Fatal outcome of Bacillus cereus septicaemia.

Bacillus cereus is a ubiquitous environmental micro-organism which is often a contaminant of clinical cultures. Infections due to B. cereus are described, but mostly in immunocompromised patients. We report a fatal outcome of B. cereus septicaemia in an immunocompetent patient with a mechanical mitral valve.

Sustained low prevalence of meticillin-resistant Staphylococcus aureus upon admission to hospital in The Netherlands.

The prevalence of meticillin-resistant Staphylococcus aureus (MRSA) carriage at hospital admission in The Netherlands was 0.03% in 1999-2000. The aim of the present study was to assess whether the prevalence of MRSA carriage in The Netherlands has changed over the last few years. In five Dutch hospitals, 6496 unique patients were screened for nasal S. aureus carriage at hospital admission by microbiological culture between 1 October 2005 and 7 June 2007. In total, 2036 of 6496 (31.3%) patients carried S. aureus in their nose, and seven of 6496 (0.11%) patients were nasal carriers of MRSA. Compared with 1999-2000, the prevalence of MRSA carriage in the Dutch population at hospital admission has increased more than three fold; however, this increase was not significant (P=0.06, Fisher's exact test). This prevalence is still among the lowest in the world, probably as a result of the stringent Dutch infection control policy, and the restrictive use of antibiotics in The Netherlands.

Slaughter pigs are commonly infected by closely related but distinct gastric ulcerative lesion-inducing gastrospirilla.

An association between (unculturable) gastrospirillum-like organisms (GLO) and ulcerative lesions in the pars oesophagea in stomachs of swine has been claimed. In dogs GLO detected by microscopy may represent several Helicobacter species or subspecies. Therefore we investigated which Helicobacter spp. are present in stomachs of swine and their possible association with ulcerative lesions of the pars oesophagea. The presence of Helicobacter spp. in the antrum and pars oesophagea in 122 stomachs of slaughter swine was determined by microscopy (n = 122), by culture on selective and nonselective media (n = 112), and by a genus-specific 16S ribosomal DNA (rDNA) PCR (n = 80). GLO could not be cultured. Phylogenetic analysis of 43 16S rDNA fragments (out of 54 PCR-positive biopsy specimens) revealed the presence of Helicobacter heilmannii type 1 in 42 of them. This correlated with the presence of bacteria with GLO morphology. Helicobacter bilis 16S rDNA was amplified directly from one sample harboring bacteria with H. bilis morphology. The association between Helicobacter spp. and gastric lesions was investigated with a second group of 41 pigs with (n = 21 cases) or without (n = 20 controls) gastric lesions. Fifteen of the 21 cases were positive by PCR or microscopy, compared to 7 of 20 of the controls (P = 0.03). 16S rDNA sequence analysis of 7 of 14 PCR-positive cases revealed the presence of H. heilmannii type 1. Microscopy showed bacteria with GLO morphology. One sample (cases) was culture negative but PCR positive for Helicobacter pullorum-related 16S rDNA. In conclusion, our findings indicate that H. heilmannii type 1 is the predominant Helicobacter spp. in the stomachs of pigs and that its presence is associated with ulcerative lesions in the pars oesophagea.

Chlamydia pneumoniae and Mycoplasma pneumoniae in children with acute respiratory infection in general practices in The Netherlands.

In this retrospective study Chlamydia pneumoniae and Mycoplasma pneumoniae infections were detected by polymerase chain reaction (PCR) in samples (n = 457) from children presenting with acute respiratory infection to general practitioners during 1992-97. Samples were collected in autumn and winter, and from 1994 onwards in spring and summer also. Overall, C. pneumoniae and M. pneumoniae were detected in throat or nasal samples by PCR in 3.1% and 2.4% of the cases, respectively. The proportion of both C. pneumoniae and M. pneumoniae infections varied between 0% and 6.9% over the years studied, whereas seasonal proportions varied from 1.8 to 9.1% and 1.2 to 4.5%, respectively. For both microorganisms the lowest proportion was detected during winter and the highest in summer. C. pneumoniae could already be detected by PCR in patients under 4 y of age, an observation not made in sero-epidemiological studies. In conclusion, both C. pneumoniae and M. pneumoniae infections play a minor role in children presenting with acute respiratory infection.

