PCR of peripheral blood for diagnosis of meningococcal disease

Abstract

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 prehospitalization 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.

False positive diagnosis of meningococcal infection by the IS1106 PCR ELISA

“…The IS1106 PCR ELISA was performed as described by Davison et al [2] but using the primers and probe which were developed by Newcombe et al [3] whilst the annealing temperature was decreased from 63 to 61³C.…”

“…Ampli¢cation of bacterial DNA from CSFs from meningitis cases of nonmeningococcal aetiologies was shown to yield a 596bp product by this method so the assay was re¢ned by the development of a solution hybridisation assay incorporating hybridisation with a speci¢c biotinylated oligonucleotide probe for increased sensitivity and speci¢city [2]. Newcombe et al [3] subsequently further increased the sensitivity and speci¢city of the assay by primer/probe redesign and this assay was evaluated at the MRU for an 18-month period from late 1995 for the non-culture con¢rmation and diagnosis of meningococcal infection. The meningococcal IS1106 PCR has proved to be highly sensitive identifying 91 cases following evaluation during the last quarter of 1995 [4].…”

Epitope Imprinting of Outer Membrane Protein of Neisseria Meningitidis

“…Timely diagnosis of this disease will be a boon for targeted population. It is currently being diagnosed by various diagnostic methods (Deivanayagam et al 1993, Sippel et al 1984, Surinder et al 2007, Tan et al 1987, Gray et al 1992, Dunbar et al 1998, Kilian et al 1979, Radstrom et al 1994, Neewcombe et al 1996, Kotilinen et al 1998, Carrol et al 2003, Baethegan et al 2003Patel et al 2010, but these can diagnose the disease only at a later advanced stage when it has spread throughout the affected organs. Its early symptoms often lead to misdiagnosis leading to high rate of mortality.…”

Evaluation of touch-down real-time PCR based on SYBR Green I fluorescent dye for the detection ofNeisseria meningitidisin clinical samples

“…During the past decade, methods based on polymerase chain reaction (PCR) for detection of meningococcal DNA have provided valuable tools for diagnosis of IMD [6][7][8][9][10][11][12][13][14][15][16][17]. Increasing demand for more sensitive and rapid diagnostic procedures, such as fluorescence-based PCR has prompted the routine use of automated liquidhandling systems in the clinical laboratory [18].…”