National Committee For Clinical Laboratory Standards Method Research Articles

Antibiotic susceptibility of potentially probiotic vaginal lactobacilli.

Objective. To study the antimicrobial susceptibility of six vaginal probiotic lactobacilli. Methods. The disc diffusion method in Müeller Hinton, LAPTg and MRS agars by the NCCLS (National Committee for Clinical Laboratory Standards) procedure was performed. Due to the absence of a Lactobacillus reference strains, the results were compared to those of Staphylococcus aureus ATCC29213. Minimal Inhibitory Concentration (MIC) with 21 different antibiotics in LAPTg agar and broth was also determined. Results. LAPTg and MRS agars are suitable media to study antimicrobial susceptibility of lactobacilli. However, the NCCLS procedure needs to be standardized for this genus. The MICs have shown that all Lactobacillus strains grew at concentrations above 10 μg/mL of chloramphenicol, aztreonam, norfloxacin, ciprofloxacin, ceftazidime, ceftriaxone, streptomycin and kanamycin. Four lactobacilli were sensitive to 1 μg/mL vancomycin and all of them were resistant to 1000 μg/mL of metronidazole. Sensitivity to other antibiotics depended on each particular strain. Conclusions. The NCCLS method needs to be standardized in an appropriate medium to determine the antimicrobial susceptibility of Lactobacillus. Vaginal probiotic lactobacilli do not display uniform susceptibility to antibiotics. Resistance to high concentrations of metronidazole suggests that lactobacilli could be simultaneously used with a bacterial vaginosis treatment to restore the vaginal normal flora.

Modified double-disc test for detection of extended-spectrum β-lactamases in Escherichia coli and Klebsiella pneumoniae

This study evaluates the performance of a modified double-disc test (MDDT) for the detection of extended-spectrum β-lactamases (ESBLs) in clinical isolates of Escherichia coli and Klebsiella pneumoniae. Ninety-six isolates of E. coli and 40 K. pneumoniae are studied for ESBL production by the National Committee for Clinical Laboratory Standards (NCCLS) combination disc tests and MDDT. A total of 112 (82%) isolates (80 [83%] E. coli, 32 [80%] K. pneumoniae) were positive for ESBL by MDDT compared to 102 (75%; 72 [75%] E. coli and 30 [75%] K. pneumoniae) by the NCCLS method. In 10 (7.4%) isolates, ESBLs were detected only by MDDT. Twenty-four (17.6%) isolates were negative for ESBL by both methods. The protocol described in this study provides a more sensitive approach than does the NCCLS method for ESBL detection in E. coli and K. pneumoniae.

Increased frequency of non- fumigatus Aspergillus species in amphotericin B– or triazole–pre-exposed cancer patients with positive cultures for aspergilli

Invasive aspergillosis (IA) can occur despite prior prophylactic or empiric use of triazoles or amphotericin B (AMB). Although profound immunosuppression may account for breakthrough IA, resistance of Aspergillus to antifungals may also play a role. To examine this question, we measured the minimal inhibitory concentration of 105 Aspergillus isolates recovered from 105 cancer patients (64 with IA, 41 with Aspergillus colonization) to AMB, itraconazole (ITC), and voriconazole (VRC) using the National Committee for Clinical Laboratory Standards (NCCLS) M38-A microdilution and E-test methods. We also determined the minimal fungicidal concentration (MFC) of these agents and the minimal effective concentration (MEC) of caspofungin (CAS) using standardized methods. We then collected information regarding pre-exposure to AMB or triazoles (fluconazole, ITC, VRC) within 3 months before Aspergillus isolation. Pre-exposure of cancer patients to AMB or triazoles was associated with increased frequency of non- fumigatus Aspergillus species. Aspergillus isolates recovered from patients who previously received AMB exhibited higher E-test AMB MICs compared with isolates from patients without prior AMB exposure ( P = 0.01). In addition, the AMB MICs by E-test were higher in triazole–pre-exposed patients compared with those not exposed to triazoles ( P = 0.001). The ITC and VRC MICs by E-test were not affected by prior AMB or triazole exposure. Finally, neither the AMB, ITC, and VRC MICs and MFCs by NCCLS method nor CAS MECs showed such changes. In conclusion, cancer patients with positive Aspergillus cultures who are pre-exposed to AMB or triazoles have high frequency of non- fumigatus Aspergillus species. These Aspergillus isolates were found to be AMB-resistant by the more sensitive E-test method.

