Efficacy and Safety of the RBD-Dimer-Based Covid-19 Vaccine ZF2001 in Adults.

BACKGROUND: The ZF2001 vaccine, which contains a dimeric form of the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 and aluminum hydroxide as an adjuvant, was shown to be safe, with an acceptable side-effect profile, and immunogenic in adults in phase 1 and 2 clinical trials. METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 3 trial to investigate the efficacy and confirm the safety of ZF2001. The trial was performed at 31 clinical centers across Uzbekistan, Indonesia, Pakistan, and Ecuador; an additional center in China was included in the safety analysis only. Adult participants (≥18 years of age) were randomly assigned in a 1:1 ratio to receive a total of three 25-µg doses (30 days apart) of ZF2001 or placebo. The primary end point was the occurrence of symptomatic coronavirus disease 2019 (Covid-19), as confirmed on polymerase-chain-reaction assay, at least 7 days after receipt of the third dose. A key secondary efficacy end point was the occurrence of severe-to-critical Covid-19 (including Covid-19-related death) at least 7 days after receipt of the third dose. RESULTS: Between December 12, 2020, and December 15, 2021, a total of 28,873 participants received at least one dose of ZF2001 or placebo and were included in the safety analysis; 25,193 participants who had completed the three-dose regimen, for whom there were approximately 6 months of follow-up data, were included in the updated primary efficacy analysis that was conducted at the second data cutoff date of December 15, 2021. In the updated analysis, primary end-point cases were reported in 158 of 12,625 participants in the ZF2001 group and in 580 of 12,568 participants in the placebo group, for a vaccine efficacy of 75.7% (95% confidence interval [CI], 71.0 to 79.8). Severe-to-critical Covid-19 occurred in 6 participants in the ZF2001 group and in 43 in the placebo group, for a vaccine efficacy of 87.6% (95% CI, 70.6 to 95.7); Covid-19-related death occurred in 2 and 12 participants, respectively, for a vaccine efficacy of 86.5% (95% CI, 38.9 to 98.5). The incidence of adverse events and serious adverse events was balanced in the two groups, and there were no vaccine-related deaths. Most adverse reactions (98.5%) were of grade 1 or 2. CONCLUSIONS: In a large cohort of adults, the ZF2001 vaccine was shown to be safe and effective against symptomatic and severe-to-critical Covid-19 for at least 6 months after full vaccination. (Funded by the National Science and Technology Major Project and others; ClinicalTrials.gov number, NCT04646590.).

Crimean-Congo Hemorrhagic Fever Virus Diversity and Reassortment, Pakistan, 2017-2020.

Sporadic cases and outbreaks of Crimean-Congo hemorrhagic fever (CCHF) have been documented across Pakistan since 1976; however, data regarding the diversity of CCHF virus (CCHFV) in Pakistan is sparse. We whole-genome sequenced 36 CCHFV samples collected from persons infected in Pakistan during 2017-2020. Most CCHF cases were from Rawalpindi (n = 10), followed by Peshawar (n = 7) and Islamabad (n = 4). Phylogenetic analysis revealed the Asia-1 genotype was dominant, but 4 reassorted strains were identified. Strains with reassorted medium gene segments clustered with Asia-2 (n = 2) and Africa-2 (n = 1) genotypes; small segment reassortments clustered with the Asia-2 genotype (n = 2). Reassorted viruses showed close identity with isolates from India, Iran, and Tajikistan, suggesting potential crossborder movement of CCHFV. Improved and continuous human, tick, and animal surveillance is needed to define the diversity of circulating CCHFV strains in Pakistan and prevent transmission.

Final efficacy analysis, interim safety analysis, and immunogenicity of a single dose of recombinant novel coronavirus vaccine (adenovirus type 5 vector) in adults 18 years and older: an international, multicentre, randomised, double-blinded, placebo-controlled phase 3 trial.

