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Background . Concurrent coronavirus disease 2019 (COVID-19) and Pneumocystis jirovecii pneumonia (PJP) has been described in various reports, with a recent study describing a 9.3 % P . jirovecii detection rate in critically ill COVID-19 patients. Methods . Patients with PCR-confirmed PJP following COVID-19 infection who were admitted to Aga Khan University Hospital, Karachi, Pakistan from March 2020-June 2021 were identified through a laboratory database. Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus was performed by RT-PCR Cobas SARS-CoV-2 qualitative assay. P. jirovecii PCR was performed using the RealStar Pneumocystis jirovecii PCR kit. Clinical, radiological and laboratory data for PJP patients were recorded. Results . During the study period, 3707 patients were admitted with COVID-19 at our hospital. P. jirovecii PCR was requested for 90 patients and was positive in 10 (11 %). Five out of 10 patients were discharged from the hospital and later developed cough and dyspnoea. Five patients remained hospitalized with severe COVID-19 and developed PJP. Eight patients in our study received systemic steroids. The trends of lymphocyte counts of all patients showed a lymphocyte count of <1000 mm -3 (<1.0×10 6  cells µl -1 ) in the week of PJP diagnosis. Four patients did not survive; one of these patients did not receive co-trimoxazole due to late diagnosis, one patient had concomitant nosocomial pneumonia and bacteraemia with multidrug-resistant Acinetobacter species, and two patients had concomitant aspergillosis. Conclusion . In summary, invasive fungal infections such as PJP should be considered as a complication in COVID-19 patients, with prompt evaluation and management., Competing Interests: The authors declare that there are no conflicts of interest., (© 2023 The Authors.)

Background: Early identification of COVID-19-associated pulmonary aspergillosis (CAPA) is particularly challenging in low- middle-income countries where diagnostic capabilities are limited, and risk factors for CAPA have not been identified. It is also essential to recognise CAPA patients who are likely to have a poorer outcome to decide on aggressive management approaches. Therefore, this study aimed to identify risk factors and outcomes for CAPA among admitted moderate to critical COVID-19 patients at our centre in Pakistan., Methods: An unmatched case-control study with ratio of 1:2 was conducted on hospitalised adult patients with COVID-19 from March 2020-July 2021. Cases were defined according to European Confederation of Medical Mycology and the International Society for Human and Animal Mycology consensus criteria. Controls were defined as patients hospitalised with moderate, severe or critical COVID-19 without CAPA., Results: A total of 100 CAPA cases (27 probable CAPA; 73 possible CAPA) were compared with 237 controls. Critical disease at presentation (aOR 5.04; 95% CI 2.18-11.63), age ≥ 60 years (aOR 2.00; 95% CI 1.20-3.35) and underlying co-morbid of chronic kidney disease (CKD) (aOR 3.78; 95% CI 1.57-9.08) were identified as risk factors for CAPA. Patients with CAPA had a significantly greater proportion of complications and longer length of hospital stay (p-value < .001). Mortality was higher in patients with CAPA (48%) as compared to those without CAPA (13.5%) [OR = 6.36(95% CI 3.6-11)]., Conclusions: CAPA was significantly associated with advanced age, CKD and critical illness at presentation, along with a greater frequency of complications and higher mortality., (© 2022 Wiley-VCH GmbH.)

Objectives: To assess the diagnostic accuracy of different cut-off values of pleural fluid adenosine deaminase levels as a diagnostic method for tuberculous pleural effusion., Methods: The prospective study was conducted from 2014 to 2016 at the Aga Khan University Hospital, Karachi, and comprised pleural fluid samples of adult patients with and without tuberculosis which were tested for adenosine deaminase levels, and divided into tuberculosis group A and non-tuberculosis group B. Sensitivity, specificity, negative predictive value and positive predictive value were calculated using different cut-offs. Data was analysed using IBM SPSS (Statistical Package for Social Sciences) version 21.0 (IBM Corp., Armonk, NY)., Results: Of 155 patients, 46(29.7%) had tuberculosis; 30(65.2%) males and 16(34.8%) females. Those who did not have tuberculosis were 109(70.3%); 69(63.3%) males and 40(36.7%) females. The adenosine deaminase levels were elevated in group A compared to group B (p<0.001). The cut-off of 30U/L showed the highest sensitivity (71.7%) and negative predictive value (87.4%), and a specificity of 82.6%. The cut-off of 50U/L showed the highest specificity (89.9%) with sensitivity 52.2%, and the cut-off of 40U/L showed the highest positive predictive value of 68.9% with sensitivity 67.4% and specificity 87.2%., Conclusions: Pleural fluid adenosine deaminase testing for diagnosing tuberculosis pleuritis revealed highest sensitivity and moderate specificity for cut-off value of 30U/L.

Introduction: The gradual increase in caspofungin usage in Pakistan raises a concern of emergence of echinocandin resistance in local Candida glabrata strains. We sequenced and determined mutations in fks1 and fks2 genes in invasive Candida glabrata strains from Pakistan., Material and Methods: Thirty-six invasive C. glabrata strains were selected with median (min-max) minimum inhibitory concentrations (MICs) of 0.06 (0.015-0.25) mg/L for caspofungin, 0.015 (0.008-0.06) mg/L for micafungin and 0.06 (0.015-0.12) mg/L for anidulafungin. fks1 and fks2 gene fragments were sequenced using Sanger methodology. Sequences were analysed with MEGA-6 software to identify specific single-nucleotide polymorphisms (SNP) against wild-type sequences of C. glabrata., Results: In fks1 gene, non-synonymous mutation D632H was observed in one isolate with caspofungin MIC of 0.25 mg/L. Synonymous mutation at position A742 was observed in 26/36 (72%) of the isolates. 34/36 (94.5%) isolates analysed for fks2 gene were observed as wild type. A novel non-synonymous mutation at I661T was observed in fks2 gene in one isolate with caspofungin MIC of 0.12 mg/L and anidulafungin and micafungin MIC of 0.06 and 0.015 mg/L, respectively. Novel fks2 synonymous mutations at position T647, K652 and I706 were observed in 16/36 (44%), 25/36 (69%) and 23/36 (63%) isolates, respectively., Conclusion: Low frequencies of both non-synonymous and synonymous polymorphisms were observed in invasive C. glabrata strains. Since S663P in fks2 gene is associated with caspofungin resistance, a novel mutation at 661 codon identified in our study needs correlation with treatment outcome data and mandates periodic genomic surveillance., (© 2022 Wiley-VCH GmbH.)

Comorbidities in human beings signify the numerous risk factors that increase the incidences of neuro- and cardio-metabolic disorders. Experimental models depicting comorbidities are important to explore the molecular pathophysiology that can help suggest appropriate treatment strategies. Tissue-accumulating potential and pathological effects of aluminium chloride (AlCl 3 )and sodium azide (NaN 3 ) are well recognized. Hence, in the current work, we have for the first time aimed to investigate the unexplored potential of graded dose effects of AlCl 3 and NaN 3 in inducing early inflammation and cardiometabolic toxicity via comparative biochemical analysis of AlCl 3 and/or NaN 3 . Rats were allocated into seven groups ( n = 6). Group 1 was normal control. Remaining groups were given graded doses of AlCl 3 and/or NaN 3 , as LD-AlCl 3 (AlCl 3 40 mg), MD-AlCl 3 (AlCl 3 45 mg), and HD-AlCl 3 (AlCl 3 50 mg) representing low dose, medium dose, and high dose of AlCl 3 , respectively, and the remaining as LD-NaN 3 (NaN 3 13 mg), MD-NaN 3 (NaN 3 15 mg), and HD-NaN 3 (NaN 3 17 mg) representing low dose, medium dose, and high dose of NaN 3 , respectively. Serum levels of glucose, insulin, lipid profile, inflammatory mediators like IL-6 and oxidative stress marker, and malondialdehyde (MDA) were analyzed. Likewise, subacute toxicity parameters were analyzed. Immunohistochemistry (IHC) and histopathology (H&E/Masson's trichrome staining) of brain, heart, and pancreatic tissues were done. ECG pattern of all groups was observed. HD-AlCl 3 was associated with elevated levels of inflammatory biomarkers, MDA, and glycemic and lipid profiles, whereas it decreased the insulin levels. HD-NaN 3 also showed the similar effects of aggravated inflammatory biomarkers, impaired glycemic and lipid profiles, but depicted the maximum mortality rate as compared to HD-AlCl 3 . IHC showed prominent amyloid plaques and neurofibrillary tangle formation with MD-AlCl 3 and HD-AlCl 3 as compared to NaN 3 -treated groups. Likewise, in brain tissues, vacuolation of white matter, vascular congestion, and hemorrhage were seen in HD-AlCl 3 treated group, while HD-NaN 3 induced death in animals. AlCl 3 exposure resulted in an inverted QRS complex, while exposure to NaN 3 showed ST depression but with increased mortality. AlCl 3 has better controlled results as compared to NaN 3 for induction of comorbid experimental animal model depicting early neuroinflammation and cardiometabolic disruption. These determined efforts facilitate the researchers for the development of clinically effective treatment strategies using such experimental models., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Serratia marcescens , a Gram-negative bacterium, is one of the known disease-causing pathogens. It is resistant to ampicillin, macrolides, cephalosporins, cefotaxime, and ceftazidime. The only antibiotic that has been proven to be effective against S. marcescens is gentamicin. By causing epigenetic alterations, bacteria can also become resistant to all antibiotics. Many epigenetically related proteins were studied, and four proteins were selected in this regard for epitope evaluation and their subsequent use in the development of a messenger ribonucleic acid (mRNA) vaccine. A series of immune-informatics tools used to build this mRNA vaccine elicited cellular and humoral immunity. Molecular docking between epitopes and alleles of the major histocompatibility complex (MHC) was performed. The vaccine was developed using 37 epitopes, an adjuvant that is a TLR-4 agonist known as resuscitation-promoting factor E (RpfE), subcellular trafficking structures, secretion boosters, and linkers. This proposed architecture was found to cover 99.6% of the population during testing. During testing, it was proven that it was both effective and safe. To confirm our idea, we performed an in silico immunological simulation of vaccination. The codon was also optimized to ensure that the mRNA reached the cytoplasm of a human host and underwent efficient translation. TLR-4 and TLR-3 were also docked against the secondary and tertiary structures of the vaccine peptide. Furthermore, the vaccine's stability was confirmed by molecular dynamics simulation. In summary, this vaccine construct can be a potential candidate against S. marcescens and is suitable for in vitro analyses to validate its effectiveness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Naveed, Mughal, Jabeen, Aziz, Naz, Nazir, Shahzad, Alharbi, Alshammari and Sadhu.)

