Prevalence of Mycoplasma genitalium and macrolide resistance in rectal and urine samples among men who have sex with men in Sweden.

OBJECTIVES: While Mycoplasma genitalium is reported as a common rectal infection among men who have sex with men (MSM), published data refer predominantly to urethral infections. Currently, most guidelines recommend M. genitalium testing from urine in men with symptomatic, non-gonococcal urethritis. Macrolide resistance-associated mutations (MRMs) among M. genitalium have increased during the last decade especially among MSM. We aim to demonstrate the prevalence and anatomical distribution of M. genitalium infection and MRM in urine and rectal specimens among MSM in Sweden. METHODS: In this cross-sectional study in 2019, paired urine and rectal samples from symptomatic and asymptomatic MSM attending a sexually transmitted infection clinic in the south of Sweden were screened for M. genitalium, presence of MRM, Neisseria gonorrhoeae, Chlamydia trachomatis, HIV and syphilis. RESULTS: The overall prevalence of M. genitalium was 10.5% (64 of 609), rectal samples 7.6% (46 of 609) and urine samples 3.9% (24 of 609) (p=0.007). Among M. genitalium-positive cases, single rectal and single urethral infection was detected in 62.5% (40 of 64) and 28.1% (18 of 64), respectively (p<0.0001). Infection at both sites was seen in 9.4% (6 of 64). The prevalence of MRM was 67.9% (19 of 28). M. genitalium was significantly associated with HIV (OR 2.60, 95% CI 1.14 to 5.88, p=0.02). Among the MSM, 7.4% (45 of 609) were infected with N. gonorrhoeae, 6.7% (41 of 609) with C. trachomatis, 7.1% (43 of 609) with HIV and 0.7% (4 of 609) with syphilis. CONCLUSIONS: In this study, among MSM, most infections with M. genitalium were detected as rectal mono infections. The prevalence of M. genitalium among MSM was almost twofold higher in rectal samples (7.6%) compared with urine samples (3.9%). The prevalence of macrolide resistance was high with no difference between urine and rectal samples.

Increased prevalence of human papillomavirus in fresh tissue from penile cancers compared to non-malignant penile samples: a case-control study.

BACKGROUND: HPV has been detected in approximately 50% of invasive penile cancers but with a large span between 24 and 89%, most likely due to different types of tumors and various methods for HPV analysis. Most studies of HPV in penile cancer have been performed using paraffin-embedded tissue, argued to be at risk for contaminated HPV analysis. Viral activity of HPV, by the use of HPV mRNA expression is well studied in cervical cancer, but seldom studied in penile cancer. The aim was to determine prevalence of HPV types in fresh tissue of penile cancers compared to non-malignant age-matched penile controls. Additional aims were to analyze the viral expression and copy numbers of HPV16-positive tumors and 10 mm adjacent to the tumor. METHODS: Fresh tissue from penile cancer cases was biopsied inside the tumor and 10 mm outside the tumor. Controls were males circumcised for non-malignant reasons, biopsied at surgery. PCR and Luminex assays were used for identification of HPV types. HPV16-positive samples were investigated for copy numbers and expression of HPV16-mRNA. RESULTS: Among tumors (n = 135) and age-matched controls (n = 105), HPV was detected in 38.5% (52/135) and 11.4% (12/105), respectively (p < 0.001), adjusted odds ratio 12.8 (95% confidence interval 4.9-33.6). High-risk HPV types were found in 35.6% (48/135) of tumors and 4.8% (5/105) of controls (p < 0.001). Among tumors and controls, HPV16 was present in 27.4% (37/135) and 1% (1/105), respectively (p < 0.001). Among HPV16-positive penile cancers, mean HPV16 viral copy/cell was 74.4 (range 0.00003-725.4) in the tumor and 1.6 (range 0.001-14.4) 10 mm adjacent from the tumor. HPV16-mRNA analysis of the tumors and 10 mm adjacent from the tumors demonstrated viral activity in 86.5% (32/37) and 21.7% (5/23), respectively. CONCLUSIONS: The prevalence of HPV was significantly higher in penile cancer (38.5%) than among age-matched non-malignant penile samples (11.4%). HPV16 predominates (27.4%) in penile tumors. HPV16 expression was more common in penile cancer than in adjacent healthy tissue, strongly suggesting an etiological role for HPV16 in the development of penile cancer.

