Invasive fungal infections in COVID-19 patients from Pakistan

Objective: The objective of this study is to report the frequency and clinical characteristic of IFI in COVID-19 patients. Method(s): This observational study was conducted in Karachi, Pakistan from March 2020-April 2021. Patients with COVID-19 associated aspergillosis (CAPA) were diagnosed using ECMM/ISHAM criteria modified to include tracheal aspirate culture and/or Galactomannan Index (GMI) >4.5 in the possible CAPA category. COVID-19 associated candidemia (CAC) was defined by isolation of Candida species from blood cultures. COVID-19 associated mucormycosis (CAM) was defined as updated EORTC/MSG criteria with inclusion of COVID-19 as host factor. Pneumocystis jirovecii pneumonia (PJP) was defined by consistent clinical and radiological features and PCR positivity. Result(s): During the study period a total of 123 (3.3%) IFI in 3506 hospitalized COVID-19 patients were identified. This included 78 (2.2%) CAPA patients (42 probable;36 possible), 29 (0.8%) CAC (5 C. auris;24 non-C. auris), 10 (0.3%) CAM (7 pulmonary;3 rhinocerebral), 3 (0.08%) PJP and three (0.08%) cases of rare invasive fungal infections (2 C. neoformans;1 Trichosporon asahii). Outcome data was available on 117/123 patients. Of these 117 patients, 78 expired (66.7%). These include 52/74 (70%) CAPA patients, 17/27 (63%) CAC patients, 7/10 (70%) CAM patients and 2/3 (67%) PJP patients. Conclusion(s): We report a rate of 3.3% IFI amongst hospitalized COVID-19 patients at our center. We consider this rate to be an underestimate due to less bronchoscopic procedures and inclusion of only candidemia cases. We also report higher mortality rate with IFI in our patients than global data probably due to delayed diagnosis, co-infections and limited therapeutic options.

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.

Effectiveness of inactivated COVID-19 vaccines against SARS-CoV-2 infections among healthcare personnel in Pakistan: a test-negative, case-control study. (preprint)

Objective: During the COVID-19 pandemic, several vaccines that were efficacious in randomized controlled trials (RCTs) were authorized for mass vaccination. In developing countries, inactivated vaccines were widely administered. While inactivated vaccines have been deemed effective in reducing disease severity, for healthcare personnel (HCPs), effectiveness against COVID-19 infections is also essential to reduce the risk to vulnerable patients and ensure a stable healthcare workforce. In addition, there are limited studies examining effectiveness of inactivated vaccines against emerging SARS-CoV-2 variants in real-world settings. We aimed to estimate the effectiveness of inactivated vaccines (BBIBP-CorV and CoronaVac) against RT-PCR-confirmed COVID-19 infections among HCPs in the setting of emerging SARS-CoV-2 variants in Pakistan. Design, setting and participants: A retrospective matched test-negative case-control analysis of existing data of HCPs at a private healthcare system in Pakistan. Methods: HCPs tested between April 1 and September 30, 2021, were included. Each case was matched to two to six controls by the date of the RT-PCR test ({+/-} 7 days) to reduce bias. We compared demographics, reasons for testing, and vaccination status between cases and controls using chi-square for categorical variables and t-test for continuous-level data. The odds of getting a PCR-confirmed SARS-COV-2 infection were calculated using conditional logistic regression, after adjusting for age, gender, and work area. Vaccine effectiveness (VE) was calculated as percent VE using (1-OR)*100. Results: Inactivated vaccines were ineffective against COVID-19 infections [≥] 14 days after receiving the first dose [VE: 20% (95% CI: -10, 41; p=0.162)]. The vaccines showed modest effectiveness [≥] 14 days after the second dose against COVID-19 infections [VE: 33% (95% CI: 11, 50; p=0.006)], and symptomatic COVID-19 infections [VE: 36% (95% CI: 10, 54; p=0.009)]. Conclusions: Inactivated vaccines show modest effectiveness against COVID-19 infections in the setting of emerging VOCs. This builds a strong case for boosters and/or additional vaccination.

BBIBP-CorV (Sinopharm) vaccination- induced immunity is affected by age, gender and prior COVID-19 and activates responses to spike and other antigens (preprint)

Long-term solutions against SARS-CoV-2 infections require understanding of immune protection induced by different vaccine COVID-19 formulations. We investigated humoral and cellular immunity induced by Sinopharm (BBIBP-CorV) in a region of high SARS-CoV-2 seroprevalence. Levels of IgG antibodies to SARS-CoV-2 spike protein and its receptor-binding domain (RBD) were determined 24-weeks. Cellular immunity was investigated using a commercially available IFN-{gamma} release assay to SARS-CoV-2 spike (Ag1 and 2) and extended genome antigens (Ag3). Increasing IgG seropositivity to Spike protein and RBD was observed post-vaccination. Seropositivity was reduced in those over 50 years and raised in females and those with prior COVID-19. After 20 weeks post-vaccination, only one third of participants had positive T cell responses to SARS-CoV-2 antigens. Prior COVID-19 impacted IFN{gamma} responses, with reactivity enhanced in those infected earlier. The frequency of IFN{gamma} responses was highest to extended genome antigen set. Overall, BBIBP-CorV- induced antibody responses were impacted by age, gender and prior COVID-19. Cellular immunity was present in a limited number of individuals after 20 weeks but was enhanced by prior infection. This suggests the need for booster vaccinations in older individuals. BBIBP-CorV induced cellular activation is broader than to spike, requiring further study to understand how to monitor vaccine effectiveness.

