The Methods Of ESBLs Detection In Clinical Setting
Modern day medical practice encounters many challenges. Among them one of the most crucial one faced day to day is development of resistance in bacteria especially gram negative bacteria towards beta lactam antibiotics due to presence of beta lactamases enzyme, caused by extensive irrational usage of antibiotic. This development of resistance by bacteria are a cause of major failure in therapeutic treatment in hospitals as there has been a huge rise in their number resulting more and more rate of morbidity and mortality.
Beta-lactamase enzymes inactivate beta lactam ring containing antibiotics, which in the course of time has evolved to extended spectrum β- lactamases (ESBL), which confers bacteria enhanced ability to be resistant against wide range of new beta-lactams antibiotics, that are the III generation cephalosporins, and aztreonam (but sensitive to cephamycins or carbapenems). The ESBLs hydrolyse the antibiotics, but are sensitive to β-lactamase inhibitors such as clavulanic acid.
ESBLs enzymes genes are predominantly coded by plasmid. Most of them are mutant forms of temoneira (TEM) and sulfhydryl variable (SHV) enzymes. Recently other forms including cefotaximase (CTX-M) type-lactamases, OXA-type, PER-type and GES-type have been discovered that are produced by mutations that cause change in the amino acid configuration around the active site of these beta-lactamases. There are more 300 variant of ESBLs available, TEM and SHV being major type. 2Extended Spectrum Beta lactamases are classified most commonly by Ambler molecular classification and the Bush-Jacoby-Medeiros Classification.
Ambler scheme divides β-lactamases into four major classes (A to D) based upon protein homology. β-lactamases of classes A, C and D are serine β-lactamases, class B enzymes are metallo-β-lactamases. Bush-Jacoby-Medeiros classification scheme groups β-lactamases according to functional similarities, substrate and inhibitor profile. This classification is more relevant to the physician or microbiologist in a diagnostic laboratory. The emergence and variation in their occurrence over different geographical areas in different period of time necessitates to screen for their detection. 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 methods. Identification of all ESBLs producing organisms in clinical Microbiology laboratory is a major challenge because of inoculum effect and substrate specificity. 1ESBL detection is important as knowledge about its prevalence is helpful to formulate infection control measures and to prevent their spread. Their detection act as epidemiology marker of colonisation and their potential to cause nosocomial infection.
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.
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 ineffective use of antibiotics for the treatment of disease but also reduces financial burden on patient.
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, Jaipur, while HimaBindu M et al, reported ESBLs frequency of 58. 8% in isolates5. Whereas study by Bajpai T et al, determined that among gram negative isolates 36. 8% were ESBL producers. ESBL isolates obtained by Bajpai T el al were Escherichia coli 41. 6% followed by P. aeruginosa 36. 1% and K. pneumoniae 26%, HimaBindu M et al, reported 56. 4% of E. coli were ESBL producers and 60 % of Klebsiella whereas Khodare A et al, reported 86% Escherichia coli strains were positive.
Presence of ESBLs in different samples were detailed by Sharma M. et al as urine (57. 2%), blood (31. 07%), pus (48. 03%), respiratory tract (63. 83%), body fluid (52. 17%) and stool samples (59. 29%), in similar study by Singh AK et al, . they isolated ESBLs from blood (66. 67%), aspirate (65%), stool (57. 14%), wound (55%), and urine (54. 67%).
Singh N et al, reported prevalence of ESBL producers among male patients to be 59. 3% and 62. 4% among female patients.
Singh Ak et al study showed that of Total ESBLs 60. 95% were from IPD while 48% from OPD10, while Sharma M. et al reported 16. 89% ESBL producers were isolated from OPD, 64. 64% were isolated from IPD. Bajpai T el al reported resistance to antibiotics like ampicillin to be 100% and sensitivity to nitrofurantoin 31. 3%3, whereas Singh N et al in their study found nitrofurantoin was the most sensitive drug (83. 6%), in similar studies by Singh Ak et al reported sensitivity to be gentamicin 68. 29% and nitofurantoin 58. 53%. 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 to be 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. 8 Khodare A et al, study showed 76% strains gave positive result with IMTDT, and was found it 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.
In study by Shaikh el at IMTDT identified 98. 41% ESBL positive isolates. Thus it was the most sensitive test. 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 β- 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 β-lactamases like AmpC enzyme.