Susceptibility tests of oropharyngeal Candida albicans from Egyptian patients to fluconazole determined by three methods

Candida albicans frequently cause oropharyngeal candidiasis in immunocompromised patients. As some of these isolates show resistance against azoles, the clinician is wary of initiating therapy with fluconazole (FZ) until a final susceptibility report is generated. We aimed to evaluate the efficacy of rapid flow cytometry (FCM) and disc diffusion (DD) methods in comparison to reference microdilution (MD) of Clinical and Laboratory Standards Institute (CLSI) method for FZ. Thirty seven Candida albicans isolates were tested by the three methods. By both MD and FCM, 26/37 (70.3%) were sensitive with minimal inhibitory concentration (MIC) ¡Ü 8¦Ìg/ml, 5/37 (13.5%) were susceptible dose dependant (S-DD) with MIC 16-32 ¦Ìg/ml and 6/37 (16.2%) were resistant with MIC ¡Ý64¦Ìg/ml. More than 92% of isolates susceptible to FZ by the MD were susceptible by the DD methods with good agreement (81.08%, P = 0.000). However, 4/5 isolates diagnosed as S-DD by MD were resistant by DD. Interestingly, the MIC by FCM at 4 h showed excellent agreement (95.59%, P = 0.000) to that obtained by MD method at 24 h. Overall, FCM antifungal susceptibility testing provided rapid, reproducible results that are valuable alternative to MD. The DD test is recommended as a simple and reliable screening test for the detection of susceptible Candida albicans isolates to FZ.

Comparison between E-test and CLSI broth microdilution method for antifungal susceptibility testing of Candida albicans oral isolates / Comparação entre E-test e o método da microdiluição do CLSI para teste de susceptibilidade a antifúngicos de isolados orais de Candida albicans

Thirty Candida albicans isolated from oral candidosis patients and 30 C. albicans isolated from control individuals were studied. In vitro susceptibility tests were performed for amphotericin B, fluconazole, 5-flucytosine and itraconazole through the Clinical and Laboratorial Standards Institute (CLSI) reference method and E test system. The results obtained were analyzed and compared. MIC values were similar for the strains isolated from oral candidosis patients and control individuals. The agreement rate for the two methods was 66.67 percent for amphotericin B, 53.33 percent for fluconazole, 65 percent for flucytosine and 45 percent for itraconazole. According to our data, E test method could be an alternative to trial routine susceptibility testing due to its simplicity. However, it can not be considered a substitute for the CLSI reference method.

Trinta Candida albicans isoladas de pacientes portadores de candidose oral e 30 Candida albicans isoladas de indivíduos controle foram estudadas. Testes de susceptibilidade in vitro foram realizados com anfotericina B, fluconazol, 5-flucitosina e itraconazol pelo método do Clinical and Laboratorial Standars Institute (CLSI) e por E-test. Os resultados obtidos foram analisados e comparados. Os valores de CIM foram semelhantes para amostras isoladas de pacientes portadores de candidose oral e indivíduos controle. A concordância entre os dois métodos foi de 66,7 por cento para a anfotericina B, 53,33 por cento para o fluconazol, 65 por cento para a flucitosina e 45 por cento para o itraconazol. De acordo com estes resultados, o método do E-test poderia ser uma alternativa para a triagem de casos de rotina pela sua simplicidade. Entretanto, este método não pode ser considerado como um substituto para o método de referência do CLSI.

The Evaluation of Clinical Utility of ATB FUNGUS 2 for Antifungal Susceptibility Testing in Candida Species / 대한임상미생물학회지

