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.

Use of pooled urine samples and automated DNA isolation to achieve improved sensitivity and cost-effectiveness of large-scale testing for Chlamydia trachomatis in pregnant women.

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.

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.

No association of anti-Chlamydia trachomatis antibodies and severity of cervical neoplasia.

OBJECTIVE: To explore whether the presence of Chlamydia trachomatis antibodies is associated with the severity of neoplastic lesions in women with cervical dyskaryosis. METHODS: In a cross sectional study in two groups of women referred for an abnormal Papanicolaou smear (group A: 296, group B: 331 women) blood samples were analysed for antichlamydial antibodies by enzyme immunoassay. Cervical neoplasia was graded histologically. RESULTS: In group A no association was found between increasing grade of CIN and the presence of antichlamydial antibodies. The proportion (93%) of women with antichlamydial antibodies was higher in 14 women with (micro)invasive carcinoma than in women with CIN (35%). As the high prevalence of antichlamydial antibodies in women with cervical carcinoma is not consistent with prevalences reported in recent literature, we analysed a second group of women in which indeed the high prevalence was not confirmed CONCLUSION: Our results suggest that the presence of circulating antichlamydial antibodies is not associated with the severity of neoplastic lesions and it seems unlikely that C trachomatis has a role in the progression of cervical neoplasia.

Diagnosing genital ulcer disease in a clinic for sexually transmitted diseases in Amsterdam, The Netherlands.

The most common etiologic agents of genital ulcer disease (GUD) are herpes simplex virus type 1 (HSV-1), HSV-2, Treponema pallidum, and Haemophilus ducreyi. In an outpatient clinic for sexually transmitted diseases in Amsterdam, The Netherlands, specimens from 372 patients with GUD were collected from February to November 1996. Sera were collected at the time of the symptoms and, for most patients, also during follow-up visits. Swabs in viral transport medium were used for HSV culture and for detection of DNA. The most prevalent pathogen found was HSV-2, which was detected by culture in 35% of the patients and by PCR in 48% of the patients. Also, HSV-1 infection was more often detected by PCR (7.8%) than by culture (5.6%). Evidence for an active infection with T. pallidum was found in 1.9% of the patients, using serological tests. A multiplex PCR for simultaneous T. pallidum and H. ducreyi DNA detection was positive for T. pallidum in 3.3% of the samples and for H. ducreyi in only 0.9% (3 out of 368) of the samples. The sensitivity of the PCR was superior to that of culture for HSV detection and to that of serology for T. pallidum detection. Specific H. ducreyi immunoglobulin G antibodies were detected in sera of 5.2% of the patients, with no concordance between serology and PCR. In 37% of the cases, none of the tested microorganisms was detected. Performance of PCR in addition to conventional techniques significantly improved the diagnosis of GUD.

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.

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.

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.

Monitoring of Chlamydia trachomatis infections after antibiotic treatment using RNA detection by nucleic acid sequence based amplification.

AIM: To investigate the value of RNA detection by nucleic acid sequence based amplification (NASBA) for the monitoring of Chlamydia trachomatis infections after antibiotic treatment. METHODS: Cervical smears (n = 97) and urine specimens (n = 61) from 25 C trachomatis positive female patients were analysed for the presence of C trachomatis 16S ribosomal RNA (rRNA) by NASBA and C trachomatis plasmid DNA by the polymerase chain reaction (PCR) before and up to five weeks after antibiotic treatment. RESULTS: Chlamydia trachomatis RNA was found in all cervical smears taken before antibiotic treatment (n = 24) and in two smears taken one week after antibiotic treatment; no C trachomatis RNA was detected after two weeks or more. In contrast, C trachomatis DNA was found in all such specimens before treatment, and 21 of 25, six of 21, and five of 20 smears were found to be positive at one, two, and three weeks after treatment, respectively. After four weeks, only one of six smears was positive, and this smear had been negative in the two preceding weeks. Of the 61 urine samples investigated, C trachomatis DNA and C trachomatis RNA were found in all before treatment (n = 15), whereas one week after treatment four of 15 were C trachomatis DNA positive and C trachomatis RNA was detected in one sample only. CONCLUSIONS: These data show that RNA detection by NASBA can be used successfully to monitor C trachomatis infections after antibiotic treatment. Furthermore, it might be possible to use urine specimens as a test of cure because neither C. trachomatis DNA or RNA could be detected two weeks or more after treatment.

