COMPARISON OF COVID‐19 LABORATORY DIAGNOSIS BY COMMERCIAL KITS: EFFECTIVITY OF RT‐PCR TO THE RT‐LAMP

Abstract Background Despite the global roll-out of rotavirus vaccines (RotaTeq/Rotarix / ROTAVAC/Rotasiil), mortality and morbidity due to group A rotavirus (RVA) remains high in sub-Saharan Africa, causing 104,000 deaths and 600,000 hospitalizations yearly. In Cameroon, Rotarix™ was introduced in March 2014, but, routine laboratory diagnosis of rotavirus infection is not yet a common practice, and vaccine effectiveness studies to determine the impact of vaccine introduction have not been done. Thus, studies examining RVA prevalence post vaccine introduction are needed. The study aim was to determine RVA prevalence in severe diarrhoea cases in Littoral region, Cameroon and investigate the role of other diarrheagenic pathogens in RVA-positive cases. Methods We carried out a study among hospitalized children < 5 years of age, presenting with acute gastroenteritis in selected hospitals of the Littoral region of Cameroon, from May 2015 to April 2016. Diarrheic stool samples and socio-demographic data including immunization and breastfeeding status were collected from these participating children. Samples were screened by ELISA (ProSpecT™ Rotavirus) for detection of RVA antigen and by gel-based RT-PCR for detection of the VP6 gene. Co-infection was assessed by multiplexed molecular detection of diarrheal pathogens using the Luminex xTAG GPP assay. Results The ELISA assay detected RVA antigen in 54.6% (71/130) of specimens, with 45, positive by VP6 RT-PCR and 54, positive using Luminex xTAG GPP. Luminex GPP was able to detect all 45 VP6 RT-PCR positive samples. Co-infections were found in 63.0% (34/54) of Luminex positive RVA infections, with Shigella (35.3%; 12/34) and ETEC (29.4%; 10/34) detected frequently. Of the 71 ELISA positive RVA cases, 57.8% (41/71) were fully vaccinated, receiving two doses of Rotarix. Conclusion This study provides insight on RVA prevalence in Cameroon, which could be useful for post-vaccine epidemiological studies, highlights higher than expected RVA prevalence in vaccinated children hospitalized for diarrhoea and provides the trend of RVA co-infection with other enteric pathogens. RVA genotyping is needed to determine circulating rotavirus genotypes in Cameroon, including those causing disease in vaccinated children.

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Cross-contamination in molecular laboratories: A challenge to COVID-19 testing in Bangladesh

Journal of Clinical Images and Medical Case Reports ◽

10.52768/2766-7820/1371 ◽

2021 ◽

Vol 2 (5) ◽

Author(s):

Mohammad Jahidur Rahman Khan ◽

◽

Selim Reza ◽

Farzana Mim ◽

Md Abdullah Rumman ◽

...

Keyword(s):

Public Health ◽

Large Scale ◽

Diagnostic Testing ◽

Diagnostic Errors ◽

Laboratory Diagnosis ◽

Cross Contamination ◽

Rt Pcr ◽

Public Health Response ◽

Surveillance Programs ◽

The Government

Rapid and accurate laboratory diagnosis of SARS-CoV-2 infection is crucial for the management of COVID-19 patients and control of the spread of the virus. At the start of the COVID-19 pandemic, Bangladesh had only one government molecular laboratory where real-time RT-PCR will be performed to diagnose SARS-CoV-2 infection. With the increasing number of suspected cases requiring confirmation diagnostic testing, there was a requirement to quickly expand capacity for large-scale testing. The government of Bangladesh established over 100 molecular laboratories within one year to test COVID-19. To fulfil the requirement for expanded testing, the government was compelled to recruit laboratory employees with inadequate experience, technical knowledge, and skills in molecular assays, particularly in processing specimens, interpreting results, recognizing errors, and troubleshooting. As a result, the risk of diagnostic errors, such as cross-contamination, is increased, as is that the risk of false-positive results, which might risk the patient’s health and undermine the efficacy of public health policies, public health response, surveillance programs, and restrictive measures aimed toward containing the outbreak. This review article aims to explain different sources of crosscontamination in the COVID-19 RT-PCR laboratories and the way to forestall them in efficient and practical ways.

