Search

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

Introduction and objective: Pulmonary mucormycosis is a rare but rapidly progressive fatal disease. Limited data exist on the outcomes and factors associated with poor prognosis of pulmonary mucormycosis. The objective of this study was to evaluate clinical characteristics, factors associated with mortality, and outcomes of pulmonary mucormycosis at a tertiary care hospital in Pakistan. Methods: This was a retrospective observational study conducted at a tertiary care hospital in Karachi, Pakistan. Medical records of hospitalized patients diagnosed with proven or probable pulmonary mucormycosis between January 2018 and December 2022 were reviewed. Univariate and regression analyses were performed to identify factors associated with mortality. Results: Fifty-three pulmonary mucormycosis patients (69.8% male) were included, with mean age of 51.19 ± 21.65 years. Diabetes mellitus was the most common comorbidity [ n = 26 (49.1%)]. Chronic lung diseases were present in [ n = 5 (9.4%)], and [ n = 16 (30.2%)] had concurrent coronavirus disease 2019 (COVID-19) pneumonia. The predominant isolated Mucorales were Rhizopus [ n = 32 (60.3%)] and Mucor species [ n = 9 (17%)]. Main radiological findings included consolidation [ n = 39 (73.6%)] and nodules [ n = 14 (26.4%)]. Amphotericin B deoxycholate was prescribed in [ n = 38 (71.7%)], and [ n = 14 (26.4%)] of patients received combined medical and surgical treatment. The median [interquartile range (IQR)] hospital stay was 15.0 (10.0–21.5) days. Intensive care unit (ICU) care was required in [ n = 30 (56.6%)] patients, with 26 (49.1%) needing mechanical ventilation. Overall mortality was seen in 29 (54.7%) patients. Significantly higher mortality was found among patients requiring mechanical ventilation 20/29 (69%, p = 0.002). Immunosuppression ( p = 0.042), thrombocytopenia ( p = 0.004), and mechanical ventilation ( p = 0.018) were identified as risk factors for mortality on multivariable analysis. Conclusion: This study provides essential insights into the clinical characteristics, outcomes, and mortality factors associated with pulmonary mucormycosis. The mortality rate was high (54.7%), particularly in patients with immunosuppression, thrombocytopenia, and those who required mechanical ventilation.

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.

Abstract Background In 2018 Pakistan initiated its national antimicrobial resistance (AMR) surveillance aligned with Global Antimicrobial Surveillance System (GLASS). To complement this surveillance, we conducted a situational analysis of AMR rates among GLASS organisms in the country. Data from published studies and from antibiograms was compared and role of antibiograms as potential contributors to national AMR surveillance explored. Methods AMR rates for GLASS specified pathogen/antimicrobials combination from Pakistan were reviewed. Data sources included published studies (2006–2018) providing AMR rates from Pakistan (n = 54) as well as antibiograms (2011–2018) available on the Pakistan Antimicrobial Resistance Network (PARN) website. Resistance rates were categorized as follows: Very low: 0–10%, Low: 11–30%, Moderate: 30–50% and High: > 50%. Results Published data from hospital and community/laboratory-based studies report resistance rates of > 50% and 30–50% respectively to 3rd generation cephalosporins, fluoroquinolones and cotrimoxazole amongst Klebsiella pneumoniae and Escherichia coli. Carbapenem resistance rates amongst these organisms remained below 30%. High (> 50%) resistance was reported in Acinetobacter species to aminoglycosides and carbapenems among hospitalized patients. The evolution of ceftriaxone resistant Salmonella Typhi and Shigella species is reported. The data showed > 50% to fluoroquinolones amongst Neisseria gonorrhoeae and the spread of methicillin resistant Staphylococcus aureus ( 50%; 2010) in hospital settings. Resistance reported in published studies aligned well with antibiogram data. The latter also captured a clear picture of evolution of resistance over the study period. Conclusion Both published studies as well antibiograms suggest high rates of AMR in Pakistan. Antibiogram data demonstrating steady increase in AMR highlight its potential role towards supplementing national AMR surveillance efforts particularly in settings where reach of national surveillance may be limited.

