Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex...

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Main Authors: Marcio Roberto Silva, Mark Drew Crosland Guimarães, Vania Maria de Oliveira, Aline dos Santos Moreira, Ronaldo Rodrigues da Costa, Kelly Cristina Ferreira Abi-Zaid, Adalgiza da Silva Rocha, Philip Noel Suffys
Format: Article
Language:English
Published: Fundação Oswaldo Cruz (FIOCRUZ) 2011-02-01
Series:Memorias do Instituto Oswaldo Cruz
Subjects:
Online Access:http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762011000100002&lng=en&tlng=en
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author Marcio Roberto Silva
Mark Drew Crosland Guimarães
Vania Maria de Oliveira
Aline dos Santos Moreira
Ronaldo Rodrigues da Costa
Kelly Cristina Ferreira Abi-Zaid
Adalgiza da Silva Rocha
Philip Noel Suffys
author_facet Marcio Roberto Silva
Mark Drew Crosland Guimarães
Vania Maria de Oliveira
Aline dos Santos Moreira
Ronaldo Rodrigues da Costa
Kelly Cristina Ferreira Abi-Zaid
Adalgiza da Silva Rocha
Philip Noel Suffys
author_sort Marcio Roberto Silva
collection DOAJ
description A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5%) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.
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spelling doaj.art-1aef1d27567140cb920266bf66e02f952023-09-02T16:26:06ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz1678-80602011-02-011061091510.1590/S0074-02762011000100002S0074-02762011000100002Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in BrazilMarcio Roberto Silva0Mark Drew Crosland Guimarães1Vania Maria de Oliveira2Aline dos Santos Moreira3Ronaldo Rodrigues da Costa4Kelly Cristina Ferreira Abi-Zaid5Adalgiza da Silva Rocha6Philip Noel Suffys7Ministério da Agricultura, Pecuária e AbastecimentoUniversidade Federal de Minas GeraisMinistério da Agricultura, Pecuária e AbastecimentoLaboratório de Genômica Funcional e BioinformáticaUniversidade Federal de Juiz de ForaFundação Hospitalar do Estado de Minas GeraisFundação Oswaldo CruzFundação Oswaldo CruzA cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5%) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762011000100002&lng=en&tlng=enoxyR pseudogenepolymerase chain reactionsputum smearMycobacterium tuberculosisMycobacterium bovismolecular sequence data
spellingShingle Marcio Roberto Silva
Mark Drew Crosland Guimarães
Vania Maria de Oliveira
Aline dos Santos Moreira
Ronaldo Rodrigues da Costa
Kelly Cristina Ferreira Abi-Zaid
Adalgiza da Silva Rocha
Philip Noel Suffys
Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil
Memorias do Instituto Oswaldo Cruz
oxyR pseudogene
polymerase chain reaction
sputum smear
Mycobacterium tuberculosis
Mycobacterium bovis
molecular sequence data
title Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil
title_full Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil
title_fullStr Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil
title_full_unstemmed Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil
title_short Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil
title_sort identification of mycobacterium tuberculosis complex based on amplification and sequencing of the oxyr pseudogene from stored ziehl neelsen stained sputum smears in brazil
topic oxyR pseudogene
polymerase chain reaction
sputum smear
Mycobacterium tuberculosis
Mycobacterium bovis
molecular sequence data
url http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762011000100002&lng=en&tlng=en
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