Real-time Real-time Reverse Transcription-polymerase Chain Reaction Research Articles

Accuracy of ICD Influenza Discharge Diagnosis Codes in Hospitalized Adults From the Valencia Region, Spain, in the Pre-COVID-19 Period 2012/2013 to 2017/2018.

International Classification of Diseases (ICD) codes obtained from real-world data can be used to identify influenza cases for epidemiological research but, without validation, may introduce biases. The objective of this study was to validate ICD influenza discharge diagnoses using real-time reverse transcription-polymerase chain reaction (RT-PCR) laboratory-confirmed influenza (LCI) results. The study was conducted during six influenza seasons (2012/2013-2017/2018) in the Valencia Hospital Surveillance Network for the Study of Influenza (VAHNSI). Patients aged 18+ years were identified via active-surveillance and had to meet an influenza-like illness (ILI) case definition to be included. All patients were tested for influenza by real-time RT-PCR. Main and secondary influenza discharge diagnosis codes were extracted from hospital discharge letters. Positive predictive values (PPVs) and the complementary of the sensitivities (1-Sensitivity) of ICD codes with corresponding 95% credible intervals (CrIs) were estimated via binomial Bayesian regression models. A total of 13,545 patients were included, with 2257 (17%) positive for influenza. Of 2257 LCI cases, 1385 (61%) were not ICD-coded as influenza. Overall, 74.73% (95% CrI: 63.24-84.44) of LCI were not-ICD coded as influenza (1-Sensitivity) after adjustment. Sensitivity improved across seasons and with increasing age. Average PPV was 74.02% (95% CrI: 68.58-79.17), ranging from 43.71% to 81.57% between seasons. Using only main and secondary discharge diagnosis codes for influenza detection markedly underestimates the full burden of influenza in hospitalized patients. Future studies, including post-COVID context, using prospective surveillance for ILI are required to assess the validity of hospital discharge data as a tool for determining influenza-related burden of disease.

ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

Real-time reverse transcription polymerase chain reaction (RT-PCR), a standard method recommended for the diagnosis of coronavirus disease 2019 (COVID-19) requires 2–4 h to get the result. Although antigen test kit (ATK) is used for COVID-19 screening within 15–30 min, the drawback is its limited sensitivity. Hence, a rapid one-step quadruplex real-time RT-PCR assay: termed ƩS COVID-19 targeting ORF1ab, ORF3a, and N genes of SARS-CoV-2; and Avocado sunblotch viroid (ASBVd) as an internal control was developed. Based on strategies including designing high melting temperature primers with short amplicons, applying a fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing/extension temperatures; the assay could be accomplished within 25 min. The limit of detection of ORF1ab, ORF3a, and N genes were 1.835, 1.310, and 1 copy/reaction, respectively. Validation was performed in 205 combined nasopharyngeal and oropharyngeal swabs. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.8%, 100%, 100%, and 97.1%, respectively with 96.7% accuracy. Cohen’s Kappa was 0.93. The newly developed rapid real-time RT-PCR assay was highly sensitive, specific, and fast, making it suitable for use as an alternative method to support laboratory diagnosis of COVID-19 in outpatient and emergency departments.

Clinical performance of the ImmuneMed Dengue NS1 Ag Rapid I test for the diagnosis of dengue fever: a diagnostic accuracy study

Background: Dengue virus (DENV) is transmitted by mosquitoes and is becoming a global threat owing to an increase in the number of cases and mortality, especially in low- and middle-income tropical countries. Rapid and easy-to-use diagnostic tests are required to differentiate dengue fever from other febrile illnesses. Methods: We evaluated the clinical performance of the ImmuneMed Dengue NS1 Ag Rapid I test (ImmuneMed, Inc.) using positive and negative sera collected from patients with confirmed DENV infection and healthy individuals, respectively. The AccuPower® ZIKV (DENV, CHIKV) multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) assay (Bioneer) was used as the reference standard to confirm DENV infection. Results: One hundred DENV-positive and 161 DENV-negative samples were evaluated. Overall, the sensitivity and specificity were 100% (95% confidence interval [CI], 96%–100%) and 100% (95% CI, 98%–100%), respectively. The sensitivity and specificity were 100% for both DENV-1 and DENV- 2. The sensitivity was the same (100%) for sera collected < 3 days and ≥ 3 days from symptom onset. The performance of the ImmuneMed Dengue NS1 Ag Rapid I and realtime RT-PCR tests showed strong overall agreement. Conclusion: The ImmuneMed Dengue NS1 Ag Rapid I test was highly specific for DENV and as sensitive as RT-PCR. These findings suggest that the ImmuneMed Dengue NS1 Ag Rapid I test may be a useful point-of-care test for dengue fever.