BACKGROUND: Although the National Committee for Clinical Laboratory Standards (NCCLS) defined a standard reference broth microdilution method for testing the susceptibility of Candida species to antifungal drugs, many clinical laboratories require easier but reliable alternatives for routine antifungal susceptibility testing. We evaluated ATB FUNGUS 2 (bioMerieux, France.; ATB) compared to the method recommended by the NCCLS (NCCLS). METHODS: A total of 28 strains of Candida species consecutively isolated from blood and CSF cultures at Asan Medical Center from April to June 2004 were tested. In addition, 12 strains comprising C. krusei (3), C. glabrata (7) and C. guilliermondii (2) from the collection of Chonnam National University Hospital were included in the study. These strains were tested for minimum inhibitory concentrations (MICs) against flucytosine (FC), fluconazole (FZ), itraconazole (IZ) and amphotericin B (AB) by both of ATB and NCCLS. In NCCLS, MICs were read using a spectrophotometer after 24 and 48 hour-incubation. RESULTS: The concordance rates of MICs between ATB and NCCLS after 24 hour-incubation were 100%, 75%, 89% and 96% within two-fold dilution and 100%, 97%, 97%, 100% within four-fold dilution for FC, FZ, IZ and AB, respectively. For C. krusei, all three FC and FZ-resistant strains were either intermediate or SDD and one IZ-resistant strain was SDD in ATB, respectively. One C. tropicalis strain resulted in AB MICs of 0.5 microgram/mL in NCCLS, but 2 microgram/mL in ATB. CONCLUSIONS: ATB showed good concordance rates with NCCLS after 24 hour-incubation. ATB appears to be a useful alternatives to NCCLS for routine antifungal susceptibility tests. However, ATB needs further evaluation with more clinical strains, especially those resistant to antifungal agents.

Quality control for microbiological culture media: Is it enough to follow the NCCLS M22-A2 procedures?

Stringent quality control protocols must be used in order to guaranty that a particular medium is able to recover all sort of organism that may be present in clinical samples. Our aim was to evaluate an alternative protocol that would allow us to detect medium failure to yield quantitative growth of selected pathogens, and compare with the document M22-A2 from NCCLS. The detection limit of Haemophilus influenzae was significantly different depending on media source. We conclude that for some fastidious microorganisms, quantitative verification of the growth capacity of the culture medium is advised.

O uso de protocolos de controle de qualidade com padrões rígidos devem ser utilizados para garantir o isolamento dos microrganismos. Nosso objetivo foi o de avaliar um procedimento alternativo que nos permitisse detectar falhas de crescimento bacteriano em termos quantitativos, e comparação com o protocolo estabelecido pelo documento M22-A2 do NCCLS. Haemophilus influenzae apresentou diferença significativa de crescimento entre meio de cultivo de fontes distintas. Nos concluímos que, para alguns microrganismos fastidiosos, seja feita uma verificação quantitativa da capacidade de crescimento de cada meio de cultivo.

Quality control for microbiological culture media: Is it enough to follow the NCCLS M22-A2 procedures?

Stringent quality control protocols must be used in order to guaranty that a particular medium is able to recover all sort of organism that may be present in clinical samples. Our aim was to evaluate an alternative protocol that would allow us to detect medium failure to yield quantitative growth of selected pathogens, and compare with the document M22-A2 from NCCLS. The detection limit of Haemophilus influenzae was significantly different depending on media source. We conclude that for some fastidious microorganisms, quantitative verification of the growth capacity of the culture medium is advised.

O uso de protocolos de controle de qualidade com padrões rígidos devem ser utilizados para garantir o isolamento dos microrganismos. Nosso objetivo foi o de avaliar um procedimento alternativo que nos permitisse detectar falhas de crescimento bacteriano em termos quantitativos, e comparação com o protocolo estabelecido pelo documento M22-A2 do NCCLS. Haemophilus influenzae apresentou diferença significativa de crescimento entre meio de cultivo de fontes distintas. Nos concluímos que, para alguns microrganismos fastidiosos, seja feita uma verificação quantitativa da capacidade de crescimento de cada meio de cultivo.

Comparison of MicroScan and Vitek ESBL test with NCCLS ESBL Confirmatory Test / 대한임상미생물학회지