Incidence of penile intraepithelial neoplasia and treatment strategies in Sweden 2000-2019.

OBJECTIVES: To analyse the incidence, treatment strategies and complications associated with penile intraepithelial neoplasia (PeIN) in Sweden over a period of 20 years. MATERIALS AND METHODS: Data on PeIN from the Swedish National Penile Cancer Register were analysed regarding treatment in relation to age, size of the PeIN lesion, localization of the PeIN lesion and complications using chi-squared tests and logistic regression. The incidence of PeIN was calculated and age-standardized according to the European Standard population. RESULTS: Between 2000 and 2019 a total of 1113 PeIN cases were reported. The age-standardized incidence of PeIN was 1.40 per 100 000 men (95% confidence interval [CI] 1.32-1.49). An increase in incidence over time was seen, with a standardized incidence rate of 2.37 (95% CI 1.56-3.70) in 2019 compared to the baseline year, 2000. Surgical or topical treatments were given in 75.0% and 14.6% of cases, respectively. The complication rate was higher in laser surgery (12.1%, 7/58) compared to local surgery (4.6%, 16/348; P = 0.03) with an age-adjusted odds ratio (OR) of 2.82 (95% CI 1.10-7.19; P = 0.03). Local surgery was more common than laser surgery in the last 5 years compared to the first 5 years of the study period: OR 5.75 (95% CI 2.94-11.27). Treatments with imiquimod and topical 5-fluorouracil (5-FU) were more common than destructive methods such as photodynamic therapy, cryotherapy, curettage and electrocautery in the last 5 years compared to the first 5 years: OR 9.48 (95% CI 2.29-39.24). CONCLUSIONS: A twofold increase in the age-standardized incidence of PeIN was seen in Sweden over 20 years. Complications were three times more common in laser surgery compared to local surgery. Changes in treatment showed an increase of treatment strategies such as local surgery and treatment with imiquimod and topical 5-FU over time.

Penile intraepithelial neoplasia, penile cancer precursors and human papillomavirus prevalence in symptomatic preputium: a cross-sectional study of 351 circumcised men in Sweden.

OBJECTIVES: To investigate the prevalence of pathological disease and spectrum of human papillomavirus (HPV) types among symptomatic foreskin tissue. PATIENTS AND METHODS: Consecutively excised symptomatic foreskins from 351 men were sent for histopathological evaluation. During the surgical procedure, a fresh biopsy was taken for HPV analysis by modified general primer polymerase chain reaction. A medical questionnaire regarding medication, smoking habits, number of lifetime sexual partners, former diseases and surgery performed on penis was completed by all participants. RESULTS: The most common clinical diagnosis and cause for circumcision was phimosis, seen in 85.2%. Histopathologically inflammatory dermatological conditions were present in 87% of the men. The most common histopathological diagnosis was lichen sclerosus (LS) observed among 58.7%. Notably, penile intraepithelial neoplasia (PeIN) was present in 2% without former clinical suspicion. Overall, HPV was detected in 17.1% of the men and 28 different HPV types were found. High-risk (HR) HPV types were identified in 9.1% and HPV16 was present in 2.3%. Current smoking increased the risk of HPV (crude odds ratio [OR] 2.8, confidence interval [CI] 1.4-5.6; P = 0.005). Having >15 lifetime sexual partners increased the risk of HPV (crude OR 2.6, 95% CI 1.4-5.1; P = 0.003) and when adjusted for current smoking the OR was substantially increased (OR 6.0, 95% CI CI 2.2-16.8; P < 0001). CONCLUSIONS: Histopathological evaluation of circumcised symptomatic foreskin revealed PeIN in 2% of the men without any clinical suspicion of malignancy and that treatable dermatological conditions were present in 87%, LS being the most common. HR-HPV types were present in 9%. Due to risk of malignant development both in PeIN and in inflammatory skin diseases we recommend sending all excised foreskins from patients with symptoms for histopathological evaluation as guidance for further clinical management.

Risk Factors for Penile Intraepithelial Neoplasia: A Population-based Register Study in Sweden, 2000-2012.

