In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific for Propionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results.

Detection of Prosthetic Hip Infection at Revision Arthroplasty by Immunofluorescence Microscopy and PCR Amplification of the Bacterial 16S rRNA Gene

Meningococcal disease is normally suspected on clinical grounds and is confirmed by isolation of Neisseria meningitidis bacteria from blood or cerebrospinal fluid or, more recently, by serology or PCR of cerebrospinal fluid. Achieving confirmation of a clinical diagnosis of meningococcal disease has become more difficult in the last few years. The pre-hospitalization administration of parenteral benzylpenicillin normally renders blood cultures sterile, and lumbar puncture is undertaken less frequently, especially in young children. We evaluated PCR for the detection of meningococcal DNA in 80 blood samples taken from patients with known or suspected meningococcal disease or from patients with other diagnoses (negative controls). Both the sensitivity and the specificity of the test were 100% for patients with confirmed cases of meningococcal disease when the blood buffy coat was used (83 to 100% sensitivity and 87 to 100% specificity with 95% confidence limits). Positive PCR results could be obtained from both blood buffy coat and serum samples. Sensitivity was unaffected by prior antibiotic treatment. PCR is a rapid, sensitive test that may be used to confirm a diagnosis of meningococcal disease by using peripheral blood samples. Introduction of this test into clinical laboratories may in some cases obviate the need for lumbar puncture to be performed on patients with suspected meningococcal disease. Our results demonstrate that a substantial number of cases of meningococcal disease are not confirmed by conventional techniques and remain undiagnosed. If the PCR test described here was widely applied, the number of cases of meningococcal disease ascertained might rise by as much as 60% greater than that recognized at present. It is likely that we are in a prevaccination era for meningococcal disease. Better case ascertainment is urgently required to assess the need for vaccines, to determine their costs and benefits, and to monitor their efficacies.

PCR of peripheral blood for diagnosis of meningococcal disease.

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis.

Diagnosis of Meningococcal Meningitis by Broad-Range Bacterial PCR with Cerebrospinal Fluid

OBJECTIVE: To study changes in the epidemiology and management of meningococcal disease in one health district during a period of high local incidence of disease. DESIGN: Prospective case ascertainment and data collection over 14 years, with retrospective analysis of cases. SETTING: West Gloucestershire (population 320,000). SUBJECTS: Residents developing meningococcal disease between 1 January 1982 and 31 December 1995. RESULTS: 252 cases of invasive meningococcal disease were identified, of which 102 (40%) were officially notified and 191 (76%) were confirmed by culture from a deep site. The observed disease incidence of 5.6/100,000/year was about 2.7 times the national incidence (as measured by either statutory notifications or reference laboratory reports). The period 1983-90 was characterised by a prolonged localised outbreak due to serogroup B serotype 15 sulphonamide resistant (B15R) strains. General practitioners gave benzylpenicillin before hospital admission to 18% of patients who presented with meningococcal disease in the first half of the study period and to 40% who presented in the second half. The overall case fatality rate was 6.7% (17/252). Four deaths were directly or indirectly related to lumbar puncture. Of 120 patients whose lumbar puncture yielded meningococci, nine (8%) showed no abnormality on initial examination. CONCLUSIONS: Neither laboratory records nor formal notifications alone can give an accurate estimate of the incidence of meningococcal disease. Because of the dangers of lumbar puncture, the frequency of misleading negative initial findings, and the advent of new diagnostic techniques, the need for samples of cerebrospinal fluid should be critically questioned in each case of suspected meningococcal disease.

/pdf/epidemiology-and-clinical-management-of-meningococcal-duvdv0hqwu.pdf

Epidemiology and clinical management of meningococcal disease in west gloucestershire: retrospective, population based study

PorA is an important component in a vaccine against infection with Neisseria meningitidis. However, porA-negative meningococci were isolated from patients, thereby potentially limiting the role of PorA-mediated immunity. To analyze the mechanism by which the porA deletion occurred, the regions upstream and downstream of porA from three meningococcal strains (H44/76, H355, and 860183) were sequenced. The porA upstream region in strain 860183 contains a cluster of 22 repetitive palindromic RS3 core sequences (ATTCCC-N8-GGGAAT) and 10 RS3 core sequences (ATTCCC) in direct orientation. The cluster is flanked by neisserial repeats, so-called Correia elements, and can be subdivided into three repeats of 518 bp followed by a truncated repeat. The porA upstream region of the other two strains showed deletions, probably caused by a recombination between RS3 core sequences. The porA downstream region of H44/76 and H355 contains the IS1106 element followed by a cluster of 10 palindromic RS3 core sequences, 4 RS3 core sequences, and 1 other RS3 core sequence (GGGAAT) and is followed by a Correia element. This cluster can be subdivided into four direct repeats of 370 bp. Strain 860183 had two such repeats instead of four. Sequence analysis of the porA-negative variants indicated that the deletion of porA occurred via a recombination between two copies of the 116-bp region, containing two palindromic RS3 core sequences and a single RS3 core sequence. This region is homologous in the upstream and downstream clusters.

/pdf/deletion-of-pora-by-recombination-between-clusters-of-362y9qns05.pdf

Deletion of porA by Recombination between Clusters of Repetitive Extragenic Palindromic Sequences in Neisseria meningitidis

Use of pcr in the diagnosis of meningococcal meningitis

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Use of pcr in the diagnosis of meningococcal meningitis