Evaluation of the MicroSeq 500 16S rDNA-based gene sequencing for the diagnosis of culture-negative bacterial meningitis

Gene amplification using 16S rDNA primers has been proposed as a strategy for the diagnosis of bacterial meningitis. The aim of this study was to evaluate the performance of the MicroSeq 500 16S ribosomal DNA test (Applied Biosystems) from patients with suspected bacterial meningitis and CSF negative-culture in comparison to traditional methods. Twelve purulent culture-negative CSF samples were collected between January 2005 and January 2007. For DNA extraction, 500 microl of CSF samples were treated using the QIAamp mini kit (QIAGEN). The extracted DNA was examined amplifying 500 bp at the 5' end of 16S rRNA gene using MicroSeq500 16S rDNA Bacterial Identification PCR kit and the sequencing reactions were performed with the MicroSeq500 16S rDNA Bacterial Identification Sequencing kit (Applied Biosystems). The sequences were compared with those available in GenBank. For the culture-negative CSF samples the MicroSeq 500 16S rDNA yielded a positive result in 9 cases (75.0%): three samples were identified as Streptococcus. pneumoniae, three as Neisseria meningitidis, and the remaining 3 as Haemophilus influenzae, Abiotrophia defectiva and Porphyromonas gingivalis. The MicroSeq 500 16S ribosomal DNA test may improve the microbiological diagnosis of bacterial meningitis, especially when spinal fluid samples are obtained after the administration of antimicrobial therapy.     

The prospects for the diagnosis of bacterial meningitis]

The samples of spinal fluid arriving to the Clinical Infectious Hospital in 1994-1996 with the clinical diagnosis "generalized form of meningococcal infection" or "purulent meningitis of unclear etiology" were studied. The etiological agent was bacteriologically identified in 35% of 487 patients (in 25% of cases Neisseria meningitidis, in 7% of cases Streptococcus pneumoniae and in 2% of cases Haemophilus influenzae, type b, were detected). The method of latex agglutination, used in this study, was highly specific (100%) and moderately sensitive (67%); this method made it possible to diagnose 25% of cases additionally (N. meningitidis in 15% of cases, S. pneumoniae in 5% of cases and H. influenzae in 3% of cases). Diagnostics with the use of PCR was characterized by high specificity (> 97%) and sensitivity (> 85%) relatively to the "golden standard" of microbiological diagnostics. There were few false positive results (3 samples), caused probably by contamination at the moment of taking the samples. For this reason the results obtained by PCR could be used for diagnostic purposes even in cases of negative results given by other methods. Tests with the use of PCR made it possible to diagnose 29% more cases additionally (in 26% of cases N. meningitidis DNA and in 3% of cases S. pneumoniae DNA were detected. Thus the complex of methods used in this study permitted the detection of the etiological agent altogether in 87% of cases.     

Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis--rapid separation of 16S rRNA PCR amplicons without the need for cloning

AIMS: The aim of this study was to develop a polyacrylamide gel electrophoresis (PAGE) method for the rapid separation of 16S rRNA PCR amplicons from aetiological agents of acute meningitis. METHODS AND RESULTS: Blood samples from 40 patients with suspected acute meningococcal meningitis were examined for the presence of causal agents, including Neisseria meningitidis employing two methods: (i) broad-range 16S rRNA PCR in conjunction with PAGE and automated sequencing and (ii) species-specific PCR employing ABI TaqMan technology for N. meningitidis. Analysis of clinical specimens employing 16S rRNA PCR yielded 33/40 (82.5%) positive for the presence of bacterial DNA. Species-specific PCR yielded 30/40 (75%) clinical specimens positive for N. meningitidis. Prior to separation by PAGE, only 6/33 (18.2%) amplicons were able to be identified by sequence analysis, the remaining amplicons (n=27) did not yield an identification due to the presence of mixed 16S rRNA PCR amplicons. Following separation, amplicons were re-amplified and sequenced, yielding 24/27 (88.9%) positive for N. meningitidis and three specimens positive for Acinetobacter sp., Staphylococcus aureus and Streptococcus pneumoniae. One specimen was positive for both N. meningitidis and Streptococcus spp. and another specimen was positive for N. meningitidis and Pseudomonas sp., by broad-range PCR. Seven clinical specimens were negative for N. meningitidis and other eubacteria using both detection techniques. CONCLUSIONS: Clinical specimens including blood and cerebrospinal fluid from patients with suspected acute bacterial meningitis, may become contaminated with commensal skin flora, resulting in difficulties in downstream sequencing of pathogen plus contaminant DNA. This study allows for the rapid separation of amplified pathogen from contaminant DNA. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrated the usefulness of the rapid separation of multiple 16S rRNA PCR amplicons using a combination of PAGE and automated sequencing, without the need of cloning. Adoption of this technique is therefore proposed when trying to rapidly identify pathogens in clinical specimens employing broad-range 16S rRNA PCR.     

