Simultaneous Detection of Omicron and Other SARS-CoV-2 Variants by Multiplex PCR MassARRAY Technology (preprint)

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/ µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 hours. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

Homologous or Heterologous COVID-19 Boosters Regimens Significantly Impact Sero-neutralization of SARS-CoV-2 Virus and Variants (preprint)

We determined the levels of neutralizing antibodies against the SARS-CoV-2 ancestral strain, Delta and Omicron variants of concern (VOCs) in 125 healthcare workers, who received Coro-naVac as their primary vaccination and later received either a single ChAdOx1 or a combination of two consecutive boosters using either two ChAdOx1 doses or a ChAdOx1 or BNT162b2 as the primary and second boosters, respectively, or 2 doses of BNT162b2. The titers 12 weeks after primary vaccination were inadequate to neutralize the all strains. After a single ChAdOx1 boost-er, the levels of neutralization at Day 30 varied significantly, only a small proportion of partici-pants developing neutralizing titers against Omicron at Day 7 and 30. The two doses of ChA-dOx1 as the booster induced lowest activity. A combination ChAdOx1 and BNT162b2 induced greater neutralization than by two doses of ChAdOx1. Two doses of BNT162b2 as the booster had the maximal activity against Omicron VOC.

Dynamics of Neutralizing Antibody and T-cell Responses to SARS-CoV-2 and Variants of Concern After Primary Immunization With CoronaVac and Booster With BNT162b2 or ChAdOx1 in Health Care Workers (preprint)

Inactivated SARS-CoV-2 vaccine (CoronaVac) is commonly used in national immunization programs. However, the immune response significantly declined within a few months. Our study assessed the immune response against SARS-CoV-2 after receiving booster shots of BNT162b2 or ChAdOx1 among health care workers who previously received CoronaVac as their primary immunization. Fifty-six participants received ChAdOx1 and forty-two participants received BNT162b2 were enrolled into this study which evaluated the immune responses including anti-SARS-CoV-2 spike total antibodies (Elecsys®), surrogated viral neutralization test (sVNT) to ancestral strain (cPass™; GenScript) and five variants of concern (Alpha, Beta, Gamma, Delta, and Omicron) (Luminex; multiplex sVNT) and the ELISpot with spike (S1 and S2) peptide pool against the ancestral SARS-CoV-2 strain. The samples were analyzed at baseline, 4, and 12 weeks after primary immunization as well as 4 and 12 weeks after receiving the booster. This study showed a significantly higher B-cell response among the BNT162b2 than the ChAdOx1 booster group, particularly against the Omicron variant, as well as a trend of good T-cell immune response in the BNT162b2 group. Moreover, the immune response rapidly declined at 12 weeks after the booster. A fourth dose or a second booster should be recommended, especially for reducing Omicron severity.

Benefits of Early Combination Antiviral Treatment Containing Favipiravir for COVID-19 in Thailand (preprint)

We report the clinical outcomes following implementation of initial COVID-19 treatment guidelines in Thailand. A composite poor outcome was defined as death, ICU admission, requiring intubation, or high-flow oxygen. 744 COVID-19 patients (48.8% male) were included, median (IQR) age was 37 (27-48) years [8.4% >60 years] and 21.4% had pneumonia at admission. Admission <4 days from symptom onset had a reduced risk of poor outcome. In a subgroup analysis, favipiravir use reduced the risk of a poor outcome for patients admitted <4 days from symptom onset (OR 0.320 (0.152-0.662), P=0.003). Thai guidelines now include favipiravir to treat all symptomatic COVID-19 patients.

Differential escape of neutralizing antibodies by SARS-CoV-2 Omicron and pre-emergent sarbecoviruses (preprint)

The SARS-CoV-2 B.1.1.529 lineage, Omicron variant, was first detected in November 2021 and carries 32 amino acid mutations in the spike protein (15 in RBD) and exhibits significant escape of neutralizing antibodies targeting the parental SARS-CoV-2 virus. Here, we performed a high-resolution multiplex (16-plex) surrogate virus neutralization assay covering all major SARS-CoV-2 variants and pre-emergent ACE2-binding sarbecoviruses against 20 different human serum panels from infected, vaccinated and hybrid immune individuals which had vaccine-breakthrough infections or infection followed by vaccination. Among all sarbecoviruses tested, we observed 1.1 to 4.7-, 2.3 to 10.3- and 0.7 to 33.3-fold reduction in neutralization activities to SARS-CoV-2 Beta, Omicron and SARS-CoV-1, respectively. Among the SARS-CoV-2 related sarbecoviruses, it is found that the genetically more distant bat RaTG13 and pangolin GX-P5L sarbecoviruses had less neutralization escape than Omicron. Our data suggest that the SARS-CoV-2 variants emerged from the changed immune landscape of human populations are more potent in escaping neutralizing antibodies, from infection or vaccination, than pre-emergent sarbecoviruses naturally evolved in animal populations with no or less immune selection pressure.

