Anneke van der Zee

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii for Clinical Diagnosis

ABSTRACT PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, with detection by molecular beacons. The real-time PCR for IS481 detects both B. pertussis and Bordetella holmesii, and the real-time PCR for IS1001 detects both B. parapertussis and B. holmesii. By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis, 10 CFU/ml for B. parapertussis, and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.

Qiagen DNA Extraction Kits for Sample Preparation for Legionella PCR Are Not Suitable for Diagnostic Purposes

Several reports describe the use of Qiagen columns as part of a Legionella PCR ([1][1], [2][2]). However, workers in two laboratories in The Netherlands have found that the Qiagen columns are contaminated with Legionella species. Nucleic acid sample preparation kits are commercially available from

Diagnosis of atypical pathogens in patients hospitalized with community-acquired respiratory infection.

The object of our study was to determine the proportion of atypical respiratory pathogens among patients hospitalized with a community-acquired respiratory infection. From September 1997 to May 1999, 159 patients (57% male, median age 55, range 1-88 y) admitted to 3 regional hospitals for a community acquired respiratory infection, were enrolled in the study. Microbiological diagnosis for the atypical pathogens Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila was performed with PCR on a throat swab, sputum and/or broncho alveolar lavage (BAL). In addition, Legionella species other than L. pneumophila (L. non-pneumophila species) were detected by PCR. Two serum samples were collected and processed for M. pneumoniae and C. pneumoniae serology. In total, 27 patients (17%) were diagnosed with an atypical pathogen. Infection with M. pneumoniae was detected in 19 patients (12%) (PCR positive n = 7), with C. pneumoniae in 5 patients (3%) (PCR positive n = 0) and with L. pneumophila in 4 patients (2.5%) (PCR positive n = 4). In 54 (34%) patients routine microbiological investigations revealed aetiological agents other than the 3 atypical pathogens, the most frequently diagnosed pathogens being Streptococcus pneumoniae (n = 18), Haemophilus influenzae (n = 17), Gram-negative rods (n = 13), Moraxella catarrhalis (n = 6) and Staphylococcus aureus (n = 6). More than 1 pathogen was found in 13 patients. Atypical pathogens were found more often in the young age group (0-18 y), in contrast to bacterial pathogens that were found more often in the older age groups (> or = 65 y). Atypical pathogens were found less often in patients with a clinical presentation of atypical pneumonia. Legionella species other than L. pneumophila were found by PCR in 13 patients (8%), and in 6 patients in combination with another pathogen. An atypical pathogen (M. pneumoniae, C. pneumoniae or L. pneumophila) was found in 17% of the patients hospitalized with a community acquired respiratory infection, predominantly in the young age group. The role of Legionella non-pneumophila species as pathogen in community acquired respiratory infection needs to be determined. The clinical presentation does not predict the type of pathogen found.

Dynamics of the population structure of Bordetella pertussis as measured by IS 1002-associated RFLP: comparison of pre- and post-vaccination strains and global distribution

The effect of temporal and geographic factors on the population structure of Bordetella pertussis was studied using IS1002-based RFLP analysis. Among the 106 strains analysed, 36 different RFLP types were observed. For the Dutch strains, there was evidence for a shift in the population structure in time since the majority of strains were found in different families of related strains in successive periods. Most pronounced were the differences observed between 1950-1954 and later periods. This difference may have been caused by the introduction of the whole-cell vaccine in 1953, resulting in the expansion of strains which are less affected by vaccine-induced immunity. Strains with RFLP types identical to the vaccine strains were observed in the period 1950-1954, but not later, suggesting a decreased frequency of such strains due to vaccination. Analysis of strains from the 1994 Dutch pertussis epidemic revealed that 71% of the strains belonged to two RFLP types, indicating that pertussis epidemics are caused by clonal expansion. IS1002-based RFLP analysis of strains from different countries suggested a partial geographic isolation of B. pertussis populations. One RFLP type was found to have a wide distribution in time and in space.

Novel PCR-probe assay for detection of and discrimination between Legionella pneumophila and other Legionella species in clinical samples.

