Prevalence of phylogroups and O25/ST131 in susceptible and extended-spectrum ß-lactamase-producing Escherichia coli isolates, the Netherlands.

To assess the distribution of phylogroups and O25/ST131 in the Netherlands, we performed a real-time polymerase chain reaction (PCR) on a collection of 108 wild-type Escherichia coli (WT-EC) and 134 extended-spectrum ß-lactamase-producing E. coli (ESBL-EC). Phylogroup B2 was predominant, but ESBL-EC were less likely to belong to this phylogroup (48.5%) than were WT-EC (66.7%; p = 0.005). In WT-EC, phylogroups B2 and D seem to be more virulent, having a higher prevalence among midstream urine isolates and blood culture isolates, than in catheter-related urine isolates (83.3% and 87.9% vs. 61.9%; p 0.048). O25/ST131 is associated with ESBL production, being almost absent among phylogroup B2 WT-EC (61.5% vs. 5.6%; p < 0.001).

The cost-effectiveness of ESBL detection: towards molecular detection methods?

Correct detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control and antibiotic choice. We performed a study to determine the cost-effectiveness of phenotypical testing, which can be inaccurate, and genotypical tests, which are considered to be more reliable but also more expensive. All patients that had been in isolation in the Amphia hospital because of the detection of ESBL according to the ESBL Etest were included in the survey. All strains were retested using the double disk confirmation test (DDCT) and a genotypical method. This was a commercially available microarray (Check-Points). Discordant results were confirmed by PCR and sequencing. In total 174 patients were included. In 24 of 174 (14%) patients, ESBL carriage could not be confirmed with the microarray. This was verified with PCR and sequencing. The mean duration of isolation was 15 days, adding up to a total number of isolation days of 2571. False-positive results according to the microarray resulted in a total of 279 days of unnecessary isolation for the Etest and 151 days for the DDCT. Using Etest to detect the presence of ESBL results in a false-positive outcome in 14% of the cases. This results in unnecessary isolation of patients, which can be omitted by using a genotypic method.

Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material.

We developed a dermatophyte-specific single-tube real-time PCR assay based on internal transcribed sequences. This assay allows the rapid detection and identification of 11 clinically relevant species within the three dermatophyte genera Trichophyton, Microsporum and Epidermophyton in nail, skin and hair samples within a few hours. Analysis of 145 clinical samples (107 nail, 36 skin scale, and two hair) by both real-time PCR and a PCR-reverse line blot (PCR-RLB) assay described earlier revealed that 133 of the 145 samples had concordant real-time PCR and PCR-RLB detection results (83 positive, 49 negative, and one inhibited). Six samples were positive by real-time PCR and negative by PCR-RLB, and two were negative by real-time PCR and positive by PCR-RLB. Four samples demonstrated inhibition in one of the two PCR assays. Only one of 83 positive samples had discordant identification results between both assays (Trichophyton verrucosum and Trichophyton erinacei by real-time PCR and Trichophyton erinacei by PCR-RLB). Dermatophytes present in seven positive samples that were incompletely identified as Trichophyton sp. by PCR-RLB were identified to the species level by real-time PCR as Trichophyton interdigitale and Trichophyton rubrum in six cases and one case, respectively. One hundred and twenty of 145 samples were also analysed by conventional dermatophyte culture and by direct microscopy. Our single-tube real-time PCR assay proved to be suitable for direct detection and identification of dermatophytes in nail, skin and hair samples with minimal total assay time (4 h after overnight lysis) and hands-on time, without the need for post-PCR analysis, and with good sensitivity and specificity.

Comparison of two PCR-based methods and conventional culture for the detection of nasal carriage of Staphylococcus aureus in pre-operative patients.

Nasal carriage is an important risk factor for the development of post-operative infections with Staphylococcus aureus and pre-operative treatment with mupirocin in carriers reduces the post-operative infection rate. Therefore, it is important to identify nasal carriage rapidly. Two polymerase chain reaction (PCR) techniques were compared to conventional culture in surgical patients. In 404 consecutive patients, nasal swabs were taken for pre-operative screening for the nasal carriage of S. aureus. The performance of the Roche Staphylococcus Kit on Lightcycler (Roche; RSA) and the Becton Dickinson (San Diego, CA) GeneOhm StaphSR assay on Smartcycler (Cepheid; BDSA) were compared with semi-quantitative culture. The sensitivity for culture, RSA and BDSA compared to the gold standard was 98.2, 82.0 and 85.6%, respectively, and the specificity was 100, 98.3 and 99.3%, respectively. The lower sensitivity of both PCR techniques was associated with samples with low bacterial loads. The RSA and BDSA were similar in performance and are suitable for the pre-operative identification of nasal carriers of S. aureus.

