Desenvolvimento de uma PCR em tempo real Multiplex para a detecção de patógenos causadores de úlceras genitais: Treponema pallidum, Herpesvírus humano (HHV) 1 e 2.

Abstract

Genital ulcers, Sexually Transmitted Infections (STIs), have as their main etiological agent Treponema pallidum and Human Herpesvirus 1 and 2. Estimates of individuals infected with the major microorganisms that cause genital ulcers are increasing in Brazil as well as in other countries. The diagnosis, with material collected from the lesion, is mostly done through miscroscopy and clinical evaluation. Clinical aspects of ulcers have low sensitivity and specificity with the etiologic agent. For syphilis, dark field or direct immunofluorescence microscopy examinations are performed. For HHV 1 and 2 the cytological examination is used where morphological alterations are detected in the infected cells, lacking specificity and sensitivity. In the scope of the professional master's degree, based on criteria of biotechnological and market innovation, contribution of the theme to society and applicability, it is proposed to standardize and validate a qualitative Singleplex Real-Time Real-Time PCR using the complementary probes detection system for T. pallidum and a qualitative Multiplex Real-Time PCR for the detection of T. pallidum and HHV 1 and 2. To evaluate the technological and market innovation criteria, a survey was carried out to estimate which tests are offered for the diagnosis of these STIs in the main laboratories in Brazil. Primers and probes were selected from the literature and in silico and in vivo analyzes of the primers and probes were performed. Parameters such as size of the primers (in base pairs), G + C content and primer dissociation temperature, hairpin and hetero-dimers formation, complementarity between primers, size of the product generated, “E value” and “Query cover” were also evaluated. Variations in the concentrations of the sense and antisense primers were used to achieve optimum conditions of each reaction. PCR efficiency and limit of detection (LOD) were evaluated. Crossreactions were performed to evaluate the specificity of the primers and probes. PCR conditions were optimized, the LOD of T.pallidum was established, and cross-reactivity and interference studies were performed. The Singleplex for T.pallidum was sensitive and specific. Multiplex also proved to be sensitive and specific. Of the 88 samples tested, 2 were positive for HHV2. Molecular diagnosis of infectious diseases, besides assisting in the diagnosis of typical cases, aid in the diagnosis of atypical cases and also multiple infections. Furthermore, when compared to traditional microbiology techniques has the fastest response time and greater sensitivity. In addition, the Singleplex for T.pallidum and the Multiplex for T.pallidum and HHV1 and 2 will contribute to a greater scientific and economic growth of the company in the area of infectious diseases diagnosis, benefiting regional and national public health.