| 1. |
- Allard, A, et al.
(författare)
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Rapid typing of human adenoviruses by a general PCR combined with restriction endonuclease analysis.
- 2001
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 39:2, s. 498-505
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion). Degenerated consensus primers were designed, allowing amplification of DNA from all 51 human Ad prototype strains and altogether 44 different genome variants of Ad serotypes 1, 3, 4, 5, 7, 11, 19, 40, and 41. The 301-bp amplimer of 22 prototype strains representing all six subgenera and the genome variant was selected as a target for sequencing to look for subgenus and genome type variabilities. The sequences obtained were used to facilitate the selection of specific REs for discrimination purposes in a diagnostic assay by following the concept of cleavage or noncleavage of the 301-bp amplimer. On the basis of these results, a flowchart was constructed, allowing identification of subgenus B:2 and D serotypes and almost complete distinction of subgenus A, B:1, C, E, and F serotypes. Application of the PCR-RE digestion system to clinical samples allowed typing of 34 of 40 clinical samples positive for Ad. The genome type determined by this method was identical to that obtained by traditional RE typing of full-length Ad DNA. The remaining six samples were positive only after a nested PCR. Therefore, to reduce the risk of false-negative results, samples scored negative by the PCR-RE digestion system should be evaluated by the described nested PCR. Used in combination, the PCR-RE digestion method and the nested PCR provide a reliable and sensitive system that can easily be applied to all kinds of clinical samples when rapid identification of adenoviruses is needed.
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| 2. |
- Aspevall, O, et al.
(författare)
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Performance of four chromogenic urine culture media after one or two days of incubation compared with reference media.
- 2002
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 40, s. 1500-1503
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Tidskriftsartikel (refereegranskat)abstract
- Four chromogenic urine culture media were compared to culture on blood agar, MacConkey agar, and CLED (cysteine-, lactose-, and electrolyte-deficient) agar for detection of uropathogens in 1,200 urine specimens. After 2 nights of incubation, 96% of all isolates were recovered on blood agar, 96% were recovered on CLED agar, 92% were recovered on CPS ID2, 96% were recovered on CHROMagar Orientation from BBL, 95% were recovered on CHROMagar Orientation from The CHROMagar Company, and 95% were recovered on Chromogenic UTI Medium.
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| 3. |
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| 4. |
- Broman, T., et al.
(författare)
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Campylobacter jejuni in black-headed gulls (Larus ridibundus) : prevalence, genotypes, and influence on C. jejuni epidemiology
- 2002
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 40:12, s. 4594-4602
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Tidskriftsartikel (refereegranskat)abstract
- Campylobacteriosis is a zoonotic disease in which birds have been suggested to play an important role as a reservoir. We investigated the prevalence of Campylobacter jejuni subsp. jejuni in black-headed gulls (Larus ridibundus) in southern Sweden with the aim of examining the nature of C. jejuni infection in this bird species. Birds were sampled in four sampling series each year during 1999 (n = 419) and 2000 (n = 365). Longitudinally sampled C. jejuni isolates from individual gulls were subjected to macrorestriction profiling (MRP) by pulsed-field gel electrophoresis to investigate the genotypical stability during the natural course of infection. Furthermore, a subset (n = 76) of black-headed gull isolates was compared to isolates from broiler chickens (n = 38) and humans (n = 56) originating from the same geographic area. We found a pronounced seasonal variation in C. jejuni carriage, with the highest rates found in late autumn. MRP similarities were higher between isolates of human and broiler chicken origin, than between those of wild bird origin and either of the other two hosts. However, identical MRPs were found in two gull isolates and one human isolate after digestion with two restriction enzymes, strongly indicating that they may have been colonized by the same clone of C. jejuni. The MRPs most prevalent in gull isolates did not occur among isolates from humans and broiler chickens, suggesting the existence of a subpopulation of C. jejuni adapted to species-specific colonization or environmental survival.
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| 5. |
- 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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| 6. |
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| 7. |
- Johansson, Anders, 1966-
(författare)
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Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia
- 2000
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Ingår i: Journal of Clinical Microbiology. - Washington D.C. : ASM. - 0095-1137 .- 1098-660X. ; 38:1, s. 22-26
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Tidskriftsartikel (refereegranskat)abstract
- PCR and culture were comparatively evaluated for their abilities to demonstrate Francisella tularensis in wound specimens from tularemia patients during an outbreak in Sweden in 1998. For transport of the specimens used for PCR, a buffer solution containing a nuclease inhibitor was used, and for transport of the specimens used for culture, a commercial transport system was selected after experimental comparison of various systems. Of 40 patients with culture- and/or serology-verified ulceroglandular tularemia, PCR detected F. tularensis DNA in 30 (75%) patients, whereas culture detected bacterial growth in 25 (62%) patients. Compared to data from a previous study, the present inclusion of a nuclease inhibitor in the transport medium did not improve the sensitivity of the PCR, whereas the sensitivity of the culture procedure was significantly increased by selection of the system used for transport. Among eight patients with clinically suspected tularemia but with negative serology and culture, specimens from four patients showed detectable DNA. In three of these patients the diagnosis was verified by the demonstration of an F. tularensis-specific T-cell response in vitro. In conclusion, PCR was more sensitive than culture for demonstration of F. tularensis in wound specimens. Besides, we showed that tularemia may proceed without development of serum antibodies, and in these patients, PCR may be of special importance for verification of the diagnosis.
