No widespread dissemination of Chlamydia trachomatis diagnostic-escape variants and the impact of Neisseria gonorrhoeae positivity on the Aptima Combo 2 assay.

OBJECTIVES: A Finnish Chlamydia trachomatis (CT) new variant was detected in 2019 that escaped detection in the Hologic Aptima Combo 2 (AC2) assay due to a C1515T mutation in the CT 23S rRNA target region. Reflex testing of CT-negative/CT-equivocal specimens as well as those positive for Neisseria gonorrhoeae (NG) with the Hologic Aptima CT (ACT) assay was recommended to identify any CT variants. METHODS: From June to October 2019, specimens with discrepant AC2/ACT CT results were submitted to Public Health England and screened for detectable CT DNA using an inhouse real-time (RT)-PCR. When enough DNA was present, partial CT 23S rRNA gene sequencing was performed. Analysis of available relative light units and interpretative data was performed. RESULTS: A total of 317 discordant AC2/ACT specimens were collected from 315 patients. Three hundred were tested on the RT-PCR; 53.3% (n=160) were negative and 46.7% (n=140) were positive. Due to low DNA load in most specimens, sequencing was successful for only 36 specimens. The CT 23S rRNA wild-type sequence was present in 32 specimens, and two variants with C1514T or G1523A mutation were detected in four specimens from three patients. Of the discordant specimens with NG interpretation, 36.6% of NG-negative/CT-negative AC2 specimens had detectable CT DNA on the inhouse RT-PCR vs 53.3% of NG-positive/CT-negative specimens. CONCLUSIONS: No widespread dissemination of AC2 diagnostic-escape CT variants has occurred in England. We however identified the impact of NG positivity on the discordant AC2/ACT specimens; a proportion appeared due to NG positivity and the associated NG signal, rather than any diagnostic-escape variants or low DNA load. Several patients with gonorrhoea may therefore receive false-negative AC2 CT results. Single diagnostic targets and multiplex diagnostic assays have their limitations such as providing selection pressure for escape mutants and potentially reduced sensitivity, respectively. These limitations must be considered when establishing diagnostic pathways.

Evaluation of the SpeeDx ResistancePlus® GC and SpeeDx GC 23S 2611 (beta) molecular assays for prediction of antimicrobial resistance/susceptibility to ciprofloxacin and azithromycin in Neisseria gonorrhoeae.

BACKGROUND: Accurate molecular assays for prediction of antimicrobial resistance (AMR)/susceptibility in Neisseria gonorrhoeae (Ng) can offer individualized treatment of gonorrhoea and enhanced AMR surveillance. OBJECTIVES: We evaluated the new ResistancePlus® GC assay and the GC 23S 2611 (beta) assay (SpeeDx), for prediction of resistance/susceptibility to ciprofloxacin and azithromycin, respectively. METHODS: Nine hundred and sixty-seven whole-genome-sequenced Ng isolates from 20 European countries, 143 Ng-positive (37 with paired Ng isolates) and 167 Ng-negative clinical Aptima Combo 2 (AC2) samples, and 143 non-gonococcal Neisseria isolates and closely related species were examined with both SpeeDx assays. RESULTS: The sensitivity and specificity of the ResistancePlus® GC assay to detect Ng in AC2 samples were 98.6% and 100%, respectively. ResistancePlus® GC showed 100% sensitivity and specificity for GyrA S91 WT/S91F detection and 99.8% sensitivity and specificity in predicting phenotypic ciprofloxacin resistance. The sensitivity and specificity of the GC 23S 2611 (beta) assay for Ng detection in AC2 samples were 95.8% and 100%, respectively. GC 23S 2611 (beta) showed 100% sensitivity and 99.9% specificity for 23S rRNA C2611 WT/C2611T detection and 64.3% sensitivity and 99.9% specificity for predicting phenotypic azithromycin resistance. Cross-reactions with non-gonococcal Neisseria species were observed with both assays, but the analysis software solved most cross-reactions. CONCLUSIONS: The new SpeeDx ResistancePlus® GC assay performed well in the detection of Ng and AMR determinants, especially in urogenital samples. The GC 23S 2611 (beta) assay performed relatively well, but its sensitivity, especially for predicting phenotypic azithromycin resistance, was suboptimal and further optimizations are required, including detection of additional macrolide resistance determinant(s).

