scholarly journals Understanding the Use of Real Time Reverse Transcriptase-Polymerase Chain Reaction (rRT-PCR) For Covid-19 Diagnosis

2020 ◽  
Vol 4 (2) ◽  
pp. 86-97
Author(s):  
Adesola O. Olalekan ◽  
Bamidele A. Iwalokun ◽  
Olutoyin C. Adekunle ◽  
Hussaini A. Makun ◽  
Tatfeng Mirabeau ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Target Genes ◽  
Protein Gene ◽  
Rna Detection ◽  
Glycoprotein Gene ◽  
Adequate Knowledge ◽  
Alternative Testing ◽  
Polymerase Chain ◽  
Testing Protocols

Background: Adequate knowledge of real time Reverse Transcriptase-Polymerase Chain Re-action (rRT-PCR) is critical for accurate implementation of the assay, interpretation of results and report-ing. This mini-review describes the principles, procedures, and level of development of rRT-PCR assays for the control of the COVID-19 pandemic. Methods: A narrative review was carried out to describe the principles of rRT-PCR, provide an update on the landscape of rRT-PCR protocols and elucidate the process control involved in pre-analytical, analytical and post-analytical stages of COVID-19 testing . Review Findings: The rRT-PCR is currently considered to be the acceptable standard for confirming COVID-19 diagnosis based on SARS-CoV-2 RNA detection via conversion to cDNA and amplification of target genes in real time using sequence specific TaqMan® probes. Available evidence indicates that different rRT-PCR protocols varying in number and type of target genes within SARS-CoV-2 genome are currently available for validation and emergency use approval (EUA) in pandemic countries. A total of 1 – 3 target genes, comprising the ORF1a, ORF1b, RNA dependent RNA polymerase (RdRp), Nucleoplasid protein gene (N), Spike glycoprotein gene (S) and Envelope protein gene (E) are detected by these proto-cols. Conclusion: rRT-PCR remains the most sensitive method for confirming, monitoring and managing COVID-19 disease in the ongoing pandemic in all affected countries. The need for validation of every rRT-PCR protocol prior to deployment for COVID-19 testing and research into the development of alternative testing protocols are strongly recommended

scholarly journals AB0691 ANALYSIS OF SARS-COV-2 ANTIBODIES IN NON-COVID-19 PATIENTS: COMPARISON BETWEEN SYSTEMIC SCLEROSIS PATIENTS AND HEALTHY CONTROLS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1379.1-1379
Author(s):  
L. Giardullo ◽  
C. Rotondo ◽  
A. Corrado ◽  
N. Maruotti ◽  
R. Colia ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Systemic Sclerosis ◽  
Autoimmune Disease ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Polymerase Chain Reaction Assay ◽  
Nuclear Antigen ◽  
Healthy Controls ◽  
Chain Reaction ◽  
Polymerase Chain

Background:Previous study evidenced a cross-reactivity between Sars-Cov-2 antibodies and autoimmune tissue antigen involved in connective tissue diseases, as nuclear antigen (NA), extractable nuclear antigen (ENA), histone and collagen (1). No study has been published about the titer of Sars-Cov-2 antibodies in non-infected patients with autoimmune disease.Objectives:To evaluate the titer of SARS-CoV-2 antibodies in non-COVID-19 patients and compare it between systemic sclerosis (SSc) patients and healthy controls (HC).Methods:A total of 58 patients with SSc (who fulfilled ACR/EULAR 2013 SSc classification criteria) and 18 HC were enrolled. Sera of all participants were collected, and SARS-CoV-2 antibodies (IgG and IgM) were evaluated by means ELISA. In all participants swabs for SARS-CoV-2 by real-time reverse-transcriptase-polymerase-chain-reaction assay were reported negative. Demographic, clinical, and autoimmune serological characteristics of SSc patients were recorded. The normal distribution was assessed using the Shapiro–Wilk’s test. Exclusion criteria was previous or actual Sars-Cov-2 infection. Comparisons between study groups of patients were evaluated by the Student’s t-test or Mann – Whitney U-test as appropriate. The differences between categorial variables were assessed by Pearson chi-square or Fisher’s exact test, as opportune. Statistical significance was set at p ≤ 0.05.Results:We observed significant differences between SSc patients and HC in serum levels of Sars-Cov-2 antibodies (IgG: 1,4±2,1 AU/ml vs 0,36±0,19 AU/ml respectively (p=0,001); and IgM: 2,5±3,1 AU/ml vs 0,8±0,7 AU/ml (p=0,022)). In 5 SSc patients was found titer of Sars-Cov-2 antibodies (IgG) exceeding the cut-off, but the control of swabs for SARS-CoV-2 by real-time reverse-transcriptase-polymerase-chain-reaction assay were negative. No significative differences in Sars-Cov-2 autoantibodies titer were found in subgroup of SSc patients with or without ILD or PAH, limited or diffuse skin subset, and different autoantibodies profile. Furthermore, antibodies titer was not associated with different drugs (steroid, methotrexate, mofetil-mycophenolate and bosentan) in use.Conclusion:A cross mimicking between Sars-Cov-2 antibodies and antinuclear antibodies or anti ENA could be hypothesized. Further studies are necessary to unravel the reliability of Sars-Cov-2 antibodies detection in autoimmune disease.References:[1]Vojdani, A., Vojdani, E., & Kharrazian, D. (2021). Reaction of human monoclonal antibodies to SARS-CoV-2 proteins with tissue antigens: Implications for autoimmune diseases. Frontiers in Immunology, 11, 3679Disclosure of Interests:None declared

Diagnosis and Causative Species of Visceral Leishmaniasis in Southwest Saudi Arabia

2021 ◽  
Author(s):  
Aymen Abdelhaleem ◽  
Nabil Dhayhi ◽  
Mohamed Salih Mahfouz ◽  
Ommer Daffalla ◽  
Mansour Mubarki ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Visceral Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Donovani ◽  
Target Genes ◽  
Sample Volume ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Robust Evidence

Visceral leishmaniasis (VL) is the most severe clinical form of the disease and has been reported in the Jazan region of southwest Saudi Arabia. This study aimed to diagnose VL by real-time polymerase chain reaction (PCR) and the direct agglutination test (DAT) and to identify the causative Leishmania species. A total of 80 participants, including 30 suspected VL patients, 30 healthy endemic control individuals, and 20 malaria disease controls, were enrolled in this study. Blood samples were collected and tested for Leishmania DNA by real-time PCR and for antibody by the DAT. Sequencing of some amplified PCR products was used to identify the causative Leishmania species. The diagnosis of VL was successfully achieved by both real-time PCR and by DAT with 100% sensitivity. Leishmania donovani and Leishmania infantum species were detected by sequencing both by the kDNA and ITS1 target genes, followed a BLASTn search. The detection of VL antibody by the DAT followed by the confirmatory detection of Leishmania DNA in patient blood by PCR could promote the adoption of the much less invasive and more sensitive methods for the routine diagnosis of VL. Further study with high sample volume to evaluate the PCR and the DAT are needed, to generate more robust evidence. Based on the sequencing results, emerging studies on VL should focus on the causative Leishmania species, reservoirs, and vectors that are important in the study area.

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