Comparison of resazurin microtiter assay performance and BACTEC MGIT 960 in the susceptibility testing of Brazilian clinical isolates of Mycobacterium tuberculosis to four first-line drugs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin B and itraconazole

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison between E-test and CLSI broth microdilution method for antifungal susceptibility testing of Candida albicans oral isolates

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% para a anfotericina B, 53,33% para o fluconazol, 65% para a flucitosina e 45% 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.

In vitro susceptibility testing of Microsporum gypseum isolated from healthy cattle and soil samples against itraconazole, terbinafine, fluconazole and topical veterinarian drugs

The present study evaluated in vitro susceptibility testing of dermatophytes isolates from healthy cattle and soil samples against three antifungal agents and three topical veterinarian drugs. Itraconazole and terbinafine showed a higher in vitro fungicidal activity than fluconazole. The veterinarian drugs LEPECID (R) and iodine 5% were more active in vitro than the UNGUENTO (R) spray. All drugs showed fungicidal activity against Microsporum gypseum, and they may be considered as efficient agents for the topical treatment of dermatophytoses in cattle.

Microplate alamarblue assay for paracoccidioides susceptibility testing

CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs. © 2013, American Society for Microbiology.

Stable expression of Escherichia coli beta-glucuronidase A (GusA) in Giardia lamblia: application to high-throughput drug susceptibility testing

OBJECTIVES: In order to create a suitable model for high-throughput drug screening, a Giardia lamblia WB C6 strain expressing Escherichia coli glucuronidase A (GusA) was created and tested with respect to susceptibility to the anti-giardial drugs nitazoxanide and metronidazole. METHODS: GusA, a well-established reporter gene in other systems, was cloned into the vector pPacVInteg allowing stable expression in G. lamblia under control of the promoter from the glutamate dehydrogenase (gdh) gene. The resulting transgenic strain was compared with the wild-type strain in a vitality assay, characterized with respect to susceptibility to nitazoxanide, metronidazole and -- as assessed in a 96-well plate format -- to a panel of 15 other compounds to be tested for anti-giardial activity. RESULTS: GusA was stably expressed in G. lamblia. Using a simple glucuronidase assay protocol, drug efficacy tests yielded results similar to those from cell counting. CONCLUSIONS: G. lamblia WB C6 GusA is a suitable tool for high-throughput anti-giardial drug screening.

Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs. old Clinical and Laboratory Standards Institute clinical breakpoints: a 6-year prospective candidaemia survey from the fungal infection network of Switzerland

We analyzed the species distribution of Candida blood isolates (CBIs), prospectively collected between 2004 and 2009 within FUNGINOS, and compared their antifungal susceptibility according to clinical breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2013, and the Clinical and Laboratory Standards Institute (CLSI) in 2008 (old CLSI breakpoints) and 2012 (new CLSI breakpoints). CBIs were tested for susceptiblity to fluconazole, voriconazole and caspofungin by microtitre broth dilution (Sensititre® YeastOne™ test panel). Of 1090 CBIs, 675 (61.9%) were C. albicans, 191 (17.5%) C. glabrata, 64 (5.9%) C. tropicalis, 59 (5.4%) C. parapsilosis, 33 (3%) C. dubliniensis, 22 (2%) C. krusei and 46 (4.2%) rare Candida species. Independently of the breakpoints applied, C. albicans was almost uniformly (>98%) susceptible to all three antifungal agents. In contrast, the proportions of fluconazole- and voriconazole-susceptible C. tropicalis and F-susceptible C. parapsilosis were lower according to EUCAST/new CLSI breakpoints than to the old CLSI breakpoints. For caspofungin, non-susceptibility occurred mainly in C. krusei (63.3%) and C. glabrata (9.4%). Nine isolates (five C. tropicalis, three C. albicans and one C. parapsilosis) were cross-resistant to azoles according to EUCAST breakpoints, compared with three isolates (two C. albicans and one C. tropicalis) according to new and two (2 C. albicans) according to old CLSI breakpoints. Four species (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis) represented >90% of all CBIs. In vitro resistance to fluconazole, voriconazole and caspofungin was rare among C. albicans, but an increase of non-susceptibile isolates was observed among C. tropicalis/C. parapsilosis for the azoles and C. glabrata/C. krusei for caspofungin according to EUCAST and new CLSI breakpoints compared with old CLSI breakpoints.

