Comprehensive global genome dynamics of Chlamydia trachomatis show ancient diversification followed by contemporary mixing and recent lineage expansion.

Chlamydia trachomatis is the world's most prevalent bacterial sexually transmitted infection and leading infectious cause of blindness, yet it is one of the least understood human pathogens, in part due to the difficulties of in vitro culturing and the lack of available tools for genetic manipulation. Genome sequencing has reinvigorated this field, shedding light on the contemporary history of this pathogen. Here, we analyze 563 full genomes, 455 of which are novel, to show that the history of the species comprises two phases, and conclude that the currently circulating lineages are the result of evolution in different genomic ecotypes. Temporal analysis indicates these lineages have recently expanded in the space of thousands of years, rather than the millions of years as previously thought, a finding that dramatically changes our understanding of this pathogen's history. Finally, at a time when almost every pathogen is becoming increasingly resistant to antimicrobials, we show that there is no evidence of circulating genomic resistance in C. trachomatis.

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.

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.

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.

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.

Granuloma Annulare and Morphea: Correlation with Borrelia burgdorferi Infections and Chlamydia-related Bacteria.

A retrospective study of 109 skin biopsies with granuloma annulare (GA) or morphea histology from patients with suspected tick bite was performed. Biopsies were tested for cutaneous Borrelia burgdorferi DNA using PCR. The same biopsies were analysed for tick-borne novel agents, Chlamydia-related bacteria (members of the Chlamydiales order), using a PCR-based method. Borrelia DNA was detected in 7/73 (9.6%) biopsies with GA and in 1/36 (2.8 %) biopsies with morphea, while Chlamydiales DNA was found in 53/73 (72.6%) biopsies with GA and 25/34 (73.4%) biopsies with morphea. All Borrelia DNA-positive GA samples were also positive for Chlamydiales DNA. The Chlamydiales sequences detected in GA were heterogeneous and contained Waddliaceae and Rhabdochlamydiaceae bacteria, which are also present in Ixodes ricinus ticks, while the Chlamydiales sequences detected in morphea closely resembled those found in healthy skin. In conclusion, tick-mediated infections can trigger GA in some cases, while correlation of either Borrelia or Chlamydiales with morphea is unlikely.

Evolution of acute infection with atypical bacteria in a prospective cohort of children with community-acquired pneumonia receiving amoxicillin.

Background: Atypical bacteria are treatable causative agents of community-acquired pneumonia (CAP). However, there is no conclusive evidence that a child with CAP should receive empirical treatment against such agents. Objectives: We assessed the possibility of association between clinical failure and acute infection by these bacteria among children with CAP treated with amoxicillin. Patients and methods: Patients aged 2-59 months with non-severe CAP received amoxicillin during prospective follow-up. Acute and convalescent blood samples were collected. Probable acute infection by Mycoplasma pneumoniae (specific IgM antibodies), by Chlamydia pneumoniae or Chlamydia trachomatis (specific IgM antibodies and/or IgG/IgA titre change) was investigated. Outcomes were assessed during follow-up at 2, 5 and 14-28 days. Treatment failure included development of danger signs, persistent fever, tachypnoea or death. ClinicalTrials.gov: NCT01200706. Results: Of 787 children, 86 (10.9%; 95% CI = 8.9%-13.3%) had acute M. pneumoniae infection. C. pneumoniae acute infection was found in 79 of 733 (10.8%; 95% CI = 8.7%-13.2%) and C. trachomatis was found in 3 of 28 (10.7%; 95% CI = 2.8%-26.5%) <6 months old. Among patients with or without treatment failure at 2 days, acute M. pneumoniae infection (11.7% versus 10.7%; P = 0.7), acute C. pneumoniae infection (8.5% versus 11.3%; P = 0.3) and acute C. trachomatis infection (16.7% versus 9.1%; P = 0.5) were found. No significant differences were found with regard to treatment failure at the 5 day evaluation. Overall, amoxicillin was substituted in 3.5% versus 2.7% among patients with or without acute infection by one of these bacteria ( P = 0.6). Conclusions: The overall substitution rate of amoxicillin was very low. It is not necessary to give an empirical non-ß-lactam antibiotic as a first-line option to treat every child between 2 and 59 months old with non-severe CAP.