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

Objective: To assess the diagnostic accuracy of different cut-off values of pleural fluid adenosine deaminase levels as a diagnostic method for tuberculous pleural effusion. Method: 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

BackgroundMycetoma 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.MethodThis 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.ResultDuring 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.ConclusionThis 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.

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. Keywords: Wounds and Injuries, Fungal Infections, Diabetic Foot, Infections, Skin and Soft Tissue Infections.

IntroductionQuality-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).MethodologySeven 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.ResultsA 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).ConclusionsOur study suggests a promising potential for open online courses (OOCs) toward addressing knowledge gaps in laboratory practice in resource limited settings.

Abstract 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

Abstract Background Burden of aspergillosis is reported to be significant from developing countries including those in South Asia. The estimated burden in Pakistan is also high on the background of tuberculosis and chronic lung diseases. There is concern for management of aspergillosis with the emergence of azole resistant Aspergillus species in neighbouring countries in Central and South Asia. Hence the aim of this study was to screen significant Aspergillus species isolates at the Microbiology Section of Aga Khan Clinical Laboratories, Pakistan, for triazole resistance. Methods A descriptive cross-sectional study, conducted at the Aga Khan University Laboratories, Karachi, from September 2016–May 2019. One hundred and fourteen, clinically significant Aspergillus isolates [A. fumigatus (38; 33.3%), A. flavus (64; 56.1%), A. niger (9; 7.9%) A. terreus (3; 2.6%)] were included. The clinical spectrum ranged from invasive aspergillosis (IA) (n = 25; 21.9%), chronic pulmonary aspergillosis (CPA) (n = 58; 50.9%), allergic bronchopulmonary aspergillosis (ABPA) (n = 4; 3.5%), severe asthma with fungal sensitization (SAFS) (n = 4; 3.5%), saprophytic tracheobronchial aspergillosis (n = 23; 20.2%). Screening for triazole resistance was performed by antifungal agar screening method. The minimum inhibitory concentration (MIC) of 41 representative isolates were tested and interpreted according to the Clinical and Laboratory Standards Institute broth microdilution method. Results All the isolates were triazole-susceptible on agar screening. MICs of three azole antifungals for 41 tested isolates were found to be ≤1 ml/L; all isolates tested were categorized as triazole-susceptible, including 4 isolates from patients previously on triazole therapy for more than 2 weeks. The minimum inhibitory concentration required to inhibit the growth of 90% organisms (MIC90) of itraconazole, voriconazole and posaconazole of the representative Aspergillus isolates was 1 mg/L, 1 mg/L and 0.5 mg/L, respectively. Conclusion Triazole resistance could not be detected amongst clinical Aspergillus isolates from the South of Pakistan. However, environmental strains remain to be tested for a holistic assessment of the situation. This study will set precedence for future periodic antifungal resistance surveillance in our region on Aspergillus isolates.

Published in May - 2021

We evaluated Salmonella enterica serotype Typhi strains isolated from all body sites in Pakistan during 2013–2018. Despite an increase in overall number of localized, extensively drug-resistant Salmonella Typhi in organ infections during 2018, there was no increase in the proportion of such isolates in comparison with non–extensively drug-resistant isolates.

Summary: Background: Due to COVID-19, thousands of healthcare workers have been affected and have lost their lives in the line of duty. For the protection of healthcare workers, WHO and CDC have made standard guidelines and requirements for PPE use. N95 masks are amongst the most readily used PPE by healthcare professionals and it is highly recommended by OSHA that every make and model of N95 should go through a fit test at least once in a year. Method: A total of 30 randomly selected healthcare professionals (who were a regular user of N95 respiratory masks) were subjected to assess in-house (saccharin sodium benzoate) reagent for use for standard qualitative fit testing in our hospital. Threshold testing with the in-house reagent at three different concentrations was performed prior to establish participants' sensitivity to the reagent. After successful completion of threshold testing, fit test was performed on participants wearing an N95 mask. Results: All the participants included in the study passed the sensitivity testing with three concentrations of the reagents, while it was concluded that the concentration of the in-house reagent that was well suited for the sensitivity testing was a concentration of 1g/dl saccharin with 10g/dl sodium benzoate. For fit testing 12g/dl was found to be more appropriate. Discussion: Our study provided a low cost solution to ensure safety of healthcare workers who are regular users of N95 masks following guidelines implemented by OSHA and CDC. Conclusion: The in-house test solution prepared was found to be equally sensitive to its commercially available counterpart.