Helicobacter pylori and the birth cohort effect: evidence of a continuous decrease of infection rates in childhood.

OBJECTIVE: To investigate whether the decrease in rate of Helicobacter pylori infection in subsequent birth cohorts has continued during the last decades. METHODS: Determination by ELISA of IgG H. pylori antibodies in 314 serum samples from Dutch children (age 6-8 yr, n = 154) and young adolescents (age 12-15 yr, n = 160), collected in 1978 and 1993. RESULTS: The prevalence of H. pylori declined from 19% to 9% at age 6-8 yr and from 23% to 11% at age 12-15 yr. For the whole study population, a decline from 21% to 10% (p = 0.01) was observed between 1978 and 1993. On the basis of these data and an incidence of infection with H. pylori of 0.3% per year during the same period, a model for both past and future prevalence rates of H. pylori in the Dutch population was calculated. The outcome demonstrates a decrease from more than 50% around World War II to less than 20% for the whole population around year 2040. CONCLUSIONS: H. pylori infection rates in childhood have continued to decline until recent decades, demonstrating a persistent birth cohort effect. This decline will result in a very low prevalence of H. pylori infection in the Dutch population during the next decades, becoming even lower as the observed decline in children and young adolescents continues.

Improvement of growth of Chlamydia pneumoniae on HEp-2 cells by pretreatment with polyethylene glycol in combination with additional centrifugation and extension of culture time.

The following adaptations led to improved growth of Chlamydia pneumoniae on HEp-2 cells compared to that by the standard method: monolayer preincubation with 7% polyethylene glycol (PEG), extension of incubation time to 7 days, and extension of incubation to 7 days in combination with centrifugation on days 3, 4, and 5. These adaptations resulted in approximate increases in numbers of inclusion-forming units (IFU) of 2-, 5-, and 69-fold, respectively. A combination of preincubation with PEG, prolonged incubation, and centrifugation on days 3, 4, and 5 increased the numbers of IFU >300-fold. This is therefore recommended as the optimal method for culturing C. pneumoniae.

Practical diagnosis of Helicobacter pylori infection.

When treatment of Helicobacter pylori infection is considered, a reliable diagnosis is essential. Different biopsy-based invasive diagnostic tests are available for diagnosis in the individual patient. Non-invasive tests are of value in epidemiological studies, and have a role in the follow-up of treatment. In this review the sensitivity, specificity and appropriateness of all available tests for the diagnosis of infection in the individual patient are discussed.

Prevalence and serovar distribution of asymptomatic cervical Chlamydia trachomatis infections as determined by highly sensitive PCR.

The prevalence rates and serovar distributions of Chlamydia trachomatis cervical infections were investigated in two different groups of women. Group I consisted of 393 asymptomatic young women (aged 17 to 30 years) who were invited to participate in a C. trachomatis screening program. Group II consisted of 734 randomly selected patients (aged 17 to 68 years) attending an inner-city gynecological outpatient clinic. C. trachomatis was detected in cervical scrapes by PCR specific for endogenous plasmid. These plasmid PCR-positive samples were subsequently subjected to genotyping by C. trachomatis-specific omp1 PCR-based restriction fragment length polymorphism analysis (J. Lan, J. M. M. Walboomers, R. Roosendaal, G. J. van Doornum, D. M. MacLaren, C. J. L. M. Meijer, and A. J. C. van den Brule, J. Clin. Microbiol. 31:1060-1065, 1993). The overall prevalence rates of C. trachomatis found in patients younger than 30 years were 9.2 and 11.8% in groups I and II, respectively. A clear age dependency was seen in group II, with the highest prevalence rate (20%) found in patients younger than 20 years, while the rate declined significantly after 30 years of age (5.9%). In women younger than 30 years, the genotyping results showed that serovars E, I, and D (in decreasing order) were frequent in group I, while serovars F, E, and G (in decreasing order) were predominantly found in group II. The study shows that C. trachomatis infections are highly prevalent in asymptomatic young women. The different serovar distributions found most likely reflect the different compositions of the study groups, but additional analysis of the case histories of individual patients suggests that certain serovars might be associated with symptomatic (i.e., serovar G) or asymptomatic (i.e., serovars D and I) infections.

Recovery of Helicobacter pylori from gastric biopsy specimens is not dependent on the transport medium used.