Effect of age and prior COVID-19 on BBIBP-CorV (Sinopharm) vaccination- induced antibody responses in a region with high seroprevalence (preprint)

Introduction Sinopharm (BBIBP-CorV) inactivated virus vaccination for COVID-19 has been administered widely in Pakistan. We investigated the dynamics of BBIBP-CorV -induced antibody responses over a 24 week period in a region with a high seroprevalence.Methods Study subjects (n = 312) were followed up over a 24-week period between May and August 2021. Sera were tested for IgG antibodies to spike and the receptor binding domain (RBD).Results Study subjects were 62% female. Twenty-two percent had a prior history of COVID-19. At 4-, 8- 16- and 24-weeks post-vaccination, the rate of IgG antibodies positive to spike was 57%, 87%, 66% and 90% of individuals, compared with to RBD which was 48%, 62%, 68% and 85% of subjects, respectively,. IgG to spike and RBD showed a positive correlation at each interval (rho > 0.6, p < 0.0001). Seropositivity to both spike and RBD was reduced in those aged 50 years and over for up until 16 weeks post-vaccination (p < 0.05). Individuals with prior COVID-19 infection showed greater antibody responses for up to 16 weeks post-vaccination (p < 0.05). SARS-CoV-2 infections were observed with a mean interval of 16 weeks post-vaccination. Antibody responses did not wane for up to 6 months post-vaccination.Conclusions Sinopharm vaccination-induced antibody responses were negatively impacted by age and positively impacted by prior COVID-19 for 16 weeks after vaccination. Importantly, we did not find waning of IgG antibodies to RBD over the study period. Maintenance of antibodies may be the result of continued community exposure and boosting with COVID-19 vaccination.

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.

Detection of IgG and IgM Levels in Patients with COVID-19 in Mosul Province, Iraq

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has become the most dangerous viral infection worldwide. Since its identification in late 2019, the number of medical trials to combat the infection has sharply increased. Here, we investigated the profiles of IgG and IgM in 85 patients with confirmed SARS-CoV-2 infection from day 1 after symptom onset until day 35 with 5-day intervals. Serum samples were collected and stored until use. We observed that IgM levels were detectable on day 5 post symptom onset and increased sharply, with the highest rate detected in moderate cases (32.332 ± 4.32, n=10). Subsequently, a significant reduction in IgM was observed until it was undetectable on day 35 after symptom onset. Meanwhile, IgG levels were detected on day 10 post symptom onset, and the highest rate was observed in moderate cases (8.232 ± 2.3, n=10). A significant increase in IgG rate was observed in all patients, with the highest rate in moderate cases (42.432 ± 4.34, n=67) on day 35 post symptom onset. The statistical difference between the case and control groups was significant (p≤0.001). Two out of 85 patients died during the study. [ FROM AUTHOR] Copyright of Journal of Pure & Applied Microbiology is the property of Dr. M. N. Khan and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

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.

The mystery of COVID-19 reinfections: A global systematic review and meta-analysis of 577 cases (preprint)

Background As the COVID-19 pandemic rages on, reports on disparities in vaccine roll out alongside reinfection and reactivation from previously recovered cases have been emerging. With newer waves and variants of COVID-19, we conducted a systematic review to assess the determinants and disease spectrum of COVID-19 reinfection. Methods A comprehensive search covering relevant databases was conducted for observational studies reporting Polymerase Chain Reaction (PCR) confirmed infection and reinfection cases. Quality assessment tool developed by the National Institute of Health (NIH) for assessment of case series was used. Meta-analyses were performed using RevMan 5.3 for pooled proportions of findings in first infection and reinfection with 95% confidence interval (CI). Results Eighty-one studies reporting 577 cases were included from 22 countries. The mean age of patients was 46.2±18.9 years with males accounting for 45.8% of the study population while 179 (31.0%) cases of comorbidities were reported. The average time duration between first infection and reinfection was 63.6±48.9 days. During first infection and reinfection, fever was the most common symptom (41.4% and 36.4%, respectively) whilst anti-viral therapy was the most common treatment regimen administered (44.5% and 43.0%, respectively). Overall, comparable odds of symptomatic presentation and management were reported in the two infections. However, a higher Intensive Care Unit (ICU) admission rate was observed in reinfection compared to first infection (10 vs 3). Ten deaths were reported with 565 patients fully recovering. Respiratory failure was the most common cause of death (7/10 deaths). Seventy-two studies were determined to be of good quality whilst nine studies were of fair quality. Conclusion As the first global-scale systematic review of its kind, our findings support immunization practices given increased ICU admissions and mortality in reinfections. Our cohort serves as a guide for clinicians and authorities for devising an optimal strategy for controlling the pandemic.

