COVID-19 diagnostic testing underestimated cases amongst females in Pakistan (preprint)

Objectives Understanding the impact of COVID-19 largely depended on information from PCR based diagnostic testing of SARS-CoV-2. It was recognized early in the pandemic that testing rates varied greatly between high and low income countries. Whilst total numbers of tests conducted are noted, little attention has been made to differences that may be due to gender and we examined this in the context of Pakistan. Methods We conducted a retrospective cross-sectional analysis of respiratory specimens received for SARS-CoV-2 by PCR at Aga Khan University Hospital, Karachi, Pakistan between February 2020 and February 2022. Data was analysed in six monthly intervals; P-I, February to July 2020; P-II, August 2020 to January 2021; P-III, February to July 2021 and August 2021 until February 2022. Results A total of 470,047 PCR tests were conducted. The proportion of tests conducted for females was, 35% in P-I; 40% in P-II; 44% in P-III and 46% in P-III. 21% of specimens were positive for SARS-CoV-2, only 9% of these specimens were from females. The greatest numbers of tests were conducted in males aged 31 to 45 years followed by those aged 16-30 years. The fewest tests were conducted in children aged under 15 years. The highest percentage of PCR positive tests was found in those ages 60 years and above. Compared for gender SARS-CoV-2 positivity rates were comparable across the study period. Conclusions COVID-19 data from Pakistan indicates that there are larger numbers of males as compared with females who were affected by this disease. Our results show that this may be due to a gender bias in the demographics of testing. This was especially true in the early pandemic period, leading to under- surveillance and -reporting of COVID-19 cases in females especially, in those of younger and older age groups.

Older age, lack of vaccination and infection with variants other than Omicron associated with severity of COVID-19 and in-hospital mortality in Pakistan (preprint)

Objectives: We investigated factors associated with COVID-19 disease severity and in-hospital mortality in a low-middle income setting. Methods: Records of 197 adult COVID-19 patients admitted to the Aga Khan University Hospital, Karachi between April 2021 and February 2022 were reviewed. Clinical data including, that of SARS-CoV-2 variants was collected. Results: The median age of the patients was 55 years and 51.8% were males. 48.2 % of patients had non-severe disease, while 52.8% had severe/critical disease. Hypertension (48%) and diabetes mellitus (41.3%) were most common comorbid conditions. Omicron (55.3%), Beta (14.7%), Alpha (13.7%), Delta (12.7%) and Gamma (3.6%) were identified in patients. The risk of severe disease was higher in those aged above 50 years (OR 5.73; 95%CI [2.45-13.7]) and in diabetics (OR 4.24; 95% CI[1.82-9.85]). Full vaccination (OR 0.25; 95%CI [0.11-0.58]) or infection with Omicron variants (OR 0.42; 95% CI[0.23-0.74]) reduced disease severity. Age > 50 (OR 5.07; 95%CI [1.92-13.42]) and presence of myocardial infarction (OR 5.11; 95% CI[1.45-17.93]) was associated with increased mortality, but infection with Omicron (OR 0.22 95% CI 0.10-0.53]) reduced risk. Conclusions: Vaccination was found to protect against severe COVID-19 regardless of the infecting variant and is recommended especially, in those aged over 50 years and with co-morbid conditions.

SARS-CoV-2 Variants of Concern (VOC) Alpha, Beta, Gamma, Delta, and Omicron coincident with consecutive pandemic waves in Pakistan (preprint)

Identification and monitoring of SARS-CoV-2 Variants of Concern/Interest (VOC/VOIs) is essential to guide public health measures. We report the surveillance of VOCs circulating in Karachi during the pandemic between April 2021 and February 2022. We screened 2150 SARS-CoV-2 PCR positive samples received at the AKUH Clinical Laboratories. VOC was identified using a PCR-based approach targeting lineage-specific mutations using commercially available assays. Of the SARS-CoV-2 PCR positive samples, 81.7% had VOC/VOI, while 18.3% were undetermined. Alpha variants were predominant at 82.5% and 40.3% of the cases in April and May 2021. Beta variants increased in May (29%) and June (42%) and then reduced to 6% by July. Gamma variant cases were at 14.5% and 9% in May and June, respectively. Delta variants first detected in May, increased to comprise 66% of all variants by July, remaining dominant in August, September, October, and November 2021 at 88%, 91%, 91% and 85% respectively. Omicron (BA.1) variants emerged in December, rising to 42% of cases with an increase to 81% by January 2022 and then reducing to 45% in February 2022. Delta variant prevalence was coincident with increased hospital admissions and mortality. The Omicron variant surge was associated with increased daily infections but limited COVID-19 severity. We highlight the predominance of the VOCs identified through a rapid PCR based approach. As this is important to inform a public health response, we propose that a mutation targeted approach can be a rapid, lower cost solution to aid tracking of known VOCs during pandemic waves.