Rapid Detection of Severe Fever with Thrombocytopenia Syndrome Virus in the Acute Phase of Infection by Direct Real-Time Reverse Transcription without RNA Extraction.

No specific treatment has been developed for severe fever with thrombocytopenia syndrome (SFTS). However, the prognosis can improve with early plasma exchange. Therefore, rapid and accurate detection of SFTS virus is important for diagnosis and prognosis. Direct real-time reverse transcription polymerase chain reaction (RT-PCR) testing is easier and more time-efficient than conventional real-time RT-PCR. Our study compared direct real-time RT-PCR efficiency without the RNA extraction and purification of conventional real-time RT-PCR. Samples were collected from 18 patients with SFTS and five without SFTS. A strong correlation (r = 0.774, 95% CI: 0.652-0.857, P <0.01) was found between conventional and direct real-time RT-PCR assays. Direct real-time RT-PCR showed 84.4% sensitivity and 92.0% specificity for viral detection. Direct real-time RT-PCR is an effective diagnostic tool for patients with acute phase SFTS, but further optimization is required for viral detection.

Maresin 1 alleviates neuroinflammation and cognitive decline in a mouse model of cecal ligation and puncture.

Inflammation in the central nervous system plays a crucial role in the occurrence and development of sepsis-associated encephalopathy. This study aims to explore the effects of maresin 1 (MaR1), an anti-inflammatory and pro-resolving lipid mediator, on sepsis-induced neuroinflammation and cognitive impairment. Mice were randomly assigned to 4 groups: A sham group (sham operation+vehicle), a cecal ligation and puncture (CLP) group (CLP operation+vehicle), a MaR1-LD group (CLP operation+1 ng MaR1), and a MaR1-HD group (CLP operation+10 ng MaR1). MaR1 or vehicle was intraperitoneally administered starting 1 h before CLP operation, then every other day for 7 days. Survival rates were monitored, and serum inflammatory cytokines [tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6] were measured 24 h after operation using enzyme-linked immunosorbent assay (ELISA). Cognitive function was assessed 7 days after operation using the Morris water maze (MWM) test and novel object recognition (NOR) task. The mRNA expression of TNF-α, IL-1β, IL-6, inducible nitric oxide synthase (iNOS), IL-4, IL-10, and arginase 1 (Arg1) in cortical and hippocampal tissues was determined by real-time reverse transcription PCR (RT-PCR). Western blotting was used to determine the protein expression of iNOS, Arg1, signal transducer and activator of transcription 6 (STAT6), peroxisome proliferator-activated receptor gamma (PPARγ), and phosphorylated STAT6 (p-STAT6) in hippocampal tissue. Microglia activation was visualized via immunofluorescence. Mice were also treated with the PPARγ antagonist GW9662 to confirm the involvement of this pathway in MaR1's effects. CLP increased serum levels of TNF-α, IL-1β, and IL-6, and reduced body weight and survival rates (all P<0.05). Both 1 ng and 10 ng doses of MaR1 significantly reduced serum TNF-α, IL-1β, and IL-6 levels, improved body weight, and increased survival rates (all P<0.05). No significant difference in efficacy was observed between the 2 doses (all P>0.05). MWM test and NOR task indicated that CLP impaired spatial learning, which MaR1 mitigated. However, GW9662 partially reversed MaR1's protective effects. Real-time RT-PCR results demonstrated that, compared to the sham group, mRNA expression of TNF-α, IL-1β, and iNOS significantly increased in hippocampal tissues following CLP (all P<0.05), while IL-4, IL-10, and Arg1 showed a slight decrease, though the differences were not statistically significant (all P>0.05). Compared to the CLP group, both 1 ng and 10 ng MaR1 decreased TNF-α, IL-1β, and iNOS mRNA expression in hippocampal tissues and increased IL-4, IL-10, and Arg1 mRNA expression (all P<0.05). Immunofluorescence results indicated a significant increase in Iba1-positive microglia in the hippocampus after CLP compared to the sham group (P<0.05). Administration of 1 ng and 10 ng MaR1 reduced the percentage area of Iba1-positive cells in the hippocampus compared to the CLP group (both P<0.05). Western blotting results showed that, compared to the CLP group, both 1 ng and 10 ng MaR1 down-regulated the iNOS expression, while up-regulated the expression of Arg1, PPARγ, and p-STAT6 (all P<0.05). However, the inclusion of GW9662 counteracted the MaR1-induced upregulation of Arg1 and PPARγ compared to the MaR1-LD group (all P<0.05). MaR1 inhibits the classical activation of hippocampal microglia, promotes alternative activation, reduces sepsis-induced neuroinflammation, and improves cognitive decline.