Objective: This study describes a case of disseminated phaeohyphomycotic lymphadenitis in a young female with delayed diagnosis and good clinical response after appropriate treatment. Methods: A 32-year-old female presented with erythematous to violaceous papules with oozing discharge bilaterally in her inguinal region for a few months. History revealed tuberculous meningitis 4 years earlier treated with first line anti-tuberculous therapy for 18 months, and 2 years previously she developed pigmented discharging lymph nodes bilaterally in her axillae. The histopathology of the biopsy of the axillary nodes showed chronic granulomatous inflammation with multiple branching septate fungal hyphae. She received amphotericin B for 21 days but without improvement. Biopsy from the inguinal lesions was sent for histopathology and culture. Results: Histopathology of the biopsy material showed chronic granulomatous inflammatory process with multinucleate giant cells, epithelioid cells, histiocytes, and lymphocytes with multiple branching septate fungal hyphae. Gram stain revealed moderate septate hyphae with numerous pus cells. Culture on Sabouraud dextrose agar yielded velvety olive–black colonies in the fourth week. Microscopic slide examination of culture material was suggestive of Cladophialophora species. The patient was started on voriconazole, which was continued for 6 months, and showed clinical improvement. Conclusion: Incomplete investigation of infectious lesions may delay diagnosis. Furthermore, clinical presentations are greatly influenced by the immune status of the host. Both histopathological and microbiological assessments are equally important for making a conclusive diagnosis. Anti-fungal therapy may delay the growth of fungi that normally grow within a week; thus, a longer incubation time may be warranted for fungal smear-positive samples.

Abstract Background Globally metronidazole and carbapenem resistance in anaerobic organisms is increasing necessitating continuous surveillance to guide selection of empirical treatment. In this study we have determined metronidazole resistance in anaerobes using MIC Evaluator strips (M.I.C.E strips). Carbapenem resistance was evaluated only in metronidazole resistant isolates. Material and methods The study was conducted at the Aga Khan University (AKU) Hospital laboratory, Karachi, Pakistan (2014–2017). Metronidazole and imipenem resistance was evaluated using M.I.C.E strips and minimum inhibitory concentrations (MICs) were interpreted using Clinical Laboratory Standards Institute (CLSI) criteria. Clinical details including demographics, prolonged hospital stay, malignancy, transplant, dialysis, diabetes, site of infection and outcome were analyzed for association with metronidazole resistance. Results Of the 223 clinically significant isolates, 39 (17.5%) were metronidazole resistant (excluding the inherently resistant organisms; for example Cutibacterium species). Imipenem resistance was determined in 29 metronidazole resistant isolates and of these 7 (24.1%) were found to be resistant. Proportion of metronidazole resistant strains was highest amongst Bacteroides species. A significant increase in metronidazole resistance from 12.3% in 2010–2011 to 17.5% in the current study was found. Carbapenem resistance also emerged in the period 2014–2017. Isolates from malignancy and transplant patients showed lower odds of developing metronidazole resistance (0.003(95% CI: 1.7–17.9)). Prolonged hospital stay was not associated with metronidazole resistance (1.1((95% CI: 0.5–2.5)). Conclusion The rising trend of metronidazole resistance and emergence of carbapenem resistance in anaerobic bacteria is alarming. Continued surveillance with strengthening of laboratory capacity regarding anaerobic susceptibility testing is urgently needed in Pakistan.

