Seroprevalence of SARS-CoV-2 in patients with rheumatic diseases on disease modifying antirheumatic drugs and /or immunosuppressive medications from India: a multicentric study (preprint)

There is an uncertainty regarding the effect of the SARS-CoV-2 infection on patients with chronic rheumatic diseases who are on immunosuppressive drugs. We did a multicentric cross sectional seroprevalence study were conducted in five different cities of India prior to COVID-19 immunization. Patients with a diagnosis of rheumatic disease and on DMARDs were included. Relatives of the patients, preferably staying in the same household with no known rheumatic diseases served as controls (subjects without disease). Serum IgG antibodies to SARS-CoV-2 Receptor Binding Domain (RBD) of the spike protein and nucleoprotein (NP) was assayed. Eight hundred eighty-nine sera (subjects with disease = 379 and in subjects without disease = 510) were assayed. IgG antibodies to either RBD and/or NP were positive in 135 (36%) subjects with disease as compared to 196 (38%) in subjects without disease. The seroprevalence to RBD and NP varied between different cities but was not significantly different between subjects with and without disease in Mumbai, Ahmedabad, Bengaluru and Bhubaneswar. However, the occurrence of IgG antibodies to RBD was significantly (p < 0.05) lower in subjects with disease (28/65;43%) as compared to subjects without disease (42/65;65%) in Kolkata, where the positivity rate was lower in connective tissue disease group than in inflammatory arthritis group. Overall, patients with rheumatic diseases on DMARDs have IgG antibodies to RBD and NP of SARS-CoV-2 at a comparable level with that of subjects without disease, but the level of antibodies to RBD is lower in patients with connective tissue disease on immunosuppressive drugs in one centre.

Analysis of the COVID-19 Testing Parameters and Progression of the Pandemic at the District Level- Findings from the Icmr- Hundred Million Test (HMT) Database During the First Wave in India (preprint)

Background: India has the second highest number of COVID-19 cases. We evaluated the progression of the pandemic across the lockdowns and phased reopening during the first wave in India at the district level.Methods: More than 100 million COVID-19 test results along with other parameters available in the Indian Council of Medical Research (ICMR) database during March-October, 2020, was used for the analysis. District was chosen as the unit of analysis, as it is the smallest unit of administration in India. The districts were stratified as high, moderate, and low case load districts, and data analysis was done for each phase of lockdown.Findings: Of the 110.5 million tests included in the analysis, 54.79 million tests were performed using molecular methods, 53.58 million by rapid antigen tests (RATs) and 2.13 million by the indigenous TruNat platform. Only 7.95 million (7.16%) tests were among symptomatic individuals. The positivity proportion among symptomatic individuals (22.6%) was significantly higher than asymptomatic individuals (8.6%). The tests conducted, and positivity proportions were significantly higher in high caseload districts and 58% of these tests were by molecular methods as opposed to only one-third in low case load districts. The proportion of ‘symptomatic contacts’ being tested increased significantly around the peak.Interpretation: Laboratory parameters, along with other demographic information, can help us better understand the spread of the pandemic in a country. Such information can be crucial to formulate and implement public health policies in any future waves of the pandemic.Funding: Indian Council of Medical Research (ICMR)Declaration of Interest: We declare no competing interestsEthical Approval: Approval from the ICMR Central Ethics Committee on Human Research (Ref. No.NCDIR/BEU/ICMR-CECHR/75/2020) was obtained for this study and no patient identifierswere accessed during analysis or reporting.

Persistence of antibodies against Spike glycoprotein of SARS-CoV-2 in health care workers post double dose of BBV-152 and AZD1222 vaccines (preprint)

