Assessing Colistin Resistance by Phenotypic and Molecular Methods in Carbapenem-resistant Enterobacterales in a Tertiary Care Hospital in Pakistan.

Introduction: Members of Enterobacterales are very common pathogens, which continue to show resistance to many antibiotics. Carbapenem performed well for some time. Colistin was the final hope for the carbapenem-resistant Enterobacterales, but resistance against it has virtually tied the clinician's hands, especially when it comes to treating critically ill patients. Purpose: Detection of colistin resistance by the agar method as well as by the polymerase chain reaction (mobilized colistin resistance-1 gene) in carbapenem-resistant Enterobacterales. Materials and Methods: A cross-sectional study from Dec 2019 to Dec 2020 was conducted at the Department of Microbiology, Army Medical College, National University of Medical Sciences Rawalpindi Pakistan. Antimicrobial susceptibility of Enterobacterales was determined according to the Kirby-Bauer disc diffusion method except for colistin. Colistin agar was used, in concentrations of 2 µg/mL and 4 µg/mL. Results were interpreted according to Clinical and Laboratory Standards Institute guidelines 2020. Mobilized colistin-resistant-1 gene in the carbapenem resistant Enterobacterales was detected by performing real-time polymerase chain reaction assay. Results: Among the 172 carbapenem-resistant Enterobacterales 18 isolates were resistant using the colistin agar test. Whereas by molecular method colistin resistance was detected among 10 isolates that carried mobilized colistin resistance 1 gene, making the frequency of the MCR-1 gene 5.81%. Seventy percent of isolates were from paired blood samples. Eight patients, from whom the colistin resistant gene was isolated expired. Conclusion: Colistin resistance is a very serious issue and should not be missed in a clinical microbiology laboratory. The phenotypic agar test method is an excellent option for routine use, as it combines ease of performance with affordable cost. However, molecular methods are essential for the detection of mobilized colistin resistance gene (1-9) for epidemiological purposes.

Influenza Virus Subtyping by Multiplex PCR during Winter Months of 2017-2018.

OBJECTIVE: To determine the frequency of infections caused by Influenza viruses, i.e. Influenza A (H1N1) pdm09, Influenza A (H3N2) and Influenza B in patients presenting with respiratory tract infections, i.e. influenza-like illness (ILI) and severe acute respiratory illness (SARI). STUDY DESIGN: Descriptive, cross-sectional study. PLACE AND DURATION OF STUDY: Department of Virology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from October 2017 to February 2018. METHODOLOGY: A total of 624 samples from patients with respiratory tract infections (both ILI and SARI) were included in the study. Specimens collected from the patients included nasal swabs and throat swabs, which were transported in viral transport medium (VTM) to Virology Department, AFIP. Multiplex PCR was done for Influenza A (H1N1) pdm09, Influenza A (H3N2) and Influenza B. RESULTS: A total of 200 (32%) samples were found to be positive for Influenza viruses. Out of total 624 samples analysed, 220 (35.3%) were from females and 404 (64.7%) from males. Among these, 510 (81.7%) presented with ILI and 114 (18.3%) with SARI. Among positive samples, 120 (19.2%) samples were positive for H1N1, 61 (9.8%) for H3N2 and 19 (3%) were positive for Influenza B. Highest number of positive cases occurred in the month of January, i.e. 148 (74%) cases. Only 3 (2.5%) patients out of 120 infected with H1N1 were in age group-I (0-5 years). While in age group-II (6-30 years), age group-III (31-60 years), and age group-IV (>60 years); 39 (32.5%), 63 (52.5%) and 15 (12.5%) patients were infected by H1N1, respectively. Maximum patients with H3N2 infection were in age group-III; 30 (49.2%) of the total 61. Commonest Influenza subtype in age group-IV was H3N2 found in 20 (32.8%) patients, followed by H1N1 in 15 (12.5%) patients. CONCLUSION: The dominant subtype in our set-up, during winter of 2017-2018, was Influenza A (H1N1) pdm09. Highest numbers of positive cases were recorded in the month of January. People with ILI and SARI should be tested for Influenza viruses to avoid unnecessary use of antibiotics.

Crystal violet decolorization assay for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates: A multicenter study.

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.

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.