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Objective: To determine blaCTX-M-15(Cefotaxime-Munich) gene amongst the extensively drug resistant (XDR) Salmonella typhi (S. typhi) isolates by quantitative Polymerase Chain Reaction (qPCR)., Study Design: Observational, cross-sectional study. Place and Duration of the Study: PNS Shifa Hospital and Bahria University of Health Sciences (BUHS), from January to June 2022., Methodology: All the patients clinically suspected of enteric fever, whose blood culture specimens yielded growth of S. typhi were included in this study. These samples were confirmed by serotyping and biochemical reactions. The ceftriaxone resistance was evaluated by antibiotic susceptibility test according to CLSI 2020 guidelines, whereas blaCTX-M-15 gene was detected by (PCR) using gene-specific primers., Results: Out of 149 S. typhi isolates, 87.2% were confirmed XDR S. typhi resistant to ceftriaxone (CRO). Among these, 83.9% harboured blaCTX-M-15 gene., Conclusion: There was a very high frequency of XDR S. typhi harbouring blaCTX-M-15 in Karachi, Pakistan., Key Words: blaCTX-M-15, Salmonella typhi, Third generation cephalosporin, Typhoid fever, Extensively drug resistant.

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.

Streptococcus pluranimalium, a gram-positive aerobic coccus, has been isolated primarily from several farm animals. The pathogenicity of this species is not well characterised either in animals or humans. As per the literature, cases of S. pluranimalium infection in humans have been reported only a handful of times. We report the case of cerebral abscess caused by S. pluranimalium in a patient who presented with weakness and confusion. The diagnosis of cerebral abscess was made on imaging supported by microbiological culture. Burr hole procedure for abscess drainage followed by an antibiotic regimen based on culture and sensitivity results contributed to a successful outcome. The bacteria were identified by analytical profile index for Streptococci (API Strep) and VITEK 2 gram-positive cocci panel. The case was successfully treated with vancomycin.

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.

Methicillin-Resistant Coagulase-Negative Staphylococcusposes a challenging situation in healthcare settings. The spread of resistance among such organisms against major drugs is alarming as it limits the treatment options for serious infections. Traditionally, Vancomycin had remained a mainstay of treatment of Methicillin-Resistant Staphylococci. Linezolid introduction into the clinical practice was a major breakthrough since it provided orally administered treatment of Methicillin-Resistant Staphylococci. Widespread use of Linezolid has gradually turned the impending fear of emergence of resistance against this novel drug into a reality. We report first case of Linezolid-Resistant Methicillin-Resistant Staphylococcushemolyticusfrom Pakistan, isolated from a leukemic patient. The organism caused a necrotic lesion on the right forearm at cannulation site of intravenous catheter. Ooze swab yielded the pure growth of Linezolid-Resistant Methicillin-Resistant Staphylococcushemolyticus.

Objective: To evaluate the in vitro effectiveness of multiple breakpoint concentrations of newer antituberculosis agents (Linezolid and Meropenem) against Multi Drug-Resistant Tuberculosis (MDR-TB) isolates., Study Design: A descriptive cross-sectional study., Place and Duration of Study: Microbiology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2011 to August 2013., Methodology: A total of 100 MDR-TB isolates recovered during the study period were subjected to susceptibility testing against multiple breakpoint concentrations of Linezolid (LZD) and Meropenem (MER). The breakpoint concentration used for LZD were 0.5, 1.0 and 2.0 µg/ml, while for MER were 4.0, 8.0 and 16 µg/ml. Mycobacterial Growth Indicator Tube (MGIT) 960 system was used to carry out drug susceptibility testing as per recommended protocol., Results: At break point concentration of 0.5 µg/ml, 80 out of 100 (80%) MDR-TB isolates were susceptible to LZD while at breakpoint concentration of 1.0 µg/ml and 2.0 µg/ml, 96/100, (96%) of MDR-TB isolates were susceptible. For MER, at breakpoint concentrations of 4.0 µg/ml no MDR-TB isolate was susceptible, while at 8.0 µg/ml 3/100, (3%) and at 16.0 µg/ml 11/100, (11%) of MDR-TB isolates were susceptible., Conclusion: LZD was found to have excellent in vitro efficacy as 96% of MDR-TB isolates were susceptible at breakpoint concentration of 1.0 µg/ml or more. In case of MER it was found that in vitro susceptibility improved as the break point concentrations were increased.

