An improved method for the species‐specific assessment of mycobacteria in routinely formalin‐fixed and paraffin‐embedded tissues

Abstract

A polymerase chain reaction (PCR) assay for the rapid and species-specific diagnosis of mycobacterial infections in paraffin-embedded clinical specimens was developed using oligonucleotide primers to amplify a fragment of the DNA coding for the ribosomal 16S RNA of mycobacteria. The oligonucleotide primers amplified DNA from all 14 species of mycobacteria tested. By means of a reamplification protocol, as few as one to two mycobacteria could be detected in the presence of human DNA. The method of DNA isolation and amplification was applied on sections of routinely formalin-fixed and paraffin-embedded tissues. PCR for the beta-actin gene served as a control for successful DNA isolation. Mycobacterial DNA could be detected in cases of mycobacterial infections. The mycobacterial species was determined by additional sequencing of the PCR fragment. This PCR method may be a powerful tool for the diagnosis of mycobacterial infections from histopathological material and for the assessment of those mycobacteria that cannot readily be cultured, such as Mycobacterium leprae.

PCR in the diagnosis of cutaneous tuberculosis

“…In studies of application of PCR in formalin-fixed and paraffinembedded tissues (5,12,16,22) to detect mycobacterial gene sequences that code for 65 kDa (GroEL), mtp40, 16S rDNA or IS6110 the sensitivity of PCR presented a range from 47% to 87%. This variation, probably, occurs in consequence of the histopathological fixative or time of fixation used in preparation of the paraffin-embedded tissues (1,8) or due to the chosen gene target sequence.…”

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

“…The high efficiency of this test might be related to the primers used, which amplify a 135 bp fragment instead of the 404 bp fragment described by RICHTER et al 19 . The size of the amplicon is a decisive factor for the amplification success 8 , because DNA in FFPE tissues is often degraded by fixation and embedding procedures 12,23,28 .…”

"Avaliação crítica do uso da reação em cadeia da polimerase e exames complementares no diagnóstico da tuberculose cutânea e micobacteriose atípica"

“…(Cook et al, 1994;Telenti et al, 1993;Kox, 1995;Richter et al, 1995;Degitz, 1996;Roth et al, 1997;Chuang et al, 1997;Tan et al, 1999;Quiros et al, 2000;Ena et al, 2001;Tan et al, 2001;Ogusku et al, 2002;Senturk et al, 2002;Hsiao et al, 2003;Honoré-Boukaline et al, 2003). Na prática dermatológica diária, sobretudo nos centros de referência e Hospitais Universitários, há com freqüência casos de suspeita diagnóstica de tuberculose cutânea e micobacterioses atípicas que, muitas vezes, não são elucidados.…”

“…Atualmente, o desenvolvimento de técnicas de biologia molecular permite a amplificação de seqüência de ácido desoxirribonucleico (DNA)-alvo, veio contribuir, significativamente, para a diagnose desses casos mais difíceis, além da vantagem de ser um método de alta sensibilidade, especificidade e rápida execução (Cook et al, 1994;Telenti et al, 1993;Kox, 1995;Richter et al, 1995;Degitz, 1996;Roth et al, 1997;Chuang et al, 1997;Tan et al, 1999;Quiros et al, 2000;Tan et al, 2001;Ena et al, 2001;Ogusku et al, 2002;Senturk et al, 2002;Hsiao et al, 2003;Honore-Boukaline et al, 2003). Na literatura, encontram-se vários relatos, em especial, com o uso de PCR (reação em cadeia da polimerase) para o diagnóstico da tuberculose e outras micobacterioses atípicas (Telenti et al, 1993;Cook et al, 1994;Kox, 1995;Richter et al, 1995;Degitz, 1996;Chuang et al, 1997;Roth et al, 1997;Tan et al, 1999;Quiros et al, 2000;Tan et al, 2001;Ena et al, 2001;Ogusku et al, 2002;Senturk et al, 2002;Hsiao et al, 2003;Honoré-Boukaline et al, 2003). A vantagem do método é que, por meio da amplificação da seqüência-alvo, detecta-se a presença de parte do gene da micobactéria, possibilitando o diagnóstico, mesmo quando os métodos de coloração, imunohistoquímica e cultura são negativos.…”

“…A literatura descreve métodos com emprego de genes que codificam proteínas comuns às micobactérias, como o gene da proteína do choque 65-kDa ("heat shock protein") ou 16S r RNA, ou ainda, 23S RNA e 23S -16S rRNA (Telenti et al, 1993;Cook et al, 1994;Richter et al, 1995;Tan et al, 1999;Quiros et al, 2000;Ena et al, 2001;Tan et al, 2001;Ogusku et al, 2002;Hsiao et al, 2003;Honore-Boukaline et al, 2003).…”