Evolution of COVID-19 infection in Punjab; trends during five waves of infection in the province of Punjab.

BACKGROUND: Pakistan witnessed five waves of COVID-19 infections during the pandemic. Punjab, the largest province of Pakistan, remained the epicentre due to a high infection rate. Administrative data for five waves of the pandemic was analyzed to determine the rate of infections and the significance of pharmacological and non-pharmacological interventions on the severity and duration of infection. METHODOLOGY: COVID-19 data from March 2020 to May 2023 was obtained from the Provincial Public Health Reference Laboratory (PPHRL), Punjab AIDS Control Program, Lahore. The data included samples from index cases, contacts, and recovered patients. A total of 36,252,48 cases were screened for COVID-19, and 90,923 (2.50%) were detected positive by RT-PCR, accounting for 5.69% of the cases reported positive throughout the country. RESULTS: Among the positive cases, 50.86% (n = 46,244) cases were new cases (registered for the first time), 40.41% (n = 36751) were the contact cases traced from the newly identified cases and 8.62% (n = 7842) repeated cases. The positivity rates among index cases were reported to be 2.37%, 2.34%, 4.61%, 2.09%, and 1.19%, respectively, for the five respective COVID-19 pandemic waves. Distribution by gender indicated that 64% of males and 35% of females were infected during the pandemic. The age factor demonstrated the most susceptibility to infection in women aged 19-29 years, whereas most males between the ages of 29-39 had an infection. Susceptibility to COVID-19 infection was observed to be equally likely between males and females; however, clinical outcomes indicated that infections in males were more severe and often resulted in fatalities as compared to those in females. This trend was also reflected in the viral titer as measured by the Ct values, where 40% of males had Ct values < 25 (an indicator of high viral titers) compared to 30% of females with Ct values < 25. CONCLUSION: Overall, our data indicated that infection rates remained stable throughout the pandemic except for 3rd wave, which showed a higher incidence of infection rate of 4%. Additionally, data showed a positive impact of masking, social distancing, and immunization, as indicated by the shorter window of high infection rates.

Employing computational tools to design a multi-epitope vaccine targeting human immunodeficiency virus-1 (HIV-1).

BACKGROUND: Despite being in the 21st century, the world has still not been able to vanquish the global AIDS epidemic, and the only foreseeable solution seems to be a safe and effective vaccine. Unfortunately, vaccine trials so far have returned unfruitful results, possibly due to their inability to induce effective cellular, humoral and innate immune responses. The current study aims to tackle these limitations and propose the desired vaccine utilizing immunoinformatic approaches that have returned promising results in designing vaccines against various rapidly mutating organisms. For this, all polyprotein and protein sequences of HIV-1 were retrieved from the LANL (Los Alamos National Laboratory) database. The consensus sequence was generated after alignment and used to predict epitopes. Conserved, antigenic, non-allergenic, T-cell inducing, B-cell inducing, IFN-É£ inducing, non-human homologous epitopes were selected and combined to propose two vaccine constructs i.e., HIV-1a (without adjuvant) and HIV-1b (with adjuvant). RESULTS: HIV-1a and HIV-1b were subjected to antigenicity, allergenicity, structural quality analysis, immune simulations, and MD (molecular dynamics) simulations. Both proposed multi-epitope vaccines were found to be antigenic, non-allergenic, stable, and induce cellular, humoral, and innate immune responses. TLR-3 docking and in-silico cloning of both constructs were also performed. CONCLUSION: Our results indicate HIV-1b to be more promising than HIV-1a; experimental validations can confirm the efficacy and safety of both constructs and in-vivo efficacy in animal models.

Sexually transmitted infections and associated risk factors among the transgender population of Pakistan.

