Evaluation of the Etest method for determining voriconazole susceptibilities of 312 clinical isolates of Candida species by using three different agar media

Aim: To assess the pattern of antimicrobial resistance of Helicobacter pylori isolates from peptic ulcer disease patients of Chandigarh, Delhi, Lucknow, Hyderabad and Chennai in India, and to recommend an updated anti-H. pylori treatment regimen to be used in these areas. Methods : Two hundred and fifty-nine H. pylori isolates from patients with peptic ulcer disease reporting for clinical management to the Post Graduate Institute of Medical Education and Research, Chandigarh; All India Institute of Medical Sciences, New Delhi; Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow; Deccan College of Medical Sciences and Allied Hospitals, Hyderabad; and hospitals in Chennai in collaboration with the Dr ALM Post Graduate Institute of Basic Medical Sciences were analyzed for their levels of antibiotic susceptibility to metronidazole, clarithromycin, amoxicillin, ciprofloxacin and tetracycline. The Etest, a quantitative antibiotic susceptibility testing method, was adopted in all the centers. The pattern of single and multiple resistances at the respective centers and at the national level were analyzed. Results: Overall H. pylori resistance rate was 77.9% to metronidazole, 44.7% to clarithromycin and 32.8% to amoxicillin. Multiple resistance was seen in 112/259 isolates (43.2%) and these were two/three and four drug resistance pattern to metronidazole, clarithromycin, amoxicillin observed (13.2, 32 and 2.56%, respectively). Metronidazole resistance was high in Lucknow, Chennai and Hyderabad (68, 88.2 and 100%, respectively) and moderate in Delhi (37.5%) and Chandigarh (38.2%). Ciprofloxacin and tetracycline resistance was the least, ranging from 1.0 to 4%. Conclusion: In the Indian population, the prevalence of resistance among H. pylori is very high to metronidazole, moderate to clarithromycin and amoxycillin and low to ciprofloxacin and tetracycline. The rate of resistance was higher in southern India than in northern India. The Etest emerges as a reliable quantitative antibiotic susceptibility test. A change in antibiotic policy to provide scope for rotation of antibiotics in the treatment of H. pylori in India is a public health emergency. HIGHLIGHTS: 1. The use of Etest for a challenging fastidious organism such as H. pylori that requires significant experience was successfully achieved in a multi-site study in India. 2. Important national and regional epidemiologic resistance patterns against commonly used drugs for used for H. pylori treatment could be generated using Etest. 3. Etest can aid the changes in antibiotic policy to provide scope for rotation in treatment of H. pylori in India which comprise a public health emergency. 3. Use of the Etest to assess synergy of antibiotic combinations against isolates of Burkholderia cepacia-complex from patients with cystic fibrosis. Manno G, Ugolotti E, Belli ML, Fenu ML, Romano L, Cruciani M. European Journal of Clinical Microbiology & Infectious Diseases, (2003), 22(1):28-34. Treatment of Burkholderia cepacia-complex infections in cystic fibrosis patients is problematic, since the microorganism is often resistant to most antimicrobial agents. In this study, the Epsilometer test, or Etest, was used to assess the activity of antimicrobial combinations against Burkholderia cepacia-complex. In a preliminary evaluation, the Etest was compared to the checkerboard method using 10 test organisms. Synergy testing by the Etest was then performed on 131 clinical isolates of Burkholderia cepacia-complex using various combinations of antimicrobial agents. Agreement between the Etest and the checkerboard method was 90%. The rate of resistance to individual agents ranged from 48% for meropenem to 100% for tobramycin, chloramphenicol, and rifampin. In 71.6%, 15.6%, and 12.6% of the test evaluations performed, the combinations tested resulted in additivity/indifference, synergism, and antagonism, respectively. The highest rates of synergy were observed with combinations of ciprofloxacin-piperacillin (44%), rifampinceftazidime (33%), chloramphenicol-ceftazidime (22%), cotrimoxazole-piperacillin/tazobactam (22%), and ciprofloxacin-ceftazidime (21%). Rates of antagonism for cotrimoxazole and chloramphenicol in combination with beta-lactam agents were higher than those observed for ciprofloxacin plus beta-lactam agents. These results suggest that the Etest is a valuable and practical method to be considered for improving the identification of possible therapeutic options in cystic fibrosis patients infected with organisms belonging to the Burkholderia cepacia-complex. HIGHLIGHTS: 1. The Etest method allowed for the rapid screen of the combined activity of multiple combinations of antibiotics against isolates of Burkholderia cepacia. 