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.

Prevalence of Mycoplasma genitalium and Antibiotic Resistance-Associated Mutations in Patients at a Sexually Transmitted Infection Clinic in Iceland, and Comparison of the S-DiaMGTV and Aptima Mycoplasma genitalium Assays for Diagnosis.

Mycoplasma genitalium is prevalent among attendees in sexually transmitted infection (STI) clinics, and therapy is hampered by rapidly rising levels of resistance to azithromycin and moxifloxacin. In this study, we evaluated, for the first time in Iceland, the prevalence of M. genitalium and azithromycin and moxifloxacin resistance-associated mutations and assessed the diagnostic performance of the CE/in vitro diagnosis (IVD)-marked S-DiaMGTV (Diagenode Diagnostics) versus the U.S. FDA/CE/IVD-approved Aptima MG (AMG; Hologic) for M. genitalium detection. From October 2018 to January 2019, urine and vaginal swabs were provided by male and female attendees at Iceland's only STI clinic. Specimens were tested with S-DiaMGTV and AMG, and resistance-associated mutations were determined by 23S rRNA gene and parC sequencing. Demographic and clinical data were collected from patient records. M. genitalium prevalence was 9.3% overall; 7.7% (38/491) among male and 10.9% (53/487) among female participants. Azithromycin and moxifloxacin resistance-associated mutations were found in 57.0% (45/79) and 0.0% (0/80) of evaluable specimens, respectively. Sensitivity was 72.5% and 100%, and specificity was 99.9% and 100% for S-DiaMGTV and AMG, respectively. No association was found between M. genitalium and symptoms of urethritis in men. Prevalence rates for M. genitalium and azithromycin resistance-associated genes in Iceland are among the highest reported in Europe. The significantly higher sensitivity of AMG over that of S-DiaMGTV can have important clinical implications. More information is urgently needed to clarify the significance of false-negative results obtained with S-DiaMGTV and other similarly performing widely used real-time PCR methods for diagnosis and management of this sexually transmitted 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.

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.

The 'Finnish new variant of Chlamydia trachomatis' escaping detection in the Aptima Combo 2 assay is widespread across Norway, June to August 2019.

The 'Finnish new variant of Chlamydia trachomatis' (FI-nvCT), escaping detection in the Aptima Combo 2 assay (AC2), is widespread across Norway. From June to August 2019, 84% (81/97) of available AC2/Aptima CT discordant samples from five laboratories were confirmed as FI-nvCT. Two additional CT variants (CT 23S rRNA C1514T and G1523A) also escaped AC2 detection. The high FI-nvCT proportion might indicate a long-term national spread and it cannot be excluded that FI-nvCT emerged in Norway.

Ten years transmission of the new variant of Chlamydia trachomatis in Sweden: prevalence of infections and associated complications.

OBJECTIVES: In 2006, a new variant of Chlamydia trachomatis (nvCT) was discovered in Sweden. It has a deletion in the plasmid resulting in failed detection by the single target systems from Abbott and Roche used at that time, whereas the third system used, from Becton Dickinson (BD), detects nvCT. The proportion of nvCT was initially up to 65% in counties using Abbott/Roche systems. This study analysed the proportion of nvCT from 2007 to 2015 in four selected counties and its impact on chlamydia-associated complications. METHODS: C. trachomatis-positive specimens collected from 2007 to 2015 were analysed by a specific PCR to identify nvCT cases. Genotyping was performed by multilocus sequence typing (MLST) and ompA sequencing. Ectopic pregnancy and pelvic inflammatory disease records were extracted from the national registers. RESULTS: In total, 5101 C. trachomatis-positive samples were analysed. The nvCT proportion significantly decreased in the two counties using Roche systems, from 56% in 2007 to 6.5% in 2015 (p<0.001). In the two counties using BD systems, a decrease was also seen, from 19% in 2007 to 5.2% in 2015 (p<0.001). Fifteen nvCT cases from 2015 and 102 cases from 2006 to 2009 had identical MLST profiles. Counties using Roche/Abbott systems showed higher mean rates of ectopic pregnancy and pelvic inflammatory disease compared with counties using BD systems. CONCLUSIONS: The nvCT proportion has decreased in all counties and converged to a low prevalence irrespective of previous rates. Genotyping showed that nvCT is clonal and genetically stable. Failing detection only marginally affected complication rates.