BACKGROUND: Transgender (TG) people are key drivers for sexually transmitted infections (STIs) all over the world. There is substantial evidence that STIs are associated with an increased likelihood of risky sexual behavior however little is known about the prevalence of STIs (HIV, HBV, HCV, and Syphilis) among HIV infected transgender population in Pakistan. METHODS: The current study investigated the seroprevalence of four STIs and associated socio-demographic risk factors among TGs of Punjab, Pakistan from July 2019 to June 2021. The samples were tested serologically and final confirmation was done through PCR for HIV, HBV, and HCV. RESULTS: A total of 1,562 transgenders cross-sectional descriptive records of the Punjab AIDS Control Program (PACP) were reviewed during the period from July 2019 to June 2021. The serological results evidenced that 533 (34.1%) had one pathogen, 309 (19.8%) had two or more (multiple) infections. The most predominant mono-infection among the transgender population was Syphilis 324 (20.7%) followed by HCV 114 (7.3%), HIV 69 (4.4%), and HBV 26 (1.7%). The highest proportions of Infections were found in TG residing in urban areas (68.6%) as compared to rural areas (31.4%). The seropositivity of all STIs was predominantly increased in Sex worker TGs i-e 55%, 46.5%, 38.5%, and 41.8% in HIV, HBV, HCV, and Syphilis respectively. Among 280 HIV-infected Transgender, 177 (63.2%) had Syphilis co-infections. While 87 (31%) and 47 (16.8%) HIV-infected individuals had HC and HBV co-infection respectively. CONCLUSION: Transgender is neglected population group in society. All STIs were predominantly common among sex worker transgenders, Illiterate educational groups, and TGs residing in urban areas. There is a need to spread awareness about STIs, preventive strategies, and facilitation to health care programs in this high-risk population group.

Diagnosis of Acute Dengue Virus Infection Using Enzyme-Linked Immunosorbent Assay and Real-Time PCR.

Dengue fever is a viral infection caused by the dengue virus and is a growing concern for public health worldwide, particularly in tropical and subtropical regions. This study aimed to assess the diagnostic accuracy of a commercially available NS1 ELISA kit for dengue fever in Pakistan using multiplex qRT-PCR as the gold standard. The study recruited 1236 suspected cases of dengue fever admitted to public sector hospitals in Lahore, Pakistan. Of the suspected cases, 610 (49.3%) were confirmed positive for DENV infection through qRT-PCR, with all four serotypes detected. DENV-2 was the most prevalent serotype, detected in 95.7% of cases. The NS1 ELISA kit detected 71.1% of the positive cases. However, the diagnostic accuracy of the NS1 ELISA kit was found to be only 64.89%. Of the 610 confirmed cases, 68% were male and 32% were female, with a median age of 30 years. Dengue fever was diagnosed in 91.8% of cases, while 8.2% were diagnosed with dengue hemorrhagic fever (DHF). DHF patients had a higher prevalence of abdominal pain, hemorrhagic manifestations, and thrombocytopenia. The cocirculation of all four DENV serotypes in Lahore is concerning and could lead to more severe forms of the disease, such as DHF or dengue shock syndrome, in the future. The study highlights the low diagnostic accuracy of commercially available NS1 ELISA kits and emphasizes the importance of using molecular methods to confirm acute dengue infections. Given the increasing prevalence of dengue fever in developing countries like Pakistan, more accurate and reliable diagnostic tools are needed for effective disease management and control.

DRUG RESISTANCE PATTERN AND MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM TUBERCULOSIS STRAINS IN PUNJAB, PAKISTAN.

Tuberculosis (TB) is a cause of death from a single infectious agent Mycobacterium tuberculosis (MTB), leading to approximately 2.5 million deaths annually worldwide. Information regarding prevalence and pattern of drug resistance among TB patients in Pakistan remains inadequate due to the country's limited resources. This study compared conventional diagnostic techniques with a PCRbased assay targeting IS6110 sequence. In addition, MTB drug resistant profiles against four first-line drugs (ethambutol, isoniazid, rifampin, and streptomycin) from new and retreatment cases of TB. From 101 sputum samples microscopic examination of Ziehl-Neelsen-stained smears and culturing on Lowenstein Jensen medium resulted in 96% and 100% positives, compared to 98% by PCR. Prevalence of MDR-MTB was 41.5% and 58.5% among new (n = 51) and retreatment (n = 50) cases, but 10% of the former group were sensitive to all four first-line anti-TB drugs. Thus, MDR-MTB is highly prevalent among TB patients in Punjab Province, Pakistan (where the study was conducted) and, although PCR amplification of MTB-specific IS6110 sequence was rapid, it lacked the sensitivity of the culture assay.

Unraveling the relationship between entrepreneurial networks and entrepreneurial ambidexterity: A mediation-moderation model.

Entrepreneurial networks play an important role in identifying and exploiting entrepreneurial opportunities and the growth of entrepreneurial ambidexterity. This exploratory research aimed to explore the role of entrepreneurial networks on entrepreneurial ambidexterity with the mediating effect of novelty ecosystem and the moderating role of entrepreneurial intensity. The data is collected from 347 SME owners and managers of manufacturing and service industries in Jiangsu province, China. The hypotheses are analyzed using the partial least squares structural equation modeling PLS-SEM method. The results indicate that entrepreneurial networks are significantly associated with entrepreneurial ambidexterity. Moreover, findings show that the novelty ecosystem positively influences the association between entrepreneurial networks and entrepreneurial ambidexterity. Furthermore, results show that entrepreneurial intensity significantly moderated the relationship between novelty ecosystem and entrepreneurial ambidexterity. Lastly, the discussion and implications are elaborated in this study.