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Lack of evidence of porcine reproductive and respiratory syndrome virus (PRRSV) infection in domestic swine in Brazil

Ciência Rural ◽

10.1590/s0103-84782004000200018 ◽

2004 ◽

Vol 34 (2) ◽

pp. 449-455 ◽

Cited By ~ 10

Author(s):

Janice Reis Ciacci-Zanella ◽

Cristiano Trombetta ◽

Ildara Vargas ◽

Denise Euclydes Mariano da Costa

Keyword(s):

Genetic Material ◽

Elisa Test ◽

Respiratory Syndrome Virus ◽

Rt Pcr ◽

Serum Samples ◽

Viral Isolation ◽

Culture Cells ◽

Commercial Kits ◽

Swine Herds ◽

Domestic Swine

This report describes the first prevalence of antibodies and experimental inoculation of suspected samples of porcine reproductive and respiratory syndrome virus (PRRSV) from ELISA positive pigs from swine herds in Brazil. Based on the hypothesis that this agent is present in swine herds worldwide, the objective of this work was to establish a diagnostic methodology and to investigate the occurrence of PRRSV in Brazilian swine herds. Fifty-four swine herds, the total number which imported genetic material (live pigs or swine semen) from countries where PRRS was endemic from 1990 to December 2000, from eight Brazilian States all included in this study. The sampling used was such as to detect a prevalence of infection of 5%, with a confidence level of 95%. A total of 3785 serum samples were tested for PRRSV antibodies by ELISA. Following the ELISA test, which was performed with two different commercial kits, all serum positive pigs were retested, examined and additional materials were collected. Viral isolation in permissive tissue culture cells and swine bioassays were performed. Additionally, reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR were also performed. We could not demonstrate the presence of PRRSV or RNA of PRRSV by viral isolation or RT-PCR (or nested RT-PCR), respectively in all of the analyzed samples. Furthermore, the pigs inoculated with PRRSV suspicion samples did not seroconvert nor produce characteristic PRRS lesions in the swine bioassay. Thus, our results indicate no evidence of PRRSV in the samples analyzed from swine herds in this study.

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Methods for Molecular Surveillance of Influenza Used in Macedonia

PRILOZI ◽

10.2478/prilozi-2014-0003 ◽

2014 ◽

Vol 35 (2) ◽

pp. 25-30

Author(s):

Golubinka Bosevska ◽

Elizabeta Janceska ◽

Gordana Kuzmanovska ◽

Vladimir Mikik ◽

Nikola Panovski

Keyword(s):

