Mucosal immunity and antibody anergy in COVID-exposed Covishield vaccinees

Knowledge is limited on mucosal immunity induction and longitudinal responses to vaccination against SARS-CoV2. Here, we determined serum/salivary antibodies and cytokines after three Covishield vaccine doses. Sera from 205 healthcare workers (HCWs) one-month after first- dose; one-, three- and six-months after second-dose; paired sera and stimulated whole mouth fluid (SWMF) from 10 HCWs one-, three- and six-months after third-dose were tested for anti- spike SARS-CoV2 antibodies by ECLIA and for cytokines by ELISA/cytokine bead arrays. One-month after second-dose, antibodies had increased significantly (6-fold) in COVID-naive group, but declined (1.5-fold) in those previously exposed to COVID. At one-month after first- dose, IL-10 levels were statistically higher in the previously COVID-exposed group compared to COVID-naive group (p<0.02). Breakthrough infections were 44% in COVID-naive group, while re-infections were 27% in COVID-exposed group (p<0.02). Proinflammatory cytokines-IL- 17/IL-21 at one-month after first- and second-doses, and memory cytokines-IL-7/IL-15 at three- and six-months after second-dose were minimal. Antibodies spiked at one-month after third- dose and declined by three- and six-months after third-dose similar to post-second-dose. Paired sera and SWMF at one- and six-months after third-dose lacked adaptive immunity cytokine expression. Innate immunity cytokines (MIG, MCP-1, IL-8, TNF-, IL-6, IL-1{beta}) showed a declining trend in serum, but were sustained in SWMF. Thus, our findings suggest that first-dose acts as an antibody boost, while second-dose induces antibody anergy in the previously COVID- exposed group. Rapidly declining antibodies and minimal T cell cytokines raises concerns over their durability in subsequent virus exposures. Sustained innate cytokines emanating from the oral mucosa warrant further in-depth explorations.

Povidone iodine, hydrogen peroxide and chlorhexidine mouthwashes reduce SARS-CoV2 burden in whole mouth fluid and respiratory droplets

SARS-CoV2 is transmitted primarily through oral mouth secretions and respiratory droplets. Commercial mouthwashes, povidone iodine (PI), hydrogen peroxide (HP) and chlorhexidine (CHX) have been tested in cell culture and RT-PCR studies for their efficacy to reduce SARS-CoV2 burden. Here, we evaluated SARS-CoV2 burden in whole mouth fluid (WMF) and respiratory droplets (RD) samples before and after the use of PI, HP or CHX mouthwashes in hospitalized COVID-19 patients using RT-PCR and rapid antigen test (RAT). Thirty-six SARS-CoV2 RT-PCR-positive in-patients were randomly assigned to one of the four groups: 20 and 60 minutes after 1% w/v PI or 1.5% HP; 90 and 180 minutes after 1.5% HP or 0.2% w/v CHX. WMF and RD samples were collected concurrently at baseline and after the two different time points. RD (92%) showed a higher reduction in SARS-CoV2 burden than WMF samples (50%; p=0.008). SARS-CoV2 burden was statistically lower at both 20 minutes (p=0.02) and 60 minutes (p=0.03) with PI; at 20 minutes with HP (p=0.0001); and 90 minutes with CHX (p=0.04). The overall and individual mean logarithmic reductions in the WMF and RD samples were greater than 1.0 at 20, 60 and 90 minutes after PI, HP or CHX. RAT-positive patients at 90 minutes post-treatment (n=3) demonstrated a one log increase in virus copies. Among the three RAT-negative post-treatment patients, SARS-CoV2 burden declined by one log in two while the third patient had a slight increase in RNA copies. In conclusion, we have shown for the first time that the mouthwashes, PI, HP and CHX can reduce the SARS-CoV2 burden in the concurrently collected RD and WMF samples. RAT is more appropriate than RT-PCR to evaluate the efficacy of the mouthwashes.

