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To study whether replacement of nosocomial ampicillin-resistantRetrospective, matched-cohort study.The study included 20 Dutch and Danish hospitals from 2009 to 2014.Within the study period, 63 patients with VRE bacteremia (36 Dutch and 27 Danish) were identified and subsequently matched to 234 patients with ARE bacteremia (130 Dutch and 104 Danish) for hospital, ward, length of hospital stay prior to bacteremia, and age. For all patients, 30-day mortality after bacteremia onset was assessed.The risk ratio (RR) reflecting the impact of vancomycin resistance on 30-day mortality was estimated using Cox regression with further analytic control for confounding factors.The 30-day mortality rates were 27% and 38% for ARE in the Netherlands and Denmark, respectively, and the 30-day mortality rates were 33% and 48% for VRE in these respective countries. The adjusted RR for 30-day mortality for VRE was 1.54 (95% confidence interval, 1.06-2.25). Although appropriate antibiotic therapy was initiated later for VRE than for ARE bacteremia, further analysis did not reveal mediation of the increased mortality risk.Compared to ARE bacteremia, VRE bacteremia was associated with higher 30-day mortality. One explanation for this association would be increased virulence of VRE, although both phenotypes belong to the same well-characterized core genomic lineage. Alternatively, it may be the result of unmeasured confounding.

BackgroundIn many European hospitals, ampicillin-resistant Enterococcus faecium (ARE) is endemic, while outbreaks of vancomycin-resistant E. faecium (VRE), belonging to the same genetic lineage, are increasingly reported. We studied the attributable mortality due to vancomycin resistance in patients with E. faecium bacteremia and evaluated whether this is mediated by a delay in appropriate antibiotic therapy.MethodsIn a retrospective matched cohort study, patients with VRE bacteremia occurring between 2009 and 2014 in 20 Dutch and Danish hospitals were matched to patients with ARE bacteremia, on hospital, ward, length of hospital stay prior to bacteremia, and age. The risk ratio (RR) for 30-day mortality contrasting VRE with ARE was estimated with further analytic control for confounding factors.ResultsIn all, 63 VRE and 234 ARE episodes were matched (36 and 130 for the Netherlands and 27 and 104 for Denmark). Crude 30-day mortality was 27% and 38% for ARE in the Netherlands and Denmark, respectively, and 33% and 48% for VRE in the respective countries. The adjusted RR for 30-day mortality for VRE was 1.54 (95% confidence interval (CI) 1.06-2.25). Although appropriate therapy was initiated later for VRE than for ARE bacteremia, this did not appear to mediate the increased mortality risk.ConclusionsCompared to ARE bacteremia, VRE bacteremia was associated with higher 30-day mortality. One explanation for this association would be increased virulence of VRE, although both phenotypes belong to the same well-characterized core genomic lineage. Alternatively, it may be the result of unmeasured confounding.

OBJECTIVES: In the context of a large outbreak of OXA-48-producing Enterobacteriaceae (OXA-E) in a Dutch hospital we determined risk factors for acquisition of OXA-E. PATIENTS AND METHODS: A matched case-control study was performed in which cases (culture positive for OXA-E) were matched 1:3 to controls (culture negative for OXA-E) based on hospital ward, index date (±1 week) and time exposed in the hospital (best match). Stratified analyses were performed for patients with OXA-E producing and not producing ESBL. Potential risk factors included age, gender, surgery and ICU admission within 30 days preceding the index date, presence of comorbidities and in-hospital antibiotic treatment within 30 days preceding the index date. Data analysis was performed using multivariable conditional logistic regression with Firth correction. RESULTS: In total, 73 cases were matched to 211 controls. In the multivariable conditional logistic regression model, male gender (OR 2.63, 95% CI 1.25-5.53), age (per year increase, OR 1.03, 95% CI 1.00-1.05) and use of fluoroquinolones within 30 days preceding the index date (OR 2.98, 95% CI 1.06-8.41) were risk factors for acquisition of OXA-E. In the stratified multivariable conditional logistic regression model, quinolone use was a risk factor for the acquisition of ESBL-producing OXA-E and surgery was a risk factor for the acquisition of non-ESBL-producing OXA-E. CONCLUSIONS: During a large, hospital-wide OXA-E outbreak, male gender, age and previous use of fluoroquinolones were risk factors for acquisition of OXA-E. These findings may help in optimizing screening and isolation strategies in future OXA-E outbreaks.

In an outbreak setting, we screened 16,296 samples from 3,644 patients by PCR for the presence of bla OXA-48 , bla VIM , bla IMP , bla NDM , and bla KPC . The bla OXA-48 gene was found in samples from 43 patients infected with 9 different species of Enterobacteriaceae . Five patients had Pseudomonas aeruginosa isolates containing bla VIM . The negative predictive value of screening was 100%, and the positive predictive value was 86%.

