Impact of SARS-CoV-2 viral load on restrictive spirometry patterns in mild COVID-19 recovered middle-aged individuals: a six-month prospective study.

BACKGROUND: Long term respiratory complications of Corona Virus Disease-2019 (COVID-19) are of great concern. Many studies have reported altered respiratory patterns in COVID-19 recovered individuals and most of them were from severe to critically ill patients. The association of viral load at the time of infection with symptoms of long COVID-19 specifically on pulmonary functions after months of recovery is still not known. This study was aimed to assess the impact of SARS-CoV-2 viral load during mild-moderate COVID-19 disease on pulmonary functions in middle-aged population after 6-8 months of acute infection. METHODS: This study included 300 (102 healthy controls and 198 COVID-19 recovered) individuals between age 30-60 of either gender. Mild-moderate COVID-19 recovered individuals were recruited between a period of 6-8 months post-acute infection. Spirometry was performed with MIR-Spirolab-III. The association of spirometry pattern was compared with SARS-CoV-2 viral loads during acute infection. RESULTS: We observed up to 70% of the participants presented with either shortness of breath (11.5%), body aches (23.5%), recurrent cough (4.4%), recurrent respiratory infections (9.5%) and/or fatigue (33.3%) at follow up. In our study, 35.5% of COVID-19 recovered individuals had abnormal respiratory patterns (33.5% had restrictive and 2% had obstructive patterns). Viral load ≤ 20 CT value was associated with restrictive respiratory patterns (p = 0.004). No association was found between viral load and disease severity (p = 0.23). CONCLUSION: In this study, we found one third of mild-moderate COVID-19 recovered individuals have restrictive respiratory patterns after 6-8 months of recovery. These findings had a strong association with SARS-CoV-2 viral loads during acute infection which has been reported for the first time in our study. Studying the relationship between viral load and pulmonary functions can contribute to identifying potential risk factors for long COVID and developing preventive measures to mitigate the long-term impact on lung health. CLINICAL TRIAL NUMBER: Not applicable.

Sequential viral introductions and spread of BA.1 across Pakistan provinces during the Omicron wave.

BACKGROUND: COVID-19 waves caused by specific SARS-CoV-2 variants have occurred globally at different times. We focused on Omicron variants to understand the genomic diversity and phylogenetic relatedness of SARS-CoV-2 strains in various regions of Pakistan. METHODS: We studied 276,525 COVID-19 cases and 1,031 genomes sequenced from December 2021 to August 2022. Sequences were analyzed and visualized using phylogenetic trees. RESULTS: The highest case numbers and deaths were recorded in Sindh and Punjab, the most populous provinces in Pakistan. Omicron variants comprised 93% of all genomes, with BA.2 (32.6%) and BA.5 (38.4%) predominating. The first Omicron wave was associated with the sequential identification of BA.1 in Sindh, then Islamabad Capital Territory, Punjab, Khyber Pakhtunkhwa (KP), Azad Jammu Kashmir (AJK), Gilgit-Baltistan (GB) and Balochistan. Phylogenetic analysis revealed Sindh to be the source of BA.1 and BA.2 introductions into Punjab and Balochistan during early 2022. BA.4 was first introduced in AJK and BA.5 in Punjab. Most recent common ancestor (MRCA) analysis revealed relatedness between the earliest BA.1 genome from Sindh with Balochistan, AJK, Punjab and ICT, and that of first BA.1 from Punjab with strains from KPK and GB. CONCLUSIONS: Phylogenetic analysis provides insights into the introduction and transmission dynamics of the Omicron variant in Pakistan, identifying Sindh as a hotspot for viral dissemination. Such data linked with public health efforts can help limit surges of new infections.

Humoral and T cell responses to SARS-CoV-2 reveal insights into immunity during the early pandemic period in Pakistan.