BACKGROUND: This study was designed to evaluate the ability of the Vitek and MicroScan ESBL test by comparing with NCCLS ESBL phenotypic confirmatory test by disk diffusion and to know the frequency of ESBL producers in the Seoul Veterans Hospital. METHODS: A total of 1,261 isolates(Escherichia coli 705, Klebsiella pneumoniae 502, K. oxytoca 54) from 883 patients were included in ESBL screening test by Vitek (494 strains) and MicroScan (767 strains). After excluding repetitive isolates from same patients, NCCLS ESBL confirmatory test was performed for 197 ESBL screening positives and 184 ESBL screening negatives. RESULTS: The overall frequency of ESBL screening positives was 22.3% (by MicroScan 26.2%, by Vitek 15.6%), and that of NCCLS ESBL positives was 18.9%(18.3% in E. coli, 21% in K. pneumoniae). MicroScan and Vitek ESBL test showed 100% and 92.3% sensitivity, 77.1% and 95.5% specificity, respectively. Among the 158 NCCLS ESBL positives, 17.7% showed clavulanic acid effect in cefotaxime only, 10.1% in ceftazidime only, and 72.2% in both. MicroScan Neg ComboPanel Type 21 test revealed that 91.4% of suspicious ESBL producers flagged by one or two antimicrobials were erroneous. In contrast, 96.2% of strains flagged by all five antimicrobials were correct. CONCLUSION: Suspicious ESBL producers by MicroScan showing three or four antimicrobial flags should be retested by NCCLS ESBLconfirmatory test. But strains with two or less flags and strains with all 5 flags can be reported as Non-ESBL producers and ESBL producers, respectively.

Comparison of Vitek ESBL Test and Other Methods for Detecting Extended-Spectrum -Lactamase-Producing Escherichia coli and Klebsiella Species / 대한임상병리학회지

BACKGROUND: Because extended-spectrum -lactamase (ESBL) producing strains can frequent-ly cause therapeutic failure and infectious outbreaks in hospitals, rapid and accurate detection of these strains are important. We compared the Vitek ESBL test with the NCCLS ESBL phenotypic confirmatory test by disk diffusion (NCCLS ESBL test) and double disk synergy test (DDST). METHODS: For a total of 316 clinical isolates composed of Escherichia coli (184), Klebsiella pneu-moniae (120) and Klebsiella oxytoca (12), we performed the Vitek ESBL test and the NCCLS ESBL test. For sixty-eight ESBL producing isolates, the Vitek ESBL test was compared with the NCCLS ESBL test and the DDST. The ESBL producer was defined as an organism showing an increase in the inhibited zone diameter of >or=5 mm for either cefotaxime or ceftazidime in combination with clavu-lanic acid versus its single test. The DDST was performed with 20 mm and 30 mm for interdisk diam-eter. For seven false negative isolates in the Vitek ESBL test, the DDST of cefepime was performed. RESULTS: Compared with the NCCLS ESBL test, the Vitek ESBL test showed one false positive (specificity, 99.6%), seven false negatives (sensitivity, 89.7%) and 97.5% agreement. Seven false negative isolates of the Vitek ESBL test were the cefoxitin-resistant ESBL producer. In positivity for the NCCLS ESBL test of 68 ESBL producing isolates, cefotaxime-clavulanic acid and ceftazidime-clavulanic acid were 94% and 91%. Cefotaxime, ceftazidime, aztreonam and ceftriaxone showed 95/90%, 100/55%, 100/85% and 95/80% positivity in double-disk synergy with amoxicillin-clavulanic acid (AMC) for 20/30 mm of the interdisk diameter respectively. For seven false negative isolates of the Vitek ESBL test, cefepime showed a distinct synergic effect with AMC. CONCLUSIONS: The Vitek ESBL test may be a useful method for clinical laboratories due to its easy, rapid and sensitive method but its method was less sensitive to cefoxitin-resistant ESBL. For these cases, if the NCCLS ESBL test or DDST with cefepime are added, the detection rate of the ESBL pro-ducer can be augmented.

Comparison of mecA Gene Detection with Susceptibility Testing Methods in Coagulase Negative Staphylococcus According to the New NCCLS Guidelines(1999) / 대한임상미생물학회지