Abstract Background An outbreak of Candida auris began globally in 2014 including Pakistan and since then it has emerged as a nosocomial multi-drug resistant pathogen. The aim of this study was to assess the clinical spectrum and outcome of patients, from a single center in Pakistan, in whom C. auris was isolated. Methods A retrospective study was conducted on 92 patients; ≥16 years with at least one culture positive for C. auris, at the Aga Khan University Hospital Karachi, Pakistan from Sept 2014-Mar 2017.Demographics, clinical history, management and outcome were studied. A logistic regression model was used to identify the risk factors for mortality. Results We identified 92 patients with C. auris (193 isolates), of whom 52.2% were males. Mean age was 54.14 ± 20.4 years. Positive cultures were obtained after a median hospital stay of 14 days. Most patients had a history of surgery (57.6%), antibiotic use (95.6%), ICU stay (44.6%), indwelling lines (88.04%) and isolation of another multi-resistant organism (52.2%).Most patients were symptomatic (70.7%). Amongst these, 38 had candidemia while 27 had non-candidemia infections. Sites of infection included central lines (35), urinary tract (19), peritonitis (4), nosocomial ventriculitis (1), empyema (1), fungal keratitis (1) otitis externa (1) and surgical site (1). Fluconazole resistance was 100% while 28.5 and 7.9% were Voriconazole and Amphotericin resistant respectively. Overall crude mortality was 42.4% while 14-day mortality was 31.5%. Both infected and colonized cases shared similar mortality (46.2% vs 33.3%; p-value = 0.25). Among infected cases mortality was high in candidemia compared to non-candidemia (60.5% vs 25.9%) in which deaths related to C. auris were 34.2% vs 22.2% respectively. On multivariate analysis candidemia (AOR 4.2, 95% CI: 1.09–16.49; p-value = 0.037) was associated with greater mortality with source control being the only protective factor for mortality (AOR 0.22, 95% CI: 0.05–0.92; p-value0.038] while ICU stay, rapidity of blood culture clearance, DM, malignancy and MDR co-infection had no impact. Conclusion Patients with C.auris from a single center in Pakistan have a wide clinical spectrum with line associated infection being the predominant site of infection. Candidemia leads to high mortality while source control improves outcome.

Background: Quantitative urine culture reporting on plates inoculated by automated streaking system (PREVI® Isola) demonstrates maximum growth only equivalent up to 104 colony-forming unit (CFU)\ml, according to the manufacturer's recommendation. Therefore, there is a probability of erroneous catheter-associated urinary tract infection (CAUTI) surveillance data as a count of ≥105 CFU/ml is required according to CAUTI definition. This study aimed to validate automated urine culture streak system for improving the surveillance of hospital-acquired CAUTIs. Materials and Methods: This was a prospective, cross-sectional study. In 1-month duration, urine samples of 1000 consecutive patients reported to the microbiology section for culture and sensitivity were included. Samples were processed simultaneously manually (1 μl loop) and with an automated streaking system. Plates streaked by automated method were arbitrarily divided into eight equally sized segments. After 24 h of incubation at similar conditions, microbial growths in each segment were noted and correlated with its manual colony count. To minimize the error, colony count and morphotype were analyzed by a senior technologist and counterchecked by a consultant. Results were recorded on an Excel sheet. Results: Growth on the 7th and 8th segments corresponded with ≥105 CFU/ml. Growth up to the 4th, 5th, and 6th segments corresponded with 104 CFU/ml, whereas the 3rd segment with >103 but

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. To study 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.

The MIC method applicable to Gram negative bacilli including Acinetobacter spp. is broth microdilution (BMD). Cost and/or availability issues limit the use of commercial MIC panels in resource limited settings. Objectives: To design and implement an in-house breakpoint BMD panel (BBMD) for colistin against Gram negative bacilli. Design: BBMD panel was prepared in 96-well plate. MIC concentrations of 1, 2, & 4 ​μg/mL for test, and 0.25, 0.5, 1, 2 & 4 ​μg/mL for control strains were selected to accommodate 19 test and 3 quality control strains per plate. Plates were frozen at −80 ​°C until testing. Validation was performed using strains from a previously published study and compared with freshly prepared MIC panel of 16–0.03 ​μg/mL. Results: Validation showed 100% agreement with the reference method and BBMD was introduced into routine laboratory practice for colistin susceptibility of carbapenem resistant Enterobacterales (CRE), Acinetobacter baumannii complex and Pseudomonas aeruginosa. From 2nd July-16th September 2018, a total of 1294 (mean 16.8 ​± ​5.5 isolates/day) clinical isolates were tested; 1157/1294 were reported (MIC ≤2 ​μg/mL) within 24-h, whereas 133 required resistance confirmation by full-range BMD. Resistance was confirmed for all but 24 isolates. These discrepancies were mostly due to contamination with bacterial genera inherently resistant to colistin. Conclusion: This BBMD plate is a high through-put and practical method that could reliably be utilized in a routine microbiology laboratory for colistin susceptibility testing of CRE, A. bauamanii complex and P. aeruginosa.

