IntroductionWith the advancement in medical practice, new problems are surfacing, one of most important and common being emergence of resistance in Gram negative bacteria towards the ? lactams due to ? lactamases enzyme.
These enzymes inactivate beta lactam ring containing antibiotics, which in the course of time has evolved to extended spectrum ?-lactamases (ESBL), which confers bacteria enhance ability to be resistant against wide variety of new beta-lactams1 including the III generation cephalosporins, and aztreonam (but not the cephamycins or carbapenems). The ESBLs hydrolysis the antibiotics, but are inhibited by ?-lactamase inhibitors such as clavulanic acid.2ESBLs are a cause of major challenge in therapeutic treatment in hospitals3 due to increase in expenditure of money on ineffective antibiotics which further develops resistance in bacteria making patient more susceptible to infection and colonization by them.4ESBL strains remain undetected as they are difficult to detect by routine susceptibility testing methods and may show false susceptibility to antibiotics by Kirby- Bauer disc diffusion methods5. Identification of all ESBLs producing organisms in clinical Microbiology laboratory is a major challenge because of inoculum effect and substrate specificity1. ESBL detection is important as knowledge about its prevalence is helpful to formulate infection control measures and to prevent their spread6. There are different methods for detection of ESBLs like phenotypic confirmatory disc diffusion test (PCDDT), Modified Double Disc Synergy Test (MDDST), Indirect Modified Three Dimensional Test (IMTDT), etc.Review of literatureThere is high prevalence of ESBLs in clinical isolates.
Sharma M. et al reported frequency of 52.49% in gram negative isolates at NIMS University, Jaipur4,while Hima Bindu M et al, reported ESBLs frequency of 58.8% in isolates7 at Mallareddy Institute of Medical Sciences, India.
Where else study by Bajpai T et al, determined that among gram negative isolate 36.8% were found to be ESBL producers8.Indirect modified 3-dimensional enzyme extract test for detection of ESBLs is sensitive and gives rapid result.
Modi D et al, reported sensitivity of IMTDT is 98% to 100% and found it to be most sensitive test among other available tests such as double disc synergy test and modified direct three dimensional test.Khodare A et al, study showed 76% strains gave positive result with IMTDT, and was found to be better than phenotypic confirmatory disc diffusion test (PCDDT) for detection of ESBLs although it was a little labour intensive and may be technically challenging. 10.In study by Shaikh el at IMTDT was found to be superior method than Modified Double Disc Synergy Test (MDDST), PCDDT and DDST for detection of production of ESBL alone or in presence of other ?- lactamases like AmpC.
PCDDT& DDST should be used in the isolates which produce only ESBL but are not useful for detection of ESBL in isolates who also produces other ?-lactamases like AmpC enzyme11.AIM & OBJECTIVEWith the advent of ESBLs which are reducing the treatment options, it is necessary to detect these with a reasonable accuracy so that appropriate treatment may be initiated in the patients. Hence this study is planned with the following objective:1. To detect ESBL producing strains using the indirect modified three dimensional test.2. To study the utility of IMTDT in detecting ESBLs in tertiary care hospitalMATERIAL AND METHODS:-The study will be carried out in the Department of Microbiology of a tertiary care hospital.
Study Type: Cross-sectional prospective study. Study Time: Two months.Sample Size: 30 strainsMethodology:All samples received for culture and sensitivity in the Microbiology will be inoculated on routine media like blood agar and Mac Conkey agar; in case of urine it will be inoculated on cysteine lactose electrolyte deficient medium (CLED). After 24 hours of incubation, the bacterial isolates, if any, will be identified by standard biochemical tests12 and antibiotic sensitivity will be performed by Kirby Bauer method as per CLSI guidelines. Also ESBL screening will be done by standard disc diffusion using cefpodoxime, cefotaxime, ceftriaxone and ceftazidime with zones of inhibition <17mm, <27mm, <25mm and <22mm respectively as probable ESBLs.