textabstractBackground & Aims The extent of entry of multidrug-resistant Escherichia coli from the community into the hospital and subsequent clonal spread amongst patients is unclear. To investigate the extent and direction of clonal spread of these bacteria within a large teaching hospital, we prospectively genotyped multidrug-resistant E. coli obtained from community- and hospital associated patient groups and compared the distribution of diverse genetic markers. Methods A total of 222 E. coli, classified as multi-drug resistant according to national guidelines, were retrieved from both screening (n = 184) and non-screening clinical cultures (n = 38) from outpatients and patients hospitalized for various periods. All isolates were routinely genotyped using an amplified fragment length polymorphism (AFLP) assay and real-time PCR for CTX-M genes. Multi-locus sequence typing was additionally performed to confirm clusters. Based on demographics, patients were categorized into two groups: patients that were not hospitalized or less than 72 hours at time of strain isolation (group I) and patients that were hospitalized for at least 72 hours (group II). Results Genotyping showed that most multi-drug resistant E. coli either had unique AFLP profiles or grouped in small clusters of maximally 8 isolates.We identified one large ST131 clade comprising 31% of all isolates, containing several AFLP clusters with similar profiles. Although different AFLP clusters were found in the two patient groups, overall genetic heterogeneity was similar (35% vs 28% of isolates containing unique AFLP profiles, respectively). In addition, similar distributions of CTX-M groups, including CTX-M 15 (40% and 44% of isolates in group I and II, respectively) and ST131 (32% and 30% of isolates, respectively) were found. Conclusion We conclude that multi-drug resistant E. coli from the CTX-M 15 associated lineage ST131 are widespread amongst both community- and hospital associated patient groups, with similar genetic diversity and similar distributions of genetic markers.

textabstractBackground The objective of our study was the development of a semi-quantitative real-time PCR to detect uropathogens. Two multiplex PCR reactions were designed to detect Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus mirabilis, Enterococcus faecalis, and Pseudomonas aeruginosa. 16S based PCR was performed in parallel to detect Gram-positive and Gram-negative bacteria. Firstly to identify non-targeted agents of infection in the same urine specimen, and secondly to quantify background flora. The method was evaluated in comparison with standard bacterial culture, and a commercial PCR kit for detection of uropathogens. Findings Analysis with a known panel of 116 clinical isolates yielded a PCR specificity of 100%. Analysis of urine specimens from 211 patients revealed a high correlation of PCR Cq values with both culture positivity and quantity. Concordance between PCR and culture was 98% when both methods yielded results. PCR was found to be more sensitive than culture. With a cut-off Cq value of 33, the negative predictive value of PCR was 94%. The 16S PCR confirmed most results. One specimen was positive by 16S PCR suggesting another cause of infection not detected by the specific PCR assays. Conclusion We conclude that it is feasible to detect and identify uropathogens by multiplex real-time PCR assay.

Epitheliocystis in leafy seadragon ( Phycodurus eques ), silver perch ( Bidyanus bidyanus ), and barramundi ( Lates calcarifer ), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae . By using in situ RNA hybridization, 16S rRNA Chlamydiales -specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent.

Mouse models have been frequently used in the study of Chlamydia pneumoniae (also known as Chlamydophila pneumoniae) infections. This gram-negative obligate intracellular bacterium causes respiratory infections, followed by dissemination of the bacterium to various organs throughout the body, including cardiovascular tissues, supporting the current hypothesis of a relationship between C. pneumoniae and atherosclerosis. Recently, clinical trials evaluated the effect of antichlamydial antibiotics on secondary cardiovascular events. Although small studies showed some effect, the large WIZARD study did not confirm these results, and the role of antichlamydial antibiotics in prevention of secondary events was questioned. To address these issues, data obtained from mouse models were systematically reviewed here. C. pneumoniae infections showed atherogenic properties in mice that were reproducible and confirmed by different research groups. However, antibiotic therapy was of limited value in these mouse models. Antibiotic therapy effectively cleared the acute infection, but did not influence the atherogenic properties of C. pneumoniae unless the therapy was started early during the acute infection. The results summarized here may help to better understand the results of the clinical antibiotic trials.

New enzyme immunoassays (EIAs) for determination of specific IgG, IgA, and IgM antibody titers to Chlamydia pneumoniae were evaluated independently in three research laboratories. Specificity of the EIAs was enhanced by removing LPS from the chlamydial antigen. The performance of these EIAs was evaluated in comparison with the microimmunofluorescence (MIF) test using specimens from: (i) a group of adult patients with community-acquired pneumonia (CAP) previously diagnosed as having an acute chlamydial infection by the complement fixation test or the whole inclusion fluorescence test; (ii) from a group of adult patients with acute respiratory tract infections; and (iii) from a group of young children consecutively presenting with acute respiratory tract infections. The MIF test and the EIAs detected acute infections in paired serum specimens from 12 of 14 patients from the first group. Eleven of these 12 patients were positive in both tests. The MIF test detected seven acute infections in single convalescence serum specimens from eight patients. Two of these were also positive in the EIAs. Paired serum specimens from the second group of adult patients (n=12) were collected during an epidemic of C. pneumoniae. The EIAs detected six acute infections. The MIF test detected two additional patients with acute infections. From the group of young children (n=30), the EIAs detected two patients with acute infections. Our conclusion from this preliminary evaluation is that these EIAs could be useful for laboratory diagnosis of acute C. pneumoniae infections. Comprehensive prospective studies should provide suitable data to calculate the sensitivity, specificity, and predictive values.