Non-tuberculous mycobacteria (NTM) are ubiquitous dwellers of environmental niches and are an established cause of natural and nosocomial infections. The incidence of NTM infections is rising owing to a growing population of immunocompromised and vulnerable individuals, complex medical and surgical procedures, as well as increased awareness and diagnostic capabilities. The prevalence of different NTM varies between continents, regions, and countries. The true global burden of pulmonary and extrapulmonary disease is unknown and estimates are subject to under and/or over-estimation. Diagnosis requires confirmation by isolation of NTM along with clinical and radiological criteria, which may be suboptimal at all levels. Susceptibility testing is complex and clinical breakpoints are not available for many of the drugs. Frequently, NTM infections are not considered until late in the course of disease. Improved and rapid detection of tuberculosis cases in high-burden countries has, however, also brought NTM infections into the limelight, and has identified a need for research efforts towards rapid diagnostic tests and the identification of biomarkers to monitor the treatment response in patients with NTM infections. Keywords: Non-tuberculous mycobacteria, Diagnosis, Epidemiology, Tuberculosis endemic regions

Background: Syncephalastrum species belong to the class Zygomycetes and order Mucorale. These are found in the environment and tropical soil, usually presenting as colonizers and rarely cause human infection. Syncephalastrum racemosum is a species of the genus Syncephalastrum and is the most commonly identified pathogen. Most cases are reported in immunocompromised individuals, such as patients on long term steroids, poorly controlled diabetes, or patients with malignancy. Case presentation: We are describing two cases of rare fungal infection by Syncephalastrum species causing invasive pulmonary manifestation. Both patients had compromised immune status and presented with worsening dyspnea to the emergency room. Both had signs and symptoms of bilateral worsening pneumonia evident by chest X-ray showing bilateral pulmonary infiltrates. Syncephalastrum species were isolated from sputum cultures. Deoxycholate amphotericin B was started and the response was monitored. One patient expired while the other improved. Syncephalastrum species belong to class Mucormycosis, rarely causing invasive infection but when they do outcome is potentially fatal. Very few cases are reported worldwide so the clinical course is still unclear. To the best of our knowledge, these are the first two cases to be reported from Pakistan. Conclusions: These two cases describe pneumonia as a result of concomitant infection by rare fungal speciesSyncephalastrum and MRSA in immunocompromised patients. Few cases are reported so limited data is available to understand complete disease implications. Mucormycosis is a therapeutic challenge because of the phylogenetic diversity, un-availability of any serological testing and invasive disease pattern.

Abstract Background The study analyzed microbiological and antimicrobial susceptibility profile of organisms isolated from patients with infective endocarditis (2015–17) and compared disease outcomes in cohorts of endocarditis patient with history of prior invasive vascular intervention (high risk group) vs those without (native valve group). We hypothesized that high risk group would be more likely to have severe disease outcomes. Methods This was a prospective cohort study (2015–17). All blood and cardiac tissue samples of enrolled patients suspected of endocarditis according to modified Duke’s criteria were followed for microbiological and antimicrobial susceptibility profile. The high risk group was compared with the native valve group with 90 day follow up to determine difference in clinical course and outcome in terms of disease severity (defined as any patient with endocarditis undergoing surgical management, readmission or dying). The data was analyzed using SPSS 21.0 software and chi-square test. 90 day mortality was calculated using Kaplan Meier survival curves. Results Total 104 patients with endocarditis were enrolled. Overall culture positivity rate was 71.2%. Streptococcus species were the most common isolate (36.7%), followed by S. aureus (17.3%) cases. In Streptococcus species, 14.2% showed intermediate susceptibility to penicillin. Thirty six patients were included in the cohort analysis. A poor outcome was seen in 85.7% high risk group as compared to 50% of native valve group. The overall mortality rate was 19.4%. Conclusions We found Streptococcus species to be the predominant pathogen for endocarditis overall. However Staphylococcus aureus predominated native valve group. High risk group showed more complicated clinical course.