Lymphogranuloma venereum (LGV) in men who have sex with men (MSM): a re-emerging problem, Malta, 2018.

From 1 January to 30 June 2018, 11 cases of Lymphogranuloma venereum (LGV; all preserved samples (n = 4) genovar L2b) were identified at the Genitourinary Clinic (GUC), Mater Dei Hospital, Msida, Malta. All cases were diagnosed in men who have sex with men (MSM); six participated in three group-sex parties. Here, we describe the outbreak and risk factors associated with LGV diagnoses in MSM in Malta in 2018.

Mycoplasma genitalium prevalence, antimicrobial resistance-associated mutations, and coinfections with non-viral sexually transmitted infections in high-risk populations in Guatemala, Malta, Morocco, Peru and South Africa, 2019-2021.

The prevalence of Mycoplasma genitalium (MG) and MG antimicrobial resistance (AMR) appear to be high internationally, however, prevalence data remain lacking globally. We evaluated the prevalence of MG and MG AMR-associated mutations in men who have sex with men (MSM) in Malta and Peru and women at-risk for sexually transmitted infections in Guatemala, South Africa, and Morocco; five countries in four WHO regions mostly lacking MG prevalence and AMR data, and estimated MG coinfections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Male urine and anorectal samples, and vaginal samples were tested for MG, CT, NG, and TV (only vaginal samples) using Aptima assays (Hologic). AMR-associated mutations in the MG 23S rRNA gene and parC gene were identified using ResistancePlus MG kit (SpeeDx) or Sanger sequencing. In total, 1,425 MSM and 1,398 women at-risk were recruited. MG was detected in 14.7% of MSM (10.0% in Malta and 20.0% Peru) and in 19.1% of women at-risk (12.4% in Guatemala, 16.0% Morocco, 22.1% South Africa). The prevalence of 23S rRNA and parC mutations among MSM was 68.1 and 29.0% (Malta), and 65.9 and 5.6% (Peru), respectively. Among women at-risk, 23S rRNA and parC mutations were revealed in 4.8 and 0% (Guatemala), 11.6 and 6.7% (Morocco), and 2.4 and 3.7% (South Africa), respectively. CT was the most frequent single coinfection with MG (in 2.6% of MSM and 4.5% of women at-risk), compared to NG + MG found in 1.3 and 1.0%, respectively, and TV + MG detected in 2.8% of women at-risk. In conclusion, MG is prevalent worldwide and enhanced aetiological MG diagnosis, linked to clinical routine detection of 23S rRNA mutations, in symptomatic patients should be implemented, where feasible. Surveillance of MG AMR and treatment outcome would be exceedingly valuable, nationally and internationally. High levels of AMR in MSM support avoiding screening for and treatment of MG in asymptomatic MSM and general population. Ultimately, novel therapeutic antimicrobials and/or strategies, such as resistance-guided sequential therapy, and ideally an effective MG vaccine are essential.

Prevalence trends of the new variant of Chlamydia trachomatis in four counties of Sweden in 2007-2011.

A new variant of Chlamydia trachomatis (nvCT) was discovered in Sweden in 2006, and it could not be detected by diagnostic systems from Abbott and Roche, whereas the third system used, from Becton Dickinson (BD), detects nvCT. We analyzed 3648 samples from 2 counties that used Roche and 2 counties that used BD methods from 2007 to 2011. After implementation of a Roche method that detects nvCT, its proportion has decreased and converged in the 4 counties but are still at different levels in Roche and BD counties. Future studies are needed to see if nvCT will decline further.