Uncovering the impact of SARS-CoV2 spike protein variants on human receptors: A molecular dynamics docking and simulation approach.

BACKGROUND: The SARS-CoV-2 pandemic, caused by the novel coronavirus, has posed a significant global health threat since its emergence in late 2019. The World Health Organization declared the outbreak a pandemic on March 11, 2020, due to its rapid global spread and impact on public health. New variants have raised concerns about their potential impact on the transmission of the virus and the effectiveness of current diagnostic tools, treatments, and vaccines. This study aims to investigate the effect of new variants in Pakistani virus strains on human receptors, specifically ACE2 and NRP1. In-silico analysis provides a powerful tool to analyze the potential impact of new variants on protein structure, function, and interactions. OBJECTIVES: The SARS-CoV-2 virus is evolving quickly. After being exposed in Wuhan, SARS-CoV-2 underwent numerous mutations, leading to several variants' emergence. These variants stabilize the interaction of spike protein with human receptors ACE2 and NRP1. The study aims to check the molecular effect of these variants on human receptors using the in-silico approach. MATERIAL AND METHODS: We use in-silico mutational tools to analyze new variants in SARS-CoV-2 and to check the molecular interaction of spike protein with human receptors (ACE2 and NRP1). Genomic sequences of 41 SARS-CoV-2 strains were sequenced using Ion Torrent (NGS) and submitted to the GISAID database. Spike protein of SARS-CoV-2 sequence trimmed and translated into a protein sequence using ExPasy. We used multiple sequence alignments to check for variants in the spike protein of strains. We utilized mutation tools such as Mupro, SIFT, SNAP2, and Mutpred2.3D structures of SARS-CoV-2 spike proteins (wild and mutated) to analyze further the mutations, ACE2 and NRP1 modelled by the ITASSER protein modelling server. Interactions of spike proteins (wild and mutant) analyzed by MD Docking, Simulation, and MMGBSA RESULTS: Variants I210T, V213G, S371F, S373P, T478K, F486V, Y505H, and D796Y were identified in SARS-CoV-2 Pakistani strains' spike protein. Variant Y505H were found to affect protein function. MD Docking, MMGBSA and MD simulation revealed that these variants increased spike protein's binding affinity with human receptors (ACE2 and NRP1). MD simulation revealed that mutated spike protein stabilized earlier than wild when interacting with ACE2 after 40 ns and interaction with NRP1 stabilized after 30 ns for mutated spike protein compared to wild. CONCLUSION: These variants in Pakistani strains of SARS-CoV-2 are increasing the stability of spike protein with human receptors. These findings provide insight into how the SARS-CoV-2 virus evolves and adapts to human hosts. This information may help develop strategies to control the virus's spread and develop effective treatments and vaccines in the future.

Molecular evidence of toxoplasmosis in people living with HIV in Pakistan.

BACKGROUND: Toxoplasma gondii is a zoonotically important parasite infecting almost all vertebrates. The parasite can easily infect humans through the fecal-oral route, particularly immunocompromised individuals, and children. Due to lack of epidemiological data regarding the occurrence of Toxoplasma gondii in people living with HIV in Pakistan, the present study was designed to estimate the prevalence of Toxoplasma gondii in people living with HIV. METHODS: Briefly, 384 blood samples were randomly collected from people living with HIV registered under Punjab AIDS Control Program. DNA of all samples was extracted and the 18S rRNA gene of T. gondii was amplified through polymerase chain reaction (PCR) by using genus-specific primers. The amplicons were spread on 1.5% agar rose gel and visualized under gel documentation system. Then the amplicons were purified from the gel and subjected to sequence and phylogenetic analysis. RESULTS: Overall prevalence of T. gondii was observed as 23.96%. The prevalence of T. gondii was significantly (p <0.05) higher in females and in middle age group (26-40 years). Similarly, the prevalence was significantly (p <0.05) higher in people with poor socioeconomic status, symptomatic patients, drugs users and people using unfiltered water for drinking. Furthermore, bodyweight (46-70 kg) and dry seasons also significantly (p <0.05) affected the prevalence of T. gondii. CONCLUSIONS: Our findings illustrated a high infection rate of toxoplasmosis in people living with HIV which could be a significant cause of mortality. More studies need to be conducted for other opportunistic infections caused by parasites and other pathogens.