Abstract Background Limited capacity of laboratories for antimicrobial susceptibility testing (AST) presents a critical diagnostic bottleneck in resource limited countries. This paper aims to identify such gaps and to explore whether laboratory networks could contribute towards improving AST in low resource settings. Methods A self-assessment tool to assess antimicrobial susceptibility testing capacity was administered as a pre-workshop activity to participants from 30 microbiology laboratories in 3 cities in Pakistan. Data from public and private laboratories was analyzed and capacity of each scored in percentage terms. Laboratories from Karachi were invited to join a support network. A cohort of five laboratories that consented were provided additional training and updates sessions over a period of 15 months. Impact of training activities in these laboratories was evaluated using a point scoring (0-11) tool. Results Results of self-assessment component identified a number of areas that required strengthening (scores of ≤60%). These included; readiness for AMR surveillance; 38 and 46%, quality assurance; 49 and 55%, and detection of specific organisms; 56 and 60% for public and private laboratories respectively. No significant difference was detected in AST capacity between public and private laboratories [ANOVA; p > 0.05]. Scoring tool used to assess impact of training within the longitudinal cohort showed an increase from a baseline of 1-5.5 (August 2015) to improved post training scores of 7-11 (October 2016) for the 5 laboratories included. Moreover, statistical analysis using paired t-Test Analysis, assuming unequal variance, indicated that the increase in scored noted represents a statistically significant improvement in the components evaluated [p

Background: With the rise in multidrug-resistant tuberculosis, there is a search for newer techniques that will rapidly detect drug-resistant Mycobacterium tuberculosis. Although molecular techniques can detect resistance, culture is still considered gold standard, especially in resource-limited settings where quick, cheap, and easy techniques are needed. The aim of the study was to evaluate microcolony method thin layer agar (TLA) for quick detection of resistance against the first- and second-line antituberculous drugs in clinical isolates. This was a cross-sectional study performed at Aga Khan University Hospital. Material and Methods: A total of 87 Z-N stain smear-positive pulmonary samples were received and indirect drug susceptibility test (ID-DST) was performed using Lowenstein–Jensen and mycobacteria growth indicator tube. Direct DST was performed using TLA on 7H10 agar. TLA was observed twice weekly under microscope for 4 weeks. Sensitivity, specificity, and accuracy were calculated for TLA using indirect susceptibility method as the gold standard. Level of agreement was calculated using Kappa score. Results: TLA showed sensitivity of 89% and 95.2% for isoniazid and rifampicin, while for ethionamide, ofloxacin, and injectable aminoglycosides, it was 96.6%, 92.1%, and 100%, respectively. Specificity for the first-line drugs was >95% while second-line drugs ranged from 70% to 100%. Mean time to positivity was 10.2 days by TLA as compared to 43.1 days by ID-DST. Conclusions: TLA is a quick and reliable method in identifying resistance, especially in resource-limited settings. However, additional liquid culture can be set up as backup, especially in patients on therapy to avoid false negative results.