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| 8. |
- Johansson, Anders, 1966-
(författare)
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Evaluation of PCR-based methods for discrimination of Francisella species and subspecies and development of a specific PCR that distinguishes the two major subspecies of Francisella tularensis.
- 2000
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Ingår i: Journal of Clinical Microbiology. - Washington D.C. : ASM. - 0095-1137 .- 1098-660X. ; 38:11, s. 4180-4185
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Tidskriftsartikel (refereegranskat)abstract
- Previous studies have demonstrated that the four subspecies of the human pathogen Francisella tularensis, despite showing marked variations in their virulence for mammals and originating from different regions in the Northern Hemisphere, display a very close phylogenetic relationship. This property has hampered the development of generally applicable typing methods. To overcome this problem, we evaluated the use of PCR for discrimination of the subspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial repetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the species level and of the four subspecies of F. tularensis. With each of these three methods, similar or identical clustering of strains was found, and groups of strains of different geographical origins or differing in virulence showed distinct patterns. The discriminatory indices of the methods varied from 0.57 to 0.65; thus, the patterns were not sufficiently discriminatory to distinguish individual strains. The sequence of a fragment generated by amplification with an arbitrary primer was determined, and a region showing interstrain heterogeneity was identified. Specific primers were designed, and a PCR was developed that distinguished strains of F. tularensis subsp. holarctica from strains of other F. tularensis subspecies, including strains of the highly virulent F. tularensis subsp. tularensis. Notably, one European isolate showed the genetic pattern typical of the highly virulent F. tularensis subsp. tularensis, generally believed to exist only in North America. It is proposed that a combination of the specific PCR together with one method generating subspecies-specific patterns is suitable as a rapid and relatively simple strategy for discrimination of Francisella species and subspecies.
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| 9. |
- Johansson, Anders, 1966-
(författare)
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Extensive allelic variation among Francisella tularensis strains in a short-sequence tandem repeat region
- 2001
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Ingår i: Journal of Clinical Microbiology. - Washington D. C. : ASM. - 0095-1137 .- 1098-660X. ; 39:9, s. 3140-3146
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Tidskriftsartikel (refereegranskat)abstract
- Members of the genus Francisella and the species F. tularensis appear to be genetically very similar despite pronounced differences in virulence and geographic localization, and currently used typing methods do not allow discrimination of individual strains. Here we show that a number of short-sequence tandem repeat (SSTR) loci are present in F. tularensis genomes and that two of these loci, SSTR9 and SSTR16, are together highly discriminatory. Labeled PCR amplification products from the loci were identified by an automated DNA sequencer for size determination, and each allelic variant was sequenced. Simpson's index of diversity was 0.97 based on an analysis of 39 nonrelated F. tularensis isolates. The locus showing the highest discrimination, SSTR9, gave an index of diversity of 0.95. Thirty-two strains isolated from humans during five outbreaks of tularemia showed much less variation. For example, 11 of 12 strains isolated in the Ljusdal area, Sweden in 1995 and 1998 had identical allelic variants. Phenotypic variants of strains and extensively cultured replicates within strains did not differ, and, for example, the same allelic combination was present in 55 isolates of the live-vaccine strain of F. tularensis and another one was present in all 13 isolates of a strain passaged in animals. The analysis of short-sequence repeats of F. tularensis strains appears to be a powerful tool for discrimination of individual strains and may be useful for a detailed analysis of the epidemiology of this potent pathogen.
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| 10. |
- Jurstrand, Margaretha, et al.
(författare)
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Characterization of Chlamydia trachomatis omp1 genotypes among sexually transmitted disease patients in Sweden
- 2001
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 39:11, s. 3915-3919
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Tidskriftsartikel (refereegranskat)abstract
- A method for detection and genotyping of genital Chlamydia trachomatis infections based on omp1 gene amplification and sequencing was developed. DNA was extracted from urogenital or urine samples using a Chelex-based method, and an approximately 1,100-bp-long fragment from the omp1 gene was directly amplified and sequenced. Genotyping was performed by BLAST similarity search, and phylogenetic tree analysis was used to illustrate the evolutionary relationships between clinical isolates and reference strains. The method was used to determine the genotypes of C. trachomatis in 237 positive urogenital and/or urine specimens collected at a Swedish sexually transmitted disease clinic during 1 year. The most common genotypes corresponded to serotypes E (47%) and F (17%). The omp1 gene was highly conserved for genotype E (106 of 112 samples without any mutation) and F (41 of 42 samples without any mutation) strains but appear slightly less conserved for genotypes G (n = 6) and H (n = 6), where the sequences displayed one to four nucleotide substitutions relative to the reference sequence. Genotyping of samples collected at the follow-up visit indicated that two patients had become reinfected, while three other patients suffered treatment failure or reinfection. One woman appeared to have a mixed infection with two different C. trachomatis strains. This omp1 genotyping method had a high reproducibility and could be used for epidemiological characterization of sexually transmitted Chlamydia infections.
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| 11. |
- Knutsson, Rickard, et al.