Rationale: Vaccine rollout in India was initiated in mid-January, 2021 and is supposed to be the only antidote against SARS-CoV-2 as of now. Objectives: To study the dynamicity of vaccine-induced IgG antibody against SARS-CoV-2. Method: ology: The present cross-sectional cohort study was undertaken to determine IgG antibody among health care workers with completed dose of either Covaxin or Covishield and were followed for 24 weeks after first dose of either vaccine to record the periodic changes in titre, concentration, clinical growth and persistence of vaccine-induced SARS-CoV-2 antibodies. Results: : Serum samples were collected from 614 participants during each follow-up and tested them in two CLIA-based platforms for testing SARS-CoV-2 antibodies both qualitatively and quantitatively. Among these participants, 308 (50.2%) were Covishield recipient and rest 306 (49.8%) took Covaxin. A total of 81 breakthrough cases was recorded among the cohort participants for whom infection post vaccination acted as booster. The rest 533 heath care workers without any history of post-vaccination infection showed significant antibody waning either from T3 (Covaxin recipient) or T4 (Covishield recipient). Conclusion: The clinical implications of waning antibody levels post vaccination are not well understood, and it remains crucial to establish S-antibody thresholds associated with protection against clinical outcomes.

Natural immunity against COVID-19 significantly reduces the risk of reinfection: findings from a cohort of sero-survey participants (preprint)

Conflicting reports on the persistence of antibody levels in individuals recovered from COVID-19 infection, suggest that the immunity against COVID-19 may not be lasting for long. In India, by 30th June, 2021, not less than 30 million people were infected with COVID-19 and 0.39 million people were reported to have lost their life to the disease in India. I the current study we followed up with a subsample of our previous sero-survey participants to assess whether natural immunity against SARS-CoV-2 was associated with a reduced risk of re-infection. We conducted telephonic interview of a total of 3038 participants, out of which 2238 participants responded and 5 participants were found to be not alive, as conveyed by their close relatives. There was a non-response rate of 26.1%. Out of the 2238 participants, 1170 were sero-positive and 1068 were sero-negative for antibody against COVID-19. Our survey found that only 3 individuals in the sero-positive group got infected with COVID-19 whereas 127 individuals reported contracting the infection the sero-negative group. Interestingly, from the 127 sero-negative individuals who later contracted COVID-19 infection, 30 needed hospitalization, out of which 12 were on oxygen therapy, four in ICU and one was on ventilator. At the other hand, from the 3 sero-positives re-infected with COVID-19, one had hospitalization, but didnnot require oxygen support or critical care. These findings reinforce the strong plausibility that development of antibody following natural infection not only protects against re-infection by the virus to a great extent, but also safeguards against progression to severe COVID-19 disease.

Breakthrough SARS-CoV-2 infections in an eastern state of India: A preliminary report (preprint)

We recorded the vaccine breakthrough cases of Covaxin and Covishield through passive and voluntary reporting at various healthcare facilities of Odisha. A total of 274 samples were found to be COVID-19 positive after 14 days of receiving complete doses of the vaccines. Almost 83.2% of the individuals were found to be symptomatic with 9.9% of those required hospitalization and were having less median antibody titers than individual in home isolation.

Effect of a single-dose of BBV-152 and AZD1222 on Spike antibody titre among healthcare workers previously infected with SARS-CoV-2 (preprint)

Objective Dissemination of vaccines among the maximum number of recipients is becoming an uphill task in many low and middle-income countries (LMIC). This study was conducted to ease the vaccination load and to reach the highest number of immunities in a population about the neutralizing antibody response.Methods We followed the vaccination status of 134 healthcare workers (HCWs) among which 59 were previously confirmed SARS-CoV-2 positive by RT-PCR test and 75 naive participants. All of them took two dosages of either BBV-152 or AZD1222 vaccine. Antibodies against SARS-CoV-2 spike and nucleocapsid proteins were measured in two CLIA-based platforms Cobas e411 and ARCHITECT i1000SR.Results From prior COVID-19 recovered HCWs, 96% were found to be seropositive and developed a median concentration of 588.0 U/mL (interquartile range [IQR], 250.0-2500.0 AU/mL) IgG antibody against SARS-CoV-2 S- protein after a single dose of vaccine whereas only 61.3% of previously non-infected HCWs developed antibody against SARS-CoV-2 S- protein after single dose with a median concentration of 7.27 U/mL (IQR, 0.4-116.5 AU/mL). A total of 82.7% HCWs having no earlier COVID-19 infection history became seropositive after the second dose of vaccine irrespective of gender and age.Conclusion We found that a single dose of BBV-152 and AZD1222 induced a high concentration of neutralizing IgG antibody in HCWs having a recovery history from COVID-19.