Dual blockade of interleukin-17A and interleukin-17F as a therapeutic strategy for liver fibrosis: Investigating the potential effect and mechanism of brodalumab

Liver fibrosis is a terminal manifestation of various chronic liver diseases. There are no drugs that can reverse the condition. Recently, the importance of interleukin-17 (IL17) in the pathophysiology has been revealed and has attracted attention as a therapeutic target. We aimed to reveal the roles of IL17A and IL17F in liver fibrosis, and to validate the potential of their dual blockade as therapeutic strategy. First, we retrospectively reviewed the longitudinal change of FIB-4 index, a clinical indicator of liver fibrosis, among psoriasis patients treated by brodalumab, which blocks IL17 receptor A (IL17RA). Next, we examined anti-fibrotic efficacy of anti-IL17RA antibody (Ab) in two murine liver fibrosis models by histopathological investigation and real-time reverse transcription polymerase chain reaction (RT-PCR). Finally, we analyzed the effect of IL17A and IL17F upon human hepatic stellate cells with RNA sequencing, real-time RT-PCR, western blotting, chromatin immunoprecipitation, and flow cytometry. Clinical data showed that FIB-4 index significantly decreased among psoriasis patients treated by brodalumab. In vivo studies additionally demonstrated that anti-IL17RA Ab ameliorates liver fibrosis induced by tetrachloride and methionine-choline deficient diet. Furthermore, in vitro experiments revealed that both IL17A and IL17F enhance cell-surface expression of transforming growth factor-β receptor II and promote pro-fibrotic gene expression via the JUN pathway in human hepatic stellate cells. Our insights suggest that IL17A and IL17F share their pro-fibrotic function in the context of liver fibrosis, and moreover, dual blockade of IL17A and IL17F by anti-IL17RA Ab would be a promising strategy for the management of liver fibrosis.

Comparative study between gold standard real-time RT-PCR assay and rapid antigen test for detection of COVID-19 in Sylhet CMH

Background: The notorious pandemic coronavirus disease 2019 (COVID-19) has been spread all over the world. Its third pandemic wave has been completed and now a fourth wave is running over many countries. Almost 60 million people have been infected and more than 5.7 million people have died. Till now almost 1.2 million people have been infected and about 30000 people have died in Bangladesh from Covid-19. It has not only taken away human lives but also has put a tremendous impact on our society and economy. So early detection of this virus and isolating the patients have a significant role to control the disease. Henceforth there is an urgent need for simple, accurate and rapid identifications of C0VID -19. Rapid antigen tests can provide timely results, which is of particular importance in a primary setting. So, Performance of the Rapid antigen detection test (RAT) should be evaluated and compared with gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19. Methods: Specimens were collected from both naso and oropharyngeal region from 1000 patients who reported to the flu centre in CMH Sylhet with the complaints of fever, cough &amp; headache for detection of COVID virus-2 RNA by rapid antigen (RAT) and RT-PCR test. We used real time RT-PCR kit (Sansure, Biotech china, gene RdRP &amp; N) and COVID-19 Ag Kit (Wondfo, Republic of China) for detection of COVID-19 RNA. Results: Out of 1000 samples 158 were positive and 842 were negative by real-time RT-PCR assay where 154 samples were positive, and 846 cases were negative by rapid antigen test for severe acute respiratory syndrome (SARS) CoV-2. The duration from the onset of symptoms to laboratory test in all suspected cases ranged from 0 to 02 days (with a median of 01 days). The rapid SARS-CoV-2 antigen detection tests sensitivity and specificity were 97.29% (95% CI, 90.06–99.89%) and 97.94% (95% CI, 97.26–98.57%), respectively. Seven samples were found negative in RAT but were found positive in RT-PCR, other three samples were found positive in RAT while they were found negative in RT-PCR. Conclusions: It is observed that most rapid antigen tests for COVID-19 are significantly comparable with RT-PCR tests and had enough sensitivity and specificity for 158 individuals, infected with severe acute respiratory syndrome-CoV-2.

Accurate detection of influenza A virus by use of a novel cross-priming isothermal amplification-based point-of-care assay.