OBJECTIVE: Atherosclerotic lesions are characterized by an immune mediated chronic inflammation. Seroepidemiological studies support a relationship between atherosclerotic disease and infection with C. pneumoniae; an association further endorsed by immunocytochemical and DNA directed studies. However, the question arises whether C. pneumoniae acts as a causal antigen, or is merely a bystander. For this reason we have analyzed the T lymphocyte population of carotid atherosclerotic plaques of symptomatic patients for their response against C. pneumoniae. METHODS: T cell lines were generated from carotid endarterectomy tissues obtained from eight patients with symptomatic disease. The response of these T cell lines against C. pneumoniae elementary bodies was analyzed by 3H-thymidine incorporation. T cell clones were generated by limiting dilution from the cell lines of three patients and tested for antigen specificity in the same manner. Furthermore, cytokine profiles (Th1/Th0/Th2) were established by measuring the production of IFN-gamma and IL-4. RESULTS: Of the eight T-cell lines five responded to C. pneumoniae. Eighteen of 69 CD4-positive clones, generated from three patients with a positive T cell lines response, responded to C. pneumoniae also. The majority (17/18, 96%) of these clones showed a Th1 cytokine profile. CONCLUSION: These results show that in a subpopulation of symptomatic patients C. pneumoniae can activate T cells within atherosclerotic plaques suggesting that a C. pneumoniae enhanced proinflammatory Th1 response contributes to plaque destabilization in these patients

Chlamydia pneumoniae is frequently found in atherosclerotic lesions, and high titers of specific antibodies are associated with increased risk for acute myocardial infarction. However, a causative relation has not been established yet. We performed a prospective study of 93 patients undergoing percutaneous transluminal coronary angioplasty (PTCA) to investigate whether angioplasty influences Chlamydia -specific antibody titers and whether there is an association with restenosis. Blood samples were obtained before and 1 and 6 months after angioplasty. Antibodies against chlamydial lipopolysaccharide and against purified C. pneumoniae elementary bodies were measured by enzyme-linked immunosorbent assay (ELISA). After angioplasty, the prevalence of antibodies to lipopolysaccharide rose from 20 to 26% for immunoglobulin A (IgA), from 53 to 64% for IgG, and from 2 to 7% for IgM ( P = 0.021, 0.004, and 0.046, respectively). There was a rapid increase of mean antibody titers of all antibody classes within 1 month of PTCA. During the following 5 months, antibody titers decreased slightly but were still higher than baseline values. Results of the C. pneumoniae -specific ELISA were essentially the same. The rise of anti- Chlamydia antibodies was not caused by unspecific reactivation of the immune system, as levels of antibodies against cytomegalovirus did not change. Neither seropositivity nor antibody titers were related to restenosis. However, increases in mean IgA and IgM titers were restricted to patients who had suffered from myocardial infarction earlier in their lives. In conclusion, we show that PTCA induces a stimulation of the humoral immune response against C. pneumoniae . These data support the idea that plaque disruption during angioplasty might make hidden chlamydial antigens accessible to the immune system.

On 31 May 2011, after notification of Klebsiella pneumoniae (KP)(OXA-48;CTX-M-15) in two patients, nosocomial transmission was suspected in a Dutch hospital. Hospital-wide infection control measures and an outbreak investigation were initiated. A total of 72,147 patients were categorised into groups based on risk of OXA-48 colonisation or infection, and 7,527 were screened for Enterobacteriaceae(OXA-48) by polymerase chain reaction (PCR). Stored KP isolates (n=408) were retrospectively tested for OXA-48 and CTX-M-1 group extended-spectrum beta-lactamases (ESBL). 285 KP isolates from retrospective and prospective patient screening were genotyped by amplified fragment length polymorphism (AFLP). 41 isolates harbouring different Enterobacteriaceae species were analysed by plasmid multilocus sequence typing (pMLST). No nosocomial transmission of Enterobacteriaceae(OXA-48) was detected after 18 July 2011. Enterobacteriaceae(OXA-48) were found in 118 patients (KP (n=99), Escherichia coli (n=56), ≥1 Enterobacteriaceae(OXA-48) species (n=52)), of whom 21 had clinical infections. 39/41 (95%) of OXA-48 containing plasmids were identical in pMLST. Minimum inhibitory concentrations (MICs) of KP(OXA-48) and E. coli(OXA-48) for imipenem and meropenem ranged from ≤1 to ≥16 mg/L, and 153/157 (97%) had MIC >0.25 mg/L for ertapenem. AFLP identified a cluster of 203 genetically linked isolates (62 KP(OXA-48;CTX-M15); 107 KP(CTX-M-15); 34 KP(OXA-48)). The ‘oldest’ KP(CTX-M-15) and KP(OXA-48) clonal types originated from February 2009 and September 2010, respectively. The last presumed outbreak-related KP(OXA-48) was detected in April 2012. Uncontrolled transmission of KP(CTX-M-15) evolved into a nosocomial outbreak of KP(OXA-48;CTX-M15) with large phenotypical heterogeneity. Although the outbreak was successfully controlled, the contribution of individual containment measures and of the hospital relocating into a new building just before outbreak notification was impossible to quantify.

