Rapid Decline of SARS-CoV-2 Viral Load in Single vs. Double-Dose (Short-Interval <6 Weeks) ChAdOx nCoV-19 Vaccinated Health-Care Workers.

The present work was carried out during the emergence of Delta Variant of Concern (VoC) and aimed to study the change in SARS CoV-2 viral load in Covishield vaccinated asymptomatic/mildly symptomatic health-care workers (HCWs) to find out the optimum isolation period. The SARS CoV-2 viral load was carried out in sequential samples of 55 eligible HCWs which included unvaccinated (UnV; n = 11), single-dose vaccinated (SDV, n = 20) and double-dose vaccinated [DDV, n = 24; short-interval (<6 weeks)] subjects. The mean load of envelope (E) gene on day 5 in SDV [0.42 × 105 copies/reaction] was significantly lower as compared to DDV [6.3 × 105 copies/reaction, P = 0.005] and UnV [6.6 × 105 copies/reaction, P = 0.001] groups. The rate of decline of SARS CoV-2 viral load in the initial 5 days of PCR positivity was significantly higher in SDV as compared to that in DDV (Mean log decline 0.39 vs. 0.19; P < 0.001). This was possibly due to interference of adenoviral immunity of first dose of adenovirus-vectored vaccine in double-dose vaccinated HCWs who had received vaccines within a shorter interval (<6 weeks).

Cycle threshold values of SARS-CoV-2 RNA in conjunctival swabs and nasopharyngeal secretions: a comparative study from a tertiary care center in India.

PURPOSE: To detect the viral RNA load of SARS-CoV-2 in conjunctival swabs of COVID-19 patients, and compare with nasopharyngeal swabs. METHODS: Conjunctival swabs of COVID-19 patients (with PCR positive nasopharyngeal swabs) were subjected to quantitative reverse transcription-polymerase chain reaction (RT-PCR) for detection of SARS-CoV-2 RNA. The cycle threshold (Ct) values of Open Reading Frame 1 (ORF 1 Ab gene) and nucleoprotein (N gene) PCRs were used to assess the viral RNA load, and compare them with the baseline values of nasopharyngeal swabs. RESULTS: Of 93 patients, 17 (18.27%) demonstrated SARS-CoV-2 RNA in conjunctival swabs. Baseline nasopharyngeal swabs were collected at a median of 2 days; while, the conjunctival swabs were collected at median 7 days, from onset of illness (p < 0.001). Despite a significant delay in conjunctival swab collection than nasopharyngeal swabs, the Ct values (ORF or N gene PCRs) were comparable between nasopharyngeal swab and conjunctival swab samples. Subsequently, during the recovery period, in four of these 17 patients (with conjunctival swab positivity), when the second nasopharyngeal swab was 'negative', the conjunctival swab was 'positive'. CONCLUSION: The conjunctival swabs demonstrated SARS-CoV-2 RNA in 17 (18.27%) of 93 COVID-19 patients. Our results may suggest a delayed or a prolonged shedding of the virus/viral RNA on the ocular surface than in nasopharyngeal mucosa.

Effect of storage temperatures simulating transport conditions of nasopharyngeal swabs on the results of a chemiluminescence immunoassay (CLIA) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen.

PURPOSE: Chemiluminescence Immunoassay (CLIA) is high throughput, rapid diagnostic test which has recently come up for the detection of SARS-CoV-2 antigen. The present study evaluated performance of CLIA antigen test in nasopharyngeal swab samples stored at different temperatures for 7 days to simulate the transport conditions and transit time across the country from remote peripheral laboratories to central facilities. MATERIALS AND METHODS: Limit of detection (LOD), sensitivity and specificity of VITROS® SARS-CoV-2 antigen assay was determined using Real-time reverse transcriptase PCR (rRT-PCR) confirmed SARS-CoV-2 positive and negative samples. To detect the effect of storage temperatures on VITROS ®SARS-CoV-2 antigen results, samples were stored at 4 â€‹°C, 25 â€‹°C & 37 â€‹°C for 7 days followed by detection of SARS-CoV-2 nucleocapsid antigen and compared with N-gene rRT-PCR. RESULTS: The VITROS® SARS-CoV-2 antigen test was found to have a sensitivity and specificity of 78.9% and 100% respectively with high sensitivity of 88.1% for samples with Ct â€‹< â€‹30. The LOD of VITROS assay was equivalent to 3800 copies of RNA per reactions as compared to 72 copies per reaction for rRT-PCR. We observed that more than 80% of samples with <30 Ct values could be detected by VITROS SARS-CoV-2 antigen assay at day 7 even when stored at 37 â€‹°C. For samples with Ct values between 26 and 30, on day 7 the positivity rate of N-antigen at 4 â€‹°C was 90.9% and 37 â€‹°C was 63.6%. CONCLUSIONS: CLIA testing can be carried out for the detection of SARS-CoV-2 N-protein in NP-swab samples transported in cold chain even with 7 days transit time, particularly for Ct â€‹< â€‹30 samples which represents cases with higher transmissibility. As drop in positivity for VITROS assay was lower as compared to rRT-PCR on day 7 in cold chain-maintained samples, the assay can be useful to screen samples received from remote peripheral areas before performing rRT-PCR.