Short-Term Immune Response After Inactivated SARS-CoV-2 (CoronaVac®, Sinovac) And ChAdOx1 nCoV-19 (Vaxzevria®, Oxford-AstraZeneca) Vaccinations in Thai Health Care Workers (preprint)

Background Inactivated SARS-CoV-2 (CoronaVac®,Sinovac, or SV) and ChAdOx1 nCoV-19 (Vaxzevria®,Oxford-Astra Zeneca, or AZ) vaccines have been administered to the health care workers (HCWs) in Thailand. Objective To determine the short-term immune response after the SV and AZ vaccinations in HCWs. Methods In this prospective cohort study, HCWs who completed a 2-dose regimen of the SV or AZ were included. Immune response was evaluated by surrogate viral neutralization test (sVNT) and anti-SARS-CoV-2 total antibody. Blood samples were analyzed at 4 and 12 weeks after the complete SV vaccination and at 4 weeks after each dose of the AZ vaccination. The primary outcome was the seroconversion rate at 4-weeks after complete immunization. Results Overall, 185 HCWs with a median (IQR) age of 40.5(30.3-55.8) years (94 HCWs in the SV group and 91 in the AZ group) were included. At 4 weeks after completing the SV vaccination, 60.6% (95%CI:50.0-70.6%) had seroconversion evaluated by sVNT(≥68%inhibition), comparable to the patients recovered from mild COVID-19 infection(69.0%), with a rapid reduction to 12.2%(95%CI:6.3-20.8) at 12 weeks. In contrast, 85.7%(95%CI:76.8-92.2%) HCWs who completed the second dose of the AZ for 4 weeks had seroconversion, comparable to the COVID-19 pneumonia patients(92.5%). When using the anti-SAR-CoV-2 total antibody level(≥132 U/ml) criteria, only 71.3% HCWs in the SV group had seroconversion, compared to 100% in the AZ group. Conclusion A rapid decline of short-term immune response in the HCWs after the SV vaccination indicates the need for a vaccine booster, particularly during the ongoing spreading of the SAR-CoV-2 variants of concern.

Antibody Responses to SARS-CoV-2 in Coronavirus Diseases 2019 Patients with Different Severity (preprint)

Background: More understanding of antibody responses in the SARS-CoV-2 infected population is useful for vaccine development. Aim: To investigate SARS-CoV-2 IgA and IgG among COVID-19 Thai patients with different severity. Methods: We used plasma from 118 adult patients who have confirmed SARS-CoV-2 infection and 49 patients under investigation without infection, 20 patients with other respiratory infections, and 102 healthy controls. Anti-SARS-CoV-2 IgA and IgG were performed by enzyme-linked immunosorbent assay from Euroimmun. The optical density ratio cut off for positive test was 1.1 for IgA and 0.8 for IgG. The association of antibody response with the severity of diseases and the day of symptoms was performed. Results: From Mar 10 to May 31, 2020, 289 participants were enrolled, and 384 samples were analyzed. Patients were categorized by clinical manifestations to mild (n=59), moderate (n=27) and severe (n=32). The overall sensitivity of IgA and IgG from samples collected after day 7 is 87.9% (95% CI 79.8-93.6) and 84.8% (95% CI 76.2-91.3), respectively. The severe group had a significantly higher level of specific IgA and IgG to S1 antigen compared to the mild group. All moderate to severe patients have specific IgG while 20% of the mild group did not have any IgG detected after two weeks. Interestingly, SARS-CoV-2 IgG level was significantly higher in males compared to females among the severe group (p=0.003). Conclusion: The serologic test for SARS-CoV-2 has high sensitivity after the second week after onset of illness. Serological response differs among patients with different severity and different sex.