Influence of entrepreneurship support programs on nascent entrepreneurial intention among university students in China.

This study aimed to investigate the impact of entrepreneurial educational support (NEES), entrepreneurial activities support (NEAS), and entrepreneurial commercialization support (NECS) on the nascent entrepreneurial intention (NEI) by mediating roles of entrepreneurial self-efficacy (NESE), opportunity recognition (OR), and the moderating effect of meaning in life (MLI). Data were gathered using a survey questionnaire from the 868 management, engineering, technical, and vocational institute students of China. The NEI model was analyzed using the partial least squares structural equation modeling through Smart-PLS software. The findings of the study reveal that NEES, NEAS, and NECS have a positive effect on NEI. Meanwhile, results indicate that NESE and OR partially mediate the relationship between entrepreneurship support programs and nascent entrepreneurial intention. Furthermore, the relationship between NESE and the NEI was insignificantly influenced by MLI, and the relationship between OR and the NEI was significantly moderated by MLI. Lastly, implications and limitations are also discussed in this article.

Evaluating the Role of Wastewaters as Reservoirs of Antibiotic-Resistant ESKAPEE Bacteria Using Phenotypic and Molecular Methods.

Introduction: Wastewaters carrying thousands of human specimens from the community and representing the diversity of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) directly from the community mirror the extent of AR spread in the community and environment. This study aimed to investigate the occurrence and distribution of antibiotic-resistant ESKAPEE bacteria in the community versus clinical settings through monitoring nonclinical and clinical wastewaters. Methodology: Seven wastewater samples were collected from different environmental sources. Isolates were obtained on general and selective media, biochemically characterized and antimicrobial-susceptibility tests performed by disk diffusion against 13 antibiotics according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines using MastDisc disk cartridges, and 16S rRNA metagenomic analysis was performed for two water samples. Results: Of 43 isolates, all representatives of the ESKAPEE group were recovered from clinical wastewaters, but Gram-positive cocci were not obtained from nonclinical wastewaters. The most predominant isolate was Pseudomonas aeruginosa (n=15; 33%), followed by Escherichia coli (n=9; 20%). Complete (100%) resistance to eleven of the tested antibiotics was observed, with only a few isolates being susceptible to clarithromycin, amikacin, and gentamicin. The lowest (79%) resistance rate was observed for linezolid. The multiple antibiotic resistance (MAR) index was calculated, and the resistance phenotype was independent of the wastewater source, indicated by x 2 (P=0.766). Metagenomic analysis replicated the results, as Pseudomonas spp., Acinetobacter spp., and Escherichia spp. were found to be predominant. The integrase gene (IntI1) was also amplified in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Conclusion: Wastewaters are significant carriers of drug-resistant ESKAPEE bacteria and play an important role in their dissemination. This study endorses the periodic surveillance of water systems to evaluate the presence and burden of antibiotic-resistant pathogens.

Dengue and Chikungunya virus co-infection in major metropolitan cities of provinces of Punjab and Khyber Pakhtunkhwa: A multi-center study.

Dengue has become endemic in Pakistan with annual recurrence. A sudden increase in the dengue cases was reported from Rawalpindi in 2016, while an outbreak occurred for the first time in Peshawar in 2017. Therefore, a multi-center study was carried out to determine the circulating dengue virus (DENV) serotypes and Chikungunya virus (CHIKV) co-infection in Lahore, Rawalpindi, and Peshawar cities in 2016-18. A hospital-based cross-sectional study was carried out in Lahore and Rawalpindi in 2016-18, while a community-based study was carried out in Peshawar in 2017. The study participants were tested for dengue NS1 antigen using an immunochromatographic device while anti-dengue IgM/IgG antibodies were detected by indirect ELISA. All NS1 positive samples were used for DENV serotyping using multiplex real-time PCR assay. Additionally, dengue samples were tested for CHIKV co-infection using IgM/IgG ELISA. A total of 6291 samples were collected among which 8.11% were NS1 positive while 2.5% were PCR positive. DENV-2 was the most common serotype (75.5%) detected, followed by DENV-1 in 16.1%, DENV-3 in 3.9% and DENV-4 in 0.7% while DENV-1 and DENV-4 concurrent infections were detected in 3.9% samples. DENV-1 was the predominant serotype (62.5%) detected from Lahore and Rawalpindi, while DENV-2 was the only serotype detected from Peshawar. Comorbidities resulted in a significant increase (p-value<0.001) in the duration of hospital stay of the patients. Type 2 diabetes mellitus substantially (p-value = 0.004) contributed to the severity of the disease. Among a total of 590 dengue positive samples, 11.8% were also positive for CHIKV co-infection. Co-circulation of multiple DENV serotypes and CHIKV infection in Pakistan is a worrisome situation demanding the urgent attention of the public health experts to strengthen vector surveillance.