The survival of six clinical isolates of Helicobacter pylori at room temperature was investigated after suspension in five different media: brucella broth with 5% lysed horse blood (BBLH), phosphate-buffered saline (PBS), 20% glucose, Stuart medium, and PBS with 10% Fildes enrichment (PBS-F). Only in BBLH and PBS-F no decrease in mean bacterial numbers was observed during the 24-h study period. No H. pylori isolates could be cultured from Stuart medium after 7 h of incubation. In contrast, the recovery rates in PBS-F or Stuart medium of H. pylori isolates from gastric tissue specimens collected from 19 H. pylori-positive patients were not significantly different even after a delay of culture of up to 24 h. Our data show that the medium composition is not critical for the survival of H. pylori within gastric tissue specimens.

Long-term sequelae of Helicobacter pylori gastritis.

Chronic Helicobacter pylori gastritis has been put forward as a risk factor for development of gastric mucosal atrophy and gastric cancer. The purpose of our study was to investigate the long-term effects of H pylori gastritis on the gastric mucosa. We prospectively studied 49 subjects negative for H pylori and 58 positive subjects for a mean follow-up of 11.5 years (range 10-13 years). Serum samples were obtained at the initial and follow-up visits for determination of H pylori IgG antibodies. Gastroscopies with biopsy sampling were done in all patients at both visits. Biopsy specimens were used for assessment of H pylori infection and histology. Development of atrophic gastritis and intestinal metaplasia occurred in 2 (4%) uninfected and 16 (28%) infected subjects. Regression of atrophy was noted in 4 (7%) infected subjects. Development of atrophic gastritis and intestinal metaplasia was significantly associated with H pylori infection (p = 0.0014; odds ratio 9.0, 95% CI 1.9-41.3). The proportion of atrophic gastritis in the study population showed an annual increase of 1.15% (0.5-1.8%). We conclude that H pylori infection is a significant risk factor for development of atrophic gastritis and intestinal metaplasia. Our findings support strongly the causative role of this infection in gastric carcinogenesis.

Presence of Helicobacter pylori in the oral cavity, oesophagus, stomach and faeces of patients with gastritis.

The presence of Helicobacter pylori in the oral cavity (6 sites), oesophagus, stomach and bowel of 20 dyspeptic patients was investigated. Samples were cultured on three selective media and analyzed by 16S rDNA polymerase chain reaction (PCR) and southern hybridization. Helicobacter pylori DNA was detected by PCR from oral-cavity samples of three (20%) and from faeces samples of only one (7%) of the patients whose stomach biopsies were positive for Helicobacter pylori. When culture was used, the microorganism's rate of recovery from the oral cavity and faeces was 13% and 7%, respectively. One patient had a Helicobacter pylori-like organism in samples collected from the tongue and palate. Both strains were urease, catalase and oxidase positive and grew microaerophilically but were negative on PCR analysis. This demonstrates the possibility of false identification of Helicobacter pylori by use of routine enzyme reactions. Interestingly, specimens collected from the cheeks of three patients were positive for Helicobacter pylori by PCR analysis. This is the first instance of detection of this microorganism in the cheek.

Importance of the fiberoptic endoscope cleaning procedure for detection of Helicobacter pylori in gastric biopsy specimens by PCR.

A 16S ribosomal DNA-based PCR appeared to be a sensitive test for the detection of infection by Helicobacter pylori in 31 patients when compared with culturing and histological and serological techniques. For five patients, PCR was the only test with a positive result. H. pylori DNA was also found in gastrointestinal equipment even after standard intensive combined manual and machine cleaning. We therefore conclude that a reliable validation of PCR for the detection of H. pylori in gastric biopsy specimens is possible only when the cleaning and disinfection method used has been proven to remove all H. pylori DNA from gastrointestinal equipment. An adequate cleaning and disinfection method for the removal of H. pylori DNA from fiberoptic endoscopes is described.

Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections.