In recent years, several PCR-based methods for detection of Legionella DNA have been described, but all PCR assays commonly lacked the ability to discriminate between Legionella pneumophila and other Legionella species ([1][1]-[3][2], [5][3]). A recent study indicated that Legionella species other

Outbreak of Infection with a Multiresistant Klebsiella pneumoniae Strain Associated with Contaminated Roll Boards in Operating Rooms

ABSTRACT An outbreak with a multiresistant Klebsiella pneumoniae (MRKP) strain among seven patients admitted to the adult intensive care unit (ICU) of a regional teaching hospital in The Netherlands was investigated. Epidemiologic investigations revealed a short delay between an operation and the acquisition of the MRKP strain. A case-control study comprising 7 cases and 14 controls was conducted to identify the risk factors associated with the acquisition of the MRKP strain. An operation at each of two operation rooms was strongly associated with the acquisition of the MRKP strain: odds ratio of 36 (95% confidence interval, 2.7 to 481.2; P = 0.003, Fisher exact two-tailed test). Cultures of environmental specimens of the operation rooms revealed contamination of the roll boards used to transport patients from the bed to the operation table with the MRKP strains. Molecular genotyping of the isolates revealed clonal similarity between the isolates of the seven cases, isolates from environmental specimen cultures, and in addition, an MRKP isolate from a repatriated ICU patient from earlier that year. The outbreak ended after cleaning and replacement of the roll boards in the operation rooms and implementation of additional barrier precautions for colonized or infected patients. It was concluded that two operation rooms played a significant role in the transmission of an MRKP strain between ICU patients during the presented outbreak.

Use of multienzyme multiplex PCR amplified fragment length polymorphism typing in analysis of outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit.

ABSTRACT We developed and optimized a new modified amplified fragment length polymorphism (AFLP) typing method to obtain a multibanding fingerprint that can be separated by agarose gel electrophoresis. Both to maximize the discriminatory power and to facilitate the computer-assisted analysis, bacterial DNA was digested with four different restriction enzymes. After ligation of adaptors to the DNA fragments, PCR testing of various single primers was performed. Two single primers that gave optimal results with regard to band resolution and discriminatory power were selected and combined. The computer-assisted analysis of fingerprint patterns was performed with Pearsons product-moment correlation values of densitometric curves, without assigning bands to peaks. Thus, the analysis is not subject to human interpretation errors. With this method, we investigated two outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit and various sporadic isolates of K. pneumoniae and Klebsiella oxytoca. Cluster analysis of isolates analyzed in different experiments and on different gels showed that fingerprint patterns clustered correctly according to subspecies or to the outbreaks. Multienzyme multiplex PCR AFLP revealed that the first outbreak was caused by two different types of strains. Outbreak two was caused by yet another strain of K. pneumoniae. In conclusion, the typing method used here is easy to perform and highly reproducible, and due to generation of complex banding patterns, it has a higher discriminatory power. Furthermore, the multienzyme multiplex PCR fingerprints are easy to analyze, and a reliable database can be stored in the computer to facilitate comparison of future isolates of Klebsiella spp. The method can be performed in every clinical microbiology laboratory.

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

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

Laboratory Diagnosis of Pertussis

SUMMARY The introduction of vaccination in the 1950s significantly reduced the morbidity and mortality of pertussis. However, since the 1990s, a resurgence of pertussis has been observed in vaccinated populations, and a number of causes have been proposed for this phenomenon, including improved diagnostics, increased awareness, waning immunity, and pathogen adaptation. The resurgence of pertussis highlights the importance of standardized, sensitive, and specific laboratory diagnoses, the lack of which is responsible for the large differences in pertussis notifications between countries. Accurate laboratory diagnosis is also important for distinguishing between the several etiologic agents of pertussis-like diseases, which involve both viruses and bacteria. If pertussis is diagnosed in a timely manner, antibiotic treatment of the patient can mitigate the symptoms and prevent transmission. During an outbreak, timely diagnosis of pertussis allows prophylactic treatment of infants too young to be (fully) vaccinated, for whom pertussis is a severe, sometimes fatal disease. Finally, reliable diagnosis of pertussis is required to reveal trends in the (age-specific) disease incidence, which may point to changes in vaccine efficacy, waning immunity, and the emergence of vaccine-adapted strains. Here we review current approaches to the diagnosis of pertussis and discuss their limitations and strengths. In particular, we emphasize that the optimal diagnostic procedure depends on the stage of the disease, the age of the patient, and the vaccination status of the patient.

Polymerase chain reaction detection of Neisseria meningitidis in the intraocular fluid of a patient with endogenous endophthalmitis but without associated meningitis

PURPOSE To report a patient with Neisseria meningitidis endophthalmitis without associated meningitis with full visual recovery, with early detection of the microorganism using polymerase chain reaction (PCR) analysis. DESIGN Retrospective, observational case report. PARTICIPANTS One patient with endogenous endophthalmitis. METHODS Polymerase chain reaction analysis and culture of the intraocular fluid sample. Polymerase chain reaction analysis was performed with a universal (16S rRNA) primer set to detect bacterial DNA, and subsequently a specific probe was used to detect Neisseria species DNA. RESULTS The 16S rRNA primers detected bacterial DNA, the specific probe detected Neisseria species DNA, and culture was positive for Neisseria meningitidis serotype C. CONCLUSIONS A universal bacterial PCR can be very helpful for the diagnosis of endogenous bacterial endophthalmitis at an early stage of the disease.