ABSTRACT
BACKGROUND: This study was undertaken to develop a sensitive and specific polymerase chain reaction (PCR)-based assay for Neisseria meningitidis and Neisseria gonorrhoeae that could ultimately be incorporated into a multiplex assay designed to screen cerebrospinal fluid (CSF) for a panel of pyogenic bacterial species associated with bacterial meningitis. METHODS: N.meningitidis-specific primers were designed from porA gene sequences with the aid of a commercial software program and were used to develop and validate a clinical assay using a liquid hybridization-gel retardation detection system. The analytic sensitivity of the assay was determined using CSF spiked with N. meningitidis and comparing the PCR-based results with culture. RESULTS: Analytic sensitivity experiments showed the assay's limit of detection to be 100 fg purified input target DNA and 0.0125 colony-forming units of N. meningitidis spiked into CSF. Specificity experiments showed the assay could detect all strains of N. meningitidis and N. gonorrhoeae tested, but did not support amplification of the commensal neisserial species or a panel of other human bacterial pathogens. CONCLUSIONS: This PCR-based assay for pathogenic neisserial species is sensitive and specific and suitable for incorporation into a multiplex assay for the clinical differentiation of aseptic and septic meningitis.
Subject(s)
DNA, Bacterial/analysis , Meningitis, Meningococcal/diagnosis , Neisseria meningitidis/isolation & purification , Polymerase Chain Reaction/methods , Bacterial Proteins/genetics , Cerebrospinal Fluid/microbiology , DNA, Bacterial/genetics , Diagnosis, Differential , Dihydropteroate Synthase/genetics , Humans , Meningitis, Aseptic/diagnosis , Meningitis, Meningococcal/cerebrospinal fluid , Meningitis, Meningococcal/microbiology , Neisseria gonorrhoeae/genetics , Neisseria gonorrhoeae/isolation & purification , Neisseria meningitidis/genetics , Sensitivity and SpecificitySubject(s)
Dihydrolipoamide Dehydrogenase/deficiency , Metabolism, Inborn Errors/complications , Myoglobinuria/etiology , Adult , Blotting, Western , DNA/analysis , DNA, Complementary , Dihydrolipoamide Dehydrogenase/genetics , Fibroblasts/enzymology , Humans , Male , Metabolism, Inborn Errors/enzymology , Mitochondria/enzymology , Myoglobinuria/enzymology , RecurrenceSubject(s)
Dihydrolipoamide Dehydrogenase/deficiency , Dihydrolipoamide Dehydrogenase/genetics , Jews/genetics , Mitochondria/enzymology , Mitochondria/genetics , Mutation , Protein Sorting Signals/genetics , Child , Child, Preschool , DNA, Mitochondrial/genetics , DNA, Mitochondrial/isolation & purification , Humans , MaleABSTRACT
Canavan disease is an infantile neurodegenerative disease that is due to aspartoacylase deficiency. The disease has been reported mainly in Ashkenazi Jews but also occurs in other ethnic groups. Determination of enzymatic activity for carrier detection and prenatal diagnosis is considered unreliable. In the present study, nine mutations were found in the aspartoacylase gene of 19 non-Jewish patients. These included four point mutations (A305E [39.5% of the mutated alleles], C218X [15.8%], F295S [2.6%], and G274R [5.3%]); four deletion mutations (827delGT [5.3%], 870del4 [2.6%], 566del7 [2.6%], and 527del6 [2.6%]); and one exon skip (527del108 [5.3%]). The A305E mutation is pan-European and probably the most ancient mutation, identified in patients of Greek, Polish, Danish, French, Spanish, Italian, and British origin. In contrast, the G274R and 527del108 mutations were found only in patients of Turkish origin, and the C218X mutation was identified only in patients of Gypsy origin. Homozygosity for the A305E mutation was identified in patients with both the severe and the mild forms of Canavan disease. Mutations were identified in 31 of the 38 alleles, resulting in an overall detection rate of 81.6%. All nine mutations identified in non-Jewish patients reside in exons 4-6 of the aspartoacylase gene. The results would enable accurate genetic counseling in the families of 13 (68.4%) of 19 patients, in whom two mutations were identified in the aspartoacylase cDNA.
Subject(s)
Canavan Disease/genetics , Alleles , Base Sequence , DNA/analysis , Europe , Female , Humans , Infant , Infant, Newborn , Jews , Male , Molecular Sequence Data , MutationABSTRACT
Pneumatocele formation, a cyst-like rarefaction that develops within the lung parenchyma, is an unusual complication of pneumonia in the neonate. It has been reported to occur with Staphlococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, and Pseudomonas aeruginosa infections. We describe a case of a premature neonate with pneumonia and subsequent pneumatocele formation caused by Enterobacter cloacae.
