Immunological Markers of Chlamydia trachomatis Infection in Epithelial Ovarian Cancer.

BACKGROUND/AIM: Pelvic inflammatory disease (PID) is a risk factor for epithelial ovarian cancer (EOC). Chlamydia trachomatis infection, a major cause of PID, may persist in some women. Serum IgG antibodies to chlamydial TroA and HtrA are more common in ascending or repeat chlamydial infection than in uncomplicated infection. The aim of this study was to explore the role of C. trachomatis infection in EOC by analyzing chlamydial TroA, HtrA and major outer membrane protein (MOMP) IgG serum antibody responses. PATIENTS AND METHODS: The study is based on the review of Oulu University Hospital medical records of 162 women diagnosed with EOC between March 2008 and May 2018. Serum IgG antibody responses to recombinant C. trachomatis TroA, HtrA and MOMP were analyzed using enzyme-linked immunoassay. Complete response to the first line therapy and the three-year survival were the study endpoints. RESULTS: Altogether, 16.7%, 11.1% and 12.3% women were C. trachomatis TroA, HtrA and MOMP IgG positive, respectively. Women with these antibodies were more likely to have a complete response to the first-line treatment, compared to women without these antibodies (63.0% vs. 34.1% for TroA IgG, 50.0% vs. 37.5% for HtrA IgG and 50% vs. 37.3% for MOMP IgG, respectively). The presence of these antibodies predicted better three-year survival. CONCLUSION: Women with EOC and positive markers of persistent C. trachomatis infection have better response to the first-line treatment and seem to have better three-year survival.

Use of gene sequences as type for naming prokaryotes: Recommendations of the international committee on the taxonomy of chlamydiae.

The International Committee on Systematics of Prokaryotes (ICSP) discussed and rejected in 2020 a proposal to modify the International Code of Nomenclature of Prokaryotes to allow the use of gene sequences as type for naming prokaryotes. An alternative nomenclatural code, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), which considers genome sequences as type material for naming species, was published in 2022. Members of the ICSP subcommittee for the taxonomy of the phylum Chlamydiae (Chlamydiota) consider that the use of gene sequences as type would benefit the taxonomy of microorganisms that are difficult to culture such as the chlamydiae and other strictly intracellular bacteria. We recommend the registration of new names of uncultured prokaryotes in the SeqCode registry.

Liposomes-In-Hydrogel Delivery System Enhances the Potential of Resveratrol in Combating Vaginal Chlamydia Infection.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infections and causes serious reproductive tract complications among women. The limitations of existing oral antibiotics and treatment of antimicrobial resistance require alternative treatment options. We are proposing, for the first time, the natural polyphenol resveratrol (RES) in an advanced delivery system comprising liposomes incorporated in chitosan hydrogel, for the localized treatment of C. trachomatis infection. Both free RES and RES liposomes-in-hydrogel inhibited the propagation of C. trachomatis in a concentration-dependent manner, assessed by the commonly used in vitro model comprising McCoy cells. However, for lower concentrations, the anti-chlamydial effect of RES was enhanced when incorporated into a liposomes-in-hydrogel delivery system, with inhibition of 78% and 94% for 1.5 and 3 µg/mL RES, respectively for RES liposomes-in-hydrogel, compared to 43% and 72%, respectively, for free RES. Furthermore, RES liposomes-in-hydrogel exhibited strong anti-inflammatory activity in vitro, in a concentration-dependent inhibition of nitric oxide production in the LPS-induced macrophages (RAW 264.7). The combination of a natural substance exhibiting multi-targeted pharmacological properties, and a delivery system that provides enhanced activity as well as applicability for vaginal administration, could be a promising option for the localized treatment of C. trachomatis infection.

Sensitivity, specificity, inclusivity and exclusivity of the updated Aptima Combo 2 assay, which provides detection coverage of the new diagnostic-escape Chlamydia trachomatis variants.