The family members of Arenaviridae include members of the genus Machupo virus, which have bi-segmented negative sense RNA inside the envelope and can be transferred to humans through rodent carriers. Machupo virus, a member of the mammarenavirus genus, causes Bolivian hemorrhage fever, its viral nucleocapsid protein being a significant virulence factor. Currently, no treatment is available for Bolivian hemorrhage fever and work to develop a protective as well as post-diagnosis treatment is underway. Adding to these efforts, this study employed a reverse-vaccinology approach to design a vaccine with B and T-cell epitopes of the viral nucleocapsid protein of the Machupo virus. Five B-cell specific, eight MHC-I restricted, and 14 MHC-II restricted epitopes were finalized for the construct based on an antigenicity score of >0.5 and non-allergenicity as a key characteristic. The poly-histidine tag was used to construct an immunogenic and stable vaccine construct and 50S ribosomal 46 protein L7/L12 adjuvant with linkers (EAAAK, GPGPG, and AYY). It covers 99.99% of the world’s population, making it highly efficient. The physicochemical properties like the aliphatic index (118.31) and the GRAVY index (0.302) showed that the vaccine is easily soluble. The overall Ramachandran score of the construct was 90.7%, and the instability index was 35.13, endorsing a stable structure. The immune simulations demonstrated a long-lasting antibody response even after the excretion of the antigen from the body in the first 5 days of injection. The IgM + IgG titers were predicted to rise to 6000 10 days post-injection and were illustrated to be stable (around 3000) after a month, elucidating that the vaccine would be effective and provide enduring protection. Lastly, the molecular interaction between the construct and the IKBKE receptor was significant and a higher eigenfactor value in MD simulations confirmed the stable molecular interaction between the receptor and the vaccine, validating our construct.

Many studies have reported the occurrence of microplastics in live shellfish intended for human consumption. However, far fewer studies have been conducted on dried shellfish from supermarkets or fishery markets. In this study, the characteristics of microplastics in six kinds of dried shellfish products following different cooking treatments were investigated. Dietary exposure to microplastics in dried shellfish was estimated using the consumption rate of seafood among different age groups. Microplastics were detected in all the uncooked, dried shellfish products, ranging from 0.3 to 4.2 items/g. Fibres accounted for more than 80% of microplastics in razor clams, winkles, and scallops. The proportion of microplastics smaller than 1 mm in size ranged from 57.1% to 89.7% of the total microplastics found in dried shellfish. The polymer types included polyethylene terephthalate (PET), rayon, polyester, nylon, polypropylene (PP), cellophane (CP), and polyethylene (PE). Principal component analysis (PCA) showed that the sizes and shapes of microplastics in scallops were more susceptible to alteration by different cooking methods. Steaming and frying significantly reduced the abundance of microplastics in razor clams. In addition, significantly fewer microplastics were found in scallop products after boiling and steaming than were found in fried scallop products. The estimated dietary intake of microplastics for infants was the highest among the age groups considered (3.05 items/kg(bw)/day). Accordingly, frying was suggested for cooking mussels, boiling for clams and winkles, and steaming for scallops. Combining risks from ingesting plastics and plastic additives, steaming is suggested as the best method to cook shellfish., Competing Interests: Declaration of competing interest Manuscript has been read and approved by all authors, and no conflict of interest exit in the submission of this manuscript. Neither the entire paper nor any part of its content has been published or has been accepted elsewhere. It is not being submitted to any other journal., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Enterobacter cloacae is mainly responsible for sepsis, urethritis, and respiratory tract infections. These bacteria may affect the transcription of the host and particularly their immune system by producing changes in their epigenetics. In the present study, four proteins of Enterobacter cloacae were used to predict the epitopes for the construction of an mRNA vaccine against Enterobacter cloacae infections. In order to generate cellular and humoral responses, various immunoinformatic-based approaches were used for developing the vaccine. The molecular docking analysis was performed for predicting the interaction among the chosen epitopes and corresponding MHC alleles. The vaccine was developed by combining epitopes (thirty-three total), which include the adjuvant Toll-like receptor-4 (TLR4). The constructed vaccine was analyzed and predicted to cover 99.2% of the global population. Additionally, in silico immunological modeling of the vaccination was also carried out. When it enters the cytoplasm of the human (host), the codon is optimized to generate the translated mRNA efficiently. Moreover, the peptide structures were analyzed and docked with TLR-3 and TLR-4. A dynamic simulation predicted the stability of the binding complex. The assumed construct was considered to be a potential candidate for a vaccine against Enterobacter cloacae infections. Hence, the proposed construct is suitable for in vitro analyses to validate its effectiveness.

Fungal wound infections are increasing worldwide. The aim of this retrospective study, conducted at the Aga Khan University laboratory, Karachi, Pakistan, was to determine the frequency of fungal isolation in wound specimens. Data of wound samples received for culture from all over the country between September and October 2018 was reviewed. Samples were processed for bacterial cultures and additionally inoculated on Sabouraud's dextrose medium. Demographic information, medical history and information on the type of wound was collected. A total of 140 cases were included, of which 87 (81%) were culture positive, while 10 (7%) cases yielded fungi. Burn and blast wounds had the highest proportion of fungal isolation, i.e. 2 out of 4 (50%). Candida species were the most common fungi (n=4), followed by Fusarium species (n=3). This study reports an alarming rate of fungal wound infections. As fungal necrotising wound infections have high morbidity and mortality, it is, therefore, important to accurately diagnose and treat such infections in local setting.

Background: Mycetoma is an important neglected tropical disease associated with debilitation, disfigurement and death if not diagnosed and treated adequately. In Pakistan, mycetoma cases have frequently been diagnosed in histopathology and microbiology laboratories. However, there is scarcity of published data from this country. Therefore, the objectives of this study were to evaluate the frequency and type of mycetoma reported in skin and soft tissue biopsies from a single center over 10 years and review of published literature from Pakistan., Method: This descriptive observational retrospective study was conducted at the Aga Khan University Hospital laboratory, Karachi, Pakistan. Laboratory data from 2009-2018 of skin and soft tissue biopsies with positive findings of mycetoma were retrieved from hospital information system. The variables for statistical analysis were age and gender of patient, anatomical site of lesion, residence of patient (geographical location) in the country, etiologic agents of mycetoma and significant gross and microscopic histopathological findings. The data was entered, and descriptive epidemiologic assessment was carried out using MS excel 2013. Geographical information system was used for mapping the location. Literature review of mycetoma cases reported from Pakistan was done on PubMed, Google search and PakMediNet from 1980 till April 2019., Result: During ten years of study period, 89 skin and soft tissue biopsies were reported as mycetoma, majority were eumycetoma [n = 66/89 (74%)] followed by actinomycetoma [n = 23/89 (26%)]. Involvement of lower limb was predominantly observed [n = 74/89 (83%)] in which foot had significant contribution [n = 65/74 (88%)]. Only 18 specimens were submitted for microbiological assessment and six grew agents of mycetoma, with Madurella mycetomatis reported in only three. Well-formed granuloma formation was observed in only 26%[n = 23/89] of cases. Specific geographical location was not identified, and cases were reported from across the country. From Pakistan, only two original papers and 7 case reports were available in published literature., Conclusion: This single center study reports a handful of cases of mycetoma from Pakistan. We conclude that the index of suspicion should remain high among treating surgeons and physicians and clinical laboratories should improve their diagnostic capacity and skills. This will have a great impact on disease outcome and patient's life., Competing Interests: The authors have declared that no competing interests exist.