Influenza Virus ◽

Predictive Value ◽

Influenza A ◽

Rna Extraction ◽

Influenza Virus Infection ◽

Laboratory Diagnosis ◽

Influenza Viruses ◽

Rt Pcr ◽

Two Samples ◽

Nose And Throat

AbstractThe aim: To present and compare different Nucleic Acid Testing assays used for laboratory diagnosis of influenza virus infection in our country.Materials and methods: Respiratory samples used were nose and throat swabs. The RNA extraction was performed with a QIAamp viral RNA kit. During the season 2009–2010 the first 25 samples were tested with: conventional gel-based RT-PCR and CDC rtRT-PCR using published specific matrix and HA gene primers and probes for influenza virus typing and subtyping.Results: Of 25 samples tested with conventional RT-PCR7(28%) were positive for influenza A, but negative for A/H1seasonal and A/H3. Retested with rtRT-PCR 9(36%) were positive for influenza A, 8(32%) were positive for A/H1pdm and 1(4%) was A/H3. Two samples positive with rtRT-PCR for influenza A were negative with RT-PCR. The sensitivity of the RT-PCR in comparison with rtRT-PCR is 100% and the specificity is 88.89%. Positive predictive value for RT-PCR is 77.78%, and negative predictive value is 100%. RT-PCR is a four-step and rtRT-PCR a one-step procedure. The turn-around time of RT-PCR is 6 hours and for rtRT-PCR it is 2 hours.Discussion and conclusion: For surveillance purposes nose and throat swabs are the more easy and practical to collect. It was proved that RT-PCR is too laborious, multi-step and time-consuming. The sensitivity of both assays is equal. The specificity of rtRT-PCR is higher. NAT assays for detection of influenza viruses have become an integral component of the surveillance programme in our country. They provide a fast, accurate and sensitive detection of influenza.

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Potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (COVID-19)

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-0285 ◽

2020 ◽

Vol 58 (7) ◽

pp. 1070-1076 ◽

Cited By ~ 179

Author(s):

Giuseppe Lippi ◽

Ana-Maria Simundic ◽

Mario Plebani

Keyword(s):

Diagnostic Accuracy ◽

Diagnostic Errors ◽

Laboratory Diagnosis ◽

Test Results ◽

Rt Pcr ◽

Viral Recombination ◽

Technical Issues ◽

Diagnostic Window ◽

Novel Coronavirus ◽

And Storage

AbstractA novel zoonotic coronavirus outbreak is spreading all over the world. This pandemic disease has now been defined as novel coronavirus disease 2019 (COVID-19), and is sustained by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As the current gold standard for the etiological diagnosis of SARS-CoV-2 infection is (real time) reverse transcription polymerase chain reaction (rRT-PCR) on respiratory tract specimens, the diagnostic accuracy of this technique shall be considered a foremost prerequisite. Overall, potential RT-PCR vulnerabilities include general preanalytical issues such as identification problems, inadequate procedures for collection, handling, transport and storage of the swabs, collection of inappropriate or inadequate material (for quality or volume), presence of interfering substances, manual errors, as well as specific aspects such as sample contamination and testing patients receiving antiretroviral therapy. Some analytical problems may also contribute to jeopardize the diagnostic accuracy, including testing outside the diagnostic window, active viral recombination, use of inadequately validated assays, insufficient harmonization, instrument malfunctioning, along with other specific technical issues. Some practical indications can hence be identified for minimizing the risk of diagnostic errors, encompassing the improvement of diagnostic accuracy by combining clinical evidence with results of chest computed tomography (CT) and RT-PCR, interpretation of RT-PCR results according to epidemiologic, clinical and radiological factors, recollection and testing of upper (or lower) respiratory specimens in patients with negative RT-PCR test results and high suspicion or probability of infection, dissemination of clear instructions for specimen (especially swab) collection, management and storage, together with refinement of molecular target(s) and thorough compliance with analytical procedures, including quality assurance.

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Challenges in Laboratory Diagnosis of the Novel Coronavirus SARS-CoV-2

Viruses ◽

10.3390/v12060582 ◽

2020 ◽

Vol 12 (6) ◽

pp. 582 ◽

Cited By ~ 61

Author(s):

Nadin Younes ◽

Duaa W. Al-Sadeq ◽

Hadeel AL-Jighefee ◽

Salma Younes ◽

Ola Al-Jamal ◽

...

