Real-time RT-PCR Test Research Articles

A-236 Rapid multiplexed PCR (polymerase chain reaction) tests for respiratory disease improve process and time to diagnosis versus laboratory-based molecular testing

Abstract Background The Infectious Disease Society of America recommends molecular testing for symptomatic patients suspected of having COVID-19. Molecular tests can be performed at the point of care or in a centralized laboratory, using different methodologies, and can be singleplex or multiplex. Limited data are available examining the use of these tests in clinical practice and impact of different test types on patient outcomes; however, common antiviral treatments for both COVID-19 and influenza are more effective when taken earlier and are not indicated for use more than 5 and 2 days after symptom onset, respectively. The aim of this study was to describe the diagnostic journey for non-inpatients who received molecular testing for respiratory pathogens in the laboratory versus those who received testing using a rapid, multiplexed real-time RT-PCR test that can be performed at the point of care. Methods De-identified administrative claims were analyzed from July 1, 2020 through September 30, 2022 for commercially insured and Medicare Advantage enrollees in the Optum Labs Data Warehouse. Patients with ≥1 medical claim for a non-inpatient visit with a procedure code for testing for SARS-CoV-2 were identified; patients who received antigen testing at index were excluded. Patients were followed for up to 90 days. Two patient groups were selected for the final study sample: 1) Patients with ≥1 claim with CPT code 0240U or 0241U with a point-of-care or laboratory place of service on the index date (proprietary laboratory analyses codes for Xpert® Xpress CoV-2/Flu/RSV, or “Xpert Xpress”); and 2) Patients with other laboratory-based molecular testing on the index date. Standardized difference of >0.10 or <-0.10 was considered a significant imbalance in proportions or means. Results In total, 51,602 patients received testing with Xpert Xpress while 317,192 received laboratory testing. Patients who were tested with Xpert Xpress were significantly older (mean 49.82 vs 42.19, SD 0.3), had a higher Charlson Comorbidity Index (mean 0.7 vs 0.35, SD 0.29), and were more likely to have a respiratory risk factor (6.09% vs 2.38%, SD 0.19). On average, patients who were tested with Xpert Xpress had significantly fewer tests for SARS-CoV-2 on the index date (mean 1.2 vs 1.58, SD -0.55), and fewer tests for SARS-CoV-2, influenza, or RSV on the index date and through the end of the 90-day fixed follow-up period (mean 1.64 vs 2.57, SD -0.45). They were also significantly more likely to be diagnosed with COVID-19 on the index date (23.31% vs 16.53%, SD 0.17). Among patients with a diagnosis for COVID-19, mean time to diagnosis was significantly shorter among patients tested with Xpert Xpress (4.85 vs 8.86 days, SD -0.27; median 0 vs 4 days). This finding was also consistent for influenza (4.05 vs 20.28 days, SD -0.74; median 0 vs 5 days). Conclusions Testing with Xpert Xpress at index resulted in fewer total tests and faster diagnosis compared to testing in the laboratory. Rapid, multiplex PCR testing provides a faster time to diagnosis and may support more appropriate and effective treatment.

Low-cost high-throughput targeted sequencing for the accurate detection of respiratory tract pathogens.

The current gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis by real-time reverse transcriptase polymerase chain reaction (RT-PCR) is limited by the number of genes that can be detected. In this study, we developed a low-cost and high-throughput next-generation sequencing technology that can overcome the limitations of real time RT-PCR. A targeted sequencing panel (TSP) consisting of approximately 500 amplicons was designed. This panel could simultaneously detect a broad range of gene loci of SARS-CoV-2, and genes for the most common infectious viruses that affect the respiratory tract, in a single run and could include up to 96 samples. Four hundred and forty-eight samples and 31 control samples were analyzed independently with both TSP and RT-PCR, and the results were compared for accuracy and other indicators. TSP identified 50 SARS-CoV-2 positive samples with a 99.33% match to RT-PCR results. It is not surprising that TSP also identified multiple infections from the 96 samples, whereas RT-PCR could not. Thus, TSP was able to accurately diagnose the samples which could not be identified based on single RT-PCR test. Our data demonstrated that TSP is a fast and accurate testing method for identifying multiple pathogen infections of the respiratory tract.

