| 1. |
- Bom, Reinier J. M., et al.
(författare)
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Evaluation of High-Resolution Typing Methods for Chlamydia trachomatis in Samples from Heterosexual Couples
- 2011
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 49:8, s. 2844-2853
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Tidskriftsartikel (refereegranskat)abstract
- We aimed to compare conventional ompA typing of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). Previously used MLST and MLVA systems were compared to modified versions that used shorter target regions and nested PCR. Heterosexual couples were selected from among persons with urogenital C. trachomatis infections visiting the sexually transmitted infection outpatient clinic in Amsterdam, The Netherlands. We identified 30 couples with a total of 65 degrees C. trachomatis-positive samples on which MLST and MLVA for eight target regions were performed. All regions were successfully sequenced in 52 samples, resulting in a complete profile for 18 couples and 12 individuals. Nine ompA genovars from D to K, with two variants of genovar G, were found. The numbers of sequence type and MLVA type profiles were 20 for MLST and 21 for MLVA, and a combination of MLST and MLVA yielded 28 profiles, with discriminatory indexes (D) ranging from 0.95 to 0.99. Partners in 17 couples shared identical profiles, while partners in 1 couple had completely different profiles. Three persons had infections at multiple anatomical locations, and within each of these three individuals, all profiles were identical. The discriminatory capacity of all MLST and MLVA methods is much higher than that of ompA genotyping (D = 0.78). No genotype variation was found within the samples of the same person or from heterosexual couples with a putative single transmission. This shows that the chlamydial genome in clinical specimens has an appropriate polymorphism to enable epidemiological cluster analysis using MLST and MLVA.
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| 2. |
- Christerson, Linus, 1985-, et al.
(författare)
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High-Resolution Genotyping of Chlamydia trachomatis by Use of a Novel Multilocus Typing DNA Microarray
- 2011
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 49:8, s. 2838-2843
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Tidskriftsartikel (refereegranskat)abstract
- Typing of Chlamydia trachomatis is important to understandingits epidemiology. Currently used methods such as DNA sequencingof the ompA gene and multilocus sequence typing (MLST) eitheroffer limited epidemiological resolution or are laborious andexpensive, or both. DNA microarray technology using the ArrayStripformat is an affordable alternative for genotyping. In thisstudy, we developed a new multilocus typing (MLT) DNA microarray,based on the target regions of a high-resolution MLST systemas well as software for easy analysis. Validation of the arraywas done by typing 80 previously MLST-typed clinical specimensfrom unselected adolescents in school. The MLT array showed100% specificity and provided 2.4-times-higher resolution thanompA sequencing, separating the commonly predominating ompAE/Bour genotype into 7 MLT array genotypes. The MLT array reproducedepidemiological findings revealed by the MLST system and showedsufficient sensitivity to work with clinical specimens. Comparedto MLST analysis, the expenses needed for testing a sample withthe MLT array are considerably lower. Moreover, testing canbe completed within 1 working day rather than 3 or 4 days, withdata analysis not requiring highly specialized personnel. Thepresent MLT array represents a powerful alternative in C. trachomatisgenotyping.
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| 3. |
- Herrmann, Björn, et al.
(författare)
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Comparison of a duplex quantitative real-time PCR assay and the COBAS Amplicor CMV Monitor test for detection of cytomegalovirus
- 2004
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 42:5, s. 1909-14
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Tidskriftsartikel (refereegranskat)abstract
- A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.
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| 4. |
- Herrmann, Björn, et al.
(författare)
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Differentiation of Chlamydia spp by sequence determination and restriction endonuclease cleavage of RNase P RNA genes
- 1996
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 34:8, s. 1897-1902
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Tidskriftsartikel (refereegranskat)abstract
- The amplification of DNA from Chlamydia trachomatis by PCR with degenerated primers yielded a 345-bp fragment of the putative RNase P RNA gene. From the deduced DNA sequence of this gene in C. trachomatis, a modified primer pair was designed. The primer pair was subsequently used to obtain the corresponding gene products from Chlamydia pneumoniae and Chlamydia psittaci. Sequence comparisons revealed similarities of 76.6% between C. trachomatis and C. pneumoniae, 79.5% between C. trachomatis and C. psittaci, and 84.7% between C. pneumoniae and C. psittaci. Furthermore, the three species were differentiated by fragment length polymorphism analysis after restriction enzyme cleavage of the PCR products. Sequence variations among 14 serotypes of C. trachomatis were confined to one purine base substitution in the putative RNase P RNA gene of lymphogranuloma venereum strains L1 to L3. Complete sequence similarity was found for nine strains of C. pneumoniae of different geographic origins. Taken together, our results indicate a possibility of the general application of this method in clinical bacteriology. Analysis of the secondary structures of the putative RNase P RNA genes from the different Chlamydia species suggested that a novel structural element in the domain of RNase P RNA is involved in base pairing with the 3'-terminal CCA motif of a tRNA precursor. This structure has not previously been found among RNase P RNAs of members of the division Bacteria.
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| 5. |
- Herrmann, Björn, et al.
(författare)
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Global Multilocus Sequence Type Analysis of Chlamydia trachomatis Strains from 16 Countries
- 2015
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 53:7, s. 2172-2179
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Tidskriftsartikel (refereegranskat)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.
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| 6. |
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| 7. |
- Innings, Åsa, et al.
(författare)
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Multiplex real-time PCR targeting the RNase P RNA gene for detection and identification of Candida species in blood
- 2007
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 45:3, s. 874-880
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a single-tube multiplex real-time PCR method for the detection of the eight most common Candida species causing septicemia: Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis. The method developed targets the RNase P RNA gene RPR1. Sequences of this geiie were determined for seven of the Candida species and showed surprisiRgly large sequence variation. C. glabrata was found to have a gene that was five times longer gene than those of the other species, and the nucleotide sequence similarity between C. krusei and C. albicans was as low as 55%. The multiplex PCR contained three probes that enabled the specific detection of C. albicans, C. glabrata, and C. krusei and a fourth probe that allowed the general detection of the remaining species. The method was able to detect 1 to 10 genome copies when the detection limit was tested repeatedly for the four species C. albicans, C. glabrata, C. krusei, and C. guilliermondii. No significant difference in the detection limit was seen when the multiplex format was compared with single-species PCR, i.e., two primers and one probe. The method detected eight clinically relevant Candida species and did not react with other tested non-Candida species or human DNA. The assay was applied to 20 blood samples from nine patients and showed a sensitivity similar to that of culture.
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| 8. |
- Klint, Markus, et al.
(författare)
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High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis
- 2007
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 45:5, s. 1410-1414
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Tidskriftsartikel (refereegranskat)abstract
- Genotyping of Chlamydia trachomatis is limited by the low sequence variation in the genome, and no adequatemethod is available for analysis of the spread of chlamydial infections in the community. We have developeda multilocus sequence typing (MLST) system based on five target regions and compared it with analysis ofompA, 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 fiveseparate target regions ranged from 8 to 16. The genetic variation in 47 clinical C. trachomatis isolates ofrepresentative 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 intoonly two by ompA analysis. The MLST system was applied to C. trachomatis specimens from a population ofmen who have sex with men and was able to differentiate 10 specimens of one predominant ompA genotype Gvariant 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.
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| 9. |
- Klint, Markus, et al.
(författare)
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Lymphogranuloma venereum prevalence in Sweden among men who have sex with men and characterization of Chlamydia trachomatis ompA genotypes
- 2006
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 44:11, s. 4066-4071
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Tidskriftsartikel (refereegranskat)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.
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| 10. |
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