Studies on risk factors for penile intraepithelial neo-plasia have been small in size, have not distinguished penile intraepithelial neoplasia from invasive cancer, and have relied on self-reported information. This study investigated risk factors for penile intraepithelial neoplasia in a cohort of 580 penile intraepithelial neoplasia cases and 3,436 controls using information from 7 Swedish registers. Cases with penile intraepithelial neoplasia had increased odds ratios (ORs) for inflammatory skin diseases (14.7, 95% CI 6.5-33.4) including lichen planus (12.0, 95% CI 3.0-48.0), indicating lichen planus to be an important risk factor. Increased ORs were also observed for diseases of the prepuce (4.0, 95% CI 2.2-7.4), immunosuppressive drugs (5.0, 95% CI 2.5-9.8), penile surgical procedures (4.8, 95% CI 2.2-10.8), balanitis (9.2, 95% CI 5.0-16.8), genital warts (9.9, 95% CI 4.3-22.7) and organ transplantation (7.0, 95% CI 2.4-20.8). This study demonstrates important risk factors for penile intraepithelial neoplasia, providing knowledge that can help prevent the development of penile cancer.

Mycoplasma genitalium and Macrolide Resistance-associated Mutations in the Skåne Region of Southern Sweden 2015.

Mycoplasma genitalium is a sexually transmitted infection ordinarily treated with azithromycin. Emerging resistance to macrolide is linked to mutations in the 23S rRNA gene. We analysed the frequency of such mutations of M. genitalium isolates from patients that were symptomatic, and from sexual partners of symptomatic individuals, from October to December of 2015, in the Skåne Region of Sweden. Mutations were analysed by the use of DNA sequencing. Overall, 11.9% (145/1,311) and 17.0% (116/704) of females and males were positive for M. genitalium, respectively. Macrolide resistant mutations were detected in 13% (31/239) of M. genitalium isolates from first-test patient samples. Twenty-one (8.8%) and 10 (4.2%) of the isolates had point mutations of the 23S-gene at position 2072 and 2071, respectively. Two different M. genitalium isolates were detected simultaneously in two cases. In summary, we found a relatively low rate of macrolide-resistant M. genitalium in the region of Southern Sweden.

Cervical myoma causing colonic obstruction in the first trimester of pregnancy - a case report.

A 39-year-old nulliparous woman with a previously known cervical myoma was admitted to the obstetrics department during the first trimester with complaints of severe abdominal pain, lack of bowel movements and the suspicion of a clinical bowel obstruction. Because no literature on this exact condition could be found, clinical decisions were based on reports and practice in similar situations. Ultrasound revealed the progression of a cervical myoma (previously 9 cm across), now 12 × 12 × 11 cm in size and a distended large bowel. Sigmoidoscopy excluded intraluminal obstruction. The patient was treated with oral laxatives and enema without success and her condition deteriorated. The myomatous cervix was examined vaginally (bimanual manoeuvre) with the patient under anaesthesia; however, attempts to dislodge the obstruction proved unsuccessful. After surgical consultation the patient was planned for an emergency laparoscopic sigmoidostomy. The post-operative course was uneventful and the patient discharged. She delivered a healthy child with caesarean section in gestation week 36. Bowel continuity was later laparoscopically restored in conjunction with a hysterectomy. This case illustrates the importance of active multidisciplinary management in a case of severe colonic obstruction caused by pregnancy-related obstruction in the small pelvis. In this case, colonic perforation and abortion of the fetus were both avoided.

Decline of the new Swedish variant of Chlamydia trachomatis after introduction of appropriate testing.

OBJECTIVE: The longitudinal epidemiological development of the new variant of Chlamydia trachomatis was studied after appropriate testing procedures had been introduced when the strain was detected in 2006. METHODS: The number of cases of the new variant of C trachomatis was followed from 2007 through 2011 from the laboratory records. Testing for C trachomatis is centralised to one laboratory with around 80-85 000 persons being tested annually in a population of 1.1 million. RESULTS: During the 5-year period, 410 973 patients were tested of which 25 723 cases were positive. The proportion of the new variant of all positive cases declined from 30% in 2007 to 6% in 2011. While the number of the new variant of C trachomatis declined, the ordinary wild-type strains remained largely unchanged. CONCLUSIONS: A selective decline of the new variant of C trachomatis has occurred after appropriate laboratory testing was introduced. A new balance point between 5% and 10% for the new variant seems to be gradually approached.

Mycoplasma genitalium in cervicitis and pelvic inflammatory disease among women at a gynecologic outpatient service.

