Molecular diagnosis of cryptococcal meningitis in cerebrospinal fluid: comparison of primer sets for Cryptococcus neoformans and Cryptococcus gattii species complex.
Marilena dos Anjos Martins, Kate Bastos Santos Brighente, Terezinha Aparecida de Matos, Jose Ernesto Vidal, Daise Damaris Carnietto de Hipólito, Vera Lucia Pereira-Chioccola
Author Information
Marilena dos Anjos Martins: Laboratório de Biologia Molecular de Parasitas e Fungos, Instituto Adolfo Lutz, Sao Paulo, SP, Brazil; Núcleo de Micologia, Instituto Adolfo Lutz, Sao Paulo, SP, Brazil.
Kate Bastos Santos Brighente: Laboratório de Biologia Molecular de Parasitas e Fungos, Instituto Adolfo Lutz, Sao Paulo, SP, Brazil.
Terezinha Aparecida de Matos: Laboratório de Líquido Cefalorraquiano, Instituto de Infectologia Emílio Ribas, Sao Paulo, SP, Brazil.
Jose Ernesto Vidal: Departamento de Neurologia, Instituto de Infectologia Emílio Ribas, Sao Paulo, SP, Brazil.
Daise Damaris Carnietto de Hipólito: Núcleo de Micologia, Instituto Adolfo Lutz, Sao Paulo, SP, Brazil.
Vera Lucia Pereira-Chioccola: Laboratório de Biologia Molecular de Parasitas e Fungos, Instituto Adolfo Lutz, Sao Paulo, SP, Brazil. Electronic address: pchioccola@gmail.com.
AIM: This study evaluated the use of polymerase chain reaction for cryptococcal meningitis diagnosis in clinical samples. MATERIALS AND METHODS: The sensitivity and specificity of the methodology were evaluated using eight Cryptococcus neoformans/C. gattii species complex reference strains and 165 cerebrospinal fluid samples from patients with neurological diseases divided into two groups: 96 patients with cryptococcal meningitis and AIDS; and 69 patients with other neurological opportunistic diseases (CRL/AIDS). Two primer sets were tested (CN4-CN5 and the multiplex CNa70S-CNa70A/CNb49S-CNb-49A that amplify a specific product for C. neoformans and another for C. gattii). RESULTS: CN4-CN5 primer set was positive in all Cryptococcus standard strains and in 94.8% in DNA samples from cryptococcal meningitis and AIDS group. With the multiplex, no 448-bp product of C. gattii was observed in the clinical samples of either group. The 695bp products of C. neoformans were observed only in 64.6% of the cryptococcal meningitis and AIDS group. This primer set was negative for two standard strains. The specificity based on the negative samples from the CTL/AIDS group was 98.5% in both primer sets. CONCLUSIONS: These data suggest that the CN4/CN5 primer set was highly sensitive for the identification of C. neoformans/C. gattii species complex in cerebrospinal fluid samples from patients with clinical suspicion of cryptococcal meningitis.
