We investigated the presence of SARS-CoV-2 variants of concern (VoC) in Karachi, Pakistan between... more We investigated the presence of SARS-CoV-2 variants of concern (VoC) in Karachi, Pakistan between April and July 2021 in specimens received at the Aga Khan University Clinical Laboratories. VoC were identified using a PCR based approach targeting lineage specific mutation. Of the 710 clinical isolates tested, 63% were VoC comprising; 36% alpha, 37% beta, 7% gamma and 21% delta variants. Alpha variants remained the majority whilst, delta strains increased to 43% of cases in July. Thirty-six per cent of all cases were admitted COVID-19 in-patient samples. Of the in-patient cases, 41% were alpha, 28% were beta, 8% were gamma and 24% were delta variants. Overall, we report an increase of delta variants in Karachi over the past two months which is concordant with the currently observed exacerbation in COVID-19 morbidity and mortality.
BackgroundCOVID-19 waves caused by specific SARS-CoV-2 variants have occurred globally at differe... more BackgroundCOVID-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.MethodsWe studied 276,525 COVID-19 cases and 1,041 genomes sequenced from December 2021 to August 2022. Sequences were analyzed and visualized using phylogenetic trees.ResultsThe highest case numbers and deaths were recorded in Sindh and Punjab, the most populous provinces in Pakistan. Omicron variants comprised 95% of all genomes, with BA.2 (34.2%) and BA.5 (44.6%) 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...
Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, e... more Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.
Tuberculosis, caused by Mycobacterium tuberculosis, is a high-burden disease in Pakistan, with mu... more 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 (< 10-SNPs difference), there was evidence of M. tuberculosis transmission, with 55 clusters formed consisting of a total of 169 isolates. Three clusters consist of M. tuberculo...
Worldwide morbidity and mortality caused by infectious diseases is high, mandating high rates of ... more Worldwide morbidity and mortality caused by infectious diseases is high, mandating high rates of antibiotic use among humans and animals. Antibiotics of anthropogenic origin often contaminate the environment. The arising ecological pressure results in alteration of bacterial “biomes,” high resistance rates in environmental microorganisms, and increase in the gene pool which contributes to antibiotic resistance. A number of such antibiotic resistance genes are carried on mobile genetic elements that can easily be exchanged between bacteria. The ecological net effect is an expanding population of resistant organisms contributing to spread of antibiotic resistance in both the clinical and the nonclinical environments. In nonclinical environments, antibiotics upset the natural symbiotic balance between microorganism and macroorganism communities. In clinical environments, while therapeutic antibiotic adverse effects are easily observed, the, impact of sub-inhibitory concentrations of an...
ObjectivesPakistan has a high infectious disease burden with about 265,000 reported cases of COVI... more ObjectivesPakistan has a high infectious disease burden with about 265,000 reported cases of COVID-19. We investigated the genomic diversity of SARS-CoV-2 strains and present the first data on viruses circulating in the country.MethodsWe performed whole-genome sequencing and data analysis of SARS-CoV-2 eleven strains isolated in March and May.ResultsStrains from travelers clustered with those from China, Saudi Arabia, India, USA and Australia. Five of eight SARS-CoV-2 strains were GH clade with Spike glycoprotein D614G, Ns3 gene Q57H, and RNA dependent RNA polymerase (RdRp) P4715L mutations. Two were S (ORF8 L84S and N S202N) and three were L clade and one was an I clade strain. One GH and one L strain each displayed Orf1ab L3606F indicating further evolutionary transitions.ConclusionsThis data reveals SARS-CoV-2 strains of L, G, S and I have been circulating in Pakistan from March, at the start of the pandemic. It indicates viral diversity regarding infection in this populous regio...
Background Healthcare providers’ (HCPs) professionalism refers to their commitment and ability to... more Background Healthcare providers’ (HCPs) professionalism refers to their commitment and ability to respond to the health needs of the communities they serve and to act in the best interest of patients. Despite attention to increasing the number of HCPs in low- and middle-income countries (LMIC), the quality of professional education delivered to HCPs and their resulting professionalism has been neglected. The Global Action Plan on Antimicrobial Resistance (AMR) seeks to reduce inappropriate use of antibiotics by urging patients to access antibiotics only through qualified HCPs, on the premise that qualified HCPs will act as more responsible and competent gatekeepers of access to antibiotics than unqualified HCPs. Methods We investigate whether weaknesses in HCP professionalism result in boundaries between qualified HCPs and unqualified providers being blurred, and how these weaknesses impact inappropriate provision of antibiotics by HCPs in two LMIC with increasing AMR—Pakistan and C...
Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious trea... more Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious treatments, validated and standardized drug susceptibility testing (DST) is required to improve patient care. This multicountry, multilaboratory external quality assessment (EQA) study aimed to validate the sensitivity, specificity, and reproducibility of provisional bedaquiline MIC breakpoints and World Health Organization interim critical concentrations (CCs) for categorizing clinical Mycobacterium tuberculosis isolates as susceptible/resistant to the drug.
Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen in Pakistan and ... more Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen in Pakistan and is emerging in the community. This is one of the first reports of the prevalent genotypes of MRSA in both hospital and community settings in Pakistan. Isolates collected in 2006–2007 were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). PFGE identified nine pulsotypes, the majority of isolates belonging to pulsotypes A (n=70) and B (n=38), which were predominant among hospital-onset MRSA (HO-MRSA) and community-onset MRSA (CO-MRSA) isolates, respectively. Among the HO-MRSA isolates, variants of SCCmec type III were prevalent, whilst SCCmec type IV or variants were predominant in the CO-MRSA isolates. MLST identified two principal sequence types, ST8 and ST239. An association was observed between ST8, PFGE pulsotype B and SCCmec type IV in the CO-MRSA (ST8-MRSA-IV). Similarly, ST239, PFGE pulsotype A and SCCmec type III w...
Background To strengthen health systems, the shortage of physicians globally needs to be addresse... more Background To strengthen health systems, the shortage of physicians globally needs to be addressed. However, efforts to increase the numbers of physicians must be balanced with controls on medical education imparted and the professionalism of doctors licensed to practise medicine. Methods We conducted a multi-country comparison of mandatory regulations and voluntary guidelines to control standards for medical education, clinical training, licensing and re-licensing of doctors. We purposively selected seven case-study countries with differing health systems and income levels: Canada, China, India, Iran, Pakistan, UK and USA. Using an analytical framework to assess regulations at four sequential stages of the medical education to relicensing pathway, we extracted information from: systematically collected scientific and grey literature and online news articles, websites of regulatory bodies in study countries, and standardised input from researchers and medical professionals familiar ...
Objective We investigated the discrepancy between clinical and PCR-based diagnosis of COVID-19. W... more 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 ef...
The global epidemiology of dengue fever (DF) and dengue haemorrhagic fever (DHF) is fast changing... more The global epidemiology of dengue fever (DF) and dengue haemorrhagic fever (DHF) is fast changing. Dengue is the most rapidly spreading mosquito-borne viral disease in the world. In the last 50 years, incidence has increased 30-fold with increasing geographic expansion to new countries and, in the present decade, from urban to rural settings. An estimated 50 million dengue infections occur annually and 1 approximately 2.5 billion people live in dengue endemic countries . Furthermore in recent years the disease regularly manifests as DHF with increasing frequency of out-breaks.
BackgroundWe investigated the genome diversity of SARS-CoV-2 associated with the early COVID-19 p... more 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 transmitte...
We investigated the presence of SARS-CoV-2 variants of concern (VoC) in Karachi, Pakistan between... more We investigated the presence of SARS-CoV-2 variants of concern (VoC) in Karachi, Pakistan between April and July 2021 in specimens received at the Aga Khan University Clinical Laboratories. VoC were identified using a PCR based approach targeting lineage specific mutation. Of the 710 clinical isolates tested, 63% were VoC comprising; 36% alpha, 37% beta, 7% gamma and 21% delta variants. Alpha variants remained the majority whilst, delta strains increased to 43% of cases in July. Thirty-six per cent of all cases were admitted COVID-19 in-patient samples. Of the in-patient cases, 41% were alpha, 28% were beta, 8% were gamma and 24% were delta variants. Overall, we report an increase of delta variants in Karachi over the past two months which is concordant with the currently observed exacerbation in COVID-19 morbidity and mortality.
BackgroundCOVID-19 waves caused by specific SARS-CoV-2 variants have occurred globally at differe... more BackgroundCOVID-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.MethodsWe studied 276,525 COVID-19 cases and 1,041 genomes sequenced from December 2021 to August 2022. Sequences were analyzed and visualized using phylogenetic trees.ResultsThe highest case numbers and deaths were recorded in Sindh and Punjab, the most populous provinces in Pakistan. Omicron variants comprised 95% of all genomes, with BA.2 (34.2%) and BA.5 (44.6%) 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...
Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, e... more Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.
Tuberculosis, caused by Mycobacterium tuberculosis, is a high-burden disease in Pakistan, with mu... more 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 (< 10-SNPs difference), there was evidence of M. tuberculosis transmission, with 55 clusters formed consisting of a total of 169 isolates. Three clusters consist of M. tuberculo...
Worldwide morbidity and mortality caused by infectious diseases is high, mandating high rates of ... more Worldwide morbidity and mortality caused by infectious diseases is high, mandating high rates of antibiotic use among humans and animals. Antibiotics of anthropogenic origin often contaminate the environment. The arising ecological pressure results in alteration of bacterial “biomes,” high resistance rates in environmental microorganisms, and increase in the gene pool which contributes to antibiotic resistance. A number of such antibiotic resistance genes are carried on mobile genetic elements that can easily be exchanged between bacteria. The ecological net effect is an expanding population of resistant organisms contributing to spread of antibiotic resistance in both the clinical and the nonclinical environments. In nonclinical environments, antibiotics upset the natural symbiotic balance between microorganism and macroorganism communities. In clinical environments, while therapeutic antibiotic adverse effects are easily observed, the, impact of sub-inhibitory concentrations of an...