Pre-existing liver disease is associated with poor outcome in patients with SARS CoV2 infection; The APCOLIS Study (APASL COVID-19 Liver Injury Spectrum Study) (preprint)

Background and Aims: COVID-19 is a dominant pulmonary disease, with multisystem involvement, depending upon co morbidities. Its profile in patients with pre-existing chronic liver disease (CLD) is largely unknown. We studied the liver injury patterns of SARS-Cov-2 in CLD patients, with or without cirrhosis. Methods: Data was collected from 13 Asian countries on patients with CLD, known or newly diagnosed, with confirmed COVID-19. Result: Altogether, 228 patients [185 CLD without cirrhosis and 43 with cirrhosis] were enrolled, with comorbidities in nearly 80%. Metabolism associated fatty liver disease (113, 61%) and viral etiology (26, 60%) were common. In CLD without cirrhosis, diabetes [57.7% vs 39.7%, OR=2.1(1.1-3.7), p=0.01] and in cirrhotics, obesity, [64.3% vs. 17.2%, OR=8.1(1.9-38.8), p=0.002) predisposed more to liver injury than those without these. Forty three percent of CLD without cirrhosis presented as acute liver injury and 20% cirrhotics presented with either acute-on-chronic liver failure [5(11.6%)] or acute decompensation [4(9%)]. Liver related complications increased (p<0.05) with stage of liver disease; a Child-Turcotte Pugh score of 9 or more at presentation predicted high mortality [AUROC-0.94, HR=19.2(95CI 2.3-163.3), p<0.001, sensitivity 85.7% and specificity 94.4%). In decompensated cirrhotics, the liver injury was progressive in 57% patients, with 43% mortality. Rising bilirubin and AST/ALT ratio predicted mortality among cirrhosis. Conclusions: SARS-Cov-2 infection causes significant liver injury in CLD patients, decompensating one fifth of cirrhosis, and worsening the clinical status of the already decompensated. The CLD patients with diabetes and obesity are more vulnerable and should be closely monitored. 

Endotoxemia and circulating bacteriome in severe COVID-19 patients (preprint)

Background: When severe, COVID-19 shares many clinical features with bacterial sepsis. Yet, secondary bacterial infection is uncommon. However, as epithelium are injured and barrier function is lost, bacterial products entering the circulation might contribute to the pathophysiology of COVID-19. Methods: We studied 19 adults, severely ill patients with COVID-19 infection, who were admitted to King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between 13th March and 17th April 2020. Blood samples on day 1, 3, and 7 of enrollment were analyzed for endotoxin activity assay (EAA), Beta-D-Glucan (BG), and 16S rRNA gene sequencing to determine the circulating bacteriome. Findings: Of the 19 patients, 14 were in intensive care and 10 patients received mechanical ventilation. We found 8 patients with high EAA ([≥] 0.6) and about half of the patients had high serum BG levels which tended to be higher in later in the illness. Although only 1 patient had a positive blood culture, 18 of 19 patients were positive for 16S rRNA gene amplification. Proteobacteria was the most abundant phylum. The diversity of bacterial genera was decreased overtime. Interpretation: Bacterial DNA and toxins were discovered in virtual all severely ill COVID-19 pneumonia patients. This raises a previously unrecognized concern for significant contribution of bacterial products in the pathogenesis of this disease.

Evaluating efficiency of pooling specimens for PCR-based detection of COVID-19 (preprint)

In the age of a pandemic, such as the ongoing one caused by SARS-CoV-2, the world faces limited supply of tests, PPE and reagents, and factories are struggling to meet the growing demands. This study aimed to evaluate the efficacy of pooling specimen for testing of SARS-CoV-2 virus, to determine whether costs and resource savings could be achieved without impacting the sensitivity of the testing. Ten specimens were pooled for testing, containing either one or two known positive specimen of varying viral concentrations. Pooling specimens did not affect the sensitivity of detecting SARS-CoV-2, and the PCR cycle threshold (Ct) between testing of pooling specimen and subsequent individual testing was not significantly different using paired t-test. This study also identified cost savings garnered from pooling of specimen for testing at 4 differing prevalence rates, ranging from 0.1-10%. Pooling specimens to test for COVID-19 infection in low prevalence areas or in low risk population can dramatically decrease the resources burden on lab operations by up to 80%. This paves the possibility for large-scale population screening, allowing for assured policy decisions by governmental bodies to ease lockdown restrictions in areas with low incidence of infection, or with lower risk populations.