Background: Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; bla(OXA-48), bla(VIM), bla(IMP), bla(NDM), and bla(KPC). Methods: A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates. Results: Both sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons. Conclusions: In this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases bla(OXA-48), bla(VIM), bla(IMP), bla(NDM), and bla(KPC), and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.

OBJECTIVE--To investigate C trachomatis serovars in contact-traced heterosexual partners. METHODS--Urogenital Chlamydia trachomatis isolates (n = 112) derived from 35 heterosexual patients (index patients) and their 37 chlamydia positive partners (contact patients) were differentiated into serovars by genotyping with restriction fragment length polymorphism (RFLP) analysis of the PCR amplified omp1 gene. In order to investigate whether different strains within the frequently prevalent serovar F were transmitted, two pairs of serovar F (n = 4) were further analysed by genomic DNA fingerprinting with arbitrary primer PCRs (AP-PCRs). RESULTS--Identical C trachomatis serovars were found in 31 of the 35 pairs, serovars E, F, D, and G being most prevalent. In the remaining four pairs different serovars (either D, E, F or G) were found between the index and the contact patients. By AP-PCR analysis the strains of serovar F were found to be identical between the index and the contact patients, but were different between the two pairs in all AP-PCRs used. CONCLUSION--A majority of heterosexual partners, once traced positive for C trachomatis infections, are infected with identical serovars. Identical strains of serovar F found in partners as found by DNA fingerprinting confirms the sexual transmission of C trachomatis.

Background A major outbreak of methicillin-resistant Staphylococcus aureus (MRSA) occurred in locations C and Z of our hospital and lasted for several years. It affected 1,230 patients and 153 personnel. Methods Outbreak management was installed according to the Dutch "search and destroy" policy. A rapid, high-throughput method for molecular screening of potential MRSA carriers was implemented. Outbreak isolates were retrospectively genotyped by pulsed field gel electrophoresis. Costs of molecular screening were compared with screening by culture. Results Genotyping results revealed 4 distinct epidemic MRSA clones. Three were present in hospital C. Because of a merger of hospitals, these clones spread to hospital Z. Another clone of MRSA affected other health care-related institutions in the region. Because of the implementation of strict containment measures of the "search and destroy" policy, the annual number of tests decreased from 100,000 to 18,000. The disposables and reagents used in polymerase chain reaction technology are more expensive than those of conventional methods. However, the clinical and economic benefits of fast results in regard to expenses of the hospital clearly outweigh the higher costs of screening. Conclusion The implementation of a rapid, high-throughput molecular screening system greatly contributed to the effectiveness of strict containment measures of the "search and destroy" policy. The major epidemic clones of MRSA in the outbreak were eradicated by this strategy.

We present the first genome sequence of Chlamydophila psittaci , an intracellular pathogen of birds and a human zoonotic pathogen. A comparison with previously sequenced Chlamydophila genomes shows that, as in other chlamydiae, most of the genome diversity is restricted to the plasticity zone. The C. psittaci plasmid was also sequenced.

Background The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST) scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease. Results A collection of 26 strains of C. trachomatis of different serovars and clinical presentation and 18 strains of C. pneumoniae were included in the study. For comparison, sequences of C. abortus, C. psittaci, C. caviae, C. felis, C. pecorum (Chlamydophila), C. muridarum (Chlamydia) and of Candidatus protochlamydia and Simkania negevensis were also included. Sequences of fragments (400 – 500 base pairs) from seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX, oppA) were analysed. Analysis of allelic profiles by eBurst revealed three non-overlapping clonal complexes among the C. trachomatis strains, while the C. pneumoniae strains formed a single group. An UPGMA tree produced from the allelic profiles resulted in three groups of sequence types. The LGV strains grouped in a single cluster, while the urogenital strains were distributed over two separated groups, one consisted solely of strains with frequent occurring serovars (E, D and F). The distribution of the different serovars over the three groups was not consistent, suggesting exchange of serovar encoding ompA sequences. In one instance, exchange of fumC sequences between strains of different groups was observed. Cluster analyses of concatenated sequences of the Chlamydophila and Chlamydia species together with those of Candidatus Protochlamydia amoebophila and Simkania negevensis resulted in a tree identical to that obtained with 23S RNA gene sequences. Conclusion These data show that C. trachomatis and C. pneumoniae are highly uniform. The difference in genetic diversity between C. trachomatis and C. pneumoniae is in concordance with a later assimilation to the human host of the latter. Our data supports the taxonomy of the order of Chlamydiales.

Objectives: This study was conducted to investigate whether serologic titers of species-specific IgA and IgG antibodies in patients with rectal chlamydial infection could discriminate between infection with serovar L2 lymphogranuloma venereum (LGV) and infection with non-LGV serovars. Methods: A total of 39 male patients with chlamydial infection of the rectum were tested for titers of IgA and IgG antibodies within 14 days after detection of the infection and 6 and 12 months after adequate treatment. Data were collected concerning demographics, sexual orientation, HIV serostatus, history of chlamydial infection, concomitant STI or HIV infection, hepatitis C virus antibodies and new STIs during follow-up. Results: Between May 2003 and November 2005, 24 males with confirmed L2 proctitis and 15 men with non-LGV rectal chlamydial infection were recruited. In multivariable analyses, both high titer of IgA within 14 days after detection of the infection and older age of the individual were significantly associated with L2 proctitis (p < 0.0005 and p = 0.001 respectively). A total sum score of seven times IgA titer plus individual9s age ≥ 50.0 resulted in an overall sensitivity of 92% and specificity of 100%. This total sum score was highly accurate for detection of LGV proctitis, with an area under the curve (AUC) in a receiver operating characteristic (ROC) curve of 0.989. Conclusions: In this study, an elevated IgA antibody response and the age of the infected individual appeared to be of possible diagnostic value for (early) detection of LGV proctitis.