Host cell Golgi anti-apoptotic protein (GAAP) and growth of Chlamydia pneumoniae.

Chlamydia pneumoniae protein CPn0809 is a type three secretion system substrate, the exact function of which in infection pathogenesis has remained unknown. In this study, we identified by yeast two-hybrid screening a potential host cell interaction partner of CPn0809, Golgi anti-apoptotic protein (GAAP), a conserved protein found in eukaryotic cells. GAAP gene is expressed at relatively constant levels and its expression remained stable also after C. pneumoniae infection. The interaction between GAAP and C. pneumoniae was suggested by transfection studies. GAAP knock-down by siRNA in infected A549 cells resulted in an increased number of C. pneumoniae genomes and growth of the bacteria as judged by quantitative PCR and inclusion counts, respectively. Silencing of GAAP did not make the A549 cells more susceptible to apoptosis per se, and infection with C. pneumoniae prevented staurosporin-induced apoptosis also in transfected cultures. Taken together, the proposed interaction between C. pneumoniae and GAAP modulates bacterial growth in A549 cells.

ABC-cassette transporter 1 (ABCA1) expression in epithelial cells in Chlamydia pneumoniae infection.

ATP-binding cassette transporter A1 (ABCA1) mediates reverse cholesterol transport and innate immunity response in different cell types. We have investigated the regulation of ABCA1 expression in response to intracellular Chlamydia pneumoniae infection in A549 epithelial lung carcinoma cells. C. pneumoniae infection decreased ABCA1 expression in A549 cells, and the activity of the ABCA1 promoter was decreased. The decreased promoter activity was dependent on its E-box and GnT-box elements of the promoter. Chlamydial growth was decreased in ABCA1-silenced epithelial lung carcinoma cells. These data indicate an important role for ABCA1 in intracellular bacterial infection.

[Fever, skin rash and blood bullae of the ears in a middle-aged woman]. / Keski-ikäisen naisen kuume, ihottuma ja korvien veribullat.

A healthcare worker sought medical advice after four days of fever, muscle pains, occipital headache and blocked ears, and was diagnosed with a high CRP level and blood bullae in the outer ear canal. In addition, skin rash appeared during hospital care, and the fever did not seem to go down upon treatment with broad-spectrum antibiotics, during which the CRP rose to a level of 413 mg/L at the highest. Haemorragic bullous otisis was confirmed diagnosis caused by Mycoplasma Pneumoniae.

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.

Flotillin-1 (Reggie-2) contributes to Chlamydia pneumoniae growth and is associated with bacterial inclusion.

Chlamydiae are obligate intracellular pathogens replicating only inside the eukaryotic host. Here, we studied the effect of human flotillin-1 protein on Chlamydia pneumoniae growth in human line (HL) and A549 epithelial cell lines. RNA interference was applied to disrupt flotillin-1-mediated endocytosis. Host-associated bacteria were detected by quantitative PCR, and C. pneumoniae growth was evaluated by inclusion counts. C. pneumoniae attachment to host cells was unaffected, but bacterial intracellular growth was attenuated in the flotillin-1-silenced cells. By using confocal microscopy, we detected flotillin-1 colocalized with the inclusion membrane protein A (IncA) in the C. pneumoniae inclusion membranes. In addition, flotillin-1 was associated with IncA in detergent-resistant membrane microdomains (DRMs) in biochemical fractioning. These results suggest that flotillin-1 localizes to the C. pneumoniae inclusion membrane and plays an important role for intracellular growth of C. pneumoniae.

Chlamydia pneumoniae entry into epithelial cells by clathrin-independent endocytosis.

