Ronza Hadad

Prevalence trends in Sweden for the new variant of Chlamydia trachomatis

In 2006, a new variant of Chlamydia trachomatis (nvCT) was discovered in Sweden that was not detectable with Abbott m2000 (Abbott) and Amplicor/COBAS Amplicor/TaqMan48 (Roche). The proportion of nvCT was 20-64% of the detected Chlamydia cases in counties using Abbott/Roche test systems. Although the ProbeTec system from Becton Dickinson (BD) could detect nvCT, the proportion of nvCT in counties using BD was 7-19%. The objective of the current study was to follow the nvCT proportions from 2007 to 2009 in two counties that used Roche and had introduced test systems able to detect nvCT in late 2006. The nvCT was also followed in two counties that used BD, and in all four counties the effect of nvCT on the serotype distribution of C. trachomatis wild-type strains was analysed. A total of 2576 specimens positive for C. trachomatis were collected in the four counties at three time points, and analysed for nvCT and serotype E. The proportion of nvCT declined significantly in the two counties using Roche, from 65% and 48% in 2007 to 24% for both counties in 2009 (p <0.001). The nvCT proportion increased in Norrbotten county, which used BD, from 9% in 2007 to 19% in 2009 (p 0.03). In Uppsala county, which also used BD but was surrounded by counties using detection systems from Roche, the proportion of nvCT declined from 24% in 2007 to 18% in 2009 (p <0.03). No major difference in the level of serotype E was seen. The proportion of nvCT seems to rapidly converge in the Swedish counties after the selective diagnostic advantage for nvCT has been lost in the Abbott/Roche counties.

Evaluation of the new COBAS TaqMan CT test v2.0 and impact on the proportion of new variant Chlamydia trachomatis by the introduction of diagnostics detecting new variant C trachomatis in Örebro county, Sweden

Background: The new variant of Chlamydia trachomatis (nvCT), discovered in Sweden in 2006, contains a 377-bp cryptic plasmid deletion, which includes the targets for the COBAS Amplicor/TaqMan C trachomatis/Neisseria gonorrhoea and Abbott m2000rt C trachomatis/N gonorrhoea tests. Objectives: To evaluate the new real-time COBAS TaqMan CT test v2.0 (CTM CT v2.0) for C trachomatis diagnostics and to investigate whether the proportion of nvCT was affected by the introduction of genetic diagnostics detecting nvCT (LightMix 480HT) in Örebro county, Sweden. Methods: CTM CT v2.0 compared with LightMix 480 HT PCR for the diagnosis of C trachomatis was evaluated. Discrepant samples were analysed using BD ProbeTec ET and Abbott m2000rt RealTime CT II. All previously LightMix and cell culture-positive samples were analysed using an nvCT-specific PCR. Results: The sensitivity, specificity, negative predictive value and positive predictive value of CTM CT v2.0 for examined samples (n  =  1058) was 100%, 99.8%, 100% and 98.2%, respectively. Of 11 577 consecutive PCR samples, 9.4% (n  =  1084) were positive and 34.3% (n  =  372) of these were nvCT. Of 2306 consecutive culture samples, 5.0% (n  =  116) were C trachomatis positive and 38.8% (n  =  45) of these were nvCT. Conclusions: CTM CT v2.0 is a sensitive and specific method for C trachomatis detection. Studies including larger numbers of symptomatic and asymptomatic patients as well as genital and extragenital samples, and in comparison with other internationally validated and, ideally, US Food and Drug Administration-approved C trachomatis nucleic acid amplification tests are imperative. The proportion of nvCT remains high in Örebro county, Sweden, despite the introduction of genetic diagnostics to detect the mutant.

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

Chlamydia trachomatis is the worlds 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 pathogens 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.

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.