Comparative evaluation of Sensititre® YeastOne vs. the NCCLS M27A protocol and E‐test for antifungal susceptibility testing of yeasts

A recently developed microdilution method (Sensititre) YeastOne) may represent a valid alternative to the National Committee for Clinical Laboratory Standards (NCCLS) method for routine testing. The Medical Mycology Committee of the Associazione Microbiologi Clinici Italiani (AMCLI) decided to evaluate its reproducibility and reliability compared with the NCCLS M27A protocol and the E-test. Nineteen strains each of Candida albicans and Ca. parapsilosis, isolated from systemic infections, were tested against amphotericin B, flucytosine, ketoconazole, itraconazole, and fluconazole. All the participating laboratories tested the YeastOne panels, while the E-test and the NCCLS method were performed by two laboratories each. Interlaboratory reproducibility showed a good correlation (from 95% for amphotericin B to 92.5% for flucytosine). The agreement between NCCLS and YeastOne ranged from 95 (ketoconazole and itraconazole) to 100% (amphotericin B and flucytosine), whereas the agreement between E-test and YeastOne ranged from 72.5 (fluconazole) to 100% (amphotericin B and flucytosine). The Sensititre YeastOne panels appear to be an excellent alternative to both the E-test and the NCCLS protocol for antifungal susceptibility testing.

In vitro susceptibility testing of filamentous fungi: comparison of Etest and reference M38-A microdilution methods for determining posaconazole MICs

The performance of the Etest for posaconazole susceptibility testing of 72 isolates of filamentous fungi was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) approved standard broth microdilution method (M38-A). The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48h at 35°C. The isolates included Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, A. versicolor, A. oryzae, A. terreus, Cladosporium spp., Curvularia sp., Exophiala sp., Fusarium spp., Paecilomyces spp., Pithomyces sp., Penicillium spp. and Scedosporium apiospermum. Overall agreement between Etest and microdilution MICs was 84% for Aspergillus spp. and 100% for the less common opportunistic molds, with the exception of Penicillium spp. (67%). Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values. The Etest method using RPMI agar appears to be a useful method for determining posaconazole susceptibilities of filamentous fungi.

Evaluation of the Etest Method for Determining Voriconazole Susceptibilities of 312 Clinical Isolates of Candida Species by Using Three Different Agar Media

Performance of the Etest for voriconazole susceptibility testing of 312 isolates of Candida spp. was assessed against that of the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and antibiotic medium 3 (AM3) agar and were read after incubation for 48 h at 35 degrees C. The Candida spp. isolates included C. albicans (n = 174), C. glabrata (n = 55), C. tropicalis (n = 31), C. parapsilosis (n = 39), C. krusei (n = 5), C. lusitaniae (n = 2), and C. guilliermondii (n = 6). The Etest results obtained using RPG correlated well with the reference MICs. Overall agreement ranged from 91% for C. glabrata to 100% for C. tropicalis, C. parapsilosis, C. guilliermondii, C. krusei, and C. lusitaniae. When CAS was used, agreement ranged from 80% for C. krusei to 100% for C. parapsilosis, C. guilliermondii, and C. lusitaniae. With AM3, agreement ranged from 58% for C. glabrata to 100% for C. lusitaniae and C. guilliermondii. The Etest method using RPG appears to be a useful method for determining voriconazole susceptibilities of Candida species.

Biotypes of oral Candida albicans isolated from AIDS patients and HIV-free subjects in Thailand.

This study was conducted to examine biotypes and antifungal susceptibility patterns of oral Candida albicans isolated from HIV-infected patients, HIV-free patients with candidiasis and healthy subjects. All isolates were biotyped using a typing system based on enzyme profiles, carbohydrate assimilation patterns and boric acid resistance. Thirty-eight biotypes were found amongst 218 oral C. albicans isolates. The major biotype found was A1S, which accounted for 32.6% of all isolates, and this biotype was the most common in all groups. There was a greater variety of biotypes of C. albicans in the HIV-infected group than in the other groups; however, there was no statistically significant difference between the groups. The minimum inhibitory concentrations (MICs) of a total of 118 isolates were determined for amphotericin B and for ketoconazole using the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method and the E-test. When the antifungal susceptibility patterns among the groups were compared, a statistically significant difference was found only with amphotericin B. The median MIC of amphotericin B in the HIV-infected group was higher than in the healthy group (P=0.013, NCCLS method; P=0.002, E-test). However, this difference in sensitivity was not restricted to any sub-type investigated. Our results showed that the biotype patterns of C. albicans isolates that colonize HIV-infected patients are similar to those of HIV-free subjects, and there is no relationship between antifungal susceptibility patterns and the biotypes.