BACKGROUND: Four new variants of Chlamydia trachomatis (nvCTs), detected in several countries, cause false-negative or equivocal results using the Aptima Combo 2 assay (AC2; Hologic). We evaluated the clinical sensitivity and specificity, as well as the analytical inclusivity and exclusivity of the updated AC2 for the detection of CT and Neisseria gonorrhoeae (NG) on the automated Panther system (Hologic). METHODS: We examined 1004 clinical AC2 samples and 225 analytical samples spiked with phenotypically and/or genetically diverse NG and CT strains, and other potentially cross-reacting microbial species. The clinical AC2 samples included CT wild type (WT)-positive (n = 488), all four described AC2 diagnostic-escape nvCTs (n = 170), NG-positive (n = 214), and CT/NG-negative (n = 202) specimens. RESULTS: All nvCT-positive samples (100%) and 486 (99.6%) of the CT WT-positive samples were positive in the updated AC2. All NG-positive, CT/NG-negative, Trichomonas vaginalis (TV)-positive, bacterial vaginosis-positive, and Candida-positive AC2 specimens gave correct results. The clinical sensitivity and specificity of the updated AC2 for CT detection was 99.7 and 100%, respectively, and for NG detection was 100% for both. Examining spiked samples, the analytical inclusivity and exclusivity were 100%, i.e., in clinically relevant concentrations of spiked microbe. CONCLUSIONS: The updated AC2, including two CT targets and one NG target, showed a high sensitivity, specificity, inclusivity and exclusivity for the detection of CT WT, nvCTs, and NG. The updated AC2 on the fully automated Panther system offers a simple, rapid, high-throughput, sensitive, and specific diagnosis of CT and NG, which can easily be combined with detection of Mycoplasma genitalium and TV.

Validation of an Aptima-format Finnish new variant of Chlamydia trachomatis (FI-nvCT) surveillance assay, 2019.

The Finnish new variant of Chlamydia trachomatis (FI-nvCT) is escaping diagnostics in Finland, Norway and Sweden. We have developed and validated an Aptima-format nucleic acid amplification test (NAAT) designed specifically to detect the FI-nvCT. This NAAT has high sensitivity (100%) and specificity (100%) for the FI-nvCT strain, enabling further investigation of the geographic distribution, prevalence and transmission of this diagnostic-escape mutant in screening populations in Europe.

The Prevalence of HSV, HHV-6, HPV and Mycoplasma genitalium in Chlamydia trachomatis positive and Chlamydia trachomatis Negative Urogenital Samples among Young Women in Finland.

Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus (HSV) and human papillomavirus (HPV) cause sexually transmitted infections. In addition, human herpesvirus 6 (HHV-6) may be a genital co-pathogen. The prevalence rates of HSV, HHV-6, HPV, M. genitalium, and the C. trachomatis ompA genotypes were investigated by PCR in urogenital samples of the C. trachomatis nucleic acid amplification test positive (n = 157) and age-, community- and time-matched negative (n = 157) women. The prevalence of HPV DNA was significantly higher among the C. trachomatis positives than the C. trachomatis negatives (66% vs. 25%, p < 0.001). The prevalence of HSV (1.9% vs. 0%), HHV-6 (11% vs. 14%), and M. genitalium DNA (4.5% vs. 1.9%) was not significantly different between the C. trachomatis-positive and -negative women. Thirteen per cent of test-of-cure specimens tested positive for C. trachomatis. The prevalence of HSV, HHV-6, HPV, M. genitalium, and the C. trachomatis ompA genotypes did not significantly differ between those who cleared the C. trachomatis infection (n = 105) and those who did not (n = 16). The higher prevalence of HPV DNA among the C. trachomatis positives suggests greater sexual activity and increased risk for sexually transmitted pathogens.

Predictive Values of Serum Chlamydia trachomatis TroA and HtrA IgG Antibodies as Markers of Persistent Infection in the Detection of Pelvic Adhesions and Tubal Occlusion.