BACKGROUND: The Ad5-nCoV vaccine is a single-dose adenovirus type 5 (Ad5) vectored vaccine expressing the SARS-CoV-2 spike protein that was well-tolerated and immunogenic in phase 1 and 2 studies. In this study, we report results on the final efficacy and interim safety analyses of the phase 3 trial. METHODS: This double-blind, randomised, international, placebo-controlled, endpoint-case driven, phase 3, clinical trial enrolled adults aged 18 years older at study centres in Argentina, Chile, Mexico, Pakistan, and Russia. Participants were eligible for the study if they had no unstable or severe underlying medical or psychiatric conditions; had no history of a laboratory-confirmed SARS-CoV-2 infection; were not pregnant or breastfeeding; and had no previous receipt of an adenovirus-vectored, coronavirus, or SARS-CoV-2 vaccine. After informed consent was obtained, 25 mL of whole blood was withdrawn from all eligible participants who were randomised in a 1:1 ratio to receive a single intramuscular dose of 0·5 mL placebo or a 0·5 mL dose of 5 × 1010 viral particle (vp)/mL Ad5-nCoV vaccine; study staff and participants were blinded to treatment allocation. All participants were contacted weekly by email, telephone, or text message to self-report any symptoms of COVID-19 illness, and laboratory testing for SARS-CoV-2 was done for all participants with any symptoms. The primary efficacy objective evaluated Ad5-nCoV in preventing symptomatic, PCR-confirmed COVID-19 infection occurring at least 28 days after vaccination in all participants who were at least 28 days postvaccination on Jan 15, 2021. The primary safety objective evaluated the incidence of any serious adverse events or medically attended adverse events postvaccination in all participants who received a study injection. This trial is closed for enrolment and is registered with ClinicalTrials.gov (NCT04526990). FINDINGS: Study enrolment began on Sept 22, 2020, in Pakistan, Nov 6, 2020, in Mexico, Dec 2, 2020, in Russia and Chile, and Dec 17, 2020, in Argentina; 150 endpoint cases were reached on Jan 15, 2021, triggering the final primary efficacy analysis. One dose of Ad5-nCoV showed a 57·5% (95% CI 39·7-70·0, p=0·0026) efficacy against symptomatic, PCR-confirmed, COVID-19 infection at 28 days or more postvaccination (21 250 participants; 45 days median duration of follow-up [IQR 36-58]). In the primary safety analysis undertaken at the time of the efficacy analysis (36 717 participants), there was no significant difference in the incidence of serious adverse events (14 [0·1%] of 18 363 Ad5-nCoV recipients and 10 [0·1%] of 18 354 placebo recipients, p=0·54) or medically attended adverse events (442 [2·4%] of 18 363 Ad5-nCoV recipients and 411 [2·2%] of 18 354 placebo recipients, p=0·30) between the Ad5-nCoV or placebo groups, or any serious adverse events considered related to the study product (none in both Ad5-nCoV and placebo recipients). In the extended safety cohort, 1004 (63·5%) of 1582 of Ad5-nCoV recipients and 729 (46·4%) of 1572 placebo recipients reported a solicited systemic adverse event (p<0·0001), of which headache was the most common (699 [44%] of Ad5-nCoV recipients and 481 [30·6%] of placebo recipients; p<0·0001). 971 (61·3%) of 1584 Ad5-nCoV recipients and 314 (20·0%) of 1573 placebo recipients reported an injection-site adverse event (p<0·0001), of which pain at the injection site was the most frequent; reported by 939 (59%) Ad5-nCoV recipients and 303 (19%) placebo recipients. INTERPRETATION: One dose of Ad5-nCoV is efficacious and safe in healthy adults aged 18 years and older. FUNDING: CanSino Biologics and the Beijing Institute of Biotechnology.

First report of coinfection and whole-genome sequencing of norovirus and sapovirus in an acute gastroenteritis patient from Pakistan.

Acute gastroenteritis is one of the most common diseases in infants and children in developing countries including Pakistan. In Pakistan, rotavirus (RVA) is known to contribute significantly to pediatric diarrheal illness, but the contribution of other viruses is still unclear. In the current study we have identified a case of mixed infection of norovirus (NoV) and sapovirus (SaV) in a 2-year-old child with acute gastroenteritis. The sample was initially processed for the detection of group A RVA through ELISA followed by NoV using RT-PCR assay. The sample tested positive for NoV RNA and was later subjected to whole-genome sequencing using meta-genome approach on Miseq (Illumina) platform. Sequencing results revealed GII.15 genotype of NoV that clustered with viruses from China and USA from 2017 to 2021. We also retrieved the complete genome of SaV (GI.1 genotype) from the same sample and phylogenetic analysis showed clustering with strains reported from Japan, South Korea, US, and Taiwan during 2012-2016. This is the first report from Pakistan that confirms coinfection of NoV and SaV and elucidates their whole genomes. We recommend initiation of NoV and SaV surveillance program to ascertain disease burden and explore genetic diversity, especially as RVA vaccines have been included in national immunization program.

