Verkooyen, R.P.A.J.

Chlamydia trachomatis respiratory infection in Dutch infants (Article)

2009-09-01T00:00:00Z

Chlamydia trachomatis is the most common bacterial pathogen causing sexually transmitted infections in Dutch adults. As prenatal screening for C trachomatis and treatment of pregnant women is not routine practice in The Netherlands, perinatal transmission of C trachomatis may therefore occur. The presence of C trachomatis in infants less than 6 months of age who presented with respiratory complaints to the Erasmus MC-Sophia hospital was evaluated. Respiratory specimens, primarily nasopharyngeal swabs, were tested for C trachomatis, respiratory viruses and Mycoplasma pneumoniae using PCR, viral isolation in cell cultures and direct immunofluorescence. C trachomatis respiratory tract infection was confirmed to be relatively common with detection in 10 of 148 (7%) infants tested. C trachomatis had not been tested for by the attending physicians, but was the second most frequently detected respiratory pathogen after human Respiratory Syncitial Virus, which was found in 41 (28%) infants.

Chlamydia trachomatis as a cause of neonatal conjunctivitis in Dutch infants (Article)

2008-02-01T00:00:00Z

BACKGROUND. Chlamydia trachomatis is the most common sexually transmitted pathogen in adults, which at delivery may be transmitted from mother to child and cause conjunctivitis and pneumonia. In the Netherlands, prenatal chlamydial screening and treatment of pregnant women is not routine practice. The contribution of C trachomatis to neonatal ophthalmic disease has not been studied in the Netherlands and remains unclear. METHODS. At the Sophia Children's Hospital and Rotterdam Eye Hospital, 2 cohorts of infants <3 months of age presenting with conjunctivitis were studied, 1 retrospectively (July 1996 to July 2001) and 1 prospectively (September 2001 to September 2002). Laboratory diagnosis was based on bacterial culture and polymerase chain reaction for C trachomatis. RESULTS. C trachomatis was detected in 27 (64%) of 42 retrospectively studied infants and 14 (61%) of 23 prospectively studied infants. Mucopurulent discharge was present in 35 (95%) of 37, swelling of the eyes in 27 (73%) of 37, conjunctival erythema in 24 (65%) of 37, respiratory symptoms in 14 (38%) of 37, and feeding problems in 5 (14%) of 37 infants respectively. Before microbiological diagnosis, general practitioners prescribed antichlamydial antibiotics locally to 5 (12%) of 41 and systemically to 4 (10%) of 41 infants who tested positive for chlamydia, and ophthalmologists prescribed to 21 (51%) of 41 and 7 (17%) of 41, respectively. CONCLUSIONS. C trachomatis was the major cause of bacterial conjunctivitis in this population. Clinically, differentiation from other pathogens was not possible. Many infants who tested positive for chlamydia did not receive appropriate antibiotic treatment. Copyright

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. (Article)

2005-09-01T00:00:00Z

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.

Reliability of nucleic acid amplification methods for detection of Chlamydia trachomatis in urine: results of the first international collaborative quality control study among 96 laboratories (Article)

2003-01-01T00:00:00Z

The first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect Chlamydia trachomatis in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, including three negative, two strongly positive, and five weakly positive samples. Ninety-six laboratories in 22 countries participated with a total of 102 data sets. Of 204 strongly positive samples 199 (97.5%) were correctly reported, and of 506 weakly positive samples 466 (92.1%) were correctly reported. In 74 (72.5%) data sets correct results were reported on all samples, and 17 data sets (16.7%) showed either one false-negative or one false-positive result. In another 11 data sets, two or more incorrect results were reported, and two data sets reported a false-positive result on one negative sample. The Roche COBAS Amplicor test was performed in 44 (43%) data sets, the Abbott LCx assay was performed in 31 (30%) data sets, the Roche Amplicor manual assay was performed in 9 (9%) data sets, an in-house PCR was performed in 9 (9%) data sets, the Becton Dickinson ProbeTec ET assay was performed in 5 (4.9%) data sets, and the GenProbe TMA assay was performed in 4 (3.9%) data sets. The results of the Roche Amplicor manual (95.6% correct), COBAS Amplicor (97.0%), and Abbott LCx (94.8%) tests were comparable (P = 0.48). The results with the in-house PCR, BD ProbeTec ET, and GenProbe TMA tests were reported correctly in 88.6, 98, and 92.5% of the tests, respectively. Freeze-drying of clinical urine specimens proved to be a successful method for generating standardized, stable, and easy-to-transport samples for the detection of C. trachomatis by using NATs. Although the results, especially the specificity, for this proficiency panel were better than most quality control studies, sensitivity problems occurred frequently, underlining the need for good laboratory practice and reference reagents to monitor the performance of these assays.

