Comparison of ELISA and RT-PCR versus Immune Electron Microscopy for Detection of Bovine Torovirus (Breda Virus) in Calf Fecal Specimens

2003 ◽  
Vol 15 (2) ◽  
pp. 100-106 ◽  
Author(s):  
Armando E. Hoet ◽  
Kyeong-Ok Chang ◽  
Linda J. Saif
Keyword(s):  
Epidemiological Survey ◽  
Epidemiological Studies ◽  
Diagnostic Techniques ◽  
Enzyme Linked Immunosorbent Assay ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Cutoff Value ◽  
Kappa Value ◽  
Sensitivity Specificity

Bovine Torovirus (BoTV) is an uncultivable enteric pathogen of cattle. Its failure to grow in vitro limits epidemiological studies, characterization of the virus, and development of diagnostic techniques. The objectives of this study were to develop and standardize an antigen-capture enzyme-linked immunosorbent assay (ELISA) and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of BoTV in fecal specimens. These assays were compared with immunoelectron microscopy (IEM) to evaluate their sensitivity, specificity, and efficiency as well as their advantages and limitations. Additionally, several methods to calculate ELISA cutoff values were used and compared using a statistical approach to obtain the optimal cutoff value for the ELISA. A plate cutoff ELISA value was determined to be the best method to calculate the cutoff value. The ELISA and RT-PCR assays developed in this study identified BoTV antigen and viral nucleic acids in feces without cross-reactions with the other calf enteric viruses examined. Both assays showed good agreement with IEM, with a Kappa value of 0.86 for ELISA and 0.85 for RT-PCR. The latter exhibited the higher analytical sensitivity. On the basis of the results obtained in this study, it is recommended that no single test should be used alone in an epidemiological survey because of the observed limitations of each assay. The fast and inexpensive ELISA combined with the highly specific and sensitive RT-PCR are a practical approach for future epidemiological studies of BoTV. These results should provide other researchers with the information needed to develop similar diagnostic assays for the study of BoTV.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

scholarly journals Design and Validation of Transcription-Mediated-Amplification Nucleic Acid Amplification Tests forMycoplasma genitalium

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Brett Kirkconnell ◽  
Barbara Weinbaum ◽  
Katherine Santos ◽  
Trisha Le Nguyen ◽  
Bryan Vinluan ◽  
...  
Keyword(s):  
Clinical Performance ◽  
Mycoplasma Genitalium ◽  
Vaginal Swab ◽  
Nucleic Acid Amplification ◽  
Clinical Validation ◽  
Analytical Sensitivity ◽  
Reference Standard ◽  
Content Type ◽  
Sensitivity Specificity

ABSTRACTMycoplasma genitaliumis a sexually transmitted bacterium linked to adverse sexual and reproductive health outcomes in women and men.M. genitaliumis difficult to culture, and in the absence of validated amplified molecular methods for diagnosis of infection, there is no reference standard available for use as a comparator for the validation of newM. genitaliumdiagnostic tests. We evaluated the analytical and clinical performance of three transcription-mediated amplification (TMA) tests forM. genitalium, each targeting unique rRNA sequences, for use as a composite comparator for clinical validation of the AptimaMycoplasma genitalium(AMG) assay, anin vitrodiagnostic (IVD) TMA test that targets 16 s rRNA ofM. genitalium. Analytical sensitivity, specificity, and strain inclusivity of all four TMA tests were determined using nine laboratory strains ofM. genitaliumand 56 nontarget bacteria, protozoa, and viruses. Analytical sensitivity of the tests forM. genitaliumranged from 0.017 to 0.040 genome equivalents/ml. None of the nontarget organisms evaluated cross-reacted with any test. A composite comparator reference standard consisting of the 3 alternate (Alt) TMA tests was used to evaluate the clinical performance of the AMG assay by testing residual vaginal swab, female urine, and male urine specimens obtained from 1,400 adult subjects from three U.S. clinical sites. Using this reference standard to establish infected specimen status, the sensitivity, specificity, and overall agreement of the AMG IVD assay were 100%, 99.9%, and 99.9%, respectively. These results demonstrate the utility of molecular composite reference standard methodology for the clinical validation of future IVD tests for this organism.