Whole-genome sequencing of Crimean-Congo hemorrhagic fever virus circulating in Pakistan during 2022.

Pakistan is an endemic country for Crimean-Congo hemorrhagic fever (CCHF) and its Balochistan province is considered a hotspot region for circulation of the virus whereas sporadic cases have been reported from other parts of the country. Our study aims to investigate the genomic diversity of the CCHF virus circulating in Punjab and Khyber Pakhtunkhwa provinces of Pakistan. Between April to September 2022, 46 samples from suspected CCHF patients were tested, with 6 (13%) showing positive RT-PCR results. Among the positive cases, all were male, aged 14-48 years among which 4 were butchers. Three CCHF patients succumbed to the disease. The complete S-M-L-gene fragments of 4 positive samples were sequenced. The S and L segments belonged to the Asia-1 clade and clustered with regional strains from Iran, India, and Afghanistan. One M segment sequence grouped into Africa-2 along with those reported from India during 2016-2019. We report the detection of a reassorted virus (NIH-PAK-CCHF-03|2022) having Asia-1-Africa-2-Asia-1 (S-M-L) segment pattern for the first time from Pakistan. Mutational analysis showed M segments harboring eight mutations (T55A, S80P, T110I, T185A, T189A, A212T, and N239I/T) in the mucin-like domain, five mutations (D250N, T333S, I375V, M401I, A433T), four mutations (N545D, Y657F, K688R, and I824V) in GP38-domain, and three mutations (T1418N, A1431V, and G1449S) in Gc-domain. These findings highlight the significance of whole-genome sequencing of indigenous strains for a better understanding of the CCHFV evolution in Pakistan.

Genomic characterization of the first mpox and varicella-zoster co-infection in Pakistan (2023) through next-generation sequencing.

The global mpox outbreak spanning 2022-2023 has affected numerous countries worldwide. In this study, we present the first report on the detection, whole-genome sequence, and coinfection of the mpox virus and varicella zoster virus (VZV) from Pakistan. During April-May 2023, samples from 20 suspected cases of mpox were tested at the National Institutes of Health, Islamabad among which 4 tested positive. All four cases had a travel history of Saudi Arabia. All the suspected samples were processed by using a Zymo research kit for DNA extraction, followed by qRT-PCR amplification by using a DaAn Gene detection kit for the mpox virus. Further, two of the positive samples with a low Ct value (<20) were subjected to whole-genome sequencing using a metagenomic approach on the iSeq (Illumina) platform. The sequencing results revealed Clade IIb and genotype A.2.1 of MPXV, which clustered with viruses from Slovenia and the UK in July and June 2022, respectively. Our analysis identified two novel nonsynonymous substitutions in mpox virus, namely V98I in OPG046 and P600S in OPG109. Furthermore, we successfully retrieved the complete genome of VZV from the same sample, belonging to Clade 5. This study represents the first positive case of MPXV in Pakistan and the coinfection of mpox and VZV by using a metagenome approach providing insights into their complete genomes. Our results highlight the importance of surveillance at the point of entries, strengthening lab capacities including next-generation sequencing, and using differential diagnosis for timely and accurate detection of mpox cases.

Nonpharmaceutical interventions reduce the incidence and mortality of COVID-19: A study based on the survey from the International COVID-19 Research Network (ICRN).

The recently emerged novel coronavirus, "severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)," caused a highly contagious disease called coronavirus disease 2019 (COVID-19). It has severely damaged the world's most developed countries and has turned into a major threat for low- and middle-income countries. Since its emergence in late 2019, medical interventions have been substantial, and most countries relied on public health measures collectively known as nonpharmaceutical interventions (NPIs). We aimed to centralize the accumulative knowledge of NPIs against COVID-19 for each country under one worldwide consortium. International COVID-19 Research Network collaborators developed a cross-sectional online survey to assess the implications of NPIs and sanitary supply on the incidence and mortality of COVID-19. The survey was conducted between January 1 and February 1, 2021, and participants from 92 countries/territories completed it. The association between NPIs, sanitation supplies, and incidence and mortality were examined by multivariate regression, with the log-transformed value of population as an offset value. The majority of countries/territories applied several preventive strategies, including social distancing (100.0%), quarantine (100.0%), isolation (98.9%), and school closure (97.8%). Individual-level preventive measures such as personal hygiene (100.0%) and wearing facial masks (94.6% at hospitals; 93.5% at mass transportation; 91.3% in mass gathering facilities) were also frequently applied. Quarantine at a designated place was negatively associated with incidence and mortality compared to home quarantine. Isolation at a designated place was also associated with reduced mortality compared to home isolation. Recommendations to use sanitizer for personal hygiene reduced incidence compared to the recommendation to use soap. Deprivation of masks was associated with increased incidence. Higher incidence and mortality were found in countries/territories with higher economic levels. Mask deprivation was pervasive regardless of economic level. NPIs against COVID-19 such as using sanitizer, quarantine, and isolation can decrease the incidence and mortality of COVID-19.

