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The role of Mycoplasma genitalium and Ureaplasma urealyticum in acute nongonococcal urethritis (NGU) in humans is not yet established (6). The pathogenetic role of M. genitalium has been suggested because it is isolated with remarkable frequency from the urogenital tract of patients with NGU (3, 14, 17). Similarly, U. urealyticum is considered a pathogen of the urogenital tract because its isolation is more prevalent in patients with NGU than in asymptomatic subjects (9). However, the presence of M. genitalium (6, 7, 13, 18) and U. urealyticum (1, 12) in the urethras of healthy volunteers, who show no signs or symptoms of a urinary tract infection, urethritis, or prostatitis during the investigation, contradicts this hypothesis.

Human immunodeficiency virus type 1 (HIV-1)-infected patients are characterized by alterations in their T-cell immune functions long before the number of circulating CD4⁺ lymphocytes starts to decline (4). In the more advanced stages of the disease, these alterations are clinically reflected in an increased frequency of opportunistic infections (5, 10, 11).

The aim of the present study was to investigate the pathogenetic roles of M. genitalium and U. urealyticum at the urethral level by studying the presence of both microorganisms in urethral swabs taken from asymptomatic HIV-1-infected male patients and male patients with full-blown AIDS; neither group had symptoms of acute urethritis. The data show that M. genitalium and U. urealyticum can be detected in a larger proportion of AIDS patients and asymptomatic HIV-1-infected patients than in the other groups of subjects studied.

Patients. One hundred eighty-seven male heterosexual HIV-1-infected patients (all intravenous-drug abusers) were enrolled at the Infectious Diseases Unit of the Hospital of Lovere, Lovere, Italy. The ages of the HIV-1-positive patients ranged from 18 to 40 years (mean age, 28 years). The clinical status of the HIV-1-infected patients was defined according to the 1993 classification system of the Centers for Disease Control and Prevention (2): 115 were asymptomatic HIV-1-positive patients without AIDS (referred to herein as non-AIDS patients) (stage A1), whereas 72 patients had full-blown AIDS (stage C3) (Table 1). The control group consisted of 114 healthy male volunteers who either were attending the Institute of Microbiology for routine checkups or were recruited from the hospital staff. They ranged in age from 20 to 41 years (mean age, 27 years). Both the HIV-1-infected and the control groups consisted of sexually active Italian subjects. The samples from healthy subjects and from HIV-1-infected patients were completely randomized during their collection from 1995 to 1998. The Ethical Committee guidelines were followed throughout this study. Both patients and healthy subjects were evaluated by a standard protocol that included a sexual history, a genital examination performed by clinicians who confirmed the absence of inflammatory processes and pathological lesions, and two urethral swab specimens for the detection of M. genitalium and U. urealyticum. All samples were examined by microscopy to exclude the presence of other pathogenetic microorganisms, spores, or leukocytes.

U. urealyticum growth conditions. Urethral swabs were stirred into 2 ml of mycoplasma-carrying broth and tested for U. urealyticum by two complementary methods, conventional culture (Mycotrim GU; Irvine Scientific, Santa Ana, Calif.) and culture by biochemical testing (MYCOFAST; DID, Milan, Italy) (12). In the conventional culture method, each Mycotrim GU flask contained agar and broth specially formulated to support the growth of U. urealyticum; positive cultures raised the pH, caused a color change in the media, and were thus identified by low-power microscopic examination of the agar surface.

M. genitalium growth conditions. Urethral swabs were stirred into 2 ml of mycoplasma transport medium (M.S. U.M.M.; International Mycoplasma, Signes, France). One milliliter of M.S. U.M.M. culture medium was added to 5 ml of SP-4 medium for M. genitalium isolation (17).

M. genitalium DNA detection. One milliliter of M.S. U.M.M. culture medium was centrifuged at 15,000 × g for 15 min at 4°C. The pellet obtained by centrifugation was resuspended in 200 µl of TE buffer (10 mM Tris hydrochloride, pH 8.0, and 1 mM EDTA), lysed by the addition of 1% sodium dodecyl sulfate (SDS), and incubated with 50 µl of proteinase K (Boehringer, Mannheim, Germany) for 4 h at 55°C. DNA was extracted twice with an equal volume of phenol saturated with TE buffer and once with chloroform-isoamyl alcohol (96:4, vol/vol). After extraction and precipitation with 3 M sodium acetate and 95% ethanol, the pellets were resuspended in 40 µl of TE buffer. M. genitalium ATCC 33530 was grown in SP-4 broth medium, collected by centrifugation for 15 min at 8,000 × g, and stored at 80°C until DNA was extracted as described above.

Results. U. urealyticum was found in the genital tracts of 23 (12.3%) HIV-1-infected patients and 8 (7.0%) healthy individuals; the number of positive specimens in non-AIDS patients totalled 10 (8.5%). Compared to healthy subjects and non-AIDS patients, a dramatic increase in the frequency of U. urealyticum colonization was observed in AIDS patients, with 13 (18%) samples testing positive (Table 2). The increase of U. urealyticum isolation between healthy subjects and AIDS patients and between non-AIDS patients and AIDS patients was statistically significant as assessed by Student's t test (P < 0.05). No statistically significant difference was observed between healthy individuals and non-AIDS patients. There was no qualitative difference in the results obtained with the two culture methods for U. urealyticum detection. All positive cultures, regardless of their patient group source, grew >10⁴ CFU/ml after 24 h of incubation.

Discussion. M. genitalium and U. urealyticum are normally harbored in the urethral tracts of healthy individuals (1, 2, 6, 7, 12, 13, 18). In our study, we found that 7.0% of our samples were positive for both of these microorganisms. In non-AIDS patients, the frequency of M. genitalium and U. urealyticum colonization was comparable to that observed in healthy subjects. In contrast, the frequency of M. genitalium and U. urealyticum isolation increased in AIDS patients. The relative occurrences of these two microorganisms may be explained by two nonexclusive phenomena. On the one hand, such data could be the result of defective humoral and cell-mediated immunity typical of AIDS patients. On the other hand, it cannot be ruled out that colonization by antibiotic-resistant M. genitalium and U. urealyticum strains in the urethral tracts of AIDS patients may be linked to alterations in the urogenital environment following antibiotic therapy, which is so extensively used with these individuals. Indeed, differences in the frequency of M. genitalium and U. urealyticum isolation in AIDS patients versus non-AIDS patients can be ascribed to the differences in the use of antibiotics in the two groups of patients; in non-AIDS patients, an expanded-spectrum antibiotic (usually sulfamethoxazole plus trimethoprim) was commonly used for long-term prophylaxis, whereas AIDS patients received broad-spectrum prophylaxis. This hypothesis is also supported by our recent study of U. urealyticum resistance to different antibiotics, including quinolones and tetracyclines, in AIDS patients (12).