Influenza virus is known to cause respiratory tract infections of varying severity in individuals of all ages. The EasyNAT Rapid Flu assay is a newly developed in vitro diagnostic test that employs cross-priming isothermal amplification (CPA) to detect and differentiate influenza A and B viruses in human nasopharyngeal (NP) swabs. The aim of this study is to determine the performance characteristics of the EasyNAT Rapid Flu assay for rapid detection of influenza virus. The limit of detection (LOD) and cross-reactivity of the EasyNAT Rapid Flu assay were assessed. The clinical performance of the assay was evaluated using NP swab samples that were tested with real-time reverse-transcription polymerase chain reaction (RT-PCR) and Xpert Xpress Flu/RSV assay. The LOD for the detection of influenza A and B using the EasyNAT Rapid Flu assay was found to be 500 copies/mL. Furthermore, the assay exhibited no cross-reactivity with other common respiratory viruses tested. For the 114 NP swab samples tested for influenza A using both the EasyNAT Rapid Flu assay and real-time RT-PCR, the two assays demonstrated a high level of agreement (κ = 0.963, P < 0.001), with a positive percentage agreement (PPA) of 97.7% and a negative percentage agreement (NPA) of 98.6%. Similarly, for the 43 NP swab samples tested for influenza A and B using both the EasyNAT Rapid Flu assay and Xpert Xpress Flu/RSV assay, the two assays showed a high level of agreement (κ = 0.933, P < 0.001), with the overall rate of agreement (ORA) of 97.7% for influenza A and 100% for influenza B. The EasyNAT Rapid Flu assay demonstrates excellent performance in the detection of influenza A, highlighted by its strong agreement with RT-PCR-based assays.IMPORTANCEThe newly developed EasyNAT Rapid Flu assay is an innovative cross-priming isothermal amplification-based method designed for detecting influenza A and B viruses at point-of-care settings. This study aims to thoroughly assess the analytical and clinical performance of the assay, offering valuable insights into its potential advantages and limitations. The findings of this research hold significant implications for clinical practice.

An Optimal Transport Medium for SARS-CoV-2 Detection in the Direct Method of Rapid Microfluidic PCR System

Recently developed rapid real-time reverse transcription PCR (RT-PCR) systems adopting microfluidic thermal cycling technology are ideal for point-of-care (POC) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because the RNA extraction step before real-time RT-PCR is rate-limiting, a direct RNA extraction method (direct method) that adopts chemical viral lysis and eliminates RNA purification steps is preferable for rapid real-time RT-PCR. In the direct method, selecting the transport medium is essential because it may be introduced into subsequent real-time RT-PCR steps, but might inhibit PCR. However, the influence of transport medium on the combination of the direct method and rapid real-time RT-PCR has been yet unstudied. In the present study, we examined the influence of various transport mediums when combining the direct method and rapid real-time RT-PCR of GeneSoC® (GeneSoC® RT-PCR), the recently developed compact PCR system that adapts novel microfluidic thermal cycling technology. To explore the influence of the transport medium on the GeneSoC® RT-PCR, the concordance of the RNA extraction and direct method was evaluated in the clinical samples collected in viral transport medium (VTM) or eSwab®. The sensitivity of GeneSoC® RT-PCR combined with the direct method was assessed using spiked samples in generic (H2O and PBS) or commercially available transport media (VTM and eSwab®). Analytical sensitivity was examined using clinical specimens collected from the VTM and eSwab®. The inhibitory effect of PCR inhibitors on clinical specimens was assessed using clinical samples diluted 1,000 times. While only 1 copy/reaction of RNA was detected in H2O and eSwab® of the spiked samples, a minimum of 5 copies/reaction was detected in PBS (-) and VTM. Among the clinical specimens tested using the direct method, the detection of viral RNA was unstable in the samples containing less than 100 copies/reaction viral RNA in VTM, whereas less than 10 copies/reaction viral RNA were detected in eSwab®. The positive, negative, and overall concordance between the RNA extraction and the direct method was 84%, 100%, and 85%, respectively, in eSwab® samples, whereas the values were 35%, 100%, and 38%, respectively, in VTM samples. When the clinical samples were diluted 1,000 times, GeneSoC® RT-PCR could detect as low as 1.15 copies/reaction RNA using direct method, and the sensitivity was comparable to that of RNA extraction. The combination of the direct method and microfluidic rapid PCR machine GeneSoC® has a high sensitivity for detecting SARS-CoV-2 RNA in clinical samples with eSwab® transport medium.

Diagnosis of COVID-19 obtained by rapid antigen test and reverse transcription–polymerase chain reaction in a medical college in Eastern India-a record-based observational descriptive study

Background: In December 2019 in Wuhan, China, a new coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appeared. Gradually, the virus started spreading around the world, which was life-threatening. Aims and Objectives: The study’s objective was to find out the positivity rate of infection, the category of patients included in the study, the percentage of positive and negative cases, comparison of rapid antigen detection in relation to real-time reverse transcription–polymerase chain reaction (RT-PCR) test, and rapid antigen detection test’s sensitivity and specificity. Materials and Methods: Eight hundred sixty-five patients’ samples and nasopharyngeal swabs were collected within 3 months as routine samples. Two hundred thirty-two samples were compared by both rapid antigen test (RAT) and RT-PCR. To rule out false negatives, those who tested negative for COVID-19 using a RAT underwent testing by RT-PCR. To rule out false positives, a small number of patients who tested positive for COVID-19 by RAT also underwent validation by RT-PCR test. Results: Two hundred thirty-two samples were obtained for rapid antigen detection, then RT-PCR testing. Both the results were compared. According to the findings, the positivity rate of COVID-19 infection was 0.86%. Two patients (0.86%) tested positive (either by one or both methods) and the number of patients who tested negative was 230 (99.14%). RAT sensitivity and specificity were 50% and 100%, respectively, compared to gold standard RT-PCR. The positive predictive value was 100% and the negative predictive value was 99%. Conclusion: We conclude that antigen detection methods are less sensitive than real-time RT-PCR but they can be used for COVID-19 mass screening and can be used as a quick diagnostic tool in remote locations without molecular testing facilities.