Validation of PCR-reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples.

A dermatophyte-specific PCR-reverse line blot (PCR-RLB) assay based on internal transcribed sequences was developed. This assay allows the rapid detection and identification of nine clinically relevant species within the three dermatophyte genera Trichophyton, Microsporum and Epidermophyton in nail, skin and hair samples within 1 day. Analysis of 819 clinical samples (596 nail, 203 skin and 20 hair) revealed a positive PCR-RLB result in 93.6% of 172 culture-positive and microscopy-positive samples. PCR-RLB was superior to culture and direct microscopy, in both detection and species identification. Comparison of identification results of 208 PCR-positive and culture-positive clinical samples showed five discrepancies (2.4%) between PCR-RLB identification and classical microscopic/biochemical identification of isolates. Comparison of PCR-RLB identification and classical identification of 98 other isolates (dermatophytes and non-dermatophytes) revealed 13 discrepancies (13.3%) and five incomplete identifications of Trichophyton spp. Sequence analysis of ITS1 regions of 23 samples with discrepant or incomplete identification results (four Centraalbureau voor Schimmelcultures dermatophyte strains, four clinical samples and 15 clinical isolates) confirmed identification results of PCR-RLB in 21 of the 23 analyzed samples. PCR-RLB proved to be extremely suitable for routine detection and identification of dermatophytes directly in nail, skin and hair samples because it is rapid, sensitive, specific and accurate.

Evaluation of an internally controlled real-time polymerase chain reaction assay targeting the groEL gene for the detection of Bartonella spp. DNA in patients with suspected cat-scratch disease.

Bartonella (B.) henselae is the causative agent of cat-scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. This study reports the development and evaluation of an internally controlled real-time polymerase chain reaction targeting the groEL gene for detection of Bartonella spp. DNA was extracted using the MagNA Pure system. The lower detection limit was 10-100 fg DNA and the in vitro sensitivity of the assay was not affected by duplexing with an internal control PCR. The real-time PCR assay detected DNA from all five B. henselae strains tested, and from B. birtlesii, B. vinsonii subsp. vinsonii, B. vinsonii subsp. arupensis and B. doshiae. The assay generated negative results with a selection of other bacteria, including several Mycobacterium spp., Streptococcus pyogenes and Staphylococcus aureus. Results of real-time PCR in clinical samples were compared with those of a conventional 16S rDNA-based PCR assay. During the period described in the Material and methods section, real-time PCR and conventional 16S PCR were performed on 73 clinical samples. Of these samples, 29 (40%) were found to give positive results and 44 (60%) gave negative results, both by real-time PCR and by conventional PCR, with a 100% agreement between the two tests. The PCR developed in this study is a rapid, sensitive, and simple method for the detection of Bartonella spp. in CSD and is suitable for implementation in the diagnostic laboratory.

Serological testing for Bartonella henselae infections in The Netherlands: clinical evaluation of immunofluorescence assay and ELISA.

Cat-scratch disease (CSD), caused by Bartonella henselae infection, can mimic malignancy and can manifest atypically. Reliable serological testing is therefore of great clinical importance. The diagnostic performance of immunofluorescence assay (IFA) and ELISA was evaluated in a group of Dutch patients with proven CSD (clinical diagnosis confirmed by PCR). Sera of 51 CSD patients and 56 controls (patients with similar symptoms, but who were B. henselae PCR-negative and had an alternative confirmed diagnosis) were tested for anti-B. henselae IgM and IgG by IFA and ELISA. A commercially available IFA test for IgM had a sensitivity of 6%. In-house assays for IgM showed specificities of 93% (IFA) and 91% (ELISA), but with low sensitivities (53% and 65%, respectively). With a specificity of 82% (IFA) and 91% (ELISA), in-house IgG testing showed a significantly higher sensitivity in IFA (67%) than in ELISA (28%, p <0.01). Sensitivity was higher for genotype I (38-75%) than for genotype II (7-67%) infections, but this was only statistically significant for IgG ELISA (p <0.05). In conclusion, detection of IgM against B. henselae by in-house ELISA and IFA was highly specific for the diagnosis of CSD. The high seroprevalence in healthy individuals limits the clinical value of IgG detection for diagnosing CSD. Given the low sensitivity of the serological assays, negative serology does not rule out CSD and warrants further investigation, including PCR. Adding locally isolated (e.g., genotype II) B. henselae strains to future tests might improve the sensitivity.