(författare)
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Modeling of 5′ nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica
- 2002
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 40:1, s. 52-60
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Tidskriftsartikel (refereegranskat)abstract
- The performance of a 5′ nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched Salmonella enterica cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, the threshold cycle (Cτ) and the fluorescence intensity by a normalized reporter value (ΔRn). The Cτ response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from S. enterica serovar Enteritidis was studied in double-distilled H2O (ddH2O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on AmpliTaq Gold or rTth. A descriptive model was proposed and fitted to the available experimental data. Equivalent PCR performances for the two PCR mixtures were obtained when DNA was diluted in ddH2O. However, the level of detection of DNA was affected when BPW was present during amplification. Use of the rTth mixture generated a 1-log-unit wider linear range of amplification, and the DNA detection levels were 2 × 10-12 g/microwell for the rTth mixture and 2 × 10-12 g/microwell for the AmpliTaq Gold mixture. To verify the improved amplification capacity of the rTth mixture, BPW was inoculated with 1 CFU of S. enterica serovar Enteritidis per ml and the mixture was incubated at 30°C. Samples for PCR were withdrawn every 4 h during a 36-h enrichment. Use of the rTth mixture resulted in an earlier PCR detection during enrichment than use of the AmpliTaq Gold mixture. For accurate detection (Cτ ≤ 30) of S. enterica serovar Enteritidis inoculated in BPW, the rTth mixture required 8.4 h of enrichment, while the AmpliTaq Gold mixture needed 11.6 h. In conclusion, the principle applied can improve the methodology of 5′ nuclease real-time PCR for numerical optimization of sample pretreatment strategies to provide automated diagnostic PCR procedures.
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| 12. |
- Kuoppa, Yvonne, et al.
(författare)
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Quantitative detection of respiratory Chlamydia pneumoniae infection by real-time PCR
- 2002
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 40:6, s. 2273-2274
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Tidskriftsartikel (refereegranskat)abstract
- Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.
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| 13. |
- Lindstrom, A., et al.
(författare)
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Pyrosequencing for detection of lamivudine-resistant hepatitis B virus
- 2004
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Ingår i: Journal of Clinical Microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 42:10, s. 4788-4795
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Tidskriftsartikel (refereegranskat)abstract
- Chronic hepatitis B virus (HBV) infection can cause severe liver disease, including cirrhosis and hepatocellular carcinoma. Lamivudine is a relatively recent alternative to alpha interferon for the treatment of HBV infection, but unfortunately, resistance to lamivudine commonly develops during monotherapy. Lamivudine-resistant HBV mutants display specific mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the viral polymerase (reverse transcriptase [rt]), which is the catalytic site of the enzyme, i.e., methionine 204 to isoleucine (rtM204I) or valine (rtM204V). The latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180 (rtL180M). In the present study, a novel sequencing method, pyrosequencing, was applied to the detection of lamivudine resistance mutations and was compared with direct Sanger sequencing. The new pyrosequencing method had advantages in terms of throughput. Experiments with mixtures of wild-type and resistant viruses indicated that pyrosequencing can detect minor sequence variants in heterogeneous virus populations. The new pyrosequencing method was evaluated with a small number of patient samples, and the results showed that the method could be a useful tool for the detection of lamivudine resistance in the clinical setting.
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| 14. |
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| 15. |
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| 16. |
- Lund, Bodil, et al.
(författare)
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Frequent transmission of enterococcal strains between mechanically ventilated patients treated at an intensive care unit
- 2002
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 40:6, s. 2084-2088
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Tidskriftsartikel (refereegranskat)abstract
- The objectives of this investigation were to study the respiratory tract colonization and transmission of enterococci between 20 patients treated with mechanical ventilation at an intensive care unit (ICU), to compare genotyping with phenotyping, and to determine the antibiotic susceptibilities of the isolated enterococci. Samples were collected from the oropharynx, stomach, subglottic space, and trachea within 24 It of intubation, every third day until day 18, and thereafter every fifth day until day 33. Enterococcal isolates (n = 170) were analyzed by pulsed-field gel electrophoresis and with the PhenePlate (PhP) system. The antimicrobial susceptibilities to five agents were determined. Seventeen of the 20 subjects were colonized with enterococci in the respiratory tract; 12 were colonized in the lower respiratory tract. Genotype analyses suggested that 13 patients were involved in a transmission event, including all patients intubated more than 12 days. In conclusion, colonization of resistant enterococci in the respiratory tract of intubated patients treated at an ICU was common. Transmission of enterococci between patients occurred frequently. Prolonged intubation period seems to be a risk factor for enterococcal cross-transmission.
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| 17. |
- Mittelholzer, C., et al.
(författare)
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Detection and Sequence Analysis of Danish and Swedish Strains of Mink Astrovirus
- 2003
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 41:11, s. 5192-5194
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Tidskriftsartikel (refereegranskat)abstract
- The sequences of mink astroviruses collected from 11 farms in Denmark and Sweden were analyzed and found to be homologous with one another but different from those of other astroviruses. A species-specific reverse transcriptase-PCR for mink astrovirus was established and shown to be suitable for the analysis of clinical samples.
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| 18. |
- Mölling, Paula, et al.