OBJECTIVE: We sought to analyze the prevalence and clinical manifestations of Mycoplasma genitalium infection in a heterogeneous population of women. STUDY DESIGN: The study was designed as a cross-sectional case-control study. Women attending a gynecological outpatient service from 2003 through 2008 were invited to participate. RESULTS: The prevalence of M genitalium was 2.1% and of Chlamydia trachomatis was 2.8% among 5519 tested women. A total of 679 women were included. Both pelvic inflammatory disease (PID) and cervicitis were independently associated with M genitalium (odds ratio, 9.00; 95% confidence interval, 1.62-49.89 and odds ratio, 3.80; 95% confidence interval, 2.06-7.03, respectively). Women with C trachomatis had a higher frequency of both PID (18.3% vs 4.9%, P < .001) and cervicitis (33.4% vs 22.3%, P < .001) than women with M genitalium. CONCLUSION: M genitalium was an independent and strong risk factor for both cervicitis and PID although, compared to C trachomatis, clinical manifestations were less frequent.

The Swedish new variant of Chlamydia trachomatis: genome sequence, morphology, cell tropism and phenotypic characterization.

Chlamydia trachomatis is a major cause of bacterial sexually transmitted infections worldwide. In 2006, a new variant of C. trachomatis (nvCT), carrying a 377 bp deletion within the plasmid, was reported in Sweden. This deletion included the targets used by the commercial diagnostic systems from Roche and Abbott. The nvCT is clonal (serovar/genovar E) and it spread rapidly in Sweden, undiagnosed by these systems. The degree of spread may also indicate an increased biological fitness of nvCT. The aims of this study were to describe the genome of nvCT, to compare the nvCT genome to all available C. trachomatis genome sequences and to investigate the biological properties of nvCT. An early nvCT isolate (Sweden2) was analysed by genome sequencing, growth kinetics, microscopy, cell tropism assay and antimicrobial susceptibility testing. It was compared with relevant C. trachomatis isolates, including a similar serovar E C. trachomatis wild-type strain that circulated in Sweden prior to the initially undetected expansion of nvCT. The nvCT genome does not contain any major genetic polymorphisms - the genes for central metabolism, development cycle and virulence are conserved - or phenotypic characteristics that indicate any altered biological fitness. This is supported by the observations that the nvCT and wild-type C. trachomatis infections are very similar in terms of epidemiological distribution, and that differences in clinical signs are only described, in one study, in women. In conclusion, the nvCT does not appear to have any altered biological fitness. Therefore, the rapid transmission of nvCT in Sweden was due to the strong diagnostic selective advantage and its introduction into a high-frequency transmitting population.

Co-evolution of genomes and plasmids within Chlamydia trachomatis and the emergence in Sweden of a new variant strain.

BACKGROUND: Chlamydia trachomatis is the most common cause of sexually transmitted infections globally and the leading cause of preventable blindness in the developing world. There are two biovariants of C. trachomatis: 'trachoma', causing ocular and genital tract infections, and the invasive 'lymphogranuloma venereum' strains. Recently, a new variant of the genital tract C. trachomatis emerged in Sweden. This variant escaped routine diagnostic tests because it carries a plasmid with a deletion. Failure to detect this strain has meant it has spread rapidly across the country provoking a worldwide alert. In addition to being a key diagnostic target, the plasmid has been linked to chlamydial virulence. Analysis of chlamydial plasmids and their cognate chromosomes was undertaken to provide insights into the evolutionary relationship between chromosome and plasmid. This is essential knowledge if the plasmid is to be continued to be relied on as a key diagnostic marker, and for an understanding of the evolution of Chlamydia trachomatis. RESULTS: The genomes of two new C. trachomatis strains were sequenced, together with plasmids from six C. trachomatis isolates, including the new variant strain from Sweden. The plasmid from the new Swedish variant has a 377 bp deletion in the first predicted coding sequence, abolishing the site used for PCR detection, resulting in negative diagnosis. In addition, the variant plasmid has a 44 bp duplication downstream of the deletion. The region containing the second predicted coding sequence is the most highly conserved region of the plasmids investigated. Phylogenetic analysis of the plasmids and chromosomes are fully congruent. Moreover this analysis also shows that ocular and genital strains diverged from a common C. trachomatis progenitor. CONCLUSION: The evolutionary pathways of the chlamydial genome and plasmid imply that inheritance of the plasmid is tightly linked with its cognate chromosome. These data suggest that the plasmid is not a highly mobile genetic element and does not transfer readily between isolates. Comparative analysis of the plasmid sequences has revealed the most conserved regions that should be used to design future plasmid based nucleic acid amplification tests, to avoid diagnostic failures.

