Evaluation of the molecular techniques of DNA microarray and LAMP for the identification of the Cryptococcus neoformans and Cryptococcus gattii species of isolates grown in culture medium and cerebrospinal fluid of patients with meningitis

Author(s)

Pamella Stivanelli

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2019-01-30

Examining board members

Maria Luiza Moretti; Celia Regina Garlipp; Gilda Maria Barbaro Del Negro

• Advisor: Maria Luiza Moretti

Abstract

Introduction. Cryptococcal meningitis is an infection caused by yeasts of the genus Cryptococcus that mainly affects, patients with HIV, immunosuppressed and organ transplanted. Species gattii and neoformans are responsible for causing meningitis in humans. Objectives. The objective of this work was to evaluate the performance of molecular microarray and Loop Mediated Isothermal amplification (LAMP) techniques in the identification of Cryptococcus in the cerebrospinal fluid of patients with cryptococcal meningitis differentiating the gattii and neoformans species. Methods. A prospective study was carried out from August 2016 to October 2018. Cerebrospinal fluid samples were positive for Cryptococcus, positive for bacteria and negative for any microorganism, where they were obtained in the infectious diseases ward and the microbiology laboratory of the Hospital de Clínicas of Unicamp. Thirty strains of C. gattii were given by the Adolfo Lutz Institute to test the methodologies studied. The DNA extracted directly from the CSF and its culture isolate was submitted to PCR using the ITS region and the CAP59 gene and later to DNA microarray, LAMP and sequencing. Results. 133 CSF were extracted, 11 positive for Cryptococcus, 15 positive for bacteria and 107 negative and 30 C. gattii strains. Eight samples were identified as C. neoformans and 2 as C. gattii and the strains were identified as C. gattii. For the microarray ITS platform, from 11 CSF samples that were extracted DNA, 10 amplified by PCR. Five CSF samples were identified, and the other 5 were not possible to obtain results. For the isolates tested on this platform, there were three samples with non-specific reactions and 7 were identified correctly. For the CAP59 platform, PCR amplification was performed on all CSF samples. There was no identification in three samples and eight were correctly identified. For the isolates of CSF, it was possible to correctly identify all the samples. Tests with different LAMP (SetA, SetD, Set1, Set2, Set3_neoformans and Set4_neoformans methods were used to identify C. neoformans and SetB/C and Set1_gattii to identify C. gattii). Two sets of primers were specific for C. neoformans and C. gattii respectively: Se3_neoformans and Set1_gattii. Of 11 CSF samples tested by LAMP, 9 were tested by Set3_neoformans and two by Set1_gattii. For the Set3_neoformans set 55.5% were identified as C. neoformans and in four samples there was no amplification and in the two C. gattii samples tested in the Set1_gattii set, it was not possible to obtain amplification. All isolates tested by Set3_neoformans amplified the LAMP methodology. The set Set1_gattii, was tested only one isolate (P285) that was identified as C. gattii, because the other the sample did not grow in culture. Negative and bacterial positive CSF samples were used as controls for our tests, and all tested negative. Conclusion. The methodologies have proven to be rapid and reliable tests in the identification of Cryptococcus, but more tests are necessary in relation to direct detection of clinical samples (AU)

• FAPESP's process: 16/12414-2 - Evaluation of the performance of molecular techniques as DNA microarray and LAMP for diagnosis and accompaniment in patients with cryptococcal meningitis
• Grantee: Pamella Stivanelli
• Support Opportunities: Scholarships in Brazil - Master