Effectiveness of COVID-19 vaccines on hospitalization and death in Guilan, Iran: a test-negative case-control study.

OBJECTIVES: The present study was conducted to estimate the effectiveness of (BBIBP)-CorV (Sinopharm), ChAdOx1-S/nCoV-19 (AZD1222, Oxford-AstraZeneca), rAd26-rAd5 (Gam-COVID-Vac, Sputnik V), and BIV1-CovIran (COVIran Barekat) and BBV152 COVAXIN (Bharat Biotech) vaccines against hospitalization and death of COVID-19 in Guilan Province of Iran from May 22 to December 21, 2021. METHODS: This test-negative case-control study was conducted on the population aged 5 years and above by extracting information from local databases (The Medical Care Monitoring Center and The Integrated Health System). A logistic regression analysis was performed to estimate the effectiveness of the vaccines against COVID-19 hospitalization and death. RESULTS: The total study population was 42,084, including 19,500 cases (with a positive Reverse Transcriptase-Polymerase Chain Reaction test admitted to hospitals in Guilan Province) and 22,586 controls (with a negative Reverse Transcriptase-Polymerase Chain Reaction test). Among the admitted patients, 1887 deaths occurred. The maximum effectiveness of BBIBP-CorV (Sinopharm) in preventing temporary hospitalization and regular hospitalization was observed 151 days after receiving the second dose, 95% (95% CI: 67-99.4%) and 85% (95% CI: 77-91%) respectively. The maximum effectiveness of the BBIBP-CorV (Sinopharm) vaccine 91-120 days after receiving the second dose against death was showed 56% (95% CI: 33-71%). The maximum effectiveness of ChAdOx1-S/nCoV-19 (AZD1222, Oxford-AstraZeneca) and BIV1-CovIran (COVIran Barekat) in preventing regular hospitalization and death was observed 121-150 and 61-90 days (respectively) after receiving the second dose, reaching 98% (95% CI: 94-99%) and 92% (95% CI: 48-99%), respectively for ChAdOx1-S/nCoV-19 and 95% (95% CI: 91-97%) and 89% (95% CI: 55-98%) respectively, for BIV1-CovIran. CONCLUSION: For almost all vaccines, the study observed an increase in effectiveness against hospitalization and death over time.

SARS-CoV-2 in infant urine and fecal samples after in utero COVID-19 exposure.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a pandemic that has and will continue to affect many pregnant women. Knowledge regarding the risk of vertical transmission is limited. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of nasopharyngeal swabs typically have been used to confirm the diagnosis among infants, but whether the virus can be detected in other biological specimens, and therefore potentially transmitted in other ways, is unknown. Positive SARS-CoV-2 RT-PCR has been reported from feces and urine from adult patients. We hypothesize that the presence of SARS-CoV-2 in infant urine and fecal samples after prenatal COVID-19 exposure is low. METHODS: We examined the presence of SARS-CoV-2 RNA using RT-PCR in urine and fecal samples among 42 infants born to SARS-CoV-2-infected mothers during different stages of pregnancy. RESULTS: A urine sample was collected from 39 of 42 infants and fecal samples from all 42 infants shortly after birth. Although the majority of the women had the symptomatic disease (85.6%), we were unable to detect the presence of SARS-CoV-2 virus from any infant urine or fecal samples. CONCLUSIONS: SARS-CoV-2 was not detected in infant urine or feces after maternal infection during pregnancy, providing further evidence for low rates of perinatal transmission. IMPACT: SARS-CoV-2 was not detected in the urine or feces of infants of mothers with COVID-19 during various time points in pregnancy. This study provides further evidence for low rates of perinatal transmission of SARS-CoV-2. Results help to provide guidance on perinatal care practices for infants exposed to COVID-19 in utero.

Visualized RNA detection of SARS-CoV-2 in a closed tube by coupling RT-PCR with nested invasive reaction.

