 (COVID-19) which is an outgoing pandemic. Test for SARS-CoV-2 is necessary not only to confirm the cases but also to control its transmission. To diagnose Covid-19, Real Time Polymerase Chain Reaction (RT-PCR) for SARS-CoV-2 is used. A retrospective, cross sectional research was conducted in Brahmanbaria Medical college to find out the prevalence of RT-PCR positivity in suspected COVID-19 patients presented from July, 2021 to August, 2021 in the Department of Microbiology. Data was collected from the registry book of the Department of Microbiology. Among the total suspected samples (n=2025), about 1145 (56.54%) cases found positive. Among the positive cases, 59% were symptomatic, and 41% were asymptomatic. Out of the total confirmed cases, 487 (42.53%) were male & 658 were (56.54) % female. Among the confirmed cases, total recovery cases were 1057 (92.3 %) and death cases were 88 (7.7%). More death was observed in age group between 51-60 years. More than half of the positive cases with a medium number of asymptomatic population indicate a high chance of disease transmission. Female being the more vulnerable group of getting infected and age group above 50 years were more prone to succumb.

scholarly journals Optimization Of Real-Time Reverse Transcriptase Polymerase Chain Reaction For Detection Of Dengue Virus

2020 ◽  
Author(s):  
Kaunara Ally Azizi ◽  
Arnold J Ndaro ◽  
Athanasia Maro ◽  
Adonira T Saro ◽  
Reginald Kavishe
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Diagnostic Methods ◽  
Virus Infections ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Objective Rapid and accurate laboratory confirmatory is very essential for control measures of dengue virus infections. However, many cases of dengue virus infections in most of the hospitals remain undiagnosed due to presence of other febrile illnesses with overlapping symptoms and lack of specificity in most of laboratory diagnostic methods. This study was set to optimize conditions for real time reverse transcriptase polymerase chain reaction (RT-PCR) for detection of dengue virus by using rapid and simple nucleic acid extraction method.Results The real time RT-PCR technique was successfully optimized using simple and rapid method for purification of nucleic acid, ‘boom method’. The technique works better when performed in a two-step procedure and can works well with all range of real time PCR machines. The optimized real time RT-PCR used in the present study is a valuable and reliable technique for routine diagnosis of dengue. Further investigation on the cost effectiveness in adopting this technique for routine screening and monitoring of the dengue infection should be done.

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

2021 ◽  
Author(s):  
Rajeev Kumar Jain ◽  
Nagaraj Perumal ◽  
Rakesh Shrivastava ◽  
Kamlesh Kumar Ahirwar ◽  
Jaya Lalwani ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Specific Gene ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Gene Target ◽  
Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

scholarly journals Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

2018 ◽  
Vol 6 (1) ◽  
pp. 2 ◽  
Author(s):  
David De la Torre ◽  
Claudete Astolfi-Ferreira ◽  
Ruy Chacon ◽  
Antonio Piantino Ferreira
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Sybr Green ◽  
Molecular Techniques ◽  
Economic Losses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Rotavirus A ◽  
Avian Nephritis Virus ◽  
Polymerase Chain

Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.

scholarly journals Evaluation of a 384–Well Format for High-Throughput Real-Time Reverse Transcription Polymerase Chain Reaction for Avian Influenza Testing

2009 ◽  
Vol 21 (5) ◽  
pp. 679-683 ◽  
Author(s):  
Pamela J. Ferro ◽  
Jason Osterstock ◽  
Bo Norby ◽  
Geoffrey T. Fosgate ◽  
Blanca Lupiani
Keyword(s):  
Polymerase Chain Reaction ◽  
Avian Influenza ◽  
Real Time ◽  
High Throughput ◽  
Reverse Transcription ◽  
Virus Isolation ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Response Planning ◽  
Polymerase Chain

As concerns over the global spread of highly pathogenic avian influenza H5N1 have heightened, more countries are faced with increased surveillance efforts and incident response planning for handling a potential outbreak. The incorporation of molecular techniques in most diagnostic laboratories has enabled fast and efficient testing of many agents of concern, including avian influenza. However, the need for high-throughput testing remains. In this study, the use of a 384–well format for high-throughput real-time reverse transcription polymerase chain reaction (real-time RT-PCR) testing for avian influenza is described. The analytical sensitivity of a real-time RT-PCR assay for avian influenza virus matrix gene with the use of both 96– and 384–well assay formats and serial dilutions of transcribed control RNA were comparable, resulting in similar limits of detection. Of 28 hunter-collected cloacal swabs that were positive by virus isolation, 26 (92.9%) and 27 (96.4%) were positive in the 96– and 384–well assays, respectively; of the 340 hunter-collected swabs that were negative by virus isolation, 45 (13.2%) and 23 (6.8%) were positive in the 96– and 384–well assays, respectively. The data presented herein supports the utility of the 384–well format in the event of an avian influenza outbreak for high-throughput real-time RT-PCR testing.

scholarly journals Collaborative Trial for Validation of a Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay for Detection of Central Nervous System Tissues as Bovine Spongiform Encephalopathy RiskMaterial: Part 1

2006 ◽  
Vol 89 (5) ◽  
pp. 1335-1340
Author(s):  
Amir Abdulmawjood ◽  
Holger Schnenbrcher ◽  
Michael BÜlte
Keyword(s):  
Central Nervous System ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meat Products ◽  
Spongiform Encephalopathy ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Heat Treated ◽  
Polymerase Chain

Abstract A collaborative trial was conducted to evaluate a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of central nervous system (CNS) tissues in meat products (e.g., sausages). The method is based on the detection of ruminant glial fibrillary acidic protein (GFAP) mRNA by applying real-time RT-PCR. The assay was evaluated through a multicenter trial involving 12 participating laboratories that received coded cDNA obtained from 3 different types of sausages. The participants used 5 different real-time detection systems. The results obtained in this validation revealed that this real-time RT-PCR assay performed well in the different laboratories with a detection limit of at least 0.1% CNS in those test materials that contained strongly heat-treated samples (sausages cooked at 120C) and the medium heat-treated samples (sausages cooked at 80C). The detection limit of liver sausages was determined to be 0.2% of CNS. Neither the samples with no CNS additive nor the bovine DNA and the negative control containing 100% swine brain gave any positive signals. The presented results indicate that the real-time RT-PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, could reliably be used for detection of bovine spongiform encephalopathy risk material in meat and meat products, and signify that it may be used with confidence in any laboratory.

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