BACKGROUND: Protection against SARS-CoV-2 is mediated by humoral and T cell responses. Pakistan faced relatively low morbidity and mortality from COVID-19 through the pandemic. To examine the role of prior immunity in the population, we studied IgG antibody response levels, virus neutralizing activity and T cell reactivity to Spike protein in a healthy control group (HG) as compared with COVID-19 cases and individuals from the pre-pandemic period (PP). METHODS: HG and COVID-19 participants were recruited between October 2020 and May 2021. Pre-pandemic sera was collected before 2018. IgG antibodies against Spike and its Receptor Binding Domain (RBD) were determined by ELISA. Virus neutralization activity was determined using a PCR-based micro-neutralization assay. T cell - IFN-γ activation was assessed by ELISpot. RESULTS: Overall, the magnitude of anti-Spike IgG antibody levels as well as seropositivity was greatest in COVID-19 cases (90%) as compared with HG (39.8%) and PP (12.2%). During the study period, Pakistan experienced three COVID-19 waves. We observed that IgG seropositivity to Spike in HG increased from 10.3 to 83.5% during the study, whilst seropositivity to RBD increased from 7.5 to 33.3%. IgG antibodies to Spike and RBD were correlated positively in all three study groups. Virus neutralizing activity was identified in sera of COVID-19, HG and PP. Spike reactive T cells were present in COVID-19, HG and PP groups. Individuals with reactive T cells included those with and without IgG antibodies to Spike. CONCLUSIONS: Antibody and T cell responses to Spike protein in individuals from the pre-pandemic period suggest prior immunity against SARS-CoV-2, most likely from cross-reactive responses. The rising seroprevalence observed in healthy individuals through the pandemic without known COVID-19 may be due to the activation of adaptive immunity from cross-reactive memory B and T cells. This may explain the more favourable COVID-19 outcomes observed in this population.

Latent M. tuberculosis infection is associated with increased inflammatory cytokine and decreased suppressor of cytokine signalling (SOCS)-3 in the diabetic host.

Tuberculosis (TB) outcomes are worsened by type II diabetes mellitus (DM). Protective immunity against Mycobacterium tuberculosis (MTB) is driven by cytokines. Latent TB (LTBi) is common but its effect on the diabetic host is not well understood. We investigated mycobacterial antigen-stimulated responses in peripheral blood mononuclear cell (PBMC) isolated from healthy endemic controls (EC), those with LTBi, DM groups with and without LTBi, as compared with TB patients. Cytokines were measured using a Luminex-based assay. Gene expression was determined by RT-PCR. In DM-LTBi cases, PPD-stimulated proinflammatory cytokines; IFN-γ, IL-6, IL-2, TNF-α and GM-CSF and anti-inflammatory cytokines, IL-5 and IL-13 were raised as compared with EC. DM-LTBi PPD-stimulated IFN-γ, IL-6 and TNF-α mRNA titres were found raised in DM-LTBi, whilst suppressor of cytokine signalling (SOCS)-3 expression was lowered. Within DM cases, stratification based on HbA1c levels revealed raised IFN-γ but lowered IL-6 gene expression in those with controlled levels as compared with uncontrolled glycaemic levels. Further, SOCS1 expression levels were found higher in DM cases with controlled glycaemia when compared with EC. Overall, we show that diabetics with LTBi manifest raised levels of inflammatory and anti-inflammatory cytokines concomitant with reduced SOCS3 mRNA expression. Reduced glycaemic control results in further inflammatory dysregulation impacting conversing impacting IFN-γ and IL-6 activation. These results suggest that dysregulated immune activation in diabetes is exacerbated by LTBi, lack of glycaemic control may further compromise immunity against MTB infection.

SARS-CoV-2 variants induce increased inflammatory gene expression but reduced interferon responses and heme synthesis as compared with wild type strains.