A gram-negative obligate intracellular bacterium, Chlamydia pneumoniae, is a common respiratory pathogen. Here, we examined the invasion and attachment of C. pneumoniae K6 into nonphagocytic HL epithelial cell line by manipulating host plasma membranes by using cholesterol-depleting methyl-beta-cyclodextrin (MßCD) and cholesterol-loading MßCD complexed cholesterol (chol-MßCD). The invasion was attenuated by MßCD-treatment while chol-MßCD augmented the attachment and invasion. In addition, the invasion was inhibited by cholesterol sequestering reagents, nystatin and filipin. Furthermore, exposure of host cells to sphingomyelinase inhibited the invasion. RNA interference was used to assay the role of clathrin and human scavenger receptor B, type I (SR-BI) in the entry of C. pneumoniae into A549 lung epithelial adenocarcinoma cells. In contrast to Chlamydia trachomatis L2, the entry of C. pneumoniae was found to be independent of clathrin. In addition, the entry was found to be SR-BI-independent, but interestingly, the chlamydial growth was attenuated in the SR-BI-silenced cells. These findings suggest that the attachment and invasion of C. pneumoniae into nonphagocytic epithelial cells is dependent on the formation of cholesterol- and sphingomyelin-rich plasma membrane microdomains, and the entry is a clathrin-independent process. In addition, our data indicate that SR-BI supports the growth of C. pneumoniae in epithelial cells.

Genotyping of Chlamydia trachomatis in rectal and pharyngeal specimens: identification of LGV genotypes in Finland.

OBJECTIVES: Lymphogranuloma venereum (LGV) infections caused by Chlamydia trachomatis L types have recently emerged in Europe among HIV-positive men having sex with men. Our aim was to introduce a genotyping strategy suitable for a diagnostic laboratory using nucleic acid amplification tests (NAATs) for detection of C trachomatis and to investigate the prevalence of LGV types in rectal and pharyngeal specimens in Finland. METHODS: Aptima Combo 2 (Gen-Probe) was used to detect C trachomatis in swabs. Altogether 140 C trachomatis NAAT-positive rectal and pharyngeal samples were genotyped by pmpH and ompA real-time PCR. RESULTS: Of the 140 NAAT-positive rectal and pharyngeal specimens, 114 (81%) were successfully typed by pmpH PCR. One hundred and four samples contained non-LGV, nine samples LGV and one sample both non-LGV and LGV C trachomatis types. The C trachomatis LGV types were mainly found in rectal samples. Six of the L types were confirmed to be genotype L2b and two were L2 with ompA PCR and sequencing. CONCLUSIONS: Our experience suggests that genotyping C trachomatis by pmpH PCR can be introduced as a function of a diagnostic laboratory already using NAAT for detection of C trachomatis. The data show that LGV infections occur also in Finland. LGV should be taken into account when considering treatment and management of rectal C trachomatis infections.

[Mycoplasma pneumoniae infections]. / Mycoplasma pneumoniae -infektiot.

Mycoplasma pneumoniae causes respiratory infections, but in as many as one out of four of those having contracted the infection symptoms may appear elsewhere, sometimes without preceding respiratory symptoms. Among these, symptoms of central nervous system origin are most the common. Specific diagnosis of the infection is demanding, and is mainly based on serology. Application of nucleic acid detection tests might lead to an earlier diagnostics. Efforts associated with diagnosis should mainly be targeted at detecting epidemics as well as diagnosing patients with severe or abnormal symptoms during the epidemy.

Analysis of Chlamydia pneumoniae infection in mononuclear cells by reverse transcription-PCR targeted to chlamydial gene transcripts.

Chlamydia pneumoniae (C. pneumoniae) is an important etiological agent of respiratory infections including pneumonia. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that monocytes can assist the spread of infection to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells, we have established an in vitro model in the human Mono Mac 6 cell line. In the present study, we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real-time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells. Furthermore, our study shows that genes related to secretion are transcribed, and secreted bacterial proteins are also translated during infection of monocytes, creating novel opportunities for the management of chlamydial infection of monocytes.