An outbreak of Legionnaire's disease among visitors to a fair in Belgium in 1999.

This paper describes an outbreak of Legionnaire's disease at Kapellen in Belgium among visitors of the annual fair. The investigation started on 13th November 1999 after a respiratory physician notified the health authorities of the province of Antwerp of presumptive cases of legionellosis. The annual commercial fair at Kapellen, a small town in northern Belgium, was held 10 days previously and attracted 50,000 visitors. Stand employees (professionals or volunteers), technical staff of the hall and visitors at the fair were affected cases. An exploratory case-control study was conducted to trace the source of the epidemic. To complete the inventory study and to evaluate other risk factors, a cohort study of exhibitors and staff was conducted. Ninety-three people met the case definition, 41 of whom were considered as confirmed, 14 as presumptive cases and 38 as possible/clinical cases. Five people died. Further testing at the reference laboratory confirmed all strains to be Legionella pneumophila serogroup 1. The sensitivity for culture was low (29.2%), and sensitivity for seroconversion was high (90.9%). For urinary antigen test, a sensitivity with Biotest EIA of 65.6% was found, and the sensitivity of polymerase chain reaction (PCR) was 85.7%. In all cases, the individual had visited the fair. Those individuals working in the central areas of the tent, near the aerosol-producing devices, were at higher risk of disease. Legionella was detected by PCR on swabs of the surfaces of the whirlpool. Although not fully proven, an aerosol-producing device was the most probable source of the outbreak.

Extraction of Chlamydia pneumoniae DNA from vascular tissue for use in PCR: an evaluation of four procedures.

The objective of this study was to compare four procedures for Chlamydia pneumoniae DNA extraction from vascular tissue. The NucliSens Kit, the QIAamp tissue DNA MiniKit, buffer-saturated phenol and the Geneclean II Kit were evaluated, based on the yield of recovered DNA, using PCR to detect C. pneumoniae in vascular tissue. The QIAamp tissue procedure had the highest detection level (0.004 inclusion-forming units/sample). All methods, except NucliSens (70 min), had a short handling time (30-40 min). Costs varied from 0.5 to 3.2 Euro.

Designation of the European Working Group on Legionella Infection (EWGLI) amplified fragment length polymorphism types of Legionella pneumophila serogroup 1 and results of intercentre proficiency testing Using a standard protocol.

The utility of amplified fragment length polymorphism (AFLP) analysis as a genotyping method for the epidemiological typing of Legionella pneumophila serogroup 1 has been previously demonstrated. This study (i). reports recommendations for the designation of the European Working Group on Legionella Infections (EWGLI) AFLP types, (ii). describes the EWGLI AFLP types identified for the 130 strains in the EWGLI culture collection, and (iii). reports the results of a newly introduced international programme of proficiency testing. Following preliminary analysis of 20 epidemiologically unrelated isolates, 16 major AFLP types were identified. A coded proficiency panel, comprising 12 additional isolates representing 9 of these 16 AFLP types, was sent to 17 centres in 14 European countries where it was analysed following a previously determined standard protocol. The identity of each coded strain (recorded as AFLP type 001-016 or untypeable) was determined by participants with reference to these 16 AFLP types, either visually or using gel analysis software where available, and reported to the coordinating centre. Nine of the 12 strains, including an epidemiologically related pair and two pairs of unrelated isolates of the same type, were correctly identified to the correct AFLP type by all or all but one of the participants. Seven laboratories correctly identified all 12 isolates, and a further seven laboratories correctly identified 11. Type identification scores ranged from 75% (1 centre), 83% (2 centres), and 92% (7 centres) to 100% (7 centres). The AFLP method as described is robust and rapid and allows the genotypic comparison of isolates of Legionella pneumophila between different testing centres without the need for exchange of the strains studied.

Correlation between detection methods of Chlamydia pneumoniae in atherosclerotic and non-atherosclerotic tissues.

Polymerase chain reaction (PCR) and immunohistochemistry (IHC) have been used to detect Chlamydia (C.) pneumoniae in vascular tissues. Discrepancies between the results of these two methods have frequently been reported. However, the correlation between PCR and IHC has not been analyzed yet. This study assesses the correlation between the detection of C. pneumoniae by PCR and IHC in 45 atherosclerotic and 50 non-atherosclerotic tissue specimens. Also, the presence of Mycoplasma (M.) pneumoniae in these 95 specimens was investigated. Correlation was found between the detection of C. pneumoniae by PCR and IHC in the atherosclerotic tissues. Both tests were positive in 10 specimens and negative in 17 specimens (p = 0.003). There was no significant correlation between PCR and IHC in non-atherosclerotic specimens (p = ns). M. pneumoniae was detected, by PCR, in one atherosclerotic specimen.The results show correlation between PCR and IHC in the detection of C. pneumoniae in atherosclerotic tissues, emphasize the association between C. pneumoniae and atherosclerosis, and support the specificity of the association between C. pneumoniae and atherosclerosis.