IgG antibody response against Nucleocapsid and Spike protein post SARS-CoV-2 infection (preprint)

Objectives: Coronavirus disease-19 (COVID-19) pandemic became the greatest public health challenge globally. Study of dynamicity and durability of naturally developed antibodies against SARS-CoV-2 are of great importance from an epidemiological viewpoint.Methods: In this observational cohort study, we have followed up the 76 individuals who tested positive for SARS-CoV-2 infection by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) for 16 weeks (post enrollment) to record the periodic changes in titre, concentration, clinical growth and persistence of naturally developed SARS-CoV-2 antibodies. We collected serum samples from these individuals for 16 weeks with a frequency of weekly and fortnightly during each follow-up and tested them in two CLIA-based platforms (Abbott Architect i1000SR and Roche Cobas e411) for testing SARS-CoV-2 antibodies both qualitatively and quantitatively.Results: We recorded the antibody magnitude of these individuals 10 times between September 2020 and February 2021. We found a waning of antibodies against nucleocapsid antigen protein but not a complete disappearance by the end of 16 weeks. Out of 76 cases, 30 cases (39.47%) became seronegative in qualitative assay, although all the sera samples (100%) remained positive when tested in quantitative assay.Conclusion: The lower persistence of anti-nucleocapsid SARS-CoV-2 antibody may not be the exact phenomenon as those cases were still seropositive against spike protein and help in neutralizing the virus.

Seroprevalence of anti-SARS-CoV-2 IgG antibody among health care workers of anaesthesia departments from various hospital settings in India (preprint)

Abstract: Background: Health care workers (HCWs) are the most susceptible group to get COVID-19 infection and this group always need special attention as they are the key human resource to contain this pandemic. Objective: To track down the seroprevalence among a particular group of HCWs working in the anaesthesia department in hospital settings. Study design: Two rounds of serosurvey were done to track the dynamicity among the 128 and 164 HCWs participants in the first round and second round, respectively. 5 mL of blood was collected and anti-SARS-CoV-2 IgG antibody was tested in Abbott Architect i1000SR. Results: The seroprevalence found in the first and second round was 12.5% and 38.4%, respectively. A significant number (n=61, 77.21%) of seropositivity came from the asymptomatic HCWs group as found in both the survey. There was no significant association among different age, gender and RT-PCR tested groups. Conclusion: Routine diagnosis of COVID-19 should be referred among HCWs to identify and act upon unrecognized SARS-CoV-2 infection.

SARS-CoV-2 IgG antibody responses in rt-PCR positive cases: first report from India (preprint)

The SARS-CoV-2 antibody responses remain poorly understood and the clinical utility of serological testing is still unclear. As it is thought to confer some degree of immunity, this study is carried out to know the relationship between demographics and ct value of confirmed rt-PCR patients. A total of 384 serum samples were collected between 4-6 weeks after confirmed SARS-CoV-2 infection. IgG positivity was found to be 80.2% (95% CI, 76.2 - 84.2). The positivity increased with the decrease in the ct value, with a highest of 87.6% positivity in individual with <20 ct value. The mean (SD) ct value of IgG positives and negatives was 23.34 (+/-6.09) and 26.72 (+/- 7.031) respectively. There was no significant difference found between the demographic characteristics such as age, sex, symptoms and antibody response. The current study is first of its kind wherein we have assessed the correlation of ct of RT-PCR with development of IgG against SARS-CoV-2. Our study showed that although Ct value might not have any relation with severity of the diseases but is associated with the antibody response among the SARS-CoV-2 infected individual.