Lipid Mediators and Cytokines/Chemokines Display Differential Profiles in Severe versus Mild/Moderate COVID-19 Patients.

Host immune responses play a key role in COVID-19 pathogenesis. The underlying phenomena are orchestrated by signaling molecules such as cytokines/chemokines and lipid mediators. These immune molecules, including anti-SARS-CoV-2 antibodies, interact with immune cells and regulate host responses, contributing to inflammation that drives the disease. We investigated 48 plasma cytokines/chemokines, 21 lipid mediators, and anti-S protein (RBD) antibodies in COVID-19 patients (n = 56) and non-COVID-19 respiratory disease controls (n = 49), to identify immune-biomarker profiles. Cytokines/chemokines (IL-6, CXCL-10 (IP-10), HGF, MIG, MCP-1, and G-CSF) and lipid mediators (TxB2, 11-HETE, 9-HODE, 13-HODE, 5-HETE, 12-HETE, 15-HETE, 14S-HDHA, 17S-HDHA, and 5-oxo ETE) were significantly elevated in COVID-19 patients compared to controls. In patients exhibiting severe disease, pro-inflammatory cytokines/chemokines (IL-6, CXCL-10, and HGF) and anti-SARS-CoV-2 antibodies were significantly elevated. In contrast, lipid mediators involved in the reduction/resolution of inflammation, in particular, 5-HETE, 11-HETE, and 5-oxoETE, were significantly elevated in mild/moderate disease. Taken together, these immune-biomarker profiles provide insight into immune responses related to COVID-19 pathogenesis. Importantly, our findings suggest that elevation in plasma concentrations of IL-6, CXCL-10, HGF, and anti-SARS-CoV-2 antibodies can predict severe disease, whereas elevation in lipid mediators peaks early (compared to cytokines) and includes induction of mechanisms leading to reduction of inflammation, associated complications, and maintenance of homeostasis.

Modelling the impact of climate change on dengue outbreaks and future spatiotemporal shift in Pakistan.

Climate change has a significant impact on the intensity and spread of dengue outbreaks. The objective of this study is to assess the number of dengue transmission suitable days (DTSD) in Pakistan for the baseline (1976-2005) and future (2006-2035, 2041-2070, and 2071-2099) periods under Representative Concentration Pathway (RCP4.5 and RCP8.5) scenarios. Moreover, potential spatiotemporal shift and future hotspots of DTSD due to climate change were also identified. The analysis is based on fourteen CMIP5 models that have been downscaled and bias-corrected with quantile delta mapping technique, which addresses data stationarity constraints while preserving future climate signal. The results show a higher DTSD during the monsoon season in the baseline in the study area except for Sindh (SN) and South Punjab (SP). In future periods, there is a temporal shift (extension) towards pre- and post-monsoon. During the baseline period, the top ten hotspot cities with a higher frequency of DTSD are Karachi, Hyderabad, Sialkot, Jhelum, Lahore, Islamabad, Balakot, Peshawar, Kohat, and Faisalabad. However, as a result of climate change, there is an elevation-dependent shift in DTSD to high-altitude cities, e.g. in the 2020s, Kotli, Muzaffarabad, and Drosh; in the 2050s, Garhi Dopatta, Quetta, and Zhob; and in the 2080s, Chitral and Bunji. Karachi, Islamabad, and Balakot will remain highly vulnerable to dengue outbreaks for all the future periods of the twenty-first century. Our findings also indicate that DTSD would spread across Pakistan, particularly in areas where we have never seen dengue infections previously. The good news is that the DTSD in current hotspot cities is projected to decrease in the future due to climate change. There is also a temporal shift in the region during the post- and pre-monsoon season, which provides suitable breeding conditions for dengue mosquitos due to freshwater; therefore, local authorities need to take adaption and mitigation actions.

Point Prevalence Survey of Antimicrobial Use in Selected Tertiary Care Hospitals of Pakistan Using WHO Methodology: Results and Inferences.