A simple, cost-effective and reliable diagnosis of pathogen nucleic acids assay is much required for controlling a pandemic of a virus disease, such as COVID-19. Our previously developed visualized detection of pathogen DNA in a single closed tube is very suitable for POCT. However, virus RNA could not be detected directly and should be reverse-transcribed into cDNA in advance. To enable this visualized assay to detect virus RNA directly, various types of reverse transcriptase were investigated, and finally we found that HiScript II reverse transcriptase could keep active and be well adapted to the one-pot visualized assay in optimized conditions. Reverse transcription, template amplification and amplicon identification by PCR coupled with invasive reaction, as well as visualization by self-assembling of AuNP probes could be automatically and sequentially performed in a closed tube under different temperature conditions, achieving "sample (RNA)-in-result (red color)-out" only by a simple PCR engine plus the naked eye. The visualized RT-PCR is sensitive to unambiguous detection of 5 copies of the N and ORFlab genes of SARS-CoV-2 RNA comparing favourably with qPCR methods (commercialized kit), is specific to genotype 3 variants (Alpha, Beta and Omicron) of SARS-CoV-2, and is very accurate for picking up 0.01% Omicron variant from a large amount of sequence-similar backgrounds. The method is employed in detecting 50 clinical samples, and 10 of them were detected as SARS-CoV-2 positive samples, identical to those by conventional RT-PCR, indicating that the method is cost-effective and labor-saving for pathogen RNA screening in resource-limited regions.

Evaluation of reverse transcriptase-polymerase spiral reaction assay for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2.

BACKGROUND AND AIM: Existing real-time reverse transcriptase PCR (RT-qPCR) has certain limitations for the point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since it requires sophisticated instruments, reagents and skilled laboratory personnel. In this study, we evaluated an assay termed the reverse transcriptase-polymerase spiral reaction (RT-PSR) for rapid and visual detection of SARS-CoV-2. METHODS: The RT-PSR assay was optimized using RdRp gene and evaluated for the detection of SARS-CoV-2. The time of 60min and a temperature of 63°C was optimized for targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2. The sensitivity of the assay was evaluated by diluting the in-vitro transcribed RNA, which amplifies as low as ten copies. RESULTS: The specific primers designed for this assay showed 100% specificity and did not react when tested with other lung infection-causing viruses and bacteria. The optimized assay was validated with 190 clinical samples in two phases, using automated RTPCR based TrueNat test, and the results were comparable. CONCLUSIONS: The RT-PSR assay can be considered for rapid and sensitive detection of SARS-CoV-2, particularly in resource-limited settings. To our knowledge, there is as yet no RT-PSR-based kit developed for SARS-CoV-2.

Laboratory and field evaluation of the STANDARD Q and Panbio™ SARS-CoV-2 antigen rapid test in Namibia using nasopharyngeal samples.

BACKGROUND: As new SARS-CoV-2 variants of concern emerge, there is a need to scale up testing to minimize transmission of the Coronavirus disease 2019 (COVID-19). Many countries especially those in the developing world continue to struggle with scaling up reverse transcriptase polymerase reaction (RT-PCR) to detect SARS-CoV-2 due to scarcity of resources. Alternatives such as antigen rapid diagnostics tests (Ag-RDTs) may provide a solution to enable countries scale up testing. METHODS: In this study, we evaluated the Panbio™ and STANDARD Q Ag-RDTs in the laboratory using 80 COVID-19 RT-PCR confirmed and 80 negative nasopharyngeal swabs. The STANDARD Q was further evaluated in the field on 112 symptomatic and 61 asymptomatic participants. RESULTS: For the laboratory evaluation, both tests had a sensitivity above 80% (Panbio™ = 86% vs STANDARD Q = 88%). The specificity of the Panbio™ was 100%, while that of the STANDARD Q was 99%. When evaluated in the field, the STANDARD Q maintained a high specificity of 99%, however the sensitivity was reduced to 56%. CONCLUSION: Using Ag-RDTs in low resource settings will be helpful in scaling-up SARS-CoV-2 testing, however, negative results should be confirmed by RT-PCR where possible to rule out COVID-19 infection.