SARS-CoV-2 variants of concern (VOC) have been associated with increased viral transmission and disease severity. We investigated the mechanisms of pathogenesis caused by variants using a host blood transcriptome profiling approach. We analysed transcriptional signatures of COVID-19 patients comparing those infected with wildtype (wt), alpha, delta or omicron strains seeking insights into infection in Asymptomatic cases.Comparison of transcriptional profiles of Symptomatic and Asymptomatic COVID-19 cases showed increased differentially regulated gene (DEGs) of inflammatory, apoptosis and blood coagulation pathways, with decreased T cell and Interferon stimulated genes (ISG) activation. Between SARS-CoV-2 strains, an increasing number of DEGs occurred in comparisons between wt and alpha (196), delta (1425) or, omicron (2313) infections. COVID-19 cases with alpha or, delta variants demonstrated suppression transcripts of innate immune pathways. EGR1 and CXCL8 were highly upregulated in those infected with VOC; heme biosynthetic pathway genes (ALAS2, HBB, HBG1, HBD9) and ISGs were downregulated. Delta and omicron infections upregulated ribosomal pathways, reflecting increased viral RNA translation. Asymptomatic COVID-19 cases infected with delta infections showed increased cytokines and ISGs expression. Overall, increased inflammation, with reduced host heme synthesis was associated with infections caused by VOC infections, with raised type I interferon in cases with less severe disease.

Substitution spectra of SARS-CoV-2 genome from Pakistan reveals insights into the evolution of variants across the pandemic.

Changing morbidity and mortality due to COVID-19 across the pandemic has been linked with factors such as the emergence of SARS-CoV-2 variants and vaccination. Mutations in the Spike glycoprotein enhanced viral transmission and virulence. We investigated whether SARS-CoV-2 mutation rates and entropy were associated COVID-19 in Pakistan, before and after the introduction of vaccinations. We analyzed 1,705 SARS-CoV-2 genomes using the Augur phylogenetic pipeline. Substitution rates and entropy across the genome, and in the Spike glycoprotein were compared between 2020, 2021 and 2022 (as periods A, B and C). Mortality was greatest in B whilst cases were highest during C. In period A, G clades were predominant, and substitution rate was 5.25 × 10-4 per site per year. In B, Delta variants dominated, and substitution rates increased to 9.74 × 10-4. In C, Omicron variants led to substitution rates of 5.02 × 10-4. Genome-wide entropy was the highest during B particularly, at Spike E484K and K417N. During C, genome-wide mutations increased whilst entropy was reduced. Enhanced SARS-CoV-2 genome substitution rates were associated with a period when more virulent SARS-CoV-2 variants were prevalent. Reduced substitution rates and stabilization of genome entropy was subsequently evident when vaccinations were introduced. Whole genome entropy analysis can help predict virus evolution to guide public health interventions.

Investigating the impact of prior COVID-19 on IgG antibody and interferon γ responses after BBIBP-CorV vaccination in a disease endemic population: A prospective observational study.

Background and Aims: COVID-19 vaccinations have reduced morbidity and mortality from the disease. Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) have been associated with immune protection. Seroprevalence studies revealed high immunoglobulin G (IgG) antibody levels to SARS-CoV-2 in the Pakistani population before vaccinations. We investigated the effect of BBIBP-CorV vaccination on circulating IgG antibodies and interferon (IFN)-γ from T cells measured in a cohort of healthy individuals, with respect to age, gender, and history of COVID-19. Methods: The study was conducted between April and October 2021. BBIBP-CorV vaccinated participants were followed up to 24 weeks. Antibodies to SARS-CoV-2 Spike protein and its receptor-binding domain (RBD) were measured. IFNγ secreted by whole blood stimulation of Spike protein and extended genome antigens was determined. Results: Study participants with a history of prior COVID-19 displayed a higher magnitude of IgG antibodies to Spike and RBD. IgG seropositivity was greater in those with prior COVID-19, aged 50 years or younger and in females. At 24 weeks after vaccination, 37.4% of participants showed IFN-γ responses to SARS-CoV-2 antigens. T cell IFN-γ release was higher in those with prior COVID-19 and those aged 50 years or less. Highest IFN-γ release was observed to extended genome antigens in individuals both with and without prior COVID-19. Conclusion: We found that IgG seropositivity to both Spike and RBD was affected by prior COVID-19, age and gender. Importantly, seropositive responses persisted up to 24 weeks after vaccination. Persistence of vaccine induced IgG antibodies may be linked to the high seroprevalence observed earlier in unvaccinated individuals. Increased T cell reactivity to Spike and extended genome antigens reflects cellular activation induced by BBIBP-CorV. COVID-19 vaccination may have longer lasting immune responses in populations with a higher seroprevalence. These data inform on vaccination booster policies for high-risk groups.