Saliva as a Potential Clinical Specimen for Diagnosis of SARS-CoV-2 (preprint)

Background: There is no sign of stopping the spread of corona virus disease- 2019 (COVID-19) pandemic since it has started in December 2019. Rapid and early detection is extremely crucial to slow down the quick spread of the virus and break the human transmission chain. There are very few studies in search of an alternate and convenient diagnostic tool which can substitute nasopharyngeal swab (NPS) specimen for detection of severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2). We aimed to analyse the comparison and agreement between the feasibility of using the saliva in comparison to NPS for diagnosis of SARS-CoV-2.Methods: A total number of 74 patients were enrolled for this study. We analysed and compared the NPS and saliva specimen collected within 48 h after the symptom onset. We used real time quantitative polymerase chain reaction (RT-qPCR), gene sequencing for the detection and determination SARS-CoV-2 specific genes. Phylogenetic tree was constructed to establish the isolation of viral RNA from saliva. We use Bland-Altman model to identify the agreement between two specimens.Findings: This study shows a lower cycle threshold (CT) mean value for the detection of SARS-CoV-2 ORF1 gene (mean 27.07; 95% CI, 25.62 to 28.52) in saliva methods than that of NPS (mean 28.24; 95% CI, 26.62 to 29.85) specimen although the difference is statistically non-significant (p>0.05). Bland-Altman analysis produces relatively smaller bias and high agreement between these two clinical specimens. Phylogenetic analysis with the RdRp and Spike gene confirmed the presence of SARS-CoV-2 in the saliva samples.Interpretation: In conclusion, our study highlights that saliva represents a promising tool in COVID-19 diagnosis and the collection method would reduce the exposure risk of frontline health workers which is one of major concerns in primary healthcare settings.Funding Statement: ICMR for provided financial grants for this study.Declaration of Interests: The authors declare that they have no competing interests.Ethics Approval Statement: The study was approved by Institutional Ethics Committee and written informed consent was obtained from the study subjects.

Feasibility of Pooled Testing in a Fully Automated High Throughput Platform for Effective Management of SARS-COV-2 in India (preprint)

Introduction: Testing for extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hit tremendously worldwide, as the scarcity of test kits has now become a major bottleneck, with the need for testing throughput growing. The study assessed the feasibility of pooled testing in the high throughput machine of Roche Cobas 6800 rapidly increasing the testing number for containing the virus spread and management of cases. Methodology: ICMR-RMRC, Bhubaneswar receives samples from various district hospitals from different districts of Odisha. Pooled testing was conducted in 2 methods. Firstly, we adopted the simple two-stage testing algorithm known as Dorfman pooling with minor modification to avoid selection bias and secondly we evaluated the ability of COBAS for detection of a single positive sample within a pool of negative samples.Results: The Cobas 6800 was able to detect the SARS-COV virus in all the samples, however, the amplified RNA reached the threshold later as the number of negative samples increased in the pool. We demonstrated strategies for pooling, which improve test efficiency and while maintaining high sensitivity in a high-throughput system. The comparison of 1410 samples tested individually and in pools of five samples/pool showed that test results were not significantly affected. Implementing the five-sample Dorfman pooling to test 1410 samples, we identified 42 (2.9%) SARS-CoV-2 positive samples, achieving a 3-fold increase in throughput with one-third of the cost.Conclusion: To the best of our knowledge this study is 1st of its kind to assess the feasibility of pooled testing in high throughput machines. As ICMR has always stressed, aggressive timely testing is the standard protocol for containing the virus spread and management of cases, these high throughput machines installed in six states with a high prevalence of Covid-19, can effectively increase the testing capacity by 2-3 fold, adopting the pooled testing strategy for successful management of SARS-CoV-2.Funding: The study was carried out with funding support from Indian Council of Medical Research.Declaration of Interest: The authors declare that they have no competing interests.Ethical Approval Statement: The study was cleared by ICMR-Regional Medical Research Centre ethical committee.