Genetic diversity of multidrug-resistant Mycobacterium tuberculosis isolates in Punjab, Pakistan.

Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries and 6th among the countries with the highest prevalence of drug-resistant TB. However, insufficient data are available on the genetic structure of M. tuberculosis strains circulating in this country. The objective of this study was to explore the genetic diversity of multidrug-resistant M. tuberculosis isolates from Punjab, Pakistan with a combination of spoligotyping and 24-loci MIRU-VNTR typing. Among a total of 127 MDR isolates studies, 53 spoligotypes were obtained, split into 14 clusters (n = 88, 69.3%, 2-29 isolates per cluster) and 39 (30.7%) unique patterns. At the phylogenetic level, the most prevalent sublineage was CAS1_DELHI (n = 53, 41.7%), mostly represented by ST 1942 (n = 29, 22.8%), followed by T1 (n = 14, 11%) and Beijing (n = 10, 7.8%). The remaining nine sublineages (CAS, MANU2, EAI5, T2, LAM10_CAM, H1, X1, H4 and CAS2) involved altogether 24 (18.9%) isolates. Twenty-six (20.5%) isolates could not be assigned to any specific clade. MIRU-VNTR typing identified 123 (96.8%), 97 (76.4%) and 65 (51.2%) unique types with a tolerance of 0, 1, and 2 locus differences between the patterns. Upon combined spoligotyping and MIRU-VNTR typing analysis, 123 (96.8%), 108 (85%), and 91 (71.7%) unique types were identified if a tolerance of 0, 1, and 2 locus differences in the MIRU-VNTR patterns was assumed, respectively. Based on the clustering results, the transmission rate for MDR-TB cases under the study was calculated at 3.2%, 15%, and 28.3%. Overall, three clades, namely CAS1_DELHI, T1, and Beijing accounted for the majority of MDR-TB cases in Pakistan. Up to a third of the cases were clustered upon combined spoligotyping and MIRU-VNTR typing, suggesting a moderate level of active transmission.

Estimation of hepatitis C prevalence in the Punjab province of Pakistan: A retrospective study on general population.

BACKGROUND: Hepatitis C virus (HCV) infections are amongst the leading public health concerns in Pakistan with a high disease burden. Despite the availability of effective antiviral treatments in the country the disease burden in general population has not lowered. This could be attributed to the asymptomatic nature of this infection that results in lack of diagnosis until the late symptomatic stage. To better estimate and map HCV infections in the country a population-based analysis is necessary for an effective control of the infection. METHODS: Serologic samples of ~66,000 participants from all major cities of the Punjab province were tested for anti-HCV antibodies. The antibody-based seroprevalence was associated with socio-demographic variables including geographical region, age, gender and sex, and occupation. RESULTS: Overall serological response to HCV surface antigens was observed in over 17% of the population. Two of the districts were identified with significantly high prevalence in general population. Analysis by occupation showed significantly high prevalence in farmers (over 40%) followed by jobless and retired individuals, laborers and transporters. A significant difference in seroprevalence was observed in different age groups amongst sex and genders (male, female and transgender) with highest response in individuals of over 40 years of age. Moreover, most of the tested IDUs showed positive response for anti-HCV antibody. CONCLUSION: This study represents a retrospective analysis of HCV infections in general population of the most populated province of Pakistan to identify socio-demographic groups at higher risk. Two geographical regions, Faisalabad and Okara districts, and an occupational group, farmers, were identified with significantly high HCV seroprevalence. These socio-demographic groups are the potential focused groups for follow-up studies on factors contributing to the high HCV prevalence in these groups towards orchestrating effective prevention, control and treatment.

Evaluation of Genotype MTBDRplus and MTBDRsl Assays for Rapid Detection of Drug Resistance in Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in Pakistan.

Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries alongside the 6th among countries with the highest burden of drug-resistant TB, including multi-drug resistant (MDR)-TB. Methods for rapid and reliable drug susceptibility testing (DST) are prerequisite for the prompt institution of effective anti-TB treatment. The aim of this study was to evaluate the efficiency of Genotype MTBDRplus and MTBDRsl assays for the detection of MDR and (pre-) extensively drug-resistant (XDR-TB) isolates in Pakistan. The study included 47 pre-XDR and 6 XDR-TB isolates, recovered from 53 patients from Pakistan. Conventional DST was performed using the standard 1% proportion method on the Löwenstein-Jensen medium. For molecular determination of drug resistance, GenoType MTBDRplus and GenoType MTBDRsl assays (Hain Lifescience, Germany) were used. To evaluate discrepancies between conventional and molecular DST results, mutation profiling was performed by amplifying and sequencing seven genetic loci, i.e., katG, inhA, and mabA-inhA promoter, rpoB, gyrA, embB, rrs. The sensitivity of Genotype MTBDRplus was 71.7% for isoniazid (INH) and 79.2% for rifampicin (RIF). Sequence analysis revealed non-synonymous mutations in 93.3 and 27.3% of isolates phenotypically resistant to INH and RIF, respectively, albeit susceptible when tested by GenoType MTBDRplus. GenoType MTBDRsl had a sensitivity of 73.6, 64.7, 20, 25, and 100% for the detection of fluoroquinolones, ethambutol, kanamycin, amikacin, and capreomycin resistance, respectively. Upon sequencing, mutations were detected in 20, 77.8%, and all isolates phenotypically resistant to aminoglycosides, ethambutol, and fluoroquinolones, respectively, yet declared as susceptible with GenoType MTBDRsl. Low sensitivities seriously impede the large-scale application of the Genotype MTBDRplus and MTBDRsl assays. Unless further optimized, the currently available line-probe assays should rather be auxiliary to the conventional, phenotype-based methods in the detection of MDR- and XDR-TB in Pakistan.

Neurochemical Alterations in Sudden Unexplained Perinatal Deaths-A Review.

Sudden unexpected perinatal collapse is a major trauma for the parents of victims. Sudden infant death syndrome (SIDS) is unexpected and mysterious death of an apparently healthy neonate from birth till 1 year of age without any known causes, even after thorough postmortem investigations. However, the incidence of sudden intrauterine unexplained death syndrome (SIUDS) is seven times higher as compared with SIDS. This observation is approximated 40-80%. Stillbirth is defined as death of a fetus after 20th week of gestation or just before delivery at full term without a known reason. Pakistan has the highest burden of stillbirth in the world. This basis of SIDS, SIUDS, and stillbirths eludes specialists. The purpose of this study is to investigate factors behind failure in control of these unexplained deaths and how research may go ahead with improved prospects. Animal models and physiological data demonstrate that sleep, arousal, and cardiorespiratory malfunctioning are abnormal mechanisms in SIUDS risk factors or in newborn children who subsequently die from SIDS. This review focuses on insights in neuropathology and mechanisms of SIDS and SIUDS in terms of different receptors involved in this major perinatal demise. Several studies conducted in the past decade have confirmed neuropathological and neurochemical anomalies related to serotonin transporter, substance P, acetylcholine α7 nicotine receptors, etc., in sudden unexplained fetal and infant deaths. There is need to focus more on research in this area to unveil the major curtain to neuroprotection by underlying mechanisms leading to such deaths.

A cross-sectional study about knowledge and attitudes toward multidrug-resistant and extensively drug-resistant tuberculosis in a high-burden drug-resistant country.

OBJECTIVE/BACKGROUND: Tuberculosis (TB) is a leading cause of death worldwide, with new threats of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. Pakistan is the fifth highest among high-burden TB countries and the fourth highest among high-burden drug-resistant-TB countries. Pakistan is the sixth most populous country in the world, and Pakistani youth is the highest population group in Pakistan and second in the world. This study was aimed at assessing the understanding, awareness, and mindset of university students toward TB, MDR TB, and XDR TB in Lahore. METHODS: A cross-sectional questionnaire-based study was performed on 1137 individuals from three major public-sector universities in Lahore, Pakistan. Information regarding their knowledge and attitude toward MDR and XDR TB was gathered using a structured questionnaire. Data collected was analyzed using SPSS version 20. RESULTS: Male (531) and female (606) students were asked about different aspects of MDR and XDR TB. Although 80.47% students had good knowledge about simple TB, a very small fraction had awareness and appropriate knowledge about MDR/XDR-TB. Considering TB as a stigma, only 9.3% students disclosed that they had household TB contact. Only 25% students knew about XDR TB. CONCLUSION: Our results indicated that a small fraction of people knew the exact definition and treatment duration of MDR TB and XDR TB in our society. There is a need to increase the awareness and knowledge status of university students about MDR and XDR TB.