Maternal and Neonatal Outcomes in Pregnant Women With Coronavirus Disease 2019 in Korea.

BACKGROUND: This study aimed to describe the maternal, obstetrical, and neonatal outcomes in pregnant women with coronavirus disease 2019 (COVID-19) and identify the predictors associated with the severity of COVID-19. METHODS: This multicenter observational study included consecutive pregnant women admitted because of COVID-19 confirmed using reverse transcriptase-polymerase chain reaction (RT-PCR) test at 15 hospitals in the Republic of Korea between January 2020 and December 2021. RESULTS: A total of 257 women with COVID-19 and 62 newborns were included in this study. Most of the patients developed this disease during the third trimester. Nine patients (7.4%) developed pregnancy-related complications. All pregnant women received inpatient treatment, of whom 9 (3.5%) required intensive care, but none of them died. The gestational age at COVID-19 diagnosis (odds ratio [OR], 1.096, 95% confidence interval [CI], 1.04-1.15) and parity (OR, 1.703, 95% CI, 1.13-2.57) were identified as significant risk factors of severe diseases. Among women who delivered, 78.5% underwent cesarean section. Preterm birth (38.5%), premature rupture of membranes (7.7%), and miscarriage (4.6%) occurred, but there was no stillbirth or neonatal death. The RT-PCR test of newborns' amniotic fluid and umbilical cord blood samples was negative for severe acute respiratory syndrome coronavirus 2. CONCLUSION: At the time of COVID-19 diagnosis, gestational age and parity of pregnant women were the risk factors of disease severity. Vertical transmission of COVID-19 was not observed, and maternal severity did not significantly affect the neonatal prognosis.

Reverse transcriptase-free detection of viral RNA using Hemo Klentaq DNA polymerase.

COVID-19 pandemic highlighted the demand for the fast and reliable detection of viral RNA. Although various methods for RNA amplification and detection have been proposed, some limitations, including those caused by reverse transcription (RT), need to be overcome. Here, we report on the direct detection of specific RNA by conventional polymerase chain reaction (PCR) requiring no prior RT step. It was found that Hemo KlenTaq (HKTaq), which is posed as DNA-dependent DNA polymerase, possesses reverse transcriptase activity and provides reproducible amplification of RNA targets with an efficiency comparable to common RT-PCR. Using nasopharyngeal swab extracts from COVID-19-positive patients, the high reliability of SARS-CoV-2 detection based on HKTaq was demonstrated. The most accurate detection of specific targets are provided by nearby primers, which allow to determine RNA in solutions affected to multiple freeze-thaw cycles. HKTaq can be used for elaboration of simplified amplification techniques intended for the analysis of any specific RNA and requiring only one DNA polymerase.

Association of mRNA Vaccination With Clinical and Virologic Features of COVID-19 Among US Essential and Frontline Workers.

