Jenny Isaksson

Comparison of species identification of endocarditis associated viridans streptococci using rnpB genotyping and 2 MALDI-TOF systems

Streptococcus spp. are important causes of infective endocarditis but challenging in species identification. This study compared identification based on sequence determination of the rnpB gene with 2 systems of matrix-assisted laser desorption ionization-time of flight mass spectrometry, MALDI Biotyper (Bruker) and VITEK MS IVD (bioMérieux). Blood culture isolates of viridans streptococci from 63 patients with infective endocarditis were tested. The 3 methods showed full agreement for all 36 isolates identified in the Anginosus, Bovis, and Mutans groups or identified as Streptococcus cristatus, Streptococcus gordonii, or Streptococcus sanguinis. None of the methods could reliably identify the 23 isolates to the species level when designated as Streptococcus mitis, Streptococcus oralis, or Streptococcus tigurinus. In 7 isolates classified to the Mitis group, the rnpB sequences deviated strikingly from all reference sequences, and additional analysis of sodA and groEL genes indicated the occurrence of yet unidentified Streptococcus spp.

Global Multilocus Sequence Type Analysis of Chlamydia trachomatis Strains from 16 Countries

ABSTRACT The Uppsala University Chlamydia trachomatis multilocus sequence type (MLST) database (http://mlstdb.bmc.uu.se) is based on five target regions (non-housekeeping genes) and the ompA gene. Each target has various numbers of alleles—hctB, 89; CT058, 51; CT144, 30; CT172, 38; and pbpB, 35—derived from 13 studies. Our aims were to perform an overall analysis of all C. trachomatis MLST sequence types (STs) in the database, examine STs with global spread, and evaluate the phylogenetic capability by using the five targets. A total of 415 STs were recognized from 2,089 specimens. The addition of 49 ompA gene variants created 459 profiles. ST variation and their geographical distribution were characterized using eBURST and minimum spanning tree analyses. There were 609 samples from men having sex with men (MSM), with 4 predominating STs detected in this group, comprising 63% of MSM cases. Four other STs predominated among 1,383 heterosexual cases comprising, 31% of this group. The diversity index in ocular trachoma cases was significantly lower than in sexually transmitted chlamydia infections. Predominating STs were identified in 12 available C. trachomatis whole genomes which were compared to 22 C. trachomatis full genomes without predominating STs. No specific gene in the 12 genomes with predominating STs could be linked to successful spread of certain STs. Phylogenetic analysis showed that MLST targets provide a tree similar to trees based on whole-genome analysis. The presented MLST scheme identified C. trachomatis strains with global spread. It provides a tool for epidemiological investigations and is useful for phylogenetic analyses.

Bubonic lymphogranuloma venereum with multidrug treatment failure

A patient with proctitis and inguinal buboes diagnosed with lymphogranuloma venereum (LGV) was treated with doxycycline 21 days, azithromycin 20 days and moxifloxacin for a further 12 days because of progressive worsening of inguinal symptoms. Despite extensive antibiotic treatment, the inguinal LGV lesions persisted; however, the patient recovered spontaneously after three months.

Comparison of multilocus sequence typing and multilocus typing microarray of Chlamydia trachomatis strains from Argentina and Chile.

This study compared conventional ompA genotyping of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus typing (MLT) DNA microarray. DNA extracts of 104 C. trachomatis positive specimens were analyzed by ompA sequencing and MLST and of these 76 by MLT array. Obtained MLST sequence types (STs) were compared to sequences in the database http://mlstdb.uu.se. The resolution obtained for MLST (35 STs) was 2.1 higher than for ompA sequencing (17 variants) and 1.3 higher than MLT array (27 MLT groups). Among the 104 samples the predominant genotype E could be divided into 5 ompA variants and 23 STs of which 16 had not been reported in previous studies. The most common STs, ST3 and ST56, were identified as founders and are common in several countries on a global scale. The MLST and the MLT array provided similar strain discrimination capacity and showed considerably higher resolution than conventional ompA sequencing.

