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Appropriate collection of respiratory samples is essential for diagnostic testing of respiratory pathogens such as, SARS-CoV-2. During the early pandemic period, there was a shortage of imports such as, nasopharyngeal (NP) swabs in Pakistan, which led to difficulties in sampling for COVID-19 tests. At the Aga Khan University, we developed a 3D-printed anterior nares nasal swab kit paired with in-house viral transport medium (VTM) to address this gap. We conducted a prospective observational study to compare sampling with 3D-printed swab kit  and standard NP commercial swab and VTM kits, in 200 individuals presenting for diagnostic testing for SARS-CoV-2 between August and September 2020. The effectiveness of the standard NP and 3D swabs were assessed using RT-PCR results of tested specimens. Results were evaluated based on cut offs of ≤ CT 35 (high to medium viral loads) or, ≤ CT 37 (low viral loads). For the former, 3D swab-based testing had a sensitivity of 93%, specificity of 99%, positive predictive value (PPV) of 98.5%, and negative predictive value (NPV) of 96.2%, respectively. For the latter, sensitivity was 88%, with specificity of 99%, with PPV of 98.5%, and NPV of 93.2%.%. In conclusion, 3D swab-based sampling of anterior nares was comparable with NP sampling using standard swabs, providing a reliable and convenient local solution for respiratory sample collection. Our work highlights the value of using 3D printing for diagnostic tools for convenient and effective sampling when items are scarce. 3D printing also provides new opportunities for rapid assay development as prototypes prior to scale up.

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

Background: From September 2014, a tertiary care hospital in Karachi, Pakistan, started diagnosing 3–5 cases/month of a yeast locally identified as Saccharomycesspp. resistant to fluconazole. US Centers for Disease Control and Prevention identified the isolates as Candida auris. The Pakistan Field Epidemiology and Laboratory Training Program (FELTP) and the hospital investigated the outbreak from April 2015 to January 2016.Objective: The aim of the outbreak investigation was to determine the risk factors and to inform measures to limit the spread of the organism in the hospital.Methods: Medical records, nursing schedules and infection control practices were reviewed. Sixty-two age- and sex-matched hospital controls from the same wards were identified.Results: Thirty cases (17 males) were identified (mean age = 51.6 years, age range = 2–91 years), case fatality was 53%. Multivariate logistic regression showed that a history of surgery within 90 days of diagnosis, admission to the emergency department and history of chronic kidney disease were significantly associated with C. aurisinfection.Discussion: This is the report of the outbreak investigation that triggered a global exploration of C. aurisas a newly identified multidrug-resistant nosocomial organism, spreading within the hospital, especially among patients with invasive procedures. Unfortunately, we could not identify any specific source of the outbreak nor stop the transmission of the organism.

ABSTRACTEarly availability of antifungal susceptibilities can ensure timely institution of targeted therapy in candidemia, which can improve patient outcomes. This study prospectively determines the agreement between the results of direct testing of antifungal susceptibilities from blood culture bottles by disk diffusion and Etest and the results of standardized susceptibility testing methods; direct testing would allow susceptibility results to be available 1 to 2 days earlier. A total of 104 blood cultures with different Candidaspecies (28% C. albicans, 27% C. parapsilosis, 26% C. tropicalis, etc.) were evaluated between January 2012 and May 2013 for agreement of fluconazole, voriconazole, and amphotericin B susceptibility results by disk diffusion. Agreement in MICs obtained by Etest was determined for fluconazole (21 isolates), voriconazole (28 isolates), amphotericin (29 isolates), and caspofungin (29 isolates). The kappa scores for categorical agreement were highest for fluconazole by disk diffusion (0.902, standard error [SE] = 0.076) and Etest (1.00, SE = 0.218) and for amphotericin B by disk diffusion (1.00, SE = 0.098). The Pearson correlation (r) of zone diameters was strongest for fluconazole (0.69) and amphotericin (0.70) and moderate for voriconazole (0.60), and the Pearson correlation of MICs was strongest for fluconazole (0.94) and caspofungin (0.88). However, the moderate correlation of amphotericin MICs with zone diameters (-0.42) precludes the use of amphotericin B disk diffusion for susceptibility testing. There were no very major errors; however, there were 1 (1%) major and 5 (4.8%) minor errors with disk diffusion and 4 (13.3%) minor errors with Etest. Thus, antifungal disk diffusion directly from blood culture bottles is a rapid and easy method for fluconazole and voriconazole susceptibility testing for timely tailoring of candidemia therapy.