(författare)
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Comparison of Serogroup W-135 Meningococci Isolated in Sweden during a 23-Year Period and Those Associated with a Recent Hajj Pilgrimag
- 2001
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 39:7, s. 2695-2699
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Tidskriftsartikel (refereegranskat)abstract
- An outbreak of serogroup W-135 meningococcal disease was reported among pilgrims returning from the annual hajj (pilgrimage to Mecca) in mid-March 2000. Molecular characterization was used to investigate the similarity of the hajj-associated W-135 strains with those isolated in Sweden during a 23-year period (1978 to 2000). The same hajj-associated genosubtype, genosubtype P1.5,2,36b, has been documented in Sweden since 1979, while pulsed-field gel electrophoresis and the sulfadiazine resistance of the W-135 isolates indicated that the outbreak was probably due to a new clone of W-135 meningococci.
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| 19. |
- Mölling, Paula, et al.
(författare)
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Direct and Rapid Identification and Genogrouping of Meningococci and porA Amplification by LightCycler PCR
- 2002
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 40:12, s. 4531-4535
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Tidskriftsartikel (refereegranskat)abstract
- Invasive meningococcal infections are usually diagnosed by culture. This approach is far from optimal due to, e.g., treatment with precollection antibiotics. Molecular-genetics methods are therefore recognized as important tools for optimal laboratory confirmation of meningococcal infections as well as characterization of meningococci (Mc). The aims of the present study were to develop real-time PCRs for identification and genogrouping (A, B, C, Y, and W-135) of Mc and porA amplification that further can be used for subsequent genosubtyping. In a first run Mc were identified. In a second run they were genogrouped and porA genes were amplified. All the Mc isolates (n = 71) but one and cerebrospinal fluid samples (n = 11) tested gave the expected positive results. The specificity, inter- and intra-assay variations, and effects of different amounts of DNA on the melting temperatures were also explored. The LightCycler PCR system was found to effectively detect and characterize Mc in a few hours. This testing, including the DNA sequencing of the porA gene to reveal the genosubtype, does not take more than a working day, and the results can be compared to those from culturing with phenotypic analysis, which takes at least a few days.
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| 20. |
- Nelson, J H, et al.
(författare)
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A novel and rapid PCR-based method for genotyping human papillomaviruses in clinical samples.
- 2000
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 38:2, s. 688-95
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Tidskriftsartikel (refereegranskat)abstract
- Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5(+)-GP6(+) consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil N-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a "T-sequencing ladder" was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.
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| 21. |
- Norén, Torbjörn, et al.
(författare)
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Molecular epidemiology of hospital-associated and community-acquired Clostridium difficile infection in a Swedish county
- 2004
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Ingår i: Journal of Clinical Microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 42:8, s. 3635-3643
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Tidskriftsartikel (refereegranskat)abstract
- All episodes of Clostridium difficile associated diarrhea (CDAD) diagnosed in a defined population of 274,000 including one tertiary and two primary hospitals and their catchment areas were studied during 12 months. The annual CDAD incidence in the county was 97 primary episodes per 100,000, and 78% of all episodes were classified as hospital associated with a mean incidence of 5.3 (range, 1.4 to 6.5) primary episodes per 1,000 admissions. The incidence among hospitalized individuals was 1,300-fold higher than that in the community (33,700 versus 25 primary episodes per 100,000 persons per year), reflecting a 37-fold difference in antibiotic consumption (477 versus 13 defined daily doses [DDD]/1,000 persons/day) and other risk factors. Three tertiary hospital wards with the highest incidence (13 to 36 per 1,000) had CDAD patients of high age (median age of 80 years versus 70 years for other wards, P < 0.001), long hospital stay (up to 25 days versus 4 days), or a high antibiotic consumption rate (up to 2,427 versus 421 DDD/1,000 bed days). PCR ribotyping of C. difficile isolates available from 330 of 372 CDAD episodes indicated nosocomial acquisition of the strain in 17 to 27% of hospital-associated cases, depending on the time interval between index and secondary cases allowed (2 months or up to 12 months), and only 10% of recurrences were due to a new strain of C. difficile (apparent reinfection). In other words, most primary and recurring episodes were apparently caused by the patient's endogenous strain rather than by one of hospital origin. Typing also indicated that a majority of C. difficile strains belonged to international serotypes, and the distribution of types was similar within and outside hospitals and in primary and relapsing CDAD. However, type SE17 was an exception, comprising 22% of hospital isolates compared to 6% of community isolates (P = 0.008) and causing many minor clusters and a silent nosocomial outbreak including 36 to 44% of the CDAD episodes in the three high-incidence wards.
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| 22. |
- Nygren, M., et al.
(författare)
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Polymorphism in the pertussis toxin promoter region affecting the DNA-based diagnosis of Bordetella infection
- 2000
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 38:1, s. 55-60
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Tidskriftsartikel (refereegranskat)abstract
- The pertussis toxin (PT) promoter region is a frequently used target for DNA-based diagnosis of pertussis and perapertussis infections. The reported polymorphism in this region has also allowed discrimination of species in mixtures with several Bordetella species by their specific PCR amplicon restriction patterns. In the present study, we investigated the degree of polymorphism in order to confirm the reliability of the assay, Five different sequence types of the amplified 239- or 249-bp region were found among the 33 Bordetella pertussis, B. parapertussis, and B. bronchiseptica American Type Culture Collection reference strains and patient isolates analyzed. According to the sequences that were obtained and according to the PT promoter sequences already available in the databases, restriction enzyme analysis with TaqI, Bg/I, and HaeII, which gave four different patterns, can be performed to reliably identify B. pertussis, B. papapeptussis, and B. bronchiseptica.