Background: College is the time where most mental health disorders are triggered. Due to an extremely strenuous curriculum, medical students are prone to develop high levels of stress. Teaching students stress management skills can be crucial for their mental and physical well being. Objectives of the study were to measure perceived stress level among undergraduate medical students of Year 5 using PSS Scale, and to explore if students find stress management activities helpful in reducing stress., Methods: It was a quasi experimental study.5th year medical students were asked to take part in a full day workshop regarding stress management. During the day they were introduced to different stress management activities. Stress levels were measured using PSS before the workshop and 1 month after the workshop., Results: On the PSS 1, 17.9% participants scored in the low stress category, 61.1% participants scored in the moderate stress category and 21.1% scored in the high stress category. On the PSS 2, 11.6% participants scored in the low Stress category, 76.8% participants scored in the moderate stress category and 11.6% scored in the high stress category. Pre and post intervention PSS stress results were statistically significant, p< 0.05., Conclusions: Majority of the final year students had moderate stress. Training undergraduate medical students in integrated stress relieving activities can help lower their perceived stress.

Background and Purpose: Influenza A and SARS-CoV-2 are risk factors for invasive pulmonary aspergillosis. Both influenza-associated pulmonary aspergillosis and COVID-19-associated pulmonary aspergillosis result in high mortality and poor clinical outcomes. No prospective study has so far compared the features, treatment, and outcomes of influenza-associated pulmonary aspergillosis and COVID-19-associated pulmonary aspergillosis within a similar time frame. Therefore, this study aimed to determine the frequency, risk factors, and outcomes of invasive pulmonary aspergillosis in critically ill patients with influenza, COVID-19, and community-acquired pneumonia., Materials and Methods: This prospective study included adult patients with pneumonia and was conducted at The Aga Khan University Hospital in Karachi, Pakistan. Patients were divided into three groups, including community-acquired pneumonia, influenza pneumonia, and COVID-19 pneumonia. The data collected included information on demographic characteristics, comorbidities, clinical features, laboratory results, treatment, and outcomes., Results: A total of 140 patients were included in this study. These included 35 (25%), 70 (50%), and 35 (25%) patients with community-acquired pneumonia, influenza pneumonia, and COVID-19 pneumonia, respectively. In addition, 20 (14.2%) patients were found to have invasive aspergillosis, of whom 10/35 (28.5%), 9/70 (12.8%), and 1/35 (2.8%) patients were in the COVID-19, influenza, and community-acquired pneumonia groups, respectively. Moreover, nine (90%) COVID-19-associated pulmonary aspergillosis patients required vasopressors, compared to three (33%) patients with influenza-associated pulmonary aspergillosis ( P=0.020 ). In total, seven (70%) COVID-19-associated pulmonary aspergillosis patients required invasive mechanical ventilation compared to four (44%) influenza-associated pulmonary aspergillosis patients ( P=0.37 ). The mean±SD length of hospital stay was highest in the COVID-19-associated pulmonary aspergillosis patients (18.3±7.28 days) compared to influenza-associated pulmonary aspergillosis patients (11.7±5.34 days) ( P=0.036 ). The number of deaths in influenza-associated pulmonary aspergillosis and COVID-19-associated pulmonary aspergillosis patients was three (33.3%) and five (50%), respectively ( P=0.526 )., Conclusion: A higher proportion of patients with COVID-19 developed invasive aspergillosis compared to those with influenza. Although the mortality rate in COVID-19-associated pulmonary aspergillosis was comparable to that in influenza-associated pulmonary aspergillosis patients, COVID-19-associated pulmonary aspergillosis patients had a significantly longer stay in the hospital., Competing Interests: The authors declare that they have no conflicts of interest to express. The abstract of this study has been submitted to TIMM (Trends in Medical Mycology) 2021 Meeting., (Copyright: © 2021, Published by Mazandaran University of Medical Sciences on behalf of Iranian Society of Medical Mycology and Invasive Fungi Research Center.)

Objectives: Coronavirus disease 2019 (COVID-19) -associated pulmonary aspergillosis (CAPA) has emerged as a complication in critically ill COVID-19 patients. The objectives of this multinational study were to determine the prevalence of CAPA in patients with COVID-19 in intensive care units (ICU) and to investigate risk factors for CAPA as well as outcome., Methods: The European Confederation of Medical Mycology (ECMM) conducted a multinational study including 20 centres from nine countries to assess epidemiology, risk factors and outcome of CAPA. CAPA was defined according to the 2020 ECMM/ISHAM consensus definitions., Results: A total of 592 patients were included in this study, including 11 (1.9%) patients with histologically proven CAPA, 80 (13.5%) with probable CAPA, 18 (3%) with possible CAPA and 483 (81.6%) without CAPA. CAPA was diagnosed a median of 8 days (range 0-31 days) after ICU admission predominantly in older patients (adjusted hazard ratio (aHR) 1.04 per year; 95% CI 1.02-1.06) with any form of invasive respiratory support (HR 3.4; 95% CI 1.84-6.25) and receiving tocilizumab (HR 2.45; 95% CI 1.41-4.25). Median prevalence of CAPA per centre was 10.7% (range 1.7%-26.8%). CAPA was associated with significantly lower 90-day ICU survival rate (29% in patients with CAPA versus 57% in patients without CAPA; Mantel-Byar p < 0.001) and remained an independent negative prognostic variable after adjusting for other predictors of survival (HR 2.14; 95% CI 1.59-2.87, p ≤ 0.001)., Conclusion: Prevalence of CAPA varied between centres. CAPA was significantly more prevalent among older patients, patients receiving invasive ventilation and patients receiving tocilizumab, and was an independent strong predictor of ICU mortality., (Copyright © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: Quality-assured antimicrobial susceptibility testing (AST) depends upon the knowledge and skills of laboratory staff. In many low- and middle-income countries (LMICs), including Pakistan, such types of knowledge and skills are limited. Therefore, the objective of this study was to use openaccess online courses to improve the knowledge of laboratory staff involved in the detection and reporting of antimicrobial resistance (AMR)., Methodology: Seven online modules comprising 22 courses aimed at strengthening the laboratory detection of Antimicrobial resistance (AMR) were developed. The courses were uploaded onto the website www.parn.org.pk. Participants had an option of selecting courses of their interest. Online registration and completion of a pre-course assessment (pre-test) were essential for enrolment. However, participation in post-course assessment (post-test) was optional. The number of registered participants and the proportion of participants who completed each course were computed. A paired t -test was used to assess the increase between mean pre- and post-test scores. The association between the participants working in public vs. private laboratories and course completion rates were determined using the chi-square test., Results: A total of 227 participants from Pakistan (March 2018 to June 2020) were registered. The largest number of registered participants and the highest completion rate were noted for AST and biosafety courses, while quality-related courses attracted a lower interest. A comparison of pre- and post-test performance using the paired mean score for the individual courses showed a statistically significant (the value of p < 0.05) improvement in 13/20 assessed courses. A higher course completion rate was observed in participants from public vs. private sector laboratories (56.8 vs. 30.8%, the value of p = 0.005)., Conclusions: Our study suggests a promising potential for open online courses (OOCs) toward addressing knowledge gaps in laboratory practice in resource limited settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saeed, Zeeshan, Farooqi, Shakoor, Jabeen, Malik, Rao and Hasan.)