Detection of Chlamydia pneumoniae DNA in buffy-coat samples of patients with abdominal aortic aneurysm.

Recent studies have suggested that Chlamydia pneumoniae infection is a risk factor for abdominal aortic aneurysm. This study explores the presence of Chlamydia pneumoniae DNA in buffy-coat samples of control subjects and of patients with abdominal aortic aneurysm. The seroepidemiological association between abdominal aortic aneurysm and Chlamydia pneumoniae was also investigated. Buffy-coat samples and serum specimens were obtained from 88 patients and 88 control subjects. Detection of Chlamydia pneumoniae DNA in buffy-coat samples and measurement of IgG antibodies to Chlamydia pneumoniae in serum specimens were performed by polymerase chain reaction and microimmunofluorescence, respectively. Chlamydia pneumoniae DNA was detected in buffy-coat samples of 18 (20%) patients and 8 (9%) control subjects (adjusted odds ratio 2.9, 95% confidence interval 1-8.5). IgG antibodies to Chlamydia pneumoniae were detected in 85 (97%) patients and 71 (81%) control subjects (adjusted odds ratio 7.2, 95% confidence interval 1.7-31). The results show an association between abdominal aortic aneurysm and either the presence of Chlamydia pneumoniae DNA in buffy-coat samples or IgG antibodies to Chlamydia pneumoniae. These findings support the hypothesis that previous infection with Chlamydia pneumoniae might be a risk factor for abdominal aortic aneurysm.

Multicenter evaluation of epidemiological typing of methicillin-resistant Staphylococcus aureus strains by repetitive-element PCR analysis. The European Study Group on Epidemiological Markers of the ESCMID.

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.

Demonstration of Bartonella grahamii DNA in ocular fluids of a patient with neuroretinitis.

We describe the clinical and laboratory features of a 55-year-old human immunodeficiency virus-negative female patient who presented with bilateral intraocular inflammatory disease (neuroretinitis type) and behavioral changes caused by a Bartonella grahamii infection. Diagnosis was based on the PCR analysis of DNA extracted from the intraocular fluids. DNA analysis of the PCR product revealed a 100% identity with the 16S rRNA gene sequence of B. grahamii. The patient was successfully treated with doxycycline (200 mg/day) and rifampin (600 mg/day) for 4 weeks. This is the first report that demonstrates the presence of a Bartonella species in the intraocular fluids of a nonimmunocompromised patient and that indicates that B. grahamii is pathogenic for humans.

Pharmacodynamics of cisplatin in human head and neck cancer: correlation between platinum content, DNA adduct levels and drug sensitivity in vitro and in vivo.

Total platinum contents and cisplatin-DNA adduct levels were determined in vivo in xenografted tumour tissues in mice and in vitro in cultured tumour cells of head and neck squamous cell carcinoma (HNSCC), and correlated with sensitivity to cisplatin. In vivo, a panel of five HNSCC tumour lines growing as xenografts in nude mice was used. In vitro, the panel consisted of five HNSCC cell lines, of which four had an in vivo equivalent. Sensitivity to cisplatin varied three- to sevenfold among cell lines and tumours respectively. However, the ranking of the sensitivities of the tumour lines (in vivo), also after reinjection of the cultured tumour cells, did not coincide with that of the corresponding cell lines, which showed that cell culture systems are not representative for the in vivo situation. Both in vitro and in vivo, however, significant correlations were found between total platinum levels, measured by atomic absorption spectrophotometry (AAS), and tumour response to cisplatin therapy at all time points tested. The levels of the two major cisplatin-DNA adduct types were determined by a recently developed and improved 32P post-labelling assay at various time points after cisplatin treatment. Evidence is presented that the platinum-AG adduct, in which platinum is bound to guanine and an adjacent adenine, may be the cytotoxic lesion because a significant correlation was found between the platinum-AG levels and the sensitivities in our panel of HNSCC, in vitro as well as in vivo. This correlation with the platinum-AG levels was established at 1 h (in vitro) and 3 h (in vivo) after the start of the cisplatin treatment, which emphasizes the importance of early sampling.

The potential of plantinum-DNA adduct determination in ex vivo treated tumor fragments for the prediction of sensitivity to cisplatin chemotherapy.