Pitfalls and fallacies of cat scratch disease serology: evaluation of Bartonella henselae-based indirect fluorescence assay and enzyme-linked immunoassay.

The diagnostic value of the detection of immunoglobulin G (IgG) and IgM by Bartonella henselae-based indirect fluorescence assay (IFA) and enzyme-linked immunoassay (EIA) for the diagnosis of cat scratch disease (CSD) was evaluated. The IFA was performed either with B. henselae that was cocultivated for a few hours with Vero cells or with noncocultivated B. henselae as the antigen. Additionally, the performance of a Bartonella PCR hybridization assay based on the 16S rRNA gene was determined and compared with those of the serologic assays. The study group consisted of 45 patients suspected of suffering from CSD by fulfilling one or more of the classical criteria. The specificities of the immunoassays were set at > or = 95% by analysis of sera from 60 healthy blood donors. It is shown that the sensitivities of the IgG assays are very low (40.9% for the IFA with noncocultivated B. henselae as antigen) and that those of the IgM assays are higher (71.4% for the EIA) for patients who fulfilled two or more criteria for CSD. The IgM EIA showed the highest sensitivity: 71.4% in patients with two or more criteria for CSD and 80.6% for patients with a positive Bartonella PCR result. The results indicate that the specificities of both IFA and EIA IgG serologies and the sensitivity of the IFA IgM serology need to be improved. The PCR hybridization assay showed a sensitivity of 86.4% for patients who fulfilled two or more criteria for CSD and 100% for seven patients who fulfilled three or more criteria. The kinetics of IgG and IgM antibody production were studied in 18 patients with CSD on the basis of a positive B. henselae IFA IgM serology. The results indicate that there is no standard course of anti-B. henselae IgG and IgM production in patients with CSD, because some patients produced high levels of both IgG and IgM, others produced only high levels of IgM, and a few patients produced only low levels of antibodies.

A microcarrier culture method for the production of large quantities of viable Chlamydia pneumoniae.

We studied the propagation of Chlamydia pneumoniae strain TW-183 in HEp2 cells grown on microcarrier beads. Infection of the cells in microcarrier culture was optimized by addition of 7.5% polyethylene glycol 4000 (PEG4000) during adsorption. The yield in microcarrier culture was similar to that of microtitre-plate culture using centrifugation-assisted infection (120 x 10(6) and 225 x 10(6) bacteria/10(6) HEp2 cells respectively), as was the burst size (505 and 449 bacteria produced/infecting bacterium respectively). However, up to 64% savings in labour time and 27% saving in culture medium were achieved if the microcarrier culture method was used instead of the microtitre-plate culture method. The optimal yield of viable bacteria could only be achieved at a narrow range of multiplicities of infection (0.24 - 1.14 inclusion-forming units/cell), independent of the mode of infection (centrifugation-assisted infection of PEG4000-facilitated infection by adsorption) and independent of incubation temperature (35 degrees C or 37 degrees C). The yield of microcarrier cultures was the same at an incubation temperature of 35 degrees C or 37 degrees C in contrast to an increased production at 35 degrees C in the microtitre-plate culture method using centrifugation-assisted infection. In conclusion, the microcarrier culture method is useful to produce large quantities of viable Chlamydia pneumoniae economically.

Comparison of two panels of monoclonal antibodies for determination of Chlamydia trachomatis serovars.

