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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.

In this study, we compared the predisposing factors, key demographic and clinical characteristics, clinical outcomes, and factors associated with poor prognosis in pneumocystis pneumonia (PCP) infection among the human immunodeficiency virus (HIV)-positive and non-HIV patient populations. This retrospective analysis was conducted at the Aga Khan University Hospital, Karachi, via the collection and analysis of patient records with a diagnosis of "pneumocystosis" between January 2015 and October 2020. Additionally, the laboratory database was evaluated, and patients with a laboratory-confirmed diagnosis of PCP were included. During the study period, 52 laboratory-confirmed hospitalized PCP patients were identified. Of these, 23 and 29 patients were diagnosed using microscopy and polymerase chain reaction, respectively. 34.6% of our patients were HIV positive, with a median CD4 count of 20.5 cells/mm3 (range: 10.7-50.5). Other conditions identified were corticosteroid use, autoimmune diseases, malignancy, radiation, and chemotherapy. On chest imaging, consolidation was found in 30%, ground-glass opacities in 24%, and nodular infiltrates in 20% of the cases. HIV-positive patients had a lower hemoglobin level and a higher level of β-D-glucan at the time of admission, whereas non-HIV patients were found to have more co-morbid conditions than HIV patients. We observed no difference in clinical outcomes between the two populations. Factors associated with a poor prognosis among our patients included concomitant infections at the time of diagnosis, the need for invasive mechanical ventilation, and a longer duration of stay in the hospital as well as the intensive care unit.

ABSTRACT Small colony variants are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata—a major intracellular fungal pathogen—produces small and slow-growing respiratory-deficient colonies, termed “petite.” Despite reports of clinical petite C. glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially encoded genes. Consistent with dual-RNAseq data, petite C. glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C. glabrata blood isolates (n = 1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0%–3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called “petite.” This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

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.

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.

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.

Background: Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. With the emergence of penicillin-resistant S. pneumoniae (PRSP), treatment has become challenging. The Clinical and Laboratory Standards Institute (CLSI) in 2008 revised its guidelines for S. pneumoniae and recommended separate penicillin breakpoints for meningeal and non-meningeal strains. Similar to penicillin’s, resistance to other classes of antibiotics has emerged globally. Objective: The objective of this study is to determine the trend of resistance to antimicrobials in S. pneumoniae infections and the impact of new CLSI guidelines on penicillin susceptibility among meningeal isolates. Methodology: Twenty-four years (1993-2016) data from S. pneumoniae isolates and their antimicrobial susceptibility was retrieved from the computerized database. Data was divided into two groups for analysis, pre-2008 and post 2008. Results: Penicillin resistance remained unchanged in non-meningeal isolates during both study periods. A significant rise in penicillin resistance in meningeal isolates was observed in the second period 2008-2016 (2.9% vs 36.2%). High resistance rates were observed for co-trimoxazole, tetracycline and erythromycin. Increased trend of multi-drug resistant (MDR) strains were also noted, from 11% in 1999 to 36% in 2016. Conclusion: The emergence of MDR strains is evident from our dataset. It seems like the rise in PRSP in meningeal isolates is due to revised CLSI guidelines. Overall low resistance to penicillin in non-meningeal isolates and no resistance to ceftriaxone is encouraging and will assist in drafting local guidelines. Cautious use of antimicrobials are essential to reduce further emergence of antimicrobial resistance in indigenous isolates.

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.

Following greater attention and follow-up of patients with treated pulmonary tuberculosis (TB), it has emerged that infections are more likely to occur in this cohort of patients. This comes as no surprise, as pulmonary TB is a destructive process that leads to cicatrization, alteration of parenchyma, bronchiectasis, and scarring of the lung, with reduction of lung volumes and an impact on pulmonary function. In addition to relapse and re-infection with TB, other pathogens are increasingly recognized in post-TB patients. This paper serves as a summary and guide on how to approach the post-TB patient with new signs and symptoms of pulmonary infection in order to ensure optimal management and rehabilitation. Keywords: Tuberculosis, Post-treatment complication, Sequelae, Pulmonary rehabilitation, NTM, Bronchiectasis

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: 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.