Chlamydia trachomatis IgG antibody testing (CAT) has been used as a screening test for tubal factor infertility (TFI), but as the CAT is only a marker of a past exposure to C. trachomatis and not of late sequelae, the positive predictive value (PPV) of the test is low. The persistence of C. trachomatis in the upper genital tract has been suggested as one of the key mechanisms in the development of TFI. Serum antibodies against C. trachomatis TroA and HtrA, proteins expressed specifically during persistent infection, have been suggested as novel biomarkers for TFI diagnostics. We studied serum IgG antibody responses against C. trachomatis TroA, HtrA and MOMP in 79 subfertile women, of whom 28 had laparoscopically proven TFI. We confirmed that the accuracy of CAT in diagnosing TFI is low, whereas TroA IgG and HtrA IgG are more accurate tests in detecting tubal occlusion and pelvic adhesions. However, the sensitivity and negative predictive value (NPV) of TroA IgG and HtrA IgG are still too low to justify their use as a screening test in clinical practice. Individual immunogenetic profiles combined with TroA and HtrA antibody responses might identify women with the highest risk for developing late complications after C. trachomatis infection.

The Finnish New Variant of Chlamydia trachomatis with a Single Nucleotide Polymorphism in the 23S rRNA Target Escapes Detection by the Aptima Combo 2 Test.

In 2019, more than 200 cases of Chlamydia trachomatis negative/equivocal by the Aptima Combo 2 assay (AC2, target: 23S rRNA) with slightly elevated relative light units (RLUs), but positive by the Aptima Chlamydia trachomatis assay (ACT, target: 16S rRNA) have been detected in Finland To identify the cause of the AC2 CT false-negative specimens, we sequenced parts of the CT 23S rRNA gene in 40 specimens that were AC2 negative/equivocal but ACT positive. A single nucleotide polymorphism (SNP; C1515T in the C. trachomatis 23S rRNA gene) was revealed in 39 AC2/ACT discordant specimens. No decrease in the number of mandatorily notified C. trachomatis cases was observed nationally in Finland in 2010-2019. When RLUs obtained for AC2 negative specimens were retrospectively evaluated in 2011-2019, a continuous increase in the proportion of samples with RLUs 10-19 was observed since 2014, and a slight increase in the proportion of samples with RLUs 20-84 in 2017-2019, indicating that the Finnish new variant of C. trachomatis might have been spreading nationally for several years. This emphasizes that careful surveillance of epidemiology, positivity rate and test performance are mandatory to detect any changes affecting detection of infections.

Finnish new variant of Chlamydia trachomatis escaping detection in the Aptima Combo 2 assay also present in Örebro County, Sweden, May 2019.

We identified the first two cases of the Finnish new variant of Chlamydia trachomatis (F-nvCT) beyond Finland in two clinical urogenital specimens in Örebro County, Sweden. These Aptima Combo 2 assay-negative specimens were Aptima Chlamydia trachomatis (CT) assay positive and had the characteristic C1515T mutation in the 23S rRNA gene. From 22 March to 31 May 2019, 1.3% (2/158) of the CT-positive cases in Örebro County were missed because of the F-nvCT. International awareness, investigations and actions are essential.

Mycoplasma pneumoniae outbreak, Southeastern Finland, 2017-2018: molecular epidemiology and laboratory diagnostic lessons.

This study characterizes a large Mycoplasma pneumoniae outbreak observed in Kymenlaakso in Southeastern Finland during August 2017-January 2018. The first part of the investigation included 327 patients, who sought healthcare consultation at local GPs or hospitals due to clinical symptoms, and were tested for M. pneumoniae antibodies (Patient cohort). The second part of the investigation, conducted approximately 4 weeks after the peak of the outbreak, consisted of school screening of pupils (N = 239) in three different school buildings by PCR on respiratory specimens and questionnaires (Screening cohort). PCR positive respiratory specimens were subsequently utilized for molecular typing. The outbreak peaked in late October 2017. Of the Patient cohort, 9/106 (8.5%) respiratory specimens were PCR positive. In contrast, 3/182 (1.6%) of the Screening cohort were PCR positive. Asymptomatic carriage was observed. Multiple-locus variable-number tandem-repeat analysis (MLVA) identified two distinct MLVA types. All typed M. pneumoniae strains belonged to P1 type 1. No mutations leading to macrolide resistance were observed. In total, 61/327 (19%) of the Patient cohort had a serological indication of recent infection. The IgM test reactivity at the time of a negative PCR test result varied from a completely non-reactive value up to very strong reactivity, highlighting the difficulty in a single specimen serodiagnosis.

