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Abstract Background Pakistan reported 1.57 million COVID-19 cases between 2020 and 2022, based on approximately 30.6 million SARS-CoV-2 RT-PCR (reverse-transcription polymerase chain reaction) tests conducted. This study utilized data from one of the largest in-country testing facilities, Aga Khan University Hospital (AKUH) in Karachi, Pakistan, to explore gender and age-related in RT-PCR testing patterns. Methods We conducted a retrospective review of SARS-CoV-2 RT-PCR test data extracted from AKUH clinical laboratory records between February 2020 and February 2022. Gender and age distributions were examined in the context of testing patterns across the period. Multivariate regression models assessed independent associations between COVID-19 positivity and key variables. Results We reviewed 470,249 RT-PCR tests, finding that most tests were in those aged 21–40 years (48.1%). Overall, COVID-19 test positivity was 20.6%. In all, 57.7% were performed for males, predominant amongst those tested across all age groups and waves. Females had significantly lower odds of testing positive for COVID-19 (OR: 0.9; 95% CI: 0.9-1.0). However, when adjusted for gender, age and pandemic phases, the positivity rates between males and females were the same. The odds of a positive result increased significantly with age; individuals aged > 80 years had 2.5 times higher odds of testing positive than those aged 0–10 years (aOR 2.5, 95% CI 2.3–2.7). Conclusions The analysis indicates a consistent male dominance in COVID-19 testing, with higher positivity rates in older age groups. Our study highlight the importance of examining demographic characteristics in disease associated data especially, representation of females amongst cohorts.

Abstract Background Linezolid-resistant Enterococcus faecium (LRE) is a global priority pathogen. Thirteen LRE were reported from clinical specimens between November 2021 and April 2023 at two laboratories in Karachi, Pakistan. We aimed to investigate the strain types and genes associated with linezolid resistance among these isolates. Whole genome sequencing (WGS) was performed and analyzed by multilocus sequence typing (MLST). The presence of linezolid resistance genes was identified using ResFinder v4.1.11 and the LRE-finder tool. Results Twelve isolates belonged to clonal complex 17 (CC17); ST80 (n = 10), ST612 (n = 1) and ST1380 (n = 1). Six isolates showed the presence of optrA gene and G2576T mutations in the 23S rRNA gene, while six showed poxtA and cfr(D) genes. One isolate showed the combination of optrA, cfr(D) and poxtA genes. Conclusion Our findings show the circulation of CC17 sequence types with a known outbreak potential and we identified molecular mechanisms of resistance that were not previously reported from Pakistan.

Introduction: Single-nucleotide variants (SNVs) in Mycobacterium tuberculosis (M. tuberculosis) genomes can predict multidrug resistance (MDR) but not all phenotype-genotype correlations can be explained. We investigated SNVs in efflux pumps (EPs) in the context of M. tuberculosis drug resistance. Methods: We analysed 2221 M. tuberculosis genomes from 1432 susceptible and 200 MDR, 172 pre-extensively drug resistant (XDR) and 417 XDR isolates. Analysis of 47 EP genes was conducted using MTB-VCF, an in-house bioinformatics pipeline. SNVs were categorized according to their SIFT/Polyphen scores. Resistance genotypes were also called using the TB-Profiler tool. Results: Genome comparisons between susceptible and drug resistant (DR) isolates identified 418 unique SNVs in EP of which; 53.5% were in MDR, 68.9% in pre-XDR and 61.3% in XDR isolates. Twenty EPs had unique SNVs with a high SIFT/PolyPhen score, comprising 38 unique SNVs. Sixteen SNVs across 12 EP genes were significantly associated with drug resistance and enriched in pre-XDR and XDR strains. These comprised 12 previously reported SNVs (in Rv0191, Rv0507, Rv0676, Rv1217, Rv1218, Rv1273, Rv1458, Rv1819, and Rv2688) and 4 novel SNVs (in Rv1877 and Rv2333). We investigated their presence in genomes of 52 MDR isolates with phenotype-genotype discrepancies to rifampicin (RIF), isoniazid (INH), or fluoroquinolones. SNVs associated with RIF and INH (Rv1217_1218, Rv1819, Rv0450, Rv1458, Rv3827, Rv0507, Rv0676, Rv1273, and Rv2333), and with fluoroquinolone (Rv2688) resistance were present in these discrepant strains. Conclusions: Considering SNVs in EPs as part of M. tuberculosis genome-based resistance interpretation may add value, especially in evaluation of XDR resistance in strains with phenotype-genotype discrepancies.

Background: COVID-19 epidemiology changed with the emergence of SARS-CoV-2 variants of concern (VOC). Pakistan administered mostly inactivated vaccines. We investigated the association between VOC and breakthrough infections in a mixed-vaccination-status population of Karachi. Methods: We investigated SARS-CoV-2 VOC tested in 392 respiratory specimens collected between May and December 2021. Data for age, sex, hospital admission, vaccinations, together with CT values of the diagnostic PCR test were analyzed. Results: The median age of COVID-19 cases tested was 40 (27−57) years and 43.4% were female. Delta variants were most common (56.4%) followed by Alpha (15.9%), Omicron (12.2%), Beta/Gamma (11.3%), and others (4.3%). Eighteen percent of cases were hospitalized whereby, predominant VOC were Beta/Gamma (40.8%), Alpha (35.2%) and Delta (22.5%). Overall, 55.4% of individuals were fully vaccinated, 7.4% were partially vaccinated and 37.2% were unvaccinated. Most (74.6%) inpatients were unvaccinated. Vaccines comprised inactivated (85.34%), single-shot vector (8.62%), two-shot vector (3.02%) and mRNA (3.02%) types. Omicron variants showed lower viral loads as compared to Alpha, Beta/Gamma, and Delta (p = 0.017). The risk of infection with Delta and Omicron variants was higher, 8 weeks after vaccination. The majority of those with breakthrough infections after receiving inactivated vaccines acquired COVID-19 within 4 months of vaccination. Conclusion: Our data highlights the shifting of VOC from Delta to Omicron during 2021 and that COVID-19 vaccinations reduced both hospitalizations and viral transmission. It informs on the increased risk of breakthrough infection within 8 weeks of vaccination, indicating the need for booster vaccinations.

Abstract 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.

Abstract 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.