A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.

Comparison of disc diffusion, Etest and broth microdilution for testing susceptibility of carbapenem-resistant P. aeruginosa to polymyxins

Abstract Background Considering the increasing use of polymyxins to treat infections due to multidrug resistant Gram-negative in many countries, it is important to evaluate different susceptibility testing methods to this class of antibiotic. Methods Susceptibility of 109 carbapenem-resistant P. aeruginosa to polymyxins was tested comparing broth microdilution (reference method), disc diffusion, and Etest using the new interpretative breakpoints of Clinical and Laboratory Standards Institute. Results Twenty-nine percent of isolates belonged to endemic clone and thus, these strains were excluded of analysis. Among 78 strains evaluated, only one isolate was resistant to polymyxin B by the reference method (MIC: 8.0 μg/mL). Very major and major error rates of 1.2% and 11.5% were detected comparing polymyxin B disc diffusion with the broth microdilution (reference method). Agreement within 1 twofold dilution between Etest and the broth microdilution were 33% for polymyxin B and 79.5% for colistin. One major error and 48.7% minor errors were found comparing polymyxin B Etest with broth microdilution and only 6.4% minor errors with colistin. The concordance between Etest and the broth microdilution (reference method) was respectively 100% for colistin and 90% for polymyxin B. Conclusion Resistance to polymyxins seems to be rare among hospital carbapenem-resistant P. aeruginosa isolates over a six-year period. Our results showed, using the new CLSI criteria, that the disc diffusion susceptibility does not report major errors (false-resistant results) for colistin. On the other hand, showed a high frequency of minor errors and 1 very major error for polymyxin B. Etest presented better results for colistin than polymyxin B. Until these results are reproduced with a large number of polymyxins-resistant P. aeruginosa isolates, susceptibility to polymyxins should be confirmed by a reference method.

In vitro susceptibility to antifungal agents of clinical and environmental Cryptococcus neoformans isolated in Southern of Brazil

The purpose of the present study was to compare the susceptibility to four antifungal agents of 69 Cryptococcus neoformans strains isolated from AIDS patients with that of 13 C. neoformans strains isolated from the environment. Based on the NCCLS M27-A methodology the Minimal Inhibitory Concentrations (MICs) obtained for amphotericin B, itraconazole and ketoconazole were very similar for clinical and environmental isolates. Clinical isolates were less susceptible to fluconazole than environmental isolates. The significance of these findings and aspects concerning the importance, role and difficulties of C. neoformans susceptibility testing are also discussed.

SUSCEPTIBILITY TEST FOR FUNGI: CLINICAL AND LABORATORIAL CORRELATIONS IN MEDICAL MYCOLOGY

SUMMARYDuring recent decades, antifungal susceptibility testing has become standardized and nowadays has the same role of the antibacterial susceptibility testing in microbiology laboratories. American and European standards have been developed, as well as equivalent commercial systems which are more appropriate for clinical laboratories. The detection of resistant strains by means of these systems has allowed the study and understanding of the molecular basis and the mechanisms of resistance of fungal species to antifungal agents. In addition, many studies on the correlation of in vitro results with the outcome of patients have been performed, reaching the conclusion that infections caused by resistant strains have worse outcome than those caused by susceptible fungal isolates. These studies have allowed the development of interpretative breakpoints for Candida spp. and Aspergillus spp., the most frequent agents of fungal infections in the world. In summary, antifungal susceptibility tests have become essential tools to guide the treatment of fungal diseases, to know the local and global disease epidemiology, and to identify resistance to antifungals.

Susceptibility to amphotericin B of Candida spp. strains isolated in Ceará, Northeastern Brazil

IntroductionAmphotericin B (AMB) is an antifungal agent used extensively in clinical medicine, yet resistance remains low. This study aims to evaluate the susceptibility of Candida spp. against AMB.MethodsFor broth microdilution susceptibility testing, 77 strains of Candida spp. were selected (32 C. albicans, 33 C. tropicalis, and 12 C. parapsilosis). The strains were considered susceptible when they exhibited MIC≤1.0µg/ml.ResultsNone of the strains showed an MIC greater than 0.25µg/ml.ConclusionsFurther works are necessary, with a higher number of strains, to assess the validity of the results used in this study.