Alternate Approach in Storing and Shipment of SARS-CoV-2 RNA Samples with the Use of FTA Cards.

Shipment of COVID-19 specimens within the country or overseas at long distances requires cold chain facility using dry ice and triple packing to prevent the risk of COVID-19 infection to the personnel involved in sample transport. The present study aimed to utilize FTA card technology as an alternate means of sample transport and storage across the country. Twenty-one SARS-CoV-2 lab confirmed samples with different Ct value (High, medium & low) were used to detect viral load in samples loaded on FTA card and further compared with VTM samples. The SARS-CoV-2 RNA was detected by rRT-PCR after storing for 14 days at 4 °C and 37 °C. The present study evaluated the utility of FTA cards for preserving the SARS CoV-2 RNA for 14-day period. A significant difference (P < 0.05) was observed in the cycle threshold (ΔCt 4-5) values obtained from FTA and VTM viral samples but it did not affect the positivity. The SARS-CoV-2 RNA could be recovered efficiently from FTA sample stored at 4 °C and 37 °C for 14 days. Thus, FTA cards could be an alternate option for transporting the samples at ambient temperature for a long time.

Validation of a noninvasive aMMP-8 point-of-care diagnostic methodology in COVID-19 patients with periodontal disease.

OBJECTIVES: The aim of this study was to validate an active matrix metalloproteinase (MMP-8) point-of-care diagnostic tool in COVID-19 patients with periodontal disease. SUBJECTS, MATERIALS, AND METHODS: Seventy-two COVID-19-positive and 30 COVID-19-negative subjects were enrolled in the study. Demographic data were recorded, periodontal examination carried out, and chairside tests run for evaluating the expression of active MMP-8 (aMMP-8) in the site with maximum periodontal breakdown via gingival crevicular fluid sampling as well as via a mouth rinse-based kit for general disease activity. In COVID-19-positive patients, the kits were run again once the patients turned COVID-19 negative. RESULTS: The overall (n = 102) sensitivity/specificity of the mouthrinse-based kits to detect periodontal disease was 79.41%/36.76% and that of site-specific kits was 64.71%/55.88% while adjusting for age, gender, and smoking status increased the sensitivity and specificity (82.35%/76.47% and 73.53%/88.24, respectively). Receiver operating characteristic (ROC) analysis for the adjusted model revealed very good area under the ROC curve 0.746-0.869 (p < .001) and 0.740-0.872 (p < .001) (the aMMP-8 mouth rinse and site-specific kits, respectively). No statistically significant difference was observed in the distribution of results of aMMP-8 mouth rinse test (p = .302) and aMMP-8 site-specific test (p = .189) once the subjects recovered from COVID-19. CONCLUSIONS: The findings of the present study support the aMMP-8 point-of-care testing (PoCT) kits as screening tools for periodontitis in COVID-19 patients. The overall screening accuracy can be further increased by utilizing adjunctively risk factors of periodontitis. The reported noninvasive, user-friendly, and objective PoCT diagnostic methodology may provide a way of stratifying risk groups, deciding upon referrals, and in the institution of diligent oral hygiene regimens.

An improved method for isolation of RNA from rat femur.

