Direct susceptibility testing on MGIT 960 TB system: a rapid method for detection of drug resistant tuberculosis

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

Evaluation of nitrate reductase assay for early detection of multi and extensively drug resistance tuberculosis in our setup

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

Dual infection by burkholderia cepacia and pseudomonas putida in an infective endocarditis case

Infective endocarditis is rarely caused by Burkholderia cepacia

Pseudomonas putida has not been reported to cause infective endocarditis so far. This is the first case of infective endocarditis being reported, that is caused by Pseudomonas putida and Burkholderia cepacia in an immunocompetent host with no predisposing factors. Aortic valve replacement surgery was carried out and antibiotics were given, to which the patient responded well and recovered

Sensitivity Pattern of Second Line Anti-Tuberculosis Drugs against Clinical Isolates of Multidrug Resistant Mycobacterium Tuberculosis

Objective: To determine the current sensitivity pattern of second line anti-tuberculosis drugs against clinical isolates of Multidrug Resistant Mycobacterium tuberculosis [MDR-TB]. Study Design: A cross-sectional study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology [AFIP], Rawalpindi, from November 2011 to April 2013. Methodology: Samples received during the study period were processed on BACTEC MGIT 960 system for Mycobacterium tuberculosis [MTB] culture followed by first line drugs susceptibility testing of culture proven MTB isolates. On the basis of resistance to rifampicin and isoniazid, 100 clinical isolates of MDR-TB were further subjected to susceptibility testing against amikacin [AMK], capreomycin [CAP], ofloxacin [OFL] and ethionamide [ETH] as per st and ard BACTEC MGIT 960 instructions. Results: Out of 100 MDR-TB isolates, 62% were from male patients and 38% from female patients. 97% were sensitive to AMK, 53% to OFL, 87% to CAP; and 87% were sensitive to ETH. Conclusion: The majority of the MDR-TB isolates showed excellent sensitivity against AMK, CAP and ETH. However, sensitivity of MDR-TB isolates against fluoroquinolones like OFL was not encouraging. Key Words: Multidrug resistant tuberculosis. Mycobacteria growth indicator tube. Second line anti-tuberculosis drugs. Amikacin. Capreomycin. Ethionamide

Rapid mycobacterium tuberculosis dna detection on fine needle aspirates from extra pulmonary lymph nodes

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 [p < 0.001]. 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

Antimicrobial susceptibility pattern of bacteria isolated from patients with urinary tract infection

To determine the antimicrobial susceptibility pattern of bacterial pathogens in the patients of urinary tract infection reporting at a tertiary care hospital. Laboratory based study. Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January to December 2012. A total of 440 culture positive bacterial isolates from 1110 urine samples; submitted over a period of one year were included in this study. Identification of bacterial isolates was done by standard biochemical profile of the organisms. The antimicrobial susceptibility of culture positive bacterial isolates was performed by disk diffusion method as recommended by Clinical Laboratory Standard Institute guidelines [CLSI]. Out of the 440 culture positive urine samples, 152 [34.6%] were from indoor patients whereas 288 [65.4%] from outdoor patients. Gram negative bacteria accounted for 414 [94%] of the total isolates while rest of the 26 [6%] were Gram positive bacteria. The most prevalent bacterial isolate was Escherichia [E.] coli 270 [61.3%] followed by Pseudomonas [P.] aeruginosa 52 [12%] and Klebsiella [K.] pneumoniae 42 [9.5%]. The susceptibility pattern of E. coli showed that 96.2% of the bacterial isolates were sensitive to imipenem, 85.1% to amikacin, 80.7% to piperacillin/tazobactam and 72.6% to nitrofurantoin. In case of P. aeruginsosa, 73% bacterial isolates were sensitive to tazobactam/piperacillin, 69.2% to sulbactam/cefoperazone and 65.38% to imipenem. The antibiogram of K. pneumoniae has revealed that 76.1% of the bacterial isolates were sensitive to imipenem and 52.3% to piperacillin/tazobactam. Nitrofurantoin and imipenem were the most effective antimicrobials amongst the Enterococcus spp. as 92.3% showed susceptibility to this bacterial isolate. Majority of the bacterial isolates were sensitive to imipenem and piperacillin/tazobactam while susceptibility to most of the commonly used oral antibiotics was very low. Among the oral antimicrobials, nitrofurantoin showed good susceptibility against Enterobacteriaceae family and Gram positive organisms

In vitro activity of fosfomycin tromethamine against extended spectrum beta-lactamase producing urinary tract bacteria

To determine the in vitro activity of Fosfomycin tromethamine against extended spectrum beta-lactamase producing uropathogens. Experimental study. Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from October 2011 to October 2012. A total of 381 culture positive ESBL producing isolates from 2400 urine samples submitted over a period of one year were included in this study. Identification of isolates was done by standard biochemical profile of the organisms. The antimicrobial susceptibility of culture positive isolates was performed by disk diffusion method as recommended by Clinical Laboratory Standard Institute guidelines [CLSI]. The antimicrobial activity of Fosfomycin to various isolates revealed that 93% of E. coli, 64% Klebsiella spp. 50% Proteus spp. 75% Enterobacter cloacae, 100% Citrobacter freundii, 100% Burkholderia spp. 100% Serratia spp. and 50% Stenotrophomonas maltophilia were susceptible to this chemical compound. Fosfomycin showed excellent effectiveness to most of the common ESBL producing bacteria such as E. coli, Klebsiella and Proteus spp