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| 23. |
- O'Meara, D., et al.
(författare)
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Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing
- 2001
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 39:2, s. 464-473
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HN-l infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.
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| 24. |
- Pulz, Matthias, et al.
(författare)
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Comparison of a Shiga Toxin Enzyme-Linked Immunosorbent Assay and Two Types of PCR for Detection of Shiga Toxin-Producing Escherichia coli in Human Stool Specimens
- 2003
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Ingår i: Journal of Clinical Microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 41:10, s. 4671-4675
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Tidskriftsartikel (refereegranskat)abstract
- Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of sporadic cases of disease as well as serious outbreaks worldwide. The spectrum of illnesses includes mild nonbloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. STEC produces one or more Stxs, which are subdivided into two major classes, Stx1 and Stx2. The ingestion of contaminated food or water, person-to-person spread, and contact with animals are the major transmission modes. The infective dose of STEC may be less than 100 organisms. Effective prevention of infection is dependent on rapid detection of the causative bacterial pathogen. In the present study, we examined 295 stool specimens for the presence of Stx-producing E. coli by three different methods: an Stx enzyme-linked immunosorbent assay, a conventional PCR assay, and a LightCycler PCR (LC-PCR) assay protocol recently developed by our laboratory at the Institute of Medical Microbiology at Hannover Medical School. Our intent was to compare these three methods and to examine the utility of the STEC LC-PCR protocol in a clinical laboratory. The addition of a control DNA to each sample to clearly discriminate inhibited specimens from negative ones enhanced the accuracy of the LC-PCR protocol. From our results, it can be concluded that LC-PCR is a very useful tool for the rapid and safe detection of STEC in clinical samples.
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| 25. |
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| 26. |
- Strålin, Kristoffer, 1969-, et al.
(författare)
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Comparison of two urinary antigen tests for establishment of pneumococcal etiology of adult community-acquired pneumonia
- 2004
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 42:8, s. 3620-3625
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Tidskriftsartikel (refereegranskat)abstract
- The Binax NOW immunochromatographic test (ICT) detecting the pneumococcal C polysaccharide and a serotype-specific latex agglutination (LA) test detecting 23 pneumococcal capsular antigens were evaluated for establishing pneumococcal etiology in community-acquired pneumonia (CAP) by use of nonconcentrated urine. ICT was considered to be strongly positive for result lines at least as intense as the control line and weakly positive for less intense result lines. When 215 adult CAP patients were tested, strong ICT, weak ICT, and LA positivity were found in 28, 24, and 16 patients, respectively; of these patients, 13 (46%), 6 (25%), and 13 (81%), respectively, had pneumococcal bacteremia and 27 (96%), 17 (71%), and 15 (94%), respectively, had Streptococcus pneumoniae isolated from blood, sputum, and/or nasopharynx. Among 108 controls tested, 2 (1.9%) were weakly ICT positive. When weak positivity was considered negative, the sensitivity of ICT decreased from 79% (19 of 24) to 54% (13 of 24), while the specificity increased from 83% (158 of 191) to 92% (176 of 191); no controls were false positive. The sensitivity and specificity of LA were 54% (13 of 24) and 98% (188 of 191), respectively. Eight of nine LA serotypes corresponded to culture serotypes. In conclusion, using nonconcentrated urine and dividing ICT-positive results into strongly and weakly positive results is a suitable way of performing ICT. While weak ICT positivity should be interpreted with caution, strong ICT positivity and LA positivity should be considered supportive of pneumococcal etiology in adult CAP. As such, these assays might have implications for antibiotic use in CAP. LA has promising potential for pneumococcal serotyping, although further evaluation is required.
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| 27. |
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| 28. |
- Thoelen, Inge, et al.
(författare)
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Analysis of the serotype and genotype correlation of VP1 and the 5' noncoding region in an epidemiological survey of the human enterovirus B species
- 2004
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Ingår i: Journal of Clinical Microbiology. - Wahington : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 42:3, s. 963-971
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Tidskriftsartikel (refereegranskat)abstract
- The sequence identity of the enterovirus VP1 gene has been shown to correlate with the serotype concept. Enterovirus molecular typing methods are therefore often based on sequencing of the VP1 genomic region and monophyletic clustering of VP1 sequences of a homologous serotype. For epidemiological surveillance, 342 enterovirus samples obtained from patients with aseptic meningitis in Belgium from 1999 to 2002 were first diagnosed as being enterovirus positive by amplification of the 5' noncoding region (5'NCR) by reverse transcription (RT)-PCR. Subsequently, samples were molecularly typed by RT-nested PCR amplification and sequencing of a portion of the VP1 gene. Phylogenetic analyses were performed to investigate enteroviral evolution and to examine the serotype and genotype correlation of the two genomic regions. Our typing results demonstrated echovirus 30, echovirus 13, echovirus 18, and echovirus 6 to be the most predominant types. Echoviruses 13 and 18 were considered to be emerging human serotypes since 2000 and 2001, respectively, as they had been rarely reported before. Several serotypes existed as multiple genotypes (subtypes) from 1999 to 2002, but genomic differences mainly resided at synonymous sites; these results strongly suggest that the subtypes exhibit similar antigenic properties. Phylogenetic analyses confirmed that VP1 is an adequate region for molecular typing. Serotype-specific clusters are not observed commonly in phylogenetic trees based on the 5'NCR, and the phylogenetic signal in the 5'NCR was found to be particularly low. However, some substructure in the 5'NCR tree made a tentative prediction of the enterovirus type possible and was therefore helpful in PCR strategies for VP1 (e.g., primer choice), provided some background knowledge on the local spectrum of enteroviruses already exists.
