Identificação de sequencias como iniciadores ("primers") especificos para a caracterização em cultura dos sorotipos A e B de Cryptococcus neoformans pela reação em cadeia da polimerase (PCR)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2000

RESUMO

1. The cryptococcosis, a cosmopolitan disease, caused by Cryptococcus neoformans , a fungus, has been known about one century. The immunossupression that occurs by the consequence with steroids treatments and other kinds of drugs, degeneratives and infectious diseases, facilitates the proliferation and the development of these infectious agents, that permit to this disease be transformed itself in an important disease, particularly, on these cases. 2. The appearance of AIOS at the 70 s end has facilitated the proliferation of cryptococcosis, since one of the effects of this syndrome is the great immunocompromise of the patients immunological defenses. 3. In this work 33 strains of Cryptococcus neoformans were isolated from 24 patients treated at the Hospital of Clinics from The State University of Campinas - State of São Paulo, Brazil. The isolation of these strains were obtained from skin, blood and specially from Central Nervous System Fluid(CNSF). 4. The 33 strains of Cryptococcus neoformans were, innitially, submitted to an identification by serotyping technique using the comercial kit named Cryptocheck(latron, Japan). Twenty four strains, from nineteen AIOS patients, were characterized like serotype A. Considering the rest of the samples, nine were from patients without AIDS, and from these nine, seven were characterized as serotype A and 2 as serotype B. 5. C. neoformans strains serotype A and B were tested by PCR method with ramdomized commercial primers denominated R 1, R2 and R3 to the determination of the genomic profile (RAPO). Using that profile, a exclusive 695 bp band were ~elected from the serotype A strains and other with 448 bp, beeing exclusive toe in the serotype B strains. These amplicons were sequenced and, after that, there was a developing of two primer pairs to be tested to the characterization of serotype A and B. 6. These two primer pairs were tested in the 33 strains of C. neoformans, previously determined like serotype A and B by the aglutination test. In the serotype A case, from ali reference strains tested, as well strains isolated from clinical cases, there was amplification of typical sequence of this serotype(695 bp band). Similarly, in the serotype B strains, a 448 bp band were amplified. The amplification of these specific sequencies, using primers studied for both serotypes was specific and sensitive, compared with the aglutination test. 7. For minimal inhibitory concentraion(MIC) study, 33 strains of C. neofonnans were tested with amphotericin B, Itraconazole, Fluconazole, 5- Fluorcytosin and commercial Amphotericin B(Fungison@), according the "National Comittee for Clinical and Laboratorial Standards"(NCCLS) proceedings. . 8. The MIC of the various studied drugs, using RPMI 1640 medium for 33 strains of C. neoformans were: Amphotericin B: 0,25 to 2 J,1g/ml; Fungison@: 0,03 to 2 J.1g/ml; Fluconazole:O,5 to 32 J.1g/ml; Itracon~zole: 0,06 to 2 J.1g/ml; 5 - Fluorcytosin: 1 to 16 J.1g/ml. 9. The results of this study showed a new approach to the characterization of C. neoformans serotype A and B based in the Protein Chain Reaction(PCR) technic, to the knowledge of C. neoformans strains sensibility collected from patients against antifungal agents and to the worried presence of strains SDD(Sensibility Dose Dependent) to the Fluconazol in patients with and without HIV infection

ASSUNTO(S)

aids (doença)

ACESSO AO ARTIGO

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