Evaluation of genotype MTBDRplus assay for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis clinical isolates from Pakistan.

BACKGROUND: GenoType MTBDRplus is a molecular assay for detection of Mycobacterium tuberculosis resistance to isoniazid (INH) and rifampicin (RMP), the two major anti-tuberculosis (TB) drugs. Identification of INH resistance is largely based on the occurrence of mutations in the katG gene, coding for the catalase-peroxidase, or in the promoter region of the inhA gene, coding for the NADH-dependent enoyl-ACP reductase. For testing the RMP resistance, mutations in the rpoB gene, coding for the RNA polymerase ß subunit, particularly in the RMP resistance determining region (RRDR) of the gene are investigated. The GenoType MTBDRplus assay has been validated in several countries. The aim of this study was to evaluate the ability of the assay to detect INH and RMP resistance among strains of M. tuberculosis, isolated from Pakistani TB patients, and phenotypically identified as multidrug-resistant (MDR), that is resistant to both INH and RMP. MATERIAL/METHODS: The study included a collection of 100 MDR M. tuberculosis strains isolated from as many Pakistani TB patients over a 9-month period (i.e. between January and September 2014). Drug susceptibility testing was performed using the standard 1% proportion method on Löwenstein-Jensen medium, with INH and RMP critical concentrations of 0.2mg/L and 40mg/L, respectively. Genomic DNA was extracted by the cetyl-trimethyl ammonium bromide (CTAB) method. The GenoType MTBDRplus assay (Hain Lifescience, Germany) was done following the manufacturer's instructions. RESULTS: In the katG gene, with MTBDRplus assay, a specific mutation associated with INH resistance (i.e. G944C transition, conferring Ser315Thr amino acid change) was detected in 66 (66%) of the strains. Thirty-four (34%) strains did not carry the katG mutation detected by the assay. Mutations in the mabA-inhA promoter region were detected in 10 (10%) strains (C-15T - in 10 strains, and T-8C - in 2 strains). Seventy-seven (77%) strains tested harboured a mutation in rpoB gene. Mutations in the rpoB gene were of four types: C1349T, A1304T, C1333G, and TC1324CA found in 63 (63%), 11 (11%), 8 (8%), and one (1%) strain, respectively. Of the 100 strains designated as MDR by the proportion method, GenoType MTBDRplus confirmed this phenotype in only 62 strains. The results of GenoType MTBDRplus and the conventional drug susceptibility method were consistent in 70% (70/100) for INH, and 77% (77/100) for RMP. CONCLUSIONS: As evidenced in this study, the major concern with the GenoType MTBDRplus assay were false negative results. In comparison to conventional drug susceptibility testing, the assay was unable to detect 30 (30/100; 30%) strains resistant to INH and 23 (23/100; 23%) strains resistant to RMP. The GenoType MTBDRplus failed to identify 38 MDR (38/100; 38%) strains. Resistance in those strains probably originate from mutations in other codons and/or genes than those covered by the test. For detecting INH and RMP resistance in TB cases, especially in high TB incidence countries, such as Pakistan, molecular approaches should still be a complement rather than areplacement to conventional drug susceptibility testing.

Diabetes mellitus among tuberculosis patients: a cross sectional study from Pakistan.

BACKGROUND: The co-occurrence of diabetes mellitus (DM) and tuberculosis (TB) is largely associated with high frequency of morbidity. OBJECTIVE: To determine the prevalence of DM among TB patients and describe the socio-demographic and behavioral factors associated with TB-DM co-occurrence. METHODS: We enrolled 500 TB patients from September, 2014 to August 2015 at four major public sector hospitals of Lahore, Pakistan. A questionnaire was used to collect information regarding associated socio-demographic and behavioral factors of the patients. We monitored the fasting blood sugar of each patient by using a semi automated clinical chemistry analyzer followed by an HbA1c level check of all hyperglycemic patients. RESULTS: The prevalence of TB-DM co-occurrence was 14.8%. The prevalence of TB-DM was higher (62.2%) among males. The >57 year age group had the highest proportion of patients (35.1%), with co-existent TB-DM. Most were illiterate (73.0%) and unemployed (48%). Moreover, among the 74 patients positive for TB-DM had a history of smoking. Age and education level were significantly associated with DM-TB while gender, occupation and smoking were not associated. CONCLUSION: The study revealed a 14.8% prevalence of DM among TB patients. This was associated with several socio-demographic factors, including age, unemployment, literacy and polluted environment. Thus, poor and unhealthy lifestyles were the factors associated with DM among immunologically compromised individuals due to TB.

