Time since last vaccine dose in PCR-positive and PCR-negative children with suspected pertussis to monitor pertussis vaccine effectiveness.

We evaluated whether the results of diagnostic polymerase chain reaction (PCR) testing combined with time since last vaccine dose could be used to monitor the effectiveness of acellular pertussis vaccines. In 258 consecutive nasopharyngeal swabs from children and adolescents with typical pertussis symptoms, 80 were positive and 178 were negative in PCR for Bordetella pertussis DNA (IS 481). Time since last vaccine dose was available for 152 patients, of which 120 were fully immunised. Among the fully vaccinated patients, the median age of 41 PCR-positive patients was 8.4 years (range 0.9-12.3) and that of 79 PCR-negative cases was 3.3 years (range 0.4-14.1) (p < 0.01). The median time since last pertussis vaccine dose was 6.05 years [95 % confidence interval (CI): 0.5-10.9] in PCR-positive cases and 2.22 years (95 % CI: 0.04-9.23) in PCR-negative cases (p < 0.001). The use of diagnostic PCR results from pertussis cases together with time since last vaccine dose permits estimates of the duration of protection after vaccination with acellular pertussis vaccines that are in keeping with more complex studies.

Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation.

Preparation of Bordetella pertussis DNA from respiratory samples for real-time PCR by commercial kits.

Bordetella pertussis is increasingly detected by real-time PCR, but most kits for extracting Bordetella-DNA from respiratory samples are not validated for this material. Respiratory clinical materials were spiked with Bordetella pertussis cells. Four ion-exchange chromatography methods from one manufacturer were used for DNA preparation. Two real-time PCRs detecting the IS481 of Bordetella pertussis and based either on a hybridisation probes format (LightCycler) or on a TaqMan format were used. All kits effectively prepared DNA for Bordetella pertussis real-time PCR. The procedures were linear over a broad range, and the lower level of sensitivity was similar. Sensitivities measured as CT values were different. Inter-assay CVs were between 6.8% and 17.3%. Two kits did not effectively remove inhibitory substances from the respiratory samples. Commercial kits are useful for preparing Bordetella pertussis DNA from respiratory samples, but even kits from one manufacturer show significant differences in effectiveness and removal of inhibitory substances.

Detection of Bordetella pertussis by determination of adenylate cyclase activity.

The adenylate cyclase activity of Bordetella pertussis in clinical isolates was measured in calmodulin-supplemented Stainer-Scholte broth by the rate of conversion of ATP to cyclic AMP. Analysis of 250 stock strains of Bordetella pertussis showed that measurable adenylate cyclase activity was produced by all strains. In clinical tests Bordetella pertussis was isolated from 135 (22%) of 605 swab samples. Increased adenylate cyclase activity was detected in 124 (92%) Stainer-Scholte broth cultures of these samples. A total of 475 swabs contained other bacteria or had no growth; only one of the Stainer-Scholte broth cultures of these swab samples contained measurable adenylate cyclase activity. The results indicate that testing for adenylate cyclase activity provides a specific and sensitive means for detecting Bordetella pertussis in clinical specimens.

Use of fosfomycin disks for isolation of diphtheroids.

A fosfomycin disk (200 micrograms) placed on blood agar was used to select diphtheroids from clinical specimens. All Corynebacterium type strains tested, representing ten different groups of diphtheroids, and 150 toxigenic strains of Corynebacterium diphtheriae from different outbreaks, overgrew the fosfomycin disk, while other aerobic gram-positive rods had a large inhibition zone. The method increased the rate of isolation of diphtheroids in 1267 clinical specimens fivefold, from 1.4% to 7.6%, especially when the diphtheroids were found concomitantly with other bacteria. Overgrowth of the disk by other microorganisms occurred in 11% of the samples tested, but such microorganisms could easily be distinguished by culture morphology or Gram stain. Fosfomycin disks provide a simple, highly sensitive but not very specific aid for isolation of Corynebacterium diphtheriae and diphtheroids from clinical specimens.

Group JK corynebacteria in skin flora of healthy persons and patients.