Pathogens causing blood stream infections and their drug susceptibility profile in immunocompromised patients

To determine the types of pathogens causing blood stream infections and their drug susceptibility profile in immunocompromised patients. Cross-sectional, observational study. Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January to September 2012. Blood culture bottles received from immunocompromised patients were dealt by two methods, brain heart infusion [BHI] broth based manual method and automated BACTEC system. The samples yielding positive growth from either of two methods were further analyzed. The identification of isolates was done with the help of biochemical reactions and rapid tests. Antimicrobial susceptibility of the isolates was carried out as per recommendations of Clinical and Laboratory Standards Institute [CLSI]. Out of the 938 blood culture specimens received from immunocompromised patients, 188 [20%] yielded positive growth. Out of these, 89 [47.3%] isolates were Gram positive and Gram negative each, while 10 [5.3%] isolates were fungi [Candida spp.]. In case of Gram positive isolates, 75 [84.3%] were Staphylococcus spp. and 51 [67%] were Methicillin resistant. Amongst Gram negative group 49 [55.1%] isolates were of enterobacteriaceae family, while 40 [44.9%] were non-lactose fermenters [NLF]. In vitro antimicrobial susceptibility of Staphylococci revealed 100% susceptibility to vancomycin and linezolid. The enterobacteriaceae isolates had better susceptibility against amikacin 85.7% compared to tigecycline 61.2% and imipenem 59.2%. For NLF, the in vitro efficacy of aminoglycosides was 72.5%.The frequency of Gram positive and Gram negative organisms causing blood stream infections in immunocompromised patients was equal. Vancomycin in case of Gram positive and amikacin for Gram negative organisms revealed better in vitro efficacy as compared to other antibiotics.

Pattern of dermatophytes among dermatological specimens at AFIP, Rawalpindi

To determine the types and prevalence of dermatophytes from the clinical specimens received at Armed Forces Institute of Pathology [AFIP]. Study design is descriptive. The study was carried out at the Department of Microbiology, AFIP Rawalpindi from June 2009 to May 2010. Total of 400 different clinical specimens were dealt during the study period. After direct microscopy, they were inoculated on Sabouraud's dextrose agar with and without antimicrobials. The plates were incubated at 220C and examined twice weekly up to four weeks for any fungal growth. Species identification was done through colony morphology and microscopic examination of lactophenol blue preparation. Out of total specimens, 221[55.25%] yielded fungal growth. The overall yield of dermatophytes from different specimens was in the order of nail [78%], followed by skin [18.3%] and hair [3.3%]. Mycological infections have growing importance because of the increasing population of immune-compromised patients warranting a high index of suspicion

Bone marrow infection with Mycobacterium fortuitum in a diabetic patient

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

Evaluation of readycult coliforms / E. coli test on water samples

To assess the diagnostic accuracy of Readycult Conforms/ E. coli test for microbiological testing of potable water using Multiple Tube Method as gold standard. Study was conducted at Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January 2008 through June 2008. This study was a continuum of a previous study. Water samples received within 2 hours of collection were divided aseptically into two equal parts; one part was used for Readycult Conforms/ E. coli test and the other for Multiple Tube Method. Results were interpreted according to WHO guidelines. The sensitivity and specificity of the Readycult Coliforms/ E. coli test was 97% and 95.2% respectively compared to the Gold standard. The positive predictive value and negative predictive value were 94.2% and 98% respectively. Readycult Coliforms/ E. coli test is an effective method for bacteriological testing of water. Results are available within 18-24 hours. This technique can be a useful tool in routine water microbiology particularly in remote areas where laboratory facilities are scarce

Status of hospital infection control measures at seven major tertiary care hospitals of northern Punjab

To determine the availability and implementation of various hospital infection control measures at tertiary care hospitals. Survery. National Institute of Science and Technology, Islamabad, from June through August 2008. Seven tertiary care very busy hospitals were selected; one from Islamabad, 5 from Rawalpindi, and one from Lahore. A detailed proforma was designed addressing all the issues pertaining to hospital infection control measures. Air sampling was done and growth yielded was identified by standard methods. Analyses revealed that all of the hospitals had an Infection Control Committee. Microbiological diagnostic facilities were adequate at all the hospitals and overall microorganism yield was very high. Antibiotic policy was claimed by most, not available on ground. Majority of the operation theatres were without proper air flow system and autoclaves were not being regularly monitored. There was no proper disposal for sharps and needles. Incineration was not the usual mode for infectious waste. The results of the present study imply availability of proper hospital infection control policies with need of strict implementation of such measures

Evaluation of BACTEC MGIT 960 system for recovery of Mycobacterium tuberculosis complex in Pakistan

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.

