Utility Of Indirect Modified Three Dimensional TestFor Detection Of Extended Spectrum Beta Lactamases In A Tertiary Care Hospital        Synopsis for ICMR STS2018  Reference ID: 2018-02235             TABLE OFCONTENTS              Content                                                                                                          PageNo.  Title                                                     ………………………………………..3 Introduction                                        ……………………………………….

.3 Review of Literature                           ………………………………………..4 Aim and Objectives                            ………………………………………..4 Material and Methods                         ……………………………………….

.5 Implication                                          ………………………………………..6 Ethical Consideration                                     ………………………………………..6 References                                          ……………………………………….

.7 Appendix                                            ………………………………………..9·        Appendix I –  Case record form               …………………………………..9         Title  Utility Of Indirect Modified ThreeDimensional Test For Detection Of Extended Spectrum Beta Lactamases In A TertiaryCare Hospital Introduction With theadvancement in medical practice, new problems are surfacing, one of mostimportant and common being emergence of resistance in Gram negative bacteriatowards the ? lactams due to ? lactamases enzyme. These enzymes inactivate betalactam ring containing antibiotics, which in the course of time has evolved to extendedspectrum ?-lactamases (ESBL), which confersbacteria enhance ability to be resistant against wide variety of new beta-lactams1including the III generation cephalosporins, and aztreonam (but not thecephamycins or carbapenems). The ESBLs hydrolysis the antibiotics, but areinhibited by ?-lactamase inhibitors such as clavulanic acid.

2 ESBLs are a causeof major challenge in therapeutic treatment in hospitals3 due toincrease in expenditure of money on ineffective antibiotics which furtherdevelops resistance in bacteria making patient more susceptible to infectionand colonization by them.4 ESBL strains remain undetected as they are difficultto detect by routine susceptibility testing methods and may show falsesusceptibility to antibiotics by Kirby- Bauer disc diffusion methods5.Identification of all ESBLs producing organisms in clinical Microbiologylaboratory is a major challenge because ofinoculum effect and substrate specificity1.  ESBL detection is important as knowledge about itsprevalence is helpful to formulate infection control measures and to preventtheir spread6. There are different methods for detection of ESBLslike phenotypic confirmatory disc diffusion test (PCDDT), ModifiedDouble Disc Synergy Test (MDDST), Indirect Modified Three Dimensional Test(IMTDT), etc.

 Review of literature There is highprevalence of ESBLs in clinical isolates. SharmaM. et al reported frequency of 52.49% in gram negative isolates at NIMSUniversity, Jaipur4,while Hima Bindu M et al, reported ESBLs frequencyof 58.8% in isolates7 at Mallareddy Institute of Medical Sciences,India.

Where else study by Bajpai T et al, determined that among gram negativeisolate 36.8% were found to be ESBL producers8. Indirect modified3-dimensional enzyme extract test for detection of ESBLs is sensitive and givesrapid result. Modi D et al, reported sensitivity of IMTDT is 98% to 100% andfound it to be most sensitive test among other available tests such as doubledisc 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 bebetter than phenotypic confirmatory disc diffusion test (PCDDT) for detectionof ESBLs although it was a little labour intensive and may be technicallychallenging. 10.

 Instudy by Shaikh el at IMTDT was found to be superior method than Modified DoubleDisc Synergy Test (MDDST), PCDDT and DDST for detection of production of ESBLalone or in presence of other ?- lactamases like AmpC. PCDDT& DDST shouldbe used in the isolates which produce only ESBL but are not useful fordetection of ESBL in isolates who also produces other ?-lactamases like AmpCenzyme11. AIMS & OBJECTIVESWith the adventof ESBLs which are reducing the treatment options, it is necessary to detectthese with a reasonable accuracy so that appropriate treatment may be initiatedin the patients.

Hence this study is planned with the following objective: 1. To detect ESBLproducing strains using the indirect modified three dimensional test.2. To study theutility of IMTDT in detecting ESBLs in tertiary care hospital    METHODOLOGY: The study willbe carried out in the Department of Microbiology of a tertiary care hospital. Study Type: Cross-sectionalprospective study. Study Time: Two months.SampleType: All samples received forculture and sensitivity in the Microbiology LaboratorySample Size:30 strains MATERIAL AND METHODS:All samples received for culture andsensitivity in the Microbiology Laboratory will be inoculated on routine medialike blood agar and Mac Conkey agar; in case of urine it will be inoculated oncysteine lactose electrolyte deficient medium (CLED). After 24 hours ofincubation, the bacterial isolates, if any, will be identified by standardbiochemical tests12 and antibiotic sensitivity will be performed byKirby Bauer method as per CLSI guidelines.

