Elsevier

The Lancet

Volume 354, Issue 9173, 10 July 1999, Pages 120-123
The Lancet

Early Report
Quantitative PCR of mycobacterial and propionibacterial DNA in lymph nodes of Japanese patients with sarcoidosis

https://doi.org/10.1016/S0140-6736(98)12310-3Get rights and content

Summary

Background

The causes of sarcoidosis are not known. The DNA of Mycobacterium tuberculosis has been detected in some sarcoid lesions. In Japan, Propionibacterium acnes has been isolated from such lesions, but whether this indigenous bacterium is related to the disease is unclear. We used PCR to estimate the number of genomes of these bacteria in sarcoid lesions, to identify any link between sarcoidosis and these two bacterial species.

Methods

We examined formalin-fixed and paraffin-embedded sections of biopsy and surgical samples from lymph nodes of 15 patients with sarcoidosis, 15 patients with tuberculosis, and 15 patients with gastric cancer (controls). Quantitative PCR was done to amplify segments of 16 S ribosomal RNA of P acnes and P granulosum and of insertion sequence 6110 of M tuberculosis. PCR products were identified and the quantities of the products were estimated in terms of the fluorescence of oligonucleotide reporter probes. The numbers of bacterial genomes in samples were estimated from standard curves of serially diluted bacterial DNA.

Findings

Genomes of M tuberculosis were found in samples from all 15 patients with tuberculosis, from three patients with sarcoidosis, and in one control sample. Genomes of P acnes were found in 12 of the 15 patients with sarcoidosis, in two tuberculosis patients, and three controls. The difference in the estimated number of P acnes genomes between individuals with and without sarcoidosis was similar to that in the number of M tuberculosis between people with and without tuberculosis. There were 5×105 P acnes genomes in sarcoidosis and 3×106 M tuberculosis genomes in tuberculosis, respectively, on average per μg of total DNA. The three patients with sarcoidosis but without P acnes all had P granulosum DNA in their biopsy samples; the number of genomes of the bacterium was 5×105.

Interpretation

These findings suggest that propionibacteria had resided or proliferated ectopically in the sarcoid lesions, whether there was a connection with the disease or not. Propionibacteria are a more likely cause than mycobacteria of sarcoidosis.

Introduction

Sarcoidosis is a systemic granulomatous disease, the cause of which is unknown. There is some evidence that the epithelioid-cell granuloma formation of sarcoidosis is an immune response to an, as yet, unidentified agent.1 Clinical, histological, and immunological features common to sarcoidosis and tuberculosis suggest that mycobacteria may cause sarcoidosis, but a mycobacterium has been isolated from sarcoid lesions of only one patient to date.2 When PCR was used to search for mycobacterial DNA in tissue samples from patients with sarcoidosis, some investigators detected mycobacteria3, 4 and others did not.5, 6

About 15 years ago, Abe and colleagues7 reported that Propionibacterium acnes was the only bacterium isolated from biopsy samples of 31 (78%) of 40 lymph nodes from 40 patients with sarcoidosis. Indeed, P acnes is a strong adjuvant, causing granulomas when injected experimentally into sensitised rats.8 However, these findings were not conclusive, because this anaerobic bacterium is commonly isolated from healthy human skin and was isolated from lymph nodes of 38 (21%) of 180 patients with diseases other than sarcoidosis in that study.

We used quantitative PCR to search for bacterial genomes of P acnes and Mycobacteria tuberculosis in histological sections of lymph nodes from patients with sarcoidosis, tuberculosis, or gastric cancer.

Section snippets

Tissue samples

We examined samples obtained by biopsy of lymph nodes from 15 patients with sarcoidosis (11 scalene, two inguinal, one cervical, and one superclavicular lymph node) and from 15 patients with tuberculous lymphadenitis (ten cervical, three axillary, one superclavicular, and one pancreatic lymph node). As controls, we examined 15 lymph nodes without metastasis from 15 patients with gastric cancer undergoing surgery. Tissue blocks fixed with 10% neutral buffered formalin and embedded in paraffin

Results

With the primers and probe for P acnes, P avidum gave a single band in the same place as that of P acnes, and a quantitative PCR value as high as that of P acnes (table 1). However, after digestion with either of the restriction enzymes, P avidum gave two bands and P acnes gave one band. With the primers and probes for M tuberculosis and P granulosum, only the bacterium in question gave a band (single), and the quantitative PCR values were more than 20 times those of other bacteria.

M

Discussion

In tuberculosis, granulomas form in reaction to a well-known causative bacterium. Quantitative PCR showed many M tuberculosis genomes in all samples from patients with tuberculosis, and a few such genomes in samples from some patients without tuberculosis.

Results of earlier studies that used PCR for the detection of mycobacterial DNA in sarcoidosis were inconsistent. The method was not quantitative, and because of its sensitivity, small amounts of DNA probably unrelated to the disease-causing

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