The current study was designed to evaluate the antifungal properties of Datura metel L. against Rizoctonia solani Kuhn. To achieve this objective, six concentrations of leaves & stem methanol extract of D. metel viz. 1%, 1.5%, 2%, 2.5%, 3% & 3.5% were tested against R. solani in vitro. Leaf extract of D. metel was found more effective as its 3.5% concentration caused 75% retardation in test fungal growth as compared to the stem extract. D. metel methanolic leaf extract was fractioned between n-butanol, n-hexane, chloroform & ethyl acetate & bioactivities of isolated fractions were tested against R. solani. The chloroform fraction was found highly effective, as its concentrations 0.1% & 0.01% caused 27% & 21% growth inhibition respectively. So, this particular chloroform fraction was further analyzed to identify various chemical constituents through GC-MS (Gas chromatography mass spectroscopic) analysis. Twelve phyto-constituents viz. eugenol, 2-pentadecanone 6,10,14 trimethyl, pentadecanoic acid, pentadecanoic acid, 1 4-methyl- methyl ester, phytol, 9,12,15-octadecatrienoic acid, heptacosane, n-hexadecanoic, 6-octadecanoic acid, 9, 12 octadecanoic acid, dodecanoic & tetradecanoic acids were identified. So, the present study concluded that the presence of these bioactive constituents make D. metel as an effective antifungal agent against R. solani.

Multisystem Inflammatory Syndrome in Children (MIS-C), representing a new entity in the spectrum of manifestations of COVID-19, bears symptomatic resemblance with Kawasaki Disease (KD). This review explores the possible associations between KD and the human coronaviruses and discusses the pathophysiological similarities between KD and MIS-C and proposes implications for the pathogenesis of MIS-C in COVID-19. Since 2005, when a case-control study demonstrated the association of a strain of human coronavirus with KD, several studies have provided evidence regarding the association of different strains of the human coronaviruses with KD. Thus, the emergence of the KD-like disease MIS-C in COVID-19 may not be an unprecedented phenomenon. KD and MIS-C share a range of similarities in pathophysiology and possibly even genetics. Both share features of a cytokine storm, leading to a systemic inflammatory response and oxidative stress that may cause vasculitis and precipitate multi-organ failure. Moreover, antibody-dependent enhancement, a phenomenon demonstrated in previous coronaviruses, and the possible superantigenic behavior of SARS-CoV-2, possibly may also contribute toward the pathogenesis of MIS-C. Lastly, there is some evidence of complement-mediated microvascular injury in COVID-19, as well as of endotheliitis. Genetics may also represent a possible link between MIS-C and KD, with variations in FcγRII and IL-6 genes potentially increasing susceptibility to both conditions. Early detection and treatment are essential for the management of MIS-C in COVID-19. By highlighting the potential pathophysiological mechanisms that contribute to MIS-C, our review holds important implications for diagnostics, management, and further research of this rare manifestation of COVID-19., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Type 2 diabetes mellitus and Alzheimer's disease (AD), both are chronic and progressive diseases. Many cardiovascular and genetic risk factors are considered responsible for the development of AD and diabetes mellitus (DM). Genetic risk factor such as apolipoprotein E (APOE) plays a critical role in the progression of AD. Specifically, APOEε4 is genetically the strongest isoform associated with neuronal insulin deficiency, altered lipid homeostasis, and metabolism, decreased glucose uptake, impaired gray matter volume, and cerebrovascular functions. In this article, we have summarized the mechanisms of cardiovascular disturbances associated with AD and DM, impact of amyloid-β aggregation, and neurofibrillary tangles formation in AD. Moreover, cardiovascular risk factors leading to insulin resistance (IR) and amyloid-β aggregation are highlighted along with the effects of APOE risk alleles on cerebral, lipid, and cholesterol metabolism leading to CVD-mediated IR. Correspondingly, the contribution of IR, genetic and cardiovascular risk factors in amyloid-β aggregation, which may lead to the late onset of AD and DM, has been also discussed. In short, IR is related to significantly lower cerebral glucose metabolism, which sequentially forecasts poorer memory performance. Hence, there will be more chances for neural glucose intolerance and impairment of cognitive function in cardiac patients, particularly APOEε4 carriers having IR. Hence, this review provides a better understanding of the corresponding crosstalk among different pathways. This will help to investigate the rational application of preventive measures against IR and cognitive dysfunction, specifically in APOEε4 carriers' cardio-metabolic patients., (© 2021 Wiley Periodicals LLC.)

After lung cancer, breast cancer is the second leading cause of death in women. If breast cancer is detected early, mortality rates in women can be reduced. Because manual breast cancer diagnosis takes a long time, an automated system is required for early cancer detection. This paper proposes a new framework for breast cancer classification from ultrasound images that employs deep learning and the fusion of the best selected features. The proposed framework is divided into five major steps: (i) data augmentation is performed to increase the size of the original dataset for better learning of Convolutional Neural Network (CNN) models; (ii) a pre-trained DarkNet-53 model is considered and the output layer is modified based on the augmented dataset classes; (iii) the modified model is trained using transfer learning and features are extracted from the global average pooling layer; (iv) the best features are selected using two improved optimization algorithms known as reformed differential evaluation (RDE) and reformed gray wolf (RGW); and (v) the best selected features are fused using a new probability-based serial approach and classified using machine learning algorithms. The experiment was conducted on an augmented Breast Ultrasound Images (BUSI) dataset, and the best accuracy was 99.1%. When compared with recent techniques, the proposed framework outperforms them.

People spend increasing amounts of time at home, yet the indoor home environment remains understudied in terms of potential exposure to toxic trace metals. We evaluated trace metal (and metalloid) concentrations (As, Cu, Cr, Mn, Ni, Pb, and Zn) and health risks in indoor dust from homes from 35 countries, along with a suite of potentially contributory residential characteristics. The objective was to determine trace metal source inputs and home environment conditions associated with increasing exposure risk across a range of international communities. For all countries, enrichments compared to global crustal values were Zn > Pb > Cu > As > Cr > Ni; with the greatest health risk from Cr, followed by As > Pb > Mn > Cu > Ni > Zn. Three main indoor dust sources were identified, with a Pb-Zn-As factor related to legacy Pb sources, a Zn-Cu factor reflecting building materials, and a Mn factor indicative of natural soil sources. Increasing home age was associated with greater Pb and As concentrations (5.0 and 0.48 mg/kg per year of home age, respectively), as were peeling paint and garden access. Therefore, these factors form important considerations for the development of evidence-based management strategies to reduce potential risks posed by indoor house dust. Recent findings indicate neurocognitive effects from low concentrations of metal exposures; hence, an understanding of the home exposome is vital.

Background: The subcontinent is famous for its variety of seasonal foods cooked in vegetable seed cooking oils at elevated heating. Oils are often of poor quality that effect to consumer health. The work, therefore, planned to examine the effects of heat on the quality of mixed canola cooking oils (MCCOs). MCCOs were analyzed by preparing volatile fatty acid methyl esters (FAMEs) and for physiochemical properties., Results: A major change was observed in the FAs composition of various MCCOs as coded K-1 to K-5. MCCOs were found rich in unsaturated 9-octadecanoic acid (oleic acid C 18:1 ) and 9, 12-octadecadienoic acid (linoleic acid C 18:2 ) along saturated octadecanoic acid (stearic acid C 18:0 ). Results reveals that canola oil samples are mixed in the range of 4-30% with other vegetable oils and animal fats. The quality of canola cooking oils further reduced after heating to 100 °C, 200 °C and 350 °C, respectively. Quality parameters of MCCOs were significantly altered after heating and found as color (510-520 nm to 570-600 nm), mass 220-237 g to 210-225 g, volume 250 mL to 239 mL, pH (6.76-6.89), specific gravity (0.87-0.92), refractive index (1.471-1.475), saponification value (SV) (0.7-2.5), un-saponifiable matter (2.4-9.8%) and acid value (AV) (1.20-5.0 mg KOH)., Conclusion: Heating of oils at elevated temperature have shown a significant effect on pH, specific gravity and un-saponifiable matter (p-value < 0.05). Large changes in the physicochemical parameters and FAs composition help to develop a conclusion that cooking at high temperatures affects the quality of mixed canola cooking oils., (© 2022. The Author(s).)