ObjectivesPakistan has a high infectious disease burden with about 265,000 reported cases of COVI... more ObjectivesPakistan has a high infectious disease burden with about 265,000 reported cases of COVID-19. We investigated the genomic diversity of SARS-CoV-2 strains and present the first data on viruses circulating in the country.MethodsWe performed whole-genome sequencing and data analysis of SARS-CoV-2 eleven strains isolated in March and May.ResultsStrains from travelers clustered with those from China, Saudi Arabia, India, USA and Australia. Five of eight SARS-CoV-2 strains were GH clade with Spike glycoprotein D614G, Ns3 gene Q57H, and RNA dependent RNA polymerase (RdRp) P4715L mutations. Two were S (ORF8 L84S and N S202N) and three were L clade and one was an I clade strain. One GH and one L strain each displayed Orf1ab L3606F indicating further evolutionary transitions.ConclusionsThis data reveals SARS-CoV-2 strains of L, G, S and I have been circulating in Pakistan from March, at the start of the pandemic. It indicates viral diversity regarding infection in this populous regio...
Background Healthcare providers’ (HCPs) professionalism refers to their commitment and ability to... more Background Healthcare providers’ (HCPs) professionalism refers to their commitment and ability to respond to the health needs of the communities they serve and to act in the best interest of patients. Despite attention to increasing the number of HCPs in low- and middle-income countries (LMIC), the quality of professional education delivered to HCPs and their resulting professionalism has been neglected. The Global Action Plan on Antimicrobial Resistance (AMR) seeks to reduce inappropriate use of antibiotics by urging patients to access antibiotics only through qualified HCPs, on the premise that qualified HCPs will act as more responsible and competent gatekeepers of access to antibiotics than unqualified HCPs. Methods We investigate whether weaknesses in HCP professionalism result in boundaries between qualified HCPs and unqualified providers being blurred, and how these weaknesses impact inappropriate provision of antibiotics by HCPs in two LMIC with increasing AMR—Pakistan and C...
Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious trea... more Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious treatments, validated and standardized drug susceptibility testing (DST) is required to improve patient care. This multicountry, multilaboratory external quality assessment (EQA) study aimed to validate the sensitivity, specificity, and reproducibility of provisional bedaquiline MIC breakpoints and World Health Organization interim critical concentrations (CCs) for categorizing clinical Mycobacterium tuberculosis isolates as susceptible/resistant to the drug.
Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen in Pakistan and ... more Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen in Pakistan and is emerging in the community. This is one of the first reports of the prevalent genotypes of MRSA in both hospital and community settings in Pakistan. Isolates collected in 2006–2007 were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). PFGE identified nine pulsotypes, the majority of isolates belonging to pulsotypes A (n=70) and B (n=38), which were predominant among hospital-onset MRSA (HO-MRSA) and community-onset MRSA (CO-MRSA) isolates, respectively. Among the HO-MRSA isolates, variants of SCCmec type III were prevalent, whilst SCCmec type IV or variants were predominant in the CO-MRSA isolates. MLST identified two principal sequence types, ST8 and ST239. An association was observed between ST8, PFGE pulsotype B and SCCmec type IV in the CO-MRSA (ST8-MRSA-IV). Similarly, ST239, PFGE pulsotype A and SCCmec type III w...
Background To strengthen health systems, the shortage of physicians globally needs to be addresse... more Background To strengthen health systems, the shortage of physicians globally needs to be addressed. However, efforts to increase the numbers of physicians must be balanced with controls on medical education imparted and the professionalism of doctors licensed to practise medicine. Methods We conducted a multi-country comparison of mandatory regulations and voluntary guidelines to control standards for medical education, clinical training, licensing and re-licensing of doctors. We purposively selected seven case-study countries with differing health systems and income levels: Canada, China, India, Iran, Pakistan, UK and USA. Using an analytical framework to assess regulations at four sequential stages of the medical education to relicensing pathway, we extracted information from: systematically collected scientific and grey literature and online news articles, websites of regulatory bodies in study countries, and standardised input from researchers and medical professionals familiar ...
Objective We investigated the discrepancy between clinical and PCR-based diagnosis of COVID-19. W... more 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 ef...
The global epidemiology of dengue fever (DF) and dengue haemorrhagic fever (DHF) is fast changing... more The global epidemiology of dengue fever (DF) and dengue haemorrhagic fever (DHF) is fast changing. Dengue is the most rapidly spreading mosquito-borne viral disease in the world. In the last 50 years, incidence has increased 30-fold with increasing geographic expansion to new countries and, in the present decade, from urban to rural settings. An estimated 50 million dengue infections occur annually and 1 approximately 2.5 billion people live in dengue endemic countries . Furthermore in recent years the disease regularly manifests as DHF with increasing frequency of out-breaks.
BackgroundWe investigated the genome diversity of SARS-CoV-2 associated with the early COVID-19 p... more 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 transmitte...
Uploads
Papers