Quality control limits for broth microdilution susceptibility tests of ten antifungal agents.

Broth microdilution susceptibility tests of Candida species have now been standardized by the National Committee for Clinical Laboratory Standards (NCCLS). An eight-laboratory collaborative study was carried out in order to document reproducibility of tests of Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 by the NCCLS method. Replicate broth microdilution tests were used to define control limits for 24- and 48-h MICs of amphotericin B, flucytosine, fluconazole, voriconazole, ketoconazole, itraconazole, caspofungin (MK 0991), ravuconazole (BMS 207147), posaconazole (SCH 56592), and LY 303366.

The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.

Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent biofilms for the assay of antibiotic susceptibilities by the standard 96-well technology. Biofilm formation was followed by quantitative microbiology and scanning electron microscopy. Susceptibility to a standard group of antibiotics was determined for National Committee for Clinical Laboratory Standards (NCCLS) reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213. Growth curves demonstrated that biofilms of a predetermined size could be formed on the CBD at specific time points and, furthermore, that no significant difference (P > 0.1) was seen between biofilms formed on each of the 96 pegs. The antibiotic susceptibilities for planktonic populations obtained by the NCCLS method or from the CBD were similar. Minimal biofilm eradication concentrations, derived by using the CBD, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to be effective at the MICs. The CBD offers a new technology for the rational selection of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic compounds.

No Increase in Frequency of Antifungal Resistance among Yeasts Isolated from Normally Sterile Sites in Patients at Foothills Hospital from 1993 to 1996

To determine the prevalence of resistance to antifungal drugs among yeasts isolated from sterile sites from patients in one hospital and the relationship of resistance to antifungal use, and to assess whether resistance was increasing. Susceptibility testing performed by National Committee for Clinical Laboratory Standards (NCCLS) (Villanova, Pennsylvania) microdilution method and by E test. Antifungal use was determined by selected chart review and from pharmacy data. SPECIMENS AND SETTING: Tertiary care adult hospital with neonatal intensive care. Distinct yeast isolates from sterile site specimens collected during the years 1993 to 1996. A total of 132 yeast isolates were studied, of which 78 (59%) were Candida albicans. The proportion of C albicans remained steady over the four-year period, and there was no trend to increased resistance among C albicans. The number of isolates of all species with fluconazole microdilution minimum inhibitory concentration (MIC) greater than 8 mg/L in each of the four years were one of 32 in 1996, three of 26 in 1994, six of 33 in 1995, and one of 41 in 1996. A single isolate had an itraconazole microdilution MIC greater than 0.5 mg/L in each year. Prior use of antifungal therapy was rare in this patient population. The increase in resistance to antifungal drugs reported by some centres did not occur in this institution over the course of the study. This experience may reflect differences in infection control practices and in patterns of use of antifungal agents. The NCCLS method was found to be superior to the E test as a routine method for testing susceptibility of yeasts.

Evaluation of the Etest method for determining fluconazole susceptibilities of 402 clinical yeast isolates by using three different agar media.

The performance of the Etest for fluconazole susceptibility testing of 402 yeast isolates was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and Mueller-Hinton agar (MHA) and were read after incubation for 48 h at 35 degrees C. The yeast isolates included Candida albicans (n = 161), Candida glabrata (n = 41), Candida tropicalis (n = 35), Candida parapsilosis (n = 29), Candida krusei (n = 32), Candida lusitaniae (n = 31), Candida species (n = 19), Cryptococcus neoformans (n = 40), and miscellaneous yeast species (n = 14). The Etest results correlated well with reference MICs. Overall agreement was 94% with RPG, 97% with CAS, and 53% with MHA. When RPG was used, agreement ranged from 89% for Candida spp. to 100% for C. krusei. When CAS was utilized, agreement ranged from 93% for Cryptococcus neoformans to 100% for C. tropicalis, C. parapsilosis, C. lusitaniae, Candida spp., and miscellaneous yeast species. With MHA, agreement ranged from 17% for C. parapsilosis to 90% for C. krusei. Both RPG and CAS supported growth of all yeast species, whereas growth on MHA was comparatively weaker. Etest results were somewhat easier to read on CAS. The Etest method using either RPG or CAS, but not MHA, appears to be a viable alternative to the NCCLS reference method for determining fluconazole susceptibilities of yeasts.

