Comparison of DNA Extraction Protocols and Molecular Targets to Diagnose Tuberculous Meningitis.

Flavia Silva Palomo,Martha Gabriela Celle Rivero,Fernando Pereira Pinto,Milene Gonçalves Quiles,Antonio Carlos Campos Pignatari,Antonia Maria De Oliveira Machado

doi:10.1155/2017/5089046

Flavia Silva Palomo, Martha Gabriela Celle Rivero + Show 4 more

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https://doi.org/10.1155/2017/5089046

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Abstract

Tuberculous meningitis (TBM) is a severe form of extrapulmonary tuberculosis. The aims of this study were to evaluate in-house molecular diagnostic protocols of DNA extraction directly from CSF samples and the targets amplified by qPCR as an accurate and fast diagnosis of TBM. One hundred CSF samples from 68 patients suspected of TBM were studied. Four DNA extraction techniques (phenol-chloroform-thiocyanate guanidine, silica thiocyanate guanidine, resin, and resin with ethanol) were compared and CSF samples were used to determine the best target (IS6110, MPB64, and hsp65 KDa) by qPCR. The extraction protocol using the phenol-chloroform-thiocyanate guanidine showed the best results in terms of quantification and sensitivity of PCR amplification, presenting up to 10 times more DNA than the second best protocol, the silica guanidine thiocyanate. The target that showed the best result for TBM diagnosis was the IS6110. This target showed 91% sensitivity and 97% specificity when we analyzed the results by sample and showed 100% sensitivity and 98% specificity when we analyzed the results by patient. The DNA extraction with phenol-chloroform-thiocyanate guanidine followed by IS6110 target amplification has been shown to be suitable for diagnosis of TBM in our clinical setting.

Highlights

Tuberculosis is a serious infectious contagious disease that usually affects the lungs but can affect other organs such as kidney, bone, and central nervous system (CNS) [1]
The sensibility for detection of M. tuberculosis in cerebrospinal fluid (CSF) samples can be substantially increased from 70% to 100% and the time required to release laboratory results can be significantly decreased with the use molecular methods, including the polymerase chain reaction (PCR)
From 35 clinical samples considered true positive by clinical parameters for Tuberculous meningitis (TBM) diagnosis, IS6110 PCR was able to detect 32 of them followed by culture and hsp65 KDa (16 samples) and MPB64 (12 samples). (Table 4)

Summary

Introduction

Tuberculosis is a serious infectious contagious disease that usually affects the lungs but can affect other organs such as kidney, bone, and central nervous system (CNS) [1]. In 2011, cases of extra pulmonary tuberculosis in Brazil reached almost 16% of all cases of the disease [2] and about 6.3% of these (1.3% of the total) were TBM [3]. The definitive diagnosis of TBM depends on the M. tuberculosis agent detection from cerebrospinal fluid (CSF). The survey of M. tuberculosis in CSF is carried out by conventional microbiological methods including ZiehlNeelsen smear, which has low sensitivity (0–20%) and culture, which requires until 65 days to final result [1, 6]. The sensibility for detection of M. tuberculosis in CSF samples can be substantially increased from 70% to 100% and the time required to release laboratory results can be significantly decreased with the use molecular methods, including the polymerase chain reaction (PCR). The rapid identification of TBM through molecular analysis of CSF is an important factor for proper and early institution of antimicrobial treatment [7,8,9]

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