ABSTRACTPakistan is a high-burden country for tuberculosis (TB). The emergence and increasing incidence of extensively drug-resistant (XDR) TB has been reported in Pakistan. Similarly, the prevalence of multidrug-resistant TB infections with fluoroquinolone resistance (pre-XDR) is also increasing. To treat these infections, local drug susceptibility patterns of alternate antituberculosis agents, including levofloxacin (LVX), linezolid (LZD), and amoxicillin-clavulanate (AMC), is urgently needed. The aim of this study was to determine the susceptibility frequencies of drug-resistant (DR) Mycobacterium tuberculosisagainst LVX, LZD, and AMC. All susceptibilities were determined on Middlebrook 7H10 agar. A critical concentration was used for LVX (1 μg/ml), whereas MICs were determined for LZD and AMC. M. tuberculosisH37Rv was used as a control strain. A total of 102 M. tuberculosisisolates (XDR, n= 59; pre-XDR, n= 43) were tested. Resistance to LVX was observed in 91.2% (93/102). Using an MIC value of 0.5 μg/ml as a cutoff, resistance to LZD (MIC ≥ 1 μg/ml) was noted in 5.9% (6/102). Although the sensitivity breakpoints are not established for AMC, the MIC values were high (>16 μg/ml) in 97.1% (99/102). Our results demonstrate that LZD may be effective for the treatment of XDR and pre-XDR cases from Pakistan. High resistance rates against LVX in our study suggest the use of this drug with caution for DR-TB cases from this area. Drug susceptibility testing against LVX and AMC may be helpful in complicated and difficult-to-manage cases.

ABSTRACTThe increasing incidence of extensively drug-resistant (XDR) Mycobacterium tuberculosisin high-tuberculosis-burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDR M. tuberculosisstrains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the “hot-spot” regions of rpoB, katG, inhA, ahpC, gyrA, gyrB, and rrsgenes in 50 XDR M. tuberculosisstrains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDR M. tuberculosisstrains would be undetected if only these common hot-spot regions were tested. The frequencies of resistance-conferring mutations were found to be comparable among all XDR M. tuberculosisstrain families present, including the Central Asian Strain, Beijing, and East African Indian genogroups and the Unique isolates. Additional genetic loci need to be tested for detection of mutations conferring fluoroquinolone, aminoglycoside, and capreomycin resistance in order to improve molecular diagnosis of regional XDR M. tuberculosisstrains.

Undiagnosed allergic bronchopulmonary aspergillosis (ABPA) can lead to chronic persistent symptoms. In country like Pakistan where tuberculosis (TB) is endemic, a significant proportion of ABPA patients are misdiagnosed as smear negative TB before reaching a diagnosis of ABPA due to chronicity of symptoms.This lead to empiric use of ATT(Anti-tuberculous therapy) and delay in primary diagnosis. The aim of the study is to determine such proportion of ABPA patients.

ABSTRACTWe report two cases of infantile diarrhea due to multidrug-resistant, NDM-1 metallo-ß-lactamase-producing Salmonella entericaserovar Agona from Pakistan. This study alerts toward possible risk of NDM-1 transmission to enteric fever pathogens and encourages microbiologists to consider active screening of carbapenem resistance in nontyphoidal Salmonellaisolates.

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.

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.

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.

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.

ABSTRACTA fluoroquinolone (FQ) resistance rate of 5.9% is reported in 205 Mycobacterium tuberculosisisolates from patients presenting to field clinics in Karachi, Pakistan (2006 to 2009). FQ resistance among multidrug-resistant (MDR) strains was 11.1% (5/45), and it was 4.9% (5/103) in M. tuberculosisstrains susceptible to all first-line agents. Spoligotyping of resistant strains did not show dominance of one strain type. Our data reflect considerable FQ-resistant M. tuberculosisisolates and the need to consider inclusion of FQ within first-line sensitivity testing in such settings.