Background and objectives: The inappropriate use of antibiotics in hospitals can potentially lead to the development and spread of antibiotic resistance, increased mortality, and high economic burden. The objective of the study was to assess current patterns of antibiotic use in leading hospitals of Pakistan. Moreover, the information collected can support in policy-making and hospital interventions aiming to improve antibiotic prescription and use. Methodology and materials: A point prevalence survey was carried out with data abstracted principally from patient medical records from 14 tertiary care hospitals. Data were collected through the standardized online tool KOBO application for smart phones and laptops. For data analysis, SPSS Software was used. The association of risk factors with antimicrobial use was calculated using inferential statistics. Results: Among the surveyed patients, the prevalence of antibiotic use was 75% on average in the selected hospitals. The most common classes of antibiotics prescribed were third-generation cephalosporin (38.5%). Furthermore, 59% of the patients were prescribed one while 32% of the patients were prescribed two antibiotics. Whereas the most common indication for antibiotic use was surgical prophylaxis (33%). There is no antimicrobial guideline or policy for 61.9% of antimicrobials in the respected hospitals. Conclusions: It was observed in the survey that there is an urgent need to review the excessive use of empiric antimicrobials and surgical prophylaxis. Programs should be initiated to address this issue, which includes developing antibiotic guidelines and formularies especially for empiric use as well as implementing antimicrobial stewardship activities.

Genomic diversity of SARS-CoV-2 in Pakistan during the fourth wave of pandemic.

The emergence of different variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in upsurges of coronavirus disease 2019 (COVID-19) cases around the globe. Pakistan faced the fourth wave of COVID-19 from July to August 2021 with 314,786 cases. To understand the genomic diversity of circulating SARS-CoV-2 strains during the fourth wave of the pandemic in Pakistan, this study was conducted. The samples from 140 COVID-19-positive patients were subjected to whole-genome sequencing using the iSeq Sequencer by Illumina. The results showed that 97% (n = 136) of isolates belonged to the delta variant while three isolates belonged to alpha and only one isolate belonged to the beta variant. Among delta variant cases, 20.5% (n = 28) isolates were showing B.1.617.2 while 23.5% (n = 25), 17.59% (n = 19), 14.81% (n = 16), and 13.89% (n = 15) of isolates were showing AY.108, AY.43 AY.127, and AY.125 lineages, respectively. Islamabad was found to be the most affected city with 65% (n = 89) of delta variant cases, followed by Karachi (17%, n = 23), and Rawalpindi (10%, n = 14). Apart from the characteristic spike mutations (T19R, L452R, T478K, P681R, and D950N) of the delta variant, the sublineages exhibited other spike mutations as E156del, G142D, T95I, A222V, G446V, K529N, N532S, Q613H, and V483A. The phylogenetic analysis revealed the introductions from Singapore, the United Kingdom, and Germany. This study highlights the circulation of delta variants (B.1.617.2 and sublineages) during the fourth wave of pandemic in Pakistan.

Genomic surveillance reveals the detection of SARS-CoV-2 delta, beta, and gamma VOCs during the third wave in Pakistan.

SARS-CoV-2 variants of concern (VOCs) have emerged worldwide and gained significant importance due to their high transmissibility and global spread, thus meriting close monitoring. In Pakistan, limited information is available on circulation of these variants as the alpha variant has been reported the main circulating lineage. The current study was designed to detect and explore the genomic diversity of SARS-CoV-2 lineages circulating during the third wave of the pandemic in the indigenous population. From May 01 to June 09, 2021, a total of 16 689 samples were tested using TaqPath™ COVID-19 kit for the presence of SARS-CoV-2. Overall, 2562 samples (15.4%) were COVID-19 positive. Out of these positive samples, 2124 (12.7%) did not show the spike gene amplification (spike gene target failure ([SGTF]), whereas 438 (2.6%) showed spike gene amplification (non-SGTF). A subset (n = 58/438) of non-SGTF samples were randomly selected for whole-genome sequencing. Among VOCs, 45% (n = 26/58) were delta, 46% (n = 27/58) were beta, and one was gamma variant. The delta variant cases were reported mainly from Islamabad (n = 15; 58%) followed by Rawalpindi and Azad Kashmir (n = 1; 4% each). Beta variant cases originated mainly from Karachi (n = 8; 30%) and Islamabad (n = 11; 41%) and the gamma variant case was reported in a traveler from Italy. The delta, beta, and gamma variants possessed lineage-specific spike mutations. Notably, two rare mutations (E484Q and L5F) were found in the delta variant. Furthermore, in the beta variant, two significant rare non-synonymous spike mutations (A879S and K444R) were also reported. High prevalence of beta and delta variants in local population may increase the number of cases in the near future and provides an early warning to national health authorities to take timely decisions and devise suitable interventions to contain a possible fourth wave.