Changing SARS-CoV-2 variants in Karachi, Pakistan from alpha to delta through COVID-19 waves three and four (preprint)

We investigated the presence of SARS-CoV-2 variants of concern (VoC) in Karachi, Pakistan between April and July 2021 in specimens received at the Aga Khan University Clinical Laboratories. VoC were identified using a PCR based approach targeting lineage specific mutation. Of the 710 clinical isolates tested, 63% were VoC comprising; 36% alpha, 37% beta, 7% gamma and 21% delta variants. Alpha variants remained the majority whilst, delta strains increased to 43% of cases in July. Thirty-six per cent of all cases were admitted COVID-19 in-patient samples. Of the in-patient cases, 41% were alpha, 28% were beta, 8% were gamma and 24% were delta variants. Overall, we report an increase of delta variants in Karachi over the past two months which is concordant with the currently observed exacerbation in COVID-19 morbidity and mortality.

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

Objective: To investigate the possible cause of discrepancy between clinical and RT-PCR based diagnosis of patients with COVID-19.Results: Laboratory records of ten patients with mild to severe COVID-19 were examined. Initially, respiratory samples from the patients were tested using the Roche SARS-CoV-2 Cobas assay and were found to be negative. Further investigation for other viral causes of pneumonia was conducted using the Filmarray RP2.1 assay, which identified SARS-CoV-2 as the pathogen in all ten cases. To investigate whether this could be due to discrepancies between PCR assay, additional testing was conducted using the TaqPath TM 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, could be due to variation in the primer binding sites. The identification of strains with SGTF identify the possible 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.

SARS-CoV-2 genome analysis of strains in Pakistan reveals GH, S and L clade strains at the start of the pandemic (preprint)

ObjectivesPakistan has a high infectious disease burden with about 265,000 reported cases of COVID-19. We investigated the genomic diversity of SARS-CoV-2 strains and present the first data on viruses circulating in the country. MethodsWe performed whole-genome sequencing and data analysis of SARS-CoV-2 eleven strains isolated in March and May. ResultsStrains from travelers clustered with those from China, Saudi Arabia, India, USA and Australia. Five of eight SARS-CoV-2 strains were GH clade with Spike glycoprotein D614G, Ns3 gene Q57H, and RNA dependent RNA polymerase (RdRp) P4715L mutations. Two were S (ORF8 L84S and N S202N) and three were L clade and one was an I clade strain. One GH and one L strain each displayed Orf1ab L3606F indicating further evolutionary transitions. ConclusionsThis data reveals SARS-CoV-2 strains of L, G, S and I have been circulating in Pakistan from March, at the start of the pandemic. It indicates viral diversity regarding infection in this populous region. Continuing molecular genomic surveillance of SARS-CoV-2 in the context of disease severity will be important to understand virus transmission patterns and host related determinants of COVID-19 in Pakistan.

Transcriptomic profiling of disease severity in patients with COVID-19 reveals role of blood clotting and vasculature related genes (preprint)

COVID-19 caused by SARS-CoV-2 manifests as a range of symptoms. Understanding the molecular mechanisms responsible for immuno-pathogenesis of disease is important for treatment and management of COVID-19. We examined host transcriptomes in moderate and severe COVID-19 cases with a view to identifying pathways that affect its progression. RNA extracted from whole blood of COVID-19 cases was analysed by microarray analysis. Moderate and severe cases were compared with healthy controls and differentially regulated genes (DEGs) categorized into cellular pathways. DEGs in COVID-19 cases were mostly related to host immune activation and cytokine signaling, pathogen uptake, host defenses, blood and vasculature genes, and SARS-CoV-2- and other virus- affected pathways. The DEGs in these pathways were increased in severe compared with moderate cases. In a severe COVID-19 patient with an unfavourable outcome we observed dysregulation of genes in platelet homeostasis and cardiac conduction and fibrin clotting with disease progression.