Chlamydiaceae-like bacterium, but no Chlamydia psittaci, in sea birds from Antarctica

Within the growing order of Chlamydiales, there are a number of pathogens. One is Chlamydia psittaci, a zoonotic pathogen, with birds as natural hosts that may be transmitted to humans and cause severe respiratory disease, psittacosis. The prevalence of this pathogen in Antarctic birds is almost unknown as well as the ramifications of its potential spread in naïve bird populations. To investigate the prevalence of chlamydia organisms, cloacal and fecal samples were collected from 264 penguins and 263 seabirds on the Antarctic Peninsula and in Southern Chile. No C. psittaci could be detected by 23S rRNA real-time PCR. However, DNA sequencing of the 16S rRNA 298-bp signature sequence revealed a Chlamydiaceae-like bacterium previously found in seabirds from the subarctic zone, demonstrating that this not yet fully characterized bacterium is widespread. In conclusion, the prevalence of C. psittaci among wild birds on the Antarctic Peninsula seems to be low, but other types of chlamydial organisms are common. Further studies are required to taxonomically define and finally understand the role of these non-classified Chlamydiae.

Lymphogranuloma venereum rates increased and Chlamydia trachomatis genotypes changed among men who have sex with men in Sweden 2004–2016

This study aimed to determine the incidence of lymphogranuloma venereum (LGV) in Sweden since 2004 and to study in detail a consecutive number of Chlamydia trachomatis cases in men who have sex with men (MSM) during a 10 month period (September 2014 to July 2015). LGV increased from sporadic import cases in 2004 to comprise a spread within Sweden in 2016. Initially, only the L2b ompA genotype was detected, but in 2015 half of the genotyped LGV cases were L2 genotype. The changing genotype distribution in Sweden is linked to increased LGV spread in Europe. High-resolution multilocus sequence typing of 168 C. trachomatis cases from MSM in 2015 resulted in 29 sequence types, of which 3 accounted for 49 % of cases. The increased rates and different genotypes of LGV indicate that more concern for high-risk taking MSM is needed to avoid further spread of this invasive infection.

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.

P3.27 Lymphogranuloma venereum in sweden 2004–2016: increased rates among hiv-negative men who have sex with men and changed genotypes

Introduction Lymphogranuloma venereum (LGV) has become prevalent among men who have sex with men (MSM) in Europe since an outbreak in The Netherlands in 2003. The aim of this study was to describe the development of LGV in relation to HIV since 2004, and genotypes of LGV and other Chlamydia trachomatis (Ct) genovars in a MSM population in 2014/15. Methods All testing for LGV in Sweden is referred to Uppsala University Hospital. LGV-specific pmpH-gene PCR was used for detection. High-resolution genotyping based on ompA gene and multilocus sequence typing (MLST) was performed on all Ct-positive cases from an STI clinic for MSM in Stockholm between 1.9. 2014 and 1.7.2015. Results The annual numbers of detected LGV cases in Sweden were 2 in 2004 to 2006, between 5 and 20 in 2007 to 2012, and between 23 and 38 in 2013 to 2016. The number of LGV-tests increased from 68 in 2008 to 268 in 2016. During the study in 2014/15 31 LGV cases were identified in 309 patients with successful PCR-results. In 39% (12/31) LGV was unexpected and had not been detected without extended screening. The HIV-prevalence among LGV-positive patients decreased from 88% 2006–2013% to 68% 2014–2015. Of ompA genotyped LGV cases 69% were L2% and 31% were L2b type. This contrasts to earlier Swedish and European data from 2004–2009 when only L2b was found. Complete genotyping, including ompA and MLST, was obtained for 151 patients with non-LGV Ct and resulted in genovar D, 27%; E, 14%; G, 30% and J 21%. MLST resulted in 27 STs of which 3 predominated and accounted for 51%. Eight STs were new when compared to the database http://mlstdb.bmc.uu.se comprising 540 STs from >3300 specimens. Conclusion In Sweden LGV has gone from sporadic import cases to a probable endemic spread among HIV-negative MSM, which emphasises the need for LGV-testing This emphasises the need for LGV-testing within this high risk group. LGV has developed from being of clonal nature to occur as different strains among MSM.