Clinical manifestations and epidemiology of the new genetic variant of Chlamydia trachomatis.

BACKGROUND: In 2006, a new genetic variant of Chlamydia trachomatis (nvCT) was discovered in Sweden. Clinical manifestations of this infection were studied in a high-risk population. METHODS: During 2007, a prospective case-control study on sexual lifestyle and urogenital infections was performed at the Centre for Sexual Health (CSH), affiliated to Malmo University Hospital. A total of 629 C. trachomatis positive cases and 1252 negative controls were included. At Malmo University Hospital, Department of Obstetrics and Gynecology, all cases of pelvic inflammatory disease (PID) were assessed and correlated to the prevalence of nvCT. RESULTS: Patients with nvCT or wild type C. trachomatis (wtCT) infection did not differ regarding their sexual lifestyle. Men with nvCT or wtCT infection did not differ in uro-genital symptoms or clinical findings. Women with nvCT infection reported painful urination (12.2% vs. 25.8%, P = 0.02) and were diagnosed with urethritis (11.1% vs. 40.0%, P = 0.04) less often than women with wtCT infection. The ratio of lower abdominal pain in women with nvCT infection was only half of that in women with wtCT infection (13.4% vs. 27.8%, P = 0.02). PID was detected in 0.8% of women with C. trachomatis infection in Malmo. All these cases were due to wtCT infection. CONCLUSIONS: Symptomatic urethral infection and lower abdominal pain was less common in women with nvCT as compared to wtCT. Infection with nvCT was more frequently asymptomatic suggesting a possible difference in virulence between the nvCT strain and the wtCT strain.

Deoxyribonucleic acid of Chlamydia trachomatis in fresh tissue from the Fallopian tubes of patients with ectopic pregnancy.

OBJECTIVES: The role of persistent chlamydial infection of the Fallopian tubes in ectopic pregnancy is still unresolved. Therefore, we examined tissue of the Fallopian tubes from patients with ectopic pregnancy for the presence of Chlamydia trachomatis. In addition, other markers of C. trachomatis infection implicated in the pathogenesis of tubal damage were studied including antibodies to heat shock protein 60 of chlamydial and human origin. STUDY DESIGN: Fresh frozen tubal tissue from 55 patients with ectopic pregnancy in a hospital setting were examined for the presence of C. trachomatis DNA by polymerase chain reaction (PCR) and blood sample were analysed for antibodies to C. trachomatis including heat shock protein 60 (hsp60). RESULTS: Chlamydial DNA was not detected in any of the 55 tubal specimens using a commercial test, Cobas Amplicor, Roche, and an in-house real time PCR able to detect a few copies of the organism. Logistic regression showed that chlamydial IgG antibodies were more common in a subgroup of patients with previous PID than in controls (OR=7.84, CI 1.78-34.6). Specific antibodies to hsp60 of chlamydial (OR=7.00, CI 1.50-32.6) but not of human origin (OR=2.13, CI 0.14-31.6) were associated with ectopic pregnancy in this group. CONCLUSIONS: No evidence of persistent infection of C. trachomatis in the fallopian tubes at the time of ectopic pregnancy was found in this study.

Plasmid deficiency in urogenital isolates of Chlamydia trachomatis reduces infectivity and virulence in a mouse model.

We hypothesized that the plasmid of urogenital isolates of Chlamydia trachomatis would modulate infectivity and virulence in a mouse model. To test this hypothesis, we infected female mice in the respiratory or urogenital tract with graded doses of a human urogenital isolate of C. trachomatis, serovar F, possessing the cognate plasmid. For comparison, we inoculated mice with a plasmid-free serovar F isolate. Following urogenital inoculation, the plasmid-free isolate displayed significantly reduced infectivity compared with the wild-type strain with the latter yielding a 17-fold lower infectious dose to yield 50% infection. When inoculated via the respiratory tract, the plasmid-free isolate exhibited reduced infectivity and virulence (as measured by weight change) when compared to the wild-type isolate. Further, differences in infectivity, but not in virulence were observed in a C. trachomatis, serovar E isolate with a deletion within the plasmid coding sequence 1 when compared to a serovar E isolate with no mutations in the plasmid. We conclude that plasmid loss reduces virulence and infectivity in this mouse model. These findings further support a role for the chlamydial plasmid in infectivity and virulence in vivo.