Implications in the quantitation of SARS-CoV2 copies in concurrent nasopharyngeal swabs, whole mouth fluid and respiratory droplets

ImportanceThe nasopharyngeal swab (NPS) is considered the ideal diagnostic specimen for Covid-19, while WMF is recently promoted due to collection simplicity and importance in disease transmission. There is limited knowledge on the relative viral load in these samples - NPS, whole mouth fluid (WMF) and respiratory droplets (RD; another important source in transmission), on how the loads vary with disease severity and on how much virus is shed. ObjectiveTo quantify and compare SARS-CoV2 copies in the NPS, WMF and RD samples, and correlate with disease severity. DesignCross sectional study. SettingTertiary care multi-speciality hospital with limited resources in a low-to-middle income country. ParticipantsEighty suspected COVID-19 patients were recruited from the COVID-19 out-patient clinic and hospital isolation wards. InterventionConcurrent NPS, WMF and RD samples were collected from all the recruited patients and tested for SARS-CoV2 copies by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Main outcomes and measuresThe main outcome was COVID-19 measured by SARS-CoV2 quantitative RT-PCR in NPS samples. COVID-19 disease severity was determined according to NIH criteria. Virus shedding was defined as the presence of SARS-CoV2 copies in the WMF and RD samples. ResultsSARS-CoV2 was detected in 55/80 (69%) of the NPS samples. Of these 55, WMF and RD samples were positive in 44 (80%) and 17 (31%), respectively. The concordance of WMF with NPS was 84% (p=0.02). SARS-CoV2 copy numbers were comparable in the NPS (median: 8.74x10^5) and WMF (median: 3.07x10^4), but lower in RD samples (median: 3.60x10^2). Patients with mild disease had higher copies in the NPS (median: 3.46x10^6), while patients with severe disease had higher copies in the WMF (median: 1.34x10^6) and RD samples (median: 4.29x10^4). The 25-75% interquartile range of NPS SARS-CoV2 copies was significantly higher in the WMF (p=0.0001) and RD (p=0.01) positive patients. Conclusion and relevanceSARS-CoV2 copies are highest in NPS samples. WMF is a reliable surrogate sample for diagnosis. High copy numbers in the NPS imply initial virological phase and higher risk of virus shedding via WMF and RD. Key pointsO_ST_ABSQuestionC_ST_ABSHow the numbers of SARS-CoV2 copies in nasopharyngeal swab (NPS) samples might reflectvirus shedding from the whole upper aerodigestive tract and indicatedisease severity? FindingsIn this cross-sectional study involving 80 suspected COVID-19 patients, the data indicate higher SARS-CoV2 copies in NPS samples of patients with mild disease,and in the whole mouth fluid (WMF) and respiratory droplet (RD) samples of patients with severe disease. Patients with higher SARS-CoV2 copies in the NPS shed the virus in the WMF and RD samples at statistically higher levels. MeaningHigh SARS-CoV2 copies in NPS samples imply initial virological phase withhigh levels of shedding through both WMF and RD.

Detection of SARS-CoV2 antigen in human saliva may be a reliable tool for large scale screening

IntroductionSARS-CoV2, the aetiological agent of the current COVID-19 pandemic, has been detected in saliva and recently implicated in several oral diseases. Collection of nasopharyngeal swabs (NPS) and detection by reverse transcriptase-polymerase chain reaction (RT-PCR) requires medical / technical expertise. A reliable and easy to handle point-of-care (POC) test is highly desirable, especially to curb transmission. Therefore, in this study, we evaluated a commercially available POC rapid antigen test (RAT) for the detection of SARS-CoV2 antigens in the saliva of RT-PCR confirmed positive and negative patients. MethodsThirty saliva samples of 10 saliva RT-PCR negative and 20 saliva RT-PCR positive patients were tested by RAT. ResultsRAT was negative in 10/10 (100%) RT-PCR-negative samples; positive in 9/20 (45%) RT-PCR-positive samples; concordance was 63% (p=0.001). Patients with positive RAT had higher virus copies in their NPS samples compared to the RAT-negative patients. This difference was also statistically significant (p=0.01). ConclusionThus, the POC RAT may be used to detect SARS-CoV2 as a reliable tool for self-testing, large-scale population screening and emergency medical/dental screening. Patients negative by RAT should be confirmed by RT-PCR.

Concentration of the cellular material in the nasopharyngeal swabs increases the clinical sensitivity of SARS-CoV2 RT-PCR

Severe acute respiratory syndrome - coronavirus 2 (SARS-CoV2) is detected by a highly sensitive molecular method, reverse transcriptase-polymerase chain reaction (RT-PCR) from nasopharyngeal swab (NPS) samples collected in 2-3ml of viral transport medium (VTM). Unlike body fluids, NPS samples are undermined by high variability in the amount of cells that get suspended into the VTM. Hence, the cell density used for RNA extraction becomes an important analytical variable that contributes to the overall sensitivity of the RT-PCR. In this study, we compared the sensitivity of SARS-CoV2 RT-PCR in 50 NPS samples collected from in-patients of the COVID wards using the concentration and direct methods. The concentration method detected the viral RNA in all 50 samples, while the direct method was positive in only 41 (82%) samples (p=0.003). Additionally, the Ct values were lower in the direct method compared to concentration method among the 41 positive samples (p=0.03 for N gene and p=0.04 for RdRp gene). The mean CV% was also [≥]10%. Thus, the concentration of the cells prior to RNA extraction drastically improves the sensitivity of detection of SARS-CoV2 in NPS samples.

