Patients and sample preparation

In March 2009, 497 HIV-1-infected men at the Jiangsu Centers for Diseases Prevention and Control were recruited for this study. Written or verbal informed consent was obtained from each individual or their legal representatives before study enrolment. The HIV infection was confirmed by Western Blot test at Jiangsu Centers for Diseases Prevention and Control.

For each subject, 3 ml of blood was drawn into a 5-ml collection tube containing 5% EDTA. At the same time, the first-void urine (FVU) specimens (15–30 ml) were collected in sterile containers and then transferred to a sterile plastic centrifuge tube. All the specimens were transported to the laboratory within 4 h. The FVU specimens were centrifuged at 3500 g for 15 min. The pellet was then suspended in 1 ml 0·9% sodium chloride solution and equated to two parts, one was immediately used to culture U. urealyticum and M. hominis, the other was stored at −70°C for subsequent PCR detection.

CD4⁺ T cell counts

Absolute CD4⁺ T cell counts were determined in EDTA-treated peripheral blood samples using an Ortho Cytoron Absolute flow cytometer (Ortho Diagnostic Systems Inc., USA) and anti-CD4-FITC monoclonal antibodies (Ortho Diagnostic System Inc.), according to the manufacturer's instructions. Three-colour flow cytometry was also performed for supplemental CD4⁺ T lymphocyte subset identification. After staining of whole blood with monoclonal antibodies, samples were lysed with FACS lysing solution (Becton Dickinson, USA). Ten thousand lymphocytes were analysed with the FACSCalibur (Becton Dickinson) system. The absolute counts of the above-mentioned CD4⁺ T cells were obtained using the TRUCount system (Becton Dickinson).

Culture of M. hominis and U. urealyticum

Detection of M. hominis and U. urealyticum was performed by culture according to the manufacturer's instructions (Identifying Culture Medium, Liming Co., China).

Detection of M. genitalium, M. fermentans, M. pirum, and M. penetrans by PCR DNA extraction

The saline suspensions described above were used for DNA extraction. Briefly, 0·5 ml of the suspension was centrifuged at 15 000 g for 15 min at 4°C. The pellet was washed three times with PBS at pH 7·3 and then centrifuged again as described above. The sediment obtained was re-suspended in 100 μl TE buffer [10 mm Tris–HCl (pH 8·0), and 1 mm EDTA], and then lysed by the addition of 1% sodium dodecyl sulfate (SDS). The mixture was then incubated with 40 μl proteinase K (Boehringer, Germany) at 55°C for 4 h. The samples were subsequently heated for 10 min at 95°C to inactivate the proteinase K. The DNA extracted was quantified by the use of a nucleic acid fixed-quantity machine at 260 wavelengths. The samples were then frozen and stored at −20°C. For PCR, 5 μl of the mixture was used.

PCR assays of M. pirum, M. genitalium, and M. penetrans

A nested PCR was designed for the amplication of M. pirum, M. genitalium, and M. penetrans. In order to improve the sensitivity and the specificity, the primers were designed against the conserved region of 16S rRNA. A two-step procedure was used, first to amplify the general genus and then to amplify the specific species. The primers are listed in Table 1.

Table 1.

Primers used in this study

PCR amplification was performed at a final volume of 50 μl. PCR buffer (10 mm Tris–HCl (pH 8·3), 50 mm KCl, 0·1% Triton X-100) containing 1·5 mm MgCl₂, 200 μm of each dNTP (Shanghai Shenergy Biocolor, China), 0·5 μm of each primer (Invitrogen Life Technologies, China), and 1 U Taq polymerase (Shanghai Shenergy Biocolor). Five microlitres of each sample were added to a final volume of 50 μl per reaction tube. The samples were heated at 95°C for 2 min. This was followed by 35 cycles of denaturation at 93°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min and a final cycle of 5 min at 72°C. Three microlitres of the first PCR reaction product were used for the second-step PCR. The second-step PCR conditions were the same as for the first step.

PCR assays of M. fermentans

The primers were designed against the insert sequence (IS) of M. fermentans (Table 1). The reaction mixture was the same as the nested PCR described above, except that the MgCl₂ concentration was 2·0 μm. The samples were heated at 95°C for 2·5 min, followed by 45 cycles of denaturation at 94°C for 35 s, annealing at 55°C for 45 s, extension at 72°C for 50 s and a final cycle of 5 min at 72°C. The products were incubated at 4°C until electrophoresis.

Detection of DNA products

Ten microlitres of the second-step PCR product and 15 μl of the 16S rRNA PCR product were electrophoresed in a 2·0% agarose gel for 45–60 min at 75 V in TAE buffer (pH 8·0). The gel was stained with ethidium bromide and the products were visualized with a UV transilluminator and photographed (UVP GDS-8000). Controls consisted of known mycoplasmas and the DNA markers were provided by Tiangen Company (China).

Rigorous precautions were taken to avoid contamination of the PCR mixture, including reagent aliquoting, the use of positive displacement pipettes, the isolation of PCR reagents and products, and the physical separation of the DNA extraction site and Mycoplasma culture.

Every step of the experiments included both negative and positive controls. Type strains of U. urealyticum (ATCC 27618), M. penetrans (ATCC 55252), M. hominis (ATCC 23114), M. fermentans (incog.), M. genitalium G37 (ATCC 33530), and M. pirum (ATCC 25960), were kindly provided by Statens Seruminstitut (Denmark).

Statistical analysis

Statistical analysis was performed using SAS 9.0 software (SAS Institute Inc., USA). The prevalence of the mycoplasmas is presented as a percentage. Categorization variables were analySed using χ² test. Continuous variables were analysed by the U test. All reported P values were two-tailed, and a P value <0·05 was considered significant.