CLINICAL PRESENTATION, IMMUNE CELL RESPONSE AND GENETICS CHARACTERIZATION OF PATIENTS WITH CHROMOBLASTOMYCOSIS IN THE STATE OF MARANHÃO
Conceição de M P S Azevedo¹, Renata R Gomes², Tatiana S Herman², Johnny Ramos do Nascimento¹, Edith M M Batista¹, Sirlei Garcia Marques¹, Matheus C Silva³, Daniel Wagner C L Santos4 e Vania Aparecida Vicente². 1- UFMA, 2-UFPR, 3-CEUMA, 4-UNIFESP
OBJECTIVE: To evaluate the cellular immune response in patients with chromoblastomycosis and to analyze the patients with severe disease and a most reserved prognosis regarding the mutation in the CARD9 gene. METHODS: A total of 89 patients were followed at the Reference Center for Infectious Diseases (RECIPD), with diagnosis of chromoblastomycosis by direct examination, culture and histopathology. IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α and IL-17A cytokines were quantified by Cytometric Bead Array (CBA) Mouse Th1 / Th2 / Th17 cytokines in 89 patients. The extraction of genomic DNA was performed in 28 patients, using the Purelink Genomic DNA kit (Invitrogen). After DNA extraction and primer construction, standardization of the polymerase chain reaction (PCR) was performed to amplify the coding region CARD9. The sequencing reaction was performed using BigDye Terminator Cycle Kit v.3.1 (Applied Biosystems, Foster City, CA, USA). The editing and analysis of the sequences obtained was performed with the aid of BioEdit (HALL, 1999) and MEGA version 7 (Kumar, Stecher, and Tamura 2015), which were compared with the wild-type CARD9 gene sequence (Ensemb number: ENSG00000187796 ) and sequences from the GenBank database (NCBI -http: //www.ncbi.nlm.nih.gov/), with the aid of the MAFFT program (MISAWA; MIYATA, 2002), alignment and visual inspection with aid of the MEGA program version 7. The Q295X mutation in the CARD9 gene, characterized by the exchange of C by T in codon 295 of exon 6, was evaluated. RESULTS: It was observed that the majority of patients were male (79 patients (88.76%)); with a mean age at diagnosis of 58.1 years (ranging from 33 to 87 years) and standard deviation of 11.49. The majority presented, at diagnosis, moderate or severe disease (75.3%). In the patients with the mild form, IL-6 was identified in 22 patients, IL-10 in 15, IFN-γ in 3 and TNF-α in 1. In those with the moderate form, IL-6 was identified in 34 patients, IL -10 in 18, INF-γ in 5 and TNF-α in 13. In those with severe form, IL-6 was identified in 31 patients, IL-10 in 12, INF-γ in 5 and TNF-α in 9. Mutation in Exon 6 of the CARD9 gene was not observed in the patients. CONCLUSION: The fact that IL-6 and TNF-α, acute phase markers, are present in high titers in patients with moderate and severe forms, infers an intense pro-inflammatory activity in all stages of the disease, denoting its recurrent character. The association of the Th1 pattern with milder forms of the disease and the Th2 pattern with severe forms was observed again, whereas the Th17 response, characteristic of patients with chronic infections, was not evidenced in this series. Mutation in CARD9 was not observed, indicating that other mechanisms may be involved in the abstention of Th17.