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.

Role of awake prone positioning in patients with moderate-to-severe COVID-19: an experience from a developing country.

There is limited evidence on the efficacy of awake prone positioning (PP) in non-ventilated patients with COVID-19 who have hypoxemia. We, therefore, aim to describe our experience with the use of early proning in awake, non-intubated patients with confirmed COVID-19. In our retrospective observational study, 23 patients with confirmed positive PCR test results for Severe Acute respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and hypoxemia that required oxygen therapy with or without non-invasive ventilation were treated with PP. Patients were classified into mild, moderate and severe COVID-19 disease. There were no targeted number of hours for proning per day and patients were kept in prone position according to their tolerance. The primary outcome measure was the avoidance of intubation and secondary outcomes were in-hospital mortality, length of hospital stays and complications related to PP. The mean (standard deviation) age of our cohort was 54.5 (11.7) years, and the majority were males (21/23, 91.3%). Sixty-one per cent (14/23) of the patients were suffering from severe disease and 82.6% (19/23) had bilateral lung involvement with interstitial infiltrates. Majority of the patients were prone positioned for a median of 6 days (IQR 4 - 8). Only one patient required transfer to ICU for mechanical ventilation and subsequently died due to severe ARDS. All 22 patients showed progressive improvement in oxygen requirement and PF ratio, mostly after 3-5 days of proning. The mean length of hospital stay was 12 days. All patients, except one, were discharged in stable conditions, on room air or on a minimal oxygen requirement of 1-2 liters. No major complication of PP was recorded. Awake prone positioning is a valuable and safe therapeutic adjunct that can be applied in patients with moderate-to-severe COVID-19. It can also be included in the home-based management protocols of COVID-19 to improve patient outcomes and mitigate the burden on health care facilities.

Are Doctors Protected Enough During COVID-19? A Cross-Sectional Study of Physician Mortality Numbers and Characteristics in South Asia (preprint)

Background:The highly contagious nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) places physicians in South Asia at a high risk of contracting the infection. Accordingly, we conducted this review to provide an up to date account of physician deaths in South Asia during the COVID-19 pandemic and to analyze and compare the different characteristics associated with physician mortality amongst the countries of the region.Study Design:Cross-Sectional StudyMethodology:We performed a review study by using published news reports on the websites of news agencies from 9 selected countries in South Asia. Our study included only those physicians and doctors who died after contracting COVID-19 from their respective workplaces. All available data about the country of origin, type of, sex, age, medical or surgical specialty, and date of death were included.Results:The total number of physician deaths reported due to COVID-19 in our study was 170, with half (87/170, 51%) of the deaths reported from Iran. Male physicians' death was reported to be 145 (145/170=85%). Internal Medicine (58, 43%) was the most severely affected sub-specialty. The highest physician mortality rate in the general population recorded in Afghanistan (27/1000 deaths).ConclusionAn increased number of physician deaths, owing to COVID-19, is seen in South Asia. This could be due to decreased personal protective equipment and the poor health care management systems of the countries in the region to combat the pandemic. Future studies should provide a detailed account of characteristics associated with physician mortalities along with the main complications arising due to the virus.

Treatment of ARDS and hyperinflammation in COVID-19 with IL-6 antagonist Tocilizumab: a tertiary care experience from Pakistan (preprint)

Cytokine release syndrome in COVID-19 is characterized by hyperinflammation which manifests as ARDS, multi-organ failure, and high inflammatory parameters. Tocilizumab, an IL-6 antagonist has been used in COVID-19 acute respiratory distress syndrome (ARDS) with conflicting results from different parts of the world. We conducted a retrospective descriptive study from Feb 2020 to May 2020 on COVID-19 patients with ARDS and hyperinflammation characterized by raised CRP and/or ferritin. A total of 244 patients with COVID-19 were admitted out of which 107 had ARDS. Thirty patients had both ARDS and hyperinflammation and received tocilizumab. The mean age was 62.5 years (SD: 13.5) and the majority were male (83%). The mean CRP pre-treatment was 217.5 mg/L and post 48 to 72 hours of tocilizumab treatment was 98.5 mg/L. Twenty-one patients (70%) also received concomitant intravenous methylprednisolone. Of the 30 patients, 7 died and 20 recovered. Ten patients required intensive care unit admission and nine developed nosocomial infections. COVID-19 associated aspergillosis was diagnosed in three patients post tocilizumab treatment. Mortality was significantly higher in patients who developed a nosocomial infection and who required intermittent positive pressure ventilation (IPPV). Our study is the first to describe the treatment outcomes with tocilizumab from a low-middle income country. The availability and cost of tocilizumab in our region which makes it imperative to understand its potential for use in our setting. Our study supports the use of tocilizumab in a select patient population with COVID-19 and recommends monitoring of nosocomial infections and opportunistic infections.

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.