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Objective: To discover the frequency of vancomycin resistant enterococci (VRE) fecal colonization and subsequent bacteremia in patients with hematological diseases in a bone marrow transplant center. Study Design: Cross-sectional study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology (AFIP), in collaboration with Armed Forces Bone Marrow Transplant Center, Rawalpindi, from Jan 2016 to Dec 2019. Methodology: Stool specimens/anal swabs from all enrolled patients were collected aseptically and transported to the laboratory without delay. Blood cultures were collected aseptically from only those enrolled patients who developed signs and symptoms of bacteremia. Stool and blood cultures were processed as per standard microbiological protocols. Enterococci were identified to species level by colony morphology and biochemical tests. Modified Kirby Bauer disc diffusion method and VITEK-2 system (Version-8.01 bio Merieux, France) were used to perform antimicrobial sensitivity of each isolate. Results: A total number of 246 patients were studied. Among them, 67 (27%) patients had fecal colonization by vancomycin resistant enterococci. We report a statistically significant association of recent hospitalization, prolonged exposure to antimicrobial therapy, chemotherapy exposure and use of indwelling devices during the hospital stay with vancomycin resistant enterococci colonization. Vancomycin resistant enterococci bacteremia was detected in 57 (23%) patients. Among these 57 patients, 53 (93%) were vancomycin resistant enterococci colonizers. Vancomycin resistant enterococci colonization was significantly associated with vancomycin resistant enterococci bacteremia. Conclusion: A considerable burden of vancomycin resistant enterococci fecal colonization exists among patients with hematological diseases. vancomycin resistant enterococci colonization poses a considerable risk of vancomycin........

Objective: To evaluate the cytospin slide microscopy method for detection of acid fast bacilli (AFB) in bronchoalveolar lavage (BAL) fluid comparing it with concentrated smear microscopy. Study Design: Cross-sectional validation study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from Dec 2016 to Sep 2018. Methodology: BAL specimens from suspected tuberculosis patients who were sputum smear negative, submitted to AFIP for diagnosis were included in the study. Smears for microscopy were prepared with the modified cytospin method as well as the standard concentrated technique. The prepared smears from both methods were stained with Ziehl–Neelsen (ZN) staining method and examined under 100 × oil immersion lens. TB culture performed on BACTEC MGIT 960 automated culture system (Becton Dickinson, USA) was taken as gold standard for TB diagnosis. The two methods were compared in terms of sensitivity, specificity, positive predictive value and negative predictive value. Results: Out of the 130 samples tested, 62 (47.7%) were positive on culture using MGIT 960 system. The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of the modified cytospin method for pulmonary TB diagnosis was found to be 68.3%, 100%, 100%, 77% and 84.6%, respectively. Conclusion: The sensitivity of the modified cytopsin smear method was significantly higher than that of the concentrated method. The study concludes that this is a simpler and more accurate method for BAL fluid microscopy.

INTRODUCTION: Typhoid remains a major healthcare problem in low and middle-income countries. The emergence of extremely drug-resistant (XDR) typhoid strains from the Indian subcontinent has led to very limited therapeutic options. Azithromycin being the only oral option for XDR typhoid faces a threat of rapid resistance due to its overuse after the COVID-19 pandemic. OBJECTIVE: To evaluate the reliability of azithromycin disc diffusion testing against clinical isolates of typhoidal salmonellae in comparison with E-test minimum inhibitory concentrations (MICs). STUDY DESIGN: This is a cross-sectional validation study. Place and duration of the study: The Department of Microbiology, Pakistan Navy Ship Shifa hospital, Karachi from June 1 to December 31, 2020. METHODOLOGY: Antimicrobial susceptibility was performed by Kirby Bauer disc diffusion method for 60 isolates including Salmonella enterica ser. Typhi and Paratyphi A using Clinical Laboratory Standard Institute (CLSI) guidelines. MICs by the E-test method were determined for Azithromycin only. RESULTS: A significant proportion of the isolates (55%) had high azithromycin MIC in the wild-type distribution range (8-16 µg/ml). Ten (16.6%) isolates showed false resistance, i.e., zone diameter

