Aplicações da PCR em tempo real no diagnóstico laboratorial da Febre Maculosa Brasileira

Abstract

Brazilian Spotted Fever (BSF) is the main tick borne disease with impact
on public health in Brazil. Early symptoms are nonspecific, but the disease
may progress rapidly to Febrile Acute Hemorrhagic Syndrome (SFHA)
with occurrence of death within a few days. The purpose of this study was
to investigate the applications of real-time PCR in the routine laboratory
for the etiologic diagnosis of BSF, in the biological samples sent to the
laboratory. Two real-time PCR protocols were tested: one for detecting
the genus Rickettsia spp (gltA-TaqMan®) with detection by TaqMan®
probe and another for specific detection of Spotted Fever Group species
(OmpA-SYBR) by SYBR Green detection. The qPCR protocol for RNaseP
was used as endogenous internal control. Samples used were blood, serum,
blood clot and biopsy from skin lesion of fatal and nonfatal clinically suspected
of BSF. The results showed that ompA-SYBR protocol suffers interference
from the biological matrix and the best performance was obtained with
serum samples. Protocols OmpA-SYBR and gltA-TaqMan® showed results
concordance up to 90%. The protocol gltA-TaqMan® was more sensitive
than OmpA-SYBR protocol, but less specificity, particularly for samples with
TC> 36. The best performance for BSF qPCR assay was obtained when
combining three qPCR protocols (OmpA-SYBR, gltA-TaqMan® and RnaseP),
named FMPCR, were used in serum samples to detection BSF in fatal cases.
FMPCR showed low sensitivity for detecting non-fatal cases positive by IFA,
the positivity was 21%. The results of this study indicate that FMPCR has
sensitivity and specificity to be used as a diagnostic tool for elucidation of
fatal BSF.