First National Genomic Epidemiological Study of Neisseria gonorrhoeae Strains Spreading Across Sweden in 2016.

The increasing transmission and antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global health concern with worrying trends of decreasing susceptibility to also the last-line extended-spectrum cephalosporin (ESC) ceftriaxone. A dramatic increase of reported gonorrhea cases has been observed in Sweden from 2016 and onward. The aim of the present study was to comprehensively investigate the genomic epidemiology of all cultured N. gonorrhoeae isolates in Sweden during 2016, in conjunction with phenotypic AMR and clinical and epidemiological data of patients. In total, 1279 isolates were examined. Etest and whole-genome sequencing (WGS) were performed, and epidemiological data obtained from the Public Health Agency of Sweden. Overall, 51.1%, 1.7%, and 1.3% resistance to ciprofloxacin, cefixime, and azithromycin, respectively, was found. No isolates were resistant to ceftriaxone, however, 9.3% of isolates showed a decreased susceptibility to ceftriaxone and 10.5% to cefixime. In total, 44 penA alleles were found of which six were mosaic (n = 92). Using the typing schemes of MLST, NG-MAST, and NG-STAR; 133, 422, and 280 sequence types, respectively, and 93 NG-STAR clonal complexes were found. The phylogenomic analysis revealed two main lineages (A and B) with lineage A divided into two main sublineages (A1 and A2). Resistance and decreased susceptibility to ESCs and azithromycin and associated AMR determinants, such as mosaic penA and mosaic mtrD, were predominantly found in sublineage A2. Resistance to cefixime and azithromycin was more prevalent among heterosexuals and MSM, respectively, and both were predominantly spread through domestic transmission. Continuous surveillance of the spread and evolution of N. gonorrhoeae, including phenotypic AMR testing and WGS, is essential for enhanced knowledge regarding the dynamic evolution of N. gonorrhoeae and gonorrhea epidemiology.

A Chlamydia trachomatis 23S rRNA G1523A variant escaping detection in the Aptima Combo 2 assay (Hologic) was widespread across Denmark in July-September 2019.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection globally, and nucleic acid amplification tests (NAATs) are recommended for highly sensitive and specific diagnosis. In early 2019, the Finnish new variant of Chlamydia trachomatis (FI-nvCT) was identified. The FI-nvCT has a C1515T mutation in the 23S rRNA gene, making it escaping detection in the Aptima Combo 2 (AC2; Hologic) NAAT, and the FI-nvCT has been subsequently reported in Sweden and Norway. In the present study, we investigated the presence of the FI-nvCT and other AC2 diagnostic-escape CT mutants in July-September 2019 in Denmark. The FI-nvCT was present but rare in Denmark. However, another AC2 diagnostic-escape CT mutant (with a 23S rRNA G1523A mutation) was found to be widespread across Denmark, accounting for 95% (76/80) of AC2 diagnostic-escape nvCT samples from five Danish CT-diagnostic laboratories. This nvCT-G1523A has previously only been detected in one single sample in the United Kingdom and Norway, respectively. It is vital to monitor the continued stability of the NAAT targets in local, national and international settings and monitor as well as appropriately analyse incidence, unexplained shifts in diagnostics rates and/or annual collections of samples diagnosed as negative/equivocal using NAATs with different target(s). Furthermore, diagnostic CT NAATs with dual target sequences are crucial, and fortunately, an updated Hologic AC2 assay including one additional target sequence is in advanced development.

The Neisseria gonorrhoeae population in Sweden during 2005-phenotypes, genotypes and antibiotic resistance.

