Development of a quantitative real-time PCR assay for detection of Mycoplasma genitalium

Mycoplasma genitalium is known to cause nonchlamydial, nongonococcal urethritis in men and to be associated with pelvic inflammatory disease in women. Specific and sensitive PCR methods are needed for diagnosis of this bacterium because it is very difficult to culture from patient samples. To determine the bacterial load in patients' specimens, a quantitative real-time LightCycler PCR was developed. The housekeeping gene gap encoding glyceraldehyde-3-phosphate dehydrogenase was chosen as the target gene. The assay could consistently detect five genome copies per reaction. To evaluate the PCR, we tested 246 selected urethral swab samples from men attending a clinic for sexually transmitted diseases. Eighty-two of the samples were found positive for M. genitalium by a conventional 16S rRNA gene PCR assay, whereas 164 samples were randomly chosen among those tested negative. Of the positive samples, 78 (95.1%) were found positive, whereas 6 (3.7%) of the negatives were found positive by the LightCycler assay. The patient samples were also tested with a quantitative TaqMan assay, and the bacterial load was compared to the LightCycler results. A good linear correlation between the LightCycler and the TaqMan assays was found with a correlation coefficient of 0.89 and a slope of 0.99. Significantly more M. genitalium-positive men had urethritis, discharge, and dysuria than had M. genitalium-negative men. The M. genitalium DNA load in samples from patients with urethritis was significantly higher than in samples from those without (61 and 2.9 copies/microl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods.     

Longitudinal quantitative detection by real-time PCR of Mycoplasma genitalium in first-pass urine of men with recurrent nongonococcal urethritis

By using a TaqMan assay we monitored longitudinal changes in Mycoplasma genitalium loads in five men with recurrent M. genitalium-positive nongonococcal urethritis. We observed regrowth of M. genitalium persisting in hosts after treatment and a possible association of the increase in the M. genitalium load with emergence of symptoms and signs of nongonococcal urethritis in four of these patients.     

Quantitative detection of Mycoplasma genitalium from first-pass urine of men with urethritis and asymptomatic men by real-time PCR

We developed a TaqMan-based real-time PCR assay for quantifying Mycoplasma genitalium. This assay is able to specifically quantify concentrations of the M. genitalium 16S rRNA gene ranging from 10(7) to 10 copies/reaction. Using the TaqMan assay, we quantified the M. genitalium 16S rRNA gene in first-pass urine of men with urethritis and asymptomatic men who were positive for M. genitalium by PCR- and phylogeny-based assay. Of 130 men with gonococcal urethritis (GU), five were positive for M. genitalium. The mycoplasma load for each specimen was <5 x 10 copies/ml. Of 84 men with chlamydial non-GU (CNGU), seven were positive for M. genitalium. One man had an M. genitalium load of <5 x 10 copies/ml, and six men had loads ranging from 1.1 x 10(7) to 2.7 x 10(2) copies/ml. Of 86 men with nonchlamydial NGU (NCNGU), 17 were positive for M. genitalium. The mycoplasma loads for these men ranged from 3.3 x 10(6) to 2.3 x 10(2) copies/ml. Of 76 asymptomatic men, only two were positive for M. genitalium. For these men, the loads were 2 x 10(2) and <5 x 10 copies/ml. The patients with NGU had significantly higher concentrations of M. genitalium in their first-pass urine than did men with GU (P < 0.01) or asymptomatic men (P < 0.05). In addition, M. genitalium loads were significantly higher in men with NCNGU than those in asymptomatic men (P < 0.05). The quantitative assessment of M. genitalium loads by the TaqMan assay will provide useful information for understanding the pathogenicity of this mycoplasma in the urogenital tract.     

A 5' nuclease PCR (TaqMan) high-throughput assay for detection of the mecA gene in staphylococci

In an effort to find a rapid, efficient, and reliable method of screening large numbers of bacterial isolates for specific antimicrobial resistance genes, we compared conventional PCR results to the results generated using the TaqMan 5' nuclease PCR kit in conjunction with an ABI Prism 7700 Sequence Detector for detecting the mecA gene in various species of staphylococci. DNA was extracted using two techniques. The first used a high-salt extraction method suitable for conventional PCR but resulted in a 7.2% rate of PCR inhibition with the TaqMan technique. PCR inhibition could be overcome by diluting samples 1:5 prior to testing. The second method used the Qiagen QIAamp Tissue Kit; no instances of PCR inhibition were encountered with this method. A total of 197 (96%) of the 206 samples with no inhibition showed agreement between the two methods. Eight of the nine disagreements were likely the result of low-level DNA cross contamination caused by frequent specimen handling. Target DNA in all eight of these samples was first detected in the initial tests only after >30 PCR cycles, and all were negative upon repeat testing even after 40 PCR cycles using freshly extracted DNA. Among those positive samples in agreement, target DNA was invariably detected before 30 PCR cycles. The TaqMan assay eliminated the need to load, run, stain, and read agarose gels and provided the advantage of instant detection of PCR product by laser-activated fluorescence. Thus, final results were obtained 2 h after PCR was initiated, as opposed to a requirement of 2 days to examine 96 samples by agarose gel electrophoresis.     

Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis,Mycoplasma hominis,Ureaplasma urealyticum,and Mycoplasma genitalium in infertile women

PurposeSexually Transmitted Diseases (STDs) can cause sterility and many other problems for women planning pregnancy. Currently, almost 340 million people worldwide suffer from Sexually Transmitted Infections (STIs). This study made attempts to quickly identify STDs' most critical infectious agents using dedicated primers and probes.MethodsThe present study was done on the cervical samples of 200 infertile women. After extracting the total DNA of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium, quantitative methods were employed to determine the rate of target bacteria using multiplex real-time PCR.ResultsThe multiplex qPCR showed the rates of 47%, 16%, 46%, and 16.5% for Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium in infertile women, respectively. In some patients, there were co-infections with two or three bacteria. The diagnostic approach used in our research could be employed as an alternative detection tool to identify the four most common STD-associated bacterial agents while detecting mixed infections.ConclusionsInfertile women with no biological problems could have their genital tract checked using this newly designed identification technique and get proper treatment for their infections as quickly as possible.     

Development of a 5' fluorogenic nuclease-based real-time PCR assay for quantitative detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

A 5' nuclease TaqMan PCR was developed for the quantitative detection of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The relative numbers of bacteria were measured by the comparative threshold cycle method. This simplified method is a way of obtaining the relative quantities of these organisms from specimens and of monitoring the effect of therapy.     

Development of a real-time PCR assay for quantitative detection of Encephalitozoon intestinalis DNA

A new real-time PCR assay for quantitation of Encephalitozoon intestinalis DNA was developed which used a TaqMan fluorescent probe for specific detection. Serial dilutions of E. intestinalis spore suspensions obtained from tissue culture were used as external standards. The detection limit of the technique was 20 spores per ml, with a good interassay reproducibility (coefficient of variation of 7.1% for the suspension containing 20 spores/ml, 5.0% for the suspension containing 75 spores/ml and below 3.5% for higher concentrations). Quantitative detection of E. intestinalis DNA was similar whether the serial dilutions of spores were made in distilled water or in a stool suspension, allowing the use of the assay for stool specimens. The assay was then applied to 14 clinical specimens from 8 immunocompromised patients with proven E. intestinalis infection. The quantitation of the parasitic burden was achieved in stools, blood, urine, tissue biopsies, and bronchopulmonary specimens. The highest parasitic burdens were noted in stools, urine, and bronchopulmonary specimens, reaching 10(5) to 10(6) spores/g or ml. Dissemination of the infection was also evidenced in some patients by demonstration of E. intestinalis DNA in blood and serum. We conclude that real-time PCR is a valuable tool for quantitation of E. intestinalis burden in clinical specimens.     

Development of a 5' nuclease-based real-time PCR assay for quantitative detection of cariogenic dental pathogens Streptococcus mutans and Streptococcus sobrinus

A 5' nuclease TaqMan PCR assay was developed for the quantitative detection of the major cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. The absolute and relative numbers of bacteria were measured by this method. This assay will be useful for quantifying these organisms in oral specimens and for analyzing biofilm formation.     

Molecular detection of linezolid resistance in Enterococcus faecium and Enterococcus faecalis by use of 5' nuclease real-time PCR compared to a modified classical approach

A nucleotide transversion from guanine to uracil in the 23S rRNA confers linezolid resistance. We describe a real-time PCR using two Taqman probes that detects a single mutated allele among the genomes of Enterococcus faecium and Enterococcus faecalis. Results were confirmed by a classical approach involving LabChip technology assayed with an Agilent Bioanalyzer 2100.     

Use of a public sexually transmitted disease clinic by known HIV-positive adults: decreased self-reported risk behavior and increased disease incidence

High-risk sexual behavior by HIV-positive individuals is an important factor contributing to the spread of the HIV epidemic. We conducted a retrospective chart review to compare self-reported sexually transmitted disease (STD) risk behavior and clinic diagnoses of known HIV-positive clients attending Miami-Dade County STD clinics with those of uninfected controls. One hundred ninety-one HIV-positive clients and 191 HIV-negative controls, 130 (68.1%) men and 61 (31.9%) women, were included in the analysis. HIV-positive clients were more likely than controls to report no sexual activity in the last 2 months (odds ratio [OR] = 2.6, 95% confidence interval [CI]: 1.5-4.5) or, if active, to report condom use at last sexual intercourse (OR = 3.1, CI: 1.9-5.3). However, HIV-positive clients were more likely to be diagnosed with infectious syphilis (OR = 13.0, CI: 1.6-99.4) and/or gonorrhea (OR = 2.1, CI: 1.1-4.2) than controls. This may be a result of overreporting of condom use or sexual activity in high-risk sexual networks with inefficient use of condoms. Ongoing sexual risk behavior and access to HIV primary care are important issues in this population.     