A panel of monoclonal antibodies was developed for serovar typing of clinical isolates of Chlamydia trachomatis. The panel could distinguish all 15 established serovars from one another, although the hybridomas of the panel were developed by fusions of myeloma cells and spleen cells from mice immunized with antigen derived from the urogenital serovars D to L3. The typing assay was based on a dot enzyme immunoassay, and the monoclonal antibodies that were included in the panel reacted strongly in this assay. A collection of 289 clinical isolates from The Netherlands was typed. The observed serovar frequency distribution was 51 isolates of serovar D (17.6%), 103 isolates of serovar E (35.6%), 62 isolates of serovar F (21.5%), 28 isolates of serovar G (9.9%), 14 isolates of serovar H (4.8%), 2 isolates of serovar I' (0.7%), 20 isolates of serovar J (6.9%), and 9 isolates of serovar K (3.1%). These results were confirmed by typing these isolates with a panel of monoclonal antibodies purchased from the Washington Research Foundation, Seattle. No strain variation was observed within serovar D with both panels. However, restriction fragment length polymorphism analysis of the gene encoding the major outer membrane protein showed that 32 isolates were similar to the prototype D and 17 were similar to the variant D-. The two others showed a new restriction pattern. Our panel of monoclonal antibodies contained one monoclonal antibody that divided the serovar G isolates into two groups. This differentiation was confirmed by restriction fragment length polymorphism analysis, confining this difference to a known sequence variation in variable domain IV. These data support the subdivision of serovar G into serovars G (prototype strain UW-57) and Ga (prototype strain IOL-238).

Detection of amplified Chlamydia trachomatis DNA using a microtiter plate-based enzyme immunoassay.

A microtiter plate-based method to detect amplified DNA was developed. The method uses on biotin-labeled primer in the polymerase chain reaction (PCR) mixture. The labeled amplicon is bound to streptavidin-coated microtiter plates, denatured and hybridized to a digoxigenin-labeled probe. The specificity of the hybridization reaction was optimized by varying the temperature of the subsequent washing step and adding urea to the washing buffer. The digoxigenin label was detected using an enzyme immunoassay (EIA). This PCR-EIA was compared with a standard PCR assay that uses gel electrophoresis, blotting and hybridization to detect the amplicon, with isolation in cell culture, and with an antigen detection EIA (Chlamydiazyme) in the diagnosis of Chlamydia trachomatis infection in 309 female patients attending a sexually transmitted diseases outpatient clinic. The prevalence of Chlamydia trachomatis infection as determined by isolation in cell culture, EIA, PCR-EIA and standard PCR assay was 9.1%, 8.7%, 12.3%, and 12.9%, respectively. Compared with results of a reference set of confirmed-positive cases (defined by a positive result in two or more independent assay after analysis of discrepancies), the sensitivity and specificity was 71.1% and 99.6% for cell culture, 65.8% and 99.3% for the EIA, 92.1% and 98.9% for the PCR-EIA, and 97.4% and 98.9% for the standard PCR assay. It is concluded that the PCR-EIA described is a fast, sensitive and specific method for detecting Chlamydia trachomatis in clinical specimens.

Chlamydia keratoconjunctivitis determination of Chlamydia trachomatis specific secretory immunoglobulin A in tears by enzyme immunoassay.

Determination of Chlamydia trachomatis specific antibodies in serum and tears, isolation of C. trachomatis in cell culture and C. trachomatis antigen detection by direct immunofluorescence test were evaluated for the laboratory diagnosis of Chlamydia trachomatis infection in 30 patients with chronic conjunctivitis. C. trachomatis specific secretory immunoglobulin A (s-IgA) was detected in the tears of all 8 patients with a positive result in culture and the direct immunofluorescence test. In 4 additional patients, the s-IgA assay of tears was also positive. A presumptive clinical diagnosis of Chlamydia conjunctivitis was correlated with the presence of s-IgA in tears. S-IgA was not detected in the serum of any of the patients, indicating that s-IgA is locally produced and not transudated from the serum. For the diagnosis of chronic Chlamydia conjunctivitis, we recommend the determination of s-IgA in tears in addition to a test for Chlamydia detection.