Desenvolvimento e validação de método de amplificação isotérmica para detecção de leptospiras patogênicas

Abstract

Leptospirosis is a zoonotic disease caused by pathogenic species of the genus Leptospira, which affects both humans and animals. Due to similarity of clinical signs with other febrile diseases, the diagnosis is a challenge. The current direct and indirect methods are bacterial culture and sorological tests. These, on the other hand, presents a variety of disadvantages such as, time-consuming technique and indirect detection of the bacteria by antibodies. So, it is necessary to search for alternative methods for diagnosing leptospirosis. Although there are molecular tests, as PCR, which are efficient and sensitive, they require sophisticated equipment, making its use not viable in underdeveloped countries. The aim of this work was to develop loop-mediated isothermal amplification (LAMP) tests for three different gene targets (LIC13162, LIC20239 and lipL32) in order to detect pathogenic leptospira. Primer constructions were made to the three sequences, and tested for different time and enzyme concentration. Analytic performance was evaluated through dilution curve of pure culture of Leptospira, with a PROBIT statistical analyses and DNA of various bacteria species. Diagnostic performance was done with 63 (44 negative and 19 positive) samples of hamster renal tissue. Both analyses had their results compared to real-time PCR. LAMP tests for LIC13162 and LIC20239 could not be implemented successfully. However, both showed to be present only in pathogenic leptospira by PCR. The lipL32 LAMP was standardized in a 30min reaction and with 4 U of enzyme. Its limit of detection was 156 cells/ml, showing the same numeric value of qPCR. Analytic sensibility of qPCR was 16,5 cells/µl (probit 0,5) and e 282 cells/µl (probit 0,95). For the LAMP test, these values were 25,7 cells/µl (probit 0,5) e 562 cells/µl (probit 0,95). Diagnostic performance of lipL32 LAMP was 84,2% sensitivity and 93,2% specificity. Results showed the identification of two novel genes of pathogenic leptospira that were not yet described in literature, and also the reassurance of lipL32 as an excellent target gene for diagnosing leptospirosis.