Hypothetical proteins (HPs) are non-predicted sequences that are identified only by open reading frames in sequenced genomes, but their protein products remain uncharacterized by any experimental means. The genome of every species consists of HPs that are involved in various cellular processes and signaling pathways. Annotation of HPs is important as they play a key role in disease mechanisms, drug designing, vaccine production, antibiotic production, and host adaptation. In the case of bacteria, 25-50% of the genome comprises HPs, which are involved in metabolic pathways and pathogenesis. The characterization of bacterial HPs helps to identify virulent proteins that are involved in pathogenesis. This can be done using in-silico studies, which provide sequence analogs, physiochemical properties, cellular or subcellular localization, structure and function validation, and protein-protein interactions. The most diverse types of virulent proteins are exotoxins, endotoxins, and adherent virulent factors that are encoded by virulent genes present on the chromosomal DNA of the bacteria. This review evaluates virulent HPs of pathogenic bacteria, such as Staphylococcus aureus, Chlamydia trachomatis, Fusobacterium nucleatum, and Yersinia pestis. The potential of these HPs as a drug target in bacteria-caused infectious diseases, along with the mode of action and treatment approaches, has been discussed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

We compared candidemia due to Candida auris and other non-C.auris cases in hospitalized COVID-19 patients over a period of 9 months at our institution. Candidemia cases in all admitted patients (with or without COVID-19) from April to December 2020 were identified. Electronic records were accessed to record clinical data of COVID-19 patients with candidemia. For statistical analysis, independent samples Mann-Whitney U test was used for continuous and Fisher's exact test was used for categorical variables.A total of 26 candidemia cases (four C.auris, 22 non-C.auris) in 2438 admitted COVID-19 (10.7 per 1000 admissions) and 59 candidemia cases (six C.auris, 53 non-C.auris) in admitted non-COVID patients (8.2 per 1000 admission) were identified. The proportion of C.auris candidemia in COVID-19 and non-COVID-19 patients was 15.4 and 10%, respectively. 4/26 of COVID-19 candidemia patients were aged ≤ 15 years (10 months--15 years). Comparison of C.auris and non-C. auris candidemia cases reveal significant difference in prior antifungal exposure, present in 100% C. auris candidemia versus 27% non-C. auris candidemia patients (P-value 0.014). Although not statistically significant, C. auris candidemia patients had a longer stay in hospital before candidemia (20 vs. 9 days), higher isolation rate of multidrug resistant bacteria (100 vs. 50%), increased rate of prior colonization of Candida species (50 vs. 14%) and lower mean beta-d-glucan levels (48.73 pg/ml vs. 138.146 pg/ml). Both C. auris and non-C. auris COVID-19 patients had similar mortality rate (67 vs. 65%). A significant number of critically ill COVID-19 patients developed candidemia in our study highlighting the need for prompt diagnosis and management., Lay Summary: 26 candidemia cases (4 Candida auris;22 non-C. auris) in COVID-19 patients (April-December 2020) are reported from Pakistan. Compared to non-C. auris, C. auris candidemia patients had higher prior antifungal exposure, longer hospital stay, higher rates of MDR bacteria and Candida colonization., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Blue mold pathogen, isolated from infected Allium cepa L., was identified as a Penicillium species through morphological and molecular characterisation. Internal Transcribed spacer (ITS) region of ribosomal DNA (rDNA) was utilised for DNA sequencing. Basic Local Alignment Search Tool (BLAST) analysis has found the maximum similarity index of the fungus to be 82.39% with the Uncultured Penicillium clone (Accession: MF535522). So, the isolated Penicillium specie is the first reported specie of the genus that infects onion. A phylogenetic tree was constructed to establish a relationship of the isolated fungus with the most relevant species reported on GenBank. Extracts of Pennisetum flaccidum Griseb. were evaluated against the isolated fungus as a potential biocontrol agent. Among the five tested methanol concentrations (0.5%, 1%, 1.5%, 2% and 2.5%) of each plant part (root, inflorescence and foliage), 0.5% root extract showed maximum growth retardation, i.e. 89%. For bioassay-guided fractionation, the root extract was partitioned in n -hexane, chloroform, n -butanol and ethyl acetate. Ethyl acetate (1%) was proved to be the most potent one. Phytochemical screening has confirmed the occurrence of terpenoids, tannins, saponins and alkaloids. The applied molecular approach has deduced that the Penicillium specie collected from Pakistan might be novel. This study can be concluded that P. flaccidum contains potent phytochemicals which might be used as antifungal agent against Penicillium species., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of King Saud University.)

Objective: To determine the trend of resistance to antimicrobials in Streptococcus pneumoniae infections, and the impact of new Clinical and Laboratory Standards Institute guidelines on 1211 among meningeal isolates., Methods: The descriptive observational retrospective study was conducted at the Aga Khan University Hospital laboratory in Karachi, and comprised Streptococcus pneumoniae isolation and antimicrobial susceptibility data over a period of 24 years, from 1993 to 2016, which was compared in terms of pre-2008 and post-2008 data, which was analysed using SPSS 19., Results: Of the 7415 non-duplicate isolates identified, 4700(63.4%) were from male patients and 2,715(36.6%) were from female patients. The overall mean age of the patients was 38±27 years. Penicillin resistance in non-meningeal isolates during the two periods was not significantly different (p>0.05), but a significant rise in penicillin resistance in meningeal isolates was observed in the second period (p<0.05). High resistance rates were observed for co-trimoxazole, tetracycline and erythromycin, and an increased trend of multi-drug resistant strains was also noted from 1999 {n=35/317(11%)} to 2016 {n=110/314 (36%)}., Conclusions: The emergence of multi-drug resistant strains was evident. The spike in penicillin-resistant Streptococcus pneumoniae in meningeal isolates may have been due to the revised guidelines by the Clinical and Laboratory Standards Institute.

Introduction. Invasive infections with Candida glabrata are a global concern due to poor clinical outcomes and propensity to acquire resistance to antifungal agents . Hypothesis/Gap Statement. Monitoring emerging resistance and trends in Candida glabrata , an important agent of candidemia in Pakistan, is critical for patient management; data that is missing from Pakistan . Aim. Thus, this study evaluated antifungal resistance and MICs) distribution in invasive C. glabrata isolates from Pakistan . Methods. This cross-sectional and retrospective study was conducted from January 2009 to March 2020 at a clinical laboratory in Pakistan that has a nation-wide network. Antifungal susceptibility data of 277 candidemia, deep organ and soft tissue (invasive) C. glabrata sensu lato isolates against fluconazole, itraconazole, voriconazole, posaconazole, anidulafungin, micafungin, caspofungin and amphotericin B was retrieved. Susceptibility testing was performed using colorimetric broth microdilution and interpreted using CLSI criteria. Demographics, clinical history and outcome were studied. Chi-square test was used to demonstrate association between antifungal resistance and clinical characteristics of the patients . Results. We identified 277 patients with invasive C. glabrata infection. Of which 48 (18.4%) isolates were resistant to fluconazole (MIC ≥64 mg l -1 ), one isolate each was resistant to amphotericin (MIC=2 mg l -1 ), anidulafungin (MIC=1 mg l -1 ) and micafungin (MIC=0.5 mg l -1 ). MIC 90 for fluconazole was 64 mg l -1 and other triazoles 2 mg l -1 , caspofungin 0.12 mg l -1 , anidulafungin 0.06 mg l -1 , micafungin 0.03 mg l -1 and amphotericin 0.5 mg l -1 . Fluconazole MIC ≥64 mg l -1 , caspofungin MIC >0.06 mg l -1 and amphotericin MIC >0.25 mg l -1 (above MIC 50 ) were significantly associated with patient being alive at the time of reporting, no use of healthcare devices, nor infection with other fungi. Fluconazole resistance was significantly associated with prior antifungal use by the patient . Conclusion. Surveillance data of antifungal resistance among common Candida species should be monitored closely for identification of resistant strains.

The required adjustments precipitated by the coronavirus disease 2019 crisis have been challenging, but also represent a critical opportunity for the evolution and potential disruptive and constructive change of medical education. Given that the format of medical education is not fixed, but malleable and in fact must be adaptable to societal needs through ongoing reflexivity, we find ourselves in a potentially transformative learning phase for the field. An Association for Medical Education in Europe ASPIRE Academy group of 18 medical educators from seven countries was formed to consider this opportunity, and identified critical questions for collective reflection on current medical education practices and assumptions, with the attendant challenge to envision the future of medical education. This was achieved through online discussion as well as asynchronous collective reflections by group members. Four major themes and related conclusions arose from this conversation: Why we teach: the humanitarian mission of medicine should be reinforced; what we teach: disaster management, social accountability and embracing an environment of complexity and uncertainty should be the core; how we teach: open pathways to lean medical education and learning by developing learners embedded in a community context; and whom we teach: those willing to take professional responsibility. These collective reflections provide neither fully matured digests of the challenges of our field, nor comprehensive solutions; rather they are offered as a starting point for medical schools to consider as we seek to harness the learning opportunities stimulated by the pandemic.

