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Emergence of resistance among Escherichia coli (E.coli) isolates against therapeutic options for UTIs (Urinary tract infections) has led to renewed interest in older antibiotics like Fosfomycin. In this study we evaluated diagnostic accuracy of Rapid Fosfomycin NP test based on glucose metabolism for rapid Fosfomycin susceptibility testing among urinary E.coli isolates.In a cross-sectional validation study conducted in the Microbiology Department, Armed Forces Institute of Pathology, Rawalpindi, Pakistan from 15th March to 15th September 2020, 149 consecutive urine specimens were included as per selection criteria. Rapid Fosfomycin NP test was performed as per protocol of Nordmann P et al on urinary E.coli isolates for detection of Fosfomycin resistance and results were compared with reference modified Kirby-Bauer disk diffusion method.Out of total 149 E.coli isolates from 149 urine specimens, 80 were classified as Fosfomycin susceptible and 69 as Fosfomycin resistant by reference disk diffusion method. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of rapid Fosfomycin NP test was found to be 94.2%, 98.75%, 98.48%, 95.2% and 96.64%, respectively. In our study reliable results were achieved after 2.5 hours of incubation.The rapid Fosfomycin NP test is valid and user-friendly technique which can be performed with minimal technical expertise. It is less time consuming than disk diffusion and Etest strips and easy to perform as compared to agar dilution method. It can be useful as alternative to agar dilution in urinary E.coli isolates which would help in selecting appropriate therapeutic option for UTIs.

Acinetobacter baumannii has emerged as one of the leading causes of multidrug resistant nosocomial infections worldwide. It is able to survive in hospital environment and build up diverse resistance mechanisms making it difficult to treat with current antibiotics. Objective: It was to determine the frequency and patterns of Acinetobacter baumannii in intensive care units (ICU) settings.A cross sectional study was carried out in the Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from 1st July 2017 to 30th June 2019. A total of 603 non-duplicate clinical specimens were received from intensive care units. Specimens yielding growth of multidrug resistant Acinetobacter baumannii, were evaluated as per standard protocol. The antimicrobial sensitivity testing was performed as per Clinical and Laboratory Standard Institute guidelines (2017-2018).Among Acinetobacter baumannii (310 isolates), 5% were multidrug resistant, 93% extensively drug resistant and 1% pan drug resistant. Percentage of carbapenem resistant strains was 92%. In drugs like tigecycline and polymyxin, resistance was noted as 73% and 1% respectively. High yield of this superbug was mainly obtained from respiratory specimens (43.5%), whereas 24% were detected from wound infections and 29% from other samples. .This study showed a rapidly increasing resistance in Acinetobacter baumannii. Therefore, polymyxin remains the only option in our intensive care units, but its usage as empirical therapy in our setting has led to the emergence of resistance to this drug. Implementing infection control practices, antimicrobial stewardship and restricted use of polymyxin can play a significant role in reducing health care burden.

Polymyxins play a significant role against carbapenem-resistant Enterobacteriaceae (CRE). A total of 121 clinical samples yielded growth of CRE that were included in the study. Rapid Polymyxin NP test was performed on all the isolates as described by Nordmann P et al. and results were compared with broth microdilution method. Majority of the isolates were Klebsiella pneumoniae (70.2%) followed by Escherichia coli (17.4%). A total of 71 isolates were found resistant and 50 as susceptible by broth microdilution. Sensitivity and specificity of rapid polymyxin NP test were found to be 97.2% and 100%, respectively. Our study concluded that rapid polymyxin NP test is reliable and can be used as an alternative to broth microdilution in resource limited settings.

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 a direct antibiotic susceptibility testing method for blood culture.. Methods The cross-sectional comparative study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from December 2016 to October 2017. Direct antimicrobial susceptibility testing was performed from positive blood culture bottles. Bacterial identification was done by using API 10S. Different antimicrobial panels were employed for Gram-negative rods (GNRs), gram-positive cocci (like suspected Staphylococci and Enterococci). Results were compared with conventional disk diffusion testing and very major, major and minor errors were calculated. Result agreement and kappa coefficient scores were generated for categorical agreement. SPSS 24 was used for data analysis. Results Of the 101 bacterial isolates, 82(81.2%) were Gram negative rods and 19(18.8%) were Grampositive cocci. Among 781 bacteria-antibiotic comparisons, the number of very major errors was 3(0.4%), major errors were 7(0.9%) and minor errors were 12(1.5%), while, 759(97.2%) comparisons yielded the same results. The kappa coefficient was 0.946, showing almost perfect agreement. Direct identification of Gram negative rods was successful in 53(64.6%) cases. Conclusions Direct susceptibility testing of blood culture produced reliable results for majority of the antimicrobials.

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.

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.Seventy-two M. tuberculosis isolates were tested in two phases by CVDA.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.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.

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.

Objective To determine the pattern of blood stream infections and their antibiotic susceptibility profile with infectivity predictors in a neonatal setting. Methods The descriptive cross-sectional study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from December 1, 2016,to April 30, 2018, and comprised blood culture samples received in Bactec/BactAlert paediatric bottles from neonates aged 0-30 days admitted in the neonatal intensive care unit. The samples were processed as per the standard guidelines. Antibiotic susceptibility was checked as per guidelines of the Clinical and Laboratory Institute. VITEK 2 system was used for rapid identification and minimum inhibitory concentrations of the drugs. SPSS 24 was used for data analysis. Results Out of 640 samples, 172(27%) were culture-positive. Among them, 98(57%) were gramnegative rods, 50(29%) gram-positive cocci and 24(14%) were fungi. Of the 172 pathogens identified, Klebsiella pneumoniae was 39(22.7%) followed by Candida species 24(14%) and methicillin-resistant Coagulase-negative staphylococci 20(11.6%). Of Klebsiella pneumoniae isolates, 26(58%) were extended spectrum -lactamase producers. Among Acinetobacterbaumanii, 11(58%) were extensively drug resistant and Carbapenem-resistant strains were 20(91%). Also, 4(8%) isolates of Enterococcus faecium were vancomycin-resistant. Conclusions Majority of the isolates causing blood stream infections in neonatal intensive care unit were multi drug resistant, posing a therapeutic challenge for the neo natologists .

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.

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.

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.

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

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.