Mycoplasma genitalium serum antibodies in infertile couples and fertile women.

OBJECTIVES: The association between Mycoplasma genitalium (M. genitalium) serum antibodies and infertility in women and men, as well as infertility subtypes, was investigated. METHODS: Stored serum was obtained from two patient cohorts: infertile couples (239 women and 243 men) attending a gynaecological outpatient clinic between October 1997 and February 2001 and 244 age-matched spontaneously pregnant women. An enzyme immunoassay was used to detect serum immunoglobulin G (IgG) antibodies to M. genitalium in these samples. Patient's Chlamydia trachomatis seropositivity had been previously determined. Risks were calculated using multivariate logistic regression. RESULTS: M. genitalium serum IgG was more common among women of infertile couples (5.4%) than among fertile controls (1.6%) (OR (95%CI) 3.45 (1.10 to 10.75)), adjusting for C. trachomatis IgG (adjusted OR=3.00 (0.95 to 9.47)). Of the women with tubal factor infertility (TFI) 9.1% had M. genitalium IgG compared with 4.6% of women without TFI (OR=2.07 (0.60 to 7.05)); (AOR=1.20 (0.32 to 74.40)). In patients IgG positive to both microorganisms the OR for having TFI was increased (OR=4.86 (1.22 to 19.36)) compared with those positive to C. trachomatis IgG only (AOR=3.14 (1.58 to 6.20)). No associations were found with other infertility diagnoses. Only two men of the infertile couples were M. genitalium IgG positive (0.8%). CONCLUSIONS: M. genitalium serum IgG was associated with infertility in women, however insignificant after adjustment for C. trachomatis IgG, but not with infertility subtypes within this study. M. genitalium IgG seroprevalence among men was very low and not associated with male factor infertility.

The new variant of Chlamydia trachomatis was present as early as 2003 in Orebro County, Sweden, but remained undetected until 2006.

OBJECTIVES: In 2006, a new variant of Chlamydia trachomatis (nvCT) was reported in Sweden. Because of a cryptic plasmid deletion, the nvCT was undetectable in several of the genetic diagnostic systems used worldwide at the time. This study aimed to evaluate whether the nvCT was present in specimens obtained from patients attending the outpatient sexually transmitted infection (STI) clinic at Örebro University Hospital, Örebro, Sweden already in 2002-2003. METHODS: In 2012, archival (-20 °C freezer) urogenital specimens (2002 (n=1083) and in 2003 (n=1143)) obtained from men (2002 (n=398) and 2003 (n=486)) and women (2002 (n=301) and 2003 (n=408)) were analysed with Cobas TaqMan CT test V.2.0. All C trachomatis positive specimens were subsequently examined using a duplex PCR assay that simultaneously detects the deletion on the nvCT cryptic plasmid and the ompA gene of C trachomatis genotype E. RESULTS: In total, 68 patients (9.7%) in 2002 and 61 (6.8%) in 2003 were C trachomatis positive. The duplex PCR assay identified 26 C trachomatis genotype E positive patients in 2002 (38%) and 25 in 2003 (41%). No nvCT was found in 2002, but one specimen obtained from a 23-year-old man in June 2003 was positive for the nvCT. CONCLUSIONS: The nvCT was present as early as 2003 in Örebro County, Sweden, which concurs with previously reported statistical estimations of its emergence. Accordingly, the nvCT spread undetected for at least 3 years, explaining the high proportion (38%) in Örebro County when it was first detected in late 2006.

Chlamydia trachomatis and Mycoplasma genitalium plasma antibodies in relation to epithelial ovarian tumors.

OBJECTIVE: To assess associations of Chlamydia trachomatis and Mycoplasma genitalium antibodies with epithelial ovarian tumors. METHODS: Plasma samples from 291 women, undergoing surgery due to suspected ovarian pathology, were analyzed with respect to C. trachomatis IgG and IgA, chlamydial Heat Shock Protein 60-1 (cHSP60-1) IgG and M. genitalium IgG antibodies. Women with borderline tumors (n=12), ovarian carcinoma (n=45), or other pelvic malignancies (n=11) were matched to four healthy controls each. RESULTS: Overall, there were no associations of antibodies with EOC. However, chlamydial HSP60-1 IgG antibodies were associated with type II ovarian cancer (P=.002) in women with plasma samples obtained >1 year prior to diagnosis (n=7). M. genitalium IgG antibodies were associated with borderline ovarian tumors (P=.01). CONCLUSION: Chlamydial HSP60-1 IgG and M. genitalium IgG antibodies are in this study associated with epithelial ovarian tumors in some subsets, which support the hypothesis linking upper-genital tract infections and ovarian tumor development.

Emergence of the new variant of Chlamydia trachomatis in a defined area of Sweden before 2002?