Microplastic pollution has received substantial international attention in terrestrial and marine environments and in the atmosphere. In this study, we assessed microplastic pollution and analyzed the accumulation of microplastics in the gills of fish caught in the Zhoushan fishing ground from September 2017 to March 2018. The average abundance of microplastics was 0.49 ± 0.54 particles/gill and frequently found chemical polymers was polyethylene terephthalate. We also conducted experiments on microplastic pollution in the water and the working solutions used during the analytical process and found that the source of microplastic contamination was the solutions used. Moreover, we evaluated and scored experimental quality control and quality assurance adapted from the 'Total Accumulated Score' method. Valuable steps are recommended in order to produce reliable results and improve the quality of results in microplastic analyses., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Undiagnosed allergic bronchopulmonary aspergillosis (ABPA) can lead to chronic persistent symptoms. In country like Pakistan where tuberculosis (TB) is endemic, a significant proportion of ABPA patients are misdiagnosed as smear negative TB before reaching a diagnosis of ABPA due to chronicity of symptoms.This lead to empiric use of ATT(Anti-tuberculous therapy) and delay in primary diagnosis. The aim of the study is to determine such proportion of ABPA patients., Material and Methods: This retrospective study was conducted at the outpatient pulmonology clinic of a tertiary care hospital in Karachi, Pakistan from January 2017 to December 2018. Xpert MTB/Rif, TB smear and culture were performed in all patients to rule out active TB., Results: A total 167 of ABPA patients were included. Mean age of the patients was 41.9 ± 13.0 years, 91(54.5%) were female and 71 (42.5%) patients had received ATT in past. Out of these 71 patients, 63 (88.7%) patients were diagnosed as smear negative TB and received empiric ATT. Among 63 patients, 52 (82.5%) patient had received ATT once, 8 (12.6%) twice and 3 (4.7%) patients had received empiric ATT thrice. Of these 27 (16.16%) patients had already developed long term complications at the time of diagnosis of ABPA and 17 (62.96%) patients were in empiric TB treatment group., Conclusion: Patient with ABPA frequently received empiric ATT as smear negative TB in high TB burden country. This results in over diagnosis of TB and unnecessary use of global resource. When Gene Xpert negative alternate diagnosis should be considered., Competing Interests: None to declare., (© 2021 Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd.)

Microplastic (MP) pollution in marine environments and organisms has received substantial international attention. However, long-term field studies of MPs are scarce. Here, we assessed the seasonal variation in MP abundance in the Zhoushan fishing ground (ZFG), one of the most abundant and productive fishing grounds worldwide, and analyzed the long-term MP accumulation in fish gastrointestinal tracts from September 2017 to June 2018. The most common MP particles in the ZFG were polyethylene terephthalate and polypropylene. After four seasons of continuous monitoring, we did not find accumulation of MPs in the fish after 10% KOH digestion. In total, 254 MP particles were removed from the gastrointestinal tracts of all fish. The average number of particles per fish was lower than that reported in previous global marine studies. There were significant differences among species. Moreover, this study provides the calculation of the weight of MPs ingested by fish and an estimate of the load of accumulated MPs in fish. According to the estimation, the load of MPs ingested by fish annually was approximately 3 kg in ZFG. These findings provide the long-term evidence of MP contamination in biota from the ZFG. The amounts of MPs ingested by fish require more detailed and improved investigation and estimation in further studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: To compare serum β-D-glucan (BDG) levels in candidaemia with different Candida species, especially C. auris., Methods: Aga Khan University clinical laboratory database was retrospectively reviewed from January 2015 to December 2019. Blood culture positive cases with any Candida species and concomitant BDG level were included., Results: Among the 192 cases included in our study, 48 were C. albicans, 54 C. auris, eight C. glabrata, 32 C. parapsilosis, 43 C. tropicalis and seven other Candida species. The level of BDG was significantly lower in C. auris (median 62.43, interquartile range (IQR) 12.80-182.94 pg/mL) compared to C. albicans (median 266.83, IQR 66.29-523.43 pg/mL) and C. tropicalis (median 324.41, IQR 105.20-523.44 pg/mL). The sensitivity of serum BDG was significantly lower for C. auris (43.75%, 95% CI 29.5-58.8%) than C. tropicalis (79.07%, 95% CI 64.0-90.0%)., Discussion: Serum BDG has lower sensitivity in patients with suspected C. auris candidaemia in our setting. Considering that C. auris has higher morbidity and mortality than other species, a more sensitive test is required., (Copyright © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Neonatal candidemia leads to high morbidity and mortality in developing countries. We studied the trends, spectrum and antifungal resistance in neonatal candidemia isolates from the year 2014 to 2019., Methods: This was a cross-sectional study conducted at the Aga Khan University, Pakistan. Neonates with positive blood cultures with Candida species were retrospectively identified from the laboratory database (2014-2018) and prospectively in 2019 where clinical information was also collected as part of routine laboratory reporting., Results: We identified 669 neonates with Candida species in blood cultures. Three hundred forty-six neonates had early-onset disease (EOD age ≤7 days) and 323 had late-onset disease (LOD age >7 days). Non-albicans Candida species (86.7%) were predominant versus C. albicans (13.3%; P-value 0.024) with Candida tropicalis being most common in both EOD and LOD. Candida pelliculosa and Candida guilliermondii were associated with EOD and C. albicans with LOD. Isolation of fluconazole nonsusceptible non-albicans Candida species was significantly higher in early-onset (5.9%) versus late-onset (2%) neonatal candidemia (P-value 0.005; crude odds ratio [COR] 2.73, 95% CI: 1.34-5.53). LOD in neonates was more likely associated with the use of vancomycin (COR 3.89, 95% CI: 1.39-10.89). EOD was more likely seen in patients with vaginal delivery (COR 4.16, 95% CI: 1.42-12.23) and in neonates with respiratory distress leading to intensive care unit admission (COR 3.31, 95% CI: 1.05-10.42)., Conclusions: Non-albicans Candida species were increasingly isolated from neonates with candidemia during recent years from Pakistan. Amphotericin remains first-line option for neonatal candidemia in our setting as fluconazole nonsusceptible Candida species are commonly isolated., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: To explore epidemiology, clinical profiles and contribution of reproductive and non-reproductive risk factors in breast cancer development., Methods: The case-control study was conducted from October 2017 to March 2018 at Quaid-i-Azam University, Islamabad, Pakistan, and comprised breast cancer patients and age-matched controls recruited from the Bahawalpur Institute of Nuclear Medicine and Oncology, and the Bahawal Victoria Hospital, Bahawalpur. Socio-demographic data, family history of cancer, reproductive health and lifestyle factors were recorded using a structured questionnaire. Data was analysed using SPSS 21 and Stata/IC 14.1., Results: Of the 326 women, 163(50%) each were cases and controls. The mean age for both the groups was identical at 46.04±10.62 years. Positive family history and hypertension were significantly linked to increased breast cancer risk (p<0.05), while intense physical activity, increased anthropometric measurements and breastfeeding per child in months were inversely associated with the risk (p<0.05)., Conclusions: Established risk factors for breast cancer were reaffirmed.

Background and Objectives: The Covid-19 pandemic has caused large-scale disruption in almost all educational programs across the world. Planning and rapid implementation of assessment through an online format presents the next set of novel challenges that must be addressed by academic administrations across the globe., Methods: This cross-sectional study was conducted between March to August 2020 at the Aga Khan University Medical College. Two hundred medical students of year 1 and 2 participated in the study. We describe the planning, processes, and outcomes of online assessments using video communication platforms conducted at a private university in Pakistan. Standardized protocols were written and piloted, extensive training of student, proctors and staff for preparation and conduct of online assessments were developed. Feedback was recorded after each session and suggestions were incorporated in subsequent high-stakes assessments., Results: A total of three pilot assessments were conducted to identify issues and process refinement. Commercially available lockdown browser and ZOOM were used in the first pilot; 80% of the class was unable to launch lockdown browser and laptops required repeated reload/reboot. For the second pilot assessment, University's VLE page & MS Teams was trailed. Issues with internet connectivity, VLE page slowdown, and suboptimal recording feature in MS Teams were identified. For the final pilot assessment, phased launching of VLE page with single test item per page was implemented with success. The students reported that attempting the online exam on VLE with ZOOM support was user friendly. Ninety percent of the class was supportive of the continuing with the online assessments., Conclusion: In order to device an effective protocol for e-assessments conducting multiple trial runs, and incorporating feedback from all stakeholders is a necessity., Competing Interests: Conflict of interest: None., (Copyright: © Pakistan Journal of Medical Sciences.)

