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Improved Detection and Accuracy of Mycobacterium Species Identification from Paraffin Embedded Tissues of Patients by Using Multigene Targeted PCR and Sequencing
BACKGROUND: Prompt and accurate identification and differentiation of Mycobacterium tuberculosis-complex (MTBC) from non-tuberculous mycobacteria (NTM) is crucial for the selection of antimicrobial treatment and appropriate public health response. Diagnosis and characterization of mycobacteria is ch...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630882/ http://dx.doi.org/10.1093/ofid/ofx163.1639 |
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author | Bhatnagar, Julu DeLeon-Carnes, Marlene Rabeneck, Demi Rowlett, Veronica Hale, Gillian Martines, Roosecelis Muehlenbachs, Atis Gary, Joy Ritter, Jana M Shieh, Wun-Ju Zaki, Sherif |
author_facet | Bhatnagar, Julu DeLeon-Carnes, Marlene Rabeneck, Demi Rowlett, Veronica Hale, Gillian Martines, Roosecelis Muehlenbachs, Atis Gary, Joy Ritter, Jana M Shieh, Wun-Ju Zaki, Sherif |
author_sort | Bhatnagar, Julu |
collection | PubMed |
description | BACKGROUND: Prompt and accurate identification and differentiation of Mycobacterium tuberculosis-complex (MTBC) from non-tuberculous mycobacteria (NTM) is crucial for the selection of antimicrobial treatment and appropriate public health response. Diagnosis and characterization of mycobacteria is challenging due to diverse clinical presentations, lack of sensitivity of smear microscopy, and fastidious culture identification. Moreover, because of clinical suspicion of noninfectious conditions, specimens are often not processed for culture and formalin-fixed, paraffin-embedded (FFPE) tissues are the only specimens available. For rapid and accurate identification of Mycobacterium spp. from patient tissues, sensitive and specific molecular assays combined with other tissue-based methods are vital. METHODS: We extracted DNA from FFPE tissues from 931 patients with clinical and histopathological suspicion of mycobacterial infection (received during 2013–2016) and evaluated by multistage, multigene targeted Mycobacterium-genus, complexes-and species-specific PCR assays (targets including 16S rRNA, rpoB, groEL, IS6110, RLEP) and sequencing. Tissues were also examined by acid-fast bacilli (AFB) stains and mycobacteria immunohistochemistry (IHC). Assays to detect mutations associated with drug resistance were performed on MTBC cases. RESULTS: A Mycobacterium species was detected in 465 (50%) cases by PCR and sequencing. Of these, 380 (82%) were positive by Mycobacterium PCR targeting 16S rRNA. 85 cases (18%), including 9 MTBC, 12 M. avium complex and 3 M. leprae, were positive by other PCRs. Co-infection of MTBC and NTM spp. was detected in 5 cases. Of 465 PCR positive cases, 327 (70%) showed immunostaining and 223 (48%) were AFB-positive. Molecular markers for drug resistance were detected in 9 out of 88 (10%) tested MTBC cases. CONCLUSION: FFPE tissue analysis by multigene targeted PCR assays expands the opportunities for rapid identification of Mycobacterium species, allows differentiation of MTBC from NTM, and helps to detect co-infections. Using multigene targeted PCRs in combination with histopathology and IHC improve the accuracy of diagnosis, particularly in the presence of commensal and environmental pathogens. DISCLOSURES: All authors: No reported disclosures. |
format | Online Article Text |
id | pubmed-5630882 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-56308822017-11-07 Improved Detection and Accuracy of Mycobacterium Species Identification from Paraffin Embedded Tissues of Patients by Using Multigene Targeted PCR and Sequencing Bhatnagar, Julu DeLeon-Carnes, Marlene Rabeneck, Demi Rowlett, Veronica Hale, Gillian Martines, Roosecelis Muehlenbachs, Atis Gary, Joy Ritter, Jana M Shieh, Wun-Ju Zaki, Sherif Open Forum Infect Dis Abstracts BACKGROUND: Prompt and accurate identification and differentiation of Mycobacterium tuberculosis-complex (MTBC) from non-tuberculous mycobacteria (NTM) is crucial for the selection of antimicrobial treatment and appropriate public health response. Diagnosis and characterization of mycobacteria is challenging due to diverse clinical presentations, lack of sensitivity of smear microscopy, and fastidious culture identification. Moreover, because of clinical suspicion of noninfectious conditions, specimens are often not processed for culture and formalin-fixed, paraffin-embedded (FFPE) tissues are the only specimens available. For rapid and accurate identification of Mycobacterium spp. from patient tissues, sensitive and specific molecular assays combined with other tissue-based methods are vital. METHODS: We extracted DNA from FFPE tissues from 931 patients with clinical and histopathological suspicion of mycobacterial infection (received during 2013–2016) and evaluated by multistage, multigene targeted Mycobacterium-genus, complexes-and species-specific PCR assays (targets including 16S rRNA, rpoB, groEL, IS6110, RLEP) and sequencing. Tissues were also examined by acid-fast bacilli (AFB) stains and mycobacteria immunohistochemistry (IHC). Assays to detect mutations associated with drug resistance were performed on MTBC cases. RESULTS: A Mycobacterium species was detected in 465 (50%) cases by PCR and sequencing. Of these, 380 (82%) were positive by Mycobacterium PCR targeting 16S rRNA. 85 cases (18%), including 9 MTBC, 12 M. avium complex and 3 M. leprae, were positive by other PCRs. Co-infection of MTBC and NTM spp. was detected in 5 cases. Of 465 PCR positive cases, 327 (70%) showed immunostaining and 223 (48%) were AFB-positive. Molecular markers for drug resistance were detected in 9 out of 88 (10%) tested MTBC cases. CONCLUSION: FFPE tissue analysis by multigene targeted PCR assays expands the opportunities for rapid identification of Mycobacterium species, allows differentiation of MTBC from NTM, and helps to detect co-infections. Using multigene targeted PCRs in combination with histopathology and IHC improve the accuracy of diagnosis, particularly in the presence of commensal and environmental pathogens. DISCLOSURES: All authors: No reported disclosures. Oxford University Press 2017-10-04 /pmc/articles/PMC5630882/ http://dx.doi.org/10.1093/ofid/ofx163.1639 Text en © The Author 2017. Published by Oxford University Press on behalf of Infectious Diseases Society of America. http://creativecommons.org/licenses/by-nc-nd/4.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Abstracts Bhatnagar, Julu DeLeon-Carnes, Marlene Rabeneck, Demi Rowlett, Veronica Hale, Gillian Martines, Roosecelis Muehlenbachs, Atis Gary, Joy Ritter, Jana M Shieh, Wun-Ju Zaki, Sherif Improved Detection and Accuracy of Mycobacterium Species Identification from Paraffin Embedded Tissues of Patients by Using Multigene Targeted PCR and Sequencing |
title | Improved Detection and Accuracy of Mycobacterium Species Identification from Paraffin Embedded Tissues of Patients by Using Multigene Targeted PCR and Sequencing |
title_full | Improved Detection and Accuracy of Mycobacterium Species Identification from Paraffin Embedded Tissues of Patients by Using Multigene Targeted PCR and Sequencing |
title_fullStr | Improved Detection and Accuracy of Mycobacterium Species Identification from Paraffin Embedded Tissues of Patients by Using Multigene Targeted PCR and Sequencing |
title_full_unstemmed | Improved Detection and Accuracy of Mycobacterium Species Identification from Paraffin Embedded Tissues of Patients by Using Multigene Targeted PCR and Sequencing |
title_short | Improved Detection and Accuracy of Mycobacterium Species Identification from Paraffin Embedded Tissues of Patients by Using Multigene Targeted PCR and Sequencing |
title_sort | improved detection and accuracy of mycobacterium species identification from paraffin embedded tissues of patients by using multigene targeted pcr and sequencing |
topic | Abstracts |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630882/ http://dx.doi.org/10.1093/ofid/ofx163.1639 |
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