Comparison of diagnostic accuracy of rapid antigen tests for COVID-19 compared to the viral genetic test in adults: a systematic review and meta-analysis.

The objective of this review was to determine the diagnostic accuracy of the currently available and upcoming point-of-care rapid antigen tests (RATs) used in primary care settings relative to the viral genetic real-time reverse transcriptase polymerase chain reaction (RT-PCR) test as a reference for diagnosing COVID-19/SARS-CoV-2 in adults. Accurate COVID-19 point-of-care diagnostic tests are required for real-time identification of SARS-CoV-2 infection in individuals. Real-time RT-PCR is the accepted gold standard for diagnostic testing, requiring technical expertise and expensive equipment that are unavailable in most primary care locations. RATs are immunoassays that detect the presence of a specific viral protein, which implies a current infection with SARS-CoV-2. RATs are qualitative or semi-quantitative diagnostics that lack thresholds that provide a result within a short time frame, typically within the hour following sample collection. In this systematic review, we synthesized the current evidence regarding the accuracy of RATs for detecting SARS-CoV-2 compared with RT-PCR. Studies that included nonpregnant adults (18 years or older) with suspected SARS-CoV-2 infection, regardless of symptomology or disease severity, were included. The index test was any available SARS-CoV-2 point-of-care RAT. The reference test was any commercially distributed RT-PCR-based test that detects the RNA genome of SARS-CoV-2 and has been validated by an independent third party. Custom or in-house RT-PCR tests were also considered, with appropriate validation documentation. The diagnosis of interest was COVID-19 disease and SARS-CoV-2 infection. This review considered cross-sectional and cohort studies that examined the diagnostic accuracy of COVID-19/SARS-CoV-2 infection where the participants had both index and reference tests performed. The keywords and index terms contained in relevant articles were used to develop a full search strategy for PubMed and adapted for Embase, Scopus, Qinsight, and the WHO COVID-19 databases. Studies published from November 2019 to July 12, 2022, were included, as SARS-CoV-2 emerged in late 2019 and is the cause of a continuing pandemic. Studies that met the inclusion criteria were critically appraised using QUADAS-2. Using a customized tool, data were extracted from included studies and were verified prior to analysis. The pooled sensitivity, specificity, positive predictive, and negative predictive values were calculated and presented with 95% CIs. When heterogeneity was observed, outlier analysis was conducted, and the results were generated by removing outliers. Meta-analysis was performed on 91 studies of 581 full-text articles retrieved that provided true-positive, true-negative, false-positive, and false-negative values. RATs can identify individuals who have COVID-19 with high reliability (positive predictive value 97.7%; negative predictive value 95.2%) when considering overall performance. However, the lower level of sensitivity (67.1%) suggests that negative test results likely need to be retested through an additional method. Most reported RAT brands had only a few studies comparing their performance with RT-PCR. Overall, a positive RAT result is an excellent predictor of a positive diagnosis of COVID-19. We recommend that Roche's SARS-CoV-2 Rapid Antigen Test and Abbott's BinaxNOW tests be used in primary care settings, with the understanding that negative results need to be confirmed through RT-PCR. We recommend adherence to the STARD guidelines when reporting on diagnostic data. PROSPERO CRD42020224250.

Detection and typing of influenza viruses in vaccinated and non- vaccinated children in central Greece during 2018-2019

During the 2018-2019 influenza season in central Greece, 388 respiratory samples were collected from children presented with influenza symptoms and tested for the detection of influenza viruses. Real-time RT-PCR testing of the samples revealed the presence of influenza A (H1N1) (pdm09), influenza A (H3N2), seasonal influenza A (H1N1), and influenza B viruses. All samples tested were found negative for influenza A(H5) HA gene sequence. The majority of influenza viruses detected were type A (90%), of which the majority, (61%), were influenza A (H1N1) (pdm09) viruses, indicating that these viruses were the dominant influenza subtype circulating in central Greece during the winter and early spring of the aforementioned season.