Keyword(s):

Point Of Care ◽

Accurate Determination ◽

High Sensitivity ◽

Laboratory Diagnosis ◽

The Novel ◽

Viral Shedding ◽

Diagnostic Capacity ◽

Commercial Kits ◽

Novel Coronavirus ◽

Diagnostic Technologies

The recent outbreak of the Coronavirus disease 2019 (COVID-19) has quickly spread worldwide since its discovery in Wuhan city, China in December 2019. A comprehensive strategy, including surveillance, diagnostics, research, clinical treatment, and development of vaccines, is urgently needed to win the battle against COVID-19. The past three unprecedented outbreaks of emerging human coronavirus infections at the beginning of the 21st century have highlighted the importance of readily available, accurate, and rapid diagnostic technologies to contain emerging and re-emerging pandemics. Real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) based assays performed on respiratory specimens remain the gold standard for COVID-19 diagnostics. However, point-of-care technologies and serologic immunoassays are rapidly emerging with high sensitivity and specificity as well. Even though excellent techniques are available for the diagnosis of symptomatic patients with COVID-19 in well-equipped laboratories; critical gaps still remain in screening asymptomatic people who are in the incubation phase of the virus, as well as in the accurate determination of live viral shedding during convalescence to inform decisions for ending isolation. This review article aims to discuss the currently available laboratory methods and surveillance technologies available for the detection of COVID-19, their performance characteristics and highlight the gaps in current diagnostic capacity, and finally, propose potential solutions. We also summarize the specifications of the majority of the available commercial kits (PCR, EIA, and POC) for laboratory diagnosis of COVID-19.

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Preventive Effects of Evodiamine on Dexamethasone-Induced Osteoporosis in Zebrafish

BioMed Research International ◽

10.1155/2019/5859641 ◽

2019 ◽

Vol 2019 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Heng Yin ◽

Jianwei Wang ◽

Mao Wu ◽

Yong Ma ◽

Shanfu Wang ◽

...

Keyword(s):

Western Blot ◽

Mapk Pathway ◽

Bone Mineralization ◽

Tartrate Resistant Acid Phosphatase ◽

Mrna Levels ◽

Rt Pcr ◽

Alizarin Red ◽

Calcium Phosphorus ◽

Commercial Kits ◽

Preventive Effects

The aim of this study was to investigate the effect of evodiamine (EV) on dexamethasone-induced osteoporosis in zebrafish. Zebrafish larvae were exposed to different concentrations of dexamethasone to obtain the osteoporosis in zebrafish. Calcium, phosphorus, and alizarin red staining determination were performed to evaluate the effects of EV on bone mineralization. Alkaline phosphatase (ALP), hydroxyproline (HP), and tartrate resistant acid phosphatase (TRAP) were also measured by commercial kits. The expression of MMP3-OPN-MAPK pathway in zebrafish was measured by Western blot. RT-PCR was used to determine mRNA levels of MMP3, OPN, and MAPK. EV could significantly increase the content of calcium and phosphorus. The results of alizarin red staining showed that EV could significantly increase the calcium sink of horse fish, increasing the area of bone formation. EV could increase the content of hydroxyproline in zebrafish. EV also increased ALP and TRAP in zebrafish. Western blot and RT-PCR results showed that EV restored the MMP3-OPN-MAPK pathway in zebrafish. In conclusion, we found that EV can alleviate dexamethasone-induced osteoporosis in zebrafish. The mechanism is related to activating MMP3-OPN-MAPK pathway and then activating bone remodeling.

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Evaluation of RT-PCR Assay for Routine Laboratory Diagnosis of Rabies in Post Mortem Brain Samples from Different Species of Animals

Indian Journal of Virology ◽

10.1007/s13337-012-0109-9 ◽

2012 ◽

Vol 23 (3) ◽

pp. 392-396 ◽

Cited By ~ 8

Author(s):

R. P. Aravindh Babu ◽

S. Manoharan ◽

P. Ramadass ◽

N. D. J. Chandran

Keyword(s):

Laboratory Diagnosis ◽

Routine Laboratory ◽

Post Mortem ◽

Rt Pcr ◽

Pcr Assay ◽

Post Mortem Brain

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Development and application of a triplex TaqMan RT-PCR assay for simultaneous detection of Feline calicivirus, Feline Herpesvirus 1 and Feline parvovirus