Employment of broad range 16S rDNA PCR and sequencing in the detection of aetiological agents of meningitis

To employ partial 16S rDNA PCR and automated sequencing technique to identify non-culturable causal agents of bacterial meningitis, 73 peripheral blood samples and 413 culture-negative and eight culture-positive CSF clinical specimens from patients with suspected acute meningitis were examined for the presence of bacterial genomic DNA employing broad range 16S rDNA PCR followed by sequencing of the amplicons. In blood samples, 63/73 specimens were PCR positive (86.3%) and after direct sequencing of the PCR amplicons, only 12.7% (8/63) gave clear sequencing results and 55/63 (87.3%) were mixed with more than one organism detected. The mixed PCR amplicons were separated by using PAGE and mixed amplicons from 29/55 (52.7%) specimens were successfully identified through sequencing. Of the CSF samples, 8/8 culture-positive samples were also PCR positive and 45/413 (10.9%) of culture-negative gave a strong PCR signal and 88/413 (21.3%) specimens yielded a weak PCR signal. The remaining 280 culture-negative specimens were also PCR negative. Nested PCR was set up for the 88 weak positive samples and yielded 72/88 (81.8%) strong positives, with the remainder failing to amplify 133/413 (32.2%) culture-negative samples were PCR positive. In this study, the most common bacteria identified from blood specimens were Neisseria meningitidis, 13/63 (20.6%); Streptococcus spp, 5/63 (7.9%); Acinetobacter spp and Pseudomonas spp 4/63 (6.3%). From culture-negative CSF, the pattern was different in that Staphylococcus spp (13/58, 22.4%), Neisseria meningitidis (9/58, 15.52%) and Pseudomonas spp (8/58, 14.79%), were the most frequent. Overall, 16S rRNA broad-range PCR combined with direct DNA sequencing is a valuable molecular tool to aid with the detection as well as identification of non-culturable aetiological agents of acute bacterial meningitis and can augment cultural methods in the diagnosis of central nervous system infections in patients who have been treated with antibiotics. However, this study demonstrates that contamination is an important complication of the molecular assay, which should be attempted to be eliminated through careful laboratory controls. Hence there should be careful interpretation of any molecular finding, in tandem with other laboratory findings, such as culture, immunological and biochemical markers, and the clinical scenario of the patient.     

Different meningitis-causing bacteria induce distinct inflammatory responses on interaction with cells of the human meninges

The interactions of bacterial pathogens with cells of the human leptomeninges are critical events in the progression of meningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the interactions of the meningeal pathogens Escherichia coli K1, Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. A rank order of association with meningioma cells was observed, with N. meningitidis showing the highest levels of adherence, followed by E. coli, S. pneumoniae and H. influenzae. Neisseria meningitidis and H. influenzae did not invade meningioma cells or induce cell death, but induced a concentration-dependent secretion of inflammatory mediators. Neisseria meningitidis induced higher levels of IL-6, MCP-1, RANTES and GM-CSF than H. influenzae, but there was no significant difference in the levels of IL-8 induced by both pathogens. Streptococcus pneumoniae was also unable to invade meningioma cells, but low concentrations of bacteria failed to stimulate cytokine secretion. However, higher concentrations of pneumococci led to cell death. By contrast, only E. coli K1 invaded meningioma cells directly and induced rapid cell death before an inflammatory response could be induced. These data demonstrate that the interactions of different bacterial pathogens with human meningeal cells are distinct, and suggest that different intervention strategies may be needed in order to prevent the morbidity and mortality associated with bacterial meningitis.     

