Risk factors for the acquisition of OXA-48-producing Enterobacteriaceae in a hospital outbreak setting: a matched case-control study.

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.

Successful control of a hospital-wide outbreak of OXA-48 producing Enterobacteriaceae in the Netherlands, 2009 to 2011.

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.

Chlamydia pneumoniae induces neointima formation in coronary arteries of normal pigs.

OBJECTIVES: We evaluated the role of intracoronary, intrapulmonary and macrophage-mediated delivery of C. pneumoniae (Cp) on coronary lesion formation. METHODS: Pigs were allocated to one of three coronary protocols (intracoronary, macrophage or control groups) or to a fourth-a pulmonary group. In the intracoronary group Cp was injected into the wall of the left anterior descending (LAD) and right coronary arteries (RCA) and vehicle into the circumflex (CX). In the macrophage group autologous macrophages preincubated with Cp or not were injected into the LAD and CX wall, respectively. Animals in the control group received vehicle in LAD and CX. In the pulmonary group aerosolised Cp was given intrabronchially, after a single injection of vehicle into the LAD wall. Delivery into the coronary artery wall was performed with a balloon catheter with low-profile injector ports. RESULTS: Seroconversion occurred in the following proportions: 5/6 (intracoronary group), 4/5 (macrophage group), 0/6 (control group), and 1/6 (intrapulmonary group). Significantly higher maximal intimal thickness (MIT) was observed in LADs of intracoronary and pulmonary groups when compared to corresponding CXs. The presence of Cp antigen was associated with higher MIT (r=0.73; P<0.0001). Injection of macrophages into the coronary artery wall did not induce proliferation. Arteries without coronary interventions were morphologically normal. CONCLUSIONS: Intracoronary and intrapulmonary but not macrophage-mediated Cp inoculation were associated with moderate intimal proliferation in the absence of a lipid-rich diet. Pre-existing coronary lesions seem a prerequisite for Cp-induced proliferation.

Unstable atherosclerotic plaques contain T-cells that respond to Chlamydia pneumoniae.

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.

Antibodies to Chlamydia pneumoniae and clinical course in patients with unstable angina pectoris.

Inflammation is one of the most important mechanisms that contribute to coronary artery disease (CAD). One of the micro-organisms that is mentioned as a source of the inflammation is Chlamydia pneumoniae. In this study, we investigated the relationship between titres of IgG and IgA antibodies to C. pneumoniae and the clinical course, during hospitalisation and during an 18-month follow-up, in 211 patients admitted to hospital with unstable angina pectoris. Slightly more patients who were refractory during their hospitalisation were positive for C. pneumoniae antibodies than patients who could be stabilised by drug treatment (53 vs. 43%, for IgG and 16 vs. 11% for IgA, respectively)(n.s.). In logistic regression analysis no significant predictive values were observed for the relationship between antibody titres and clinical course. The antibody titres to C. pneumoniae were lower in the unstable angina patients who had plasma levels of interleukin-10 (IL-10) above 5 pg/ml than in the patients with levels below 5 pg/ml, and higher in smokers than in non-smokers. No associations were observed between antibody titres to C. pneumoniae and C-reactive protein (CRP), interleukin-6 (IL-6), age, total cholesterol levels, fibrin degradation products (FDP), plasminogen activator inhibitor-1 (PAI-1) and erythrocyte sedimentation rate (ESR). In conclusion, there was no significant association between antibody titres to C. pneumoniae and risk of events during hospitalisation and the 18-month follow-up period in patients admitted for unstable angina pectoris.

An enzyme immunoassay to detect specific antibodies to protein and lipopolysaccharide antigens of Chlamydia trachomatis.

We have developed an enzyme immunoassay to measure antibodies to the proteins and lipopolysaccharide (LPS) of Chlamydia trachomatis. Antibodies to proteins could be differentiated from antibodies to lipopolysaccharide (LPS) by treatment of the antigen with periodate or Triton X-100. Some important parameters of the oxidation by periodate were studied by comparing the response of several monoclonal antibodies. Four types of response could be observed: type I, a reduced response after mild or strong oxidation; type II, a normal response after mild oxidation, but reduced after strong oxidation; type III, not affected; type IV, an increased response after oxidation. Treatment with Triton X-100 had the same effect as mild oxidation and confirmed the response types I, III, and IV. Treatment of antigen with periodate reduced the IgG response measured in sera from patients with evidence of Chlamydia psittaci infection.

Detection of microorganisms in vessel wall specimens of the abdominal aorta: development of a PCR assay in the absence of a gold standard.