Development of a real-time PCR targeting the yidC gene for the detection of Mycoplasma hominis and comparison with quantitative culture

Mycoplasma hominis is an opportunistic human mycoplasma species that can be either commensal or pathogenic. Its detection by culture is considered to comprise the reference technique. Previously reported PCR techniques target the 16S rRNA or the gap gene, although sequence variations among clinical isolates may lead to variations in clinical sensitivity. The present study aimed to develop a specific TaqMan quantitative real-time PCR assay, targeting a gene conserved in all M. hominis isolates, and to compare it with quantitative culture. With the knowledge of the M. hominis PG21 genome sequence, the yidC gene, encoding a membrane protein translocase, was chosen as target. Its intraspecies heterogeneity was checked at the nucleotide level using 31 reference or clinical strains. The limit of detection, the analytical specificity and the reproducibility of the assay were assessed. Moreover, PCR and culture results were compared using 153 urogenital specimens. The limit of detection was seven copies/μL. The analytical specificity was 100%, with good inter- and intra-assay reproducibility. Among the 153 urogenital specimens, the yidC PCR and culture allowed detection of 55 and 45 M. hominis-positive samples, respectively. Comparison of the bacterial load among the 45 specimens found to be M. hominis-positive by both techniques revealed a statistically significant association between the quantitative results obtained. In conclusion, we developed a specific, sensitive and reproducible real-time PCR to detect all M. hominis clinical isolates. This PCR was shown to have higher sensitivity than culture, although both methods were correlated for quantification of M. hominis loads in urogenital specimens.     

Sensitive and specific detection of proviral bovine leukemia virus by 5' Taq nuclease PCR using a 3' minor groove binder fluorogenic probe

Sensitive assays are required to detect proviral bovine leukemia virus (BLV) in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5' Taq nuclease assays using 3' minor groove binder DNA probes (TaqManMGB) were developed and compared to conventional PCR assays for sensitive detection of Australian BLV. Seven beef and dairy herds were screened using DNA prepared by a variety of protocols to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserted BLV sequences. Animals were also screened by the BLV standard agar-gel immunodiffusion test (AGID) and commercial enzyme linked immunosorbent assays (ELISA) for antibodies, and an ELISA for detecting viral antigens expressed (VAE) in lymphocyte cultures. The TaqMan MGB assay based on the pol region was the most sensitive and specific for the detection of BLV. This is the first report of a sensitive BLV 5' Taq nuclease assay.     

Comparison of a new quantitative ompA-based real-Time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays

Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10(-6) inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompA-based real-time protocol produced the most consistent positive results for all replicates tested down to 10(-6) IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n = 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10(8) particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n = 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.     

Development of a duplex real-time TaqMan PCR assay with an internal control for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from commercial and backyard poultry

In this study, we report the development and validation of a duplex real-time polymerase chain reaction (PCR) assay with an internal control using TaqMan-labelled probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae (duplex MGMS PCR). The MGMS PCR was highly specific with a sensitivity of 7 and 1 colony-forming units/ml for M. gallisepticum and M. synoviae, respectively, using dilution of pure culture that corresponds to 34 and 29 DNA copies per reaction. Validation of the assay was completed with 260 and 27 pooled samples (tracheal swabs) from commercial chickens and turkeys, respectively, with potential M. gallisepticum and M. synoviae involvement and 42 samples (palatine cleft swabs) from backyard geese and ducks. Using isolation as the gold standard, the MGMS PCR was more sensitive than isolation and the analytical sensitivity was 0.944 and 0.958 for M. gallisepticum and M. synoviae, respectively. In comparison with a gapA-based assay (gapA PCR) and a 16S rRNA-based assay (16S PCR) for M. gallisepticum and M. synoviae, respectively, the results agreed for 94.5% and 96.6%, respectively. The use of the internal control allowed monitoring of proper extraction and inhibition of amplification that was detected in 12 samples. The duplex MGMS PCR was shown to be superior to the presently reported real-time PCR assays in terms of combination of sensitivity, specificity and capacity of detection of more than one target in a single tube. In conclusion, the duplex MGMS PCR was highly specific, sensitive, and reproducible and could be used on clinical samples from commercial chickens, turkeys and backyard poultry including ducks and geese.     