Prospective study of bacterial meningitis in North East Thames region, 1991-3, during introduction of Haemophilus influenzae vaccine.

OBJECTIVE--To describe the epidemiology of primary bacterial meningitis in the North East Thames region over a three year period before and during the introduction of the vaccine for Haemophilus influenzae type b. DESIGN--Analysis of information on cases of primary bacterial meningitis identified by microbiology laboratories in the region, with collection of case data by questionnaire. MAIN OUTCOME MEASURES--Annual incidence rates for types of meningitis according to age and ethnic group. RESULTS--The annual incidence rates for the three major causes of bacterial meningitis in the general population were 1.9/100,000 for Neisseria meningitidis, 1.6/100,000 for Haemophilus influenzae before vaccination, and 1.0/100,000 for Streptococcus pneumoniae. Higher rates of H influenzae meningitis were found in Asians compared with white people (3.6/100,000 v 1.5/100,000, P = 0.01). As a result of the vaccine programme introduced in October 1992 the number of cases of H influenzae meningitis in children under 5 years has fallen by 87%. CONCLUSIONS--Bacterial meningitis is a serious problem especially in preschool children. There are differences in the incidence of some causes of bacterial meningitis in different ethnic groups; with H influenzae type b being significantly more common among black and Asian people than among white people. The immunisation programme for H influenzae type b in the North East Thames region has been successful in reducing the incidence of this type of meningitis in Asian and white populations. The numbers were too small to evaluate in the black population.     

Incorporation of real-time PCR into routine public health surveillance of culture negative bacterial meningitis in São Paulo, Brazil

Real-time (RT)-PCR increases diagnostic yield for bacterial meningitis and is ideal for incorporation into routine surveillance in a developing country. We validated a multiplex RT-PCR assay for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in Brazil. Risk factors for being culture-negative, RT-PCR positive were determined. The sensitivity of RT-PCR in cerebrospinal fluid (CSF) was 100% (95% confidence limits, 96.0%-100%) for N. meningitidis, 97.8% (85.5%-99.9%) for S. pneumoniae, and 66.7% (9.4%-99.2%) for H. influenzae. Specificity ranged from 98.9% to 100%. Addition of RT-PCR to routine microbiologic methods increased the yield for detection of S. pneumoniae, N. meningitidis, and H. influenzae cases by 52%, 85%, and 20%, respectively. The main risk factor for being culture negative and RT-PCR positive was presence of antibiotic in CSF (odds ratio 12.2, 95% CI 5.9-25.0). RT-PCR using CSF was highly sensitive and specific and substantially added to measures of meningitis disease burden when incorporated into routine public health surveillance in Brazil.     

Antimicrobial susceptibility profiles associated with bacterial meningitis among children: a referral hospital-based study in Iran

Bacterial meningitis continues to be associated with high morbidity and mortality rate worldwide, especially in the pediatric age group. This study was performed to identify the microbial etiologies of meningitis among 31 children, who were admitted in the Emergency Ward of a referral pediatric hospital in Iran. Culture identification showed that Streptococcus pneumoniae (12 subjects), Haemophilus influenzae (11 subjects) were the most common bacteria, followed by Escherichia coli (7 cases) and Neisseria meningitidis (only one case). Antibiotic susceptibility tests revealed that vancomycin had the best effect on S. pneumoniae in comparison with other antibiotics, whereas H. influenzae and E. coli were more susceptible to ceftriaxone, ceftazidime, and ceftizoxime than other antibiotics. In conclusion, despite the advances in antibiotic therapy and vaccine development, bacterial meningitis still is a health problem. S. pneumoniae, H. influenzae, and N. meningitidis are the main sources of bacterial meningitis, but other organisms such as E. coli should also be suspected, when a case is admitted to a referral pediatric hospital.     