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| 29. |
- Unemo, Magnus, 1970-, et al.
(författare)
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Comparison of serologic and genetic porB-based typing of Neisseria gonorrhoeae : consequences for future characterization
- 2003
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 41:9, s. 4141-4147
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Tidskriftsartikel (refereegranskat)abstract
- Due to temporal changes in the epidemiology of gonorrhea, a precise characterization of Neisseria gonorrhoeae is essential. In the present study genetic heterogeneity in the porB genes of N. gonorrhoeae was examined, and serovar determination was compared to porB gene sequencing. Among 108 N. gonorrhoeae isolates, phylogenetic analysis of the entire porB alleles (924 to 993 bp) identified 87 unique sequences. By analyzing only the four to six most heterogeneous porB gene regions (174 to 363 bp), 86 out of these 87 genetic variants were identified. Consequently, analysis of shorter highly variable regions of the porB gene generates high-level discriminatory ability as well as fast, objective, reproducible, and portable data for epidemiological characterization of N. gonorrhoeae. Regarding putative antigenic epitopes of PorB for Genetic Systems monoclonal antibodies (MAbs), some of the previous findings were confirmed, but new findings were also observed. For several of the MAbs, however, the precise amino acid residues of PorB critical for single-MAb reactivity were difficult to identify. In addition, repeated serovar determination of 108 N. gonorrhoeae isolates revealed discrepancies for 34 isolates, mostly due to nonreproducible reactivity with single MAbs. Thus, the prospects of a genetic typing system with congruent translation of the serovar determination seem to be limited. In conclusion, analysis of short highly variable regions of the porB gene could form the basis for a fast molecular epidemiological tool for the examination of emergence and transmission of N. gonorrhoeae strains within the community.
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| 30. |
- Unemo, Magnus, 1970-, et al.
(författare)
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Molecular epidemiology of Neisseria gonorrhoeae : sequence analysis of the porB gene confirms presence of two circulating strains
- 2002
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 40:10, s. 3741-3749
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Tidskriftsartikel (refereegranskat)abstract
- The phenotypic and genotypic characteristics of Neisseria gonorrhoeae strains fluctuate over time both locally and globally, and highly discriminative and precise characterization of the strains is essential. Conventional characterization of N. gonorrhoeae strains for epidemiological purposes is mostly based on phenotypic methods, which have some inherent limitations. In the present study sequence analysis of porB1b gene sequences was used for examination of the genetic relationships among N. gonorrhoeae strains. Substantial genetic heterogeneity was identified in the porB genes of serovar IB-2 isolates (8.1% of the nucleotide sites were polymorphic) and serovar IB-3 isolates (5.2% of the nucleotide sites were polymorphic) as well as between isolates of different serovars. The highest degree of diversity was identified in the gene segments encoding the surface-exposed loops of the mature PorB protein. Phylogenetic analysis of the porB1b gene sequences confirmed previous findings that have indicated the circulation of one N. gonorrhoeae strain each of serovar IB-2 and serovar IB-3 in the Swedish community. These strains caused the majority of the cases in two domestic core groups comprising homosexual men and young heterosexuals, respectively, and were also detected in other patients. The phylogenetic analyses of porB gene sequences in the present study showed congruence, but not complete identity, with previous results obtained by pulsed-field gel electrophoresis of the same isolates. In conclusion, porB gene sequencing can be used as a molecular epidemiological tool for examination of genetic relationships among emerging and circulating N. gonorrhoeae strains, as well as for confirmation or discrimination of clusters of gonorrhea cases.
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| 31. |
- Unemo, Magnus, 1970-, et al.
(författare)
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Molecular typing of Neisseria gonorrhoeae isolates by pyrosequencing of highly polymorphic segments of the porB gene
- 2004
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 42:7, s. 2926-2934
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Tidskriftsartikel (refereegranskat)abstract
- For prevention and control of gonorrhea, an objective, highly discriminating, and reproducible molecular epidemiological characterization of Neisseria gonorrhoeae is essential. In the present study, in pursuance of providing such qualities, pyrosequencing technology, a fast real-time DNA sequence analysis, was applied to six short, highly polymorphic porB gene segments, with subsequent genetic variant (genovar) determination of the bacterial isolates. The sequencing templates were obtained by real-time PCR amplification, which also included fluorescence melting curve analysis of the entire porB gene in order to determine the genogroup (porB1a or porB1b allele) prior to pyrosequencing analysis. The PSQ 96 MA system used allowed rapid (in approximately 1.5 h) determination of 96 sequences of 20 to 65 correct nucleotides each. The results were reproducible and mostly in concordance with the results of conventional Sanger dideoxy sequencing, with the exception of shorter read lengths and some uncertainty in determining the correct number of identical nucleotides in homopolymeric segments. The number of sequence variants identified in each of the six highly polymorphic segments of the porB1a and porB1b alleles (encoding surface-exposed amino acid loops of the mature PorB protein) ranged from 5 to 11 and from 8 to 39, respectively. Among porB1a isolates (n = 22) and porB1b isolates (n = 65), 22 and 64 unique genovars, respectively, were identified. All isolates were typeable. The present results provide evidence of a high discriminatory ability, practically the same as that for sequencing of the entire porB gene. In conclusion, the fast and high-throughput pyrosequencing technology can be used for molecular epidemiological characterization of N. gonorrhoeae.