Molecular identification and characterization of measles virus genotypes circulating in Afghanistan, 2008-2018.

The measles virus (MV) remains a leading cause of morbidity and mortality in children under 5 years of age. Molecular identification of circulating wild-type MV) strains is a vital component of the measles elimination program. We received 159 oral swab samples from Afghanistan during 2008-2018. Viral RNA was extracted, followed by one-step RT-PCR and positive amplicons were subject to sequencing for genotype identification. Out of 159 total samples, 52% (83/159) were detected positive by RT-PCR. Genotype D4 was identified from 2.4% (2/83), genotype H1, 4.8% (4/83), and genotype B3, 92.7% (77/83) cases, respectively.

The global case fatality rate of coronavirus disease 2019 by continents and national income: A meta-analysis.

The aim of this study is to provide a more accurate representation of COVID-19's case fatality rate (CFR) by performing meta-analyses by continents and income, and by comparing the result with pooled estimates. We used multiple worldwide data sources on COVID-19 for every country reporting COVID-19 cases. On the basis of data, we performed random and fixed meta-analyses for CFR of COVID-19 by continents and income according to each individual calendar date. CFR was estimated based on the different geographical regions and levels of income using three models: pooled estimates, fixed- and random-model. In Asia, all three types of CFR initially remained approximately between 2.0% and 3.0%. In the case of pooled estimates and the fixed model results, CFR increased to 4.0%, by then gradually decreasing, while in the case of random-model, CFR remained under 2.0%. Similarly, in Europe, initially, the two types of CFR peaked at 9.0% and 10.0%, respectively. The random-model results showed an increase near 5.0%. In high-income countries, pooled estimates and fixed-model showed gradually increasing trends with a final pooled estimates and random-model reached about 8.0% and 4.0%, respectively. In middle-income, the pooled estimates and fixed-model have gradually increased reaching up to 4.5%. in low-income countries, CFRs remained similar between 1.5% and 3.0%. Our study emphasizes that COVID-19 CFR is not a fixed or static value. Rather, it is a dynamic estimate that changes with time, population, socioeconomic factors, and the mitigatory efforts of individual countries.

Associations of the COVID-19 pandemic with the reported incidence of important endemic infectious disease agents and syndromes in Pakistan.

BACKGROUND: Persons in Pakistan have suffered from various infectious diseases over the years, each impacted by various factors including climate change, seasonality, geopolitics, and resource availability. The COVID-19 pandemic is another complicating factor, with changes in the reported incidence of endemic infectious diseases and related syndromes under surveillance. METHODS: We assessed the monthly incidence of eight important infectious diseases/syndromes: acute upper respiratory infection (AURI), viral hepatitis, malaria, pneumonia, diarrhea, typhoid fever, measles, and neonatal tetanus (NNT), before and after the onset of the COVID-19 pandemic. Administrative health data of monthly reported cases of these diseases/syndromes from all five provinces/regions of Pakistan for a 3-year interval (March 2018-February 2021) were analyzed using an interrupted time series approach. Reported monthly incidence for each infectious disease agent or syndrome and COVID-19 were subjected to time series visualization. Spearman's rank correlation coefficient between each infectious disease/syndrome and COVID-19 was calculated and median case numbers of each disease before and after the onset of the COVID-19 pandemic were compared using a Wilcoxon signed-rank test. Subsequently, a generalized linear negative binomial regression model was developed to determine the association between reported cases of each disease and COVID-19. RESULTS: In late February 2020, concurrent with the start of COVID-19, in all provinces, there were decreases in the reported incidence of the following diseases: AURI, pneumonia, hepatitis, diarrhea, typhoid, and measles. In contrast, the incidence of COVID was negatively associated with the reported incidence of NNT only in Punjab and Sindh, but not in Khyber Pakhtunkhwa (KPK), Balochistan, or Azad Jammu & Kashmir (AJK) & Gilgit Baltistan (GB). Similarly, COVID-19 was associated with a lowered incidence of malaria in Punjab, Sindh, and AJK & GB, but not in KPK and Balochistan. CONCLUSIONS: COVID-19 was associated with a decreased reported incidence of most infectious diseases/syndromes studied in most provinces of Pakistan. However, exceptions included NNT in KPK, Balochistan and AJK & GB, and malaria in KPK and Balochistan. This general trend was attributed to a combination of resource diversion, misdiagnosis, misclassification, misinformation, and seasonal patterns of each disease.