A procedure in which the "Invitrogen Easy-DNA" kit was followed by a silica-based method for the isolation of DNA was developed for extraction of PCR-inhibitor-free DNA from up to 300 mg of human vessel wall tissue. Optimally designed PCR assays were developed for the detection of at least one infected cell in this amount of tissue. Details of the procedure are given for the detection of DNA of Chlamydia pneumoniae, cytomegalovirus and herpes simplex virus type 1 and type 2 in human vessel walls. The procedure can serve as a reference method or as a gold standard when a high-performance method is needed.

Prevalence and correlates of herpes simplex virus type 2 infection: evaluation of behavioural risk factors.

OBJECTIVE: To examine the prevalence and correlates of infection with herpes simplex virus type 2 (HSV-2) among sexually transmitted disease (STD) clinic attenders, we studied the 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 in Amsterdam. Seropositivity for HSV-2 was determined in 1798 serum samples by means of a monoclonal antibody-blocking enzyme-linked immunoassay. RESULTS: The prevalence of HSV-2 antibodies was higher than expected: 32.3% in a population in which 3% had current genital herpes and 8% gave a history of genital herpes. 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 was found for sexual behaviour, more specifically: homosexual orientation, increasing number of years of sexual activity, increasing number of lifetime partners, number 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 behaviour and, for both sexes, with receptive anal intercourse. HSV-2 antibodies may be used as a surrogate marker of sexual risk behaviour in comparing different populations over time.

Chlamydia pneumoniae in vitro and in vivo: a critical evaluation of in situ detection methods.

AIMS: There is a considerable discrepancy between data from the detection of Chlamydia pneumoniae in atherosclerotic lesions obtained by means of immunocytochemistry and data obtained using the polymerase chain reaction. This study evaluated methods for the in situ detection and assessment of the viability of C pneumoniae bacteria. METHODS: Chlamydia pneumoniae membrane protein, heat shock protein 60, and lipopolysaccharide were detected by immunocytochemistry, and genomic DNA and 16S rRNA by in situ hybridisation in paraffin wax embedded sections of cultured HEp2 cells infected with C pneumoniae and of lungs from mice infected intranasally with C pneumoniae. RESULTS: Inclusions reactive for all three antigens, DNA, and 16S rRNA were seen in infected HEp2 cells, in all positive bronchus and alveolar epithelial cells, and in some of the positive infiltrate cells in the lungs of mice up to seven days after infection. In all alveolar macrophages and in the infiltrate cells positive for antigens only, the staining pattern was granularly dispersed throughout the cytoplasm up to seven days after infection. At 21 days after infection, only this granular staining pattern was seen for antigens in infiltrate cells and macrophages in the alveoli and bronchus associated lymphoid tissue. At this time point, DNA or 16S rRNA were detected sporadically, but always as inclusion-like staining. CONCLUSIONS: Because antigens with an inclusion-like staining were detected only together with DNA and 16S rRNA, this type of staining pattern suggested the presence of viable bacteria. Thus, the granular staining pattern of antigens in the absence of staining for DNA and 16S is most likely caused by non-viable bacteria. In conclusion, these methods are suitable for the in situ detection of C pneumoniae and the assessment of its viability.

Chlamydia pneumoniae antigens, rather than viable bacteria, persist in atherosclerotic lesions.

AIMS: To evaluate the nature of the presence of Chlamydia pneumoniae or of other members of the order Chlamydiales in atherosclerotic lesions. METHODS: Consecutive sections of 13 carotid artery specimens obtained at necropsy and of C pneumoniae infected HEp2 cells were analysed using: (1) immunocytochemistry (ICC) to detect C pneumoniae membrane protein; (2) in situ hybridisation (ISH) using a polymerase chain reaction (PCR) fragment of the omp1 gene to detect C pneumoniae specific DNA; (3) ISH using an oligonucleotide probe to detect Chlamydiales specific 16S rRNA; (4) PCR to detect C pneumoniae 16S rDNA; and (5) in situ DNA nick and labelling (TUNEL) to detect fragmented DNA. RESULTS: Staining by ICC and ISH of infected HEp2 cells showed characteristic inclusions. Chlamydia pneumoniae membrane protein was demonstrated in macrophages in advanced atherosclerotic lesions (six of six), but not in fatty streaks (none of two), or normal arteries (none of five). ISH assays using both probes and PCR were all negative, indicating the absence of both specific C pneumoniae DNA and Chlamydiales specific 16S rRNA. Only after treatment with DNAse I were uniformly sized dots demonstrated by the TUNEL assay in inclusions of infected HEp2 cells. The TUNEL assay showed a similar staining pattern in macrophages in five carotid artery specimens, of which four were also positive for C pneumoniae membrane protein. Both macrophage populations were morphologically similar and were similarly distributed. CONCLUSIONS: No evidence was obtained for the involvement of other members of the order Chlamydiales in atherosclerosis. The presence of C pneumoniae antigen in the absence of DNA and 16S rRNA suggests that antigens, rather than viable bacteria, persist in atherosclerotic lesions.