Binding of tissue-type plasminogen activator (t-PA) to Neisseria meningitidis and Haemophilus influenzae

Abstract Forty-nine bacterial strains representing five species known to interact with human plasminogen were tested for the ability to bind the two major human plasminogen activators, t-PA and urokinase. The bacterial species tested included Haemophilus influenzae, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus equisimilis and human group G streptococci. All N. meningitidis and 11 of 14 H. influenzae strains displayed substantial binding of t-PA with values in the range of 20–46%. On the contrary, none of the streptococcal strains bound significant amounts of tPA. With urokinase no binding could be found for any of the bacterial species tested. Scatchard analysis with a selected H. influenzae strain (HI23354) demonstrated 10 000 receptors per bacterium for t-PA with a K _d value of about 20 nmol l⁻¹. The corresponding values with a selected N. meningitidis strain (Mo 52) was 8500 receptors per bacterium and 70 nmol l⁻¹. t-PA binding could be reduced about 40% by the addition of 10 nmol l⁻¹ of the lysine analogue ϵ-aminocaproic acd (EACA) whereas no inhibitory effect could be demonstrated with arginine. Addition of 2 μmol l⁻¹ of plasminogen which is enough to occupy all bacterial sites for plasminogen did not interfere with the t-PA binding, suggesting that the receptors for t-PA and plasminogen are distinct. Using very high plasminogen concentrations however, t-PA binding could be reduced by about 50% possibly due to an interaction between t-PA and plasminogen in the fluid phase. Our results demonstrate the occurrence of a previously unknown type of bacterial receptor that is capable of specifically binding t-PA.     

Genetic variation in the β2-adrenocepter gene is associated with susceptibility to bacterial meningitis in adults

Recently, the biased β2-adrenoceptor/β-arrestin pathway was shown to play a pivotal role in crossing of the blood brain barrier by Neisseria meningitidis. We hypothesized that genetic variation in the β2-adrenoceptor gene (ADRB2) may influence susceptibility to bacterial meningitis. In a prospective genetic association study we genotyped 542 patients with CSF culture proven community acquired bacterial meningitis and 376 matched controls for 2 functional single nucleotide polymorphisms in the β2-adrenoceptor gene (ADRB2). Furthermore, we analyzed if the use of non-selective beta-blockers, which bind to the β2-adrenoceptor, influenced the risk of bacterial meningitis. We identified a functional polymorphism in ADRB2 (rs1042714) to be associated with an increased risk for bacterial meningitis (Odds ratio [OR] 1.35, 95% confidence interval [CI] 1.04-1.76; p?=?0.026). The association remained significant after correction for age and was more prominent in patients with pneumococcal meningitis (OR 1.52, 95% CI 1.12-2.07; p?=?0.007). For meningococcal meningitis the difference in genotype frequencies between patients and controls was similar to that in pneumococcal meningitis, but this was not statistically significant (OR 1.43, 95% CI 0.60-3.38; p?=?0.72). Patients with bacterial meningitis had a lower frequency of non-selective beta-blockers use compared to the age matched population (0.9% vs. 1.8%), although this did not reach statistical significance (OR 1.96 [95% CI 0.88-4.39]; p?=?0.09). In conclusion, we identified an association between a genetic variant in the β2-adrenoceptor and increased susceptibility to bacterial meningitis. The potential benefit of pharmacological treatment targeting the β2-adrenoceptor to prevent bacterial meningitis in the general population or patients with bacteraemia should be further studied in both experimental studies and observational cohorts.     