The aim of this study is to see the frequency, clinical presentation, and therapeutic response of extensively drug-resistant Salmonella enterica serovar Typhi and current susceptibility pattern of typhoidal Salmonella strains in our setup. This study was carried out at the Department of Medical Microbiology and Immunology and Department of Medicine, Pakistan Navy Ship (PNS) Shifa Hospital, Karachi, from January 1 to December 31, 2018. All the blood culture samples of patients (indoor and outdoor) with suspicion of enteric fever were processed. Isolates were cultured and identified using standard microbiological procedures. The antimicrobial sensitivity against the typhoidal Salmonellae was determined using Kirby-Bauer disc diffusion method as per the guidelines of Clinical and Laboratory Standards Institute (2018) and all the extensively drug-resistant (XDR) isolates were confirmed by Vitek 2 system. Clinical presentation and response to treatment of patients were followed. A total of 292 typhoidal Salmonella isolates were cultured. Resistance to ciprofloxacin against both Salmonella Typhi and Salmonella Paratyphi A was found to be very high (91%). Percentage of multidrug-resistant (MDR) isolates in Salmonella Typhi was 76% (182 isolates) and in Salmonella Paratyphi it was 34% (18 isolates). XDR isolates in Salmonella Typhi were significant that is 48% (115 isolates). Only 10 cases were given azithromycin who responded to treatment in mean 4.3 days. Out of 115 cases of XDR Salmonella Typhi, 103 patients were given parenteral meropenem and clinical response was seen in mean 5 days. The emergence and rapid spread of extensively drug-resistant Salmonella Typhi is alarming and highlights the significance of strict antimicrobial susceptibility surveillance programs with antimicrobial stewardship.

Objective To evaluate performance of thin layer agar (TLA) 7H11 method for detection of ofloxacin (OFX) and kanamycin (KM) resistance in smear positive clinical specimens of patients with tuberculosis comparing the results with gold standard MGIT 960 system. Study design Cross-sectional validation study. Place and duration of study Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from April to September 2017. Methodology Acid fast bacilli (AFB) smear positive specimens submitted at the study place, were inoculated on TLA 7H11 agar. Growth was examined along with susceptibility of OFX and KM and compared with gold standard MGIT 960 system. Results One hundred and sixty specimens were evaluated. Sensitivity and specificity of TLA for OFX was found to be 100% and 99.3%, respectively; and PPV and NPV was found to be 90.9% and 100%, respectively. Overall diagnostic accuracy was 99.38%. Sensitivity and specificity of TLA for KM was found to be 80% and 100%, respectively. PPV and NPV was found to be 100% and 99.36%, respectively. Overall diagnostic accuracy was 99.38%. Conclusion Thin layer agar is reliable, easy to perform and cost effective technique not only for rapid detection of MTB but also for drug susceptibility (DST) of second line anti TB agents. It is a suitable alternative to culture on LJ medium and can also be alternative to MGIT 960 system in resource-poor settings.

Objective To evaluate the performance of nitrate reductase assay on smear positive pulmonary specimens for detection of multi and extensively drug resistant tuberculosis simultaneously. Study design Cross-sectional analytical study. Place and duration of study Microbiology Department, Armed Forces Institute of Pathology, Rawalpindi from June to December 2016. Methodology Smear positive pulmonary samples were processed both by nitrate reductase method on Lowenstein Jenson medium and also inoculated on gold standard Bactec MGIT 960 TB system. All the specimens were first digested and decontaminated according to standard protocol before inoculation. Results Out of total 76 samples, three did not give color and, therefore, were excluded from the final data analysis. Among the remaining 73 samples, mycobacterial index was: 28 specimens were having 1+ (1-9 bacilli/100 fields), 26 samples were 2+ (1-9 bacilli/ field), and 19 samples were having 3+ index (>9 bacilli/field). The respective sensitivity and specificity were 84% and 100% for isoniazid (INH); 82% and 100% for rifampin (RIF); 67% and 100% for amikacin (AK); and both 100% for ofloxacin (OFX). Overall agreement in case of INH, RIF, AK, and OFX was 94.5%, 97.2%, 98.6% and100%, respectively. Overall average agreement was 97.5%. Conclusion Nitrate reductase assay is a reliable, low cost and accurate method that can be used for early for diagnosis of multi and extensively drug resistant tuberculosis.