Isolation, molecular characterization, and genetic diversity of recently isolated foot-and-mouth disease virus serotype A in Egypt.

Foot-and-mouth Disease (FMD) is a highly contagious viral disease affecting all hoof-cloven animals. Serotypes A, O and SAT 2 of the foot-and-mouth disease virus (FMDV) are circulating in Egypt. The present study aimed to identify and molecularly characterize the FMDV strains circulating in Northern Egypt during an epidemic that struck the nation in 2022. RNA was extracted from the epithelial specimens, vesicular fluid from affected cattle. The samples were screened using real-time reverse-transcription polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. Positive samples underwent individual serotype-specific amplification using primers designed for VP1 of O, A, and SAT 2 serotypes. Subsequently, direct sequencing was performed on the positive samples. The real-time RT-PCR detected positive samples from epithelial and vesicular fluid samples, but not in the blood of infected animals. Out of the 16 samples, seven tested positive for FMDV serotype A. Of these seven positive samples, six were categorized as serotype A-African topotype-G-IV, and these positive samples were isolated in BHK-21 cells, yielding an overt cytopathic effect caused by the virus. In conclusion, it is necessary to sustain continuous surveillance of the evolution of circulating FMDV strains to facilitate the assessment and aid in the selection of vaccine strains for the effective control of FMDV in Egypt.

Performance evaluation of the cobas SARS-CoV-2 Duo, a novel qualitative and quantitative assay, for the detection of SARS-CoV-2 RNA.

Viral load quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a critical measure for monitoring clinical responses of patients with COVID-19. The cobas SARS-CoV-2 Duo (Roche Molecular Systems, Inc.) uses the cobas 6800/8800 platform and is an automated real-time reverse transcription PCR (RT-PCR) assay for the qualitative detection of SARS-CoV-2 RNA (ORF1a and ORF1a/b non-structural regions) in nasal and nasopharyngeal specimens. This assay also performs quantitation of the SARS-CoV-2 RNA level in the collected specimen. The precision, accuracy, and linearity of the cobas SARS-CoV-2 Duo test were assessed and correlated with the cycle threshold (Ct) value of the cobas SARS-CoV-2 assay, and the cobas Liat SARS-CoV-2 and influenza A/B assay (Inf A/B) (cobas Liat) in 201 clinical nasopharyngeal swab specimens. The analytical measurement range of the cobas SARS-CoV-2 Duo has been verified to be 1.0E + 02 IU/mL to 1.0E + 09 IU/mL. The between- and within-run precision evaluation and the linearity results (R 2 = 0.9961) of the cobas SARS-CoV-2 Duo were acceptable based on the verification criteria. A total of 201 nasopharyngeal specimens were evaluated. The positive, negative, and total agreements between the cobas SARS-CoV-2 Duo and the cobas SARS-CoV-2 assay were 93.5% (157 of 168), 75% (33 of 44), and 92.5% (186 of 201), respectively. The qualitative results (Ct values) from the cobas SARS-CoV-2 and the cobas Liat demonstrated good agreement with the quantitative results from the cobas SARS-CoV-2 Duo assay. In conclusion, the diagnostic performance of the cobas SARS-CoV-2 Duo assay is appropriate. It is a precise, accurate, and sensitive method for the detection of SARS-CoV-2 RNA and is comparable to two qualitative assays. IMPORTANCE Quantitative SARS-CoV-2 tests for viral load are necessary to guide patient treatment, as well as to determine infection control measures and policies. Although the real-time RT-PCR assays can report the Ct value to estimate the viral load, there are several serious concerns regarding the use of Ct values. Importantly, Ct values can vary significantly among between- and within-run methods. The diagnostic performance of the cobas SARS-CoV-2 Duo is appropriate. It is a precise, accurate, and sensitive method for the detection of SARS-CoV-2 RNA and is comparable to two qualitative assays (the cobas SARS-CoV-2 and the Liat cobas SARS-CoV-2 and Inf A/B). In contrast, using the Ct value to estimate viral load is not reliable, and utilization of a quantitative detection test, such as the cobas SARS-CoV-2 Duo, to accurately measure the viral load is needed.