Tuberculosis (TB) and sarcoidosis are both granulomatous diseases. Here, we compared the immunological microenvironments of granulomas from TB and sarcoidosis patients using in situ sequencing (ISS) transcriptomic analysis and multiplexed immunolabeling of tissue sections. TB lesions consisted of large necrotic and cellular granulomas, whereas “multifocal” granulomas with macrophages or epitheloid cell core and a T-cell rim were observed in sarcoidosis samples. The necrotic core in TB lesions was surrounded by macrophages and encircled by a dense T-cell layer. Within the T-cell layer, compact B-cell aggregates were observed in most TB samples. These B-cell clusters were vascularized and could contain defined B-/T-cell and macrophage-rich areas. The ISS of 40–60 immune transcripts revealed the enriched expression of transcripts involved in homing or migration to lymph nodes, which formed networks at single-cell distances in lymphoid areas of the TB lesions. Instead, myeloid-annotated regions were enriched in CD68, CD14, ITGAM, ITGAX, and CD4 mRNA. CXCL8 and IL1B mRNA were observed in granulocytic areas in which M. tuberculosis was also detected. In line with ISS data indicating tertiary lymphoid structures, immune labeling of TB sections expressed markers of high endothelial venules, follicular dendritic cells, follicular helper T cells, and lymph-node homing receptors on T cells. Neither ISS nor immunolabeling showed evidence of tertiary lymphoid aggregates in sarcoidosis samples. Together, our finding suggests that despite their heterogeneity, the formation of tertiary immune structures is a common feature in granulomas from TB patients.

Abstract 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.

Abstract Background and Aims COVID‐19 morbidity and mortality varied globally through the pandemic. We studied the relationship of SARS‐CoV‐2 variants of concern (VOC) with COVID‐19 severity and mortality among hospitalized patients in Pakistan. Methods A retrospective review of clinical, laboratory, and vaccination data of 197 COVID‐19 adult patients at the Aga Khan University Hospital, Karachi between April 2021, and February 2022 was performed. SARS‐CoV‐2 VOC identified in respiratory samples were analyzed. Univariate and multivariate analysis was conducted to identify factors associated with COVID‐19 outcomes. Results The median age of cases was 55 years and 51.8% were males. Twenty‐four percent of females were pregnant. Of COVID‐19 cases, 48.2% had nonsevere disease, while 52.8% had severe/critical disease. Hypertension (48%) and diabetes mellitus (41%) were common comorbids. SARS‐CoV‐2 VOC identified comprised; Omicron (55.3%), Beta (14.7%), Alpha (13.7%), Delta (12.7%), and Gamma (3.6%) variants. Most (59.7%) study subjects were unvaccinated. Of vaccines, 88% had received inactivated virus COVID‐19 vaccines. Increased risk of severe disease was associated with age ≥50 years (odds ratio [OR]: 5.73; 95% confidence interval [CI]: [2.45–13.7]), as well as with diabetes mellitus (OR: 4.24; 95% CI: [1.82–9.85]). Full vaccination (OR: 0.25; 95% CI: [0.11–0.58]) or infection with Omicron (OR: 0.42; 95% CI: [0.23–0.74]) was associated with reduced disease severity. The risk of mortality increased with age ≥50 years (OR: 5.07; 95% CI: [1.92–13.42]) and a history of myocardial infarction (OR: 5.11; 95% CI: [1.45–17.93]) whilst, infection with Omicron was found to reduce the risk (OR: 0.22; 95% CI: [0.10–0.53]). Conclusion Our study describes the relationship between the severity of COVID‐19, in‐hospital mortality in relation to SARS‐CoV‐2 variants, and the impact of COVID‐19 vaccination in Pakistan. Outcomes were more favorable in younger individuals, after vaccinations and with Omicron variant infections. Most cases received inactivated virus vaccines therefore these data highlight the protection provided against severe COVID‐19.

Abstract Background Enteric fever is an acute systemic infectious disease associated with substantial morbidity and mortality in low- and middle-income countries (LMIC), with a global burden of 14.3 million cases. Cases of enteric fever or paratyphoid fever, caused by Salmonella enterica serovar Paratyphi A (S. Para A) have been found to rise in many endemic and non-endemic countries. Drug resistance is relatively uncommon in S. Para A. Here we report a case of paratyphoid fever caused by ceftriaxone resistant S. Para A from Pakistan. Case presentation A 29-year-old female presented with a history of fever, headache, and shivering. Her blood culture revealed a S. Para A isolate (S7), which was resistant to ceftriaxone, cefixime, ampicillin and ciprofloxacin. She was prescribed oral Azithromycin for 10 days, which resulted in resolution of her symptoms. Two other isolates of S. Para A (S1 and S4), resistant to fluoroquinolone were also selected for comparison. DST and whole genome sequencing was performed for all three isolates. Sequence analysis was performed for identification of drug resistance and phylogeny. Whole Genome Sequencing (WGS) of S7 revealed the presence of plasmids, IncX4 and IncFIB(K). blaCTX-M-15 and qnrS1 genes were found on IncFIB(K). The gyrA S83F mutation conferring fluoroquinolone resistance was also found present. Multi-locus sequence typing (MLST) showed the S7 isolate to belong to ST129. S1 and S4 had the gyrA S83Y and S83F mutations respectively. Conclusions We highlight the occurrence of plasmid-mediated ceftriaxone resistant strain of S. Para A. This is of significance as ceftriaxone is commonly used to treat paratyphoid fever and resistance in S. Para A is not known. Continuous epidemiological surveillance is required to monitor the transmission and spread of antimicrobial resistance (AMR) among Typhoidal Salmonellae. This will guide treatment options and preventive measures including the need for vaccination against S. Para A in the region.

Abstract Background and Aims COVID‐19 vaccinations have reduced morbidity and mortality from the disease. Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) 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.