Objective: To evaluate the diagnostic efficacy of two rapid methods i.e. Mycobacterium tuberculosis (MTB) Polymerase Chain Reaction (PCR) on Fine Needle Aspiration (FNA) samples by comparing with cytology of respective site sample., Study Design: Cross-sectional comparative study., Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan, from July 2010 through November 2013., Methodology: A total of 105 extra pulmonary lymph nodes aspirates obtained through fine needle aspiration were processed. Cytology and PCR were done on each specimen. Cytology was taken as gold standard., Results: Out of the total 105 samples, 71 (67.6%) were positive for the MTB PCR while 34 (32.4%) showed negative status. According to FNA cytology (FNAC) results, 72 (68.6%) cases were positive for the disease while 33 (31.4%) were negative. Sensitivity of PCR was 90.3%, specificity 81.8%, positive predictive value (PPV) 91.5%, negative predictive value (NPV) 79.4%, with diagnostic accuracy of 87.6%. Area under the curve was 0.860 (p < 0.001)., Conclusion: PCR is a sensitive tool for detection of MTB on FNA samples from EPTB cases. The results are available within few hours which is helpful for the clinicians to initiate therapy.

Objective: To find out the frequency of Extensively Drug Resistant (XDR) and pre-XDR tuberculosis in clinical isolates of Multi-Drug Resistant (MDR) Tuberculosis (TB) by determining the susceptibilities against Levofloxacin and Amikacin (classical second line antituberculosis drugs)., Study Design: Adescriptive cross-sectional study., Place and Duration of Study: Microbiology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2011 to August 2013., Methodology: Amikacin (AK) and Levofloxacin (LEVO) were obtained in chemically pure form from Sigma (Taufkirchen, Germany). The breakpoint concentration used for AK was 1.0 µg/ml and for LEVO 2.0 µg/ml. Mycobacterial Growth Indicator Tube (MGIT) 960 system was used to carry out drug susceptibility testing as per recommended protocol., Results: A total of 3 MDR-TB isolates (3%) turned out to be XDR-TB based upon simultaneous resistance to injectable second line antituberculosis drug AK and one of the fluoro-quinolones (LEVO). A total of 24 MDR-TB isolates (24%) were found to be pre-XDR based upon resistance to LEVO alone. Treatment status record of patients with XDR and pre-XDRTB isolates revealed that majority of patients had received fluoroquinolones (FQs) during the course of treatment., Conclusion: XDR-TB has started to emerge in MDR-TB isolates in our set up. The worrying sign is the high frequency of pre-XDR tuberculosis. Urgent steps need to be taken to stem the tide of pre-XDR-TB in our population. It is thus recommended to develop facilities to carry out drug susceptibility testing to monitor the status of pre-XDR and XDR-TB in our population.

Objective: To evaluate the yield of polymerase chain reaction (PCR) for detection of Mycobacterium tuberculosis from different clinical specimens., Study Design: Observational study., Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January 2001 to December 2010., Methodology: Different clinical specimens received for Mycobacterium tuberculosis PCR were dealt during the study period. Contaminated samples like sputum were processed by the standard N-acetyl L-cysteine (NALC)-NaOH method. PCR protocols were followed as per manufacturer's manual. PCR was performed using a Thermal Cycler (Master Cycler, Eppendorf, Germany): an initial denaturation step at 94°C for 3 minutes was followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 30 seconds and a final extension at 72°C for 7 minutes. The products were held at 4°C and later run on 1% agarose gel, stained with Ethidium bromide and visualized in ultraviolet (UV) transilluminator., Results: Out of a total 4620 samples for PCR, 299 were positive for Mycobacterium tuberculosis (6.5%). The percentage of samples from male patients were 63.2%. The mean age of patients was 38+11.5 years. Blood was the most frequent specimen received for PCR (46.66%), followed by body fluids (18.41%) and CSF (10.64%). Yield for different clinical samples was 63/471 for sputum (13.4%), 3/29 for endobronchial washings (10.3%), 59/851 for body fluids (6.9%) and 24/400 for urine (6%). Positive yield from blood was the lowest (101/2156, 4.7%)., Conclusion: PCR for Mycobacterium tuberculosis is a rapid and reliable method for the diagnosis of both pulmonary and extrapulmonary tuberculosis. The highest positive yield was obtained from sputum and lowest from blood specimens.

Incidence and prevalence of Mycobacterium fortuitum infection vary greatly by location and death is very rare except in disseminated disease in immunocompromised individuals. We present what we believe is the first case of bone marrow infection with Mycobacterium fortuitum in an HIV negative patient. Bone marrow examination revealed presence of numerous acid fast bacilli which were confirmed as Mycobacterium fortuitum on culture and by molecular analysis. Patient was managed successfully with amikacin and ciprofloxacin.

This case report describes an outbreak of multidrug resistant Acinetobacter baumannii in the intensive care unit of a tertiary care hospital. Three patients were infected on the same day from an Ambu bag which was used on all the patients. The outbreak was immediately identified and the source was traced within one week. Appropriate measures were taken and a continuous surveillance was carried out resulting in reporting of no such case from the intensive care unit in the last 6 months.