Shift in SARS-CoV-2 variants of concern from Delta to Omicron was associated with reduced hospitalizations, increased risk of breakthrough infections but lesser disease severity.

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.

Exploring the relationship between SARS-CoV-2 variants, illness severity at presentation, in-hospital mortality and COVID-19 vaccination in a low middle-income country: A retrospective cross-sectional study.

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.

Substitution spectra of SARS-CoV-2 genome from Pakistan reveals insights into the evolution of variants across the pandemic.

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.

Sequential viral introductions and spread of BA.1 across Pakistan provinces during the Omicron wave.

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.

Tracking SARS-CoV-2 variants through pandemic waves using RT-PCR testing in low-resource settings.

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<0.0001). We observed VOC through the pandemic waves using spike mutation specific RT-PCR assays. We show the spike mutation specific RT-PCR assay is a rapid, low-cost and adaptable for the identification of VOC as an adjunct approach to NGS to effectively inform the public health response. Further, by associating the VOC with CT values of its diagnostic PCR we gain information regarding the viral load of samples and therefore the level of transmission and disease severity in the population.

Whole-genome sequencing reveals genotypic resistance in phenotypically susceptible Mycobacterium tuberculosis clinical isolates.

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.

Ceftriaxone resistant Salmonella enterica serovar Paratyphi A identified in a case of enteric fever: first case report from Pakistan.

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.

Bedaquiline resistant Mycobacterium tuberculosis clinical isolates with and without rv0678 mutations have similar growth patterns under varying BDQ drug pressure.

Resistance associated mutations have been reported to alter the growth of Mycobacterium tuberculosis (MTB) isolates under drug pressure. However, there is little information on the growth characteristics of bedaquiline (BDQ) resistant isolates in the presence of BDQ. To further understand the role of rv0678, we aimed to study whether the presence of rv0678 variants in BDQ resistant isolates alters the killing effect of BDQ. We, therefore, selected BDQ resistant clinical MTB isolates with (n = 6) and without (n = 3) variants in rv0678 gene. Using time kill assays, growth inhibition; taken as the relative change in log average colony forming unit (CFU)/ml at selected time points (24-96 h), was studied at Minimum Inhibitory Concentrations (MICs): 0x, 1x, 2.5x, 5x, 7.5x, 10x for these isolates. Growth inhibition was then analyzed using Kruskal Wallis and Kolmogorov Smirnov tests in PRISM vr.9. During the 24-96 h lag phase isolates with and without variants in rv0678 showed a similar growth inhibition pattern. No difference was noted in growth inhibition between BDQ resistant isolates and H37Rv. These findings suggest that role of alternate mechanisms in contributing to BDQ tolerance needs to be explored.

Evolutionary history and introduction of SARS-CoV-2 Alpha VOC/B.1.1.7 in Pakistan through international travelers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and their identification is important for the public health response to coronavirus disease 2019 (COVID-19). Genomic sequencing provides robust information but may not always be accessible, and therefore, mutation-based polymerase chain reaction (PCR) approaches can be used for rapid identification of known variants. International travelers arriving in Karachi between December 2020 and February 2021 were tested for SARS-CoV-2 by PCR. A subset of positive samples was tested for S-gene target failure (SGTF) on TaqPathTM COVID-19 (Thermo Fisher Scientific) and for mutations using the GSD NovaType SARS-CoV-2 (Eurofins Technologies) assays. Sequencing was conducted on the MinION platform (Oxford Nanopore Technologies). Bayesian phylogeographic inference was performed integrating the patients' travel history information. Of the thirty-five COVID-19 cases screened, thirteen had isolates with SGTF. The travelers transmitted infection to sixty-eight contact cases. The B.1.1.7 lineage was confirmed through sequencing and PCR. The phylogenetic analysis of sequence data available for six cases included four B.1.1.7 strains and one B.1.36 and B.1.1.212 lineage isolate. Phylogeographic modeling estimated at least three independent B.1.1.7 introductions into Karachi, Pakistan, originating from the UK. B.1.1.212 and B.1.36 were inferred to be introduced either from the UK or the travelers' layover location. We report the introduction of SARS-CoV-2 B.1.1.7 and other lineages in Pakistan by international travelers arriving via different flight routes. This highlights SARS-CoV-2 transmission through travel, importance of testing, and quarantine post-travel to prevent transmission of new strains, as well as recording detailed patients' metadata. Such results help inform policies on restricting travel from destinations where new highly transmissible variants have emerged.

Variants associated with Bedaquiline (BDQ) resistance identified in Rv0678 and efflux pump genes in Mycobacterium tuberculosis isolates from BDQ naïve TB patients in Pakistan.

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.

A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum.

BACKGROUND: Individuals 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. METHODS: The 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. RESULTS: Out 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. CONCLUSION: We 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.