scholarly journals Validation of a Methodology for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva by Real-Time Reverse Transcriptase-PCR

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniel F. Escobar ◽  
Pablo Díaz ◽  
Diego Díaz-Dinamarca ◽  
Rodrigo Puentes ◽  
Pedro Alarcón ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Reverse Transcriptase ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Health Authorities ◽  
Specific Alternative ◽  
Pcr Method ◽  
Sensitivity Specificity ◽  
Study Participants

In January 2021, the Chilean city of Concepción experienced a second wave of coronavirus 2019 (COVID-19) while in early April 2021, the entire country faced the same situation. This outbreak generated the need to modify and validate a method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva, thereby expanding the capacity and versatility of testing for COVID-19. This study was conducted in February 2021 in the Chilean city of Concepción during which time, the town was under total quarantine. The study participants were mostly symptomatic (87.4%), not hospitalized, and attended care centers because of their health status rather than being asked by the researchers. People coming to the health center in Concepción to be tested for COVID-19 (via reverse transcriptase polymerase chain reaction [RT-PCR]) from a specimen of nasopharyngeal swab (NPS) were then invited to participate in this study. A total of 131 participants agreed to sign an informed consent and to provide saliva and NPS specimens to validate a method in terms of sensitivity, specificity, and statistical analysis of the cycle threshold (Ct) values from the RT-PCR. Calculations pertaining to the 127 participants who were ultimately included in the analysis showed sensitivity and specificity at 94.34% (95% CI: 84.34–98.82%) and 98.65% (95% CI: 92.70–99.97%), respectively. The saliva specimen showed a performance comparable to NPS as demonstrated by the diagnostic parameters. This RT-PCR method from the saliva specimen is a highly sensitive and specific alternative compared to the reference methodology, which uses the NPS specimen. This modified and validated method is intended for use in the in vitro diagnosis of SARS-CoV-2, which provides health authorities in Chile and local laboratories with a real testing alternative to RT-PCR from NPS.

Agreement of Enzyme-Linked Immunosorbent Assay With Rapid Plasma Reagin and Treponema pallidum Hemagglutination Assay for Screening and Diagnosis of Syphilis at National Blood Bank Service in Addis Ababa, Ethiopia

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S153-S154
Author(s):  
Jemal Ibrahim ◽  
Ejigayehu Afework ◽  
Mintewab Hussein
Keyword(s):  
Positive Predictive Value ◽  
Predictive Value ◽  
Addis Ababa ◽  
Treponema Pallidum ◽  
Blood Bank ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rapid Plasma Reagin ◽  
Hemagglutination Assay ◽  
Kappa Value ◽  
Sensitivity Specificity

Abstract Objectives To determine the agreement of enzyme-linked immunosorbent assay (ELISA) with rapid plasma reagin (RPR) and Treponema pallidum hemagglutination assay (TPHA) at the National Blood Bank Service in Addis Ababa, Ethiopia. Methods The study was conducted from January to June 2016 on 190 syphilis ELISA positive and 190 negative samples stored at the National Blood Bank Service (NBBS) laboratory, Addis Ababa, Ethiopia, from July 2015 to December 2015. A systematic random sampling method was used to select samples. The data were analyzed by SPSS version 20 software. The overall percent agreement, kappa value, sensitivity, specificity, positive predictive value, and negative predictive value of the tests were calculated. Results From 190 positive sera, 151 (80%) were confirmed as positive by TPHA, and 39 (20%) were found false positive; 59 (31.1%) of them were positive and 131 (68.9%) were false positive by RPR. From 190 negative sera, all were negative by RPR and TPHA. The sensitivity, specificity, positive predictive value, and negative predictive value of TPHA were 99.9%, 85%, 79%, and 100%, respectively, while RPR was 62%, 99%, 100%, and 63%, respectively. Overall percent agreement of ELISA with TPHA was 90% and corresponding kappa value was 0.795, and ELISA with RPR was 66% with a kappa value of 0.375. Conclusion TPHA was very sensitive; there was substantial agreement with ELISA. Whereas RPR was highly specific and less sensitive, there was fair agreement with ELISA. TPHA can be used interchangeably with ELISA to screen blood in blood bank laboratories.

scholarly journals Comparison of Three Enzyme-Linked Immunosorbent Assays for Serologic Diagnosis of Contagious Agalactia in Sheep