Background: Whole-genome sequencing (WGS) data of Mycobacterium tuberculosis (MTB) complex strains have revealed insights about genetic variants associated with drug resistance (DR). Rapid genome-based diagnostics are being sought for specific and sensitive identification of DR; however, correct prediction of resistance genotypes requires both informatics tools and understanding of available evidence. We analyzed WGS datasets from phenotypically susceptible MTB strains using MTB resistance identification software. Methods: WGS data for 1526 MTB isolates classified as phenotypically drug susceptible were downloaded from the ReSeqTB database. The TB-Profiler software was used to call Single Nucleotide Variants (SNV) associated with resistance to rifampicin (RIF), isoniazid (INH), ethambutol (EMB), pyrazinamide, fluoroquinolone (FLQ), streptomycin (STR), and aminoglycosides. The SNV were further matched against the 2021 World Health Organization (WHO) catalogue of resistance mutations. Results: Genome analysis of 1526 MTB strains susceptible to first-line drugs revealed 39 SNV associated with DR to be present in across 14 genes in 5.9% (n = 90) isolates. Further interpretation of SNV based on the WHO catalog of mutations revealed resistance that 21 (1.4%) MTB isolates were resistant to first-line (4 to RIF, 14 to INH, 3 to EMB) drugs. While, 36 (2.6%) isolates were resistant to second-line (19 to STR, 14 to FLQ, and three to capreomycin) agents. The most frequent predictive SNV were; rpoB Ser450 Leu for RIF; katG Ser315Thr, inhA Ser94Ala, fabG1-15C >T (for INH); gyrA Asp94Gly for FLQ; embB Met306 Leu for EMB; rpsL Lys43Arg for STR; and tlyA Asn236 Lys for Capreomycin. Conclusions: Our study highlights the value of WGS-based sequence data for identifying resistance in MTB. It also shows how MTB strains may be misclassified simply on phenotypic drug susceptibility testing, and that correct genome interpretation is key for correct interpretation of resistance genotypes that can be used to guide clinical treatment.

Objective During the COVID-19 pandemic, several vaccines that were efficacious in randomised controlled trials were authorised 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 (HCP), effectiveness against SARS-CoV-2 infections is essential to reduce the risk to vulnerable patients and ensure a stable healthcare workforce. There are limited studies examining inactivated vaccines’ effectiveness against SARS-CoV-2 variants of concern (VOCs) in real-world settings. We estimated the effectiveness of inactivated vaccines (BBIBP-CorV and CoronaVac) against reverse transcription PCR (RT-PCR)-confirmed SARS-CoV-2 infections among HCP in the setting of emerging SARS-CoV-2 VOCs in Pakistan.Design A retrospective matched, test-negative case–control analysis using existing data from an Employee Health database on HCP at a large, private healthcare system in Pakistan.Participants 4599 HCP were tested between 1 April and 30 September 2021. Each case (PCR positive) was matched to two to six controls (PCR negative) by the date of the RT-PCR test (±7 days) to reduce bias.Primary and secondary outcome measures The primary outcome was vaccine effectiveness (VE) against SARS-CoV-2 infection. The secondary outcome was VE against symptomatic SARS-CoV-2 infection. Per cent VE was calculated using (1-OR)*100, with the OR of getting a PCR-confirmed SARS-COV-2 infection estimated using conditional logistic regression, after adjusting for age, gender, work area and history of SARS-CoV-2 infection.Results Inactivated vaccines were ineffective against SARS-CoV-2 infections after receiving the first dose (VE 17%, 95% CI −10, 39; p=0.261). They showed modest effectiveness ≥14 days after the second dose against SARS-CoV-2 infections (VE 30%, 95% CI 7, 48; p=0.015) and symptomatic SARS-CoV-2 infections (VE 33%, 95% CI 6, 52; p=0.002).Conclusions Inactivated vaccines show modest effectiveness against SARS-CoV-2 infections in the setting of emerging VOCs. This builds a strong case for boosters and/or additional vaccination.

Abstract Tuberculosis, caused by Mycobacterium tuberculosis, is a high-burden disease in Pakistan, with multi-drug (MDR) and extensive-drug (XDR) resistance, complicating infection control. Whole genome sequencing (WGS) of M. tuberculosis is being used to infer lineages (strain-types), drug resistance mutations, and transmission patterns—all informing infection control and clinical decision making. Here we analyse WGS data on 535 M. tuberculosis isolates sourced across Pakistan between years 2003 and 2020, to understand the circulating strain-types and mutations related to 12 anti-TB drugs, as well as identify transmission clusters. Most isolates belonged to lineage 3 (n = 397; 74.2%) strain-types, and were MDR (n = 328; 61.3%) and (pre-)XDR (n = 113; 21.1%). By inferring close genomic relatedness between isolates (

Abstract Background Mutations in the Rv0678, pepQ and atpE genes of Mycobacterium tuberculosis (MTB) have been reported to be associated with reduced antimycobacterial susceptibility to bedaquiline (BDQ). Resistance conferring mutations in treatment naïve MTB strains is likely to have implications for BDQ based new drug regimen that aim to shorten treatment duration. We therefore investigated the genetic basis of resistance to BDQ in MTB clinical isolates from BDQ naïve TB patients from Pakistan. In addition, mutations in genes associated with efflux pumps were investigated as an alternate mechanism of resistance. Methods Based on convenience sampling, we studied 48 MTB clinical isolates from BDQ naïve TB patients. These isolates (from our strain bank) included 38 MDR/pre-XDR/XDR (10 BDQ resistant, 8 BDQ intermediate and 20 BDQ susceptible) and 10 pan drug susceptible MTB isolates. All strains were subjected to whole genome sequencing and genomes were analysed to identify variants in Rv0678, pepQ , atpE, Rv1979c, mmpLS and mmpL5 and drug resistance associated efflux pump genes. Results Of the BDQ resistant and intermediate strains 44% (8/18) had variants in Rv0678 including; two reported mutations S63R/G, six previously unreported variants; L40F, R50Q and R107C and three frameshift mutations; G25fs, D64fs and D109fs. Variants in efflux pumps; Rv1273c (G462K), Rv0507c (R426H) and Rv1634c (E198R) were found to be present in drug resistant isolates including BDQ resistant and intermediate isolates. E198R in efflux pump gene Rv1634c was the most frequently occurring variant in BDQ resistant and intermediate isolates (n = 10). Conclusion We found RAVs in Rv0678 to be commonly associated with BDQ resistance. Further confirmation of the role of variants in efflux pump genes in resistance is required so that they may be incorporated in genome-based diagnostics for drug resistant MTB.

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

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.