Chlamydia trachomatis samples testing falsely negative in the Aptima Combo 2 test in Finland, 2019.

Since February 2019, over 160 Chlamydia trachomatis (CT) cases testing negative or equivocal by Aptima Combo 2 (AC2) but positive by Aptima CT test run with Panther instruments occurred in Finland. The AC2 test targets chlamydial 23S rRNA while the CT test targets 16S rRNA. Sequencing of 10 strains revealed a nucleotide substitution in 23S rRNA. The significance of this for the failure of the AC2 test to detect the variant is not yet known.

Parachlamydia acanthamoebae Detected during a Pneumonia Outbreak in Southeastern Finland, in 2017⁻2018.

Community-acquired pneumonia (CAP) is a common disease responsible for significant morbidity and mortality. However, the definite etiology of CAP often remains unresolved, suggesting that unknown agents of pneumonia remain to be identified. The recently discovered members of the order Chlamydiales, Chlamydia-related bacteria (CRB), are considered as possible emerging agents of CAP. Parachlamydia acanthamoebae is the most studied candidate. It survives and replicates inside free-living amoeba, which it might potentially use as a vehicle to infect animals and humans. A Mycoplasma pneumoniae outbreak was observed in Kymenlaakso region in Southeastern Finland during August 2017-January 2018. We determined the occurrence of Chlamydiales bacteria and their natural host, free-living amoeba in respiratory specimens collected during this outbreak with molecular methods. Altogether, 22/278 (7.9%) of the samples contained Chlamydiales DNA. By sequence analysis, majority of the CRBs detected were members of the Parachlamydiaceae family. Amoebal DNA was not detected within the sample material. Our study further proposes that Parachlamydiaceae could be a potential agent causing atypical CAP in children and adolescents.

Transcriptional Expression of the ompA, cpaf, tarp, and tox Genes of Chlamydia trachomatis Clinical Isolates at Different Stages of the Developmental Cycle.

The transcriptional gene expression patterns of Chlamydia trachomatis have mainly been studied using reference strains propagated in cultured cells. Here, using five low-passage-number C. trachomatis clinical isolates that originated from asymptomatic or symptomatic female patients, the in vitro expression of the ompA, cpaf, tarp, and tox genes was studied with reverse transcriptase real-time PCR during the chlamydial developmental cycle. We observed dissimilarities in the gene expression patterns between the low-passage-number clinical isolates and the reference strains. The expression of ompA and the peak of the tox expression were observed earlier in the reference strains than in most of the clinical isolates. The expression of cpaf was high in the reference strains compared with the clinical isolates at the mid-phase (6-24 hours post infection) of the developmental cycle. All of the strains had a rather similar tarp expression profile. Four out of five clinical isolates exhibited slower growth kinetics compared with the reference strains. The use of low-passage-number C. trachomatis clinical isolates instead of reference strains in the studies might better reflect the situation in human infection.

Correction: Hokynar, K. et al. Chlamydia-Like Organisms (CLOs) in Finnish Ixodes ricinus Ticks and Human Skin. Microorganisms 2016, 4, 28.

The authors wish to make the following modification to this paper [...].

Predicting tubal factor infertility by using markers of humoral and cell-mediated immune response against Chlamydia trachomatis.