Analysis of real-time RT-PCR and rapid antigen test and its correlation with clinical characteristics, investigation profile, and imaging of SARI-suspected COVID-19 infections

Abstract Background: Coronavirus disease 2019 (COVID-19) continues to be a significant global public health issue. Aims and Objectives: This study aims to describe the demographic characteristics, comorbidities, clinical presentations, inflammatory biomarkers, and chest computed tomography (CT) findings of patients with severe acute respiratory infection (SARI) at a tertiary care hospital in Odisha, India. Additionally, the study aims to evaluate different COVID-19 diagnostic methods. Materials and Methods: A prospective observational study was conducted at a tertiary care hospital in Odisha, India. All admitted SARI patients underwent COVID-19 testing. Data on demographics, clinical presentations, laboratory investigations, and outcomes at 72 h were collected. COVID-19 diagnostic methods were also analyzed. Statistical Analysis: The statistical analysis was performed using GraphPad Prism 9.5. Results: Among the 830 patients studied, 305 tested positive for COVID-19. The rapid antigen test (RAT) demonstrated a sensitivity of 93.85% and a specificity of 97.43% compared to the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. Discrepancies with RAT results were noted in cases with higher cycle threshold (Ct) values (>25). A male predominance was observed, with a mean age of 43.30 ± 15.19 years in the COVID-19-positive group and 40.95 ± 17.15 years in the COVID-19-negative group. Patients with multiple comorbidities (15.68%) had a higher susceptibility to COVID-19 infection. The COVID-19-positive group showed a significantly elevated neutrophil-to-lymphocyte ratio (NLR) (P = 0.0001) and thrombocytopenia (P = 0.001). Inflammatory biomarkers, including CRP, LDH, ferritin, D-dimer, and IL-6, were markedly elevated in swab-positive cases. Bilateral chest involvement and ground glass opacities were common radiological findings in both groups. Conclusion: In resource-limited settings, the RAT is considered an alternative screening tool for COVID-19. Initial laboratory and radiological investigations may aid clinicians in effective management of such patients.

Molecular Methods for the Simultaneous Detection of Tomato Fruit Blotch Virus and Identification of Tomato Russet Mite, a New Potential Virus–Vector System Threatening Solanaceous Crops Worldwide

Tomato fruit blotch virus (ToFBV) (Blunervirus solani, family Kitaviridae) was firstly identified in Italy in 2018 in tomato plants that showed the uneven, blotchy ripening and dimpling of fruits. Subsequent High-Throughput Sequencing (HTS) analysis allowed ToFBV to be identified in samples collected in Australia, Brazil, and several European countries, and its presence in tomato crops was dated back to 2012. In 2023, the virus was found to be associated with two outbreaks in Italy and Belgium, and it was included in the EPPO Alert list as a potential new threat for tomato fruit production. Many epidemiologic features of ToFBV need to be still clarified, including transmission. Aculops lycopersici Massee (Acariformes: Eriophyoidea), the tomato russet mite (TRM), is a likely candidate vector, since high population densities were found in most of the ToFBV-infected tomato cultivations worldwide. Real-time RT-PCR tests for ToFBV detection and TRM identification were developed, also as a duplex assay. The optimized tests were then transferred to an RT-ddPCR assay and validated according to the EPPO Standard PM 7/98 (5). Such sensitive, reliable, and validated tests provide an important diagnostic tool in view of the probable threat posed by this virus–vector system to solanaceous crops worldwide and can contribute to epidemiological studies by simplifying the efficiency of research. To our knowledge, these are the first molecular methods developed for the simultaneous detection and identification of ToFBV and TRM.

Application of multiplex realtime PCR detection for hemorrhagic fever syndrome viruses

BackgroundMultiplex real-time PCR is a quick and cost effective method for detection of various gene simultaneously. HFSV (Hemorrhagic Fever Syndrome Virus) is a newly emerging infectious disease because of globalization and climate change. We tried to develop a molecular diagnostic technique for various causative viruses and evaluate its usefulness for improving public health. MethodsMolecular diagnostic test method that qualitatively detects viruses causing viral hemorrhagic fevers hired Taq-Man Real-time RT-PCR technique. The Ct value was experimentally observed three or more times at the RNA concentration before and after the detection limit. After designing a multiplex real-time RT-PCR test for target gene of selected 17 viruses, the detection limit for each target and the presence or absence of cross-reaction and interference reaction were evaluated to determine its availability. ResultsSix kinds of viruses, including Crimean-Congo hemorrhagic fever virus, Omsk hemorrhagic fever virus, Sabia virus, Chapare virus, Yellow fever virus, and Variola virus (A4L gene, B12R gene), were able to confirm the detection limit of 0.5 copies/μl, and other Ebola virus, Marburg virus, Rift Valley fever virus, Kyasanur Forest disease virus, Junin virus, Guanarito virus, Machupo virus, Chikungunya virus, Hantavirus, Dengue virus types 1–4, and Lassa virus (L gene, GPC gene), and 11 kinds of viruses, the detection limit was confirmed at 5 copies/μl. No cross-reaction or interference between detected genes was observed. ConclusionThe virus test method developed through this study using multiplex is expected to be used for public health and quarantine as a test method that can be used when a hemorrhagic fever virus of unknown cause is introduced.