Importance: Data on the epidemiology of mild to moderately severe COVID-19 are needed to inform public health guidance. Objective: To evaluate associations between 2 or 3 doses of mRNA COVID-19 vaccine and attenuation of symptoms and viral RNA load across SARS-CoV-2 viral lineages. Design, Setting, and Participants: A prospective cohort study of essential and frontline workers in Arizona, Florida, Minnesota, Oregon, Texas, and Utah with COVID-19 infection confirmed by reverse transcriptase-polymerase chain reaction testing and lineage classified by whole genome sequencing of specimens self-collected weekly and at COVID-19 illness symptom onset. This analysis was conducted among 1199 participants with SARS-CoV-2 from December 14, 2020, to April 19, 2022, with follow-up until May 9, 2022, reported. Exposures: SARS-CoV-2 lineage (origin strain, Delta variant, Omicron variant) and COVID-19 vaccination status. Main Outcomes and Measures: Clinical outcomes included presence of symptoms, specific symptoms (including fever or chills), illness duration, and medical care seeking. Virologic outcomes included viral load by quantitative reverse transcriptase-polymerase chain reaction testing along with viral viability. Results: Among 1199 participants with COVID-19 infection (714 [59.5%] women; median age, 41 years), 14.0% were infected with the origin strain, 24.0% with the Delta variant, and 62.0% with the Omicron variant. Participants vaccinated with the second vaccine dose 14 to 149 days before Delta infection were significantly less likely to be symptomatic compared with unvaccinated participants (21/27 [77.8%] vs 74/77 [96.1%]; OR, 0.13 [95% CI, 0-0.6]) and, when symptomatic, those vaccinated with the third dose 7 to 149 days before infection were significantly less likely to report fever or chills (5/13 [38.5%] vs 62/73 [84.9%]; OR, 0.07 [95% CI, 0.0-0.3]) and reported significantly fewer days of symptoms (10.2 vs 16.4; difference, -6.1 [95% CI, -11.8 to -0.4] days). Among those with Omicron infection, the risk of symptomatic infection did not differ significantly for the 2-dose vaccination status vs unvaccinated status and was significantly higher for the 3-dose recipients vs those who were unvaccinated (327/370 [88.4%] vs 85/107 [79.4%]; OR, 2.0 [95% CI, 1.1-3.5]). Among symptomatic Omicron infections, those vaccinated with the third dose 7 to 149 days before infection compared with those who were unvaccinated were significantly less likely to report fever or chills (160/311 [51.5%] vs 64/81 [79.0%]; OR, 0.25 [95% CI, 0.1-0.5]) or seek medical care (45/308 [14.6%] vs 20/81 [24.7%]; OR, 0.45 [95% CI, 0.2-0.9]). Participants with Delta and Omicron infections who received the second dose 14 to 149 days before infection had a significantly lower mean viral load compared with unvaccinated participants (3 vs 4.1 log10 copies/µL; difference, -1.0 [95% CI, -1.7 to -0.2] for Delta and 2.8 vs 3.5 log10 copies/µL, difference, -1.0 [95% CI, -1.7 to -0.3] for Omicron). Conclusions and Relevance: In a cohort of US essential and frontline workers with SARS-CoV-2 infections, recent vaccination with 2 or 3 mRNA vaccine doses less than 150 days before infection with Delta or Omicron variants, compared with being unvaccinated, was associated with attenuated symptoms, duration of illness, medical care seeking, or viral load for some comparisons, although the precision and statistical significance of specific estimates varied.

A comparative study on the efficiency of commercial reverse transcriptase-Polymerase chain reaction kits for the detection of severe acute respiratory syndrome coronavirus 2 infections.

Background: Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) kits have been reliably employed for the diagnosis of coronavirus disease 2019 (COVID-19) by the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA since the beginning of the disease outbreak. In consideration of reliable diagnosis, apart from RT-PCR, the isothermal nucleic acid amplification-based point-of-care automated kits have also been tagged as a simpler and rapid alternative to the conventional techniques. Currently, the availability of a better diagnostic method for COVID-19 when compared to RT-PCR is nil. The most important step in the detection of SARS-CoV-2 in a RT-PCR diagnostic laboratory is to identify and employ RT-PCR kits with higher sensitivity as well as specificity. Objectives: This study aimed to study commercially available RT-PCR kits for the detection of SARS-CoV-2 infections. Methods: The performance of seven different RT-PCR kits from different manufacturers used for diagnosis of COVID-19 in Govt Theni Medical College and Hospital, Theni, Tamil Nadu were analysed. Nasopharyngeal and oropharyngeal swabs were collected from patients and subjected to RT-PCR using these kits. Results and Conclusion: The sensitivities and batch effects of the assessed kits were slightly different for different targets, for SARS-CoV-2 detection in nasopharyngeal swab specimens. Examination of COVID-19 kits should be done using currently employed kits in routine diagnosis for better efficiency.