Bronchiectasis unrelated to cystic fibrosis (non-CF bronchiectasis) has become a major respiratory disease in developing nations. The dilated mucus filled airways promote bacterial overgrowth followed by chronic infection, bronchial inflammation, lung injury and re-infection. Accurate pathogen identification and antimicrobial susceptibility allowing appropriate treatment, in turn, may break this vicious cycle.  This study aimed to gain kowledge about the spectrum and antimicrobial spectrum of pathogen yielded from respiratory specimens in adult patients with acute exacerbation of non-cystic fibrosis (CF) bronchiectasis. This cross-sectional study was performed at the pulmonology clinics of the Aga Khan University, Karachi, Pakistan from 2016-2019. Respiratory specimens were collected from adult patients with acute exacerbation of non-CF bronchiectasis presenting in pulmonology clinics. Microbial cultures were performed using standard methodology. Susceptibility testing was performed and interpreted using Clinical Laboratory Standard Institute criteria.  A total of 345 positive cultures from 160 patients presenting with acute exacerbation were evaluated. The most frequent organisms were Pseudomonas aeruginosa (n=209) followed by Hemophilus influenzae (n=40) and Staphylococcus aureus (n=24). High rates of antimicrobial resistance were found in all these pathogens. Proportion of Pseudomonas aeruginosa strains resistant to ciprofloxacin, imipenem, ceftazidime and piperacillin-tazobactam were 27.1%, 16.8%, 14.8% and 13.1% respectively. 65% of Hemophilus influenzae strains were resistant to cotrimoxazole and ciprofloxacin and 66.7% of Staphylococcus aureus strains were resistant to methicillin. High antimicrobial resistance in non-CF bronchiectasis patients against commonly used antimicrobials is a concern and highlight need for urgent community level interventions to improve clinical outcome in these patients.

Burn patients are at great risk of developing fungal wound infections. Fungi are frequently cultured from burn tissue specimens alone or in combination with bacteria. However, the spectrum of fungi in burn patients from Pakistan has not been reported previously. A retrospective laboratory-based study was conducted and data on all positive cultures from burn wounds were included. During the study period, specimens were cultured on appropriate media and incubated for anaerobic and aerobic growth. Clinical and demographic information recorded during clinical reporting was also collected. Seventy tissue cultures were positive for microorganisms. Of these, 27 (39%) had growth of either filamentous mold (17 cases) or Candida species (14 cases). Two cases had growth of both mold and yeast. Aspergillus flavus was the most common mold (9) followed by Fusarium species (3). Candida tropicalis was the most common yeast (7) followed by Candida parapsilosis (4). There was concomitant bacterial growth in 19 cases, predominantly of Staphylococcus aureus (13), Pseudomonas aeruginosa (8), and other Gram-negative rods (6). Of the 35 patients in which treatment history was available, 33 were receiving broad-spectrum antibiotics. A high incidence of fungal isolation was seen in this study. Their findings are consistent with the global increase in fungal infections in burn wounds. High index of suspicion by clinicians and revision of culture protocols in burn patients may be warranted for optimal patient management., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Burn Association. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Family with sequence similarity 26, member F (FAM26F) is an important innate immunity modulator playing a significant role in diverse immune responses, however, the association of FAM26F expression with HBV infection is not yet known. Thus, the current study aims to explore the differential expression of FAM26F in vitro in HepAD38 and HepG2 cell lines upon HBV infection, and in vivo in HBV infected individuals. The effects of antioxidant and calcium inhibitors on the regulation of FAM26F expression were also evaluated. The expression of FAM26F was simultaneously determined with well-established HBV infection markers: IRF3, and IFN-β., Methods: The expression of FAM26F and marker genes was analyzed through Real-time qPCR and western blot., Results: Our results indicate that the differential expression of FAM26F followed the same trend as that of IRF3 and IFN-β. The in vitro study revealed that, in both HBV infected cell lines, FAM26F expression was significantly down-regulated as compared to uninfected control cells. Treatment of cells with N-acetyl-L-cysteine (NAC), EGTA-AM, BAPTA-AM, and Ru360 significantly upregulated the expression of FAM26F in both the cell lines. Moreover, in in vivo study, FAM26F expression was significantly downregulated in all HBV infected groups as compared to controls (p = 0.0007). The expression was higher in the HBV recovered cases, probably due to the decrease in infection and increase in the immunity of these individuals., Conclusion: Our study is the first to show the association of FAM26F with HBV infection. It is proposed that FAM26F expression could be an early predictive marker for HBV infection, and thus is worthy of further investigation.

Objective: To compare the knowledge, attitude and practice regarding diabetes mellitus among diabetics and non-diabetics., Methods: The cross-sectional study was conducted at the Government College University, Faisalabad, Pakistan, from December 2017 to April 2018, and comprised subjects recruited randomly from different cities of Punjab, Pakistan. Data was collected using a predesigned structured questionnaire regarding socio-demographic characteristics, general knowledge about diabetes, perception regarding indication, risk factors, diagnosis, and complications, and practices followed for treatment and management of diabetes., Results: Of the 2,000 subjects, 972(48.6%) had family history of diabetes, 1338(66.9%) were living in urban areas, 1068(53.4%) were university graduates, 804(40.2%) were employed and 1152(57.6%) belonged to socio-economically balanced families. Composite knowledge score was significantly associated with age and socio-economic status (p<0.05). A highly significant association was observed regarding family history (p<0.001), level of education (p<0.0001) and occupation (p<0.001) with composite knowledge score., Conclusions: The knowledge level about diabetes was seen to be average.

Smoking is considered as one of the major reasons behind genetic variations in cardiometabolic disorders. However, effect of nicotine via smoking on Pakistani population still needs to be elucidated. This study was aimed to investigate genetic variation among PPAR-γ, FTO, and ABCC8 genes in cardiometabolic patients along with their biochemical parameters. A total of 472 CVD patients were enrolled in this study and divided into three groups; n = 144 for PPAR-γ (C/G) variation and n = 164 in each group to investigate FTO (T/A) and ABCC8 (G/T) variation, respectively. Polymorphisms within groups were identified by using Tetra and/or Tri ARM-PCR. This study showed positive association among genetic polymorphisms in PPAR-γ, FTO, and ABCC8 groups with altered metabolic parameters in CVD patients. Findings showed that smoking is major contributory factor for genetic polymorphism that was strongly associated with elevated blood glucose and serum TGs accompanying PPAR-γ, FTO, and ABCC8 genetic polymorphism in 25%, 24%, and 20% in smokers and 11%, 10%, and 5% in non-smoker CVD patients, respectively. However, highest polymorphism occurred in PPAR-γ both in smokers and non-smoker CVD patients that show that smoking-mediated gene polymorphism might be a contributory factor in provoking CVD risk approximately twice in smokers as compared to that in non-smoker CVD patients.

Insulin resistance (IR) and type 2 diabetes mellitus (T2DM) is a leading cause of deaths due to metabolic disorders in recent years. Molecular mechanisms involved in the initiation and development of IR and T2DM are multiples. The major factors include mitochondrial dysfunction which may cause incomplete fatty acid oxidation (FAO). Oleic acid upregulates the expression of genes causing FAO by deacetylation of PGC1α by PKA-dependent activation of SIRT1-PGC1α complex. Another potent factor for the development of IR and T2DM is endothelial dysfunction as damaged endothelium causes increased release of inflammatory mediators such as TNF-α, IL-6, IL-1β, sVCAM, sICAM, E-selectin and other proinflammatory cytokines. While, on the other hand, oleic acid has the ability to regulate E-selectin, and sICAM expression. Rest of the risk factors may include inflammation, β-cell dysfunction, oxidative stress, hormonal imbalance, apoptosis, and enzyme dysregulation. Here, we have highlighted how oleic acid regulates underlying causatives factors and hence, keeps surpassing effect in prevention and treatment of IR and T2DM. However, the percentage contribution of these factors in combating IR and ultimately averting T2DM is still debatable. Thus, because of its exceptional protective effect, it can be considered as an improved therapeutic agent in prophylaxis and/or treatment of IR and T2DM.