The incidence of chronic pulmonary aspergillosis (CPA) is especially increasing in high tuberculosis (TB) burden countries. Despite a high estimated CPA burden in Pakistan, actual data on CPA are not available. The aim of the current study is to determine the underlying conditions and clinical spectrum of CPA at a tertiary care hospital in Karachi, Pakistan. This is a retrospective chart review study in patients admitted with CPA from January 2012 to December 2017. A total of 67 patients were identified during the study period. Mean age of CPA patients was 45.9 ± 15 years, 44 (65.7%) were male and 19 (28.4%) had diabetes. The most common type of CPA was simple aspergilloma (49.2%) followed by chronic cavitary pulmonary aspergillosis (CCPA) (44.7%). TB was the underlying cause of CPA in 58 (86.6%) patients followed by bronchiectasis caused by allergic bronchopulmonary aspergillosis (ABPA) 8 (11.9%). Aspergillus flavus was identified in 17 (47.2%), followed by A. fumigatus in 13 (36.1%) CPA patients. Isolation of multiple Aspergillus species was found in 10 (25.6%) patients. Itraconazole was given in 27 (40.3%) patients and a combination therapy of itraconazole and surgery was given in 21 (31.34%) patients. We found aspergilloma and CCPA as the most prevalent forms of CPA in our setting. Further large prospective studies using Aspergillus specific immunoglobulin G (IgG) antibodies testing are required for better understanding of CPA in Pakistan.

Chronic pulmonary aspergillosis (CPA) is a recognized complication of pulmonary tuberculosis (TB). In 2015, the World Health Organization reported 2.2 million new cases of nonbacteriologically confirmed pulmonary TB; some of these patients probably had undiagnosed CPA. In October 2016, the Global Action Fund for Fungal Infections convened an international expert panel to develop a case definition of CPA for resource-constrained settings. This panel defined CPA as illness for >3 months and all of the following: 1) weight loss, persistent cough, and/or hemoptysis; 2) chest images showing progressive cavitary infiltrates and/or a fungal ball and/or pericavitary fibrosis or infiltrates or pleural thickening; and 3) a positive Aspergillus IgG assay result or other evidence of Aspergillus infection. The proposed definition will facilitate advancements in research, practice, and policy in lower- and middle-income countries as well as in resource-constrained settings.

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.

Fluoroquinolones (FQ) play an essential role in the treatment and control of multidrug-resistant tuberculosis (MDR-TB). They are also being evaluated as part of newer regimens under development for drug-sensitive TB. As newer FQ-based regimens are explored, knowledge of FQ resistance data from high TB burden countries becomes essential. We examine available FQ resistance data from high TB burden countries and demonstrate the need for comprehensive surveys to evaluate FQ resistance in these countries. The factors driving FQ resistance in such conditions and the cost of such resistance to weak healthcare systems are discussed. The need for a comprehensive policy for addressing the issue of FQ resistance is highlighted.

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.

Frequency of extensively drug-resistant tuberculosis in Pakistan increased from 1.5% in 2006 to 4.5% in 2009 (p

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.

Aims and objective: Despite effective treatment of pulmonary tuberculosis (TB) patients, destruction of lung parenchyma may lead to complications including repeated infections. These infections are often misdiagnosed or wrongly identified as TB recurrence, and hence are not treated effectively. The frequency and severity of these infections vary with the extent of damage, and are much more prominent in patients with post-TB bronchiectasis and fibrocavitary diseases. This presentation will focus on the epidemiology, treatment, and management of post-TB infections and challenges, and the impact of these infections on public health in high-TB-burden countries. Methods: Published literature and review articles were evaluated to address this objective. Results: Apart from conventional agents of pneumonia, patients with post-TB bronchiectasis and post-TB fibrocavitary diseases are prone to develop chronic pulmonary aspergillosis and nontuberculous mycobacterial infections. A high burden of chronic pulmonary aspergillosis has been reported in TB-endemic countries. Similarly, prior TB increases the risk of acquiring nontuberculous mycobacterial infections. Diagnosis and management of chronic pulmonary aspergillosis and nontuberculous mycobacterial infections require expertise and high-level care. Conclusion: Limited diagnostic and therapeutic capacities compounded by nonavailability of essential antimicrobials in most high-TB-burden countries pose great challenges to physicians involved in the management of these infections. These infections affect the overall outcome and lead to high cost for public health systems.