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| 32. |
- Vasquez, Alejandra, et al.
(författare)
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Vaginal Lactobacillus flora of healthy Swedish women
- 2002
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Ingår i: Journal of clinical microbiology. - 0095-1137 .- 1098-660X. ; 40:8, s. 2746-2749
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Tidskriftsartikel (refereegranskat)abstract
- Species of the Lactobacillus acidophilus complex are generally considered to constitute most of the vaginal Lactobacillus flora, but the flora varies between studies. However, this may be due to difficulties in identifying the closely related species within the L. acidophilus complex by using traditional methods and to variations in the vaginal status of the participants. Two hundred two isolates from the vaginal fluids of 23 Swedish women without bacterial vaginosis, as defined by the criteria of Nugent et al. (R. P. Nugent, M. A. Krohn, and S. L. Hillier, J. Clin. Microbiol. 29:297-301, 1991), were typed by randomly amplified polymorphic DNA (RAPD) analysis and identified to the species level by temporal temperature gradient gel electrophoresis, multiplex PCR, and 16S ribosomal DNA sequencing. The vaginal flora of most participants was dominated by a single RAPD type, but five of them harbored two RAPD types representing two different species or strains. The most frequently occurring species were Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii. L. iners has not previously been reported as one of the predominant Lactobacillus species in the vagina.
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| 33. |
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| 34. |
- Wolrath, Helen, 1971-, et al.
(författare)
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Analysis of bacterial vaginosis-related amines in vaginal fluid by gas chromatography and mass spectrometry
- 2001
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 39:11, s. 4026-4031
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Tidskriftsartikel (refereegranskat)abstract
- The presence of various amines in vaginal fluid from women with malodorous vaginal discharge has been reported before. The investigations have used several techniques to identify the amines. However, an optimized quantification, together with a sensitive analysis method in connection with a diagnostic procedure for vaginal discharge, including the syndrome of bacterial vaginosis, as defined by the accepted “gold standard,” has not been done before. We now report a sensitive gas chromatographic and mass spectrometric method for identifying the amines isobutylamine, phenethylamine, putrescine, cadaverine, and tyramine in vaginal fluid. We used weighted samples of vaginal fluid to obtain a correct quantification. In addition, a proper diagnosis was obtained using Gram-stained smears of the vaginal fluid that were Nugent scored according to the method of Nugent et al. (R. P. Nugent et al., J. Clin. Microbiol., 29:297–301, 1991). We found that putrescine, cadaverine, and tyramine occurred in high concentrations in vaginal fluid from 24 women with Nugent scores between 7 and 10. These amines either were not found or were found only in very low concentrations in vaginal fluid from women with Nugent scores of 0 to 3. There is a strong correlation between bacterial vaginosis and the presence of putrescine, cadaverine, and tyramine in high concentrations in vaginal fluid.
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| 35. |
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| 36. |
- Aabenhus, Rune, et al.
(författare)
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Lectin Typing of Campylobacter concisus
- 2002
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Ingår i: Journal of Clinical Microbiology. - 1098-660X. ; 40:2, s. 715-717
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Tidskriftsartikel (refereegranskat)abstract
- A total of 44 clinical isolates and the type strain of the putative pathogen Campylobacter concisus were grouped based on their reactions with plant lectins. The optimized lectin typing system used C. concisus strains proteolytically pretreated and subsequently typed by using a panel of four lectins. The system grouped all 45 strains into 13 lectin reaction patterns, leaving no strain untypeable due to autoagglutination. Lectin types were both stable and reproducible.
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| 37. |
- Abate, Getahun, et al.
(författare)
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Direct colorimetric assay for rapid detection of rifampin-resistant Mycobacterium tuberculosis
- 2004
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Ingår i: Journal of Clinical Microbiology. - 1098-660X. ; 42:2, s. 871-871
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Tidskriftsartikel (refereegranskat)abstract
- The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was standardized for direct detection of rifampin-resistant Mycobacterium tuberculosis in sputum samples. The sensitivity and specificity of the direct MTT assay matched those of the standard indirect susceptibility assay on 7H10 medium, and interpretable results were obtained for 98.5% of the samples within 2 weeks. Traditional methods of in vitro drug susceptibility testing are time consuming and laborious. Susceptibility tests on clinical isolates require 6 to 9 weeks, and tests conducted directly on smear-positive samples take about 3 weeks (International Union Against Tuberculosis and Lung Disease, The public health service national tuberculosis reference laboratory and the national laboratory network. Minimum requirements, role and operation in a low-income country, Paris, France, 1998, and P. T. Kent and G. P. Kubica, Public health mycobacteriology. A guide for the level III laboratory, Centers for Disease Control and Prevention, Atlanta, Ga., 1985). More-rapid methods are available but are very expensive for routine use under program conditions in countries with high levels of tuberculosis endemicity.
