Iycee Charles de Gaulle Summary Introduction Dimensional Test (IMTDT), etc. Review of

Introduction Dimensional Test (IMTDT), etc. Review of



With 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.2

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!

order now


ESBLs 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.4


ESBL 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 literature


There 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.


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



With 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 hospital




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.

Type: All samples received for
culture and sensitivity in the Microbiology Laboratory

Sample Size:
30 strains



All samples received for culture and
sensitivity in the Microbiology Laboratory 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.13   Indirect 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.9 E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 will be used as a negative control and positive control respectably.9   Inclusion 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.   IMPLICATION: This study provides an insight on ESBLs prevalance and statistical data on their efficient detection in clinical laboratory with precision and accurary.Study helps in contributing to the knowledge about the sensitivity and specificity of IMTDT for detection of ESBLs and it as an option  to be used for routine analysis to provide accurate result so effective treatment and proper care could be provided to the patient. Early and accurate detection of ESBLs are key factor in influencing the prognosis of the patient , as it could range from uncomplicated urinary tract infections to life-threatening sepsis. By early detection, it would not just rule out the use of ineffective antibotics for the treatment of disease but also reduces financial burden on patient.1   ETHICAL CONSIDERATIONS: Ethical clearance will be obtained from Institutional Ethics Committee (IEC).           Reference   1. 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):90–10   3. 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):2173–77   4. 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):96–8   5. 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;7:1–8.   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 ,2014   9. 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):348-53   10. 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):244-247   11. 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): 10-14   12. J Vandepitte and J Verhaegen et al. 2003. Basic Laboratory procedure in Clinical Bacteriology, 2nd Edn, WHO, Geneva   13. Performance Standards for Antimicrobial Susceptibility testing, 27th Informational Supplement, CLSI Document, M100-S27, Wayne PA: Clinical and Laboratory Stabdards Institute 2017.