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.

In this study we aimed to characterize the ompA gene by sequencing DNA from all detected cases of Chlamydia trachomatis infection in a Swedish county during 2001, in order to improve the efficiency of contact tracing. Approximately 990 bp of the ompA gene was amplified, and sequence analysis was achieved in 678 (94%) of 725 C. trachomatis -positive cases in this unselected population. The most prevalent genotype was serotype E (39%), followed by F (21%), G (11%), D (9%), K (9%), J (7%), H (2%), B (1%), and Ia (1%). Serotype E was found in five genotype variants, with the reference sequence comprising 96% of all E cases. Serotype D was the most variable, and of seven sequence variants, three were identified as recombinants with serotype E. Altogether 29 genetic variants were detected, and mutations and recombination events are discussed. Clinical manifestations were not associated with genotypes. Sequence variation was linked to sexual networks identified by contact tracing and improved epidemiological knowledge but was of limited benefit.

A nested reverse transcription (RT)-PCR was developed for simultaneous detection and typing of influenza viruses A and B. The detection limit for influenza virus A subtypes H1 and H3 and that for influenza virus B were between 1 and 4 target gene copies per reaction for each type. The clinical benefit of the RT-PCR method was evaluated by comparing the results with virus isolation and direct immunofluorescence (IF) assays on 215 nasopharyngeal aspirates from patients with suspected influenza virus infection. The RT-PCR detected 83 cases of influenza A, compared to 66 cases detected by virus isolation and 68 cases detected by IF assay. The corresponding figures for the detection of influenza B were 15, 12, and 11 cases, respectively. In total, 16 out of 98 RT-PCR-positive specimens were negative by virus isolation and IF. An optical immunoassay for rapid detection of influenza A and B (FLU OIA; Bio Star Inc., Boulder, Colo.) was compared to RT-PCR and IF on 105 nasopharyngeal aspirates and 79 swabs. The sensitivity for the OIA was 40.4% compared to PCR and 48.8% compared to IF assay, when nasopharyngeal aspirates were examined. The specificities were 94.3 and 93.9%, respectively. The sensitivity was higher for OIA on nasopharyngeal swabs, 77.5% and 86.6% compared to PCR and IF, respectively, while the specificity was lower, 82.0% and 75.5%, respectively. The RT-PCR provides a sensitive and specific method for detecting and typing influenza viruses A and B. The rapid OIA is useful as a complementary test, but it cannot replace established methods without further evaluation.

The rnpB gene is universally present in bacterial species and encodes the RNA subunit of endoribonuclease P. In this study, rnpB was sequenced in 50 type strains and 29 additional strains of the genus Streptococcus. Putative secondary-structure models and possible interactions in RNase P RNA molecules are discussed. Phylogenetic relationships were studied and Bayesian, maximum-parsimony and minimum-evolution analyses supported six main clades that comprised 22 of the 50 species analysed. Phylogenetic inference was also studied for the 16S rRNA gene; it indicated a similar tree topology, but with weaker support values than for rnpB. Combined analysis of rnpB and 16S resulted in a phylogeny with significantly better support. Variability in the rnpB and 16S genes among all type strains, calculated as Shannon–Wiener information index values, was 0·45 for rnpB and 0·15 for 16S. Intraspecies proximity was assessed by principal coordinate analysis of rnpB for 32 strains of six closely related species (two clades) and showed species-specific clusters, but heterogeneity occurred in two species. It can be concluded that the rnpB gene is suitable for phylogenetic analysis of closely related taxa and has potential as a tool for species discrimination.

Background: Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. Results: The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 10⁵ genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively. In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria. Conclusions: The PCR provides increased sensitivity and the multiplex format facilitates diagnosis of S. pneumoniae, H. influenzae and N. meningitidis and the assay enable detection after antibiotic treatment has been installed. Quantification increases the specificity of the etiology for pneumonia.

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.