The sensitivities of three methods of detection of Mycoplasma pneumoniae by a 16S rDNA PCR were compared by using a serial dilution of M. pneumoniae. These methods consisted of a PCR performed directly on cells after a proteinase K pretreatment (direct PCR), a PCR after purification of nucleic acids (DNA-PCR), and a PCR with rRNA sequences as the target after reverse transcription. The direct PCR and the reverse transcription PCR had a sensitivity of 1.5 CFU (approximately 250 genomes). By purification, a 10-fold loss of target DNA occurred, as shown by a 10-fold decrease in sensitivity (15 CFU) of the DNA-PCR. The presence of an excess of human background DNA did not influence the sensitivity of either PCR. The direct PCR was evaluated on samples from patients with respiratory complaints. Direct PCR amplification was possible in 94.9% of the samples, which were tested by amplification of a 326-bp fragment of the beta-globin gene, which was performed to test for the suitability of amplification. Nucleic acid purification was performed on the beta-globin-negative samples, after which only 2% remained negative. A positive correlation between the direct M. pneumoniae PCR and serology, as tested by the microparticle agglutination assay (MAG assay), was found in 88.1% of the cases. A positive MAG assay result was found for samples from 10 (17%) of the patients; samples from 6 (10.2%) of these patients were also positive by PCR. Samples from three patients were found to be positive by the M. pneumoniae PCR and negative by the MAG assay. Persistence of M. pneumoniae, as detected by PCR was observed in three patients. These results indicate that the direct PCR with 16S rDNA could prove to be useful in the detection of M. pneumoniae in respiratory tract samples, although more studies are needed to evaluate the correlation between clinical symptoms and positive test results.

Microtiter enzyme-linked immunosorbent assay using recombinant derived antigens versus western blot in the confirmation of presence of antibodies against the human immunodeficiency virus type 1.

A microtiter enzyme-linked immunosorbent assay using recombinant derived antigens was compared with the Western blot (Dupont) in the confirmation of the presence of antibodies against the human immunodeficiency virus type 1 (HIV-1). Of 104 sera (104 individuals) that were negative by a screening ELISA, 91 were also negative by both confirmation assays. In three sera only the microtiter assay was found to be indeterminate, and in nine other sera only Western blot. The only microtiter assay positive serum was from a male patient at risk for infection with HIV. 279 sera from 83 patients were found positive by screening. Of these, 223 sera were positive in both confirmation assays, and no serum was negative. Only one serum was indeterminate by the microtiter ELISA in contrast to 55 sera, including follow-up samples from 25 patients, most of whom had AIDS, by Western blot (Dupont criteria). However, the number of Western blot indeterminate sera decreased substantially applying less stringent criteria for interpretation. In conclusion, the microtiter ELISA performed well as a confirmation test for the presence of antibodies against HIV-1. In addition, the results demonstrate that in the microtiter assay the envelope peptide kp41 is highly discriminative in detecting anti-HIV-1 negative and anti-HIV-1 positive sera.

Comparison of different primer sets for detection of Chlamydia trachomatis by the polymerase chain reaction.

The sensitivity and specificity of the polymerase chain reaction (PCR) method was studied in vitro with HeLa cells infected with Chlamydia trachomatis serovar L2. Three different primer sets were studied; they were derived from the endogenous plasmid, the nonvariable part of the MOMP gene and the 16S ribosomal RNA (rRNA) gene. The plasmid primers were the most sensitive in the PCR method and detected at least 0.1 infectious unit of C. trachomatis in the presence of a superfluous amount of human DNA. Application of this plasmid PCR to 13 C. trachomatis culture-positive cervical smears containing < 10- > 200 inclusion-forming units showed that it was the most sensitive of the three methods and detected C. trachomatis in all samples. This correlates with the observation that the plasmid PCR method could detect C. trachomatis in cervical smears of four symptomatic patients for up to 3 weeks after the start of treatment with doxycycline. In contrast, the MOMP gene- and rRNA gene-directed PCR, as well as culture and direct immunofluorescence, gave negative results within 1 week. Therefore, we conclude that the plasmid primers are the best candidates for use in the PCR method in C. trachomatis screening programmes and clinical follow-up studies.

Direct detection and genotyping of Chlamydia trachomatis in cervical scrapes by using polymerase chain reaction and restriction fragment length polymorphism analysis.

