Tracking SARS-CoV-2 variants through pandemic waves using RT-PCR testing in low-resource settings.

COVID-19 resulted in extensive morbidity and mortality worldwide. SARS-CoV-2 evolved rapidly, with increasing transmission due to Variants of Concern (VOC). Identifying VOC became important but genome submissions from low-middle income countries (LMIC) remained low leading to gaps in genomic epidemiology. We demonstrate the use of a specific mutation RT-PCR based approach to identify VOC in SARS-CoV-2 positive samples through the pandemic in Pakistan. We selected 2150 SARS-CoV-2 PCR positive respiratory specimens tested between April 2021 and February 2022, at the Aga Khan University Hospital Clinical Laboratories, Karachi, Pakistan. Commercially available RT-PCR assays were used as required for mutations in Spike protein (N501Y, A570D, E484K, K417N, L452R, P681R and deletion69_70) to identify Alpha, Beta, Gamma, Delta, and Omicron variants respectively. Three pandemic waves associated with Alpha, Delta and Omicron occurred during the study period. Of the samples screened, VOC were identified in 81.7% of cases comprising mainly; Delta (37.2%), Alpha (29.8%) and Omicron (17.1%) variants. During 2021, Alpha variants were predominant in April and May; Beta and Gamma variants emerged in May and peaked in June; the Delta variant peaked in July and remained predominant until November. Omicron (BA.1) emerged in December 2021 and remained predominant until February 2022. The CT values of Alpha, Beta, Gamma and Delta were all significantly higher than that of Omicron variants (p<0.0001). We observed VOC through the pandemic waves using spike mutation specific RT-PCR assays. We show the spike mutation specific RT-PCR assay is a rapid, low-cost and adaptable for the identification of VOC as an adjunct approach to NGS to effectively inform the public health response. Further, by associating the VOC with CT values of its diagnostic PCR we gain information regarding the viral load of samples and therefore the level of transmission and disease severity in the population.

Using rapid antigen testing for early, safe return-to-work for healthcare personnel after SARS-CoV-2 infection in a healthcare system in Pakistan: A retrospective cross-sectional study.

Anticipating staff shortage during the Omicron variant surge, we modified the US Centers for Disease Control and Prevention's contingency guidelines at a healthcare system in Pakistan. Infected staff had a SARS-CoV-2 rapid antigen test after 5-7 days of isolation, to decide a safe return-to-work. This led to signifcant cost savings without compromising patient/staff safety.

IgG antibodies to SARS-CoV-2 in asymptomatic blood donors at two time points in Karachi.

INTRODUCTION: An estimated 1.5 million cases were reported in Pakistan until 23 March, 2022. However, SARS-CoV-2 PCR testing capacity has been limited and the incidence of COVID-19 infections is unknown. Volunteer healthy blood donors can be a control population for assessment of SARS-CoV-2 exposure in the population. We determined COVID-19 seroprevalence during the second pandemic wave in Karachi in donors without known infections or symptoms in 4 weeks prior to enrollment. MATERIALS AND METHODS: We enrolled 558 healthy blood donors at the Aga Khan University Hospital between December 2020 and February 2021. ABO blood groups were determined. Serum IgG reactivity were measured to spike and receptor binding domain (RBD) proteins. RESULTS: Study subjects were predominantly males (99.1%) with a mean age of 29.0±7.4 years. Blood groups were represented by; B (35.8%), O (33.3%), A (23.8%) and AB (7%). Positive IgG responses to spike were detected in 53.4% (95% CI, 49.3-37.5) of blood donors. Positive IgG antibodies to RBD were present in 16.7% (95% CI; 13.6-19.8) of individuals. No significant difference was found between the frequency of IgG antibodies to spike or RBD across age groups. Frequencies of IgG to Spike and RBD antibodies between December 2020 and February 2021 were found to be similar. Seropositivity to either antigen between individuals of different blood groups did not differ. Notably, 31.2% of individuals with IgG antibodies to spike also had IgG antibodies to RBD. Amongst donors who had previously confirmed COVID-19 and were seropositive to spike, 40% had IgG to RBD. CONCLUSIONS: Our study provides insights into the seroprevalence of antibodies to COVID-19 in a healthy cohort in Karachi. The differential dynamics of IgG to spike and RBD likely represent both exposure to SARS-CoV-2 and associate with protective immunity in the population.