Performance of saline and water gargling for SARS-CoV-2 reverse transcriptase PCR testing: a systematic review and meta-analysis.

The performance of gargling for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcriptase (RT)-PCR testing has not been previously reviewed. This review systematically assessed the performance of saline and water gargling for SARS-CoV-2 RT-PCR testing in the settings of diagnosing and monitoring viral shedding.We included original studies comparing the performance of gargling and (oropharyngeal-)nasopharyngeal swabs for SARS-CoV-2 RT-PCR testing. Studies conducted in either suspected individuals or confirmed cases were included and analysed separately. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were examined using random-effects models.Gargles achieved a high overall sensitivity (91%), specificity (97%), PPV (95%) and NPV (91%) for SARS-CoV-2 RT-PCR testing. Studies using saline gargle and water gargle have an overall sensitivity of 97% and 86%, respectively. The sensitivity values were largely maintained for saline and water gargling on stratified analysis, for both diagnosis (96% and 92%) and viral shedding monitoring (98% and 78%). A higher sensitivity was also reported by studies using sterile saline (100%), a smaller amount of gargling solution (92% versus 87%) and a longer gargling duration (95% versus 86%).Our results supported the use of gargling as a sampling approach for SARS-CoV-2 RT-PCR testing, which achieved a high sensitivity for both diagnosis and viral shedding monitoring purposes. Further investigation on the comparative performance of different gargling mediums is needed to draw a definitive conclusion.

Durability of Immune Response After COVID-19 Booster Vaccination and Association With COVID-19 Omicron Infection.

Importance: The BNT162b2 two-dose vaccine (BioNTech/Pfizer) has high effectiveness that wanes within several months. The third dose is effective in mounting a significant immune response, but its durability is unknown. Objective: To compare antibody waning after second and third doses and estimate the association of antibody kinetics with susceptibility to infection with the Omicron variant of SARS-CoV-2. Design, Setting, and Participants: In a prospective longitudinal cohort study in a tertiary medical center in Israel, health care workers who received the BNT162b2 vaccine were followed up monthly for IgG and neutralizing antibody levels. Linear mixed models were used to compare antibody titer waning of second and third doses and to assess whether antibody dynamics were associated with Omicron transmission. Avidity, T cell activation, and microneutralization of sera against different variants of concern were assessed for a subgroup. Exposure: Vaccination with a booster dose of the BNT162b2 vaccine. Main Outcomes and Measures: The primary outcome was the rate of antibody titer change over time, and the secondary outcome was SARS-CoV-2 Omicron variant infection, as confirmed by reverse transcriptase-polymerase chain reaction. Results: Overall, 4868 health care workers (mean [SD] age, 46.9 [13.7] years; 3558 [73.1%] women) and 3972 health care workers (mean [SD] age, 48.5 [14.1] years; 996 [74.9%] women) were followed up for 5 months after their second and third vaccine doses, respectively. Waning of IgG levels was slower after the third compared with the second dose (1.32%/d [95% CI, 1,29%/d to 1.36%/d] vs 2.26% [95% CI, 2.13%/d 2.38%/d]), as was waning of neutralizing antibody levels (1.32%/d [95% CI, 1.21%/d to 1.43%/d] vs 3.34%/d [95% CI, 3.11%/d to 3.58%/d]). Among 2865 health care workers assessed for Omicron incidence during an additional 2 months of follow-up, lower IgG peak (ratio of means 0.86 [95% CI, 0.80-0.91]) was associated with Omicron infection, and among participants aged 65 years and older, faster waning of IgG and neutralizing antibodies (ratio of mean rates, 1.40; [95% CI, 1.13-1.68] and 3.58 [95% CI, 1.92-6.67], respectively) were associated with Omicron infection. No waning in IgG avidity was observed 112 days after the third dose. Live neutralization of Omicron was lower compared with previous strains, with a geometric mean titer at the peak of 111 (95% CI, 75-166), compared with 942 (95% CI, 585-1518) for WT, 410 (95% CI, 266-634) for Delta; it demonstrated similar waning to 26 (95% CI, 16-42) within 4 months. Among 77 participants tested for T cell activity, mean (SD) T cell activity decreased from 98 (5.4) T cells/106 peripheral blood mononuclear cells to 59 (9.3) T cells/106 peripheral blood mononuclear cells. Conclusions and Relevance: This study found that the third vaccine dose was associated with greater durability than the second dose; however, Omicron was associated with greater resistance to neutralization than wild type and Delta variants of concern. Humoral response dynamics were associated with susceptibility to Omicron infection.