Also ESBL screening will be done bystandard disc diffusion using cefpodoxime, cefotaxime, ceftriaxone andceftazidime with zones of inhibition <17mm, <27mm, <25mm and <22mmrespectively as probable ESBLs.13  Indirect modified three dimensional test :Crude enzyme extract of the test strain willbe prepared by freeze-thawing of approximately 15 mg of the isolate suspendedin 0.5 ml of peptone water. Lawn culture of Escherichia coli (ATCC 25922) willbe 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 thisa 4mm well will be punched out. Into this well, 30 µl of the crude extract willbe filled and incubated at 370C for 24 hours. A heart shapeddistortion of the zone of inhibition around the cefotaxime disc will be takento a positive test.

9E. coli ATCC25922 and Klebsiella pneumoniae ATCC 700603 will be used as a negative controland positive control respectably.9 Inclusion Criteria: All samples that will yield ESBL producingstrains by standard disc diffusion method during the study period will beincluded in the study.  Exclusion Criteria: All strainsthat are sensitive to the antibiotics as well as all Gram positive isolates.  Planned procedure to analyze data:Alldata will be maintained in Microsoft office Excel.

All statistical analysiswill be carried out using Excel and Appropriate Statistical tools will beapplied wherever required. IMPLICATION:This study provides an insight on ESBLs prevalanceand statistical data on their efficient detection in clinical laboratory withprecision and accurary.Study helps in contributing to the knowledge about thesensitivity and specificity of IMTDT for detection of ESBLs and it as an option to be used for routine analysis to provideaccurate result so effective treatment and proper care could be provided to thepatient. Early and accurate detection of ESBLs are keyfactor in influencing the prognosis of the patient , as it could range from uncomplicated urinary tract infections tolife-threatening sepsis. By early detection, it would not just rule out the use of ineffectiveantibotics for the treatment of disease but also reduces financial burden onpatient.

1 ETHICAL CONSIDERATIONS:Ethicalclearance will be obtained from Institutional Ethics Committee (IEC).      Reference 1. Rawat D, Nair D. Extended-spectrum ?-lactamases inGram 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, epidemiologyand treatment. Saudi Journal of Biological Sciences. 2015;22(1):90–10 3.

Sharma M. Prevalence and antibiogram of ExtendedSpectrum ?-Lactamase (ESBL) producing Gram negative bacilli and furthermolecular characterization of ESBL producing Escherichiacoli and Klebsiella spp. Journal of Clinical and Diagnostic Research.2013;7(10):2173–77 4. Harakuni S, Mutnal M, Karadesai S, Metgud S.Prevalence of extended spectrum ?-lactamase-producing clinical isolates of Klebsiellapneumoniae inintensive care unit patients of a tertiary care hospital. Ann Trop MedPublic Health. 2011;4(2):96–8 5.

Singh AK, Kumar D, Ali M, Chander Y. Antimicrobialsusceptibility profile of extended spectrum ?-Lactamase (ESBL) producing escherichiacoli from various clinical samples.Infectious Diseases: Research and Treatment. 2014;7:1–8. 6. Singh N, Pattnaik D, Neogi DK, Jena J, Mallick B.

Prevalence of ESBL inEscherichia coli IsolatesAmong ICU Patients in a Tertiary Care Hospital.Journal of Clinical andDiagnostic Research, JCDR,2016;10(9):DC19-DC22.doi:10.7860/JCDR/2016/21260.

8544. 7. Hima Bindu M1 , Kasturi ,Prevalence of ESBLProduction in Escherichia coli and Klebsiella spp from Different ClinicalSamples A Study in a Teaching Hospital in Telangana, India, ijcmas(2015) 8. Bajpai T Prevalence of extended spectrumbeta-lactamase producing uropathogens and their antibiotic resistance profilein patients visiting a tertiary care hospital in central India, Indian J Pathol Microbiol ,2014 9. Modi D, Patel D, Patel S, Jain M, Bhatt S, Vegad M.Comparison of various methods for the detection of extended spectrum betalactamase in klebsiella pneumoniae isolated from neonatal intensive care unit,Ahmedabad. National journal of medical research 2012;2(3):348-53 10. Khodare A, Mutha A, Purohit M.

Prevalence ofextended-spectrum ?-lactamases producing Escherichia coli in urinary specimensand their phenotypic detection by modified three-dimensional enzyme extracttesh: Comparison with the Phenotypic confirmatory disc diffusion test. Indian JMicrobiol Res 2017;4(3):244-247 11. Shaikh N. K , Comparison of different phenotypicmethods for the detection of extended spectrum b- lactamase  (ESBL) in bacterial isolates from tertiarycare center , IJCRR. 2016; 8(11): 10-14 12. J Vandepitte and J Verhaegen et al.

2003. BasicLaboratory procedure in Clinical Bacteriology, 2nd Edn, WHO, Geneva 13. Performance Standards for AntimicrobialSusceptibility testing, 27th Informational Supplement, CLSI Document, M100-S27,Wayne PA: Clinical and Laboratory Stabdards Institute 2017.    Case Record Form   Name : Age : Gender : Sample Id no.

: Sample type : Isolate: Antibiotic susceptibility test: Result of IMTDT :          Positive / Negative       

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