Pneumococcal meningitis in childhood: a longitudinal prospective study

After implementation of programmes for active immunization against Haemophilus influenzae b, Streptococcus pneumoniae and Neisseria meningitidis became the most common agents of bacterial meningitis in childhood. Over a 9-year period, children showing clinical and laboratory findings of meningitis on the basis of their positive cultures of blood or cerebro-spinal fluid (CSF) for S. pneumoniae were enrolled. Predisposing conditions, clinical and laboratory findings, and microbiological and imaging studies were considered. Meningitis-related death or neurological sequelae defined an unfavourable outcome. Sixty-four patients met the inclusion criteria. Thirty-one (48%) children had predisposing conditions to pneumococcal meningitis. Fever and neck stiffness were the main symptoms; 14 patients (22%) reported seizures before admission. Twenty-one patients required treatment in the intensive care unit (ICU). Streptococcus pneumoniae strains were penicillin susceptible in 54 cases (84%). Forty-eight children (75%) showed complete recovery. Two patients (3%) died, and 14 (22%) had sequelae. Patients with a low CSF cell count, low neutrophils, early admission to ICU or infection by penicillin-nonsusceptible strains of S. pneumoniae had an unfavourable outcome more frequently. Low blood neutrophils, low CSF cell count, early admission to ICU and infection by penicillin-nonsusceptible strains are the main factors predicting an unfavourable outcome in children with pneumococcal meningitis.     

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

Antibiotic treatment prior to transport or admission of patients to hospital has reduced the proportion of patients with invasive meningococcal disease (IMD) from whom Neisseria meningitidis can be isolated by standard microbiological techniques. Assays to detect the crgA gene were used to detect meningococcal DNA by both conventional polymerase chain reaction (PCR) and real-time PCR (RTPCR) in relation to microbiological diagnosis of cases over two years between 2002 and 2003. The sensitivity of both PCR assays for culture-confirmed cases was 93% and the specificity was 98.6%. Agreement between the two PCR assays was 96.2%. The inter- and intra-assay variations and effects of different amounts of DNA on the melting temperatures were examined. The touch-down RTPCR based on SYBR Green I fluorescent dye detected and characterized N. meningitidis in clinical samples within one hour.     

Mouse models of infection for Neisseria meningitidis B,2b and Haemophilus influenzae type b diseases

A disseminated and fatal infection was established in C57BL mice, injected intraperitoneally with either Neisseria meningitidis B,2b or Haemophilus influenzae type b bacteria plus enhancement factors. The effects of mucin, hemoglobin, and iron dextran as enhancement of bacterial infectivity in mice were evaluated individually and in combination. A mixture of mucin and hemoglobin was most effective in enhancing the virulence of the pathogens. Inbred mouse lines were more susceptible than outbred ones. Relative virulence of a number of bacterial strains was also compared in one selected mouse line. Neisseria meningitidis B,2b and Haemophilus influenzae type b strains were more virulent than non-B,2b and nontypable strains. Finally, the course of bacteremia for the two infections in mice was followed by quantitative blood cultures. The animals succumbed to the generalized condition within 72 h. In the case of Neisseria meningitidis B,2b, 10 organisms with 4% mucin and 1.6% hemoglobin were sufficient to kill 50% of the animals. For Haemophilus influenzae type b, 300 bacteria with 5% mucin and 2% hemoglobin were necessary to obtain similar effects.     

Rapid detection of Haemophilus influenzae by hel gene polymerase chain reaction

AIMS: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. METHODS AND RESULTS: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (65.9%) of 91 samples in contrast to 62 (68.12%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCR-positive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. CONCLUSIONS: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. SIGNIFICANCE AND IMPACT OF THE STUDY: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods.     

Comparison of conventional and molecular methods for the detection of bacterial pathogens in sputum samples from cystic fibrosis patients

The nature of the micro-flora present in sputa of six different cystic fibrosis (CF) patients was assessed using routine microbiological culture and molecular methods. Bacterial genes for the small subunit ribosomal RNA (ssu rDNA) were specifically amplified from DNA extracted from the sputum samples, cloned and characterised by hybridisation and DNA sequencing. A large number of clones from six sputa were screened. Initially, oligonucleotide hybridisation was performed with five probes, specific for Gram-positives and Gram-negatives in general and the main pathogens for the CF patient (Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae). For a single sputum sample, the results were fully congruent when culture and molecular methods were compared. In the other five sputa, discrepancies for S. aureus and/or H. influenzae were documented. Although S. aureus DNA and H. influenzae DNA was detected in three and four sputa, respectively, strains could not be cultured. Although the PCR approach is not capable of distinguishing viable from dead bacteria, all of the CF patients had a history of S. aureus infections, while one of the CF patients once had cultivable H. influenzae in the sputum as well. A number of clones for probe-unidentified Gram-negative or Gram-positive bacterial species were further analysed by sequencing and additional potential pathogens were identified. Although routine culture of sputum frequently points to mono-specific exacerbations, our molecular data indicate that the other CF-related pathogens appear to be persistently present as well. We conclude that routine culture for bacterial pathogens from CF sputa yields limited microbiological information since it frequently fails to identify a number of pathogenic bacterial species that are potentially present in a viable status in the lungs of these patients.     