Testing for Chlamydia trachomatis (Ct) is less accepted in people of non-Dutch ethnicity than Dutch people. We offered additional Ct and gonorrhoea testing through our outreach sexually transmitted infections (STI) prevention programme to determine whether this intervention strategy is feasible and efficient.Outreach workers offered test kits to women and men aged 15-29 years, in group and street settings and in a vocational training school. Demographic and behavioural data and characteristics of non-responders were assessed. DNA was isolated (using the MagNA Pure LC system) from urine and tested using the Cobas Amplicor test.Among sexually active people, the test rate differed by venue (groups 80% (74/93), school 73% (49/67), street 17% (49/287); p0.001). There was no difference in test rate between group and school settings by gender or ethnicity. Ct positivity was 14.5% (25/172); women 20.2% (20/99) versus men 6.8% (5/73); p = 0.01. Ct positivity was highest at school (24.5% (12/49)) and among Surinamese/Antillean people (17.5% (14/80)). Treatment rate of index cases and current partners was 100% and 78%, respectively.We found a high acceptance of chlamydia testing in group and school settings in both men and women of non-Dutch ethnicity. The prevalence indicates that we have accessed high risk people. Outreach testing and is feasible and most efficient in school and group settings. School screening may have an impact on community prevalence of Ct infections.

In December 2004, a patient presented at the sexually transmitted infection unit of Barcelona with anorectal syndrome.

The success of large-scale screening for Chlamydia trachomatis depends on the availability of noninvasive samples, low costs, and high-quality testing. To evaluate C. trachomatis testing with pregnant women, first-void urine specimens from 750 consecutive asymptomatic pregnant women from the Rotterdam area (The Netherlands) were collected. Initially, we investigated the performance of three different DNA isolation methods with 350 of these urines and 70 pools of 5 of the same subset of urine samples. The routinely used COBAS AMPLICOR test was compared to the COBAS AMPLICOR test with prior DNA isolation by use of the MagNA Pure large-volume kit and the MagNA Pure bacterial DNA isolation kit. The latter combination provided the best DNA test for pooled urines, with a sensitivity twice that of the other methods. Next, using all 750 urines, the COBAS AMPLICOR performance for individual testing was compared to pooled testing with the standard COBAS AMPLICOR procedure and subsequently to pooled testing with COBAS AMPLICOR in combination with the MagNA Pure bacterial DNA isolation kit. The sensitivity of COBAS AMPLICOR was 65% on individual and 42% on pooled urines but improved to 92% on pooled urines with the MagNA Pure bacterial DNA isolation kit, making this combination the best screening method. The C. trachomatis prevalence in this population appeared to be 6.4%. Additionally, the cost of the combined MagNA Pure bacterial DNA isolation kit and COBAS AMPLICOR method on pooled urines was only 56% of the cost of the standard COBAS AMPLICOR test applied to individual urines. Costs per positive case detected in the combined method were 39% of standard costs.

Mouse models have been frequently used in the study of Chlamydia pneumoniae (also known as Chlamydophila pneumoniae) infections. This gram-negative obligate intracellular bacterium causes respiratory infections, followed by dissemination of the bacterium to various organs throughout the body, including cardiovascular tissues, supporting the current hypothesis of a relationship between C. pneumoniae and atherosclerosis. Recently, clinical trials evaluated the effect of antichlamydial antibiotics on secondary cardiovascular events. Although small studies showed some effect, the large WIZARD study did not confirm these results, and the role of antichlamydial antibiotics in prevention of secondary events was questioned. To address these issues, data obtained from mouse models were systematically reviewed here. C. pneumoniae infections showed atherogenic properties in mice that were reproducible and confirmed by different research groups. However, antibiotic therapy was of limited value in these mouse models. Antibiotic therapy effectively cleared the acute infection, but did not influence the atherogenic properties of C. pneumoniae unless the therapy was started early during the acute infection. The results summarized here may help to better understand the results of the clinical antibiotic trials.