BACKGROUND: Coagulase negative staphylococcus (CNS) spp. is a major pathogenic organism of nosocomial and community-acquired urianry tract infections, and causes infrctions in the immunocompromised host, and in particular, bloodstream infetions in patent with indwelling devices. High prevalance of methicillin resistance has been noticed in CNS which also have been recongnized as an important multidrug resistant pathogen. The optimal phenotypic method for detecting methicillin resistance still remains controversial, and new guidelines for detecting methicillin resistance of CNS was proposed by NCCLS in January 1999. We evaluated the relationship between mecA gene by PCR method and antimicrobial susceptibility tests according to the new NCCLS guidelines. METHODS: A total of 82 CNS isolates were examined for MICs and penicillin MICs by disk diffusion and agar dilution method according to NCCLS guidelines, and detections, and detection of mecA gene by PCR. RESULT: In disk diffusion method, 66 strains (80.5%) and 63 strains (76.8%) showed resistance to penicillin and oxacillin, respectively, and in agar dilution method, 71 strains(86.6%) and 53 strains (64.6%), respectively. In PCR method, mecA genes were detected in 49 strains(59.8%). Comparing with mecA gene detection by PCR method, the sensitivity of disk diffusion and agar dilution method was 95.8% and 89.8%, repectively. However, the sensitivity of disk diffusion and agar dilution method was 65.3% and 75.5%, respectively using previous NCCLS criteria. CONCLUSION: The new criteria of NCCLS detects the methicillin resistance induced by mecA gene more sensitively than previous one.

A Trial Establishment of Reference Interval of Serum Lipids Levels for the Elderly and Effects of Aging on Total Cholesterol, HDL-C, TG and LDL-C Levels / 日本農村医学会雑誌

The purpose of this study was to establish by way of trial reference interval of serum lipids (TC, HDL-C, TG and LDL-C) for old people aged 65 or over based on the document published by America's National Committee for Clinical Laboratory Standards. For this purpose, we used the results of the mass health screening tests run on a total of 14, 738 residents in Shimane Prefecture. We selected reference sample groups by excluding those examinees who had been undergoing medical treatment, those who had taken their meal less than 12 hours before, habitual drinkers and smokers, and those whose systolic blood pressure was more than 160mmHg, diastolic blood pressure upwards of 95mmHg, obesity level downwards -20% or upwards of +20%.<BR>T-C values peaked in the age group of 50s for men, while for women in the age group of 60s. Regarding HDL-C values, women were generally high compared withmen, but mean values for women in their 60s were significantly low (p<0.01) compared with those for women in their 50s. TG values were higher in men than in women. LDLC levels showed the same tendency as T-C levels.<BR>We compared the mean values of adults aged 64 or below and elders aged 65 or above. In the elders, the mean values for T-C, TG and LDL-C were significantly higher (p<0.01) than in the adults, and the mean HDL-C values were significantly lower (p<0.01) than in the adults.<BR>These results indicated that the reference interval of elders of T-C, HDL-C, TG and LDL-C could be from 147 to 289mg/dl, from 37 to 99mg/dl, from 40 to 209mg/dl and from 70 to 200mg/dl, respectively.

E-test for Antifungal Susceptibility Testing of Candida Species / 대한임상병리학회지

BACKGROUND: Although standardized broth dilution methods for antifungal susceptibility testing are available, easier testing procedures are desirable. We evaluated the E-test (AB disk, Sweden) as a possible alternative instead of NCCLS (National Committee for Clinical Laboratory Standards) broth macrodilution method. METHODS: Fifty-two bloodstream isolates of Candida spp. (including 11 C. albicans, 13 C. tropicalis, 18 C. parapsilosis, 1 C. glabrata, 4 C. krusei, 2 C. pelliculosa, 2 C. lipolytica, and 1 C. guilliermondii) were tested. Amphotericin B and fluconazole MICs for each isolate were determined by both NCCLS broth macrodilution method and E-test. The results of E-test for Candida spp. were compared with those of NCCLS macrodilution method. For selecting plating media for E-test, we compared E-test results in two different media (RPMI and Casiton medium) using five ATCC Candida strains. RESULTS: As E-test media, we selected RPMI medium for amphotericin B and Casitone medium for fluconazole because of higher agreement with NCCLS method. The E-test and NCCLS method of 52 Candida spp. yielded a very narrow range of MICs (0.064-2.0 microgram/mL) for amphotericin B and a broad range of MICs (0.5-64 microgram/mL) for fluconazole. The agreements of E-test within one doubling dilutions of the macrodilution reference were 90.4% (24h and 48h) for amphotericin B, and 90.4% (24h) and 96.2% (48h) for fluconazole. CONCLUSION: The E-test is a valuable alternative to the NCCLS macrodilution method for amphotericin B and fluconazole susceptibility testing of Candida species.