Findings from serological surveys (in August 2020) to assess the exposure of adult population to SARS Cov-2 infection in three cities of Odisha, India. (preprint)

Background: There is always an uncertainty of epidemiological , serological infectivity and virulence of the emerging novel coronavirus. Antibody test can be used for assessing whether immunity has developed in the infected person after 5-7 days of illness and understand cumulative exposure levels to the infection, make inferences on the actual burden of infection, its geographical spread, effect on specific demographic/risk groups, gaps in testing and infection fatality rates. Objective- To estimate and compare the sero-prevalence, hidden prevalence and determine the demographic risk factors associated with SARS-CoV-2 infection among adults in three largest cities of Odisha, India. Methodology: This was a population based cross sectional serological survey carried out in August 2020 in the three largest cities of the state of Odisha. Sample size per city was estimated to be 1500 and participants were enrolled from the community using multi-stage random sampling from 25 clusters from each city. Data was collected using ODK based tools by household visits and 3-4 ml of blood samples were collected after informed consent. Samples were transported to testing lab where Serum was separated and tested for anti- SARS CoV-2 antibodies using automated CLIA platform. Statistical analysis was done using R-software packages. Results: A total of 4146 participants from the 3 cities of Bhubaneswar (BBS), Berhampur (BAM) and Rourkela (RKL) participated. A total of 5635 households were approached and the average non response rate in the community was 17.4%. The gender weighted seroprevalence across the three cities was 20.78% (95% CI: 19.56%-22.05%). Seroprevalence was highest in BAM at 31.14% (95% CI: 28.69-33.66%) followed by 24.59% (95% CI: 22.39-26.88%) in RKL and 5.24% (95% CI: 4.10-6.58%) in BBS. While females reported a higher seroprevalence (22.8%) as compared to males (18.8%), there was no significant difference in seroprevalence across age groups. A majority of the seropositive participants were asymptomatic (93.87%). Among those who reported symptoms, the most common symptom was fever (68.89%) followed by cough (46.06%) and myalgia (32.67%). The case to infection ratio on the date of serosurvey was 1: 6.6 in BBS, 1:61 in BAM and 1:29.8 in RKL. Conclusion: The study found a high seroprevalence against COVID-19 in urban Odisha as well as high numbers of asymptomatic infections.

Diagnostic comparison of three fully automated chemiluminescent immunoassay platforms for the detection of SARS-CoV-2 antibodies (preprint)

The whole world is battling against coronavirus disease-19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Various strategies are taken to curb the spread of the virus and to move out from the enforced lockdown stage. Serological tests are the neediest diagnostic and surveillance tool to complement the gold standard molecular diagnostic method to track down the transmission rate of SARS-CoV-2. Automated chemiluminescent immunoassay (CLIA) based analyzers become highly demanding platforms both to clinicians and policy makers for the detection anti-SARS-CoV-2 antibodies. In this study, serum from 594 patients positive for COVID-19 and 100 samples from pre-COVID cases were tested by three automated platforms: Abbott architect i2000SR, Roche cobas e411 and Yhlo iFlash 1800 and their diagnostic accuracy were compared. All three platforms showed high specificity as claimed by manufacturer. Clinical sensitivities of the machines were calculated as 64.48% (58.67-70.3) for Abbott, 80.48% (76.62-84.34) for Roche and 76.94% (72.65-81.23) for Yhlo. The Cohens kappa value was determined from 0.69-0.89 when inter-rater agreements were calculated. The area under the curves (AUC) values demonstrated Roche Cobas e411 as the diagnostically most accurate platform among the three CLIA analyzers.

Saliva as a potential clinical specimen for diagnosis of SARS-CoV-2 (preprint)

Background It is almost nine months, still there is no sign to stop the spreading of the COVID-19 pandemic. Rapid and early detection of the virus is the master key to cease the rapid spread and break the human transmission chain. There are very few studies in search of an alternate and convenient diagnostic tool which can substitute nasopharyngeal swab (NPS) specimen for detection of SARS-CoV-2. We aimed to analyse the comparison and agreement between the feasibility of using the saliva in comparison to NPS for diagnosis of SARS-CoV-2. Methods A total number of 74 patients were enrolled for this study. We analysed and compared the NPS and saliva specimen collected within 48 h after the symptom onset. We used real time quantitative polymerase chain reaction (RT-qPCR), gene sequencing for the detection and determination SARS-CoV-2 specific genes. Phylogenetic tree was constructed to establish the isolation of viral RNA from saliva. We use Bland-Altman model to identify the agreement between two sampling methods. Findings This study shows a lower CT mean value for the detection of SARS-CoV-2 ORF1 gene (27.07; 95% CI, 25.62 to 28.52) in saliva methods than that of NPS (28.24; 95% CI, 26.62 to 29.85) sampling method. Bland-Altman analysis produces relatively smaller bias and high agreement between these specimen tools. Phylogenetic analysis with the RdRp and Spike gene confirmed the presence of SARS-CoV-2 in the saliva samples. Interpretation: In conclusion, our study highlights that saliva represents a promising tool in COVID-19 diagnosis and would reduce the exposure risk of frontline health workers which is one of biggest concern in primary healthcare settings.