2. The modified Etest technique for synergy studies is simple to use, time efficient and reproducible and is now increasingly being used. 3. This study support further evaluation of Etest for improving therapeutic options for pulmonary exacerbations of cystic fibrosis associated with Burkholderia cepacia and other microorganisms. EXAMPLES OF NOTABLE ETEST REFERENCES -2004 1. Pharmacokinetic/pharmacodynamic modeling can help guide targeted antimicrobial therapy for nosocomial gram-negative infections in critically ill patients. Mohr JF, Wanger A, Rex JH. Diagnostic Microbiology and Infectious Disease, 2004 February; 48(2): 125-30. Critically ill patients have altered pharmacokinetics (PK) that needs to be considered when choosing and dosing antibiotics. We conducted a prospective, observational study to assess clinical and microbiologic response rates in 19 critically ill patients with nosocomial Gram-negative infections. Antibiotics were dosed based on a mathematical pharmacodynamic (PD) model accounting for these altered kinetic parameters. The average APACHE II score +/SE on intensive care unit admission and at the time of infection was 13.6 +/1.2 and 14.6 +/1.1, respectively. With targeted antimicrobial therapy adjusted to achieve an optimal PD profile, 17/19 (89%) patients had a clinical cure or improvement and 16/19 (84%) had either microbiologic eradication or presumed eradication. Modeling PD in these critically ill patients resulted in good clinical and microbiologic outcomes. HIGHLIGHTS: 1. Etest was used to generate the exact MIC values for pharmacodynamic modeling to help target antibiotic therapy of serious Gram negative nosocomial infections in critically ill patients 2. Based on the MIC-PK/PD models, the most potent agent within the class could be utilised and this would increase the probability of achieving the PK/PD target while eliminating kinetic variability. 3. Optimization of antimicrobial therapy based on MIC-PK/PD modelling was well received by the primary physicians. 4. High rates of clinical cure and microbiologic eradication was achieved using MIC targeted therapy. 2. Vancomycin-resistant Staphylococcus aureus New York, 2004. Kacica M, McDonald LC. Morbidity and Mortality Weekly Report, April 23, 2004; 53(15): 322-323. Staphylococcus aureus is a common cause of hospitaland community-acquired infections (1,2). The development of vancomycin-resistant enterococci in 1988 led the way to the emergence of vancomycinresistant S. aureus (VRSA) (minimum inhibitory concentration [MIC] [greater than or equal to] 32 [micro]g/mL [3]), first recognized in 2002 (4-7). This report describes the third documented clinical isolate of VRSA from a patient in the United States and provides evidence of failure to detect this VRSA by commonly used automated antimicrobial susceptibility testing. HIGHLIGHTS: 1. The isolate tested using Microscan® overnight panels gave a vancomycin result of MIC 4 μg/mL while Etest showed the isolate to be highly resistant to vancomycin (MIC >256 μg/mL). 2. After notification and subsequent analysis by the New York State Department of Health (NYSDOH), the isolate was forwarded to CDC where it was confirmed to be VRSA (vancomycin resistant S. aureus); MIC = 64 μg/mL. 2. Although the VRSA isolate contained the vanA resistance gene, the vancomycin MIC appeared low when tested initially by an automated method. 3. Additional testing at CDC indicated that Microscan® and Vitek® testing panels and cards available in the United States did not detect vancomycin resistance in this VRSA isolate. 4. Consequently, it may be possible that additional VRSA infections might have occurred but were undetected by laboratories using automated methods. 5. Potential VRSA isolates should be saved for confirmatory testing, and clinical microbiology laboratories must ensure that they are using susceptibility testing methods that will detect VRSA. 6. “The most accurate form of vancomycin susceptibility testing for staphylococci is a non automated MIC method (e.g., broth microdilution, agar dilution, or agar-gradient (Etest) diffusion) in which the organisms are incubated for a full 24 hours before reading results.” EXAMPLES OF NOTABLE ETEST REFERENCES -2005 1. Breakpoints for predicting Pseudomonas aeruginosa susceptibility to inhaled tobramycin in cystic fibrosis patients: use of high-range Etest strips. Morosini MI, Garcia-Castillo M, Loza E, Perez-Vazquez M, Baquero F, Canton R. Journal of Clinical Microbiology, 2005 Sep;43(9):4480-5. Inhaled administration of tobramycin assures high concentrations in cystic fibrotic lungs, improving the therapeutic ratio over that of parenteral tobramycin levels, particularly against Pseudomonas aeruginosa. Conventional Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) breakpoints only consider parenteral levels and do not take into account these high antimicrobial concentrations. The Spanish Antibiogram Committee (The MENSURA Group) has tentatively defined specific breakpoint values for inhaled tobramycin when testing P. aeruginosa isolates from cystic fibrosis (CF) patients (susceptible, < or =64 microg/ml; resistant, > or =128 microg