In Sweden, the gonorrhoea incidence has significantly increased since an all-time low in 1996. We aimed to phenotypically and genotypically characterise N. gonorrhoeae isolates (n=180) transmitted in Sweden during 2005. All isolates were susceptible to cefixime, ceftriaxone, and spectinomycin. However, 2%, 50% and 75% displayed intermediate susceptibility or resistance to azithromycin, ciprofloxacin and ampicillin, respectively. The isolates were assigned to 28 different serovars using Genetic Systems monoclonal antibodies (Mabs) (discriminatory index, 91.0%) and 46 different serovars using Pharmacia Mabs (index, 94.4%). Furthermore, they displayed 95 porB sequences (index, 97.8%) and 95 N. gonorrhoeae multiantigen sequence typing (NG-MAST) sequence types (STs) (index, 98.0%). 51 (54%) of these STs have not been previously described. 14 ST clusters, comprising between 3 and 15 isolates, were identified that indicate the existence of several transmission chains. The high number of unique STs (n=63) may be associated with import of strains from abroad, local emergence of new STs, incomplete epidemiological surveillance, and/or suboptimal diagnostics, including contact tracing. Overall, the Swedish N. gonorrhoeae population was remarkably diversified. Comprehensive knowledge regarding transmission, phenotypes (including antibiotic resistance), but also in many cases highly discriminative and precise genotypic characteristics of the N. gonorrhoeae strains circulating in our societies, is crucial.

Macrolide and fluoroquinolone resistance in Mycoplasma genitalium in two Swedish counties, 2011-2015.

Mycoplasma genitalium, causing non-gonococcal non-chlamydial urethritis and associated with cervicitis, has developed antimicrobial resistance (AMR) to both the macrolide azithromycin (first-line treatment) and the fluoroquinolone moxifloxacin (second-line treatment). Our aim was to estimate the prevalence of resistance, based on genetic AMR determinants, to these antimicrobials in the M. genitalium population in two Swedish counties, Örebro and Halland, 2011-2015. In total, 672 M. genitalium positive urogenital samples were sequenced for 23S rRNA and parC gene mutations associated with macrolide and fluoroquinolone resistance, respectively. Of the samples, 18.6% and 3.2% in Örebro and 15.2% and 2.7% in Halland contained mutations associated with macrolide and fluoroquinolone resistance, respectively. The predominating resistance-associated mutations in the 23S rRNA gene was A2059G (n = 39) in Örebro and A2058G (n = 13) and A2059G (n = 13) in Halland. The most prevalent possible resistance-associated ParC amino acid alterations were S83I (n = 4) in Örebro and S83N (n = 2) in Halland. Resistance-associated mutations to both macrolides and fluoroquinolones were found in 0.7% of samples. Our findings emphasize the need for routine AMR testing, at a minimum for macrolide resistance, of all M. genitalium-positive samples and regular national and international surveillance of AMR in M. genitalium, to ensure effective patient management and rational antimicrobial use.

Protection against genital tract Chlamydia trachomatis infection following intranasal immunization with a novel recombinant MOMP VS2/4 antigen.

The asymptomatic nature of most Chlamydia trachomatis infections and the lack of appropriate effects by current prevention and management call for vaccine development. We evaluated a recombinant subunit vaccine candidate based on the major outer membrane protein variable segments 2 and 4 (MOMP VS2/4). To achieve maximal immunogenicity and ease of production and purification, MOMP VS2/4 was constructed by using highly immunogenic sequences of MOMP only, thereby minimizing the presence of hydrophobic regions, and spacing the immunogenic epitopes with a flexible amino acid sequence. A purification tag was also added. The MOMP VS2/4 was given intranasally, with or without intravaginal boost, with cholera toxin (CT) adjuvant to C57BL/6 mice, which were screened for immunogenicity and protection against a live challenge infection with C. trachomatis serovar D. Bacterial shedding, cell-mediated responses, and antibody responses were monitored. Immunized mice exhibited significantly less bacterial shedding and were better protected against infertility as compared to unimmunized control mice. Immunizations stimulated both systemic and local specific antibody (IgG1, IgG2c, and IgA) responses, and primed T cells that produced interferon-γ and interleukins 13 and 17 upon challenge with recall antigen. Thus, MOMP VS2/4, in combination with CT adjuvant, stimulated Th1, Th2, and Th17 effector cells, and generated protective immunity associated with less pathology. We regard MOMP VS2/4 as a promising candidate for further development into a mucosal chlamydial vaccine.