Emergence of mixed infection of Beijing/Non-Beijing strains among multi-drug resistant Mycobacterium tuberculosis in Pakistan.

Tuberculosis (TB) remains as one of the deadliest diseases after HIV globally with 95 % of deaths confined to low-and-middle income countries. Pakistan is fifth among the 22 high-burden TB countries with the incidence rate of 230/100,000 persons, however, studies related to prevalent Mycobacterium tuberculosis strains and their spread, drug resistance pattern and evolutionary genetics are inadequate. The present study was undertaken to highlight the circulation of M. tuberculosis strains causing drug resistant TB in our community by targeting the molecular marker IS6110 and then characterization of these strains as Beijing and Non-Beijing genotypes. Sputum samples from 102 MDR TB suspects from different cities of Punjab were collected and their record was stored in a database. Sputum samples were evaluated by Ziehl Neelson staining and cultured on Lownstein Jensen medium by Modified Petroff's method. DST was performed for first-line anti-mycobacterial drugs by indirect proportion method. Mycobacterium tuberculosis isolates were investigated for the presence of IS6110 and further identification as Beijing, Non-Beijing or mixed genotype. Percentage of male and female patients was found to be 58.8 and 41.2 % respectively. DST showed resistance of 93 % of isolates to isoniazid and rifampicin. All of the isolates showed positive results for IS6110 amplification. Based on PCR amplification of Beijing and non-Beijing primer sets 4.9 % of the patients showed infection with pure Beijing isolates, 14.7 % with both Beijing and non-Beijing isolates and 80.3 % with pure non-Beijing isolates. Analysis of IS6110 and Beijing sequences showed the presence of putative transposase conserved domain while non-Beijing sequences were epitomized with RAMP_I_III superfamily domain (CRISPR-associated protein family). TB in Pakistan is predominantly caused by Non-Beijing genotypes, but Beijing strains showed incessant circulation in our community as both single and mixed (co-infecting Non-Beijing and Beijing) strains.

Characteristics and Antibiotic Resistance of Urinary Tract Pathogens Isolated From Punjab, Pakistan.

BACKGROUND: Urinary tract infection (UTI) is deemed the most prevalent infectious disease in that it has now touched the overall incidence of 18/1000 persons per year in the general population. OBJECTIVES: This study sought to determine the characteristics of isolates from patients with UTI and their susceptibility to commonly used antibiotics in Punjab, Pakistan. PATIENTS AND METHODS: Totally, 1429 urine samples were analyzed from UTI patients for the isolation of uropathogens at Chughtai's Lahore Lab, Lahore, Pakistan, during a period of 14 months. The antimicrobial susceptibility test was performed via the disc diffusion method for the isolates obtained from 392 (26%) positive cultures. RESULTS: The highest percentage (67%) of isolates was from females in comparison to males (33%). The frequency of Escherichia coli was the highest (62%) in culture-positive urine samples, followed by E. faecalis (15%), Candida (14%), Pseudomonas (6%), Klebsiella spp. (1%), Proteus (1%), and Staphylococcus aureus (1%). E. coli was highly resistant to antimicrobial drugs, viz. cephalexin (95%), cephradine (95%), pipemidic acid (92%), amikacin (91%), and nalidixic acid (91%). Most of the routine ß-lactam antibiotics like amoxicillin/clavulanic acid, ampicillin, and aztreonam were also ineffective against E. coli, with resistance rates of 84%, 84%, and 72%, correspondingly. This pathogen showed maximum susceptibility (97%) against three drugs, namely imipenem, meropenem, and cefoperazone. Piperacillin and fosfomycin also provided significant results against E. coli with respective susceptibility rates of 96% and 90%. CONCLUSIONS: Our results showed that broad-spectrum antibiotics such as imipenem, meropenem, fosfomycin, cefoperazone/sulbactam, and vancomycin would be the first line and the most effective drugs for the empirical treatment of urinary tract pathogens due to their higher resistance rates against other drugs like cephalexin, cephradine, ciprofloxacin, levofloxacin, and norfloxacin.