Primary HIV infection education: knowledge and attitudes of HIV-negative men who have sex with men attending a public health sexually transmitted disease clinic

BACKGROUND: Although many individuals who acquire HIV are symptomatic, primary HIV infection (PHI) is infrequently diagnosed, even after the integration of RNA testing into HIV screening programs. Until more individuals with PHI seek evaluation, the public health impact of RNA testing is likely to be small. OBJECTIVE: To describe knowledge of PHI and attitudes toward health care-seeking behavior for symptoms consistent with PHI in men who have sex with men (MSM). METHODS: Between April 2004 and March 2005, HIV-negative MSM attending the Public Health-Seattle and King County sexually transmitted disease (STD) clinic completed an anonymous, self-administered, written questionnaire. RESULTS: Ninety-six (64%) of 150 subjects named > or =1 symptom associated with PHI. Only 18 (39%) of 46 men who knew PHI could resemble influenza would seek care for flu-like symptoms. Fifteen (65%) of 23 men reporting a week-long febrile illness with rash in the preceding year sought health care, but only 7 (30%) were tested for HIV. CONCLUSIONS: Although most subjects identified some symptoms of PHI, relatively few would seek care for such symptoms. MSM seeking attention for febrile illnesses were infrequently tested for HIV. Increased symptom recognition, health care-seeking behavior, and routine HIV RNA testing are needed if PHI screening programs are to have meaningful impact.     

Prevalence of antibodies to human immunodeficiency virus type 1 and condom use among outpatients at a sexually transmitted disease clinic in rome

To assess the prevalence of HIV-1 infection and study selected risk factors among patients attending a clinic for sexually transmitted diseases in Rome, 1442 outpatients seen consecutively between 20 February and 12 December 1989 were anonymously tested for anti-HIV-1. An evaluation of the trend of the HIV-1 infection was attempted by comparing the results of the present study with those obtained from a similar sample studied in 1986 in the same clinic. The overall estimated prevalence of anti-HIV-1 was 1.2 % among heterosexual non-drug user subjects and 16.1 % among homosexual or bisexual men. The anti-HIV-1 seropositivity was significantly higher in heterosexual subjects who reported sexual contact with intravenous drug users, as compared with those who did not report such exposure (12.5 % vs 0.8 %, p<0.005). Comparing the present data with those of a study conducted in 1986 in the same clinic, a lower prevalence of anti-HIV-1 was found among heterosexual subjects (1.2 % in 1989 vs 6.0 % in 1986, p<0.001). The availability after 1986 of several outpatient facilities attracting seropositive subjects and a change in the sexual behaviour of anti-HIV-1 positive subjects could explain this finding. Twenty percent of the heterosexual subjects and 62 % of the homosexual or bisexual men reported consistent use of condoms. In both heterosexual subjects and homosexual/bisexual men only the number of sexual partners in the previous year seemed to be related to the use of condoms, a higher proportion of subjects with two or more partners reporting the use of condoms, as compared with monogamous subjects (29.5 % vs 11.5 %, p<0.001 and 68.0 % vs 37.0 %, p<0.005). The infrequent use of condoms, in particular among heterosexual subjects, suggests that education campaigns conducted so far were partly effective.     

Use of the genomic subtractive hybridization technique to develop a real-time PCR assay for quantitative detection of Prevotella spp. in oral biofilm samples

Genomic subtractive hybridization was used to design Prevotella nigrescens-specific primers and TaqMan probes. Based on this technique, a TaqMan real-time PCR assay was developed for quantifying four oral black-pigmented Prevotella species. The combination of real-time PCR and genomic subtractive hybridization is useful for preparing species-specific primer-probe sets for closely related species.     

Overcoming barriers to HIV testing: preferences for new strategies among clients of a needle exchange,a sexually transmitted disease clinic,and sex venues for men who have sex with men

OBJECTIVE: To determine strategies to overcome barriers to HIV testing among persons at risk. METHODS: We developed a survey that elicited testing motivators, barriers, and preferences for new strategies among 460 participants at a needle exchange, three sex venues for men who have sex with men, and a sexually transmitted disease clinic. RESULTS: Barriers to testing included factors influenced by individual concern (fear and discrimination); by programs, policies, and laws (named reporting and inability to afford treatment); and by counseling and testing strategies (dislike of counseling, anxiety waiting for results, and venipuncture). The largest proportions of participants preferred rapid testing strategies, including clinic-based testing (27%) and home self-testing (20%); roughly equal proportions preferred oral fluid testing (18%), urine testing (17%), and standard blood testing (17%). One percent preferred home specimen collection. Participants who had never tested before were significantly more likely to prefer home self-testing compared with other strategies. Blacks were significantly more likely to prefer urine testing. CONCLUSIONS: Strategies for improving acceptance of HIV counseling and testing include information about access to anonymous testing and early treatment. Expanding options for rapid testing, urine testing, and home self-testing; providing alternatives to venipuncture; making pretest counseling optional; and allowing telephone results disclosure may encourage more persons to learn their HIV status.