Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis

Abstract

The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.

FIG. 1.

Standard curves of real-time (TaqMan) PCR for bacterial and human DNA. Eight samples of a given amount of bacterial DNA (from 5 × 10⁶ to 5 × 10⁻² genomes per PCR) or human DNA (from 5 × 10⁵ to 5 × 10⁻² genome equivalents per PCR) were amplified by 50 cycles of TaqMan PCR. The cycle threshold for each sample was calculated. Some datum points overlap. Abbreviations: N, negative control; n, exponent of 10.

FIG. 2.

TaqMan measurement of bacterial DNAs from lymph nodes of patients with sarcoidosis or tuberculosis or from control lymph nodes. The y axis shows the numbers of bacterial genomes in 500 ng of total tissue DNA extracted from samples collected at different institutes. Some datum points overlap. Groups were compared as follows (by the Mann-Whitney U test): P. acnes and M. tuberculosis, P < 0.001 in all institutes; P. granulosum and M. tuberculosis, P < 0.001 in all institutes; P. acnes, sarcoidosis, and tuberculosis, P = 0.0040 in Tokyo, P = 0.0029 in Germany, and P < 0.001 in Kumamoto, Italy, and England; P. acnes, sarcoidosis, and control, P = 0.0033 in Tokyo, P = 0.0090 in Germany, P = 0.0013 in England, and P < 0.001 in Kumamoto and Italy; P. granulosum, sarcoidosis, and tuberculosis, P = 0.23 in Germany, P = 0.085 in England, and P < 0.001 in Tokyo, Kumamoto, and Italy; P. granulosum, sarcoidosis, and control, P = 0.17 in Germany, P = 0.017 in England, and P < 0.001 in Tokyo, Kumamoto, and Italy. Abbreviations: PA, P. acnes; PG, P. granulosum; TB, M. tuberculosis; EC, E. coli.