2003 ◽  
Vol 15 (3) ◽  
pp. 281-285 ◽  
Author(s):  
Michel Pépin ◽  
Philippe Dufour ◽  
Maurice Lambert ◽  
Michel Aubert ◽  
Aurèle Valognes ◽  
...  
Keyword(s):  
False Negative ◽  
Good Alternative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Analytical Sensitivity ◽  
Diagnostic Specificity ◽  
True Negative ◽  
Serologic Diagnosis ◽  
Contagious Agalactia ◽  
Kappa Value ◽  
Kinetics Of

Serologic diagnosis of ovine contagious agalactia ( Mycoplasma agalactiae) with the enzyme-linked immunosorbent assay (ELISA) developed by Agence Française de Séceurité Sanitaire des Aliments (AFS-SA) may produce a few false-positive (FP) and false-negative (FN) results. When the prevalence of disease is low, these erroneous results may generate problems for eradication schemes. To prevent this, 2 commercial ELISAs were compared with the AFSSA ELISA. Flocks of known status were selected and classified into 4 categories: true positive (TP), FP, true negative (TN), and FN; 20 sheep per flock were submitted for blood sampling. A flock was considered positive when at least 1 out of 20 sera was positive or 2 sera were doubtful. In the flock, the diagnostic sensitivity of the 3 kits was very good (100%), and the diagnostic specificity showed an improvement from 46% (AFSSA test) to 88% and 92% (commercial tests). Considering individual animals, very few positive ewes were detected within TN or FP flocks; the proportion of positive ewes varied greatly from one kit to another (48% to 82%) within TP flocks. The kinetics of antibody response in sheep experimentally infected with various field strains of M. agalactiae were quite similar with all 3 ELISAs. The agreement between the 3 tests, assessed using the kappa value, varied from moderate to good (respective values of 0.56, 0.61, and 0.86). The 2 commercial ELISAs showed better performances, probably because of a superior analytical sensitivity, and are a good alternative for the serodiagnosis of contagious agalactia in sheep.

scholarly journals Feline immunodeficiency virusinfection: a comparative study of different diagnostic techniques

2004 ◽  
Vol 56 (1) ◽  
pp. 13-18
Author(s):  
E. Mortola ◽  
G. Oliva ◽  
M. Risso ◽  
M. Pecoraro ◽  
M.C. Venturini
Keyword(s):  
High Sensitivity ◽  
Epidemiological Survey ◽  
Epidemiological Studies ◽  
Diagnostic Techniques ◽  
Immunofluorescence Assay ◽  
Feline Immunodeficiency Virus Infection ◽  
Immunodeficiency Virus ◽  
Pcr Method ◽  
Specific Reactivity ◽  
Polymerase Chain

This study evaluated an indirect immunofluorescence assay (IFA) to detect feline immunodeficiency virus infection (FIV) antibody in a comprehensive epidemiological survey of FIV in Argentina. IFA modified in our laboratory, was compared with two other immunoassays, western blot (WB) and a sandwich immunochromatographic commercial kit (SI), and also with a direct polymerase chain reaction (PCR) method that detects proviral DNA. IFA showed to be a test with high sensitivity and specificity, and could be useful as a diagnostic tool in epidemiological studies. The presence of a low percentage of results with non-specific reactivity in the IFA could be resolved with further testing or use of an alternative method.

scholarly journals Accuracy of cone-beam CT in detecting vertical root fractures in teeth with post-endodontic restorations: an in vitro study

2021 ◽  
Vol 43 ◽  
pp. e55832
Author(s):  
Fernanda Chiguti Yamashita ◽  
Amanda Lury Yamashita ◽  
Irma Milena Menck Romanichen ◽  
Elen de Souza Tolentino ◽  
Sérgio Sábio ◽  
...  
Keyword(s):  
Cone Beam Ct ◽  
In Vitro Study ◽  
Cone Beam ◽  
Control Group ◽  
Endodontically Treated Teeth ◽  
Kappa Value ◽  
Diagnostic Capacity ◽  
Root Fractures ◽  
Sensitivity Specificity