The presence of group JK corynebacteria in the skin flora of the axilla and inguinal regions was monitored for several months in three groups comprising 45 oncological patients, 49 dialysis patients and 36 healthy staff members. Oncology patients were colonised significantly more often (51%) than dialysis patients and staff members (33% and 36% respectively). The frequency of isolation did not differ between male patients and staff whereas female patients were colonised significantly more often than female staff members. The inguinal region was colonised more often than the axilla. Oncological patients had the highest proportion of antibiotic resistant group JK corynebacteria strains on their skin and staff members the lowest proportion.

Serological diagnosis of whooping cough.

Cooperating with pediatricians in private practice and in hospitals, we tried to evaluate the diagnostic relevance of three serological methods for the detection of antibodies to Bordetella pertussis. The tests employed were a microagglutination, a complement fixation and an enzyme-linked immunoassay (ELISA) to measure specific IgG, IgM, IgA and IgE with whole B. pertussis phase I cells as an antigen. In addition, sera were tested for complement-fixing antibodies to respiratory-syncytial (RS) virus. Microbiological procedures also were used to detect B. pertussis in pernasal swabs. Single sera from 259 children and adults with suspected whooping cough were tested. Of these 117 samples did not contain any measurable antibodies to B. pertussis (45%) and only four sera of this group exhibited complement-fixing antibodies to RS-virus (3%). Antibodies to B. pertussis could be found in the sera of 142 patients. In this group, complement-fixing antibodies to RS-virus were detected in 23 sera (16%). Comparing the methods used to measure antibodies to B. pertussis showed that the ELISA was the most sensitive test, followed by complement fixation and microagglutination. In 21 additional cases, paired sera and several swabs could be obtained from individual patients. These data allowed us to follow the antibody response to B. pertussis during whooping cough, which seemed to be characterized by a relatively late onset with a subsequent rapid increase of initially IgM and IgA and then IgG antibodies. Analogously, the titers of complement-fixing and agglutinating antibodies increased during the later stages of the disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance of gnotobiotic nude mice to Listeria infection.

Unlike the acute and short-term course of experimental listeriosis in phenotypically normal (nu/+) mice, Listeria monocytogenes infection in conventionally raised athymic Nude mice results in chronic inflammation with persistence of bacteria and lesions in the internal organs such as liver and spleen. Although it could be shown that germfree Nude mice had no intestinal microbial flora and lacked measurable serum immunoglobulins, neither the course nor the histomorphology of experimental listeriosis differed significantly from that of their conventionally raised littermates. The results indicate that the typical response of Nude mice to Listeria infection is primarily determined by inborn factors and not by prior antigenic stimulation.

Isolation of Corynebacterium group JK from clinical specimens with a semiselective medium.

A semiselective medium for the detection of multiresistant lipophilic corynebacteria is described. The medium consisted of tryptose agar, supplemented with Tween 80, lecithin, histidine, glycerol, sodium thiosulfate, fosfomycin, ticarcillin, and 5-fluorocytosine. The medium was tentatively called lecithin-Tween medium (LT medium). It promoted mainly the growth of Corynebacterium group JK, which has recently been identified as a cause of serious infection in immunocompromised patients. The application of LT medium to 6,859 routine clinical specimens increased the percentage of Corynebacterium group JK isolation from 0.1 to 1.0%. Although a total of 72 isolates were found in 65 patients, on the basis of clinical criteria only 2 patients were regarded as having an infection with these bacteria. Consequently, Corynebacterium group JK infection can only be diagnosed through a combination of clinical and microbiological findings. In high-risk areas, however, the use of LT medium for surveillance may facilitate the early detection of these potentially dangerous organisms.

Biological activity of Bordetella pertussis in lipopolysaccharide-resistant mice.

Effects of Bordetella pertussis organisms, such as adjuvanticity, induction of hypersplenia, and leukocytosis as well as modification of nonspecific resistance to infection and typical morphological response of lymphatic organs, were studied in the lipopolysaccharide-resistant C3H/HeJ mouse strain. It was shown that B. pertussis exerted all of these effects in C3H/HeJ mice, although the morphological response, hypersplenia, and modification of resistance to infection with Listeria monocytogenes in such animals were less pronounced than those in lipopolysaccharide-sensitive mouse strains. This indicated that the biological activity of B. pertussis as determined in the present studies, is due partly to structural components other than lipopolysaccharide.