BACKGROUND: Response to cisplatin-therapy is assumed to be related to the formation of platinum (Pt)-DNA adducts. Measurement of these adducts prior to therapy could be of value to improve cisplatin based cancer therapy. MATERIALS AND METHODS: We determined Pt-GG and Pt-AG adduct levels by use of 32P-postlabeling after ex vivo cisplatin treatment of fragments of head and neck squamous cell carcinoma (HNSCC) xenografts (five lines), and of tumor biopsies from patients with HNSCC (n = 8) and testicular cancer (n = 8). RESULTS: Adduct levels in fragments (3 x 3 x 3 mm) exposed to 10 to 80 microM cisplatin for one hour, showed positive correlations with the in vivo response to cisplatin treatment (P < 0.05), as well as with the xenograft adduct levels observed after in vivo cisplatin treatment (P < 0.02). After an additional five-hour drug-free incubation period the correlations were absent. When patient tumor fragments were exposed ex vivo to 80 microM cisplatin for one hour, adduct levels were similar in HNSCC and testicular cancer. Persistence of adducts was observed for testicular cancer in the additional drug-free period. The adduct levels in the samples of two HNSCC patients who received cisplatin chemotherapy were in line with the hypothesis that higher adduct levels are associated with a better response. CONCLUSION: Our preliminary results show that analysis of DNA adducts following ex vivo drug treatment is a feasible approach towards a predictive assay, which warrants further investigation.

Molecular genotyping of Staphylococcus aureus strains: comparison of repetitive element sequence-based PCR with various typing methods and isolation of a novel epidemicity marker.

Repetitive sequence-based (Rep)-PCR genotyping as described here is based on the presence of homologues of Mycoplasma pneumoniae repeat-like elements in Staphylococcus. In this study we comparatively evaluated the usefulness of rep-PCR typing with two sets of well-defined collections of Staphylococcus aureus strains. Rep-PCR analysis of the first collection of S. aureus strains (n = 59) and one Staphylococcus intermedius strain showed 14 different rep-PCR patterns, with each pattern harboring 6 to 15 DNA fragments. The discriminatory power of rep-PCR typing compared well to those of arbitrarily primed PCR (average of 20 types) and pulsed-field gel electrophoresis (11 types). S. aureus strain collection I comprised four outbreak-related groups of isolates. The isolates in only one group were found to have identical rep-PCR profiles. However, in an analysis of isolates from three additional independent local outbreaks (n for outbreaks 1 and 2 = 5, n for outbreak 3 = 12), identical rep-PCR types were found among strains isolated during each outbreak. Therefore, we conclude that rep-PCR genotyping may be an easy and fast method for monitoring of the epidemiology of nosocomial Staphylococcus infections. Rep-PCR analysis of strain collection II, which consisted of epidemic and nonepidemic methicillin-resistant S. aureus (MRSA) strains, revealed that a cluster of similar rep-PCR profiles was found among MRSA isolates which were more frequently isolated and which were most often associated with outbreaks.

Clinical spectrum of infections due to the newly described Actinomyces species A. turicensis, A. radingae, and A. europaeus.

Over a 7-year period, we isolated 294 Actinomyces-like organisms (ALOs) which were not clearly identifiable. Using well-defined probes coding for sequences specific for recently described Actinomyces species (A. turicensis, A. radingae, and A. europaeus), we were able to identify 128 strains. The majority belonged to the A. turicensis species. A. radingae was found only in patients with skin-related pathologies. A. europaeus was also detected in patients with urinary tract infections. The main sources of A. turicensis were genital infections, followed by skin-related and urinary tract infections. Additional clinical pictures were appendicitis, cholecystitis, ear, nose, and throat infections, and bacteremia. In a small number of patients these ALOs were found as the only pathogen. Strains of the three species were tested by two widely used biochemical identification methods. A. turicensis was easily identifiable by both these methods. We conclude that these ALOs are not infrequent pathogens and are found in a wide range of human infections. At least A. turicensis is easily identifiable by clinical diagnostic laboratories.

Characterization of Actinomyces turicensis and Actinomyces radingae strains from human clinical samples.

Whole-organism protein electrophoresis was used to compare and group unidentified coryneform bacteria resembling Gardnerella vaginalis and various Actinomyces and Arcanobacterium species. The obtained clusters of strains were further characterized by whole-cell fatty acid analysis and a variety of biochemical tests. Species-specific oligonucleotide probes based on 16S rRNA gene sequences were designed. The results demonstrate that the majority of the isolates belonged to Actinomyces turicensis; the other strains belonged to Actinomyces radingae. The descriptions of both species are emended.