- Volume 54, Issue 4, 2005

Automated amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) techniques with fluorescently labelled primers were used to track differences among isolates of the eight known species of the Bordetella genus. Eighty-one representative strains of these species from international and Polish bacterial collections were genotyped according to RAPD protocols using primer 1254 or 1247, and AFLP involving EcoRI/MseI or newly designed SpeI/ApaI restriction/ligation/amplification procedures. By comparing AFLP and RAPD data, it was concluded that the discriminatory power of AFLP is higher in comparison with RAPD for both intra- and inter-species differentiation of isolates of the Bordetella genus. The most precise level of inter-species discrimination and the highest level of intra-species discrimination of the Bordetella isolates of the eight species were observed in the AFLP EcoRI/MseI and SpeI/ApaI sets, respectively. Both techniques might provide alternative tools for the identification of Bordetella at the genomic species and strain levels, and thus may be valuable in human and veterinary diagnostics as well as in epidemiology. By applying the AFLP technique presented in this article, more precise data on the emergence of newly acquired and/or on expanded clones and transmission routes of isolates of the Bordetella genus in the human and animal environments might be obtained.

The aim of this study was to evaluate the performance of the EVIGENE VRE Detection kit and compare it with PCR, considered the gold standard for detection of vancomycin-resistant enterococci (VRE). The correlation between the MIC values of vancomycin and teicoplanin using the epsilon test was also determined. In the EVIGENE VRE Detection kit, DNA probes specific for bacterial target DNA sequences are bound to microwell plates. A hundred and ten VRE (104 Enterococcus faecium and six Enterococcus faecalis) and 45 vancomycin-susceptible E. faecium were tested. All VRE strains were found to be positive for the vanA genotype using the EVIGENE VRE Detection kit. All results obtained with the EVIGENE VRE Detection kit were confirmed by PCR. MIC results for the strains also correlated highly with the PCR and kit results. The EVIGENE VRE Detection kit should be used in preference to other methods for detecting resistance genes in all strains, since it is less time-consuming, does not require the handling of hazardous chemicals and has the same specificity as PCR.

In a previous study, a molecular capsular type (MCT) prediction system for 51 Streptococcus pneumoniae serotypes was developed based on a combination of partial cpsA–cpsB sequencing and serotype(s)/group(s)-specific PCR. In this study, another 169 S. pneumoniae isolates were added to the existing database of 427 isolates, including representatives of all 39 serotypes not previously studied. In addition to the authors’ own limited sequence data for all 90 serotypes, cpsA–cpsB sequence data published by the S. pneumoniae capsular loci-sequencing group (http://www.sanger.ac.uk/Projects/S_pneumoniae/CPS/) at the Sanger Institute or available from GenBank were incorporated into the database. All serotypes, except 25A, were represented by at least two isolates. The number of sequence types identified was 138, of which 110 corresponded to single conventional serotypes (CSs); of these, 57 were represented by two or more isolates. Twenty-six sequence types were shared by between two and four CSs. To resolve these shared cpsA–cpsB sequence types and increase the discriminatory power of our system, the genes encoding the capsular polysaccharide flippase (wzx) and polymerase (wzy) were annotated and 24 new serotype(s)/group(s)-specific PCRs targeting wzy and two targeting wzx were designed. Using both cpsA–cpsB sequencing and wzx/wzy PCR, MCT correctly predicted the CSs of 516 (73 %) and the serogroup of an additional 155 (22 %) of the 708 isolates evaluated. For 5 % of isolates, MCT could not distinguish between members of five serotype pairs (37 isolates) containing members of different serogroups. Although further study of the relationship between MCT and CS is needed, this system now allows serotype or serogroup identification of 95 % of S. pneumoniae isolates.

The Gen-Probe APTIMA Combo 2 assay has previously been reported to have a high sensitivity. The end point of this assay was evaluated using highly purified chlamydial elementary bodies (EBs). The performance of the APTIMA Combo 2 assay was compared with a commercially available PCR kit, AMPLICOR Chlamydia trachomatis. The number of inclusions of C. trachomatis at the end point of the APTIMA Combo 2 assay was 0.005 inclusion-forming units (i.f.u.) ml−1, which was equivalent to 0.008 EBs per assay. The end point of the AMPLICOR kit was 5 i.f.u. ml−1 (equivalent to 0.5 EB per assay). The efficacy of the AMPLICOR C. trachomatis assay was inhibited by phosphate or Fe2+ ion, while these had no effect on the APTIMA Combo 2 assay. In conclusion, the APTIMA Combo 2 assay appears to have a greater sensitivity than the AMPLICOR C. trachomatis assay for detection of C. trachomatis, while demonstrating few problems with inhibitory substances such as phosphate and Fe2+ ion.