Feasibility of Pooled Testing in a Fully Automated High Throughput Platform for Effective Management of SARS-CoV-2 in India (preprint)

Introduction: Testing for extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hit tremendously worldwide, as the scarcity of test kits has now become a major bottleneck, with the need for testing throughput growing. The study assessed the feasibility of pooled testing in the high throughput machine of Roche Cobas 6800 rapidly increasing the testing number for containing the virus spread and management of cases.Methodology: ICMR-RMRC, Bhubaneswar receives samples from various district hospitals from different districts of Odisha. Pooled testing was conducted in 2 methods. Firstly, we adopted the simple two-stage testing algorithm known as Dorfman pooling with minor modification to avoid selection bias and secondly we evaluated the ability of COBAS for detection of a single positive sample within a pool of negative samples.Results: The Cobas 6800 was able to detect the SARS-COV virus in all the samples, however, the amplified RNA reached the threshold later as the number of negative samples increased in the pool. We demonstrated strategies for pooling, which improve test efficiency and while maintaining high sensitivity in a high-throughput system. The comparison of 1410 samples tested individually and in pools of five samples/pool showed that test results were not significantly affected. Implementing the five-sample Dorfman pooling to test 1410 samples, we identified 42 (2.9%) SARS-CoV-2 positive samples, achieving a 3-fold increase in throughput with one-third of the cost.Conclusion: To the best of our knowledge this study is 1st of its kind to assess the feasibility of pooled testing in high throughput machines. As ICMR has always stressed, aggressive timely testing is the standard protocol for containing the virus spread and management of cases, these high throughput machines installed in six states with a high prevalence of COVID-19, can effectively increase the testing capacity by 2-3 fold, adopting the pooled testing strategy for successful management of SARS-CoV-2.

SARS-CoV2 genome analysis of Indian isolates and molecular modelling of D614G mutated spike protein with TMPRSS2 depicted its enhanced interaction and virus infectivity (preprint)

COVID-19 that emerged as a global pandemic is caused by SARS-CoV-2 virus. The virus genome analysis during disease spread reveals about its evolution and transmission. We did whole genome sequencing of 225 clinical strains from the state of Odisha in eastern India using ARTIC protocol-based amplicon sequencing. Phylogenetic analysis identified the presence of all five reported clades 19A, 19B, 20A, 20B and 20C in the population. The analyses revealed two major routes for the introduction of the disease in India i.e. Europe and South-east Asia followed by local transmission. Interestingly, 19B clade was found to be much more prevalent in our sequenced genomes (17%) as compared to other genomes reported so far from India. The haplogroup analysis for clades showed evolution of 19A and 19B in parallel whereas the 20B and 20C appeared to evolve from 20A. Majority of the 19A and 19B clades were present in cases that migrated from Gujarat state in India suggesting it to be one of the major initial points of disease transmission in India during month of March and April. We found that with the time 20A and 20B clades evolved drastically that originated from central Europe. At the same time, it has been observed that 20A and 20B clades depicted selection of four common mutations i.e. 241 C>T (5UTR), P323L in RdRP, F942F in NSP3 and D614G in the spike protein. We found an increase in the concordance of G614 mutation evolution with the viral load in clinical samples as evident from decreased Ct value of spike and Orf1ab gene in qPCR. Molecular modelling and docking analysis identified that D614G mutation enhanced interaction of spike with TMPRSS2 protease, which could impact the shedding of S1 domain and infectivity of the virus in host cells.