Brucellosis is endemic in livestock and humans in Uganda and its transmission involves a multitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR) loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections.

A general aim was to combine molecular typing methods with clinical background information to increase epidemiological knowledge about Chlamydia trachomatis infections. An outbreak of Lymfogranuloma venereum (LGV), caused by a more invasive variant of C. trachomatis, was reported from the Netherlands in 2003 among men who have sex with men (MSM). All Chlamydia positive specimens from a venereal disease clinic for MSM in Stockholm during one year were genotyped. No spread of LGV was found, apart from three symptomatic cases. The same ompA genotypes were found among MSM in Melbourne, but the genotype distribution was different compared to findings among the heterosexual population in Sweden. The standard method for genotyping of Chlamydia is ompA-sequencing, but it has low resolution because one genotype predominates. A multilocus sequence typing (MLST) system based on five targets was developed. In a sample of 47 specimens, 32 variants were found with MLST, but only 12 variants with ompA-sequencing. The polymorphisms in the hctB gene, one MLST target, are caused by an element of 108 bp that is present in two to four repetitions and in different variants. Although the DNA-binding function of Hc2 that is encoded by hctB has been studied, our findings of a considerable size variation show that new studies are needed. In 2006, specimens with a 377 bp deletion in the cryptic plasmid covering the target region for diagnostic test systems from Abbott and Roche were discovered in Sweden. Applying MLST to these specimens indicated that there was a single clone, denoted nvCT. The proportion of nvCT in all detected Chlamydia cases was higher (20% to 65%) in counties using Abbott/Roche compared to counties using the BectonDickinson test system (7% to 20%). The proportions of nvCT converge in counties with high or low levels when detection systems were adjusted to detect nvCT.

BACKGROUND:In the past, contradictory results have been obtained linking Chlamydia trachomatis serovars (ompA gene) to different clinical courses of infection.METHODS:A high resolution multilocus sequence typing (MLST) system was used to genotype 6 genetic regions, including ompA, in 70 Dutch urogenital C. trachomatis strains from patients with different degrees of defined clinical symptoms (asymptomatic, symptomatic, and lower abdominal pain), to determine if MLST genotypes correlated with clinical manifestations of infection.RESULTS AND CONCLUSIONS:We identified 46 MLST types, with only a small overlap to Swedish MLST types. This study found no correlation between MLST profiles and symptomatology. To understand the clinical course of infection, future studies should not only consider bacterial factors but also look on the immunogenetics of the host.

Objectives In 2006 a new variant of Chlamydia trachomatis (nvCT), with a deletion in the cryptic plasmid, was reported in Sweden. This deletion included the targets for the genetic diagnostic systems used in many clinical laboratories and resulted in thousands of false-negative results. The aim of this study was to characterise consecutive Chlamydia tissue culture-positive samples from 2006 in Orebro County, after identification of the nvCT, and to compare the results from samples collected in the same county in 1999-2000. The study also aimed to evaluate the discriminatory capacity of multilocus sequence typing (MLST) compared with ompA sequencing.Methods ompA sequencing and MLST was used to characterise 100 consecutive Chlamydia tissue culture-positive samples.Results A significant (p < 0.001) increase of genotype E, from 47% in 1999-2000 to 69% in 2006, was detected. All 41 nvCT isolates from 2006 displayed an identical ompA genotype E and MLST profile. Excluding the nvCT isolates, the distribution of ompA genotypes is similar to the genotyping results from 1999-2000. Among the wild-type genotype E isolates from 2006, 14 unique MLST sequence types were obtained from 26 isolates while they were identical in ompA genotyping. The discriminatory power (D) of C trachomatis strains in this material was 83.5% using the MLST system compared with 49.5% utilising ompA sequencing.Conclusion In all, MLST enables improved studies of the molecular epidemiology of C trachomatis. All nvCT isolates from 2006 displayed an identical ompA genotype E and MLST profile, which strongly indicates a clonal spread of the nvCT.

Background The histone-like protein Hc2 binds DNA in Chlamydia trachomatis and is known to vary in size between 165 and 237 amino acids, which is caused by different numbers of lysine-rich pentamers. A more complex structure was seen in this study when sequences from 378 specimens covering the hctB gene, which encodes Hc2, were compared. Results This study shows that the size variation is due to different numbers of 36-amino acid long repetitive elements built up of five pentamers and one hexamer. Deletions and amino acid substitutions result in 14 variants of repetitive elements and these elements are combined into 22 configurations. A protein with similar structure has been described in Bordetella but was now also found in other genera, including Burkholderia , Herminiimonas , Minibacterium and Ralstonia . Sequence determination resulted in 41 hctB variants that formed four clades in phylogenetic analysis. Strains causing the eye disease trachoma and strains causing invasive lymphogranuloma venereum infections formed separate clades, while strains from urogenital infections were more heterogeneous. Three cases of recombination were identified. The size variation of Hc2 has previously been attributed to deletions of pentamers but we show that the structure is more complex with both duplication and deletions of 36-amino acid long elements. Conclusions The polymorphisms in Hc2 need to be further investigated in experimental studies since DNA binding is essential for the unique biphasic life cycle of the Chlamydiacae . The high sequence variation in the corresponding hctB gene enables phylogenetic analysis and provides a suitable target for the genotyping of C. trachomatis .