BACKGROUND: RNA isolation from ossified bone is a difficult and time-consuming process which often results in poor recovery of RNA. The yield is limited and might not be suitable for gene quantification studies by real time PCR. METHODOLOGY: The present study demonstrates RNA extraction from rat femur utilizing the silica column along with the trizol reagent. Quality of RNA was assessed by agarose gel analysis and its suitability for real-time PCR analysis was determined by ß-actin Ct values. RESULTS: The RNA isolated using silica columns in conjugation with trizol reagent resulted in higher yield of RNA and purity (A260/280=2.04; yield =1545.73 µg/ml) compared to the trizol method alone (A260/280=1.85; yield =571.2 µg/ml). Ct value of ß actin obtained from RNA isolated by trizol method was higher than the Ct value obtained by trizol in conjugation with the column method (31.41 and 15.41 respectively). CONCLUSION: Combination of trizol along with silica column resulted in better quality and improved yield of RNA suitable for gene quantification by Real time PCR.

Respiratory viral infections other than SARS CoV-2 among the North Indian patients presenting with acute respiratory illness during the first COVID-19 wave.

Acute respiratory infections due to viral or bacterial etiology can cause 60 deaths per one lakh population. Viral etiology is more common as compared to bacterial, but lack of definite diagnosis leads to increased usage of empirical antibiotics. During the first wave of the COVID-19 pandemic, there was a need to identify co-infections especially in severe acute respiratory illness (SARI) patients to identify it as one of the cofactors for increased severity of illness and to identify the causative agents in COVID-19 negative individuals. The SARS CoV-2 real time PCR was carried out using ICMR approved kits and the other respiratory viruses were detected using the multiplex commercially available real time  kit. A total of 186 patients presenting with either SARI (89.8%) or influenza like illness (10.2%) were included in the study. Out of these, 43 (23.1%) were positive for SARS CoV-2 RNA and 2 (4.6%) patients with SARI showed concomitant infection with either human rhinovirus or human respiratory syncytial virus . Out of 143 patients negative for SARS CoV-2, 35 (24.5%) were positive for one or more microbial infections and 28 (19.6%) infected with other respiratory viral infection most common being human rhinovirus. The results suggest that viral coinfections are significantly higher among COVID-19 negative individuals (24.5% vs 4.6%) presenting with respiratory illness as compared to COVID-19 positive individuals possibly due to viral interference and competitive advantage of SARS-CoV-2 in modulating the host immunity. Further detailed research is required for the understanding of mechanisms of viral co-infection.

The clinical association between Periodontitis and COVID-19.

OBJECTIVES: The study aimed to clinically assess the association between periodontitis and COVID-19-related outcomes. MATERIAL AND METHODS: Data pertaining to patient demographics, medical history, blood parameters, periodontal clinical examination and aMMP-8 point-of-care diagnostics (both site-level and patient-level) was recorded for eighty-two COVID-19-positive patients. COVID-19-related outcomes such as COVID-19 pneumonia, death/survival, types of hospital admission and need of assisted ventilation were also assessed. RESULTS: Males were predominantly afflicted with COVID-19, with advanced age exhibiting a greater association with the presence of periodontitis. Higher severity of periodontitis led to 7.45 odds of requiring assisted ventilation, 36.52 odds of hospital admission, 14.58 odds of being deceased and 4.42 odds of COVID-19-related pneumonia. The aMMP-8 mouthrinse kit was slightly more sensitive but less specific than aMMP-8 site-specific tests. CONCLUSIONS: Based on the findings of the present study, periodontitis seems to be related to poorer COVID-19-related outcomes. However, within the constraints of this work, a direct causality may not be established. Periodontitis, by means of skewing the systemic condition for a number of comorbidities, may eventually influence COVID-19 outcomes in an indirect manner. CLINICAL RELEVANCE: The study is the first to clinically, and by means of a validated point-of-care diagnostic methodology, assess the association between periodontal health and COVID-19-related outcomes. Assessment of the periodontal status of individuals can aid in the identification of risk groups during the pandemic along with reinforcing the need to maintain oral hygiene and seeking periodontal care.

Correlation of SARS-CoV-2 Viral Load in Different Population Subsets: A Study from a Tertiary Care North Indian Hospital.

BACKGROUND: The correlation of SARS-CoV-2 viral load with disease severity in different population subsets is still elusive. There is a scarcity of literature regarding this aspect in Indian Population. AIM: To study retrospectively the risk factors and the role of viral load with disease severity among different age groups of North Indian population. METHODS: Here we quantified the viral load of 239 positive participants and collected data retrospectively from April 2020 to May 2020 and categorised the patients as per disease severity and population subsets. RESULTS: Asymptomatic patients were found to have higher viral load than the symptomatic patients, though the difference was not found to be statistically significant. The logistic regression analysis showed that contact with laboratory confirmed cases, SARI and ILI were independent risk factors for acquiring COVID-19 infection. CONCLUSION: SARS-CoV-2 viral load is not significantly associated with disease severity among different population subsets. However, there is a need to carry out more studies with a larger number of patients to validate and confirm the above findings.