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| 38. |
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| 39. |
- Alestig, Erik, et al.
(författare)
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Phylogenetic origin of hepatitis B virus strains with precore C-1858 variant.
- 2001
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Ingår i: Journal of clinical microbiology. - 0095-1137. ; 39:9, s. 3200-3
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Tidskriftsartikel (refereegranskat)abstract
- Mutations that prevent the expression of the hepatitis B e antigen frequently emerge in the immunoreactive phase of infection. The predominant mutation, the precore G-->A-1896 mutation, is restricted by the variability at position 1858 and is rare in strains with cytosine at nucleotide 1858. The C-1858 variant is characteristic of genotype A. It also occurs in genotypes C and F, but not in B, D, or E, explaining the geographical variation in the prevalence of precore mutants. C-1858 strains have been frequently observed in southeast Asia, but have not been phylogenetically characterized. By sequencing eight complete hepatitis B virus genomes, C-1858 variants of east Asian origin were found to constitute a phylogenetic entity within genotype C that probably diverged several hundred years ago. Further study of the distribution of this variant is warranted.
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| 40. |
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| 41. |
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| 42. |
- Apfalter, Petra, et al.
(författare)
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Multicenter comparison trial of DNA extraction methods and PCR assays for detection of Chlamydia pneumoniae in endarterectomy specimens
- 2001
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Ingår i: Journal of Clinical Microbiology. - 1098-660X. ; 39:2, s. 519-524
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Tidskriftsartikel (refereegranskat)abstract
- The reported rate of detection of Chlamydia pneumoniae DNA within atherosclerotic lesions by PCR varies between 0 and 100%. In this study, identical sets of coded experimental atheroma samples (n = 15) and spiked controls (n = 5) were analyzed by 16 test methods in nine centers by means of PCR. The positive controls were correctly identified to levels of 1, 0.1, and 0.01 inclusion bodies of C. pneumoniae/ml of tissue homogenate by 16 (100%), 11 (69%), and 3 (19%) of the test methods, respectively. Three out of 16 negative controls (19%) were rated positive. Positivity rates for atheroma samples varied between 0 and 60% for the different test methods, with the maximum concordant result for positivity being only 25% for one carotid artery sample. There was no consistent pattern of positive results among the various laboratories, and there was no correlation between the detection rates and the sensitivity of the assay used.
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| 43. |
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| 44. |
- Asrat, Daniel, et al.
(författare)
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Prevalence of Helicobacter pylori vacA and cagA genotypes in Ethiopian dyspeptic patients
- 2004
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Ingår i: Journal of Clinical Microbiology. - 1098-660X. ; 42:6, s. 2682-2684
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Tidskriftsartikel (refereegranskat)abstract
- A total of 300 gastric biopsy samples and 50 Helicobacter pylori isolates were collected from Ethiopian adult dyspeptic patients. The vacA and cagA genes were detected in 90 and 79% of biopsy specimens, respectively, and in 100 and 87% of clinical isolates, respectively. Both genes were detected in 84% of the gastric biopsy samples and in 87% of the clinical isolates. Among vacA genotypes, the s1/m1 genotype was the most common in gastric biopsy samples (48%). The vacA and cagA positive H. pylori strains were detected to a higher degree in patients with chronic active gastritis (71%) than patients with other histopathological findings (29%) (P < 0.05).
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| 45. |
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| 46. |
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| 47. |
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| 48. |
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| 49. |
- Brudey, Karine, et al.
(författare)
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Molecular epidemiology of Mycobacterium tuberculosis in western Sweden.
- 2004
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Ingår i: Journal of clinical microbiology. - 0095-1137. ; 42:7, s. 3046-51
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Tidskriftsartikel (refereegranskat)abstract
- The genetic diversity of Mycobacterium tuberculosis isolates among patients from Sweden was determined by a combination of two PCR-based techniques (spoligotyping and variable number of tandem repeats analysis). It resulted in a clustering of 23.6% of the isolates and a rate of recent transmission of 14.1%. The clustered isolates mainly belonged to the Haarlem family (23.2%), followed by the Beijing (9.8%), Latin American and Mediterranean (LAM; 8%), and East African-Indian (EAI; 6.2%) families. A comparison of the spoligotypes with those in the international spoligotyping database showed that 62.5% of the clustered isolates and 36.6% of all isolates typed were grouped into six major shared types. A comparison of the spoligotypes with those in databases for Scandinavian countries showed that 33% of the isolates belonged to an ill-defined T family, followed by the EAI (22%), Haarlem (20%), LAM (11%), Central Asian (5%), X (5%), and Beijing (4%) families. Both the highest number of cases and the proportion of clustered cases were observed in patients ages 15 to 39 years. Nearly 10% of the isolates were resistant to one or more drugs (essentially limited to isoniazid monoresistance). However, none of the strains were multidrug resistant. Data on the geographic origins of the patients showed that more than two-thirds of the clustered patients with tuberculosis were foreign-born individuals or refugees. These results are explained on the basis of both the historical links within specific countries and recently imported cases of tuberculosis into Sweden.
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| 50. |
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