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Detection of Mycobacteria and Propionibacterium Acnes 16s rRNA in Sarcoidosis Tissue Reveals Genetic Ambiguity Across Species
Description
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A4818 - Detection of Mycobacteria and Propionibacterium Acnes 16s rRNA in Sarcoidosis Tissue Reveals Genetic Ambiguity Across Species
Author Block: O. S. Chioma1, W. P. Drake2; 1Infectious Diseases, Vanderbilt University Medical Center, Nashville, TN, United States, 2Pathology, Microbiology, and Immunology, Vanderbilt Univ, Nashville, TN, United States.
RATIONALE
Sarcoidosis is an inflammatory disease characterized by the presence of non-caseating granulomata in the affected organs, particularly the lungs and lymph nodes. While the etiology of sarcoidosis is unknown, several laboratories have presented evidence that implicates infectious agents particularly Mycobacteria and Propionibacterium acnes as key players in sarcoidosis. These organisms have been detected by several methods from sarcoidosis tissues including polymerase chain reaction (PCR) of the 16s rRNA gene. In this study we hypothesize that the PCR amplification of the 16s rRNA gene is not a thorough method of identification.
METHODS
20 lung samples were analyzed, 10 samples from sarcoidosis patients and 10 samples from normal lungs for the detection of Mycobacteria and P. acnes using primers specific for 16srRNA gene of both organisms. The PCR product was sequenced to properly identity the organisms. Primers specific for Mycobacteria were used to amplify all 20 samples.
RESULTS
Mycobacteria were identified in 90% of the sarcoidosis tissues and absent in the normal lung tissues. Using primers specific for P. acnes to amplify all 20 samples, P. acnes was identified in 60% of the sarcoidosis tissues and 100% of the normal lung tissue. Notably, using Mycobacterium tuberculosis (MTB) H37RV Deoxyribonucleic acid (DNA) as MTB control DNA, to amplify P. acnes specific primers for 16s rRNA gene resulted in sequence product corresponding to MTB 16s rRNA gene. Conversely, using P. acnes ATCC 11828 DNA as control DNA to amplify MTB specific primers for 16s rRNA gene resulted in sequence product corresponding to P. acnes 16s rRNA gene as confirmed by agarose gel electrophoresis and DNA sequencing suggesting that the primers designed are not exclusive to P. acnes or Mycobacteria as both organisms belong to the same order of classification. Multiple sequence alignment to compare the 16s rRNA gene of P. acnes ATCC 11828 and M. tuberculosis H37Rv, revealed that both organisms are 87.2% homologous, hence primers designed to differentiate between the two organism may overlap and cause ambiguity in the results.
CONCLUSIONS
Taken together this investigation reveals the importance of sequencing to correctly identify Mycobacteria or P. acnes DNA in sarcoidosis specimens. Dependence on identification by PCR amplification of the 16s rRNA gene alone using DNA from sarcoidosis granulomas is not a thorough method of identification. Consideration of other consecutively expressed genes is necessary to correctly allocate genus and species within sarcoid specimens.
Author Block: O. S. Chioma1, W. P. Drake2; 1Infectious Diseases, Vanderbilt University Medical Center, Nashville, TN, United States, 2Pathology, Microbiology, and Immunology, Vanderbilt Univ, Nashville, TN, United States.
RATIONALE
Sarcoidosis is an inflammatory disease characterized by the presence of non-caseating granulomata in the affected organs, particularly the lungs and lymph nodes. While the etiology of sarcoidosis is unknown, several laboratories have presented evidence that implicates infectious agents particularly Mycobacteria and Propionibacterium acnes as key players in sarcoidosis. These organisms have been detected by several methods from sarcoidosis tissues including polymerase chain reaction (PCR) of the 16s rRNA gene. In this study we hypothesize that the PCR amplification of the 16s rRNA gene is not a thorough method of identification.
METHODS
20 lung samples were analyzed, 10 samples from sarcoidosis patients and 10 samples from normal lungs for the detection of Mycobacteria and P. acnes using primers specific for 16srRNA gene of both organisms. The PCR product was sequenced to properly identity the organisms. Primers specific for Mycobacteria were used to amplify all 20 samples.
RESULTS
Mycobacteria were identified in 90% of the sarcoidosis tissues and absent in the normal lung tissues. Using primers specific for P. acnes to amplify all 20 samples, P. acnes was identified in 60% of the sarcoidosis tissues and 100% of the normal lung tissue. Notably, using Mycobacterium tuberculosis (MTB) H37RV Deoxyribonucleic acid (DNA) as MTB control DNA, to amplify P. acnes specific primers for 16s rRNA gene resulted in sequence product corresponding to MTB 16s rRNA gene. Conversely, using P. acnes ATCC 11828 DNA as control DNA to amplify MTB specific primers for 16s rRNA gene resulted in sequence product corresponding to P. acnes 16s rRNA gene as confirmed by agarose gel electrophoresis and DNA sequencing suggesting that the primers designed are not exclusive to P. acnes or Mycobacteria as both organisms belong to the same order of classification. Multiple sequence alignment to compare the 16s rRNA gene of P. acnes ATCC 11828 and M. tuberculosis H37Rv, revealed that both organisms are 87.2% homologous, hence primers designed to differentiate between the two organism may overlap and cause ambiguity in the results.
CONCLUSIONS
Taken together this investigation reveals the importance of sequencing to correctly identify Mycobacteria or P. acnes DNA in sarcoidosis specimens. Dependence on identification by PCR amplification of the 16s rRNA gene alone using DNA from sarcoidosis granulomas is not a thorough method of identification. Consideration of other consecutively expressed genes is necessary to correctly allocate genus and species within sarcoid specimens.
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