Urogenital, maternal and neonatal isolates of Haemophilus influenzae: identification of unusually virulent serologically non-typable clone families and evidence for a new Haemophilus species

A collection of 117 strains of Haemophilus influenzae, including 112 non-typable isolates recovered predominantly in the USA and France from genital, obstetric and neonatal sources, was characterized by the electrophoretic mobilities of 10 metabolic enzymes. Eighty-six distinctive multilocus chromosomal genotypes (electrophoretic types, ETs) were distinguished on the basis of allele profiles at the enzyme loci. Isolates of five allied biotype IV ETs were highly divergent from all other strains and hybridization of chromosomal DNA revealed that they undoubtedly represent a previously unrecognized species of Haemophilus. Isolates representing these ETs were recovered predominantly from obstetric infections and serious neonatal diseases and apparently possess specific tropism for the genital tract. Strains of these five ETs were present in samples from both the USA and France, but only in the USA did they cause bacteraemia and meningitis, an occurrence which probably reflects differences in patient management between the two countries. Although strains assigned to H. influenzae (sensu stricto) were strongly polymorphic in multilocus enzyme genotype, 69% of isolates recovered from patients with meningitis and/or septicaemia were assigned to only two clone families, a result suggesting that some serologically nontypable strains of H. influenzae originating from the genital tract are unusually virulent.     

Direct PCR assay for detection ofNeisseria meningitidis in human cerebrospinal fluid

A PCR amplification was performed to detectNeisseria meningitidis insertion sequence1106 (IS-1106) in the humancerebrospinalfluid (CSF) in cases of meningitis. The study included 27 CSF samples from suspected meningitis patients. Although the inflammatory response in most of the samples was slightly increased, the results showed that 7 (26%) and 8 (30%) CSF samples were diagnosed as meningococcal meningitis by Gram staining and by culture, respectively. The primers of theIS-1106 were used for direct diagnosis ofN. meningitidis in the human spinal fluid after a minor treatment of the CSF samples. The sample was diagnosed as meningococcal meningitis, if a DNA band of about 600 bp was detected in the ethidium bromide-stained agarose gel. The 27 CSF samples were analyzed in a random manner. Of these, 18 samples including the Gram staining- and culture-positive samples were also positive in PCR amplification. However, a CSF sample, which was diagnosed to be meningococcal meningitis in culture was negative in both Gram staining and PCR analysis. The specificity of theIS-1106 primers was determined to be 95%, with 100% sensitivity in comparison to Gram staining and culture. The primers were sensitive to 10 pg or more of meningococcal DNA. In addition, the PCR amplification showed high predictive values (89 and 100%) in diagnosing meningitis in patients that were negative and positive responders when tested by culture and by Gram staining. In conclusion, the PCR amplification ofIS-1106 ofN. meningitidis is specific and sensitive to both culture-positive and-negative meningococcal meningitis. Hence, PCR assay is highly recommended for use in a rapid diagnosis of suspected meningitis patients.     

Haemophilus influenzae meningitis in Manitoba and the Keewatin District,NWT: potential for mass vaccination.