Breakthrough infection among healthcare personnel following exposure to COVID-19: Experience after one year of the world’s largest vaccination drive

ABSTRACT Context: The COVID-19 vaccination drive globally was supposedly a game-changing event. However, the emerging variants of the virus and waning immunity over time posed new challenges for breakthrough infections. Standing at the frontline of defense against COVID-19, healthcare personnel (HCP) were vulnerable to such infections. Aims: This study estimates i) the vaccine breakthrough infections (VBI) among HCP following exposure to COVID-19 cases, and ii) the mean interval between the second dose of vaccine and laboratory-confirmed SARS-CoV-2 infection Materials and Methods: A cross-sectional study was conducted including 385 HCP with a history of exposure to COVID-19 cases during January and February 2022. Demographic details and clinical and vaccination history were collected from the test forms and the Web-based hospital management system. Laboratory testing of COVID-19 was carried out by real-time RT-PCR test. Results: The majority of the HCP were males (262; 68.05%) and nurses (180; 46.75%) by occupation. Two doses of vaccines were received by 278 (87.7%) HCP. VBI was confirmed in 185 (66.55%) HCP. No significant difference in VBI between the COVAXIN and COVISHIELD recipients (P = 0.69) was observed. The interval between the second dose and confirmed SARS-CoV-2 infection was significantly higher (P < 0.00001) in COVAXIN recipients (median 228 days) than in COVISHIELD recipients (median 95 days). Conclusions: The incidence of VBI was very high among the HCP, but not statistically different among the COVAXIN and COVISHIELD-recipients. Waning immunity over time suggests boosting immunity with a third dose because of emerging variants.

A case of granulomatosis of polyangiitis presenting with COVID-19 infection: False-positivity or co-existence?

Coronavirus disease 2019 (COVID-19) was declared a global pandemic and a public health emergency worldwide in March 2020.
 COVID-19 presents with non-specific symptoms of the upper airway and pulmonary system, which can overlap with other diseases
 involving the respiratory system as granulomatosis with polyangiitis (GPA). Both diseases have high morbidity and mortality rates
 and it is important to promptly differentiate and treat them. Real-time reverse transcriptase polymerase chain reaction (RT-PCR)
 is currently the recommended method for diagnosing COVID-19. Antibody-based tests are used to diagnose both pat and current
 COVID-19 infections.
 We present a previously healthy thirteen-year-old girl who was admitted with upper airway symptoms and pulmonary involvement,
 and progressed to acute kidney failure. Laboratory findings showed leukocytosis, anemia, elevated kidney function tests and 2+
 proteinuria. Computed tomography (CT) of the lungs showed multiple nodules, cavities, and ground-glass opacities (GGOs). We
 performed RT-PCR tests for COVID-19 for three times. Results were all negative, but the COVID-19 immunoglobulin (Ig)M test
 sent simultaneously was positive. Based on the cytoplasmic antineutrophilic cytoplasmic antibody (c-ANCA) positivity, upper airway,
 pulmonary, and renal involvement, she was diagnosed as GPA.
 This report highlights that COVID-19 antibody tests can be false-positive in patients with autoimmune diseases including GPA.

Orthohantavirus infections in humans and rodents in the Yichun region, China, from 2016 to 2021.

