QUANTIFICAÇÃO DO DNA DE Mycobacterium leprae EM DIFERENTES CAMADAS DA PELE: ESTUDO SOBRE ELIMINAÇÃO TRANSEPIDÉRMICA, PROGRESSÃO PARA CURA E TRANSMISSÃO
: Leprosy, Mycobacterium leprae, real-time polymerase chain reaction, transmission, diagnostics.
The progress of molecular techniques was essential for the previous diagnosis of leprosy. However, current studies in the area have not been able to clarify the performance of these techniques in clinical practice yet. The main objective of this project was to test in clinical practice the accuracy, sensitivity and specificity of a new pair of primers developed for the repetitive element target (RLEP) of Mycobacterium leprae. Furthermore, as a secondary objective, was sought to identify the transmission potential of patients with multibacillary and paucibacillary leprosy. Thus, a crosssectional cohort study of diagnostic accuracy was carried out with a total of one hundred patients with a clinical picture compatible with leprosy. In this study, real-time Polymerase Chain Reaction (qPCR) was used to quantify M. leprae in different layers of the skin and in nasal swabs. Skin samples were divided into four layers: epidermis, upper dermis, lower dermis and hypodermis. The results of quantification of the bacillus in nasal swab samples were related to the number of household contacts. As a positivity criterion for the qPCR reaction, three cutoff points were established: 1 - any quantifiable bacillus DNA, 2 - quantification greater than or equal to 0.1 bacillus, 3 - quantification greater than or equal to 1 bacillus. Validation of the in vitro quantification of the reaction resulted in a quantification limit of 0.03 bacilli. The best sensitivity was observed in the upper dermis according to the first established cut-off point [sensitivity = 59.26% (95% CI = 45.97 – 71.32)]. In the epidermis, the third cutoff point resulted in 100% specificity (95% CI = 92.29 - 100). The best accuracy was found in the hypodermis at the first cutoff point [accuracy = 68.09% (95% CI = 58.11 – 76.64)]. For swab samples, the best sensitivity was 20.83% (95% CI = 11.73 – 34.26), and was achieved at the first cutoff point. At the three tested cutoff points, 100% specificity was obtained in the swab samples. The number of bacilli found in nasal swabs was not significantly related to the number of household contacts also diagnosed with leprosy. Paucibacillary patients tested positive only for bacillus fragments in nasal swabs, but not for the entire bacillus. This fact demonstrates that paucibacillary patients may not be a relevant source of disease transmission. It was concluded that different types of skin samples have little influence on the accuracy of the qPCR test. However, in contrast to this result, different cutoff points may influence the accuracy, sensitivity and specificity of the qPCR test. xviii Keywords: L