P3.268 A Pilot Study of Genotyping Extrarectal Lymphogranuloma Venereum Strains

Background The first case of LGV in Barcelona was diagnosed in 2005 and since then around 200 cases have been notified up to 2012. All cases have been diagnosed among MSM, 80% of them coinfected with HIV and 97% of the cases had proctitis. Since 2008 some cases have appeared with extrarectal manifestations. Objective To compare the molecular epidemiology profiles of extrarectal LGV cases diagnosed in Barcelona with profiles reported in rectal cases. Methods A convenient 14 samples from 9 confirmed LGV cases in 2012 with extrarectal involvement were selected for LGV typing. DNA was extracted from samples using a semi automated system and kept al –80º C. The strains were further analysed by genotyping using a multilocus sequence typing (MLST) based on 5 highly variable gene regions, in addition the ompA gene was sequenced. Results DNA quality for MLST was suboptimal in some samples. The genotyping pattern showed one single MLST-5 profile (27, 13, 17, 13, 28) among all the samples. In ompA there were two variants (22 and 28), in the 2 cases with ompA variant 22 the samples were obtained from inguinal ganglia. Discussion The MLST-5 profile in LGV cases from Barcelona is the same as the predominating sequence type found in rectal cases. This is in line with the spread of a single clone, without specific tissue tropism. In ompA the 2 cases with variant 22 were identical to ompA in the reference strain L2/434/Bu, but differed from the currently predominating variant L2b among MSM. Considering the difference in ompA is minor it is more probable that L2b is a classical L2 isolate that has been circulating for a long time but showing now a new spectrum of manifestations. Our study does not support any difference in LGV strains obtained from extrarectal sites or from rectum.

P4-S1.06 High-resolution Multilocus Sequence Typing (MLST) of Chlamydia trachomatis from three catchment areas in North and Central Norway

Background The C trachomatis incidence rate in the county of Finnmark in North Norway is twice as high as in the rest of the country. This study genetically characterises the C trachomatis strains in Finnmark and compares them with strains from two other counties in Norway. Methods DNA sequencing of ompA, which encodes the major outer membrane protein MOMP, and a high-resolution multilocus sequence typing (MLST) system, which is based on PCR amplification and DNA sequencing of five highly variable genetic regions (not housekeeping genes as in conventional MLST), were used. In total, 80 specimens from adolescents mainly aged 15–19 were collected in five high schools (n=60) and in the laboratory routine (n=20) in Finnmark (extended county with sparse population in minor municipalities) and compared to 80 laboratory specimens from Tromsø county (capital of North Norway) and 88 laboratory specimens from Trondheim in Central Norway. Results Sequencing of ompA detected 11 genotypes in these 248 specimens. The MLST analysis displayed 50 sequence types (STs), providing five times higher resolution than ompA. Thirty-one (62%) of the STs were novel. The study revealed overlapping STs between the different counties, but 64% of the STs were unique for specific counties (Abstract P4-S1.06 figure 1). The MLST genetic diversity was lower in Finnmark (0.26 ST per specimen compared to 0.34 in Tromsø and 0.32 in Trondheim, respectively), but the geographical differences were not statistically significant.The common ompA genovar E predominated in Tromsø and Trondheim (55%) whereas genovar G (36%) was most frequent in Finnmark. Abstract P4-S1.06 Figure 1 Distribution of sequence types (n=50) of 248 C trachomatis cases in the counties of Finnmark (n=80), Tromsø (n=80), and Trondheim (n=88). Conclusions Our MLST provided a significantly higher differentiation capacity than conventional ompA typing, which has predominated genotyping of C trachomatis for decades. Thus high-resolution MLST is a useful tool for molecular epidemiology of chlamydial infections.