13Indirect modified three dimensional test:Crude enzyme extract of the test strain will be prepared by freeze-thawing of approximately 15 mg of the isolate suspended in 0.5 ml of peptone water. Lawn culture of Escherichia coli (ATCC 25922) will be prepared on Muellar Hinton Agar with turbidity matching McFarland 0.5 standard. A cefotaxime disc will be placed on the plate and at distance of 2mm from this a 4mm well will be punched out. Into this well, 30 µl of the crude extract will be filled and incubated at 370C for 24 hours.
A heart shaped distortion of the zone of inhibition around the cefotaxime disc will be taken to a positive test.9Inclusion Criteria: All samples that will yield ESBL producing strains by standard disc diffusion method during the study period will be included in the study. Exclusion Criteria: All strains that are sensitive to the antibiotics as well as all Gram positive isolates. Planned procedure to analyze data:All data will be maintained in Microsoft office Excel. All statistical analysis will be carried out using Excel and Appropriate Statistical tools will be applied wherever required.
ETHICAL CONSIDERATIONS:Ethical clearance will be obtained from Institutional Ethics Committee (IEC). Reference1. Rawat D, Nair D. Extended-spectrum ?-lactamases in Gram Negative Bacteria.Journal of Global Infectious Diseases. 2010;2(3):263-274. doi:10.
4103/0974-777X.68531.2. Shaikh S, Fatima J, Shakil S, Rizvi SMD, Kamal MA.
Antibiotic resistance and extended spectrum beta-lactamases: Types, epidemiology and treatment. Saudi Journal of Biological Sciences. 2015;22(1):90–103. Sharma M. Prevalence and antibiogram of Extended Spectrum ?-Lactamase (ESBL) producing Gram negative bacilli and further molecular characterization of ESBL producing Escherichia coli and Klebsiella spp.
Journal of Clinical and Diagnostic Research.2013;7(10):2173–774. Harakuni S, Mutnal M, Karadesai S, Metgud S. Prevalence of extended spectrum ?-lactamase-producing clinical isolates of Klebsiella pneumoniae in intensive care unit patients of a tertiary care hospital.
Ann Trop Med Public Health. 2011;4(2):96–85. Singh AK, Kumar D, Ali M, Chander Y. Antimicrobial susceptibility profile of extended spectrum ?-Lactamase (ESBL) producing escherichia coli from various clinical samples.Infectious Diseases: Research and Treatment. 2014;7:18.
6. Singh N, Pattnaik D, Neogi DK, Jena J, Mallick B. Prevalence of ESBL inEscherichia coli Isolates Among ICU Patients in a Tertiary Care Hospital.Journal of Clinical and Diagnostic Research, JCDR,2016;10(9):DC19-DC22.
doi:10.7860/JCDR/2016/21260.8544.7. Hima Bindu M1 , Kasturi ,Prevalence of ESBL Production in Escherichia coli and Klebsiella spp from Different Clinical Samples A Study in a Teaching Hospital in Telangana, India, ijcmas (2015)8. Bajpai T Prevalence of extended spectrum beta-lactamase producing uropathogens and their antibiotic resistance profile in patients visiting a tertiary care hospital in central India, Indian J Pathol Microbiol ,20149.
Modi D, Patel D, Patel S, Jain M, Bhatt S, Vegad M. Comparison of various methods for the detection of extended spectrum beta lactamase in klebsiella pneumoniae isolated from neonatal intensive care unit, Ahmedabad. National journal of medical research 2012;2(3):348-5310.
Khodare A, Mutha A, Purohit M. Prevalence of extended-spectrum ?-lactamases producing Escherichia coli in urinary specimens and their phenotypic detection by modified three-dimensional enzyme extract tesh: Comparison with the Phenotypic confirmatory disc diffusion test. Indian J Microbiol Res 2017;4(3):244-24711. Shaikh N. K , Comparison of different phenotypic methods for the detection of extended spectrum b- lactamase (ESBL) in bacterial isolates from tertiary care center , IJCRR. 2016; 8(11): 10-1412.
J Vandepitte and J Verhaegen et al. 2003. Basic Laboratory procedure in Clinical Bacteriology, 2nd Edn, WHO, Geneva13. Performance Standards for Antimicrobial Susceptibility testing, 27th Informational Supplement, CLSI Document, M100-S27, Wayne PA: Clinical and Laboratory Stabdards Institute 2017.