OBJECTIVES: To compare the proportions of Chlamydia trachomatis-positive specimens detected by Cobas Amplicor CT/NG (CA PCR) with C trachomatis positives in cell culture from 1999 to 2006 in order to estimate when the new variant of C trachomatis (nvCT) with a deletion in the cryptic plasmid (in the target region for CA PCR that resulted in false-negative results) emerged in Örebro County, Sweden. METHODS: The annual number of specimens analysed using CA PCR in 1999-2006 ranged from 5077 to 11,622 and using cell culture (McCoy cells) from 5201 to 7425. Logistic regression was applied to evaluate the change in the proportion of C trachomatis-positive tests over the years between the two methods. The statistical interaction effect of year and method was estimated using both unadjusted and adjusted (age, gender and clinic) models. RESULTS: From 2002, the proportion of C trachomatis-positive specimens identified using CA PCR decreased annually, whereas the proportion of culture-positive specimens increased annually. Logistic regression showed a statistically significant interaction effect between periods (1999-2006) and groups of specimens analysed using CA PCR or cell culture. A statistically significant association between the interaction of CA PCR/cell culture and period was observed in the unadjusted and adjusted models. CONCLUSION: This study indicates that in Orebro County, Sweden, nvCT was already present before 2002, that is, when the difference between the proportions of C trachomatis-positive specimens identified by CA PCR compared with cell culture-positive specimens began to show a statistically significant decline.

Characterisation of Chlamydia trachomatis by ompA sequencing and multilocus sequence typing in a Swedish county before and after identification of the new variant.

OBJECTIVES: In 2006 a new variant of Chlamydia trachomatis (nvCT), with a deletion in the cryptic plasmid, was reported in Sweden. This deletion included the targets for the genetic diagnostic systems used in many clinical laboratories and resulted in thousands of false-negative results. The aim of this study was to characterise consecutive Chlamydia tissue culture-positive samples from 2006 in Orebro County, after identification of the nvCT, and to compare the results from samples collected in the same county in 1999-2000. The study also aimed to evaluate the discriminatory capacity of multilocus sequence typing (MLST) compared with ompA sequencing. METHODS: ompA sequencing and MLST was used to characterise 100 consecutive Chlamydia tissue culture-positive samples. RESULTS: A significant (p<0.001) increase of genotype E, from 47% in 1999-2000 to 69% in 2006, was detected. All 41 nvCT isolates from 2006 displayed an identical ompA genotype E and MLST profile. Excluding the nvCT isolates, the distribution of ompA genotypes is similar to the genotyping results from 1999-2000. Among the wild-type genotype E isolates from 2006, 14 unique MLST sequence types were obtained from 26 isolates while they were identical in ompA genotyping. The discriminatory power (D) of C trachomatis strains in this material was 83.5% using the MLST system compared with 49.5% utilising ompA sequencing. CONCLUSION: In all, MLST enables improved studies of the molecular epidemiology of C trachomatis. All nvCT isolates from 2006 displayed an identical ompA genotype E and MLST profile, which strongly indicates a clonal spread of the nvCT.

Chlamydia trachomatis, Mycoplasma genitalium, Neisseria gonorrhoeae, human papillomavirus, and polyomavirus are not detectable in human tissue with epithelial ovarian cancer, borderline tumor, or benign conditions.

OBJECTIVE: We sought to analyze the presence of the microorganisms Chlamydia trachomatis, Mycoplasma genitalium, Neisseria gonorrhoeae, human papillomavirus (HPV), and the polyomaviruses BK virus (BKV) and JC virus (JCV) in ovarian tissues of women with ovarian carcinomas, borderline tumors, and benign conditions. STUDY DESIGN: Ovarian tissue, snap-frozen and stored at -80 degrees C, from 186 women with benign conditions, borderline tumors, and epithelial ovarian cancer, as well as tissue from the contralateral ovary of 126 of these women, were analyzed regarding presence of C trachomatis and N gonorrhoeae (transcription mediated amplification), M genitalium (real-time polymerase chain reaction [PCR]), HPV (PCR), and BKV and JCV (PCR). RESULTS: All the tissue samples studied were found negative for the microorganisms analyzed. CONCLUSION: C trachomatis, M genitalium, N gonorrhoeae, HPV, and the polyomaviruses BKV and JCV are not detectable in ovarian tissues either from women with benign conditions and borderline tumors or from women with ovarian cancer.

Real-time PCR of the 16S-rRNA gene in the diagnosis of neonatal bacteraemia.