Evolutionary history and introduction of SARS-CoV-2 Alpha VOC/B.1.1.7 in Pakistan through international travelers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and their identification is important for the public health response to coronavirus disease 2019 (COVID-19). Genomic sequencing provides robust information but may not always be accessible, and therefore, mutation-based polymerase chain reaction (PCR) approaches can be used for rapid identification of known variants. International travelers arriving in Karachi between December 2020 and February 2021 were tested for SARS-CoV-2 by PCR. A subset of positive samples was tested for S-gene target failure (SGTF) on TaqPathTM COVID-19 (Thermo Fisher Scientific) and for mutations using the GSD NovaType SARS-CoV-2 (Eurofins Technologies) assays. Sequencing was conducted on the MinION platform (Oxford Nanopore Technologies). Bayesian phylogeographic inference was performed integrating the patients' travel history information. Of the thirty-five COVID-19 cases screened, thirteen had isolates with SGTF. The travelers transmitted infection to sixty-eight contact cases. The B.1.1.7 lineage was confirmed through sequencing and PCR. The phylogenetic analysis of sequence data available for six cases included four B.1.1.7 strains and one B.1.36 and B.1.1.212 lineage isolate. Phylogeographic modeling estimated at least three independent B.1.1.7 introductions into Karachi, Pakistan, originating from the UK. B.1.1.212 and B.1.36 were inferred to be introduced either from the UK or the travelers' layover location. We report the introduction of SARS-CoV-2 B.1.1.7 and other lineages in Pakistan by international travelers arriving via different flight routes. This highlights SARS-CoV-2 transmission through travel, importance of testing, and quarantine post-travel to prevent transmission of new strains, as well as recording detailed patients' metadata. Such results help inform policies on restricting travel from destinations where new highly transmissible variants have emerged.

A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum.

BACKGROUND: Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. METHODS: The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro-neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. RESULTS: Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. CONCLUSION: We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.

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.

Discrepancy between PCR based SARS-CoV-2 tests suggests the need to re-evaluate diagnostic assays.

OBJECTIVE: We investigated the discrepancy between clinical and PCR-based diagnosis of COVID-19. We compared results of ten patients with mild to severe COVID-19. Respiratory samples from all cases were tested on the Roche SARS-CoV-2 (Cobas) assay, Filmarray RP2.1 (bioMereiux) and TaqPath™ COVID19 (Thermofisher) PCR assays. RESULTS: Laboratory records of ten patients with mild to severe COVID-19 were examined. Initially, respiratory samples from the patients were tested as negative on the SARS-CoV-2 Roche® assay. Further investigation using the BIOFIRE® Filmarray RP2.1 assay identified SARS-CoV-2 as the pathogen in all ten cases. To investigate possible discrepancies between PCR assays, additional testing was conducted using the TaqPath™ COVID19 PCR. Eight of ten samples were positive for SARS-CoV-2 on the TaqPath assay. Further, Spike gene target failures (SGTF) were identified in three of these eight cases. Discrepancy between the three PCR assays could be due to variation in PCR efficiencies of the amplification reactions or, variation at primer binding sites. Strains with SGTF indicate the presence of new SARS-CoV-2 variant strains. Regular modification of gene targets in diagnostic assays may be necessary to maintain robustness and accuracy of SARS-CoV-2 diagnostic assays to avoid reduced case detection, under-surveillance, and missed opportunities for control.

COVID-19 age-dependent immunology and clinical outcomes: implications for vaccines.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to acute respiratory distress syndrome (ARDS). Understanding the evolution of the virus, and immune-pathogenic processes are critical for designing future therapeutic interventions. In this review, we collate information on the structure, genome, viral life cycle, and adult and pediatric host immune responses in response to SARS-CoV-2. The immunological responses are a prototype of the developmental origins of health and disease (DOHaD) hypothesis to explain the socio-geographic differences impacting the severity and mortality rates in SARS-CoV-2 infections. The DOHaD hypothesis identifies the relevance of trained innate immunity, age groups, and geography for effective vaccinations. As COVID-19 vaccines are being rolled out, it may be pertinent to assess population-based immunological responses to understand the effectiveness and safety across different populations and age groups.

Comparison of clinical characteristics and outcomes between COVID-19 pneumonia and H1N1 influenza.

INTRODUCTION: The COVID-19 pandemic has been likened to the 2009 H1N1 influenza pandemic. We aim to study the similarities and differences between patients hospitalized with COVID-19 and H1N1 influenza in order to provide better care to patients, particularly during the co-circulation of Influenza A Subtype H1N1 and SARS-CoV-2. MATERIAL AND METHODS: A retrospective cohort study was conducted in order to compare clinical characteristics, complications, and outcomes of hospitalized patients with PCR-confirmed H1N1 influenza pneumonia and COVID-19 at a tertiary care center in Karachi, Pakistan. RESULTS: A total of 115 patients hospitalized with COVID-19 were compared with 55 patients with H1N1 Influenza A pneumonia. Median age was similar in both COVID-19 patients (54 years) and in patients with H1N1 influenza (59 years), but there was male predominance in COVID-19 patients (OR = 2.95; 95% CI: 1.12-7.79). Patients with COVID-19 pneumonia were 1.34 (95% CI: 1.14-1.62) times more likely to have a greater duration of illness prior to presentation compared to H1N1 influenza patients. COVID-19 patients were 4.59 times (95% CI: 1.32-15.94) more likely to be admitted to a general ward compared to H1N1 pneumonia patients. Moreover, patients with COVID-19 were 7.62 times (95% CI: 2.42-24.00) more likely to be treated with systemic steroids compared to patients with H1N1 pneumonia. The rate of nosocomial infections as well as mortality was similar in both H1N1 and COVID-19 pneumonia. CONCLUSION: Our study found a male predominance and longer duration of illness in hospitalized patients with COVID-19 compared to H1N1 influenza patients but no difference in outcomes with either infection.