Pulmonary cryptosporidiosis in a bone marrow transplant recepient

Cryptosporidium, a protozoan parasite of Phylum Apicomplexa, causes acute short term intestinal infection in immunocompetent individuals. However, in immunocompromised patients, it causes prolonged and life threatening watery diarrhea, rarely with extra-intestinal involvement. We present a case of Cryptosporidium parvum with pulmonary involvement who was managed with azithromycin and co-trimoxazole combination. This is the second reported case in the world in HIV negative patient undergoing bone marrow transplantation

Rapid detection of methicillin resistant staphylococci from the nasal screening swabs by mannitol salt agar with cefoxitin and oxacillin disks

To assess the reliability of Manitol salt agar [MSA] for directly identifying Methicillin Resistant Staphylococcus aureus [MRSA] and Methicillin Resistant Coagulase negative Staphylococci, [MRCoNS] in nasal swabs for screening purposes using cefoxitin and oxacillin disks. Descriptive and Quasi-experimental. The study was done in the two surgical units of Combined Military Hospital, Rawalpindi and all the samples were processed at the Department of Microbiology, Armed forces Institute of Pathology, Rawalpindi during July 2007. A total of eighty four duplicate swabs were taken from the anterior nares of various staff members of the two surgical units and were directly inoculated on Mannitol salt agar with Cefoxitin disc 30 micro g [MSAFOX] and oxacillin disc 1 micro g [MSAOX]. All the samples were simultaneously inoculated on blood and MacConkey agar for conventional testing, using standard conditions, and confirmed as MRSA or MRCoNS by oxacillin disk diffusion technique. The staphylococcal isolates were later confirmed as MRSA/MRCoNS by polymerase chain reaction [PCR] for mec A gene analysis. There were 45 staphylococci which revealed mec A gene [40 MRCoNS and 5 MRSA] by PCR. Both the disks with MSA effectively identified the methicillin resistance. MSA with cefoxitin could identify 40 methicillin resistant staphylococci [35 MRCoNS and 5 MRSA] where as MSA with oxacillin could identify 39 methicillin resistant staphylococci [34 MRCoNS and 4 MRSA]. There was no significant difference between the two disks in sensitivity, specificity, positive and negative predictive values and overall efficacy of the procedures. MSA with cefoxitin 30 micro g and oxacillin 1 micro g appear to be highly accurate, easy to perform and beneficial for quick and reliable detection of methicillin resistant staphylococci from the nasal carriers in a routine microbiology laboratory

Invasive infection in a young immunocompetent soldier caused by scytalidium dimidiatum

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

Comparison of two different sensitivity testing agars for detecting methicillin resistance in Staphylococcus aureus

To compare the accuracy of Mueller-Hinton agar and Isosensitest agar using cefoxitin disc for detecting methicillin resistant Staphylococcus aureus using mecA gene PCR assay as gold standard. Comparative study. Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from May 2006 to January 2007. One hundred clinical isolates of Staphylococcus aureus were evaluated; 64 MRSA [methicillin resistant Staphylococcus aureus] and 36 MSSA [methicillin sensitive Staphylococcus aureus] by mecA PCR assay All the isolates were tested with cefoxitin 30 micro g disc using semi-confluent growth on Mueller-Hinton agar as well as on Iso-sensitest agar in ambient air at 35-37°C after an overnight incubation as per recommendations of Clinical and Laboratory Standard Institute. Following diameters provided the best sensitivity and specificity without substantial overlapping between the zones of resistant and sensitive isolates; Mueller-Hinton agar: R /= 22 mm [sensitivity 97.2% and specificity 100%], and Iso-sensitest agar: R /= 26 mm [sensitivity 100% and specificity 100%]. High accuracy was obtained with cefoxitin disc on both media. Performance of both media was equally convincing for reliable prediction of methicillin resistance in Stapfylococcus aureus by placing cefoxitin 30 micro g disc on either of these in routine susceptibility testing

Enterococcus: enemy within

Enterococci have speedily emerged not only as organisms associated with nosocomial infections but have also created therapeutic problems. Presently in United States they are placed second in the category of nosocomial pathogens. A study was carried out at Armed Forces Institute of Pathology to determine the susceptibility pattern of enterococci to vancomycin and to find out whether vancomycin resistant enterococci colonize intestine, in our set up. One hundred enterococci collected from various clinical specimens were identified at species level. Modified Kirby-Bauer method was employed for disk diffusion and minimum inhibitory concentrations were determined by dilution method, according to the recommendations of National Committee for Control of Laboratory Standards [NCCLS]. The results were very encouraging as the tested isolates demonstrated excellent invitro susceptibility to vancomycin. Minimum inhibitory concentration [MIC]9o of the isolates was 4 mg/L and no isolate was found to be fully resistant to vancomycin. Vancomycin-resistant enterococci were not isolated from faecal specimens. Fortunately according to this study vacomycin resistant enterococci are not prevalent in our set-up, but one has to be on a continuous watch