Novel meningococcal 4CMenB vaccine antigens - prevalence and polymorphisms of the encoding genes in Neisseria gonorrhoeae

The first cross‐protective Neisseria meningitidis vaccine (focus on serogroup B), the protein‐based 4 component meningococcus serogroup B (4CMenB), includes the New Zealand outer membrane vesicle and three main genome‐derived neisserial antigens (GNAs). These GNAs are fHbp (fused to GNA2091), NHBA (fused to GNA1030) and NadA. In this study, the prevalence and polymorphisms of the nucleotide and amino acid sequences of the 4CMenB antigens in a temporally and geographically diverse collection of N. gonorrhoeae isolates (n = 111) were investigated. All the examined GNA genes, except the nadA gene, were present in all gonococcal isolates. However, 25 isolates contained premature stop codons in the fHbp gene and/or the nhba gene, resulting in truncated proteins. Compared with the 4CMenB antigen sequences in reference strain MC58, the gonococcal strains displayed 67.0–95.4% and 60.9–94.9% identity in nucleotide sequence and amino acid sequence, respectively, in the equivalent GNA antigens. The absence of NadA, lack of universal expression of fHbp and NHBA and the uncertainty regarding the surface exposure of fHbp as well as the function of NHBA in N. gonorrhoeae will likely limit the use of the identical 4CMenB antigens in a gonococcal vaccine. However, possible cross‐immunity of 4CMenB with gonococci and expression and function of the equivalent gonococcal GNAs, as well as of more appropriate GNAs for a gonococcal vaccine, need to be further examined.

Oral delivery of plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses.

During early infection with human immunodeficiency virus type 1 (HIV-1), there is a rapid depletion of CD4(+) T-cells in the gut-associated lymphoid tissue (GALT) in the gastrointestinal tract. Therefore, immediate protection at these surfaces is of high priority for the development of an HIV-1 vaccine. Thus, transgenic plants expressing HIV-1 antigens, which are exposed to immune competent cells in the GALT during oral administration, can be interesting as potential vaccine candidates. In the present study, we used two HIV-1 p24 antigen-expressing transgenic plant systems, Arabidopsis thaliana and Daucus carota, in oral immunization experiments. Both transgenic plant systems showed a priming effect in mice and induced humoral immune responses, which could be detected as anti-p24-specific IgG in sera after an intramuscular p24 protein boost. Dose-dependent antigen analyses using transgenic A. thaliana indicated that low p24 antigen doses were superior to high p24 antigen doses.

First reported case of the Swedish new variant of Chlamydia trachomatis (nvCT) in Eastern Europe (Russia), and evaluation of Russian nucleic acid amplification tests regarding their ability to detect nvCT.

First Reported Case of the Swedish New Variant of Chlamydia trachomatis (nvCT) in Eastern Europe (Russia), and Evaluation of Russian Nucleic Acid Amplification Tests Regarding Their Ability to Detect nvCT

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.

P2.087 In Vitro Antimicrobial Synergy Testing, Using Etest Methodology, of Neisseria Gonorrhoeae For Evaluation of Susceptibility When Using Dual Antimicrobial Therapy?

Background Antimicrobial resistance in Neisseria gonorrhoeae is a major public health problem worldwide. Recently, the first gonococcal isolates with high-level resistance to extended-spectrum cephalosporins, including ceftriaxone, were reported and gonorrhoea may become untreatable in certain circumstances. As a response, dual antimicrobial therapy (mainly ceftriaxone+azithromycin) has been introduced in the USA and Europe. The aim of this study was to apply a method for in vitro synergy testing, using Etest methodology, of various combinations of antimicrobials, i.e. currently used or of potential interest for future dual antimicrobial therapy. Methods The eight WHO 2008 N. gonorrhoeae reference strains and 51 clinical N. gonorrhoeae isolates were investigated by synergy testing using Etest of in total 15 combinations of ceftriaxone, cefixime, azithromycin, moxifloxacin, spectinomycin, and gentamicin. Results Highest levels of synergistic and/or additive effects, without any observed antagonistic effects, were observed for the combinations cefixime+gentamicin (in total 80% of isolates), azithromycin+gentamicin (65%), and cefixime+azithromycin (63%). The combination of ceftriaxone+azithromycin, currently recommended in the dual antimicrobial therapy, also showed substantial synergistic and/or additive effects (34%), without any observed antagonistic effects. Nevertheless, the results of in vitro antimicrobial synergy testing need to be interpreted with some caution, because these may not absolutely correspond to the in vivo situation. Conclusion This study demonstrates in vitro synergy between several of the antimicrobials currently used or potentially considered for dual antimicrobial therapy of gonorrhoea and this is also the first study using Etest as an objective, easily performed and reproducible in vitro method for dual antimicrobial synergy testing of N. gonorrhoeae. Such method might be crucial if susceptibility testing for combination antimicrobial therapy will be performed prior to treatment of gonorrhoea.

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.