When to test for COVID-19 using real-time reverse transcriptase polymerase chain reaction: a systematic review.

OBJECTIVES: The aim of this study was to evaluate the time in days between symptom onset and first positive real-time reverse transcriptase polymerase chain reaction (RT-PCR) result for COVID-19. METHODS: This systematic review was conducted in the MEDLINE (PubMed), Embase, and Scopus databases using the following descriptors: "COVID-19", "SARS-CoV-2", "coronavirus", "RT-PCR", "real time PCR", and "diagnosis". RESULTS: The included studies were conducted in 31 different countries and reported on a total of 6831 patients. The median age of the participants was 49.95 years. The three most common symptoms were fever, cough, and dyspnea, which affected 4012 (58.68%), 3192 (46.69%), and 2009 patients (29.38%), respectively. Among the 90 included studies, 13 were prospective cohorts, 15 were retrospective cohorts, 36 were case reports, 20 were case series, and six were cross-sectional studies. The overall mean time between symptom onset and positive test result was 6.72 days. Fourteen articles were analyzed separately for the temporal profile of RT-PCR test results; the best performance was on days 22-24, when 98% of test results were positive. CONCLUSION: These findings corroborate the RT-PCR COVID-19 testing practices of some health units. In addition, the most frequently described symptoms of these patients can be considered the initial symptoms of infection and used in decision-making about RT-PCR testing.

An isothermal lab-on-phone test for easy molecular diagnosis of SARS-CoV-2 near patients and in less than 1 hour.

OBJECTIVES: The performance of a new point-of-care CE-IVD-marked isothermal lab-on-phone COVID-19 assay was assessed in comparison to a gold standard real-time reverse transcriptase-PCR method. METHODS: The study was conducted following a nonprobability sampling of ≥16-year-old volunteers from three different laboratories, using direct mouthwash (N = 24) or nasopharyngeal (N = 191) clinical samples. RESULTS: The assay demonstrated 95.19% sensitivity and 100% specificity for detection of SARS-CoV-2 in direct nasopharyngeal crude samples and 78.95% sensitivity and 100% specificity in direct mouthwash crude samples. It also successfully detected currently predominant SARS-CoV-2 variants of concern (Beta B.1.351, Delta B.1.617.2, and Omicron B.1.1.529) and demonstrated to be inert against potential cross-reactions of other common respiratory pathogens that cause infections that present similar symptoms to COVID-19. CONCLUSION: This lab-on-phone pocket-sized assay relies on an isothermal amplification of SARS-CoV-2's N and E genes, taking just 50 minutes from sample to result, with only 2 minutes of hands-on time. It presents good performance when using direct nasopharyngeal crude samples, enabling a low-cost, real-time, rapid, and accurate identification of SARS-CoV-2 infections at the point of care, which is important for both clinical management and population screening, as a tool to break the chain of transmission of COVID-19 pandemic, especially in low-resources environments.