Rodents are the predominant natural hosts of orthohantavirus and the source of human infection, hemorrhagic fever with renal syndrome (HFRS) caused by orthohantavirus is a severe public health problem in the Yichun region, Jiangxi Province, China. However, little information is known about the infection of orthohantavirus in humans and rodents, and the genetic characteristics of the epidemic orthohantavirus in the region. The clinical data of HFRS cases in 2016-2021 was analyzed. Virus infection in rodents was analyzed by orthohantavirus antigen detection using immunofluorescent assay, and the species of orthohantaviruses in rodents and patients were identified by real-time RT-PCR and gene sequencing. The S and M segments of orthohantaviruses from rodents and patients were recovered and analyzed. A total of 1,573 HFRS cases were reported in the Yichun region from 2016 to 2021, including 11 death cases. HFRS cases peaked twice each year: in winter from November to January and early summer from May to June. Farmers constituted the predominant population suffering from HFRS. The orthohantavirus antigen was identified in five species of rodents: Apodemus agrarius (A. agrarius), Rattus norvegicus (R. norvegicus), Sorex araneus, Rattus losea (R. losea), and Niviventer confucianus (N. confucianus). The real-time RT-PCR test and genetic analysis results showed that Hantaan orthohantavirus (HTNV), Seoul orthohantavirus (SEOV), and Dabieshan orthohantavirus (DBSV) were circulated in the rodents. HTNV, SEOV, and DBSV from the rodents were distantly related to other known orthohantaviruses and belonged to novel genetic lineages. SEOV and HTNV were found in HFRS patients, but 97.8% (90/92) of the infections were caused by HTNV. Winter and early summer peaks were both caused by HTNV. The HTNV sequences recovered from HFRS cases were closely related to those from A. agrarius. In the Yichun region, the orthohantaviruses transmitted in rodents include HTNV, SEOV, and DBSV, which have obvious genetic characteristics and high genetic diversity. At the same time, this region is an HFRS mixed epidemic area dominated by HTNV, with two peaks every year, which deserves our high attention.

Performance of MassARRAY system for the detection of SARS-CoV-2 compared to real-time PCR.

Early identification of COVID-19 (coronavirus disease of 2019) by diagnostic tests played an important role in the isolation of infectious patients and management of this pandemic. Various methodologies and diagnostic platforms are available. The current "gold standard" for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) diagnosis is real-time reverse transcriptase-polymerase chain reaction (RT-PCR). To overcome the limitations posed by the short supply experienced early during the pandemic and to increase our capacity, we assessed the performance of the MassARRAY System (Agena Bioscience). MassARRAY System (Agena Bioscience) combines RT-PCR (reverse transcription-polymerase chain reaction) with high-throughput mass spectrometry processing. We compared the MassARRAY performance to a research-use-only E-gene/EAV (Equine Arteritis Virus) assay and RNA Virus Master PCR. Discordant results were tested with a laboratory-developed assay using the Corman etal. E-gene primers and probes. 186 patient specimens were analyzed using the MassARRAY SARS-CoV-2 Panel. The performance characteristics were as follows: the positive agreement was 85.71%, 95% CI (78.12 - 91.45), and the negative agreement was 96.67%, 95% CI (88.47 - 99.59). 19/186 (10.2%) results were found to be discordant and assessed by a different assay with the exception of 1, where the sample was not available for repeat testing. 14 out of 18 agreed with the MassARRAY after testing with the secondary assay. The overall performance after discordance testing was as follows: the positive agreement was 97.3%, 95% CI (90.58 - 99.67), and the negative agreement was 97.14%, 95% CI (91.88 - 99.41). Our study demonstrates that the MassARRAYSystem is an accurate and sensitive method for SARS-CoV-2 detection. Following the discordant agreement with an alternate RT-PCR test, the performance was found to have sensitivity, specificity, and accuracy exceeding 97%, making it a viable diagnostic tool. It can be used as an alternative method during periods when real-time RT-PCR reagent supply chains are disrupted.

Assessing the comparability of cycle threshold values derived from five external quality assessment rounds for omicron nucleic acid testing

BackgroundA variety of open-system real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays for several acute respiratory syndrome coronavirus 2 are currently in use. This study aimed to ensure the quality of omicron nucleic acid testing and to assess the comparability of cycle threshold (Ct) values derived from RT-PCR.MethodsFive external quality assessment (EQA) rounds using the omicron virus-like particles were organized between February 2022 and June 2022.ResultsA total of 1401 qualitative EQA reports have been collected. The overall positive percentage agreement was 99.72%, the negative percentage agreement was 99.75%, and the percent agreement was 99.73%. This study observed a significant variance in Ct values derived from different test systems. There was a wide heterogeneity in PCR efficiency among different RT-PCR kits and inter-laboratories.ConclusionThere was strong concordance among laboratories performing qualitative omicron nucleic acid testing. Ct values from qualitative RT-PCR tests should not be used for clinical or epidemiological decision-making to avoid the potential for misinterpretation of the results.