Evaluation of the Performance of the COVID-19 Rapid Antigen Tests in a Tertiary Level Hospital in Bangladesh

The tests providing high sensitivity and specificity are essential to identify and manage COVID-19 patients.There is aheavy demand for low-cost rapid antigen tests (RATs) for COVID-19 with a decent diagnostic value. Globally,several RATs for COVID-19 have been developed, but their clinical efficacy has not been well recognized.Thepurpose of the study was to evaluate the performance of RATs. We conducted this prospective observational study atShaheed Suhrawardy Medical College hospital from February 2021 to April 2021 in Dhaka, Bangladesh. This studyincluded the patients admitted in this hospital at the COVID-19 isolation unit or referred from the triage facility of theoutdoor department of this hospital suspected as COVID-19 case. Two nasopharyngeal samples were collectedsimultaneously. We used one sample on the spot for the RATs and the other was sent to the adjacent ShaheedSuhrawardy Medical College COVID-19 RT-PCR laboratory for real-time reverse transcription PCR (RT-PCR). Theperformance of the RATs was evaluated by consuming the real-time RT-PCR results as a reference. In thisinvestigation, SARS-CoV-2 was identified in 84 (30.7%) of the 223 patients who participated. Of these 84 patients, 9(10.7%) were asymptomatic. The overall sensitivity and specificity of RATs were 78.6% and 99.3%, respectively. Thesensitivity was 81.3% in symptomatic cases and 55.6% in asymptomatic cases. There were 18 false-negativespatients,including 3 asymptomatic cases, who had a low viral load (cycle threshold(Ct) &gt; 30). When the Ct value was up to24, the detection rate of RATs was 100%. The detection rate was 42.3% when the Ct was &gt;29. When symptoms beganwithin three days, the detection rate was 92.3%. When the start of symptoms was over 7 days, the detection rate was33.3%. RATs for COVID-19 used in this study delivered an acceptable performance in patients with high viral loadand within the first week of the onset of symptoms. This method can be a supplementary method to RT- PCR for thediagnosis of symptomatic COVID-19 patients. Bangabandhu Sheikh Mujib Med. Coll. J. 2022;1(2):58-64

Development and evaluation of an external quality control and internal quality control containing real-time RT-PCR assay for the detection of o'nyong-nyong virus

O'nyong-nyong fever is a mosquito-borne tropical viral disease while few molecular diagnostic tools have been established for its surveillance until now. In the current study, a single-step, dual-color real-time reverse transcription polymerase chain reaction (RT-PCR) assay which contained both external quality control (EQC) and internal quality control (IQC) prepared by armored RNA technique was developed and evaluated for the detection of o'nyong-nyong virus (ONNV). Results showed that the assay was established successfully without cross-reaction with genetically related or symptom-alike diseases, which showed high specificity of the assay. The coefficient of variation of the assay was 0.97%, far less than 5%, indicating good repeatability of the assay. The lower limit of detection of the assay could reach as low as 100 copies of genome equivalent. During evaluation, the assay could correctly detect ONNV from spiked human serum samples and Anopheles species mosquito samples, while no ONNV positive was observed either from serum samples of patients with acute febrile illness or from local Anopheles species mosquitoes, suggesting no ONNV had been transmitted locally. In conclusion, the assay could potentially provide a valuable platform for ONNV molecular detection, which may improve the preparedness for future o'nyong-nyong fever outbreaks.

ESTIMATION OF PREVALENCE OF HEPATITIS C VIRUS INFECTION BY SEROLOGICAL TEST AND REAL TIME RT-PCR TEST AMONG SUSPECTED VIRAL HEPATITIS CASES ATTENDING TERTIARY CARE HOSPITAL, KOLKATA

Objective: Hepatitis C virus (HCV) infection is a major public health problem in India and worldwide. Majority remain asymptomatic until they develop serious complications like liver cirrhosis or hepatocellular carcinoma with fatal outcome. Hence, early diagnosis of active HCV infection and prompt initiation of treatment is important. Treatment with directly acting antivirals (DAAs) resulted in high sustained virological response (SVR) rates of &gt;95% globally. This study was done to estimate seroprevalence and prevalence of active HCV infection among study population. After initiation of treatment, SVR rates were estimated. Methods: This was hospital-based, cross-sectional, and observational study. Screening was done by third-generation Enzyme-linked immunosorbent assay to detect anti-HCV antibody, then confirmatory real-time reverse transcription-polymerase chain reaction (RT-PCR) test was done for detection of active cases and determining their viral load. Treatment of 12-week duration was initiated by DAAs and followed up to estimate SVR by doing real-time RT-PCR after 12 weeks of treatment completion. Result: Among 17,752 consecutive non-repetitive blood samples, seroprevalence was 1.78%. The prevalence of active cases was 1.52%. HCV active infection was prevalent more among male (64.21%) and among 40–60 years age group. History of multiple blood transfusion (58%) was the most common risk factor, followed by multiple sex partners (13.3%). Coinfections with Hepatitis B virus and Human immunodeficiency virus were seen in 13.65% of cases. About 92% of patients completed their treatment. SVR was 97.87%. Conclusion: High SVR of 97.87% is evidence-based data that support that proper treatment can eliminate HCV infection. Detection by real-time RT-PCR and highly effective DAA has made a paradigm shift to approach of HCV diagnosis and management in recent times

COVID-19 scent dog research highlights and synthesis during the pandemic of December 2019-April 2023.