Background. Lymphogranuloma venereum (LGV) is a sexually transmitted disease (STD) and is rare in the Western world. Recently, 3 men who have sex with men presented with LGV proctitis at the Erasmus Medical Center, Rotterdam, The Netherlands. We investigated a possible outbreak in a sexual network of men who have sex with men (MSM). Methods. After active case finding, a total of 15 men presented and were investigated. Serum antibody titers to Chlamydia trachomatis were determined. Urine and rectum specimens were analyzed by polymerase chain reaction (PCR) for the presence of C. trachomatis. C. trachomatis‐positive specimens were genotyped to detect the specific C. trachomatis serovars. All subjects underwent routine STD screening. Sociodemographic, clinical, and endoscopic characteristics were evaluated. Results. Thirteen subjects had high immunoglobulin (Ig) G and IgA titers to C. trachomatis, suggesting an invasive infection. Rectal specimens of 12 subjects were PCR-positive for C. trachomatis. All urine specimens were negative. Genotyping revealed serovars L2 ( ) and L1 ( ). An ulcerative proctitis was found in all subjects n p 8 n p 1 obtaining sigmoidoscopy ( ). Eleven of 13 subjects with an LGV diagnosis were seropositive for human n p 9 immunodeficiency virus (HIV), 6 had another concomitant STD, and 1 had recently acquired a hepatitis C virus infection. Further sexual contacts were reported from The Netherlands, Germany, Belgium, the United Kingdom, and France. Conclusions. We revealed an outbreak of LGV proctitis among MSM in The Netherlands. The ulcerous character favors transmission of HIV, other STDs, and blood-borne diseases. From a public health perspective, it seems important to increase the awareness of possible LGV in MSM with symptomatic proctitis.

To investigate the correlation between the incA I47T mutation in Chlamydia trachomatis and the nonfusogenic phenotype, the incA genes of 25 isolates were sequenced. Four major sequence types were identified. Seven isolates (28%) had the I47T mutation. Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion. In conclusion, the incA I47T mutation is not associated with the nonfusogenic phenotype.

In this paper we present a case history of a homosexual HIV-positive male with a painless nodule on the penis. Screening for sexually transmitted diseases did not detect any infection until the node perforated spontaneously. A diagnosis of lymphogranuloma venereum was made when chlamydia trachomatis type L2 DNA was extracted from the lesion. This case illustrates that standard screening may not be sufficient for making the diagnosis of lymphogranuloma venereum.

Amplified fragment length polymorphism (AFLP) fingerprinting of clinical isolates of Chlamydia trachomatis serovars D, E, and F showed a low percentage of genetic heterogeneity, but clear differences were found. Isolates from index patients and partners had identical AFLP patterns and AFLP markers. Characterization of these AFLP markers could give more insight into the differences in virulence and clinical course of C. trachomatis infections.

Chlamydiae are obligate intracellularly growing bacteria. They are widespread throughout the world and infect both humans and animals. Currently, four species, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, and Chlamydia pecorum, belonging to the genus Chlamydia of the family Chlamydiaceae within the order Chlamydiales, are recognized (10, 11, 35). C. pneumoniae and C. trachomatis are primarily human pathogens. C. pneumoniae has been recognized as a major cause of respiratory infections. In addition, C. pneumoniae infection has been associated with new-onset asthma, exacerbation of chronic asthma, atherosclerotic disease, and, recently, Alzheimer’s dementia (2, 22). C. trachomatis is a major cause of sexually transmitted diseases and trachoma in humans (18). C. psittaci and C. pecorum are primarily animal pathogens, but C. psittaci may cause zoonotic infections (44). Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified genes has been used to characterize chlamydial isolates. Using PCR-RFLP analysis of different genes, Chlamydia could be differentiated at the species level, and C. trachomatis and C. psittaci could be differentiated at a strain level corresponding to serovars and types (4, 12–14, 24, 29, 31, 53). However, C. pneumoniae isolates originating from all over the world could not be differentiated by this technique (4, 13, 29). Furthermore, the available sequence data for C. pneumoniae shows complete or nearly complete conservation for omp1, omp2, 16S rRNA, domain I of the 23S rRNA, RNase P RNA, the genes for the 53-kDa protein and the 76-kDa protein, dnaK, and waaA (kdtA) and the 16S-23S ribosomal DNA intergenic spacer (7, 13, 15, 17, 21, 26, 30, 39, 40, 55). By analysis of the whole genome using RFLP (1, 5, 9, 27, 28, 37, 38, 42), random amplification of polymorphic DNA (RAPD) (41, 45), or hybridization (5, 6, 9), the four species could be differentiated, and subgroups could be recognized within the C. trachomatis and C. psittaci species. These findings are in agreement with the power of discrimination of RFLP and RAPD at the species-to-strain level and of DNA-DNA hybridization at the genus-to-subspecies level (52). RFLP analysis of the genome of C. pneumoniae showed only two nearly identical patterns. One extra band was observed in two of eight C. pneumoniae isolates (5). However, DNA-DNA hybridization experiments showed 94 to 96% relatedness among C. pneumoniae isolates, suggesting at least some genomic variation (6). Recently, a novel high-resolution technique has been introduced for whole-genome analysis: amplified fragment length polymorphism (AFLP) (54). This technique requires relatively low amounts of genomic DNA. The DNA is digested by a combination of a restriction enzyme that has a high number of restriction sites in DNA and a restriction enzyme that has an average number of restriction sites in DNA. Selected sets of restriction fragments are amplified and analyzed on gels. This technique has proven its usefulness as a tool in bacterial taxonomy and epidemiology (16, 20, 43) and has also been applied in C. trachomatis research (32). Here, we report on the application of AFLP to analyze the differences among Chlamydia species and, within the species, among subgroups.