Upregulated type I interferon responses in asymptomatic COVID-19 infection are associated with improved clinical outcome.

Understanding key host protective mechanisms against SARS-CoV-2 infection can help improve treatment modalities for COVID-19. We used a blood transcriptome approach to study biomarkers associated with differing severity of COVID-19, comparing severe and mild Symptomatic disease with Asymptomatic COVID-19 and uninfected Controls. There was suppression of antigen presentation but upregulation of inflammatory and viral mRNA translation associated pathways in Symptomatic as compared with Asymptomatic cases. In severe COVID-19, CD177 a neutrophil marker, was upregulated while interferon stimulated genes (ISGs) were downregulated. Asymptomatic COVID-19 cases displayed upregulation of ISGs and humoral response genes with downregulation of ICAM3 and TLR8. Compared across the COVID-19 disease spectrum, we found type I interferon (IFN) responses to be significantly upregulated (IFNAR2, IRF2BP1, IRF4, MAVS, SAMHD1, TRIM1), or downregulated (SOCS3, IRF2BP2, IRF2BPL) in Asymptomatic as compared with mild and severe COVID-19, with the dysregulation of an increasing number of ISGs associated with progressive disease. These data suggest that initial early responses against SARS-CoV-2 may be effectively controlled by ISGs. Therefore, we hypothesize that treatment with type I interferons in the early stage of COVID-19 may limit disease progression by limiting SARS-CoV-2 in the host.

Higher entropy observed in SARS-CoV-2 genomes from the first COVID-19 wave in Pakistan.

BACKGROUND: We investigated the genome diversity of SARS-CoV-2 associated with the early COVID-19 period to investigate evolution of the virus in Pakistan. MATERIALS AND METHODS: We studied ninety SARS-CoV-2 strains isolated between March and October 2020. Whole genome sequences from our laboratory and available genomes were used to investigate phylogeny, genetic variantion and mutation rates of SARS-CoV-2 strains in Pakistan. Site specific entropy analysis compared mutation rates between strains isolated before and after June 2020. RESULTS: In March, strains belonging to L, S, V and GH clades were observed but by October, only L and GH strains were present. The highest diversity of clades was present in Sindh and Islamabad Capital Territory and the least in Punjab province. Initial introductions of SARS-CoV-2 GH (B.1.255, B.1) and S (A) clades were associated with overseas travelers. Additionally, GH (B.1.255, B.1, B.1.160, B.1.36), L (B, B.6, B.4), V (B.4) and S (A) clades were transmitted locally. SARS-CoV-2 genomes clustered with global strains except for ten which matched Pakistani isolates. RNA substitution rates were estimated at 5.86 x10-4. The most frequent mutations were 5' UTR 241C > T, Spike glycoprotein D614G, RNA dependent RNA polymerase (RdRp) P4715L and Orf3a Q57H. Strains up until June 2020 exhibited an overall higher mean and site-specific entropy as compared with sequences after June. Relative entropy was higher across GH as compared with GR and L clades. More sites were under selection pressure in GH strains but this was not significant for any particular site. CONCLUSIONS: The higher entropy and diversity observed in early pandemic as compared with later strains suggests increasing stability of the genomes in subsequent COVID-19 waves. This would likely lead to the selection of site-specific changes that are advantageous to the virus, as has been currently observed through the pandemic.