Background: Effective control of tuberculosis is achieved by early diagnosis and drug susceptibility testing for initiation of appropriate treatment. The performance of crystal violet decolorization assay (CVDA) for susceptibility testing of Mycobacterium tuberculosis to isoniazid (INH) and rifampicin (RIF) was compared in a multicenter study. Methods: Seventy-two M. tuberculosis isolates were tested in two phases by CVDA. Results: In Phase I, the specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and agreement for INH were 100%, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 98.2%, 100%, 94.1%, 100%, and 98.6%, respectively. In Phase II, specificity, sensitivity, PPV, NPV, and agreement were 98%, 100%, 95.4%, 100%, and 98.6% for INH, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 96.3%, 88.2%, 88.2%, 96.3%, and 94.4%, respectively. Results in the study were obtained on average 10.9 ± 3.1 days in Phase I and 9.8 ± 2.2 days in Phase II. Conclusion: CVDA can be performed for drug susceptibility testing in developed and developing countries. In addition, further studies with larger sample size are needed for evaluation of this method. Fil: Coban, Ahmet Yilmaz. Akdeniz Üniversitesi; Turquía Fil: Alakbarova, Gumral. Medical Microbiology Laboratory. Thalassemia Center; Azerbaiyán Fil: Satti, Luqman. Armed Forces Institute of Pathology; Pakistán Fil: Tayyab, Nadia. Armed Forces Institute of Pathology; Pakistán Fil: Zaman, Gohar. Armed Forces Institute of Pathology; Pakistán Fil: Morcillo, Nora Susana. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; Argentina Fil: Imperiale, Belén Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Wainmayer, Ingrid. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Símboli, Norberto Fabián. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina

Objective To assess the utility of galactomannan and beta-D-glucan assays in the diagnosis of invasive aspergillosis in clinically suspected cases, and to compare their diagnostic potential to determine whether a combination of the two may result in an early and specific diagnosis. Methods The descriptive cross-sectional case-control study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from April 1, 2017, to March 31, 2018, and comprised serum samples from clinically suspected invasive aspergillosis patients and healthy controls. The sera were tested for galactomannan and beta-D-glucan detection. Proven, probable and possible categories of invasive aspergillosis according to European Organisation for Research and Treatment of Cancer / Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group criteria. Galactomannan antigen was detected using a one-stage immunoenzymatic sandwich microplate assay. Beta-D-Glucan antigen was detected using a protease zymogen-based colorimetric assay. Sensitivity and positive / negative likelihood ratio of both the cases and the controls were calculated and compared. Results Of the 178 subjects, 119(67%) were cases and 59(33%) were controls. Beta-D-glucan assay was more sensitive than galactomannan assay (91.6% versus 80.67%) whereas galactomannan assay was more specific than beta-D-glucan assay (86.44% versus 76.27%) in the diagnosis of invasive aspergillosis. The sensitivities of both assays decreased with decreasing probability of invasive aspergillosis, i.e., maximum sensitivities of both beta-D-glucan and galactomannan assays were for proven cases (100% versus 87.5%), followed by probable cases (89.29% versus 85.71%), and possible cases (91.57% versus 78.31%). Conclusions Both beta-D-glucan and galactomannan assays seemed to play an encouraging role in the diagnosis of invasive aspergillosis in high-risk clinically suspected cases, with the former assay being more sensitive and the latter assay being more specific.