Health care associated Clostridioides difficile infection and colonization in patients admitted at tertiary care hospital Pakistan.

Objectives: To evaluate the epidemiology of clostridioides difficile infections and colonisation in a tertiary-care setting. METHODS: The cross-sectional study was conducted at the Combined Military Hospital, Rawalpindi, Pakistan, from June 1, 2017, to October 31, 2019, and comprised adult patients admitted in high-risk units of the hospital for any disease experiencing watery stools after 48 hours of hospital admission and passing more than 3 stools per day with no other recognised aetiology. Stool samples of the participants, diagnosed with antibiotic associated diarrhoea, were submitted for glutamate dehydrogenase antigen assay and clostridioides toxin A/B assay detected by enzyme-linked immunosorbent assay and clostridioides difficile toxin gene detection by polymerase chain reaction. Clostridium difficile-associated diarrhoea was diagnosed by a positive toxin assay or polymerase chain reaction. Data was analysed using SPSS25. RESULTS: Of the 715 subjects, 322(45%) were males and 393(55%) were females. The overall mean age was 56.64±8.57 years, and 488(68.3%) were aged <60 years, while 227(31.7%) were aged >60 years. The incidence of clostridioides difficile-associated diarrhoea was found in 10(1.4%) patients and was highest in oncology unit 3(4.3%). No positive case was detected from the high dependency unit and the surgical ward. All the10(1.4%) positive cases were on >2 antibiotics with a combination of oral vancomycin and intravenous metronidazole. Mortality rate was significantly higher in the positive cases compared to those with clostridioides difficile colonisation (p<0.05). CONCLUSIONS: The incidence of clostridioides difficile-associated diarrhoea was found to be low.

Epidemiology of Chikungunya virus isolates 2016-2018 in Pakistan.

The chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, which has infected millions of people in Africa, Asia, Americas, and Europe since it remerged in India and Indian Ocean regions in 2005-2006. The purpose of this study was to evaluate the genetic diversity and evolutionary changes in CHIKV from 2016 to 2018 in Pakistan. Blood specimens were collected and processed following the Centers for Disease Control and Prevention Trioplex Protocol. Sequencing and phylogenetic analysis of complete coding sequence of representative isolates from the CHIKV outbreak was carried out during December 2016 to July 2018, a total of 1549 samples were received, out of which 50% (n = 774) were found positive for CHIKV RNA. Mean age of chikungunya positive patients was 31.8 ± 15.7 years and most affected were between 21 and 40 years of age. The Pakistan CHIKV strains clustered with the Indian Ocean sublineage of East/Central/South African with cocirculation of some variants In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Key substitutions in the neutralizing epitopes site and a few changes indicative of adaptive to other insect cells were also detected in Pakistani strains. This study provides the emerging trend of CHIKV in the country for early identification of potential variants of high virulence and preventive measures for vector borne disease especially in the endemic areas.

A comprehensive review on clinical and mechanistic pathophysiological aspects of COVID-19 Malady: How far have we come?

Since its outbreak in 2019, the coronavirus disease (COVID-19) has become a pandemic, affecting more than 52 million people and causing more than 1 million mortalities globally till date. Current research reveals a wide array of disease manifestations and behaviors encompassing multiple organ systems in body and immense systemic inflammation, which have been summarized in this review. Data from a number of scientific reviews, research articles, case series, observational studies, and case reports were retrieved by utilizing online search engines such as Cochrane, PubMed, and Scopus from December 2019 to November 2020. The data for prevalence of signs and symptoms, underlying disease mechanisms and comorbidities were analyzed using SPSS version 25. This review will discuss a wide range of COVID-19 clinical presentations recorded till date, and the current understanding of both the underlying general as well as system specific pathophysiologic, and pathogenetic pathways. These include direct viral penetration into host cells through ACE2 receptors, induction of inflammosomes and immune response through viral proteins, and the initiation of system-wide inflammation and cytokine production. Moreover, peripheral organ damage and underlying comorbid diseases which can lead to short term and long term, reversible and irreversible damage to the body have also been studied. We concluded that underlying comorbidities and their pathological effects on the body contributed immensely and determine the resultant disease severity and mortality of the patients. Presently there is no drug approved for treatment of COVID-19, however multiple vaccines are now in use and research for more is underway.

