Margaretha Jurstrand

Characterization of Chlamydia trachomatis omp1 Genotypes among Sexually Transmitted Disease Patients in Sweden

ABSTRACT A method for detection and genotyping of genital Chlamydia trachomatis infections based on omp1 gene amplification and sequencing was developed. DNA was extracted from urogenital or urine samples using a Chelex-based method, and an approximately 1,100-bp-long fragment from the omp1 gene was directly amplified and sequenced. Genotyping was performed by BLAST similarity search, and phylogenetic tree analysis was used to illustrate the evolutionary relationships between clinical isolates and reference strains. The method was used to determine the genotypes ofC. trachomatis in 237 positive urogenital and/or urine specimens collected at a Swedish sexually transmitted disease clinic during 1 year. The most common genotypes corresponded to serotypes E (47%) and F (17%). The omp1 gene was highly conserved for genotype E (106 of 112 samples without any mutation) and F (41 of 42 samples without any mutation) strains but appear slightly less conserved for genotypes G (n = 6) and H (n = 6), where the sequences displayed one to four nucleotide substitutions relative to the reference sequence. Genotyping of samples collected at the follow-up visit indicated that two patients had become reinfected, while three other patients suffered treatment failure or reinfection. One woman appeared to have a mixed infection with two different C. trachomatis strains. Thisomp1 genotyping method had a high reproducibility and could be used for epidemiological characterization of sexually transmitted Chlamydia infections.

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.

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.

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.

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.

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.

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 Örebro 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.

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.

Genotyping of Chlamydia trachomatis Would Improve Contact Tracing

Background The reported number of genital Chlamydia trachomatis 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 Örebro 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.