Background: MTBDRplus line probe assay (LiPA) has been endorsed by the World Health Organization for pulmonary tuberculosis (TB) diagnosis. However, its value for Mycobacterium tuberculosis (MTB) detection in bronchoalveolar lavage (BAL) needs exploration. This study determined the diagnostic performance of MTBDRplus in BAL for MTB complex detection and isoniazid/rifampicin resistance in smear-negative and sputum scarce patients. Materials and Methods: Retrospective evaluation of data (January–December 2013) from patients who underwent bronchoscopy was done. Of these, patients with high TB suspicion with available data on acid-fast bacilli (AFB) smear/culture and MTBDRplus were selected. Results of MTBDRplus were compared with AFB smear/culture and drug susceptibility. Sensitivity and specificity of MTBDRplus was determined using TB culture as gold standard. Results: Data on 383 patients who underwent bronchoscopy were collected. Of these, 154 previously untreated TB suspect patients that were either smear negative on sputum microscopy or sputum scarce were selected. Out of 154 patients, 11 were smear positive and 34 patients were AFB culture positive. MTBDRplus detected MTB in 23/34 cases, the sensitivity and specificity being 67.6% and 85% (P < 0.001) versus 32.4% and 100% (P < 0.001) compared to smear microscopy. All smear-positive cases (n = 11) were detected by MTBDRplus. There were no discrepancies between phenotypic drug susceptibility testing and LiPA for isoniazid and rifampicin resistance in patients. Two cases of multidrug-resistant TB were detected. Conclusion: MTBDRplus detected TB more rapidly and accurately than smear microscopy with significant accuracy for isoniazid and rifampicin resistance. Its use in clinical practice would lead to rapid detection and effective management.

Background: This study aimed to compare the accuracy of Candida species identification by direct inoculation of positive blood culture specimens into CornMeal Tween™ (CMT) 80 agar with the conventional methods. Methods: This study was performed in two phases: Phase 1: Inoculation of direct CMT from artificially spiked blood culture bottles with identified Candida species. Phase 2: Direct inoculation of smear-positive bottles on CMT agar for identification. Routine identification method was carried out using urease production, sensitivity to cycloheximide, CMT inoculation, and colony morphology on BiGGY agar. All isolates were also evaluated for sugar assimilation on the biochemical test panel Analytical profile index (API) 20C AUX (BioMerieux). Statistical Analysis Used: The correlation between both methods was determined as percentage agreement rate using kappa score. Results: In the first phase 50 artificially spiked blood culture bottles were included in the study. The sample set consisted of 24% C albicans, 38% C tropicalis, and 22% C glabrata isolates. There was 100% agreement between the directly inoculated and CMT inoculated from colonies at 48 hours. Phase 2: The overall agreement between directly inoculated CMT and conventional method of identification at 72 h was 92%. Conclusion: Direct inoculation of CMT agar can easily differentiate common Candida species microscopically on the basis of chlamydospores, blastopores, and arrangement of pseudo hyphae. However, identification of rare species from specimens containing two different species using direct method is challenging. Therefore, conventional methods including macroscopic/microscopic characteristics and biochemical profile with the aid of API with API 20 AUX would be a better choice for such cases.

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.

A ventricular septal defect (VSD) patch infection with Acremonium species isolated from vegetation and blood culture is described. Antifungal treatment was discontinued after 3 months and patient developed relapse. Surgery with prolonged oral voriconazole was instituted with recovery. We emphasize importance of surgery and prolonged therapy to treat such infections.

Background: Duration of digestion/decontamination has a considerable impact on yield of mycobacteria especially from sterile body fluids and pus specimens. Additionally, duration of digestion/decontamination affects the contamination rates. This study evaluates the effect of digestion/decontamination protocol for 15 and 20min versus inoculation of media directly from the sample on contamination rates as well as the yield of mycobacteria from pus and sterile fluids other than cerebrospinal fluids. Methods: Pleural fluid (n = 60), pus (n = 48) and ascitic fluid (n = 12) specimens were cultured for mycobacteria and evaluated for contamination and mycobacterial yield using three different processing methodologies: without digestion/decontamination with 5% NaOH-NALC (D/D), D/D for 15min and D/D for 20min. All samples >3 mL in volume were spun at 3000 RCF for 15min, whereas those less than 3 mL were used as is. They were simultaneously processed using the three different methods as mentioned above, and inoculated on LJ media and MGIT. Smear was made from samples treated for 20min and stained with fluorescent stain. Kinyoun staining was done on smears with dubious findings. Mycobacterial culture yield and contamination rates were recorded at 6 weeks as recommended by the Global Laboratory Initiative (GLI) laboratory manual 2014. Results: Pleural fluid and pus contamination rates were substantially lowered by increasing decontamination time from 15 to 20min, but it did not have any effect for ascitic fluid (Table 1). The 5-min difference in the decontamination procedure improved mycobacterial culture yield for pus samples by 10%, but there was no substantial effect on pleural and ascitic fluids. Prolonged decontamination did not compromise the culture yield in any of the mentioned specimens. Conclusion: In areas where specimen delay is common and sterility of collection procedure cannot be ensured, digestion/decontamination with NaOH-NALC for up to 20min can reduce contamination rates without considerably compromising mycobacterial culture yield. However, one should be alert to the possibility of decreased viability, and culture should be supplemented with molecular methods.