COVID-19 infection in a HIV positive health care worker: first case report from a tertiary care hospital of North India.

People living with Human Immunodeficiency Virus (PLHIV) are at greater risk of developing prolonged illness due to COVID 19 leading to longer duration of virus shedding owing to their underlying immune defects. The present study compared SARS-CoV-2 infection developing at the same time among two health care workers living with and without a history of HIV and working in the same ward of a tertiary care hospital of North India. A higher viral load was reported in the SARS-CoV-2 infected worker who was immunocompromised as compared to immunocompetent patient (19,193 copies/µL vs 9.4 copies/µL). In this preliminary case report, no difference was observed in the clinical presentation of both patients at the time of diagnosis. Further studies are required to investigate the COVID-19 susceptibility and severity among HIV-infected patients.

An innovative and cost-effective way to estimate alkaline phosphatase activity in in vitro cellular model systems.

Alkaline phosphatase is an enzyme that converts para-nitrophenyl phosphate to para-nitrophenol (yellow coloured) in 2-amino, 2-methyl, 1-propanol buffer at pH 10.5. However, when this protocol is applied to the in vitro cellular model systems to estimate alkaline phosphatase activity, it tends to generate clumps of genomic DNA, leading to inaccurate pipetting for protein estimation. The aim of the study was to introduce minor modifications in the existing protocol to make it simple, cost-effective, with minimal labor-intensive procedures while estimating alkaline phosphatase activity in cellular model systems. The genomic DNA clumps were dissolved by depurination (adding 0.2 N HCl) and fragmentation (adding 0.2 N NaOH) during enzyme estimation. Moreover, these minor modifications have been standardized and optimized extensively by using serum samples (rich source of alkaline phosphatase), hFOB/ER9 (human Fetal osteoblastic cell) and HepG2 cells. Our results suggest that the modification incorporated in previously published method was robust enough to estimate ALP activity and protein concentration accurately. There was no significant variation in ALP activity estimated after modification (P > 0.05). This innovative approach could be beneficial for a researcher by providing an easy, cost effective and less labor-intensive solution for estimation of enzymatic activity in cellular model systems.

Validation of SARS CoV-2 detection by real-time PCR in matched pooled and deconvoluted clinical samples before and after nucleic acid extraction: a study in tertiary care hospital of North India.

The diagnosis of coronavirus disease-19 (COVID-19) relies on the detection of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA by real-time reverse-transcription polymerase chain reaction in respiratory samples. Rapid increase in the COVID-19 cases across the world requires fast and efficient testing as testing capacity is a bottleneck in diagnosis. In this context, pooling strategy can be opted for rapid testing in a cost-effective manner. In this study, the authors have optimized and compared the effect of pooling (5 and 10 samples) before and after nucleic acid extraction. It was concluded that there was no significant difference in the SARS CoV-2 RNA detection in the pools prepared at sample or RNA level. Even after pooling, 10-fold dilution was detectable with 3-cycle threshold value change in both type of pools when compared with individual samples. Hence, sample pool size of 10 can be used in low-prevalent areas, and testing capacity can be substantially increased.

Anosmia and ageusia as presenting complaints of coronavirus disease (COVID-19) infection.

The recently discovered SARS-CoV2 virus produces a influenza like illness named Coronavirus disease 2019 (COVID-19). The usual presentation is with upper/lower respiratory tract symptoms and rarely gastrointestinal symptoms. Although some of the clinical features of this novel disease like fever, dry cough, and shortness of breath have been well documented in literature, we report hitherto infrequently reported clinical features of this disease, namely Anosmia and Ageusia.

Cloning of hok gene into anhydrotetracycline inducible pASK75 vector reveals potent antimicrobial effect of 19 amino acid long N-terminal fragment of hok peptide.