Summary: Respiratory tract infections in man may be caused by several members of the genus Chlamydia and also by two Chlamydia-like strains, ‘Simkania negevensis’ (Z-agent) and ‘Parachlamydia acanthamoebae’ (Bn9). To facilitate diagnostic procedures a PCR assay able to detect all known Chlamydiaceae sequences in one reaction was developed. For this purpose, primers were selected to amplify a fragment of the 16S rRNA gene. Characterization of the amplified fragments was done by hybridization with specific probes and by sequencing. PCR assays were carried out using DNA isolated from nose/throat specimens or from peripheral blood mononuclear cells of patients with respiratory tract infections, and from vessel wall specimens of abdominal aneurysms. Six of the 42 nose/throat swab specimens analysed yielded strong bands and one yielded a faint band. Three of these bands were identified as Chlamydia pneumoniae and one as Chlamydia trachomatis by sequencing. Analysis of the three other bands yielded two different new sequences. DNA isolated from peripheral blood mononuclear cells of one patient yielded a third new sequence. DNA isolated from peripheral blood mononuclear cells of four healthy controls was negative. One of the abdominal aneurysm specimens also yielded a strong band. Sequencing revealed a fourth new sequence. All negative controls included during specimen processing and PCR analysis remained negative. The typical secondary structure of microbial 16S genes was present in all four new sequences indicating the validity of the sequence data. All four new sequences were distinct from other bacteria and clustered together with known Chlamydiaceae sequences. Phylogenetic analysis suggested a new lineage, separating the four new sequences, ‘S. negevensis’ and ‘P. acanthamoebae’ from the genus Chlamydia with the four known chlamydial species. In conclusion, this study provides evidence for the existence of several new members of the order Chlamydiales. Since the source of the Chlamydia-like strains has not been identified and serological and/or molecular cross-reactivities may be expected, results of identification of infecting recognized organisms should be interpreted cautiously.

Objective To examine the prevalence and correlates of infection with herpes simplex virustype 2 (HSV-2) among sexually transmitted disease (STD) clinic attenders, we studiedthe prevalence of antibodies to HSV-2 and their association with risk behaviour.Methods Data were collected in a cross-sectional study among STD clinic attenders inAmsterdam. Seropositivity for HSV-2 was determined in 1798 serum samples bymeans of a monoclonal antibody-blocking enzyme-linked immunoassay.Results The prevalence of HSV-2 antibodies was higher than expected: 32.3% in a popula-tion in which 3% had current genital herpes and 8% gave a history of genitalherpes. Of those with HSV-2 antibodies, only 18% had a history of genital herpes.A strong independent association with the presence of HSV-2 antibodies wasfound for sexual behaviour, more specifically: homosexual orientation, increasingnumber of years of sexual activity, increasing number of lifetime partners, num-ber of past gonococcal infections, having receptive anal and (or) vaginal contact.Conclusion The presence of HSV-2 antibodies had a strong association with past sexual be-haviour and, for both sexes, with receptive anal intercourse. HSV-2 antibodies maybe used as a surrogate marker of sexual risk behaviour in comparing differentpopulations over time.Keywords Herpes genitalis, seroprevalence, epidemiology, risk factors, sexual behaviour,sexually transmitted diseasesAccepted 27 June 1997Type-specific serological tests for herpes simplex virus type 2(HSV-2) have been used to assess the prevalence of HSV-2 inpopulations, '~

Urogenital isolates ( n = 93) of Chlamydia trachomatis were differentiated into serovars and variants by serotyping with monoclonal antibodies and genotyping by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified omp1 gene, respectively. The types of 87 of the 93 isolates (94%) were identical, as determined by both methods. Among these 87 isolates, 3 isolates were identified as the recently described new serovariant Ga/IOL-238 by omp1 nucleotide sequence analysis of the variable domains. Of the remaining six isolates, three isolates serotyped as both L2 and Ba but were identified as Ba/A-7 by genotyping by RFLP analysis of omp1 . The omp1 nucleotide sequences of variable domains VD1, VD2, and VD4 of these urogenital Ba strains were identical to the sequences of the variable domains of Ba/J160, an ocular Ba type. The three remaining isolates were serotyped as J, but the patterns obtained by RFLP analysis of omp1 , which were identical for the three isolates, differed from that of prototype serovar J/UW36. omp1 nucleotide sequence analysis revealed that these strains are genovariants of serovar J/UW36. Nucleotide sequence differences between serovar J/UW36 and this J genovariant, designated Jv, were found in both variable and constant domains. In conclusion, this study shows that the PCR-based genotyping of clinical C. trachomatis isolates by RFLP analysis of omp1 has a higher discriminatory power and is more convenient than serotyping. Variants of C. trachomatis serovars Ba, G, and J were identified and characterized.