The aim of this study was to evaluate the accuracy of cone-beam CT (CBCT) for the detection of artificially created vertical root fractures (VRF) in extracted teeth restored with metallic (MP) and fiberglass (FGP) posts. After root canal obturation, 60 extracted human premolars were scanned by using the highest-resolution settings of a CBCT unit. The prepared roots were randomly divided into three groups: group C (control group): non-restored and non- endodontically treated teeth (n = 20); group MP (n = 20); group FGP (n = 20). In all groups, 10 teeth were artificially fractured. Two external and independent observers blindly recorded the presence or absence of VRF. Sensitivity, specificity, accuracy, intra- and interobserver agreement were calculated. Kappa value for inter- and intraobserver agreement was 0.82 and 0.84 respectively, demonstrating good agreement. The highest values for sensitivity (0.80 - 0.90), specificity (1.00) and accuracy (0.90 - 0.95) were found in the control group. The teeth with FGP restoration presented the lowest sensitivity (0.30 - 0.50) and accuracy (0.60 - 0.65) values. Both examiners had a good performance in the diagnosis of fractures in teeth with MP, with accuracy ranging between 0.85 - 0.90. The presence of MP did not influence accuracy; however, the presence of FGP reduced the diagnostic capacity of CBCT.

Target Testing and Specificity of Nucleic Acid Based Diagnostics for COVID-19

2021 ◽  
Vol 1 (2) ◽  
Author(s):  
Ghazala Rubi ◽  
Musbih ul Qayyum Zia ◽  
Muhammad Yousaf Rana ◽  
Ayaz Akbar ◽  
Zainab Javiad
Keyword(s):  
Nucleic Acid ◽  
Diagnostic Testing ◽  
Rt Pcr ◽  
Likelihood Ratios ◽  
International Importance ◽  
Amplification Process ◽  
Group 2 ◽  
Novel Coronavirus ◽  
Sensitivity Specificity

Objective: The seek of this study is to provide an indication on the features of diagnostic testing of SARS-CoV-2 by RT-PCR, including parameters of sensitivity, specificity, positive and negative likelihood ratios. Background: Coronavirus Disease is the fifth international emergency after 1918 Spanish flu pandemic, triggered by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2). On 30 January, the WHO acknowledged COVID-19 to be a global health disaster of international importance and a pandemic on 11 March 2020. In vitro analyses of the data shows that for SARS-CoV-2 the RT-PCR test is highly specific, as it is not counter react with nucleic acid of other viruses. Methods: Oropharyngeal and nasopharyngeal swabs were collected into a 3 ml viral transport media (VTM) and transported to Laboratory. Extraction of the viral RNA was done by Qiasymphony DSP Virus/ Pathogen mini kit (Qiagen GmbH, Germany). For amplification process of RT-PCR qualitative detection of SARS-CoV-2 RNA utilizing with SYSTAAQ 2019-Novel Coronavirus (COVID-19) Real time PCR kit using a BIORAD-CFX 96. Results: Out of 15,049, 3195 samples were positive for covid-19 qPCR. Ratio of the Males patients were greater than females. 63.7% males and 36.3% females were effected with Covid-19. Symptom wise analysis shows 62% patient were asymptomatic, 22.7% mild, 1.7% moderate, 12.7% stable, 0.6% severe and 0.2% were critical. Our analysis reveals age group 1 (4.9%), group 2 (55.5%), group 3 (27.5%), and group 4 (12.1%) were effected with SARS-nCoV-2. Our result shows 3.0 % patients were deceased and 97% were recovered. Conclusion: Our findings contribute to the evolving understanding of the sophisticated interaction between this emerging SARS-CoV-2 virus and nucleic acid based target testing of COVID-19.

ASSESSMENT OF THE POSSIBILITY OF APPLICATION IN LABORATORY PRACTICE OF REAGENT KIT FOR DIAGNOSIS OF GLANDERS AND MELIOIDOSIS BY REAL-TIME POLYMERASE CHAIN REACTION

2019 ◽  
Vol 64 (11) ◽  
pp. 700-704
Author(s):  
L. V. Lemasova ◽  
G. A. Tkachenko ◽  
E. V. Prokhvatilova ◽  
L. I. Belitskaya ◽  
D. V. Viktorov ◽  
...  
Keyword(s):  
Real Time ◽  
Diagnostic Value ◽  
Medical Laboratory ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Laboratory Practice ◽  
Environmental Objects ◽  
Polymerase Chain ◽  
State Examination

The reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR is designed for detecting in vitro diagnostics and differentiate the DNA of glanders and melioidosis pathogens by real-time multiplex PCR in biological (clinical) material and cultures of microorganisms, as well as environmental objects and solid food products (rice). During clinical testing diagnostic value of reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR has been studied. Based on the results obtained, a high analytical sensitivity (1×103 microbe cells/ml) and specificity (100%) of PCR-RT with the developed reagent kit were established, regardless of the type of material being studied. The diagnostic sensitivity of PCR-RT using a set of reagents was at least 98.0% and specificity at least 99%. The stages of state examination have been completed, a registration certificate has been obtained at Roszdravnadzor, production, sale and use of reagent kit in medical laboratory practice have been permitted.

scholarly journals In vitroeffects of fluticasone propionate on IL-13 production by mitogen-stimulated lymphocytes

2002 ◽  
Vol 11 (3) ◽  
pp. 187-190 ◽  
Author(s):  
Gabriele Di Lorenzo ◽  
Maria Luisa Pacor ◽  
Maria Esposito Pellitteri ◽  
Sebastiano Gangemi ◽  
Patrizia Di Blasi ◽  
...  
Keyword(s):  
Fluticasone Propionate ◽  
Healthy Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complete Medium ◽  
Therapeutic Effects ◽  
Rt Pcr ◽  
Corticosteroid Administration ◽  
Asthmatic Patients ◽  
Pcr Products

Background: Corticosteroid administration produces multiple immunomodulatory effects, including down-regulation of cytokine production by CD4 T lymphocytes. Fluticasone propionate (FP) (Glaxo Smith&Kline, Greenford, UK), a highly lipophilic topical corticosteroid, has been shown to be safe and effective in the treatment of asthma and of both seasonal and perennial rhinitis.Aims: To gain insight into the mechanisms of FP therapeutic effects, we evaluated interleukin (IL)-13 (a type 2 cytokine that seemingly plays a pivotal role in allergic mechanisms) production by mitogen-stimulated peripheral blood mononuclear cells (MNC)in vitro, treated or not with FP.Methods: MNC from 10 healthy subjects and 10 asthmatic atopic patients with Parietaria allergy were stimulated v/v with phytohaemagglutinin (PHA) (50 γ/ml) or with complete medium alone as a control. Culture supernatants,in vitrotreated or not with 10-7or 10-8M FP, were collected after 48 or 72 h incubation. IL-13 production was assessed by enzyme-linked immunosorbent assay. In random selected samples, after 4 or 24 h of cell cultures, RNA was extracted and IL-4 and IL-5 reverse transcriptase-polymerase chain reaction (RT-PCR) products analyzed.Results: At 48 h, there were no differences in IL-13 concentration in PHA-stimulated cultures between healthy subjects and asthmatic patients (93.6 ± 18.9 versus 111.0 ± 25.1 pg/ml). At 72 h, similar results were obtained (63.9 ± 3.0 versus 73.3 ± 2.5 pg/ml, respectively). At this time, however, IL-13 concentrations were significantly decreased versus 48 h both in asthmatics (p<0.001) and in controls (p<0.001). Treatment with 10-7M FP significantly reduced IL-13 production in healthy subjects and asthmatic patients both at 48 h (93.6 ± 18.9 versus 50.50 ± 10.6 pg/ml,p<0.001, and 111.0 ± 25.1 versus 59.3 ± 13.6 pg/ml,p<0.001, respectively) and at 72 h (63.9 ± 9.6 versus 35.5 ± 4.4 pg/ml,p<0.001, and 73.3 ± 8.0 versus 40.7 ± 4.5 pg/ml,p<0.001, respectively). Similar results were obtained with 10-8M FP at 48 and 72 h. Accordingly, evaluation of RT-PCR products from selected cell samples showed a FP dosage-dependent inhibition of IL-4 and IL-5 mRNA production both for healthy subjects and asthmatic patients.Conclusions: FPin vitroimpairs IL-13 production by PHA-stimulated MNC from asthmatic and control subjects. This strengthens previous suggestions that IL-13 inhibition by steroids may, at least in part, account for their therapeutic effects.

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