Introduction. Infective endocarditis (IE) is an important clinical condition with significant morbidity and mortality among the affected population. A single etiological agent is identifiable in more than 90 % of the cases, however, polymicrobial endocarditis (PE) is a rare find, with a poor clinical outcome. Here we report a case of native valve dual pathogen endocarditis caused by Burkholderia cepacia and Aspergillus flavus in an immunocompetent individual. It is among unique occurrences of simultaneous bacterial and fungal etiology in IE. Case presentation. A 30-year-old male was admitted to a cardiology institute with complaints of low grade intermittent fever and progressive shortness of breath for last two months. He was a known case of rheumatic heart disease and had suffered an episode of IE three years ago. On the basis of clinical presentation and the results of radiological investigations, a diagnosis of infective endocarditis was made. Paired blood samples for culture and sensitivity, sampled before the commencement of antimicrobial therapy, yielded growth of Burkholderia cepacia which was highly drug resistant. Sensitivity results-directed therapy consisting of tablet Trimethoprim–Sulfamethoxazole, two double-strength tablets 12 hourly, and Meropenem, 1 g IV every 8 h, was commenced. Despite mild relief of fever intensity, overall clinical condition did not improve and double valve replacement therapy was carried out. Excised valves were sent for microbiological analysis. Burkholderia cepacia was grown on tissue culture with a similar antibiogram to that previously reported from the blood culture of this patient. Direct microscopy of section of valvular tissue with 10 % KOH revealed abundant fungal hyphae. Patient serum galactomannan antigen assay was also positive. Histopathological examination of vegetations also revealed hyphae typical of species of the genus Aspergillus. The patient was successfully treated with meropenem, trimethoprim–sulfamethoxazole and voriconazole. Conclusion. The hallmark of successful treatment in this case was exact identification of pathogens, antibiogram-directed therapy and good liaison between laboratory experts and treating clinicians.

Objective To evaluate direct drug susceptibility testing on MGIT 960 system for detection of multidrug resistant tuberculosis from smear positive pulmonary specimens. Study design Cross-sectional analytical study. Place and duration of study Microbiology Department, Armed Forces Institute of Pathology, Rawalpindi, from July 2016 to September 2017. Methodology Smear positive specimens were pretreated according to guidelines and then tested on MGIT 960 TB system for direct drug susceptibility testing (DST) of isoniazid and rifampin. Samples were also processed by gold standard indirect method, which comprises culture and then DST from positive growth by MGIT 960 TB system. Results Out of 108 specimens, 95 (88%) DST results were reportable. Out of 95 reportable specimens, 17 isolates were resistant to both isoniazid (INH) and rifampin (RIF) by direct DST. The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy for INH were 92%, 93%, 82%, 97% and 92.6%, respectively; and 95%, 96%, 86.3%, 98.6% and 95.7%, respectively for RIF. Average time to report DST by indirect method was 23.6 ±3.9 days, while it was 11.4 ±2.7 days for the direct method. Conclusion Direct susceptibility testing on MGIT 960 system showed very good agreement when compared with indirect method. Time saving is crucial factor in initiation of early effective therapy, especially in drug resistant cases. Further studies on large scale are required for more accurate evaluation of this method.

Summary In this study we evaluated the performance of colorimetric nitrate reductase assay (NRA) on Middlebrook 7H11 agar instead of Lowenstein–Jensen medium for detection of isoniazid (INH) and rifampin (RIF) resistance directly on 114 smear positive sputum specimens and compared the results with direct proportion method on LJ medium. The results of both methods were in 100% agreement for detection of RIF resistance while agreement for INH was 96.4%. The average turnaround time for NRA was 18.6 days and majority of the specimens gave positive results within 21 days. Thus direct NRA testing on smear positive sputum specimens by using 7H11 agar could be used as a fast, reliable and inexpensive method in resource starved settings.

We evaluated nutrient agar using the microcolony detection method for the recovery of Mycobacterium tuberculosis on 37 acid-fast bacilli (AFB) positive sputum specimens, and compared it with conventional Lowenstein-Jensen (LJ) medium. Nutrient agar detected 35 isolates compared to 34 on LJ medium. The mean time to detection of mycobacteria on nutrient agar and LJ medium was respectively 9.6 and 21.4 days. The contamination rate on nutrient agar and LJ medium was respectively 5.4% and 2.7%. Nutrient agar detects M. tuberculosis more rapidly than LJ medium, and could be an economical, rapid culture method in resource-poor settings, provided our findings are confirmed by further studies.