The diagnostic significance of enzyme linked immuno-sorbent assay for herpes simplex, varicella zoster and cytomegalovirus retinitis.

PURPOSE: To evaluate the diagnostic usefulness of enzyme linked immuno-sorbent assay (ELISA) in single serum samples to associate herpes simplex virus (HSV), varicella zoster virus (VZV) or cytomegalovirus (CMV) with viral retinitis as against polymerase chain reaction (PCR) on intraocular specimens. It was also designed to study the seroprevalence in normal healthy individuals, and the genomic prevalence of HSV, VZV and CMV in patients without an active viral inflammatory process. METHODS: PCR for the detection of HSV, VZV and CMV genomes was done on 33 and 90 intraocular fluids from viral retinal patients and non-viral controls respectively. ELISA was done on 30 and 100 serum samples from viral retinitis patients and normal healthy controls respectively. RESULTS: PCR did not detect HSV, VZV and CMV genomes except one, in which VZV-DNA was detected. ELISA showed prevalence rates of 28%, 83% and 90% for antibodies against HSV, VZV and CMV respectively in the normal population. In the 30 viral retinitis patients, PCR detected HSV-DNA in 2 (6.7%), VZV-DNA in 7 (23.3%) and CMV-DNA in 6 (20.0%) patients, while ELISA detected antibodies against HSV, VZV and CMV in 13 (43.3%), 24 (80.0%) and 23 (76.7%) patients respectively. ELISA was of value in indirect diagnosis only in 6 (20.0%) as compared to 15 (50.0%) of 30 patients by PCR, this difference was statistically significant (McNemar test, P value = 0.005). CONCLUSION: Serology by ELISA is no longer a useful diagnostic tool to associate HSV, VZV and CMV viruses with viral retinitis.

Isolation of a vesicular virus belonging to the family rhabdoviridae from the aqueous humor of a patient with bilateral corneal endotheliitis.

PURPOSE: To report bilateral corneal endotheliitis caused by a vesicular virus (family Rhabdoviridae). METHODS: Case report of a 49-year-old man with a complaint of sudden onset of decreased vision in both eyes had diffuse corneal stromal edema with extensive folds in Descemet's membrane and was diagnosed as having bilateral viral endotheliitis. Virologic investigations were performed using aqueous humor from the right eye. RESULTS: An ether- and chloroform-sensitive cytopathic agent was isolated in Vero and BHK-21 cell lines from the aqueous humor. It was identified as a vesicular virus belonging to the family Rhabdoviridae by electron microscopy. Neutralizing antibody was demonstrated at a titer greater than 1 in 4,096 dilutions in the convalescent serum. Neurologic complications included loss of hearing and postinfectious polyradiculopathy affecting both lower limbs. Best-corrected visual acuity was 20/120 OD and 20/20 OS. Six months later, he developed glaucoma in the right eye. Trabeculectomy with intraoperative application of 5-fluorouracil was performed. CONCLUSION: This is the first report of bilateral endotheliitis caused by a vesicular virus and confirmed by virus isolation from the aqueous humor of the affected eye.

Association of herpesviruses in the aqueous humor of patients with serpiginous choroiditis: a polymerase chain reaction-based study.

PURPOSE: To determine the presence of herpesvirus DNA in the aqueous humor (AH) of patients with serpiginous choroiditis using polymerase chain reaction (PCR). METHODS: AH from nine patients previously diagnosed with serpiginous choroiditis were investigated for herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) by conventional virological methods and PCR. The PCR-positive DNA was gel-purified, extracted, and sequenced using a dye-based Applied Biosystems procedure. The sequences were processed through the National Cancer Institute's BLAST inquiry for species identification. RESULTS: Culture and cytological examination of AH from all nine patients were negative for HSV, VZV, and CMV. Five were positive for VZV, one was positive for HSV, and three were wholly negative using PCR. Subsequent DNA sequencing of the positive samples authenticated the presence of VZV and HSV DNA in the respective patients. CONCLUSION: VZV and HSV DNA were detected in a subset of patients with serpiginous choroiditis, suggesting that these viruses may function in the pathogenesis of this disease.