Evaluation of SARS-CoV-2 isolation in cell culture from nasal/nasopharyngeal swabs or saliva specimens of patients with COVID-19

It has been revealed that SARS-CoV-2 can be efficiently isolated from clinical specimens such as nasal/nasopharyngeal swabs or saliva in cultured cells. In this study, we examined the efficiency of viral isolation including SARS-CoV-2 mutant strains between nasal/nasopharyngeal swab or saliva specimens. Furthermore, we also examined the comparison of viral isolation rates by sample species using simulated specimens for COVID-19. As a result, it was found that the isolation efficiency of SARS-CoV-2 in the saliva specimens was significantly lower than that in the nasal/nasopharyngeal swab specimens. In order to determine which component of saliva is responsible for the lower isolation rate of saliva specimens, we tested the abilities of lactoferrin, amylase, cathelicidin, and mucin, which are considered to be abundant in saliva, to inhibit the infection of SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pv). Lactoferrin and amylase were found to inhibit SARS-CoV-2pv infection. In conclusion, even if the same number of viral genome copies was detected by the real-time RT-PCR test, infection of SARS-CoV-2 present in saliva is thought to be inhibited by inhibitory factors such as lactoferrin and amylase, compared to nasal/nasopharyngeal swab specimens.

Molecular Characterization of Influenza A/H3N2 Virus Isolated from Indonesian Hajj and Umrah Pilgrims 2013 to 2014.

The Hajj and Umrah are the annual mass gatherings of Muslims in Saudi Arabia and increase the transmission risk of acute respiratory infection. This study describes influenza infection among pilgrims upon arrival in Indonesia and the genetic characterization of imported influenza A/H3N2 virus. In total, 251 swab samples with influenza-like illness were tested using real-time RT-PCR for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and influenza viruses. Complete sequences of influenza A/H3N2 HA and NA genes were obtained using DNA sequencing and plotted to amino acid and antigenicity changes. Phylogenetic analysis was performed using a neighbour-joining method including the WHO vaccine strains and influenza A/H3N2 as references. The real-time RT-PCR test detected 100 (39.5%) samples positive with influenza with no positivity of MERS-CoV. Mutations in the HA gene were mainly located within the antigenic sites A, B, and D, while for the NA gene, no mutations related to oseltamivir resistance were observed. Phylogenetic analysis revealed that these viruses grouped together with clades 3C.2 and 3C.3; however, they were not closely grouped with the WHO-recommended vaccine (clades 3C.1). Sequences obtained from Hajj and Umrah pilgrims were also not grouped together with viruses from Middle East countries but clustered according to years of collection. This implies that the influenza A/H3N2 virus mutates continually across time.

SG-APSIC1054: Sputnik-V postvaccination immunologic responses in nasal mucosa: A prospective cohort study in Kazakhstan

Objectives: Sputnik-V (Gam-COVID-Vac) is a recombinant adenoviral (rAdv) vector-based, COVID-19 vaccine now used in >70 countries. Mucosal immunity is thought to be important for protection against COVID-19. We did a prospective cohort study to assess Sputnik-V–elicited mucosal SARS-CoV-2 antibody responses. Methods: We divided 82 COVID-19–free participants into prior COVID-19 and no prior COVID-19 groups and followed them at day 21 after Sputnik-V dose 1′ (rAd5) and dose 2′ (rAd26). Nasopharyngeal swabs and blood were collected to perform SARS-CoV-2 diagnostic and immunologic assays. SARS-CoV-2 spike-specific IgG and IgA ELISAs were performed on both nasal swabs and blood. SARS-CoV-2 real-time RT-PCR testing was performed to exclude infectious influencing. Results: Nasal S-IgG levels increased 25-fold after dose 1′ (P < .001) and remained high after dose 2 in all participants. Prior COVID-19 exposure was associated with both elevated baseline mucosal IgG and IgA and higher postvaccination IgG, but not IgA, boost. Nasal IgA levels increased 16.5-fold after dose 1′ (P < .001) and remained high after dose 2’ in all participants. Compared to dose 1′, Sputnik-V dose 2′ did not further increase either mucosal IgG levels (P = .626) or IgA levels (P = .609). Conclusions: A single dose of Sputnik-V boosted mucosal SARS-CoV-2 immunity. The effects of Sputnik-V dose 2′ on mucosal immunity were minimal. These findings indicate (1) that intramuscularly administered adenoviral vaccines enhance SARS-CoV-2 immunity via both systemic and mucosal routes and (2) that cost-effectiveness and the efficacy of Sputnik-V vaccination could be improved by adjusting the current prime-booster regimen and extending the 21-day interval between the doses. Trial registration: Registered on ClinicalTrials.gov (no. NCT04871841).