Detection and genotyping of Chlamydia trachomatis were optimized by using a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis performed directly with crude cells of cervical scrapes. Different PCR pretreatment methods were evaluated on samples which were positive for C. trachomatis by cell culture. In comparison with DNA extraction and different proteolytic digestion methods, a simple pretreatment of 10 min of boiling appeared to be optimal for PCR amplification. Crude samples (n = 209) were first screened for C. trachomatis by both cell culture and plasmid PCR. Subsequently, positive samples found by plasmid PCR were subjected to a direct omp1 PCR-based RFLP analysis to differentiate C. trachomatis serovars A to K, Ba, Da, and L1 to L3 and serovariant D-. All cervical scrapes that were found positive for C. trachomatis by cell culture (n = 30) were also positive by plasmid PCR and omp1 PCR and could be easily genotyped. In addition, of the culture-negative group, eight samples were found positive by plasmid PCR. Five of these eight samples were also positive by omp1 PCR; of these five, two were positive by a nested omp1 PCR. Genotyping by RFLP analysis of the 35 omp1 PCR-positive samples showed that serovars D, E, and F are the most prevalent types found in cervical scrapes, while serovariant D- was also detected. This study shows that direct PCR and PCR-based RFLP analysis are feasible for detection and genotyping of C. trachomatis in cervical scrapes and are more sensitive than culture-based serotyping.

Impact of the duration of infection on the activity of ceftazidime, gentamicin and ciprofloxacin in Klebsiella pneumoniae pneumonia and septicemia in leukopenic rats.

An experimental Klebsiella pneumoniae pneumonia and septicemia in leukopenic rats was used to study the impact of the duration of infection on the bactericidal activity of ceftazidime, gentamicin and ciprofloxacin. It appeared that the number of bacteria persisting after a single intravenous injection progressively increased with delay of antibiotic administration up to 3 h after bacterial inoculation with each of the drugs tested. This effect was most pronounced for ciprofloxacin. An inoculum effect could not explain this decrease in bacterial killing. It was also observed that a single injection with a particular dose of each of the respective drugs did not kill all the Klebsiella pneumoniae organisms in the lung. Persisting bacteria did not represent a preexisting less susceptible subpopulation selected after antibiotic administration. In further experiments the impact of delay of the start of treatment on the efficacy of ceftazidime or ciprofloxacin after administration for a period of four days with intramuscular injections at 6 h intervals was investigated. Treatment was started at 5, 12 or 24 h after bacterial inoculation. The therapeutic efficacy of both drugs decreased with the increase of duration of infection, which may be at least in part due to the progressive number of bacteria persisting after antibiotic administration. These data underline the need to start antimicrobial treatment as soon as possible.

Impact of dosage schedule of antibiotics on the treatment of serious infections.

Experimental studies suggest that the importance of the antibiotic dosage schedule for therapeutic efficacy in severe infection and when host defences are impaired is related to the class of antibiotic. The efficacy of beta-lactams is mainly dependent on the maintenance of adequate antibiotic concentrations in plasma during the entire treatment interval, and not on high peak concentrations. The efficacy of aminoglycosides is related to the total dose administered, i.e., the area under the concentration-time curve, irrespective of the frequency of administration. This difference in efficacy between beta-lactams and aminoglycosides in relation to the dosage schedule correlate well with differences between both classes of antibiotics in kinetics of antibacterial activity in vitro and in vivo. Another factor relevant in this respect is the post-antibiotic effect (PAE) which means the suppression of bacterial regrowth at the end of the period of exposure to antibiotic.

Impact of the antibiotic dosage schedule on efficacy in experimental lung infections.

In agreement with results obtained in the thigh infection model for which there is no human equivalent, the impact of the dosage schedule on efficacy in various clinically relevant experimental lung infections is related to the class of antibiotic. The efficacy of beta-lactams increases with increasing frequency of administration. Sustained antibiotic concentrations in serum at a relatively low level are more effective than high peak concentrations at intervals. In contrast, the dosing interval has little impact on the activity of aminoglycosides, which is mainly dependent on the total amount of antibiotic in serum during treatment, either as short lasting high peaks or as long lasting relatively low concentrations. Limited data available on quinolones suggest a slight increase in efficacy with increasing dosing intervals. These differences in efficacy correlate with differences in the pharmacodynamics of antibiotics of different classes as seen in vitro as well as in the lungs of infected animals. Knowledge about both the pharmacokinetics and pharmacodynamics of antibiotics is required for the correct interpretation of comparative studies on antibiotic efficacy in experimental infections, and also for the evaluation of experimental data as support for the proper design of clinical trials.