10.21203/rs.3.rs-205583/v1 ◽

2021 ◽

Author(s):

Xiyu Zhang ◽

Zhihui Tang ◽

Haoyan Niu ◽

Liping Yan ◽

suquan song

Keyword(s):

Simultaneous Detection ◽

Fold Increase ◽

Feline Calicivirus ◽

Rt Pcr ◽

Pcr Assay ◽

Coefficients Of Variation ◽

Feline Herpesvirus ◽

Repeatability And Reproducibility ◽

Pcr Method ◽

Commercial Kits

Abstract The feline calicivirus (FCV), feline herspesvirus 1 (FHV-1) and feline panleukemia virus (FPV) are heavily threaten the health of cats. In this study, a triplex TaqMan real-time polymerase chain reaction (RT-PCR) assay (triplex assay) was developed to detect these viruses. The optimized concentration of primers was 0.5 µM of each, probes concentration was 0.1 µM for FCV and FHV-1, 0.05 µM for FPV. The annealing temperature was set at 54 ℃. The triplex RT-PCR assay was carefully validated. The detection limit for FPV, FCV, and FHV-1 was 5×101 copies/µL, which showed a 10-100-fold increase in the sensitivity compared with the conventional PCR. The coefficients of variation (CV) of the intra-assay variability of the test were < 1.86%, and that of inter-assay was < 3.19%, indicating excellent repeatability and reproducibility of the triplex assay. Additionally, the assay has perfect specificity. In a pilot study, samples from 48 cats were analyzed using the triplex RT-PCR method and the commercial kits, and further confirmed by sequencing. The positive rates for the samples analyzed with these two methods were 70.83% and 62.5%, respectively, which demonstrated that the developed method was more accurate than the commercial kits in clinical diagnosis.

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Comparison of SARS-CoV-2 antigen electrochemiluminescence immunoassay to RT-PCR assay for laboratory diagnosis of COVID-19 in Peshawar

Diagnosis ◽

10.1515/dx-2021-0078 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Bilal Iqbal ◽

Maria Khan ◽

Noman Shah ◽

Mirza Muhammad Dawood ◽

Valeed Jehanzeb ◽

...

Keyword(s):

Laboratory Diagnosis ◽

Cross Sectional Study ◽

Antigen Test ◽

Rt Pcr ◽

Pcr Assay ◽

Cross Sectional ◽

Viral Loads ◽

Testing Algorithms ◽

Frequent Testing ◽

Antigen Testing

Abstract Objectives Antigen based rapid diagnostic tests possesses a potential to be utilized along with Gold standard methods to detect Covid-19 infection to cope with the demand of testing. The aim of this study was to determine diagnostic accuracy of electrochemiluminescence based automated antigen detection immunoassay comparing with molecular based test RT-PCR (Covid-19). Methods It was a cross-sectional study conducted in RMI Peshawar, from 1st April 2021 till 30th April 2021. The study comprised 170 individuals who were suspected of having Covid-19. Nasopharyngeal samples taken from suspected individuals were analyzed by RT-PCR and automated antigen test (Elecsys SARS-CoV-2 Antigen) simultaneously. The correlation of SARS-CoV-2 antigen with PCR positive and negative cases was analyzed for specificity, sensitivity respectively. Results The ECLIA based Elecsys antigen test (Roche) revealed overall sensitivity 72%, specificity 95% and accuracy of 94.9%. Sensitivity of antigen test progressively declined from 94.3% in Ct <25 to 70.8% in Ct 26–29 and then to 47.2% in Ct 30–35. Conclusions Based on the findings of our study we conclude that automated antigen testing (Elecsys SARS-CoV-2 Antigen) cannot replace molecular based testing like RT PCR. Elecsys SARS-CoV-2 Ag test should be used complementary to RT-PCR in testing algorithms. Frequent testing strategy should be adopted while using automated antigen testing to overcome its limitation in individuals with low viral loads.

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