Pulmonary parenchymal involvement secondary to subcutaneous injection of silicone gels: a case report

Pulmonary parenchymal involvement secondary to the subcutaneous injection of silicone gels is an unusual condition which occurs more frequently in women aged between 22 and 55 years. Although different theories have been put forward about its etiology, it is unknown and the condition may cause local and systemic complications and even have a fatal outcome. Few cases have been reported in South America and there is no report of this unique entity in Peru. We present the case of a previously healthy 28-year-old male transgender patient who, after an illegal subcutaneous injection of silicone gels in the gluteal region given by a non-healthcare professional, showed progressive respiratory distress and stabbing chest pain of approximately 7 out of 10 on the pain scale within the first 24 hours. Upon admission to the emergency room,respiratory failure was objectively evidenced since the patient had an oxygen saturation of 72 % at a FiO₂ of 21 %, as well as pulmonary parenchymal involvement both in the CT scan and chest X-ray with signs highly suggestive of this pathology. Using a SARS-CoV-2 RNA real-time RT-PCR test performed on a respiratory specimen, COVID pneumonia, immunodeficiency disorders and pulmonary embolism were ruled out. Since there is no standard treatment, the patientwas given relevant support measures such as the administration of supplemental oxygen at a low flow rate by binasal cannula, intravenous systemic corticosteroids and antibiotic therapy, thus achieving good progress with resolution of the initial clinical presentation. Then, after 10 days of intrahospital treatment, the patient was discharged.

Diagnosis of COVID-19 cases on X-Ray images using CNN

COVID-19 is a viral disease that has killed more than 10 million people worldwide and infected millions of people. Therefore, it has become necessary to screen large numbers of people to detect infected individuals and reduce the spread of the disease. Maximum spread for confirming a virus is estimated with RT-PCR test. PCR (Polymerize Chain Response) is a popular device for predicting pathological examination. A key issue with real-time RT-PCR testing is the risk of generating false-negative and false-positive results. As an adjunct to RT-PCR, Computed Tomography (CT) can be used to diagnose COVID-19. In this article, using CXR scans, we proposed a deep-layered convolutional neural network (CNN) for accurate COVID-19 detection. Our model yields 97% accuracy.

West Nile Virus in Culex Mosquitoes in Central Macedonia, Greece, 2022.

In 2022, Greece was the second most seriously affected European country in terms of the West Nile virus (WNV), after Italy. Specifically, Central Macedonia was the region with the most reported human cases (81.5%). In the present study, 30,816 female Culex pipiens sensu lato mosquitoes were collected from May to September 2022 in the seven regional units of Central Macedonia; they were then grouped into 690 pools and tested for WNV, while next-generation sequencing was applied to the samples, which showed a cycle threshold of Ct < 30 in a real-time RT-PCR test. WNV was detected in 5.9% of pools, with significant differences in the detection rate among regional units and months. It is of interest that in the Thessaloniki regional unit, where most of the human cases were observed, the virus circulation started earlier, peaked earlier, and lasted longer than in the other regional units. All sequences clustered into the Central European subclade of WNV lineage 2, and the virus strain differed from the initial Greek strain of 2010 by 0.52% and 0.27% at the nucleotide and amino acid levels, respectively. Signature substitutions were present, such as S73P and T157A in the prM and E structural proteins, respectively. The screening of mosquitoes provides useful information for virus circulation in a region with a potential for early warning, while the availability of whole-genome sequences is essential for further studies, including virus evolution.