Emergence and spread of drug resistant Neisseria gonorrhoeae is global concern. We evaluated trends of antimicrobial resistance in Neisseria gonorrhoeae over years 1992–2009 in Pakistan. Resistance rates were compared between years (2007–2009) and (1992–2006). Antimicrobial susceptibility testing was performed and interpreted according to Clinical Laboratory Standards Institute (CLSI) criteria using the disk diffusion methodology against penicillin, ceftriaxone, tetracycline and ofloxacin. Additional antibiotics tested in 100 strains isolated during 2007–2009, included cefotaxime, cefoxitin, cefuroxime, cefipime, ceftazidime, ceftizoxime, cefixime, cefpodoxime, spectinomycin and azithromycin. Neisseria gonorrhoeae ATCC 49226 was used as control. Chi-square for trend analysis was conducted to assess resistance trend over the study period. During study period significant increase in combined resistance to penicillin, tetracycline and ofloxacin was observed (P value

Background: Pakistan has an annual tuberculosis (TB) incidence rate of 233 per 100,000 population and ranks sixth among the high TB burden countries worldwide. Tuberculous pleural effusion (TPE) occurs in up to 25% of TB patients. Owing to the pauci-bacillary nature of the pleural fluid, the diagnosis of TPE is a challenge. Objective: The aim of the present study is to evaluate the performance of MTBDRplus line probe assay in terms of its sensitivity and specificity for the rapid TB diagnosis in TPE and frequency of multidrug-resistant TB (MDR-TB) in TPE through the detection of rifampicin (Rif) and isoniazid (INH) resistance. Methods: This study is an ongoing prospective cross-sectional study being performed at Aga Khan University Hospital. Patients aged 18 years or more with suspected TPE were asked to participate. In addition to a preformed questionnaire which gathered information regarding clinical history and previous laboratory investigations, already tapped pleural effusion samples are tested for pleural fluid composition, TB smear and culture, MTBDRplus line probe assay and ADA analysis. Results: So far, 22 patients with a mean age of 49.81±21.75 (range: 18–86; IQR: 29.25, 68.00) years have been enrolled. Almost two-thirds are males (n = 15, 68.2%). The majority of the patient population belongs to Karachi (n = 18, 81.8%), followed by Quetta (n = 2, 9.1%), Faisalabad (n = 1, 4.5%) and Sukkur (n = 1, 4.5%). Half of the patients had a history of TB contact (n = 11, 50.0%), whereas only two (9.1%) had a past history of TB. Fifty percent (n = 11) had a right-sided pleural effusion. Effusion size was moderate in 36.4% (n = 8), mild in 27.3% (n = 6) and massive in 22.5% (n = 5). Pleural fluid MTBDRplus positively detected TB in 9.1% (n = 2) of cases. INH resistance was detected in one of these two cases. Pleural fluid culture was positive in two cases. Pleural fluid AFB smear was negative in all cases. Conclusion: Given the study is still under way, it is hoped that the results of this study may investigate the relevance, sensitivity and specificity of MTBDRplus line probe assay as a tool in the rapid detection of MTB in TPE and the early detection of MDR-TB through the detection of Rif and INH resistance. It may also provide an estimate of prevalence of drug resistant strains in TPE, which would be useful for guiding empirical anti-tuberculous therapy in TPE.

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.