Development and Validation of an In-House Real-Time Reverse-Transcriptase Polymerase Chain Reaction Assay for SARS-CoV-2 Omicron Lineage Subtyping between BA.1 and BA.2.

In order to rapidly differentiate sublineages BA.1 and BA.2 of the SARS-CoV-2 variant of concern Omicron, we developed a real-time reverse-transcriptase polymerase chain reaction to target the discriminatory spike protein deletion at amino acid position 69-70 (S:del69-70). Compared to the gold standard of whole genome sequencing, the candidate assay was 100% sensitive and 99.4% specific. Sublineage typing by RT-PCR can provide a rapid, high throughput and cost-effective method to enhance surveillance as well as potentially guiding treatment and infection control decisions.

Expression of Codon-Optimized Gene Encoding Murine Moloney Leukemia Virus Reverse Transcriptase in Escherichia coli.

Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L-1 to 0.175 g L-1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.

Factors associated with weak positive SARS-CoV-2 diagnosis by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR).

During the COVID-19 pandemic, the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay has been the primary method of diagnosis of SARS-CoV-2 infection. However, RT-qPCR assay interpretation can be ambiguous with no universal absolute cut-off value to determine sample positivity, which particularly complicates the analysis of samples with high Ct values, or weak positives. Therefore, we sought to analyse factors associated with weak positive SARS-CoV-2 diagnosis. We analysed sample data associated with all positive SARS-CoV-2 RT-qPCR diagnostic tests performed by the Victorian Infectious Diseases Reference Laboratory (VIDRL) in Melbourne, Australia, during the Victorian first wave (22 January 2020-30 May 2020). A subset of samples was screened for the presence of host DNA and RNA using qPCR assays for CCR5 and 18S, respectively. Assays targeting the viral RNA-dependent RNA polymerase (RdRp) had higher Ct values than assays targeting the viral N and E genes. Weak positives were not associated with the age or sex of individuals' samples nor with reduced levels of host DNA and RNA. We observed a relationship between Ct value and time post-SARS-CoV-2 diagnosis. High Ct value or weak positive SARS-CoV-2 was not associated with any particular bias including poor biological sampling.

Charge Engineering Improves the Performance of Bst DNA Polymerase Fusions.

The charge states of proteins can greatly influence their stabilities and interactions with substrates, and the addition of multiple charges (supercharging) has been shown to be a successful approach for engineering protein stability and function. The addition of a fast-folding fusion domain to the Bacillus stearothermophilus DNA polymerase improved its functionality in isothermal amplification assays, and further charge engineering of this domain has increased both protein stability and diagnostics performance. When combined with mutations that stabilize the core of the protein, the charge-engineered fusion domain leads to the ability to carry out loop-mediated isothermal amplification (LAMP) at temperatures up to 74° C or in the presence of high concentrations of urea, with detection times under 10 min. Adding both positive and negative charges to the fusion domain led to changes in the relative reverse transcriptase and DNA polymerase activities of the polymerase. Overall, the development of a modular fusion domain whose charged surface can be modified at will should prove to be of use in the engineering of other polymerases and, in general, may prove useful for protein stabilization.

Diagnostic Accuracy of SARS-CoV-2 Nucleocapsid Antigen Self-Test in Comparison to Reverse Transcriptase-Polymerase Chain Reaction.