An important toxin-antitoxin (TA) system hok/sok, encoded by R1 plasmid of Escherichia coli, is involved in the post segregation killing of cells that have lost the plasmid. The lethal properties of hok protein have been utilized for the environmental containment of microbes and the development of potential vaccine candidates. This study aimed to demonstrate the potent anti-microbial property of a 19 amino acid (AA) long N-terminal fragment of hok peptide. This was accomplished by designing a conditional suicide system based on hok gene expression cloned in an anhydrotetracycline (aTc) inducible vector - pASK75. Heat shock and electroporation were utilized for the transformation of Escherichia coli and Vibrio cholerae cells, respectively. The minimal induction concentration (MId C) of aTc, determined by analyzing the expression of green fluorescent protein cloned separately into pASK75 vector, was 30 ng/mL. As hok gene was synthesized de novo (using recombinant polymerase chain reaction) in our study, various random sized hok fragments were generated (as a result of the error-prone nature of Taq polymerase). The smallest hok fragment able to bring about effective antimicrobial killing was a 19 AA long N-terminal fragment of hok having the wild type sequence, except for the carboxy terminus AA residue. The MId C of aTc in our experiments was 6-fold lower than previously reported, making our bacterial clones suitable for use in mammalian systems as potential vaccine candidates. Based on our experiments, we hypothesize the 19 AA long N-terminal fragment of hok peptide to be the smallest possible hok fragment sufficient to bring about effective antimicrobial killing.

Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling.

BACKGROUND & OBJECTIVES: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India. METHODS: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (Ct) values was analyzed. RESULTS: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the Ct value ≤30 cycles and 95.5 per cent for Ctvalues ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with Ct values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools. INTERPRETATION & CONCLUSIONS: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.

Novel 2019-coronavirus on new year's Eve.

An ongoing apocalyptic outbreak of a new virus causing pneumonia-like clusters in Wuhan city, China, has gleamed the world. The outbreak, confirmed on the New Year's Eve 2020, has known no boundaries since then. The number has surpassed that of Severe Acute Respiratory Syndrome (SARS) and Middle East respiratory syndrome (MERS), and is uninterruptedly escalating. Being an RNA virus, it has a propensity to mutate due to the low proofreading capacity of RNA-dependent RNA polymerase. Step-wise mutations have led to the gradual spillover of virus and after crossing the inter-species interface, the virus has adapted itself for a stable human-to-human transmission. The disease caused by severe acute respiratory syndrome coronavirus (CoV)-2 (SARS-CoV-2) can prove deadlier if the so-called 'super-spreading events' emerge with time. Recent research has shown the maximum homology of 99% of SARS-CoV-2 to pangolins associated coronavirus, owing to which these can serve as potential intermediate host. India is responding swiftly to the emergency situation, and the whole of the country is under lockdown since 25 March 2020, to ensure social distancing. All the international flights are padlocked and the travellers are being screened at airports and seaports via thermal sensors, and quarantine for a period of 14 days is recommended. Three hundred and forty-five patients across the country tested positive with six fatalities as of 22 March 2020. No specific anti-CoV drugs are currently available. Patients are being treated with protease drugs are inhibitors, remdesivir, chloroquine, angiotensin-converting enzyme 2 inhibitors, ivermectin, sarilumab and tocilizumab, though none of these is Food and Drug Administration approved and are undergoing trials. Preventive measures such as social distancing, quarantine, cough etiquettes, proper hand washing, cleaning and decontaminating the surfaces are the mainstay for curbing the transmission of this virus. The present review highlights the update of novel SARS-CoV-2 in context to the Indian scenario.

Resveratrol restores the level of key inflammatory cytokines and RANKL/OPG ratio in the femur of rat osteoporosis model.

Resveratrol's effect on bone mineral density (BMD) and expression of cytokines in ovariectomized rats (postmenopausal osteoporosis model) was studied. The study was conducted on 3-month-old Sprague-Dawley rats that were (a) sham-operated, (b) ovariectomized, (c) ovariectomized and treated with ß-estradiol (487.5 µg/kg weight/day), and (d) ovariectomized and treated with resveratrol (625 µg/Kg body weight/day). The treatment was for 4 weeks. After sacrifice BMD and gene expression (RANKL, OPG, IL-23, and IL-17A, IL-1ß, and TNFα) were measured in tibia and femur respectively. Resveratrol could restore RANKL/OPG ratio, slightly increase BMD, and moderately but significantly reduce IL-23, IL-17A, IL-1ß, and TNF-α cytokine expression levels.