OBJECTIVE: To evaluate a real-time PCR assay for the diagnosis of neonatal bacteraemia. PATIENTS AND METHODS: Two hundred ninety-five plasma samples from 288 newborns with suspected neonatal sepsis were collected prospectively for the purpose of polymerase chain reaction (PCR)-based bacterial detection. A real-time PCR targeting the bacterial gene for 16S-rRNA gene combined with four specific probes designed to detect Gram-negative bacteria, Staphylococcus aureus and coagulase-negative staphylococci (CoNS) was developed. All samples positive in the universal PCR were further sequenced for bacterial identification. RESULTS: When applied to a material from 50 patients with positive blood culture and 245 patients with negative blood culture, the universal PCR showed a sensitivity of 42% (28-57), a specificity of 95% (92-97), a positive predictive value of 64% (45-80), and a negative predictive value of 89% (84-92) (95% confidence intervals in brackets). CONCLUSION: A new real-time PCR technique was for the first time applied to a well-defined prospectively and consecutively enrolled material of newborns with suspected sepsis, combining the benefits of real-time PCR with specific probes and sequencing. The method managed to detect bacteraemia with high specificity even though the sensitivity was low. Factors causing the low sensitivity are identified and further strategies to develop the method are described.

A comparative study of three different PCR assays for detection of Mycoplasma genitalium in urogenital specimens from men and women.

The aim of this study was to compare conventional 16S rRNA gene PCR, real-time 16S rRNA gene PCR and real-time Mycoplasma genitalium adhesin protein (MgPa) gene PCR as detection methods for M. genitalium infection. The study also determined the prevalence of M. genitalium in male and female patients attending a sexually transmitted infections clinic in a rural area in the west of Sweden. First void urine (FVU) and/or urethral swabs were collected from 381 men, and FVU and/or cervical swabs and/or urethral swabs were collected from 298 women. A total of 213 specimens were used in the PCR comparative study: 98 consecutively sampled specimens from patients enrolled in the prevalence study, 36 consecutively sampled specimens from patients with symptoms of urethritis and 79 specimens from patients positive for M. genitalium by real-time MgPa gene PCR in the prevalence study. A true-positive M. genitalium DNA specimen was defined as either a specimen positive in any two PCR assays or a specimen whose PCR product was verified by DNA sequencing. The prevalence of M. genitalium infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %), respectively. In the PCR comparative study, M. genitalium DNA was detected in 61/76 (80.3 %) of true-positive specimens by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Real-time MgPa gene PCR thus had higher sensitivity compared with conventional 16S rRNA gene PCR and had considerably increased sensitivity compared with real-time 16S rRNA gene PCR for detection of M. genitalium DNA. Real-time MgPa gene PCR is well suited for the clinical diagnosis of M. genitalium.

A serological study of the role of Mycoplasma genitalium in pelvic inflammatory disease and ectopic pregnancy.

OBJECTIVES: Establishing a connection between the emerging urogenital tract pathogen Mycoplasma genitalium and upper genital tract infection in women would be of major importance. The aim of this study was to evaluate the association between M genitalium antibodies and pelvic inflammatory disease (PID) and ectopic pregnancy (EP) using a lipid-associated membrane protein-enzyme immunoassay (LAMP-EIA) method. METHODS: The LAMP-EIA was used to analyse sera obtained from patients with clinical PID and EP collected in Sweden between February 1984 and April 1986. Sera from healthy pregnant women (Ctrl) collected during approximately the same period were used as controls. Evidence of chlamydial infection was investigated using a commercial anti-Chlamydia trachomatis EIA assay. RESULTS: The LAMP-EIA was specific as determined by a lack of cross-reactivity with other Mycoplasma species. The LAMP-EIA showed that 17% (33/193) of the PID patients were M genitalium positive as compared to 18% (15/82) of the EP patients and 15% (36/246) of the Ctrl women. No significant association could be demonstrated between M genitalium antibodies and PID or EP in crude or adjusted logistic regression. Antibodies against C trachomatis were demonstrated in 54% of the PID and 57% of the EP patients, and also in 37% of the Ctrl women, showing a statistically significant association. CONCLUSION: No statistically significant association between PID or EP and M genitalium antibodies could be found using the LAMP-EIA, although a slight tendency toward association was found when focusing on younger individuals.

Detection of Mycoplasma genitalium in urogenital specimens by real-time PCR and by conventional PCR assay.