We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s, including a variant from Europe.

OBJECTIVES The nvCT has a 377 bp plasmid deletion that has caused commonly used nucleic acid amplification tests to generate false negative results. In Swedish counties nvCT has been reported to account for up to 65% of all chlamydia cases in 2007. The aim was to evaluate the CTQ DNA amplification assay for detection of the new Chlamydia trachomatis variant (nvCT) in urine specimens from a laboratory in Sweden. METHODS In this study, 199 unscreened urine specimens were collected and tested using the BD ProbeTec ™ ET CT assay and then tested with the BD ProbeTec ™ CTQ assay on the BD Viper System in extracted mode. Discrepant cases were tested with real time PCR. 1 The CTQ assay targets a region of the Cryptic Plasmid outside the 377 bp deletion found in nvCT strains. An additional 30 urine specimens collected in March 2008 and identified as positive with nvCT PCR 2 were also tested on the CTQ assay for verification of detection accuracy. RESULTS Of the 199 unscreened urine samples, 34 (17%) were positive in the routine BD ProbeTec ™ ET CT compared to 36 (18%) tested positive in the CTQ assay (Table 1). Three cases were positive only with BD ProbeTec CTQ assay and two of these were positive also with real time PCR. One case was only positive by BD ProbeTec ET CT assay and was also negative in the real time PCR. In the collection of 30 urine specimens with nvCT all were detected by the CTQ assay. Pos Neg BD ProbeTec CTQ Pos 33 3 Neg 1 162 CONCLUSION The BD ProbeTec CTQ assay can adequately detect nvCT and wild type strains of C. trachomatis.

A variant of Chlamydia trachomatis that had escaped detection by commonly used systems was discovered in Sweden in 2006. In a nationwide study, we found that it is now prevalent across Sweden, irrespective of the detection system used. Genetic analysis by multilocus sequence typing identified a predominant variant, suggesting recent emergence.

Genotyping of Chlamydia trachomatis is limited by the low sequence variation in the genome, and no adequate method is available for analysis of the spread of chlamydial infections in the community. We have developed a multilocus sequence typing (MLST) system based on five target regions and compared it with analysis of ompA, the single gene most extensively used for genotyping. Sequence determination of 16 reference strains, comprising all major serotypes, serotypes A to L3, showed that the number of genetic variants in the five separate target regions ranged from 8 to 16. The genetic variation in 47 clinical C. trachomatis isolates of representative serotypes (14 serotype D, 12 serotype E, 11 serotype G, and 10 serotype K strains) was analyzed; and the MLST system detected 32 variants, whereas 12 variants were detected by using ompA analysis. Specimens of the predominant serotype, serotype E, were differentiated into seven genotypes by MLST but into only two by ompA analysis. The MLST system was applied to C. trachomatis specimens from a population of men who have sex with men and was able to differentiate 10 specimens of one predominant ompA genotype G variant into four distinct MLST variants. To conclude, our MLST system can be used to discriminate C. trachomatis strains and can be applied to high-resolution molecular epidemiology.

ABSTRACT An outbreak of lymphogranuloma venereum (LGV) infections has recently been reported from The Netherlands and other European countries. The Swedish surveillance system has identified three LGV cases since 2004, all with clinically suspected infection in men who have sex with men (MSM). In order to assess the prevalence of LGV in a high-risk group of MSM and include clinically atypical cases, retrospective analysis of 197 Chlamydia trachomatis -infected men was performed. Sequencing of the ompA gene showed a different serotype distribution compared to recent Swedish studies in heterosexual populations. The most common types were G (45%), D (27%), and J (26%), whereas the normally predominant type E accounted for only 4% of the chlamydia cases. Furthermore, certain ompA genotype variants of the dominant serotypes were highly prevalent among MSM, and the reason for this is discussed. No additional case of LGV was detected by retrospective analysis of the high-risk MSM population. This indicates that, thus far, LGV in Sweden is only a result of sporadic import from infected MSM clusters abroad.

Chlamydophila psittaci was detected in 10% of 431 fulmars examined from the Faroe Islands. Analysis of ompA showed a sequence almost identical to that of the type strain. The origin of C. psittaci outbreaks in fulmars is discussed. Despite a high level of exposure, the risk for transmission of C. psittaci to humans is low.