OBJECTIVE--To determine the prevalence of Chlamydia trachomatis infection in women attending a family planning clinic in Papua New Guinea, in the period between April and June 1991. SETTING--The outpatient department of Obstetrics and Gynaecology of Port Moresby General Hospital, Port Moresby, Papua New Guinea, the departments of Dermato-Venereology and Clinical Microbiology of the Erasmus University, Rotterdam, The Netherlands and the National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands. PATIENTS--A total of 254 consecutive women who attended the family planning clinic at Port Moresby General Hospital, Papua New Guinea were enrolled into this study. METHODS--Cervical infections with C trachomatis were diagnosed using the direct immunofluorescent assay (DFA) and the polymerase chain reaction (PCR). Serum IgM and IgG antibodies directed against C trachomatis were detected using the enzyme-linked fluorescent assay (ELFA). RESULTS--The prevalence of C trachomatis was 14.6% using the PCR, 9.1% using the DFA and 17.3% when the results of the PCR and the DFA were combined. An elevated IgM titre was observed in 14.2% of the women, whereas 44.1% had an elevated IgG titre. The titres of IgM or IgG were significantly higher in women who were positive using the PCR or the DFA than in those who were negative in both the PCR and the DFA (p = 0.032 and p = 0.0046, respectively). CONCLUSION--Cervical infection by C trachomatis can be considered a major health problem in at least the studied population in Papua New Guinea. The prevalence of C trachomatis infection is at least comparable with that in groups with a high prevalence in industrialized countries. Effective screening and treatment programmes are imperative to combat this problem.

To determine the role of Bartonella (formerly Rochalimaea) species and Afipia felis in cat scratch disease (CSD), two polymerase chain reaction (PCR) hybridization assays were developed to detect DNA from these organisms. These assays were applied on 89 pus aspirates from skin test-positive CSD patients (group 1) and on 137 pus and lymph node specimens from CSD suspects (group 2). Bartonella DNA was detected in 96% of the samples from group 1 patients and in 60% of group 2 samples; however, A. felis DNA could not be detected in any clinical samples. These results suggest that CSD is caused by bartonellae and that A. felis does not play a significant role in this zoonosis. A strong correlation between Bartonella PCR positivity and Bartonella henselae antibody titer was found. Comparison of CSD skin test results with those obtained by Bartonella PCR suggests a low sensitivity of the skin test.

Findings To facilitate automation, a novel DNA extraction method for MRSA was adopted. The MRSA specific chromosome-SCCmec PCR was adapted, additional primers were added, and the performance was validated. From various laboratories in The Netherlands we received a total of 86 MRSA clinical isolates, that were negative in commercially available tests. We identified 14 MRSA strains with new variant chromosome-SCCmec junctions by sequence analysis. These MRSA strains appeared to carry SCCmec sequences with a high degree of homology to SCC regions of S. epidermidis and S. haemolyticus. All were included for detection in chromosome-SCCmec based PCR. Background Efficient management of Methicillin Resistant Staphylococcus aureus (MRSA) in the hospital is needed to prevent dissemination. It is important that MRSA can be rapidly identified, and effective infection control measures can be initiated. Equally important is a rapid MRSA negative report, especially for patients in isolation. For negative screening we implemented fully automated high through-put molecular screening for MRSA. Conclusions Fourteen variant chromosome-SCCmec junctions in MRSA, that are not detected in commercially available MRSA detection kits were added to our PCR to detect all currently known variant SCC-mec types of MRSA.

Background It has been reported that Chlamydophila (C.) pneumoniae is involved in the initiation and promotion of asthma and chronic obstructive pulmonary diseases (COPD). Surprisingly, the effect of C. pneumoniae on airway function has never been investigated. Methods In this study, mice were inoculated intranasally with C. pneumoniae (strain AR39) on day 0 and experiments were performed on day 2, 7, 14 and 21. Results We found that from day 7, C. pneumoniae infection causes both a sustained airway hyperresponsiveness and an inflammation. Interferon-γ (IFN-γ) and macrophage inflammatory chemokine-2 (MIP-2) levels in bronchoalveolar lavage (BAL)-fluid were increased on all experimental days with exception of day 7 where MIP-2 concentrations dropped to control levels. In contrast, tumor necrosis factor-α (TNF-α) levels were only increased on day 7. From day 7 to 21 epithelial damage and secretory cell hypertrophy was observed. It is suggested that, the inflammatory cells/mediators, the epithelial damage and secretory cell hypertrophy contribute to initiation of airway hyperresponsiveness. Conclusion Our study demonstrates for the first time that C. pneumoniae infection can modify bronchial responsiveness. This has clinical implications, since additional changes in airway responsiveness and inflammation-status induced by this bacterium may worsen and/or provoke breathlessness in asthma and COPD.

Background In Dutch laboratories molecular detection of B. pertussis and B. parapertussis is commonly based on insertion sequences IS 481 and IS 1001, respectively. Both IS elements are more widely spread among Bordetella species. Both Bordetella holmesii, and B. bronchiseptica can harbour IS 481. Also, IS 1001 is found among B. bronchiseptica. IS 481, and IS 1001 based PCR thus lacks specificity when used for detection of specific Bordetella spp. Findings We designed a PCR based on IS 1002, another IS element that is present among Bordetella species, and exploited it as a template in combination with PCR for IS 481, and IS 1001. In combining the PCRs for IS 481, IS 1001, and IS 1002, and including an inhibition control, we were able to detect and discriminate all clinically relevant Bordetella species. Conclusions We developed an improved PCR method for specific detection of B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica.