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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.A descriptive cross-sectional study.Microbiology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2011 to August 2013.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.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.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.

Thrombocytopenia is often seen in the patients of malaria infected with Plasmodium vivax. We studied patients admitted in hospital having coexisting thrombocytopenia and malaria, and recorded the response to anti-malarial therapy.In this cross-sectional descriptive study, a total of 120 patients admitted in medical ward with Plasmodium vivax malaria and co-existing thrombocytopenia were studied.Out of total 120 slide positive Malaria patients who had low platelet count (150x10(9) /L), platelet count increased to ≥150x10(9) /L in 73 (60.8%) patients after five days of anti-malarial therapy while in 47 (39.2%) patients thrombocytopenia persisted. After ten days of anti-malarial therapy, platelet count in all the patients recovered to ≥ 150x10(9) L. None of the patients required platelet transfusion.In majority of the patients of Plasmodium vivax malaria having thrombocytopenia, platelet count returns to normal within five to ten days of start of anti-malarial treatment and nlatelet transfusion is not required.

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.Cross-sectional comparative study.Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan, from July 2010 through November 2013.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.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 (p0.001).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.

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).Adescriptive cross-sectional study.Microbiology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2011 to August 2013.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.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.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.

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.

Diarrhoea is a serious threat all over the world with great economic implications especially evident in the developing world. This study was aimed at determining in vitro efficacy of Zinc (Zn) against common enteric bacterial pathogens.A total of 100 bacterial enteric pathogens: Salmonellae (n = 16), enteropathogenic Escherichia coli (EPEC) (n = 26). Shigellae (n = 28) and Vibrio cholerae (n = 30) were isolated from diarrhoeal stool specimens at Department of Microbiology, Armed Forces Institute of Pathology Rawalpindi during Aapril 2009 to Jan 2010. These isolates were tested against various concentrations of Zn supplemented in Mueller Hinton (MH) agar using a multipoint inoculator. A minimum inhibitory concentration of active Zn in ZnSO4.7H2O ranging from 0.03 mg/ml to 1 mg/ml was used.Zn completely inhibited the growth of all the tested pathogens and most of them were inhibited at a concentration of 0.06 mg/ml to 0.5 mg/ml of Zn.Zinc has an excellent antibacterial activity against enteric bacterial pathogens common in our setup which may provide basis for treatment of diarrhoea. Clinical study based on these findings is recommended.

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.

In this study, we evaluated the performance of blood agar (by macroscopic growth) and nutrient agar (by a microcolony detection method) for drug susceptibility testing of Mycobacterium tuberculosis against rifampin (RIF) and isoniazid (INH), using 67 smear-positive sputum specimens. The direct proportion method on Lowenstein-Jensen (LJ) medium was used as the “gold standard.” Compared with LJ medium, results for both media were in 100% agreement for RIF, while for INH the agreement levels for blood agar and nutrient agar were 98% and 95%, respectively. Within 2 weeks, 100% of specimens yielded results on blood agar, while 96.8% of specimens yielded results on nutrient agar. Our study showed that blood agar and nutrient agar can be used as alternative media for direct susceptibility testing of RIF and INH, especially in resource-poor settings.

To evaluate the yield of polymerase chain reaction (PCR) for detection of Mycobacterium tuberculosis from different clinical specimens.Observational study.Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January 2001 to December 2010.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.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%).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.

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.

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.

Scytalidium dimidiatum is mainly responsible for human skin and nail infections but the mould has also been reported for invasive infections in immunocompromised individuals. We report a young immunocompetent individual diagnosed with invasive non-traumatic Scytalidium dimidiatum infection involving the left orbital cavity and maxillary sinus.