We report a case of dust mite carriage in a 56-year-old gentleman. Dust mites eggs and larvae were found in a stool sample which was taken for a routine clinical examination. He was completely asymptomatic with no history of rash, airway disease or other allergic manifestations associated with dust mites. We noticed that the oval structure of mite eggs resembled helminth eggs and therefore may be misidentified during routine clinical analysis. As the patient was otherwise healthy, it was concluded that no rigorous antiparasitic therapy was necessary to eliminate dust mites from his system.

Background and objective: Pakistan is one of the high-tuberculosis (TB) burden countries (HBCs) with a considerable proportion of multidrug-resistant TB (MDR-TB) strains. Recently, high resistance to fluoroquinolone has been reported in both MDR and non-MDR-TB strains from Pakistan. This study aims to evaluate trends of resistance in Mycobacterium tuberculosis (MTB) against injectable second-line drugs (2010–July 2014) from Aga Khan University Laboratory, a technical partner of the Pakistan National TB Program and part of the WHO Supra-national Laboratory Network for TB based in Karachi, Pakistan. Methods: MTB strains were isolated using standard methods. Susceptibility testing was performed using the agar proportion method with drug concentrations as recommended by the Clinical Laboratory Institute Standards (CLSI). MTB H37Rv was used as a control with each batch of susceptibility testing. MDR-TB was defined as resistance to both isoniazid (0.2 μg/ml) and rifampicin (1.0 μg/ml). Results: During the study period, 14,711 MTB strains were isolated. Of these, 43.5% were MDR and 56.5% were non-MDR. Resistance pattern to amikacin, kanamycin and capreomycin over the years is shown in the table below. [INLINE:1] Conclusions: Rising resistance to injectable anti-tuberculous agents in MDR-TB isolates possibly reflects their increasing use in patient management. Presence of capreomycin resistance even amongst non-MDR strains is alarming for a country where fluoroquinolone resistance is also high and patient follow-up and drug compliance is a challenge.

Introduction: In 2008, the World Health Organization (WHO) approved a rapid molecular test known as Line Probe Assay (LiPA) for the diagnosis of multidrug-resistant tuberculosis (MDR-TB) in pulmonary specimens. Due to lack of available data, its use in extra-pulmonary tuberculosis (EPTB) specimens has not been determined yet. Recommendations for the use of Xpert MTB/RIF in EPTB have been issued, but alternative rapid molecular tests for EPTB diagnosis need to be evaluated. Method: LiPA (GenoType® MTBDRplus 2.0 Hain Lifescience) was performed on 97 specimens of extrapulmonary origin at the clinical laboratory at Aga Khan University (Dec 2012–Jan 2014). The results were compared with TB cultures in solid and liquid medium. Results: 97 specimens were tested simultaneously for culture and LiPA, including pleural fluid (35), CSF (22), pus (17), tissue (10) and urine (3). Concentrated smear was positive for 7 while 14 were culture positive for MTB. All 7/7 smear-positive specimens were LiPA positive, while 6/7 were culture positive. Amongst the smear-negative specimens, 8/90 were culture positive and 9/90 were LiPA positive. The overall sensitivity and specificity of LiPA for the detection of MTB in the EPTB specimens was 71.4% (95% CI 41.9–91.4) and 92.8% (95% CI 84.9–97.3), respectively. The highest sensitivity (100%) was seen in urine as 2 of 3 which were culture positive were also LiPA positive. Pus samples showed sensitivity of 85.7% and specificity of 70%. Conclusion: The study shows that LiPA has good overall sensitivity and specificity compared with culture. Although the number of samples was very small, the applicability appears to be most useful in urine and pus specimens and should be explored further as a diagnostic tool in these cases.