Risk factors associated with Campylobacter jejuni infections in Curacao, Netherlands Antilles (Article)

2003-01-01T00:00:00Z

A steady increase in the incidence of Guillain-Barre syndrome (GBS) with a seasonal preponderance, almost exclusively related to Campylobacter jejuni, and a rise in the incidence of laboratory-confirmed Campylobacter enteritis have been reported from Curacao, Netherlands Antilles. We therefore investigated possible risk factors associated with diarrhea due to epidemic C. jejuni. Typing by pulsed-field gel electrophoresis identified four epidemic clones which accounted for almost 60% of the infections. One hundred six cases were included in a case-control study. Infections with epidemic clones were more frequently observed in specific districts in Willemstad, the capital of Curacao. One of these clones caused infections during the rainy season only and was associated with the presence of a deep well around the house. Two out of three GBS-related C. jejuni isolates belonged to an epidemic clone. The observations presented point toward water as a possible source of Campylobacter infections.

Evaluation of PCR, culture, and serology for diagnosis of Chlamydia pneumoniae respiratory infections (Article)

1998-01-01T00:00:00Z

We prospectively studied 156 patients with a diagnosis of community-acquired pneumonia requiring admission. Several respiratory specimens were obtained for the detection of Chlamydia pneumoniae by cell culture and PCR. Three serum samples were obtained from each patient. Serological diagnosis of a C. pneumoniae infection was determined by the microimmunofluorescence (MIF) test, the complement fixation (CF) test, and recombinant lipopolysaccharide (LPS) enzyme-linked immunosorbent assay (ELISA; referred to as the rDNA LPS ELISA). Twenty-three patients (15%) had serological results compatible with acute C. pneumoniae infection; nine (39%) of these subjects were C. pneumoniae PCR positive. Twenty-two patients (14%) had positive PCR results without serological evidence of an acute C. pneumoniae infection. An attempt was made to calculate the sensitivities and specificities of the MIF test, rDNA LPS ELISA, and PCR for the diagnosis of chlamydial community-acquired pneumonia. Several "gold standards" were defined. Generally, the sensitivities of the rDNA LPS ELISA and MIF were comparable, while the sensitivity of the CF test was shown to be very low. Independent of the gold standard used, the best PCR results were obtained with nasopharyngeal specimens. However, the predictive value of a positive C. pneumoniae PCR result for patients with community-acquired pneumonia remains unknown and may be low. Although a widely accepted gold standard is still lacking, the rDNA LPS ELISA may currently be the preferred tool for diagnosing acute respiratory Chlamydia infections in routine clinical practice. However, the MIF test remains the method of choice for determining the prevalence of C. pneumoniae infections in a given community.

Chlamydia pneumoniae : studies on an emerging pathogen (Doctoral Thesis)

1997-11-05T00:00:00Z

The Chlamydiae are characterized as bacteria because of the composition of their cell wall and their growth by binary division. However, they are obligate intracellular bacterial parasites of eukaryotic cells that have a unique replication cycle similar to Rickettsia. Chlamydial infection of host cells is initiated by the elementary body (? = 300-400 nm) , a stable structure specifically adapted to survive the extracellular phase during transit between cells. According to the knowledge, the infectious elementary body enters the cell via endocytosis (90,118). Intracellular parasites have evolved several ways to avoid being killed by lysosomal enzymes (139). However, the mechanism by which subsequent fagosome/lysosome fusion is avoided, is not completely understood. After endocytosis of the elementary body, it will reorganize into a non-infectious, metabolic active reticulate body (? = 800 - 1000 nm), which is responsible for intracellular replication through binary fission. At both stages of development the chlamydial cell is surrounded by an envelope similar to that of gram-negative bacteria. The envelope consists of two trilalninar lnenlbranes, an outer lnelnbrane and an inner, cytoplasmic membrane (24). The fissions occur in the original phagosome that enlarges during this process until it resembles a vacuole. This vacuole is referred to as an inclusion body or in short, an inclusion. During replication, the reticulate body obtains adenosine triphosphate, amino acids and sugars fi'om the host cell (7). Each reticulate body will reorganize into one or more elementary bodies (204). After 48-72 hrs, release of infective elementary bodies will take place due to host cell lysis (Figure 1). The order Chlamydiales has one family, Chlamydiaceae, and one genus, Chlamydia. There are now four recognized species, Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae and Chlamydia pecol'1lm. Of these, the most recently described C.peCOl'llm infects sheep and cattle, and has so far not been associated with human disease (58,59). At this moment, 19 different C.tl'achomatis serovars or serotypes are known, i.e. distinguished from each other by polyclonal antibodies that were formed in mice after they have been infected with a single type Chlamydia (201).

Dissemination of a single clone of methicillin-resistant Staphylococcus aureus among Turkish hospitals (Article)

1997-01-01T00:00:00Z

A collection of 39 methicillin-resistant Staphylococcus aureus (MRSA) stains derived from six different hospitals in Ankara and one hospital in Barsa, Turkey, were analyzed by multiple genotyping. In agreement with the other genotyping assays, pulsed-field gel electrophoresis of DNA macrorestriction fragments identified genetic homogeneity among all MRSA isolates studies. It is concluded that a major clone of MRSA has spread through a large part of Turkey, causing longitudinally persistent colonization in all of the institutions surveyed.