PROBLEM: The accuracy of Chlamydia trachomatis antibody test in predicting tubal factor infertility (TFI) is limited, and more accurate methods are needed. Cell-mediated immune response (CMI) is crucial in the resolution of pathogen, but it may play an important role in the pathogenesis of C trachomatis-associated tubal damage. We studied whether combining the markers of C trachomatis-induced CMI to humoral immune response improves the accuracy of serology in TFI prediction. METHOD OF STUDY: Our prospective study consists of 258 subfertile women, of whom 22 (8.5%) had TFI. Women with other causes for subfertility served as a reference group. Serum C trachomatis major outer membrane protein (MOMP) and chlamydial heat-shock protein 60 (cHSP60) IgG antibodies were measured by ELISA. CMI was studied by lymphocyte proliferation assay in vitro. RESULTS: Serological markers were more prevalent in women with TFI than in other subfertile women (40.9% vs 12.3% for MOMP IgG and 27.3% vs 10.2% for cHSP60 IgG). The best test combination for TFI was C. trachomatis MOMP and cHSP60 antibody with an accuracy of 90.3%, sensitivity of 22.7% and specificity of 96.6%. Positive post-test probability of this combination was 54.2%, and negative post-test probability was 12.4%. Adding of the markers of CMI did not significantly improve the accuracy of serology in TFI prediction. CONCLUSION: The accuracy of TFI prediction increases when the combination of C trachomatis MOMP and cHSP60 antibody tests is used. C trachomatis-induced CMI was common in our study population, but the markers of CMI did not predict TFI.

Prevalence of Mycoplasma genitalium and mutations associated with macrolide and fluoroquinolone resistance in Finland.

The aim was to examine the prevalence of Mycoplasma genitalium and to determine the prevalence of mutations leading to resistance to macrolides and fluoroquinolones in a sexually transmitted infection clinic setting in Finland, and as a service evaluation, to validate the performance of a commercial Aptima® Mycoplasma genitalium assay. Urogenital samples were studied for M. genitalium with an automated commercial Aptima® Mycoplasma genitalium assay on the Panther® system (Hologic), and with an in-house real-time polymerase chain reaction (PCR) (mgpB). Positive specimens were further studied for mutations associated with macrolide resistance within the 23S rRNA gene and the known quinolone resistance-determining regions within genes gyrA, gyrB and parC. Altogether 17/303 (5.6%) of samples contained M. genitalium by either test. Two of the samples positive by the Aptima assay were not detected by the in-house PCR assay, although the internal control (beta-globin gene) was amplified. The Aptima assay gave an invalid result for five samples, all of which were negative by the in-house PCR. Mutations resulting in macrolide resistance were detected in 30.8% of M. genitalium-positive specimens. Prevalence of M. genitalium infections in the specimens tested is similar to that in other parts of Europe, 5.6%. The Aptima® Mycoplasma genitalium assay detected slightly more positives than the in-house PCR assay. Mutations resulting in macrolide resistance were common in M. genitalium and detection of these mutations is recommended in diagnostic laboratories to assist in selection of treatment.

Chlamydia trachomatis-induced cell-mediated and humoral immune response in women with unexplained infertility.

PROBLEM: What is the role of past Chlamydia trachomatis infection in unexplained infertility? METHOD OF STUDY: This is a prospective study of the impact of past C. trachomatis infection on pregnancy rates in 96 women with unexplained infertility. Both humoral and cell-mediated immune responses (CMI) against C. trachomatis were studied. Serum C. trachomatis IgG antibodies were analyzed using major outer membrane protein (MOMP) peptide-based ELISA. CMI was studied by lymphocyte proliferation (LP) assay in vitro. Data on given fertility treatment, time to pregnancy, and pregnancy outcome were collected. RESULTS: Altogether, 11.5% of the 96 women had C. trachomatis IgG antibodies. LP response to C. trachomatis was positive in 62.9% women. The overall pregnancy rate or live birth rate did not differ by the presence of antichlamydial antibodies or CMI against C. trachomatis. Time to spontaneous pregnancy was longer among C. trachomatis sero-positive women than among sero-negative women (2.9 years vs 2.0 years, P = .03). CONCLUSION: Past chlamydial infection does not play a major role in unexplained infertility. Women with unexplained infertility and positive immune response to C. trachomatis do not have reduced pregnancy rates, but time to spontaneous pregnancy is longer among C. trachomatis IgG sero-positive women than among sero-negative women.