Cost-analysis of real time RT-PCR test performed for COVID-19 diagnosis at India's national reference laboratory during the early stages of pandemic mitigation.

Real-time reverse transcription polymerase chain reaction (rRT-PCR) is one of the most accurate and extensively used laboratory procedures for diagnosing COVID-19. This molecular test has high diagnostic accuracy (sensitivity and specificity) and is considered as the gold standard for COVID-19 diagnosis. During COVID-19 surge in India, rRT-PCR service was encouraged and supported by the government of India through existing healthcare setup at various levels of healthcare facilities. The primary purpose of this research was to determine the per-unit cost of providing COVID-19 rRT-PCR services at the national reference laboratory at ICMR-National Institute of Virology in Pune during the early phase of COVID-19 pandemic mitigation, from the provider's perspective. The monthly cost for rRT-PCR testing as well as an estimated annual average unit cost for testing that takes account of peaks and troughs in pandemic were investigated. The time frame used to estimate unit cost was one year (July 2020-June 2021). For data collection on all resources spent during the early phase of pandemic, a conventional activity-based bottom-up costing technique was used. Capital costs were discounted and annualized over the estimated life of the item. Apportioning statistics were selected for cost heads like human resources, capital, and equipment based on time allocation, sharing of services, and utilization data. The data was also used to understand the breakdown of costs across inputs and over time and different levels of testing activity. During the initial phase of pandemic mitigation, the per unit cost of providing the COVID-19 rRT-PCR test was estimated to be ₹566 ($7.5) in the month of July 2020, where the total 56318 COVID-19 rRT-PCR tests was performed. The major proportion (87%) of funds was utilized for procuring laboratory consumables, followed by HR (10%), and it was least for stationary & allied items (0.02%). Unit cost was found to be the most sensitive to price variations in lab consumables (21.7%), followed by the number of samples tested (3.9%), salaries paid to HR (2.6%), price of equipment (0.23%), and building rental price (0.14%) in a univariate sensitivity analysis. The unit cost varies over the period of the pandemic in proportion with the prices of consumables and inversely proportional with number of tests performed. Our study would help the Government to understand the value for money they invested for laboratory diagnosis of COVID-19, budget allocation, integration and decentralization of laboratory services so as to help for achieving universal health coverage.

New insights on epidemiology of infectious bronchitis virus in Iran by comparing two genotyping methods

Different genotypes of infectious bronchitis virus (IBV) in poultry are circulating worldwide. The most efficient classification system for IBV genotypes is based on the complete sequencing of S1 gene, and the target organ can be trachea, kidney, oviduct, lung, esophagus, proventriculus, intestine, liver, spleen, bursa, cecal tonsils, cloaca, and testis. In Iran, IBV genotyping is usually performed by partial sequencing of S1 gene, and the trachea is often selected for sampling. This study applies a different genotyping method, and compares the results with the routine method of genotyping. Samples were collected from the trachea and kidneys of 50 broiler flocks with respiratory symptoms. The presence of IBV was confirmed by a real-time RT-PCR analysis targeting the 5'UTR region of the genome. Genotype of positive samples was determined by two methods of partial S1 gene sequencing and genotype-specific primers. In the real-time RT-PCR test, 88% and 90% of tracheal and kidney samples showed positive results, respectively. When the S gene was sequenced, Variant 2 (GI-23) (68.18%), 793/B (GI-13) (22.73%), Massachusetts (GI-1) (6.82%), and QX (GI-19) (2.27%) were detected in the tracheal samples, whereas QX (GI-19) and Massachusetts (GI-1) were not diagnosed in the kidney samples. In the genotypespecific method, IBV genotypes Variant 2, 793/B, Massachusetts, and QX were detected in both tracheal and kidney samples. The results of genotype-specific method also demonstrated that 70% of tracheal samples and 62% of kidney samples were infected with a single IBV genotype, while 30% of tracheal samples and 38% of kidney samples were coinfected with different IBV types. When comparing two genotyping methods, the use of genotype-specific primers was superior to partial S1 gene sequencing, not only in detecting different IBV types, but also in efficiently applying them in both trachea and kidney.