The Methods Of ESBLs Detection In Clinical Setting Essay
The Methods Of ESBLs Detection In Clinical Setting Essay
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. The Methods Of ESBLs Detection In Clinical Setting Essay.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.
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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 Methods Of ESBLs Detection In Clinical Setting Essay.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. The Methods Of ESBLs Detection In Clinical Setting Essay. 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.The Methods Of ESBLs Detection In Clinical Setting Essay. 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.
Objective: Extended-spectrum β-lactamase producing K. pneumoniae is a serious threat to the patients. This manuscript shows the comparison of phenotypic characterization methods used for ESBL K. pneumoniae and frequency distribution of these isolates in various clinical samples.
Methodology: Eleven different types of pathological samples collected on various time intervals were analyzed. K. pneumoniae were identified with API 20E system (bioMerieux) and initial screening of ESBL K. pneumoniae was performed using the ceftazidime antimicrobial disc. Double-disc synergy test (DDST) and CLSI confirmatory test were compared for the phenotypic detection of ESBL K. pneumoniae. The Methods Of ESBLs Detection In Clinical Setting Essay.
Results: A total number of 214 ESBL producing K. pneumoniae were isolated from various clinical samples. Frequency distribution of ESBL producing K. pneumoniae was found to be highest among blood 117 (54.7%) and urine 46 (21.5%) samples. Data regarding the use of various interventions among these patients showed most common presence of intravenous line 209 (97.7%) and urinary catheters 46 (21.5%). Comparison of DDST and CLSI confirmatory test showed that the DDST detected 145 (67.8%) isolates while 213 (99.5%) ESBL K. pneumoniae were characterized by CLSI confirmatory test.
Conclusion: The use of CLSI confirmatory test is very efficient in the early detection of ESBL K. pneumoniae especially when the facilities for molecular characterization are not available.
Extended-spectrum β-lactamases (ESBLs) are the enzymes, mostly encoded by plasmids in result of mutation due to which bacteria show resistance to various β-lactam antibiotics including cephalosporins and monbactams.1 Beyond one hundred and fifty various ESBLs have been described and majority of them belong to class A enzymes (SHV, TEM and CTX-M).2 ESBLs are most commonly found in bacteria (G–) especially the members of Enterobacteriaceae family. An increase in prevalence of ESBLs has been documented in recent years but it varies in different geographical areas of the world.3
Klebsiella pneumoniae is the most important and most common infectious pathogen in the hospitals environment and is mainly responsible for pneumonia, urinary tract infections, neonatal septicemia and wound infections among children.4 Apart from the other members of Enterobacteriaceae family, ESBLs are highly prevalent in K. pneumoniae.5 Global data show that the rate of ESBL producing bacteria was highest among K. pneumoniae isolated from fourty four percent in America followed by twenty two percent Asia pacific, thirty three percent in Europe and seven percent in North America.6 Many nosocomial outbreaks caused by ESBL K. pneumoniae have been reported among paediatric patients. ESBL K. pneumoniae are associated with high mortality rate among children.7 .The Methods Of ESBLs Detection In Clinical Setting Essay.
Many tests have been recommended for the detection of ESBL production in vitro. The most commonly used methods include double disc synergy test, combined disc method and E-test. Several automated systems have also been developed for detection and some laboratories use molecular methods for detection of ESBL phenomenon.8 There are limited number of antibiotic options available to treat the infections caused by ESBL producing strains.9 Present study was conducted with the objective, to evaluate the frequency of ESBL producing K. pneumoniae and comparison of detection methods among various clinical samples collected from a tertiary care children hospital. The early detection of ESBL producing K. pneumoniae strains can help to reduce high morbidity and mortality in paediatric patients.
METHODOLOGY
The study was conducted at the Microbiology Department of The Children’s Hospital and Institute of Child Health Lahore, Pakistan, from May 2010 to February 2012. Eleven pathological samples including blood, cerebro-spinal fluid (CSF), urine, sputum, peritoneal dialysis catheters, tracheal secretions and pus were collected from paediatric patients were analysed during the study period. The patient’s history regarding the use of various interventions like intravenous line, urinary catheters, endotracheal tube, lumber puncture, peritoneal dialysis catheters, exchange transfusion, nasogastric tube, surgery, central venous pressure line and tracheostomy was also noted. The Brain Heart Infusion Broth (BHI) was used to inoculate the blood samples and after a period of incubation they were sub-cultured on solid media. All of the clinical samples were cultured on different solid media of Blood, Chocolate and MacConkey agar plates (90mm) while the urine samples were cultured only on Cystine Lysine Electrolyte Deficient Medium (CLED). An overnight incubation of these culture plates was done at 37±1ºC in an incubator. The bacterial strains were identified using API 20E system (bioMerieux).Only the K. pneumoniae strains were included and processed further for ESBL detection. The Methods Of ESBLs Detection In Clinical Setting Essay.The culture media and antibiotic discs were purchased from Oxoid.
The K. pneumoniae strains were screened as ESBL screening-positive on the basis of resistance to ceftazidime. The screen positive K. pneumoniae strains were further processed for double disc synergy test (DDST) and Clinical Laboratory Standard Institute (CLSI) confirmatory test. In DDST, a disc of amoxicillin-clavulanic acid (AMC) was placed in the center of Mueller-Hinton agar plate (90mm) at 20mm distance to ceftazidime (CAZ 30μg) and cefotaxime (CTX 30μg). ESBL production was detected by the appearance of key hole effect due to the enhanced activity of ceftazidime and cefotaxime with clavulanic acid.
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Both DDST positive and negative K. pneumoniae strains were analysed by CLSI confirmatory test. The CLSI confirmatory test was performed with of ceftazidime (CAZ 30μg) and cefotaxime (CTX 30μg) alone and using a combined disc of ceftazidime-clavulanic acid and cefotaxime-clavulanic acid (CAZ/CLA and CTX/CLA 30/10 μg). The CLSI confirmatory test was considered positive when the inhibition zone produced by the combined effect of ceftazidime or cefotaxime and clavulanic acid increased ≥5 mm than ceftazidime or cefotaxime without the clavulanic acid.10
RESULTS
A total number of 710 K. pneumoniae were isolated during the study period. Out of 710 positive culture 214 (30.1%) were ESBL screening-positive K. pneumoniae and 496 (69.9%) were screening-negative positive K. pneumoniae.
Initial screening of K. pneumoniae isolates with ceftazidime showed positivity in all of the isolates. Comparison between DDST and CLSI confirmatory test showed that 145 (67.8%) isolates were identified by DDST and 213 (99.5%) by using CLSI confirmatory test (Table-I). The CLSI confirmatory test had significantly (p<0.0001) higher sensitivity. Frequency distribution of ESBL K. pneumoniae was found to be highest in the blood samples 117 (54.7%) followed by urine 46 (21.5%), endotracheal tube 13 (6.1%), cerebro-spinal fluid 13 (6.1%) and peritoneal dialysis catheters 10 (4.7%). The rest of the samples showed the lesser occurrence of ESBL producing K. pneumoniae. The Methods Of ESBLs Detection In Clinical Setting Essay.