Interpretation of trailing endpoints in antifungal susceptibility testing by the National Committee for Clinical Laboratory Standards method.

Trailing endpoints remain a problem in antifungal susceptibility testing using the National Committee for Clinical Laboratory Standards (NCCLS) method. For isolates for which trailing endpoints are found, MICs of < or = 1 microg/ml at 24 h and of > 64 microg/ml at 48 h are usually observed. In a study of human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis, we identified three patients with multiple serial isolates for which trailing endpoints were observed with fluconazole. At 24 h, MICs were generally < or = 1 microg/ml by both broth macro- and microdilution methods. However, at 48 h, MICs were > 64 microg/ml, while the organism remained susceptible by agar dilution testing with fluconazole. Most episodes of oropharyngeal candidiasis with trailing-endpoint isolates responded to doses of fluconazole as low as 100 mg/day. Two patients had both susceptible and trailing-endpoint isolates by NCCLS broth macro- and microdilution testing; these isolates were found to be the same strain by pulsed-field gel electrophoresis using restriction fragment length polymorphisms. Another patient had two different strains, one for which trailing endpoints were observed and one which was susceptible at 48 h. Trailing endpoints may be seen with selected isolates of a strain or may be a characteristic finding for most or all isolates of a strain. In addition, with isolates for which trailing endpoints are observed, reading the endpoint for the NCCLS method at 24 h may be more appropriate.

Evaluation of the Etest for susceptibility testing of anaerobic bacteria

We compared the susceptibility test results of 220 anaerobes against 14 antimicrobials using the Etest (AB Biodisk, Solna, Sweden) with those using the National Committee for Clinical Laboratory Standards (NCCLS) standard agar dilution method (Wadsworth version). The Etest medium was brucella blood (whole) agar and the inoculum size was equivalent to a no. 1 McFarland standard. Thirty-six percent of Etest results were unreadable after 24 h of anaerobic incubation compared to only 5% after 48 h. Also, there were more results with categorical agreement with the NCCLS method after 48 h (97.9%) than at 24 h (89%) and more very major errors (VMEs) at 24 h (22% of resistant organisms) than at 48 h (3.2%). VMEs and major errors occurred most frequently with clindamycin, ceftriaxone, and trospectomycin (which should not be used with the Etest) and involved the Bacteroides fragilis group and/or Clostridium most commonly. The Etest is simple to perform and is a generally reliable method that is optimally read after 48 h of incubation. It should be an acceptable alternative to the agar dilution standard, although results with certain organism-antimicrobial combinations should be read very conservatively because of the frequency of VMEs.

Improved method of determining the susceptibility of Candida albicans to fluconazole.

The MICs of fluconazole for four reference strains of Candida albicans were determined in one laboratory by the macrobroth dilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) and by a modified version of the method which incorporated microbroth dilution, RPMI 1640 medium supplemented with glucose and the reading of MIC endpoints by spectrophotometry. The results obtained by the two methods were within one two-fold dilution. In addition, the susceptibilities of 58 clinical isolates of C. albicans to fluconazole were determined in two laboratories by the modified NCCLS method. Close interlaboratory agreement was observed, 98.2% of MICs varying by no more than one two-fold dilution.

Evaluation of a rapid inoculum preparation method for agar disk diffusion susceptibility testing.

A rapid inoculum preparation method for agar disk diffusion susceptibility testing which does not require incubation before inoculation of Mueller-Hinton plates was compared with the National Committee for Clinical Laboratory Standards (NCCLS) method. A total of 326 fresh clinical isolates were tested, and the NCCLS-recommended quality control organisms were included with each test series. Randomly distributed interpretative changes occurred with 27 (0.8%) of the 3,215 test results for the clinical isolates. The quality control organisms were tested on 29 separate days, and results were consistently within tolerance limits. The rapid method was found to be equivalent to the standard NCCLS method and required less time and expense.