Development of a monoclonal antibody for use as an amboceptor in complement fixation tests.

A monoclonal antibody for use as a haemolytic amboceptor in complement fixation tests was developed. The monoclonal antibody selected was investigated in parallel with conventional rabbit amboceptor. The results with the monoclonal amboceptor were at least as good as with the rabbit amboceptor. Production of the monoclonal amboceptor was attained provided that hybridoma growth factor was added to the culture medium.

A preliminary evaluation of a new enzyme immunoassay to detect Chlamydia pneumoniae-specific antibodies.

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.

Presumed ocular bartonellosis.

BACKGROUND: The spectrum of diseases caused by Bartonella henselae continues to expand and ocular involvement during this infection is being diagnosed with increasing frequency. METHODS: The clinical features and visual prognosis for 13 patients with intraocular inflammatory disease and laboratory evidence of bartonellosis were investigated. There were nine patients with neuroretinitis and four with panuveitis with positive antibody titres against B henselae determined by an enzyme immunoassay (IgG exceeding 1:900 and/or IgM exceeding 1:250). RESULTS: Positive IgG levels were found for eight patients and positive IgM levels for five. Despite animal exposure of 10 patients, only two (IgG positive) cases had systemic symptoms consistent with the diagnosis of cat scratch disease. Pathological fluorescein leakage of the optic disc was observed in all affected eyes. At 6 months' follow up, 3/18 (17%) affected eyes had a visual acuity of less than 20/100, owing to optic disc atrophy and cystoid macular oedema. 12 patients (17 eyes) were treated with antibiotics; visual acuity improved two or more Snellen lines for 9/17 (53%) eyes. CONCLUSIONS: The possibility of B henselae infection should be considered in patients with neuroretinitis and panuveitis (especially in cases with associated optic nerve involvement) even in the absence of systemic symptoms typical for cat scratch disease.

[A cluster of lymphogranuloma venereum among homosexual men in Rotterdam with implications for other countries in Western Europe]. / Cluster van lymphogranuloma venereum onder homoseksuele mannen in Rotterdam, met grensoverschrijdende gevolgen.

In mid-December 2003, a cluster of 15 cases of lymphogranuloma venereum (LGV) among male homosexuals was reported to the Municipal Health Service in Rotterdam by the Erasmus Medical Centre's outpatient clinic for sexually transmitted infections (STI). Most patients presented with proctitis and some with constipation. All were Caucasian and between 26 and 48 years of age. Thirteen of them were HIV-positive and eight had a concomitant STI. All men reported having had unprotected insertive and receptive anal sexual contact. Many sexual contacts were anonymous and were reported to have taken place in Germany, Belgium, the United Kingdom and France. This outbreak of LGV may extend through a large part of western Europe. In view of the patients' international contacts, international warnings and alertness are needed. Concerted action of professionals in infectious disease control and curative care is called for.

2010 European guideline for the management of Chlamydia trachomatis infections.

This guideline aims to provide comprehensive information regarding the management of infections caused by Chlamydia trachomatis in European countries. The recommendations contain important information for physicians and laboratory staff working with sexually transmitted infections (STIs) and/or STI-related issues. Individual European countries may be required to make minor national adjustments to this guideline as some of the tests or specific local data may not be accessible, or because of specific laws.

Innate immunity in defense against Chlamydia trachomatis infections.

Innate immunity is of key importance in primary recognition of invading pathogens. Infected epithelial cells respond in similar, but not identical ways to different invading pathogens and the pathogens are capable of modifying the host cell response. Chlamydia trachomatis is a major cause of preventable blindness in underdeveloped countries and of sexually transmitted infections with sequelae such as infertility, pelvic inflammatory disease, and extrauterine gravidity throughout the world. Limited knowledge about molecular mediators and effectors, immunocompetent cells, and host response in chlamydial mucosal infections will be described. Recent findings of a differential response to invasive and noninvasive chlamydial infections are highlighted.

More than 25 years of urogenital Chlamydia trachomatis research in the Netherlands.

This article provides an overview of the Dutch work performed on urogenital Chlamydia trachomatis infections which started over 30 years ago. We will review past PhD research, 50% of which involved C. trachomatis as the main focus of the thesis, as well as research by current PhD fellows investigating (partially) C. trachomatis, and publications from Dutch authors or co-authors and the main discussion forums.