Genomic-based characterization of Enterococcus spp.: an emerging pathogen isolated from human gut.

BACKGROUND: Enterococci are ubiquitous microorganisms having diverse ecological niches but most prominently in gastrointestinal tract of humans and animals. Production of enterocins makes them a good probiotic candidate. However, their role as probiotics has become ambiguous in the last few years because of the presence of virulence factors and antibiotic resistance genes. These virulence traits are known to be transferred genetically, which makes them opportunistic pathogens in the gastrointestinal tract leading to serious concerns about their being used as probiotics. In the present study, Enterococcusspp. isolated from the human gut were subjected to Whole-Genome Sequencing (WGS) to determine the presence of resistance and virulence genes. METHODS AND RESULTS: Four human origins Enterococcus spp. including Enterococcus faecalis, Enterococcus casseliflavus, and two Enterococcus gallinarum were isolated from human fecal samples and further cultured on blood agar. Sanger sequencing was done using Applied Biosystems 3730xl DNA Analyzer. These strains were further subjected to WGS using oxford nanopore technology MinION. Raw data were analyzed using the free online tool epi2me. The Comprehensive Antibiotic Resistance Database (CARD) and RAST (Rapid Annotation using Subsystem Technology) software were used to look for the presence of antibiotic resistance genes in these strains. Resistance determinants for clinically important antibiotics (vancomycin) and functional virulence factor genes were detected. G-view server was used for comparative genomics of all strains. CONCLUSION: The genomic sequencing of Enterococcus suggested that E. faecalis, E. casseliflavus, and E. gallinarum strains are opportunistic pathogens, having antibiotic resistance genes. All isolates had vancomycin resistance genes, which were expressed phenotypically. Genes related to bacteriocin resistance were also present in E. casseliflavus and E. gallinarum.

Silver nanoparticles biosynthesized from secondary metabolite producing marine actinobacteria and evaluation of their biomedical potential.

Biosynthesis of silver nanoparticles (AgNPs) from marine actinobacteria offers a promising avenue for exploring bacterial extracts as reducing and stabilizing agents. We report extracellular extracts of Rhodococcus rhodochrous (MOSEL-ME29) and Streptomyces sp. (MOSEL-ME28), identified by 16S rRNA gene sequencing for synthesis of AgNPs. Ultrafine silver nanoparticles were biosynthesized using the extracts of R. rhodochrous and Streptomyces sp. and their possible therapeutic applications were studied. The physicochemical properties of nanoparticles were established by HR-SEM/TEM, SAED, UV-Vis, EDS, XRD, and FTIR. UV-Vis spectra displayed characteristic absorption at 430 nm and 412 nm for AgNPs from Streptomyces sp. (S-AgNPs) and Rhodococcus sp. (R-AgNPs), respectively. HR-SEM/TEM, XRD, EDS analysis confirmed the spherical shape, crystalline nature, and elemental formation of silver. Crystallite or grain size was deduced as 5.52 nm for R-AgNPs and 35 nm for S-AgNPs. Zeta-potential indicated electrostatic negative charge for AgNPs, while FTIR revealed the presence of diverse functional groups. Disc diffusion assay indicated the broad-spectrum antibacterial potential of S-AgNPs with the maximum inhibition of B. subtilis while R-AgNPs revealed potency against P. aeruginosa at 10 µg/mL concentration. Biogenic AgNPs revealed antileishmanial activity and the IC50 was calculated as 164 µg/mL and 184 µg/mL for R-AgNPs and S-AgNPs respectively. Similarly, the R-AgNPs and S-AgNPs revealed anti-cancer potential against HepG2 and the IC50 was calculated as 49 µg/mL and 69 µg/mL for R-AgNPs and S-AgNPs, respectively. Moreover, the antioxidant activity showed significant results. MTT assay on RD cells, L20B cells, and Hep-2C indicated intensification in viability by reducing the concentration of R-AgNPs and S-AgNPs. The R-AgNPs and S-AgNPs inhibited sabin-like poliovirus (1TCID50 infection in RD cells). Furthermore, hemocompatibility at low concentrations has been confirmed. Hence, it is concluded that biogenic-AgNPs has the potential to be used in diverse biological applications and that the marine actinobacteria are an excellent resource for fabrication of AgNPs.