Quantitative detection and phylogeny-based identification of mycoplasmas and ureaplasmas from human immunodeficiency virus type 1-positive patients.

More than 20 years ago, Mycoplasma genitalium was first isolated from men with nongonococcal urethritis (NGU). Some strains of Mycoplasma and Ureaplasma are associated with NGU or other genitourinary infections. It is reasonable to suggest that mycoplasmas and ureaplasmas may increase the development of acquired immunodeficiency syndrome in human immunodeficiency virus type 1 (HIV-1)-infected patients. This study investigated a rapid and reliable diagnostic method, based on a LightCycler SYBER green I real-time PCR assay and phylogenetic analysis, for detecting mycoplasmas and ureaplasmas in urine from HIV-1-infected patients. Primers were designed to amplify a 711-bp fragment of the 16S rRNA gene. Positive PCR-amplified products were further sequenced, and a phylogenetic tree was constructed by neighbor joining of the V4 and V5 sequences from 15 prototypes strains. PCR-positive samples were detected in 17 (9.1%) urine samples, their prototypes being as follows: 2/17 (11.2%) M. genitalium, 1/17 (5.9%) M. hominis, 12/17 (70.6%) Ureaplasma urealyticum, 1/17 (5.9%) mixed M. genitalium and M. hominis, and 1/17 (5.9%) M. hominis and U. urealyticum, respectively. Mycoplasma and Ureaplasma load for each specimen was 3.8 x 10 copies/ml to 7.2 x 10(3) copies/ml. Overall, this study demonstrates that real-time PCR and phylogenetic analysis are the fastest available methods for the detection of mycoplasmas in the urogenital tract.