Abstract Objectives This study aimed to explore mechanism of colistin resistance amongst Klebsiella pneumoniae isolates through plasmid mediated mcr-1 gene in Pakistan. Carbapenem and Colistin resistant K. pneumoniae isolates (n = 34) stored at − 80 °C as part of the Aga Khan University Clinical Laboratory strain bank were randomly selected and subjected to mcr-1 gene PCR. To investigate mechanisms of resistance, other than plasmid mediated mcr-1 gene, whole genome sequencing was performed on 8 clinical isolates, including 6 with colistin resistance (MIC > 4 μg/ml) and 2 with intermediate resistance to colistin (MIC > 2 μg/ml). Results RT-PCR conducted revealed absence of mcr-1 gene in all isolates tested. Whole genome sequencing results revealed modifications in Lipid A-Ara4N pathway. Modifications in Lipid A-Ara4N pathway were detected in ArnA_ DH/FT, UgdH, ArnC and ArnT genes. Mutation in ArnA_ DH/FT gene were detected in S3, S5, S6 and S7 isolates. UgdH gene modifications were found in all isolates except S3, mutations in ArnC were present in all except S1, S2 and S8 and ArnT were detected in all except S4 and S7. In the absence of known mutations linked with colistin resistance, lipid pathway modifications may possibly explain the phenotype resistance to colistin, but this needs further exploration.

Abstract 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.

Mycobacterium tuberculosis (MTB) is the causative agent of TB. TB incidence is high in many low resource settings where limited health systems make it difficult for screening of co-morbid conditions. Susceptibility to TB is increased with coincident diabetes mellitus (DM) or prediabetes. DM leads to chronic, subclinical inflammation in the host leading to compromised protective immunity against MTB, impacting TB treatment. This review focuses on the immunological impact of DM and prediabetes on TB infections, highlighting the importance of having effective diagnostic, treatment and management programs for early identification of hyperglycemia in TB patients to improve treatment outcomes. Further, it describes challenges in monitoring of TB and DM co-morbidity in a high-burden setting.

Abstract 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.

Summary: Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation.

Abstract Introduction Influenza viruses specifically, A and B mainly contribute to seasonal outbreaks that occur globally. However, due to limited diagnostics for influenza there is little data regarding clinical outcomes of patients with H1N1 pneumonia in our region. Our objective was to determine the clinical characteristics and outcomes of patients hospitalized with H1N1 pneumonia at a tertiary care facility in Karachi, Pakistan. Methods A retrospective study of adult patients admitted with influenza pneumonia from November 2017 to February 2018 at a tertiary care hospital in Karachi, Pakistan. Patient characteristics were compared between influenza A H1N1 and other types of influenza using multivariable logistic regression analysis and subgroup analysis for factors associated with mortality in H1N1 Pneumonia was performed. Results Out of 497 adult patients with community acquired pneumonia (CAP), 172 fulfilled the criteria for Influenza like illness (ILI). 88 patients had PCR confirmed Influenza pneumonia of whom n = 57 (65%) had Influenza A H1N1. The mean age of patients 53.5 years (SD: 17.3) and 60% were male. The overall mortality from Influenza in this study was 15.9% (n = 14); out of these 11 (78.5%) had Influenza A H1N1. Multivariable analysis showed that the increase in length of hospital admission was significantly associated with H1N1 Influenza A infection (OR: 1.47 CI: 1.2–1.8). Factors associated with mortality showed that presence of ARDS, Septic shock and multi-organ failure was highly significantly associated with death (p-value

Abstract Introduction There were 10 million new cases of tuberculosis (TB) in 2017. To eliminate TB, it is necessary to diagnose active TB and latent tuberculosis infection (LTBI). Diagnosis of paucibacillary disease and in extrapulmonary TB (EPTB) remains challenging; low mycobacterial load can be missed by microbiological or molecular based confirmation; EPTB, can be misdiagnosed due to absence of site specific specimens for testing. Interferon gamma release assays (IGRA) use T cell-based Interferon-gamma (IFN-γ) to identify infection with M. tuberculosis (MTB) but cannot discriminate between active and LTBI. We investigated how IGRA was being used in a high burden low resource setting. Methods We conducted a retrospective review of 149 consecutive cases received for QuantiFERON-TB Gold In-Tube Assay (QFT-GIT) testing in routine clinical service. Results Fifty-six cases were QFT-GIT positive and 93 were QFT-GIT negative. Thirty-six per cent of QFT-GIT tested cases had active TB. Of QFT-GIT positive cases, 59% patients had active TB; 10 with pulmonary and 23 with extra-pulmonary TB. The remaining 41% QFT-positive cases were LTBI. Of the QFT-GIT negative cases, 22% had active TB. Co-morbid conditions were present in 37% of QFT-GIT positive and 60% of QFT-GIT negative cases. Conclusions Our study shows that IGRA is being used as an adjunct test for active TB in this population. It highlights the complexity of interpreting QFT-GIT results particularly for QFT-GIT negative cases when ruling out MTB infection.

IntroductionAn 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 methodsWe 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.ResultsStudy 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.ConclusionsOur 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.

BackgroundIndividuals 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.MethodsThe 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.ResultsOut 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.ConclusionWe 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.

BackgroundWe 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 methodsWe 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.ResultsIn 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.ConclusionsThe 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.

One of the most prominent issues that occupy the world today with regard to the challenges and risks involved is research interest in the areas of disclosure of the cost of greenhouse gases. Its consequences extend to future generations in economic, environmental and social terms. With the increasing demand of stakeholders and their awareness of the importance of companies disclosing their financial reports on the strategic effects of their business on the climate, companies ought to find ways to disclose by responding to the requirements of global disclosure in international markets. However, what companies face in the Iraqi environment and with increasing greenhouse gas emissions (global warming) are challenges, most notably the weak disclosure in their annual reports and the absence of mandatory or voluntary instructions in the financial market as instructions No. (8) for the year (2011). The research aims to prepare a proposed index for the Iraqi financial market based on international treaties and protocols concerned with the disclosure of greenhouse gases through 31 paragraphs, knowing that it is possible to add any sections to these paragraphs and according to the activity of each company. The index was tested on a sample of the tourism companies listed on the Iraq Stock Exchange. The results show that there are initial initiatives to disclose greenhouse gases in the sample, even if relatively weakly.