A community-based surveillance study of all central nervous system infections was carried out in Manitoba and the Keewatin District, NWT, between Apr. 1, 1981, and Mar. 31, 1984. There were 201 cases of bacterial meningitis in Manitoba over the study period, 81 (40%) caused by Haemophilus influenzae; all but one isolate tested were type b (Hib). There were nine cases of H. influenzae meningitis in the Keewatin District. The overall annual incidence rate of H. influenzae meningitis in Manitoba was 2.5/100,000; for children under 5 years the rate was 32.1/100,000. For the Keewatin District the corresponding rates were 69.6/100,000 and 530/100,000. A total of 85% and 100% of the cases of H. influenzae meningitis occurred by 24 months of age in Manitoba and the Keewatin District respectively. The age at onset was earlier in native Indian children (22 cases) and Inuit children (9 cases) than in non-native children (59 cases) (p less than 0.005); thus, vaccine prevention of Hib meningitis will likely be more difficult in native Indian and Métis children. Without evaluating the increased potential of H. influenzae vaccines to prevent nonmeningitic forms of disease, we concluded that mass childhood vaccination with polyribosylribitolphosphate (PRP) vaccine is not warranted in Manitoba or the Keewatin District. Immunogenicity studies suggest that administration of conjugated Hib vaccines such as PRP-D in infancy may prevent approximately one-third to two-thirds of cases of H. influenzae meningitis; these vaccines warrant consideration for use in mass childhood vaccination programs.     

Evaluation of non-culture diagnosis of invasive meningococcal disease by polymerase chain reaction (PCR)

Antibiotic treatment prior to transport or admission to hospital has reduced the proportion of cases of invasive meningococcal disease (IMD) from which Neisseria meningitidis can be isolated by standard microbiological techniques. Identification of meningococci by polymerase chain reaction (PCR) was assessed in relation to microbiological diagnosis for cases over a 4-year period between 1998 and 2001. A screening assay for the IS1106 gene was used to detect meningococcal DNA and five additional assays for siaD and orf-2 genes were performed to determine the serogroup. PCR results were compared with results of bacteriological culture, other laboratory test results and clinical data. The sensitivity of the PCR assay for culture-confirmed cases was 98.5%. The specificity of the assay was 96% based on test results for patients from whom other bacteria were isolated, children with viral meningitis and afebrile negative controls. The siaD B/C/W-135 and Y as well as the orf-2 gene for serogroup A PCR assays were able to determine the serogroup for 75.2% of cases that were positive by PCR screening assay. When isolates from patients with IMD were tested by both agglutination and PCR, the results agreed in all cases. PCR is a useful tool for diagnosis of IMD when Gram stain and culture tests are negative due to antibiotic treatment prior to collection of samples for microbiological analyses.     

Carbon-Mercaptooctadecane/Carboxylated Multi-walled Carbon Nanotubes Composite Based Genosensor for Detection of Bacterial Meningitis

Human brain bacterial meningitis is a life-threatening disease mainly caused by Neisseria meningitidis, lead to several complications including damage of brain or even death. The present available methods for diagnosis of meningitis have one or more limitations. A rmpM gene based genosensor was fabricated by immobilizing 5′-amino modified 19-mer single stranded DNA probe onto carbon-mercaptooctadecane/carboxylated multi-walled carbon nanotubes composite electrode and hybridized with 2.5–40 ng/6 μL of single stranded genomic DNA (ssG-DNA) of N. meningitidis from cerebrospinal fluid (CSF) of the suspected meningitis patients. The electrochemical response was measured by using cyclic voltammetry and differential pulse voltammetry (DPV) using 1 mM methylene blue as redox indicator in 30 min (including a response time of 1 min) at 25 °C. The sensitivity of the genosensor was 3.762 (μA/cm²)/ng and limit of detection was 2 ng of ssG-DNA of N. meningitidis with DPV. The genosensor has specificity only to N. meningitidis and does not hybridize with the genomic DNA of any other possible pathogen in human CSF. The immobilization of the probe and hybridization of the ssG-DNA were characterized by using electrochemical impedance in presence of 5 mM potassium ferricyanide and scanning electron microscopy. The genosensor loses only 12 % of its original DPV current on storage at 4 °C for 6 months. Carbon composite based electrochemical array can be constructed to detect multiple bacterial meningitis suspected patient CSF samples during an outbreak of the disease.