Detection of Chlamydia trachomatis in male and female urine specimens by using the amplified Chlamydia trachomatis test (Article)

1997-01-01T00:00:00Z

The amplified Chlamydia trachomatis test (AMP-CT; Gen-Probe), a new diagnostic test for the detection of Chlamydia trachomatis, was evaluated with urine specimens from 1,000 patients visiting the outpatient department for sexually transmitted diseases at the University Hospital Rotterdam, Rotterdam, The Netherlands, by comparing the results to those of cell culture. From February 1996 to July 1996, urine samples for the AMP-CT test and urethral swabs for cell culture were collected from 544 men, while cervical swabs from 456 women were also taken for cell culture. Positive test results were obtained for 130 (13%) of the patients. AMP-CT test and cell culture results were discordant for 70 (7%) specimens. Analysis of the samples with discordant results was performed by an in-house PCR. After resolution of the discordant results, the sensitivity, specificity, and positive and negative predictive values of the AMP-CT test were 84.3, 98.8, 89.6, and 98%, respectively, for samples from females and 100, 99.2, 93.1, and 100%, respectively, for samples from males, while for cell culture these values were 72.5, 99.2, 92.5, and 98%, respectively, for samples from females and 57.4, 99.0, 86.1, and 95.4%, respectively, for samples from males. We conclude that the AMP-CT test is a fast and reliable test for the detection of C. trachomatis in urine specimens from females and, in particular, males.

Comparison of three commercially available amplification assays, AMP CT, LCx, and COBAS AMPLICOR, for detection of Chlamydia trachomatis in first-void urine (Article)

1997-01-01T00:00:00Z

We compared the Gen-Probe transcription-mediated amplification assay (AMP CT), the Abbott LCx assay, and the Roche COBAS AMPLICOR assay for the detection of Chlamydia trachomatis in a mixed population in urine samples. First-void urine, urethral specimens, and cervical specimens in females were obtained from 1,000 patients (544 males and 456 females) visiting the outpatient sexually transmitted disease clinic of our hospital. The prevalence of C. trachomatis infection was 7.7% as determined by tissue culture of urethral and cervical specimens. The sensitivities of LCx, COBAS AMPLICOR, and AMP CT compared to cell culture were 79, 86, and 78%, respectively. Sensitivity and specificity were recalculated by using a new "gold standard", i.e., a sample was considered to be true positive if two or more techniques yielded positive results. Specimens positive only by cell culture or positive in only one commercial amplification technique were retested by a previously described in-house PCR. After discordance analysis the sensitivities of LCx, COBAS AMPLICOR, and AMP CT were 84, 93, and 85%, respectively. Specificity exceeded 99% for all three assays. With each method the sensitivity was lower for urine samples from females compared to urine samples from males. By application of this new gold standard, existing differences between methods are highlighted; future evaluations of new techniques should be validated against two or more amplification assays.

Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay (Article)

1996-01-01T00:00:00Z

To determine that susceptibility of AMPLICOR Chlamydia trachomatis PCR to inhibitory factors possibly present in cervical specimens, we obtained cervical specimens from 200 gynecology patients attending our outpatient clinic. The prevalence of C. trachomatis infection was 4.1%, as determined by cell culture. All AMPLICOR specimens were tested in one procedure as described by the manufacturer, and after the specimen was spiked with C. trachomatis, several other pretreatment protocols were used. Complete inhibition of the PCR was observed in 38 (19%) cervical specimens. Heat treatment at 95 degrees C, freeze-thawing, or 10-fold dilution of the samples reduced the initial inhibition to 9, 16, or 9%, respectively. A combination of heat treatment and 10-fold dilution reduced the inhibition to 4% of the samples. A second specimen type (swabs inoculated in 0.2 M sucrose phosphate buffer [2SP]) was also evaluated. A 10-fold dilution of the spiked 2SP specimen resulted in an inhibition rate of 6%, which was comparable to that obtained by centrifugation of the 2SP specimen prior to processing. Furthermore, it was shown that the inhibition was not correlated with blood contamination. Processing the specimens on the day of collection or the day after resulted in a higher inhibition rate than did delayed processing (27.6 versus 15.5%, respectively). An inverse correlation was found between the concentration of C. trachomatis added to the sample and the rate of inhibition observed. The inhibition was partly correlated with the pH of the cervical mucosa. Decreased inhibition was found at pH values of > or = 7.5. The effects of blood, pH, and delay in processing were all evaluated by using the AMPLICOR specimen. We conclude that the susceptibility of AMPLICOR C. trachomatis PCR to inhibiting factors in cervical specimens can be significantly reduced if the pretreatment procedure includes heat treatment or the use of 2SP transport medium. Also, a 10-fold dilution of the clinical specimen followed by heat treatment will largely prevent the inhibition of this PCR.