Transformation of a plasmid-free, genital tract isolate of Chlamydia trachomatis with a plasmid vector carrying a deletion in CDS6 revealed that this gene regulates inclusion phenotype.

The development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis provides the basis for the detailed investigation of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In this study we constructed a plasmid vector with CDS6 deleted (pCDS6KO) from the original Escherichia coli/C. trachomatis shuttle vector pGFP::SW2. pCDS6KO was transformed into a clinical isolate of C. trachomatis from Sweden that is plasmid-free (C. trachomatis SWFP-). Penicillin-resistant transformants expressing the green fluorescent protein were selected. These transformants did not stain with iodine, indicating that this property is regulated by CDS6 or its gene product. In addition, mature inclusions of C. trachomatis SWFP- transformed by pCDS6KO displayed an identical morphological phenotype to the untransformed plasmid-free recipient host. In this phenotype the morphology of inclusions was altered with the chlamydiae lining the periphery of the inclusion leaving a 'hole' in the centre. These green fluorescent inclusions appear 'doughnut-shaped' with an empty centre when examined under blue light, giving rise to a characteristic 'black hole' phenotype. Our study demonstrates the power of the new genetic system for investigating chlamydial gene function using gene deletion technology.

Genetic transformation of a clinical (genital tract), plasmid-free isolate of Chlamydia trachomatis: engineering the plasmid as a cloning vector.

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle" that it is possible to "knock out" selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb ß-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed ß-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active ß-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.

Genotyping markers used for multi locus VNTR analysis with ompA (MLVA-ompA) and multi sequence typing (MST) retain stability in Chlamydia trachomatis.

We aimed to evaluate the stability of the Chlamydia trachomatis multi locus VNTR analysis (MLVA-ompA) and multi sequence typing (MST) systems through multiple passages in tissue culture. Firstly, we analyzed the stability of these markers through adaptation of C. trachomatis to tissue culture and secondly, we examined the stability of a four-locus MLVA-ompA and a five-locus MST system after multiple passages in tissue culture. Marker sequences were monitored through successive chlamydial developmental cycles to evaluate the stability of the individual DNA markers through many bacterial divisions and this, in turn, informed us of the usefulness of using such typing systems for short and long-term molecular epidemiology. Southampton genitourinary medicine (GUM) clinic isolates from endocervical swabs collected from C. trachomatis positive women were passaged through tissue culture. MLVA-ompA typing was applied to primary swab samples and to the same samples after C. trachomatis had been passaged through cell culture (eight passages). Sequence data from time-zero and passage-eight isolates were aligned with reference sequences to determine the stability of the markers. The Swedish new variant (nvCT) underwent 72 passages in cell culture and the markers of the two schemes were similarly analyzed. Analysis of genetic markers of the MLVA-ompA typing system before and after the isolates were introduced to tissue culture showed no change in the dominant sequence. The nvCT that had been passaged 72 times over the duration of a year also showed no variation in the dominant sequence for both the genotyping schemes. MLVA-ompA and MST markers are stable upon adaptation of C. trachomatis to tissue culture following isolation of strains from primary endocervical swab samples. These markers remain stable throughout multiple rounds of cell-division in tissue culture, concomitant with the incubation period and appearance of symptoms normally associated with host-infection. Both genotyping schemes are, therefore, suitable for epidemiology of C. trachomatis.

Whole-genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing.

Chlamydia trachomatis is responsible for both trachoma and sexually transmitted infections, causing substantial morbidity and economic cost globally. Despite this, our knowledge of its population and evolutionary genetics is limited. Here we present a detailed phylogeny based on whole-genome sequencing of representative strains of C. trachomatis from both trachoma and lymphogranuloma venereum (LGV) biovars from temporally and geographically diverse sources. Our analysis shows that predicting phylogenetic structure using ompA, which is traditionally used to classify Chlamydia, is misleading because extensive recombination in this region masks any true relationships present. We show that in many instances, ompA is a chimera that can be exchanged in part or as a whole both within and between biovars. We also provide evidence for exchange of, and recombination within, the cryptic plasmid, which is another key diagnostic target. We used our phylogenetic framework to show how genetic exchange has manifested itself in ocular, urogenital and LGV C. trachomatis strains, including the epidemic LGV serotype L2b.