Abstract Objective Children with tuberculosis (TB) remain underdiagnosed due to difficulty in testing for Mycobacterium tuberculosis (MTB) infection. We evaluated the Xpert MTB/RIF assay for respiratory and stool testing in children for pulmonary TB through a cross-sectional study at tertiary care facilities in Karachi, Pakistan. Fifty children aged 0–15 years screened by a modified Kenneth-Jones (KJ) score were included. Mycobacterial culture of respiratory samples was the microbiological standard against stool Xpert TB results. All positive TB cases were compared against a treatment response standard (TRS). Results Twelve study subjects were diagnosed by Xpert TB and nine by MTB culture. Compared with culture [gastric aspirates (GA)/sputum (spm)], stool Xpert TB had a sensitivity of 88.9% (95% CI 50.7–99.4) and a specificity of 95% (95% CI 81.8–99.1). Xpert TB stool versus GA/spm had sensitivity of 81.8% (95% CI 47.8–96.8) and specificity of 94.7% (95% CI 84.6–99.9). We found good agreement (kappa scores of >0.8) between stool Xpert, GA/spm Xpert and GA/spm culture. Stool Xpert PPV and NPV against TRS was 100 and 82.1% respectively. Stool Xpert TB is a relatively easy option for diagnosis for pulmonary childhood TB in a high burden low-resource setting.

Background and Objectives: Dengue virus infections (Dengue) have become increasingly common in Pakistan and can result in case fatalities if not managed appropriately. Patients with Dengue virus infection may be asymptomatic or present with Dengue fever (DF), Dengue with warning signs (DWS) or severe Dengue (SD). Severity in Dengue is coincident with an exacerbated production of lymphocyte-induced cytokines and chemokines which are associated with plasma leakage. We investigated the association of circulating levels of cytokines such as Interleukin (IL)-6, tumor necrosis factor (TNF)-alpha and CXCL-10 in Dengue patients with differing severity of disease. Materials and Methods: Dengue infection was confirmed by testing for human IgM to the Dengue virus. Dengue patients (n=58) and healthy controls (n=33) were recruited. Dengue patients were grouped into those with DF (n=39), DWS (n=15) and SD (n=4). Serum IL-6, TNFα and CXCL10 levels were tested by ELISA. The Mann Whitney U test was used for statistical analysis. Results: Circulating levels of TNFα (p≤0.001) and CXCL10 (p≤0.001) levels were increased in Dengue patients as compared with controls. When patients were stratified for disease severity, it was observed that CXCL10 was increased in DWS as compared to DF (p=0.046). IL-6 levels were increased in patients with SD as compared to those with DWS (p=0.044). TNFα levels were not found to differ between different groups of Dengue patients. Conclusion: Raised CXCL10 and TNFα levels were associated with increased clinical severity of Dengue infection and probably increased disease progression due to excessive inflammation and increased vascular changes in the patients.

ABSTRACT Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S. Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S. Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum β-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S. Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally. IMPORTANCE Typhoid fever is a severe disease caused by the Gram-negative bacterium Salmonella enterica serovar Typhi. Antibiotic-resistant S. Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR) S. Typhi clone in Sindh, Pakistan. The XDR S. Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in S. Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR S. Typhi to investigate the phylogenetic relationship between these isolates and a global collection of S. Typhi isolates and to identify multiple genes linked to antibiotic resistance. This S. Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in S. Typhi observed here adds urgency to the need for typhoid prevention measures.

Crimean-Congo hemorrhagic fever (CCHF) is endemic in Eurasian countries such as, Turkey, Pakistan, Afghanistan and Iran. CCHF virus is spread by the Hyalomma tick, which is found mainly on cattle and sheep. Muslim countries, in which these animals are sacrificed during Eid-Al-Adha, are among the countries where CCHF is endemic, and it has been observed that CCHF is associated with practices surrounding the Eid-ad-Adha festival. The dates for Eid-Al-Adha drift 10 days earlier in each year according to Georgian calendar. In previous years Eid-al-Adha occurred in autumn-winter months however in the next 10-15 years it will be take place in the summer months when CCHF is more prevalent. This may lead to a rise in the number of cases due to increased dissemination of CCHF virus with uncontrolled animal movements in and between countries. This consensus report focuses on the variable practices regarding animal handling in different regions and possible preventative measures to reduce the incidence of CCHF. Environmental hygiene and personal protection are essential parts of prevention. There is a need for international collaborative preparedness and response plans for prevention and management of CCHF during Eid-Al-Adha in countries where the disease is prevalent.

Background: Mycobacterium tuberculosis (MTB) PE_PGRS genes belong to the PE multigene family. Although the function of PE_PGRS genes is unknown, it is hypothesized that the PE_PGRS genes may be associated with antigenic variability in MTB. Material and methods: Whole genome sequencing analysis was performed on (n = 37) extensively drug-resistant (XDR) MTB strains from Pakistan, which included Lineage 1 (East African Indian, n = 2); Other lineage 1 (n = 3); Lineage 3 (Central Asian, n = 24); Other lineage 3 (n = 4); Lineage 4 (X3, n = 1) and T group (n = 3) MTB strains. Results: There were 107 SNPs identified from the analysis of 42 PE_PGRS genes; of these, 13 were non-synonymous SNPs (nsSNPs). The nsSNPs identified in PE_PGRS genes – 6, 9 and 10 – were common in all EAI, CAS, Other lineages (1 and 3), T1 and X3. Deletions (DELs) in PE_PGRS genes – 3 and 19 – were observed in 17 (80.9%) CAS1 and 6 (85.7%) in Other lineages (1 and 3) XDR MTB strains, while DELs in the PE_PGRS49 were observed in all CAS1, CAS, CAS2 and Other lineages (1 and 3) XDR MTB strains. All CAS, EAI and Other lineages (1 and 3) strains showed insertions (INS) in PE_PGRS6 gene, while INS in the PE_PGRS genes 19 and 33 were observed in 20 (95.2%) CAS1, all CAS, CAS2, EAI and Other lineages (1 and 3) XDR MTB strains. Conclusion: Genetic diversity in PE_PGRS genes contributes to antigenic variability and may result in increased immunogenicity of strains. This is the first study identifying variations in nsSNPs and INDELs in the PE_PGRS genes of XDR-TB strains from Pakistan. It highlights common genetic variations which may contribute to persistence.