MicroRNAs (miRNAs) are non-coding RNAs that are associated with adipokine homeostasis and insulin resistance. Whereas, smoking can disturb metabolic homeostasis. Present study was aimed to investigate the level of miRNA-141 in experimental animal model that were exposed with graded doses of nicotine. We further aimed to investigate the possible interplay of miRNA-141 expression change with adipokine homeostasis and occurrence of insulin resistance in nicotine-exposed experimental animals. Nicotine (0.5, 1.0, 3.0 and 6.0 mg/Kg) was administered to early adolescent; postnatal days ranging from 25 to 30 Wistar rats for one month. Serum was analyzed for leptin, adipokines, IL-6, MDA, HbA1c, insulin, G6PDH, hexokinase, and lipid profile. While miRNA-141 expression level was determined in plasma. Higher doses of nicotine were associated with higher glucose, HbA1c, leptin, IL-6, MDA and lipids levels, while, insulin, adiponectin, G6PDH, hexokinase and HDL levels were lower. Higher doses of nicotine also impaired glucose tolerance and exhibited significant increase in miR-141 expression signifying that nicotine exposure may influence adipokines regulation altering glycemic profile. This is accompanied with aggravated inflammatory responses where genetic expression of miRNA-141 can be an accessible biomarker for metabolic disturbances with insulin resistance and glucose intolerance., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Diagnosis of lower respiratory tract infections (LRTI) depends on the presence of clinical, radiological and microbiological findings. Endotracheal suction aspirate (ETSA) is the commonest respiratory sample sent for culture from intubated patients. Very few studies have compared quantitative and semi-quantitative processing of ETSA cultures for LRTI diagnosis. We determined the diagnostic accuracy of quantitative and semi-quantitative ETSA culture for LRTI diagnosis, agreement between the quantitative and semi quantitative culture techniques and the yield of respiratory pathogens with both methods., Methods: This was a cross-sectional study conducted at the Aga Khan University clinical laboratory, Karachi, Pakistan. One hundred and seventy-eight ETSA samples sent for routine bacteriological cultures were processed quantitatively as part of regular specimen processing method and semi-quantitatively. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy was calculated for both methods using clinical diagnosis of pneumonia as reference standard. Agreement between the quantitative and semi quantitative methods was assessed via the kappa statistic test. Pathogen yield between the two methods was compared using Pearson's chi-square test., Results: The quantitative and semi-quantitative methods yielded pathogens in 81 (45.5%) and 85 (47.8%) cases respectively. There was complete concordance of both techniques in 155 (87.1%) ETSA samples. No growth was observed in 45 (25.3%) ETSA specimens with quantitative culture and 37 (20.8%) cases by semi-quantitative culture. The diagnostic accuracy of both techniques were comparable; 64.6% for quantitative and 64.0% for semi-quantitative culture. The kappa agreement was found to be 0.84 (95% CI, 0.77-0.91) representing almost perfect agreement between the two methods. Although semi-quantitative cultures yielded more pathogens (47.8%) as compared to quantitative ETSA cultures (45.5%), the difference was only 2.3%. However, this difference achieved statistical (chi-square p-value < 0.001) favoring semi-quantitative culture methods over quantitative culture techniques for processing ETSA., Conclusion: In conclusion, there is a strong agreement between the performances of both methods of processing ETSA cultures in terms of accuracy of LRTI diagnosis. Semi-quantitative cultures of ETSA yielded more pathogens as compared to quantitative cultures. Although both techniques were comparable, we recommend processing of ETSA using semi-quantitative technique due to its ease and reduced processing time.

Despite a high burden of chronic pulmonary aspergillosis (CPA) in Pakistan, Aspergillus -specific IgG testing is currently not available. Establishing cut-offs for Aspergillus -specific IgG for CPA diagnosis is crucial due to geographical variation. In settings such as Pakistan, where non- Aspergillus fumigatus (mainly A. flavus ) Aspergillus species account for the majority of CPA cases, there is a need to explore additional benefit of Aspergillus flavus -specific IgG detection along with A. fumigatus -specific IgG detection. This study was conducted at the Aga Khan University, Karachi, Pakistan after ethical approval. Serum for IgG detection were collected after informed consent from healthy controls ( n = 21), diseased controls (patients with lung diseases, n = 18), and CPA patients ( n = 21). A. fumigatus and A. flavus IgG were detected using Siemens immulite assay. The sensitivity and specificity of A. fumigatus -specific IgG were 80.95% and 82.05%, respectively at a cut-off of 20 mg/L. The sensitivity and specificity of A. flavus -specific IgG were 80.95% and 79.49% at a cut-off of 30 mg/L. We report, for the first time, performance of A. flavus -specific IgG for CPA diagnosis. Although there was no statistically significant difference between the performance of both antigens, it seems contextually relevant to include A. flavus IgG in the CPA diagnostic algorithm in regions with higher non- A. fumigatus CPA infections.

Nitric oxide is a key regulating factor for physiological functions, when elevated during inflammatory conditions, NO, can lower endogenous antioxidants level. Naringenin, a bioflavonoid has shown to possess anti-inflammatory action. However, its role in NO-mediated responses has not been elucidated till date. This study was designed to investigate antioxidant potential of naringenin against inflammation-mediated nitric oxide overproduction and antioxidant status with an improved glycemic profile in diabetic rats. From total rats, Group 1 received normal saline, while remaining received single intraperitoneal injection of alloxan and were then equally divided into group 2, 3, and 4, which latter received no-treatment, metformin (50 mg kg -1  day -1 ) and naringenin (50 mg kg -1  day -1 ), respectively, for 1 month. Results showed that naringenin significantly downregulated levels of glucose (p < .05), lipid profile, inflammatory biomarkers, and nitric oxide (p < .01) in alloxan-induced diabetic rats. It also improved SOD level as compared to that of metformin treatment. This work delivers that naringenin exerts antioxidant effect by downregulating inflammation-mediated nitric oxide overproduction. PRACTICAL APPLICATIONS: Naringenin is a well-recognized member of bioflavonoids and is commonly present in citrus fruits like oranges, grapes, and berries. The foremost property of naringenin is its antioxidant potential, which aids in decreasing the burden of oxidative stress by declining the generation of free radicals. The overproduction of these oxygen or nitrogen reactive species are considered as underlying cause of undesired biological activities like O 2 - ∙ or nitrite-mediated inflammation and altered metabolic parameters. Hence, this study was designed to investigate the antioxidant potential of naringenin as natural flavone to downregulate the inflammation-mediated nitric oxide overproduction and improve glycemic profile. The therapeutic perspective of naringenin from current study against nitric oxide overproduction and to eradicate inflammation via controlling of levels of pro-inflammatory mediators suggests that naringenin holds the forthcoming vision as a supportive constituent alone or in combination with some other conventional medicinal agents against conditions like metabolic disorders., (© 2020 Wiley Periodicals LLC.)

We aimed to investigate the association of single nucleotide polymorphism of Pro/Ala (rs1801282) in peroxisome proliferator-activated receptor-gamma (PPAR-γ) gene with risk factors of diabetes mellitus (DM) in cardiovascular disease (CVD) patients. We recruited 244 participants from Faisalabad Institute of Cardiology and Department of Cardiology, Sargodha District Head Quarter Teaching Hospital, Pakistan. Out of 244 participants, 144 cases were CVD patients and 100 were healthy controls. CVD patients were further divided into 111 coronary artery disease (CAD) and 33 cardiomyopathy (CMP) patients. Assessment of variant specific polymorphism/mutation of Pro/Pro and Pro/Ala genotypes was done through amplification refractory mutation system polymerase chain reaction (ARMS-PCR). Further, serum biomarkers were measured to investigate the association among risk factors of DM and Pro/Ala polymorphism in PPAR-γ gene. About 31.5% Pro/Ala genotype was found in CVD patients out of which 22.5% were CAD patients and 9% were CMP patients. As a result, obesity, hypertension and smoking (35%, 23%, 21%, respectively) were observed to be the most critical risk factors accompanying Pro/Ala mutation in PPAR-γ particularly in CAD patients as compared to that in CMP patients. A similar pattern of association was observed among the elevated levels of glucose, cholesterol, triglyceride and ALT with Pro/Ala mutation in CAD patients. Further, CAD patients using ACE inhibitors (18%) and β-blockers (13%) were found to be the carriers of Pro/Ala genotype and also showed significant increase in glucose level. This study suggests that hyperglycaemia in CAD patients particularly obese, smokers and hypertensives having Pro/Ala polymorphism in PPAR-γ gene are at high risk of developing DM as clearly observed by hyperglycaemia in CAD patients., (© 2020 John Wiley & Sons Australia, Ltd.)

Background: From September 2014, a tertiary care hospital in Karachi, Pakistan, started diagnosing 3-5 cases/month of a yeast locally identified as Saccharomyces spp. resistant to fluconazole. US Centers for Disease Control and Prevention identified the isolates as Candida auris . The Pakistan Field Epidemiology and Laboratory Training Program (FELTP) and the hospital investigated the outbreak from April 2015 to January 2016., Objective: The aim of the outbreak investigation was to determine the risk factors and to inform measures to limit the spread of the organism in the hospital., Methods: Medical records, nursing schedules and infection control practices were reviewed. Sixty-two age- and sex-matched hospital controls from the same wards were identified., Results: Thirty cases (17 males) were identified (mean age = 51.6 years, age range = 2-91 years), case fatality was 53%. Multivariate logistic regression showed that a history of surgery within 90 days of diagnosis, admission to the emergency department and history of chronic kidney disease were significantly associated with C. auris infection., Discussion: This is the report of the outbreak investigation that triggered a global exploration of C. auris as a newly identified multidrug-resistant nosocomial organism, spreading within the hospital, especially among patients with invasive procedures. Unfortunately, we could not identify any specific source of the outbreak nor stop the transmission of the organism., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)