This review was undertaken to provide information concerning the advancement of research in the area of COVID-19 screening and testing during the worldwide pandemic from December 2019 through April 2023. In this review, we have examined the safety, effectiveness, and practicality of utilizing trained scent dogs in clinical and public situations for COVID-19 screening. Specifically, results of 29 trained scent dog screening peer-reviewed studies were compared with results of real-time reverse-transcription polymerase chain reaction (RT-PCR) and rapid antigen (RAG) COVID-19 testing methods. The review aims to systematically evaluate the strengths and weaknesses of utilizing trained scent dogs in COVID-19 screening. At the time of submission of our earlier review paper in August 2021, we found only four peer-reviewed COVID-19 scent dog papers: three clinical research studies and one preprint perspective paper. In March and April 2023, the first author conducted new literature searches of the MEDLINE/PubMed, Google Scholar, and Cochrane Library websites. Again, the keyword phrases utilized for the searches included "COVID detection dogs," "COVID scent dogs," and "COVID sniffer dogs." The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 Checklist was followed to ensure that our review adhered to evidence-based guidelines for reporting. Utilizing the results of the reviewed papers, we compiled statistics to intercompare and summarize basic information concerning the scent dogs and their training, the populations of the study participants, the types of sampling methods, the comparative tests utilized, and the effectiveness of the scent dog screening. A total of 8,043 references were identified through our literature search. After removal of duplicates, there were 7,843 references that were screened. Of these, 100 were considered for full-text eligibility, 43 were included for qualitative synthesis, and 29 were utilized for quantitative analysis. The most relevant peer-reviewed COVID-19 scent dog references were identified and categorized. Utilizing all of the scent dog results provided for this review, we found that 92.3 % of the studies reached sensitivities exceeding 80 and 32.0 % of the studies exceeding specificities of 97 %. However, 84.0 % of the studies reported specificities above 90 %. Highlights demonstrating the effectiveness of the scent dogs include: (1) samples of breath, saliva, trachea-bronchial secretions and urine as well as face masks and articles of clothing can be utilized; (2) trained COVID-19 scent dogs can detect presymptomatic and asymptomatic patients; (3) scent dogs can detect new SARS-CoV-2 variants and Long COVID-19; and (4) scent dogs can differentiate SARS-CoV-2 infections from infections with other novel respiratory viruses. The effectiveness of the trained scent dog method is comparable to or in some cases superior to the real-time RT-PCR test and the RAG test. Trained scent dogs can be effectively utilized to provide quick (seconds to minutes), nonintrusive, and accurate results in public settings and thus reduce the spread of the COVID-19 virus or other viruses. Finally, scent dog research as described in this paper can serve to increase the medical community's and public's knowledge and acceptance of medical scent dogs as major contributors to global efforts to fight diseases.

PERFORMANCE OF RAPID ANTIGEN TEST IN COMPARISON TO REAL-TIME RT-PCR IN DIAGNOSIS OF COVID-19 INFECTION: A SHORT STUDY

Objective: The global pandemic of Coronavirus Disease 2019 (COVID-19) is still spreading. Because of this, it is urgently necessary to develop quick, easy, and accurate assays to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. For the purpose of diagnosing COVID-19 cases, the quick SARS-CoV-2 antigen detection test’s performance attributes should be assessed and contrasted with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. Methods: A retrospective analysis was done during January to May, 2022 at IPGME&amp;R and SSKM Hospital, Kolkata, India. 2842 paired samples were taken from all patients reporting to the hospital for Rapid Antigen Test (RAT) and RT-PCR. The sensitivity and specificity were calculated to evaluate the performance of the RAT. Results: Of 2842 samples, 229 (8%) were positive, and 2613 (92%) were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. The rapid SARS-CoV-2 antigen detection test’s sensitivity and specificity were 77.29% (177 out of 229) and 99.54% (2601 out of 2613), respectively. Positive predictive value was 93.65% and negative predictive value was 93% with the Ct value ≤35 of real-time RT-PCR result. Conclusion: RAT-positive samples are also positive by real-time RT-PCR test though RAT-negative patient with symptom, should further test for real-time RT-PCR as sensitivity is bit low particularly in symptomatic patient respect to real-time RT-PCR result. Hence, we can conclude that specificity- and sensitivity-wise real-time RT-PCR method is best, but during pandemic/outbreak situation, RAT is good alternative for fast screening and point-of-care test of COVID-19 detection.