Mycobacterium tuberculosis (MTB) Central Asian Strain (CAS) lineage strains are predominant in South Asia. Mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing is an effective way of determining genetic diversity of strains. A maximum of 24 loci-based MIRU-VNTR typing can be used, however, it is important to investigate the relevance of specific MIRU loci for regional strains for more cost-effective MIRU typing. MIRU-VNTR typing was performed on MTB strains from Pakistan. Strains were comprised of CAS (n = 113) and non-CAS lineages (n = 87) – both multi-drug resistant (MDR) and drug susceptible. Hunter Gaston Discriminatory Index (HGDI) for each MIRU loci was interpreted as poor, moderate or highly discriminatory. Results were analyzed using Bionumerics software and miru-vntrplus database link. Clustering analysis revealed 185 different MIRU types. Eight clusters of 2 strains each were present amongst MDR (3 clusters) and drug susceptible (5 clusters) isolates. MDR clusters had orphan and Haarlem strains, whereas drug susceptible strain clusters were comprised of CAS and Beijing lineage strains. The HGDI for 15 loci-based MIRU typing of all isolates was 0.620, whereas HGDI for CAS was lower than non-CAS lineage strains (p-value: 0.023). HGDI of 8 MIRU-VNTR loci (Qub 26b, 10, 26, 4156, Mtub 04, 16, 31 and ETR-A) were all highly discriminatory. The average HGDI based on these 8 loci was significantly lower for CAS than non-CAS strains (P value: 0.03). The lower discriminatory index for CAS using both 15 and 8 MIRU loci-based analysis suggests less genetic diversity in these isolates than in other lineages. The eight highly discriminatory MIRU loci for CAS may help in monitoring the transmission of MTB strains in regions with high CAS lineage prevalence.

Introduction In this study, we describe the results of controlled synthesis and application of gold coated Fe3O4/SiO2 nano-shells combined with the optical property of gold for enhancement of selective photothermal interaction with cancerous cells based on the surface plasmon resonance (SPR) Phenomena. Materials and Methods Magnetite Nano-Particles (MNPs) were prepared by means of co-precipitation. MNPs were modified with a thin layer of Silica using the Stober method. The amino-modified Fe3O4/SiO2 nano-shells were covered with gold colloids as a self-assembeled process. In-vitro assays were performed to determine the effect of apoptosis of the cells based on the cells morphological changes. Results The biologically inert nano-shells (85 nm) with a Magnetite/Silica core and a gold shell were optically activated. A successful laser-hyperthermia based on the thermal effect of surface plasmon resonance was performed using different gold concentrations. The thermal profile effects of laser power are presented as ideal cases of nanoshell-assisted photo-thermal therapy. The thermally-induced cell death has been shown to be dependent on NPs concentration and laser power density. The power densities of 157 and 184 W/cm2 caused complete cell death at the focal point of the laser beam.Cell damage was reduced by decreasing the power density of laser. Also, a larger area of damage on cell culture plates was observed at longer intervals of laser irradiation. Conclusion An optimized laser-(SPR) hyperthermia was obtained using a concentration of gold coated Fe3O4/SiO2 nano-shells concentration=0.1 mg/ml at intensity=157 W/cm2 at 60s.

Due to difficulties in direct diagnosis of Mycobacterium tuberculosis infection where site-specific specimens are not available, indirect methods of testing for infection are required. M. tuberculosis early secreted antigen target-6 (ESAT6) induced IFN-γ responses are specific, but do not differentiate between latent and active TB. The use of adjunct biomarkers for TB diagnosis has been proposed, such as the chemokines: CXCL9, CXCL10 and CCL2. ESAT6-induced IFN-γ CXCL9, CXCL10 and CCL2 was measured in whole blood cell supernatants of patients with pulmonary tuberculosis (PTB, n = 36) and extrapulmonary TB (ETB, n = 31) and compared with healthy endemic controls (EC, n = 33). ESAT6-induced IFN-γ responses were positive in 32% of TB cases as compared with 15% of EC cases (p = 0.048). ESAT6-induced CXCL9 responses were positive in 42% of TB cases and 15% of EC cases (p = 0.006). ESAT6-induced-CXCL10 and -CCL2 responses did not discriminate between TB and EC groups. Measurement of IFN-γ or CXCL9 together diagnosed TB (53%) cases and was significant as compared with EC (p = 0.014) cases. IFN-γ and CXCL10 together did not increase the number of TB cases diagnosed. Within TB groups, ESAT6-IFN-γ/CXCL9-based detection increased to 53% in PTB (p = 0.031) and 54% in ETB (p = 0.021), with comparable diagnosis in less severe extrapulmonary TB (L-ETB, 55%) and severe disseminated extrapulmonary TB (D-ETB, 50%). Given that 47% of TB cases remained undetected, this study shown that ESAT6-induced IFNγ and CXCL9 can support diagnosis, but must be supported by clinical correlation and other relevant investigations.

A number of HIV-1 subtypes are identified in Pakistan by characterization of partial viral gene sequences. Little is known whether new recombinants are generated and how they disseminate since whole genome sequences for these viruses have not been characterized. Near full-length genome (NFLG) sequences were obtained by amplifying two overlapping half genomes or next generation sequencing from 34 HIV-1-infected individuals in Pakistan. Phylogenetic tree analysis showed that the newly characterized sequences were 16 subtype As, one subtype C, and 17 A/G recombinants. Further analysis showed that all 16 subtype A1 sequences (47%), together with the vast majority of sequences from Pakistan from other studies, formed a tight subcluster (A1a) within the subtype A1 clade, suggesting that they were derived from a single introduction. More in-depth analysis of 17 A/G NFLG sequences showed that five shared similar recombination breakpoints as in CRF02 (15%) but were phylogenetically distinct from the prototype CRF02 by forming a tight subcluster (CRF02a) while 12 (38%) were new recombinants between CRF02a and A1a or a divergent A1b viruses. Unique recombination patterns among the majority of the newly characterized recombinants indicated ongoing recombination. Interestingly, recombination breakpoints in these CRF02/A1 recombinants were similar to those in prototype CRF02 viruses, indicating that recombination at these sites more likely generate variable recombinant viruses. The dominance and fast dissemination of new CRF02a/A1 recombinants over prototype CRF02 suggest that these recombinant have more adapted and may become major epidemic strains in Pakistan.