In this study the raising and development of the immune response to Borrelia burgdorferi infection in 45 Italian patients suffering from culture-confirmed Lyme borreliosis erythema migrans was investigated. A total of 95 serially collected serum samples were tested by using three different commercial ELISAs: recomWell Borrelia (Mikrogen), Enzygnost Borreliosis (DADE Behring) and Quick ELISA C6 Borrelia (Immunetics). The sensitivities of the ELISAs were as follows: Enzygnost Borreliosis IgM, 70.5 %; Quick ELISA C6 Borrelia, 62.1 %; recomWell Borrelia IgM, 55.7 %; recomWell Borrelia IgG, 57.9 %; and Enzygnost Borreliosis IgG, 36.8 %. In order to compare the specificity values of the three ELISAs, a panel of sera obtained from blood donors (210 samples coming from a non-endemic area and 24 samples from an endemic area) was tested, as well as sera from patients suffering from some of the most common biological conditions that could result in false-positive reactivity in Lyme disease serology (n = 40). RecomWell Borrelia IgG and recomWell Borrelia IgM were the most specific (97.1 % and 98.9 %, respectively), followed by Quick ELISA C6 Borrelia (96.7 %). Enzygnost Borreliosis IgG and IgM achieved 90.1 % and 92.3 % specificity, respectively. Sera that gave discrepant results when tested by the three ELISAs were further analysed by Western blotting.

Early and rapid detection of the causative organism is necessary in tuberculosis, particularly tuberculous meningitis, as the disease affects mainly children and if untreated or improperly treated can cause severe central nervous system disorders and can often be fatal. An in-house-developed PCR technique was developed for the detection of Mycobacterium tuberculosis DNA, in which the target for amplification was a 340 bp nucleotide sequence located within the 38 kDa protein gene. The test can detect as small an amount of DNA as 10 fg, which is equivalent to two to three organisms, and is highly specific. Amplified product was detected by ethidium bromide staining after electrophoresis and Southern hybridization. Evaluation of test sensitivity and specificity was carried out using acid-fast bacilli-positive sputum samples from patients with pulmonary tuberculosis and an equal number of non-tuberculosis patient samples as negative controls. In a double-masked study 30 cerebrospinal fluid samples from tuberculous meningitis patients and 30 samples from non-tuberculous meningitis patients were investigated. Out of the 30 samples 22 were positive by ethidium bromide-stained gel electrophoresis and 27 gave positive results by Southern hybridization. All of the 30 control samples showed negative results. The sensitivity of this PCR was 90 % and specificity, 100 %.

A Crimean-Congo haemorrhagic fever (CCHF) outbreak emerged from 2001 to 2003 in the Middle Anatolia region of Turkey. This study describes the clinical characteristics and outcome features of CCHF patients admitted to four tertiary care hospitals in Turkey. Definitive diagnosis was based on the detection of CCHF virus-specific IgM by ELISA or of genomic segments of the CCHF virus by RT-PCR. Related data were collected by a retrospective chart review. Hospital costs were extracted from the final discharge bills. Univariate and multivariate analyses were conducted to determine the independent predictors of mortality. CCHF virus-specific antibodies or genomic segments were detected in the sera of 99 cases. Seven cases that were treated with ribavirin were excluded from the study. Cases were mostly farmers (83 cases, 90 %), and 60 % had a tick-bite history before the onset of fever. Impaired consciousness and splenomegaly were independent predictors of a fatal outcome.

Episodes of candida infection at a teaching hospital were investigated. During a 3-year period from 1998 to 2000, there were 53 cases of candidaemia. Candida albicans (64.2 %) was the most common causative species, followed by Candida glabrata (17.0 %) and Candida parapsilosis (15.1 %). Molecular analysis of a cluster of eight infections from a single unit was performed using Southern blotting with Ca3 probe hybridization. This showed that the patients were each infected by unrelated strains of C. albicans. On occasion, isolates were found to be closely related within individual patients. Following Southern blot analysis, it was concluded that the infections were not part of an outbreak caused by a single, epidemic strain.

In this study the aetiology of community-acquired pneumonia (CAP) in Japan was investigated and the incidence of causative pathogens in ambulatory and hospitalized patients was compared. In addition, the roles of Chlamydophila felis and Chlamydophila pecorum as causes of CAP were investigated. Five hundred and six patients with CAP who visited an outpatient clinic or were admitted to one of three different hospitals were enrolled in this study; 106 of them were outpatients and 400 were hospitalized patients. Among the 506 CAP cases, Mycoplasma pneumoniae was the most common pathogen found in the outpatients and Streptococcus pneumoniae was the most common in the hospitalized patients. No cases of Chlamydophila pecorum pneumonia were observed and only one patient had an antibody titre suggestive of recent infection with the feline strain of Chlamydophila. The incidence of infection with M. pneumoniae and Chlamydophila pneumoniae was higher among the outpatients than among hospitalized patients, whereas the incidence of infection with S. pneumoniae and Haemophilus influenzae was higher among the hospitalized patients. Recognition of these results will allow prompt and appropriate antimicrobial therapy to be provided using Japanese CAP guidelines.