A New Method to Detect Variants of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Bioluminescent Assay in Real Time (RT-LAMP-BART)

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), of which there are several variants. The three major variants (Alpha, Delta, and Omicron) carry the N501Y, L452R, and Q493R/Q498R mutations, respectively, in the S gene. Control of COVID-19 requires rapid and reliable detection of not only SARS-CoV-2 but also its variants. We previously developed a reverse transcription loop-mediated isothermal amplification assay combined with a bioluminescent assay in real time (RT-LAMP-BART) to detect the L452R mutation in the SARS-CoV-2 spike protein. In this study, we established LAMP primers and peptide nucleic acid probes to detect N501Y and Q493R/Q498R. The LAMP primer sets and PNA probes were designed for the N501Y and Q493R/Q498R mutations on the S gene of SARS-CoV-2. The specificities of RT-LAMP-BART assays were evaluated using five viral and four bacterial reference strains. The sensitivities of RT-LAMP-BART assays were evaluated using synthetic RNAs that included the target sequences, together with RNA-spiked clinical nasopharyngeal and salivary specimens. The results were compared with those of conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. The method correctly identified N501Y and Q493R/Q498R. Within 30 min, the RT-LAMP-BART assays detected up to 100–200 copies of the target genes; conventional real-time RT-PCR required 130 min and detected up to 500–3000 copies. Surprisingly, the real-time RT-PCR for N501Y did not detect the BA.1 and BA.2 variants (Omicron) that exhibited the N501Y mutation. The novel RT-LAMP-BART assay is highly specific and more sensitive than conventional real-time RT-PCR. The new assay is simple, inexpensive, and rapid; thus, it can be useful in efforts to identify SARS-CoV-2 variants of concern.

Development of a real-time RT-PCR system applicable for rapid and pen-side diagnosis of foot-and-mouth disease using a portable device, PicoGene® PCR1100

Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease, causing devastating losses to the livestock industry. A diagnostic method that enables quick decisions is required to control the disease, especially in FMD-free countries. Although conventional real-time reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive method widely used for the diagnosis of FMD, a time lag caused by the transport of samples to a laboratory may allow the spread of FMD. Here, we evaluated a real-time RT-PCR system using a portable PicoGene PCR1100 device for FMD diagnosis. This system could detect the synthetic FMD viral RNA within 20 min with high sensitivity compared to a conventional real-time RT-PCR. Furthermore, the Lysis Buffer S for crude nucleic extraction improved the viral RNA detection of this system in a homogenate of vesicular epithelium samples collected from FMD virus-infected animals. Furthermore, this system could detect the viral RNA in crude extracts prepared using the Lysis Buffer S from the vesicular epithelium samples homogenized using a Finger Masher tube, which allows easy homogenization without any equipment, with a high correlation compared to the standard method. Thus, the PicoGene device system can be utilized for the rapid and pen-side diagnosis of FMD.

RAPID DETECTION OF SARS COV-2 INFECTION IN COMPARISION WITH RT-PCR IN TERTIARY CARE HOSPITAL

Objectives: The goal of the present study was to assess the SARS-CoV-2 antigen detection test’s performance features and compare them to the real-time reverse transcription polymerase chain reaction (RT-PCR) test, the gold standard test for the diagnosis of COVID-19 cases. Methods: From October 2020 to May 2021, patients attending the OPD, including those undergoing surgery, at a Tertiary Care Teaching Hospital in Telangana provided 1000 respiratory samples, primarily nasopharyngeal swabs. A skilled technician had collected two nasopharyngeal swabs from each person in a COVID sample collection room while wearing personal protective equipment and following strict infection control procedures. One swab was used for the rapid antigen test given by the standard Q COVID-19 Ag test kit and placed into the extraction buffer tube. Second swab was kept in the viral transport medium and used for Allplex™ 2019-nCoV Assay (Seegene, Korea), which targets envelope gene (E), and RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS CoV-2, was used for SARS-CoV-2 RNA detection according to the manufacturer’s instructions. Results: Out of 1000 samples tested for COVID-19, 623 (63.7%) were males and 377 (36.3%) were females. Out of 1000 samples, 347 samples were RT-PCR positive and 653 were RT-PCR negative. Out of 347 RT-PCR samples positive, 341 were Rapid antigen test positive samples and six were negative. Overall sensitivity and specificity are 98.27% and 99.85%, respectively. Conclusion: The real-time RT-PCR assay’s sensitivity and specificity were comparable to those of the rapid assay for SARS-CoV-2 antigen detection. It can be utilized for contact tracing measures to control the COVID-19 pandemic in places such as border crossings, airports, interregional bus and train stations, and mass testing campaigns needing quick findings. This is especially true in areas with a high prevalence of the disease.