Vaccination with Bacille Calmette–Guérin (BCG) is given at birth to protect against tuberculosis (TB) in Pakistan. The country ranks 6th amongst high-burden countries worldwide and has an incidence of 231/100,000 pyopulation. This was a cross-sectional multi-center hospital-based study. TB patients (n = 218) with pulmonary (PTB, n = 120) or extrapulmonary (ETB, 98) were recruited, and the presence of a BCG vaccination scar was documented. Cases were further classified into minimal, moderate and advanced PTB or less severe (L-ETB) or severe disseminated (D-ETB) disease. The association of age, gender and severity of TB infections with BCG vaccination of the individual TB cases was investigated. No difference was found of the BCG vaccination status of PTB and ETB cases, or in relation to age or gender. Patients under 29 years of age comprised the largest group. There were more females with ETB than PTB. The largest group within ETB comprised those with tuberculous lymphadenitis (LNTB, 39%). A significantly greater number of LNTB cases had received BCG vaccinations than had those with pleural (unilateral) TB (p = 0.004), and tuberculous meningitis (p = 0.027) groups. Also, there were more immunized patients with pulmonary as compared with pleural disease (p = 0.001). LNTB represents localized granulomatous disease and the observation of higher vaccination rates in this group suggests that BCG has protected against more severe forms of TB in this high-burden region.

In the time period between the 9th -11th centuries (AH) local historiography of Guilan flourished at an overwhelming rate. The declining role of the central government and expanding role of the local rulers of that time are one of the main contributing factors attributed for this growth. To further study the local historiography of Guilan, it is imperative to evaluate three important works associated with this time period, specifically: Guilan & Deylamestan, Khani History, and Guilan History. When the political structures of these works are analyzed, it seems that the authors of ''Guilan and Deylamestan'' as well as '' Khani History'' were both influenced by the authority and persona of Kiai rulers. Thus, the political condition of that time was given a high priority in their work. Moreover the personal bias of historians such as Marashi and Lahiji to Kiai rulers is apparent in their books. As a result, a favorable view of Kiai rulers is given in their reports. Conversely, in ''Guilan History'', Foumani presents us with a different outlook regarding the political climate of that time and the fall of local rulers. Foumani presents us with a wider scope, which emphasizes social and sociological issues and paints a more complete picture of the local historiography of that era. As the main topic of this research, we try to determine what were the factors that caused Guilan's local historiographers to emphasize their political tendencies in their works. Based on this question, we think that due to political conditions as well as serving in the court of rulers, historiographers viewed their works in the liaison of those rulers.

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

Introduction: Extensively drug-resistant tuberculosis (XDR-TB) has emerged as one of the biggest threats to public health and TB control programs worldwide. XDR-TB is caused by Mycobacterium tuberculosis (MTB) strains resistant to rifampin and isoniazid, as well as to a fluoroquinolone and to at least one injectable aminoglycoside. Drug resistance in MTB has primarily been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, it has also been shown that efflux pumps may play a role in resistance of MTB. Upregulation of drug efflux pumps can decrease the intracellular concentration of drugs and reduce their efficacy. Methods: Whole genome sequencing was performed on 32 XDR-TB clinical isolates. Sequence data were used to investigate SNPs in efflux pump genes as compared with the H37Rv reference genome. Results: Of the XDR MTB strains, eight (21.62%) were wild type for rpsL, rrs (500 region), and gidB genes, but had non-synonymous (ns) SNPs (aspartic acid to histidine) in the drrA efflux pump gene at position 3273138. Three of eight (37.5%) XDR MTB strains, wild type for rpsL, rrs (500 region), gidB, and gyrB genes were phenotypically streptomycin sensitive and five (62.5%) XDR MTB strains were streptomycin resistant, while all XDR MTB strains, wild type for rpsL, rrs, gidB, and gyrB genes were resistant to fluoroquinolone (ofloxacin) and ethambutol. In addition, three XDR MTB strains wild type for rpsL, rrs, gidB, and drrA genes showed nsSNPs (isoleucine to valine) in the major facilitator superfamily, Rv1634 efflux pump gene at position 1839306. Conclusion: Our data show an nsSNP in the drrA efflux pump gene that may result in upregulation of drug efflux mechanisms in MTB strains. It is therefore imperative to understand the mechanism of efflux and its role in drug resistance, which will enable the identification of new drug targets and development of new drug regimens to counteract the drug efflux mechanism of MTB.

Objective/background: Childhood tuberculosis (TB) is largely a paucibacillary disease and difficult to diagnose. It is difficult to obtain a sputum or gastric aspirate (GA) sample, and patients are often undiagnosed and treated empirically. Stool is a noninvasive specimen not usually used for TB testing in Pakistan. We investigated the value of Xpert MTB/RIF to diagnose Mycobacterium tuberculosis (MTB) in children with pulmonary TB cases, by performing comparative testing of GA and stool samples. Method: We recruited 60 children aged 1–15 years, suspected of TB, from the Department of Pediatrics, Civil Hospital, Karachi, Pakistan and The Aga Khan University Hospital, Karachi, Pakistan. All were immunocompetent. Patients had a Kenneth Jones TB score of ≥5. Paired GA/sputum and stool samples were collected for testing. All GA samples were tested by Xpert MTB/RIF assay and MTB culture, while stool was tested by Xpert MTB/RIF. Results: The study participants included 27 males and 23 females with a mean age of 6 years and a mean TB (Kenneth Jones) score of 7. Stool was received in the laboratory within 1–2 days of the GA sample for all but one participant, who expired. The rates of MTB detection were as follows: 22% (11 cases) based on Xpert MTB testing of GA, 21% (10 cases) based on MTB culture of GA, and 21% (10 cases) based on Xpert MTB testing of stool. No rifampicin resistance was detected. Overall, there was concordance between testing of GA and stool. One case had GA with low positive Xpert and positive MTB culture, but negative stool Xpert result. In another case, there was low positive GA Xpert, positive GA MTB culture, and positive stool Xpert. A positive Xpert MTB stool test was associated with a higher TB score (>5) and a greater bacillary load. All 11 cases of TB diagnosed were put on antituberculous therapy and responded well to treatment. Conclusion: Use of Xpert MTB/RIF assay for stool-based diagnosis of pulmonary TB in immunocompetent children is useful in a resource poor setting. This is a valuable and noninvasive diagnostic alternative for the diagnosis of childhood TB and can be adapted by pediatric arms of national TB programs.

Frequency of extensively drug-resistant tuberculosis in Pakistan increased from 1.5% in 2006 to 4.5% in 2009 (p

It seems almost frivolous to say that I do "biomedical science research" in a country with a literacy rate below 30%, where the per capita income is less than $800 per annum, the cost of good healthcare is far beyond the reach of the common citizen, and government health systems are both insufficient and ineffective. Pakistan has the 6th highest global burden of tuberculosis. The immediate concern of health care professionals remains diagnosis and treatment of the disease. Therefore, although there is a real need to conduct basic research in pathogenesis of tuberculosis infections in order to study and develop new prevention, treatment and diagnostic modalities, there is limited public appreciation of "high-tech" research.