Upregulated type I interferon responses in asymptomatic COVID-19 infection are associated with improved clinical outcome.

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.

Higher entropy observed in SARS-CoV-2 genomes from the first COVID-19 wave in Pakistan.

BACKGROUND: We 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 METHODS: We 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. RESULTS: In 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. CONCLUSIONS: The 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.

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

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.

Efflux pump as alternate mechanism for drug resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) remains an important global public health issue with an approximate prevalence of 10 million people with TB worldwide in 2015. Since antibiotic treatment is one of the foremost tools for TB control, knowledge of Mycobacterium tuberculosis (MTB) drug resistance is an important component for disease control. Although gene mutations in specific loci of the MTB genomes are reported as the primary basis for drug resistance, additional mechanisms conferring resistance to MTB are thought to exist. Efflux is a ubiquitous mechanism responsible for innate and acquired drug resistance in prokaryotic and eukaryotic cells. MTB presents a large number of putative drug efflux pumps compared to its genome size. Bioinformatics-based evidence has shown an association between drug efflux and innate or acquired resistance in MTB. This review describes the recent understanding of drug efflux in MTB.

Single nucleotide polymorphisms in efflux pumps genes in extensively drug resistant Mycobacterium tuberculosis isolates from Pakistan.

It is challenging to understand mechanisms of drug resistance in Mycobacterium tuberculosis (MTB) due to the large variability in resistance associated genes. Efflux pump genes contribute to drug resistance and thus add to this complexity. Efflux pump gene protein superfamilies have been characterized by genome analysis of drug resistant strains and through in vitro transcriptional studies. However, there is limited information regarding efflux pump genes in extensively drug resistant (XDR) tuberculosis (TB) isolates. Whole genome sequencing (WGS) based analysis of 37 extensively drug resistant (XDR) and five drug sensitive (DS) MTB clinical isolates was performed. Single nucleotide polymorphisms (SNPs) in efflux pump genes Rv0194, Rv1217, Rv1218, drrA, drrB, Rv1258, Rv1634, Rv2688, Rv1273, Rv1819, Rv1458, Rv1877 and Rv1250 were determined in the clinical isolates as compared with the H37Rv reference strain. Allele frequencies of SNPs identified in XDR strains were compared with DS strains. Gene expression of Rv0194, Rv2688, Rv1634, drrA and drrB was determined in XDR -TB isolates (n = 9), DS-TB strains (n = 4) and H37Rv. We identified SNPs in XDR-TB isolates which were either unique or present at very low frequencies in DS strains; Rv0194 G170V; Rv1217 L151R; Rv1258 P369T and G391R; Rv1273 S118G and I175T; Rv1877 I534T; Rv1250 V318X/A and S333A, and Rv2688 P156T. The expression of Rv2688 and drrB was found to be raised in XDR-TB as compared with DS-TB strains. We identified unique SNPs in efflux pump genes which may be associated with increased drug resistance in the isolates. Increased levels of Rv2688 and drrB efflux pump gene expression observed in XDR strains even in the absence of antibiotics suggests that these clinical isolates may be more refractory to treatment. Further studies are required to directly associate these mutations with increased resistance in MTB.

Evaluation of Xpert MTB/RIF testing for rapid diagnosis of childhood pulmonary tuberculosis in children by Xpert MTB/RIF testing of stool samples in a low resource setting.

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.

Flaviviruses as a Cause of Undifferentiated Fever in Sindh Province, Pakistan: A Preliminary Report.

Arboviral diseases are expanding worldwide, yet global surveillance is often limited due to diplomatic and cultural barriers between nations. With human encroachment into new habitats, mosquito-borne viruses are also invading new areas. The actual prevalence of expanding arboviruses is unknown in Pakistan due to inappropriate diagnosis and poor testing for arboviral diseases. The primary objective of this study was to document evidence of flavivirus infections as the cause of undifferentiated fever in Pakistan. Through a cooperative effort between the USA and Pakistan, patient exposure to dengue virus (DENV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was examined in Sindh Province for the first time in decades. Initial results from the 2015 arbovirus season consisting of a cross-sectional study of 467 patients in 5 sites, DENV NS1 antigen was identified in 63 of the screened subjects, WNV IgM antibodies in 16 patients, and JEV IgM antibodies in 32 patients. In addition, a number of practical findings were made including (1) in silico optimization of RT-PCR primers for flavivirus strains circulating in the Middle East, (2) shipping and storage of RT-PCR master mix and other reagents at ambient temperature, (3) Smart phone applications for the collection of data in areas with limited infrastructure, and (4) fast and reliable shipping for transport of reagents and specimens to and from the Middle East. Furthermore, this work is producing a group of highly trained local scientists and medical professionals disseminating modern scientific methods and more accurate diagnostic procedures to the community.

Increased expression of efflux pump genes in extensively drug-resistant isolates of Mycobacterium tuberculosis.

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.