BACKGROUND: Currently, the rapid antigen test (RAT) and reverse transcriptase-polymerase chain reaction (RT-PCR) are considered the main stakeholders in COVID-19 diagnosis. In RT-PCR, any of at least 2 evolutionary conserved genes (RdRP, E-, N-, ORF1ab gene) and S-gene of SARS-CoV-2 are endorsed, and in RAT, the nucleocapsid antigen (N-Ag) of SARS-CoV-2 is considered due to its stability and fewer chances of mutation effects. In the present work, we evaluated the performance of the AG-Q COVID-19 N-Ag self-test kit and conducted a validation study in comparison with the RT-PCR. METHODS: AG-Q COVID-19 N-Ag rapid test kit is an Indian Council of Medical Research (ICMR) approved product developed and marketed by Agappe Diagnostics Limited. The RT-PCR assay was performed with a COVIPATH COVID-19 RT-PCR kit from Thermo Fisher Scientific. RESULTS: We observed 19 false-negative results in antigen self-tests, including samples of threshold cycle (Ct) values 22/22 (N-gene/ORF1ab-gene) in RT-PCR, indicating inadequate sampling by the patients in self-tests, leading to false-negative results and increased chances of the disease spreading. Based on the RT-PCR Ct value vs antigen self-test comparison, it is evident that proper sampling is crucial in performing antigen self-tests. Also, there were weak positive results in antigen self-tests with a Ct value of 18/19 in RT-PCR. CONCLUSIONS: Although the sensitivity and diagnostic accuracy offered by the AG-Q COVID-19 N-Antigen self-test in comparison with RT-PCR fulfills the ICMR tenets for RAT, this study recommends the laboratory/hospital-based RAT execution would be appropriate, rather than the self-test.

Pneumonia Frequency and Severity in Patients With Symptomatic COVID-19: Impact of mRNA and Adenovirus Vector Vaccines.

BACKGROUND. Additional evidence of the role of COVID-19 vaccination in reducing pneumonia frequency and severity in the setting of breakthrough infection could help combat ongoing vaccine hesitancy. OBJECTIVE. The purpose of this article was to compare the frequency and severity of pneumonia on chest CT in patients with confirmed COVID-19 between patients who are unvaccinated and those who are fully vaccinated by messenger RNA (mRNA) or adenovirus vector vaccines. METHODS. This retrospective single-center study included 467 patients (250 men, 217 women; mean age, 65 ± 17 [SD] years) who underwent chest CT between December 15, 2021, and February 18, 2022, during hospitalization for symptomatic COVID-19, confirmed by reverse transcriptase-polymerase chain reaction assay. A total of 216 patients were unvaccinated, and 167 and 84 patients were fully vaccinated (defined as receipt of the second dose at least 14 days before COVID-19 diagnosis) by the BNT162b2 mRNA vaccine or the ChAdOx1-S adenovirus vector vaccine, respectively. Semiquantitative CT severity scores (CT-SS; 0-25 scale) were determined; CT-SS of 0 indicated absence of pneumonia. Presence of bilateral involvement was assessed in patients with pneumonia. Associations were explored between vaccination status and CT findings. RESULTS. The frequency of the absence of pneumonia was 15% (32/216) in unvaccinated patients, 29% (24/84) in patients fully vaccinated with ChAdOx1-S vaccine, and 51% (85/167) in patients fully vaccinated with BNT162b2 vaccine (unvaccinated and ChAdOx1-S vs BNT162b2: p < .001; unvaccinated vs ChAdOx1-S: p = .08). Mean CT-SS was significantly higher in unvaccinated patients (9.7 ± 6.1) than in patients fully vaccinated with BNT162b2 (5.2 ± 6.1) or ChAdOx1-S (6.2 ± 5.9) vaccine (both p < .001). Full vaccination was significantly associated with CT-SS independent of patient age and sex (estimate = -4.46; p < .001). Frequency of bilateral lung involvement was significantly higher in unvaccinated patients (158/184, 86%) and in patients fully vaccinated with ChAdOx1-S vaccine (54/60, 90%) than in patients fully vaccinated with BNT162b2 vaccine (47/82, 57%) (both p < .001). CONCLUSION. Pneumonia frequency and severity were lower in patients with full vaccination by mRNA and adenovirus vector vaccines who experienced breakthrough infections in comparison with unvaccinated patients. CLINICAL IMPACT. The visual observation by radiologic imaging of the protective effect of vaccination on lung injury in patients with breakthrough infections provides additional evidence supporting the clinical benefit of vaccination.