Objectives: The global increase in drug resistance in Mycobacterium tuberculosis (MTB) strains increases the focus on improved molecular diagnostics for MTB. Extensively drug-resistant (XDR) – TB is caused by MTB strains resistant to rifampicin, isoniazid, fluoroquinolone and aminoglycoside antibiotics. Resistance to anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs), in particular MTB genes. However, there is regional variation between MTB lineages and the SNPs associated with resistance. Therefore, there is a need to identify common resistance conferring SNPs so that effective molecular-based diagnostic tests for MTB can be developed. This study investigated used whole genome sequencing (WGS) to characterize 37 XDR MTB isolates from Pakistan and investigated SNPs related to drug resistance. Methods: XDR-TB strains were selected. DNA was extracted from MTB strains, and samples underwent WGS with 76-base-paired end fragment sizes using Illumina paired end HiSeq2000 technology. Raw sequence data were mapped uniquely to H37Rv reference genome. The mappings allowed SNPs and small indels to be called using SAMtools/BCFtools. Results: This study found that in all XDR strains, rifampicin resistance was attributable to SNPs in the rpoB RDR region. Isoniazid resistance-associated mutations were primarily related to katG codon 315 followed by inhA S94A. Fluoroquinolone resistance was attributable to gyrA 91–94 codons in most strains, while one did not have SNPs in either gyrA or gyrB. Aminoglycoside resistance was mostly associated with SNPs in rrs, except in 6 strains. Ethambutol resistant strains had embB codon 306 mutations, but many strains did not have this present. The SNPs were compared with those present in commercial assays such as LiPA Hain MDRTBsl, and the sensitivity of the assays for these strains was evaluated. Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and genotypic testing, the results would be rifampicin (100%), isoniazid (89%), fluoroquinolones (95%), aminoglycoside (81%) and ethambutol (61%). This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

Introduction: Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) strains that are multidrug resistant (MDR) and also resistant to a fluoroquinolone and to one injectable aminoglycoside or capreomycin. Whilst resistance in MTB has been associated with single nucleotide polymorphisms (SNPs), efflux pumps are thought to play a role in conferring resistance to MTB but little is known about them. Methods: We studied XDR MTB (n = 10) strains characterized by whole genome sequencing (WGS; http://www.ebi.ac.uk/ena/data/view/PRJEB7798). Phenotypic susceptibility testing was performed by the MGIT 960 (Becton, Dickinson and Co., NJ, USA) method. All XDR MTB strains were resistant to at least seven drugs whilst one XDR MTB strain, X54 was resistant to isoniazid, rifampicin, pyrazinamide, streptomycin, ethambutol, fluoroquinolones, capreomycin, kanamycin, amikacin, and ethionamide. The mRNA expression of efflux candidate genes Rv0194, Rv2688c, Rv1634, drrA, and drrB was determined in XDR MTB strains as compared with the ATCC reference strain, H37Rv, and drug-susceptible (DS) MTB (n = 9) strains using the relative quantification method normalized to 16S rRNA. Results: The mRNA expression levels of efflux genes Rv2688c (p = 0.0037), Rv1634 (p = 0.0042), drrA (p = 0.0078) and drrB (p = 0.0003) were upregulated in XDR-TB strains as compared with DS MTB strains. Conclusion: The differences between XDR-TB and drug-susceptible isolates suggest that the increased expression levels of MTB efflux pump genes may contribute to drug resistance in extensively drug-resistant tuberculosis. Future studies are needed to determine whether combining efflux pump inhibitors to antitubercular drugs would be effective to treat resistant tuberculosis.

Introduction: Pakistan ranks fifth in high tuberculosis (TB)-burden countries and seventh among countries with high prevalence rates of diabetes mellitus (DM). DM is a risk factor for TB and worsens disease outcomes. Furthermore, Mycobacterium tuberculosis (MTB) infection can induce glucose intolerance and worsen glycemic control in diabetes. Suppressor of cytokine signaling (SOCS)-1 and -3 molecules regulate cytokine signaling and are important in maintaining an immune balance. In TB, interleukin (IL)-6 upregulation induces SOCS3, which is also a negative regulator of insulin signaling. This research focuses on the mechanism by which SOCS1 and SOCS3 affect insulin resistance and increased susceptibility to TB. Methods: We studied gene expression in peripheral blood cells of patients with diabetes (n = 10) and healthy endemic controls (EC, n = 11) both with and without MTB infection. Mycobacterial antigen (PPD) and mitogen-stimulated SOCS1, SOCS3, interferon-gamma (IFN-γ), IL-6, and tumor necrosis factor alpha (TNFα) mRNA expression levels were determined using real-time polymerase chain reaction. Results: MTB antigen-stimulated mRNA levels of IFN-γ was 10-fold higher, SOCS1 was 4 times greater, TNFα was 10-fold higher, and IL-6 was 2-fold greater in patients with DM than in ECs. Overall levels of PPD-stimulated IL-6 was higher in patients with DM than in ECs (p = .036). Mitogen-induced mRNA levels of IFN-γ were 30-fold higher, SOCS3 was 20 fold higher, and SOCS1 was 4-fold higher in patients with DM than in ECs. Conclusion: Increased proinflammatory cytokine production in response to MTB antigens in diabetes would lead to exacerbated pathology and reduced inflammatory control at the site of MTB infection. This would in turn hamper the resolution of inflammation, resulting in unfavorable disease outcomes.

Objective/background: Pakistan ranks sixth among high-tuberculosis (TB)-burden countries worldwide and fifth worldwide for diabetes incidence. Although both these factors are independently known, the rate of diabetes among TB patients is not well known. We aimed to determine the prevalence of diabetes among patients with TB presenting at tertiary care health centers in Karachi, Pakistan. Methods: A total of 216 patients with TB were recruited and their blood samples were taken for testing of glycosylated hemoglobin (HbA1c) and random blood sugar. Diabetes was defined as HbA1c>6.5% and random blood sugar>180 mg/dL, pre-diabetes as HbA1c 5.7–6.4%, and normoglycemia as HbA1c