DIRECT PCR ENABLES DETECTION OF MYCOPLASMA-PNEUMONIAE IN PATIENTS WITH RESPIRATORY-TRACT INFECTIONS

The sensitivities of three methods of detection of Mycoplasma pneumoni ae by a 16S rDNA PCR were compared by using a serial dilution of M. pn eumoniae. These methods consisted of a PCR performed directly on cells after a proteinase K pretreatment (direct PCR), a PCR after purificat ion of nucleic acids (DNA-PCR), and a PCR with rRNA sequences as the t arget after reverse transcription. The direct PCR and the reverse tran scription PCR had a sensitivity of 1.5 CFU (almost-equal-to 250 genome s). By purification, a 10-fold loss of target DNA occurred, as shown b y a 10-fold decrease in sensitivity (15 CFU) of the DNA-PCR. The prese nce of an excess of human background DNA did not influence the sensiti vity of either PCR. The direct PCR was evaluated on samples from patie nts with respiratory complaints. Direct PCR amplification was possible in 94.9% of the samples, which were tested by amplification of a 326- bp fragment of the beta-globin gene, which was performed to test for t he suitability of amplification. Nucleic acid purification was perform ed on the beta-globin-negative samples, after which only 2% remained n egative. A positive correlation between the direct M. pneumoniae PCR a nd serology, as tested by the microparticle agglutination assay (MAG a ssay), was found in 88.1% of the cases. A positive MAG assay result wa s found for samples from 10 (17%) of the patients; samples from 6 (10. 2%) of these patients were also positive by PCR. Samples from three pa tients were found to be positive by the M. pneumoniae PCR and negative by the MAG assay. Persistence of M. pneumoniae, as detected by PCR, w as observed in three patients. These results indicate that the direct PCR with 16S rDNA could prove to be useful in the detection of M. pneu moniae in respiratory tract samples, although more studies are needed to evaluate the correlation between clinical symptoms and a positive t est result.