Objectives: To perform a multicentre study evaluating the performance of the direct nitrate reductase assay (NRA) for the detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in sputum samples. Methods: The study was conducted in six laboratories performing tuberculosis diagnosis that were located in six different countries. The NRA was performed directly on sputum samples in parallel with the reference method used at each site. Detection of resistance was performed for rifampicin, isoniazid, ofloxacin and kanamycin. Results: Excellent agreement was obtained for all drugs tested at the majority of sites. The accuracy was 93.7%-100% for rifampicin, 88.2%-100% for isoniazid, 94.6%-100% for ofloxacin and 100% for kanamycin. The majority of NRA results were available at day 21 for sites 1, 2 and 5. Site 3 had a turnaround time of 13.9 days, at site 4 it was 18.4 days and at site 6 it was 16.2 days. The contamination rate ranged between 2.5% and 12%. Conclusions: Rapid detection of drug resistance by the direct NRA on sputum smear-positive samples was accurate and easy to implement in clinical diagnostic laboratories, making it a good alternative for rapid screening for MDR and XDR tuberculosis. Fil: Martin, Anandi. University of Ghent; Bélgica Fil: Imperiale, Belén Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; Argentina Fil: Ravolonandriana, Pascaline. Institut Pasteur de Madagascar; Madagascar Fil: Coban, Ahmet Yilmaz. Ondokuz Mayis University; Turquía Fil: Akgunes, Alper. Chest Diseases Hospital; Turquía Fil: Ikram, Aamer. Combined Military Hospital; Pakistán Fil: Satti, Luqman. Combined Military Hospital; Pakistán Fil: Odoun, Mathieu. Laboratoire de Reference des Mycobacterie; Benín Fil: Pandey, Pooja. All India Institute of Medical Sciences; India Fil: Mishra, Manvi. All India Institute of Medical Sciences; India Fil: Affolabi, Dissou. Laboratoire de Reference des Mycobacterie; Benín Fil: Singh, Urvashi. All India Institute of Medical Sciences; India Fil: Rasolofo, Voahangy. Institut Pasteur de Madagascar; Madagascar Fil: Morcillo, Nora Susana. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; Argentina Fil: Vandamme, Peter. University of Ghent; Bélgica Fil: Palomino, Juan Carlos. University of Ghent; Bélgica

Rapid advancements in the medical sciences have changed the understanding of the diseases down to the molecular level and in turn revolutionized the diagnostics and therapeutics. Similarly, architectural and engineering progression has reshaped the outlooks of the hospitals with the aim of comforting the patients. Despite all that, hospital environments remain a source of infection for the already ailing clientele. The scare of ‘super bugs’ has further aggravated the situation requiring more consolidated efforts for protection of admitted patients.

We evaluated the performance of MGIT 960 system in terms of recover rate, detection time of mycobacteria and contamination rate from various human clinical specimens and compared it with already in use BACTEC 460 TB system and conventional LJ medium. This is the first reported study on MGIT 960 and its comparison with BACTEC 460 system in Pakistan. A total of 260 different clinical specimens received for the culture of mycobacteria were dealt during the six months study period. All the specimens were digested and decontaminated according to the standard N-acetyl-Lcysteine NaOH method. All the processed specimens were inoculated on both the liquid systems and solid medium and incubated for six weeks and eight weeks consecutively. A total of 44 mycobacterial isolates (Mycobacterium tuberculosis, n=43; Mycobacteria other than tuberculosis, n=1) were recovered from 260 clinical specimens. The recovery rate of M. tuberculosis complex was 97.6% on BACTEC MGIT 960 system and 93.0% on BACTEC 460 system and 83.7% on LJ medium. The mean detection time of mycobacteria on BACTEC MGIT 960 system was 11.2 days in smear positive cases, 14.2 days in smear negative cases and 14.8 days in smear positive cases on BACTEC 460 system. Contamination rates were 9.6% and 5.6% and 3.4% for BACTEC MGIT 960, BACTEC 460 system and LJ medium respectively. The non-radiometric, fully automated BACTEC MGIT 960 system has better diagnostic ability as compared with radiometric, semi-automated BACTEC 460 system and LJ medium, so it can be used as a reliable alternative in over burden laboratories.