This technology involves yeast acid nucleic isolation, amplification by PCR and identification by electrophoresis on polyacrylamide gel with silver salt staining.ġ. neoformans and do not amplify the DNA of other yeast 17. In this work the single-step PCR technique on clinical samples was optimized by utilizing the initiator pairs CN-4/CN-5 that are specific for C. neoformans can be detected by initiators that encode the ribosomal RNA (rRNA) gene 16S-5S,8S-23S which is highly conserved 4,17. It constitutes a method of choice for early alternative diagnosis to the conventional ones and contributes to supply important subsidies to the diagnosis of this pathology mainly when there is clinical suspicion of the disease 12. For the diagnosis of neurocryptococcosis the application of more sensitive and specific laboratorial techniques are necessary in order to introduce early and specific antifungal therapy. These can be done in various clinical materials, such as blood, liquor, secretions, cutaneous scrapings, bronchial alveolar aspirate and urine 24. Molecular tests for detecting nucleic acids of infectious agents in biological samples have been developed for C. neoformans penetrates the organism, lodging primarily in the lungs and later presents a notable tropism for the central nervous system 7,9,14. It is an important opportunist systemic mycosis that involves mainly immunosuppressed individuals and starts when C. In the last decades, cryptococcosis has been assuming a prominent role at public health level due to the growing number of AIDS individual cases 2,3,20. A análise comparativa dos três métodos, tinta da china, cultura e PCR, demonstrou que o último é muito mais sensível e específico, podendo ser aplicável como importante recurso laboratorial no diagnóstico da neurocriptococose.Ĭryptococcosis is a cosmopolitan infection caused by Cryptococcus neoformans, an encapsulated yeast pathogenic to humans and animals 1,13,18. A técnica de PCR pode detectar 1 célula/mL de líquor e é altamente específica. Os resultados demonstraram que a técnica tem alta sensibilidade, superior a cultura (85,7%) e ao teste da tinta da china (76,8%). Os pacientes eram portadores de meningite criptocócica (n = 56) e meningite ocasionada por outros agentes (n = 16). Foram estudados 72 líquors obtidos de casos de pacientes com e sem AIDS. A amplificação foi realizada nas áreas ITS e 5,6S do RNA ribossomal (rRNA). Keywords: Cryptococcus neoformans PCR Diagnostic methods AIDS CryptococcosisĪ detecção de Cryptococcus neoformans em líquor foi otimizada pela técnica de PCR. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.
PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). Tel: + 55 11 30917294, Fax: +55 11 3091 7354Ĭryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis.
IIIDepartment of Microbiology, Institute of Biomedical Sciences, USP, São Paulo, SP, Brazil IIAdolfo Lutz Institute, São Paulo, SP, Brazil IUniversity of São Paulo, USP, Faculty of Pharmaceutical Sciences, Department of Clinical Analyses and Toxicology, São Paulo, SP, Brazil Regina Célia Paschoal I Mário Hiroyuki Hirata I Rosário Crespo Hirata I Márcia de Souza Carvalho Melhem II Amanda Latercia Tranches Dias III Claudete Rodrigues Paula III Neurocryptococcosis: diagnosis by PCR method
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