A real-time LightCycler PCR (LC-PCR) with hybridization probes for detection of Mycoplasma genitalium in endocervical and first void urine specimens was developed and compared to a conventional PCR. The primers for both assays were identical and designed to amplify a 427 bp fragment of the 16S rRNA gene of M. genitalium. The LC-PCR assay had a detection limit of < 5 bacterial genomes per reaction when dilutions of genomic DNA from a type strain of M. genitalium were tested. First void urine from 398 men and first void urine and endocervical specimens from 301 women attending an STD clinic were analysed by LC-PCR and by the conventional PCR. Using the conventional PCR as reference, the LC-PCR had a specificity of 99.7 % and a sensitivity of 72.2 % for the detection of M. genitalium in first void urine samples from men. There was no significant difference in the performance of the LC-PCR assay compared to the conventional PCR when endocervical swabs were considered (58 and 65 %, respectively) or with a set of endocervical swab/urine specimens for which the LC-PCR assay detected 73 % of the infections (specificity = 98.6 % and sensitivity = 68.2 %) while the conventional PCR detected 85 % of the infections. With female urine specimens there was a significant difference between the two assays (38 and 73 %, respectively; P = 0.01 McNemar's test). This illustrates the need to analyse both endocervical and urine specimens, because M. genitalium DNA was detected in only one of the two specimens in a great number of the M. genitalium-infected women. The lower sensitivity of the LC-PCR assay was probably caused by a combination of inhibition and limitations regarding the amount of template DNA. The LC-PCR assay was easy to perform and the simultaneous amplification and detection eliminated the need for further handling of PCR products. With improvement in sample preparation methods and increased volumes of the template DNA, the LC-PCR assay could be a useful routine diagnostic method.

Defective neutrophil oxidative burst in preterm newborns on exposure to coagulase-negative staphylococci.

The neutrophil oxidative burst is a product of the regulated assembly of the multicomponent oxidase enzyme. Our aim was to compare the oxidative burst in term (n = 10) and preterm newborns <31 wk gestational age (n = 10) after stimulation with coagulase-negative staphylococci in vitro. Strains of Streptococcus epidermidis with different invasive and slime-producing properties, one strain of S. haemolyticus, and one strain of group B-streptococcus were investigated. A whole-blood flow cytometric assay using the oxidation of hydroethidine to ethidium bromide was used. The oxidative activity in unstimulated neutrophil granulocytes [polymorphonuclear leukocytes (PMNLs)] was similar in term and preterm newborns, but the preterm newborns showed a significantly lower capacity to up-regulate the oxidative burst intensity after bacterial stimulation (p = 0.004). In the term but not in the preterm group, the oxidative burst intensity after bacterial stimulation correlated with the baseline oxidative burst intensity. After bacterial stimulation, there was a trend toward a greater percentage of activated neutrophils in the term group than in the preterm group, but the difference was less pronounced than that in oxidative burst intensity. Significant differences in oxidative burst response to different bacterial strains were observed (p < 0.001), but the differences could not be correlated exclusively to invasive capacity or slime-producing properties. It is concluded that the baseline oxidative activity is similar in term and preterm PMNLs but that preterm PMNLs have a decreased capacity to increase the oxidative burst in response to bacterial stimulation.

Molecular genetic methods for diagnosis and characterisation of Chlamydia trachomatis and Neisseria gonorrhoeae: impact on epidemiological surveillance and interventions.

One of the mainstays in the prevention of Chlamydia trachomatis and Neisseria gonorrhoeae infections is the availability of laboratory diagnostics with high sensitivity and specificity. Assays for diagnosis of C. trachomatis include cell culture and nucleic acid amplification tests (NAATs). The major target sequences for C. trachomatis diagnosis by NAATs are located at the cryptic plasmid and the major target used for characterisation is the omp1 gene. The gold standard for diagnosis of N. gonorrhoeae is culture. However, numerous NAATs for identification of N. gonorrhoeae and a number of molecular genetic methods for characterisation of N. gonorrhoeae have been developed. Probably no routine laboratory can attain as high sensitivity by culturing C. trachomatis or N. gonorrhoeae as by using NAATs. For that reason NAATs can be recommended for diagnosing C. trachomatis, but not as the only diagnostic assay for N. gonorrhoeae, due to lack of antibiotic susceptibility testing and specificity problems, most pronounced for pharyngeal and rectal samples. Genotyping of C. trachomatis or N. gonorrhoeae provides additional information for contact tracing. It is recommended for N. gonorrhoeae, at least in low prevalence geographic areas, but cannot today be recommended for C. trachomatis. This is due to the low genetic variability and hence the limited benefits for partner notification. However, genotyping of C. trachomatis may play an important role under special circumstances.

Genotyping of Chlamydia trachomatis would improve contact tracing.

BACKGROUND: The reported number of Chlamydia trachomatis genital infections has increased 15% annually since 1997 in Sweden. Inaccurate partner notification might be one reason. GOAL: The goals were to determine if genotyping of C trachomatis would improve partner notification and to study the duration of infection. STUDY DESIGN: Sexual networks were constructed. C trachomatis isolates from 231 individuals attending the Orebro STD clinic during 1 year were typed by sequencing of the omp1 gene. RESULTS: All individuals were traced and diagnoses were established in 30 of 161 networks. More than one genotype was seen in seven networks. The mean duration of C trachomatis infection in each network was calculated to be 23 weeks. CONCLUSION: Genotyping could be a useful tool in partner notification when there are discrepant or uncommon genotypes. Limited clinic catchment areas create information difficulties that obstruct accurate contact tracing.