Improved methods for the detection and characterization of carried Neisseria meningitidis isolates are needed. We evaluated a multiplex PCR algorithm for the detection of a variety of carriage strains in the meningitis belt. To further improve the sensitivity and specificity of the existing PCR assays, primers for gel-based PCR assays (sodC, H, Z) and primers/probe for real-time quantitative PCR (qPCR) assays (porA, cnl, sodC, H, E, Z) were modified or created using Primer Express software. Optimized multiplex PCR assays were tested on 247 well-characterised carriage isolates from six countries of the African meningitis belt. The PCR algorithm developed enabled the detection of N. meningitidis species using gel-based and real-time multiplex PCR targeting porA, sodC, cnl and characterization of capsule genes through sequential multiplex PCR assays for genogroups (A, W, X, then B, C, Y and finally H, E and Z). Targeting both porA and sodC genes together allowed the detection of meningococci with a sensitivity of 96% and 89% and a specificity of 78% and 67%, for qPCR and gel-based PCR respectively. The sensitivity and specificity ranges for capsular genogrouping of N. meningitidis are 67% - 100% and 98%-100% respectively for gel-based PCR and 90%-100% and 99%-100% for qPCR. We developed a PCR algorithm that allows simple, rapid and systematic detection and characterisation of most major and minor N. meningitidis capsular groups, including uncommon capsular groups (H, E, Z).

Bivalent rLP2086 (Trumenba), a vaccine for prevention of Neisseria meningitidis serogroup B (NmB) disease, was licensed for use in adolescents and young adults after it was demonstrated that it elicits antibodies that initiate complement-mediated killing of invasive NmB isolates in a serum bactericidal assay with human complement (hSBA). The vaccine consists of two factor H binding proteins (fHBPs) representing divergent subfamilies to ensure broad coverage. Although it is the surrogate of efficacy, an hSBA is not suitable for testing large numbers of strains in local laboratories. Previously, an association between the in vitro fHBP surface expression level and the susceptibility of NmB isolates to killing was observed. Therefore, a flow cytometric meningococcal antigen surface expression (MEASURE) assay was developed and validated by using an antibody that binds to all fHBP variants from both fHBP subfamilies and accurately quantitates the level of fHBP expressed on the cell surface of NmB isolates with mean fluorescence intensity as the readout. Two collections of invasive NmB isolates (n = 1,814, n = 109) were evaluated in the assay, with the smaller set also tested in hSBAs using individual and pooled human serum samples from young adults vaccinated with bivalent rLP2086. From these data, an analysis based on fHBP variant prevalence in the larger 1,814-isolate set showed that >91% of all meningococcal serogroup B isolates expressed sufficient levels of fHBP to be susceptible to bactericidal killing by vaccine-induced antibodies.IMPORTANCE Bivalent rLP2086 (Trumenba) vaccine, composed of two factor H binding proteins (fHBPs), was recently licensed for the prevention of N. meningitidis serogroup B (NmB) disease in individuals 10 to 25 years old in the United States. This study evaluated a large collection of NmB isolates from the United States and Europe by using a flow cytometric MEASURE assay to quantitate the surface expression of the vaccine antigen fHBP. We find that expression levels and the proportion of strains above the level associated with susceptibility in an hSBA are generally consistent across these geographic regions. Thus, the assay can be used to predict which NmB isolates are susceptible to killing in the hSBA and therefore is able to demonstrate an fHBP vaccine-induced bactericidal response. This work significantly advances our understanding of the potential for bivalent rLP2086 to provide broad coverage against diverse invasive-disease-causing NmB isolates.

In 2009, in the Active Bacterial Core surveillance sites, penicillin was not commonly used to treat meningococcal disease. This is likely because of inconsistent availability of antimicrobial susceptibility testing and ease of use of third-generation cephalosporins. Consideration of current practices may inform future meningococcal disease management guidelines.

Haemophilus haemolyticus (H. haemolyticus) has been recently discovered to have the potential to cause invasive disease. It is closely related to non-typeable Haemophilus influenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus A real-time PCR targeting purT (encoding phosphoribosylglycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR reaction; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus, and 92.8% sensitivity and 100% specificity for the identification of H. influenzae The dual-target testing scheme can be used for the diagnosis and surveillance of infection disease caused by H. haemolyticus and NT H. influenzae.

Neisseria meningitidis is an important cause of meningococcal disease globally. Sequence type (ST)-11 clonal complex (cc11) is a hypervirulent meningococcal lineage historically associated with serogroup C capsule and is believed to have acquired the W capsule through a C to W capsular switching event. We studied the sequence of capsule gene cluster (cps) and adjoining genomic regions of 524 invasive W cc11 strains isolated globally. We identified recombination breakpoints corresponding to two distinct recombination events within W cc11: a 8.4 kb recombinant region likely acquired from W cc22 including the sialic acid/ glycosyl-transferase gene, csw resulted in a C-->W change in capsular phenotype and a 13.7 kb recombinant segment likely acquired from Y cc23 lineage includes 4.5 kb of cps genes and 8.2 kb downstream of the cps cluster resulting in allelic changes in capsule translocation genes. A vast majority of W cc11 strains (497/524, 94.8%) retain both recombination events as evidenced by sharing identical or very closely related capsular allelic profiles. These data suggest that the W cc11 capsular switch involved two separate recombination events and that current global W cc11 meningococcal disease is caused by strains bearing this mosaic capsular switch.

Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824-135,982 for 5x Omni, and 168-6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0-99.9%, 97.5-99.9%, and 92.9-99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.

In this study, a multicenter evaluation of the Life Technologies TaqMan(R) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators.

Neisseria meningitidis is a leading bacterial cause of sepsis and meningitis globally with dynamic strain distribution over time. Beginning with an epidemic among Hajj pilgrims in 2000, serogroup W (W) sequence type (ST) 11 emerged as a leading cause of epidemic meningitis in the African 'meningitis belt' and endemic cases in South America, Europe, Middle East and China. Previous genotyping studies were unable to reliably discriminate sporadic W ST-11 strains in circulation since 1970 from the Hajj outbreak strain (Hajj clone). It is also unclear what proportion of more recent W ST-11 disease clusters are caused by direct descendants of the Hajj clone. Whole genome sequences of 270 meningococcal strains isolated from patients with invasive meningococcal disease globally from 1970 to 2013 were compared using whole genome phylogenetic and major antigen-encoding gene sequence analyses. We found that all W ST-11 strains were descendants of an ancestral strain that had undergone unique capsular switching events. The Hajj clone and its descendants were distinct from other W ST-11 strains in that they shared a common antigen gene profile and had undergone recombination involving virulence genes encoding factor H binding protein, nitric oxide reductase, and nitrite reductase. These data demonstrate that recent acquisition of a distinct antigen-encoding gene profile and variations in meningococcal virulence genes was associated with the emergence of the Hajj clone. Importantly, W ST-11 strains unrelated to the Hajj outbreak contribute a significant proportion of W ST-11 cases globally. This study helps illuminate genomic factors associated with meningococcal strain emergence and evolution.

BACKGROUND: Antimicrobial treatment and chemoprophylaxis of patients and their close contacts is critical to reduce the morbidity and mortality and prevent secondary cases of meningococcal disease. Through the 1990's, the prevalence of antimicrobial resistance to commonly used antimicrobials among Neisseria meningitidis was low in the United States. Susceptibility testing was performed to ascertain whether the proportions of isolates with reduced susceptibility to antimicrobials commonly used for N meningitidis have increased since 2004 in the United States. METHODS: Antimicrobial susceptibility testing was performed by broth microdilution on 466 isolates of N meningitidis collected in 2004, 2008, 2010, and 2011 from an active, population-based surveillance system for susceptibility to ceftriaxone, ciprofloxacin, penicillin G, rifampin, and azithromycin. The molecular mechanism of reduced susceptibility was investigated for isolates with intermediate or resistant phenotypes. RESULTS: All isolates were susceptible to ceftriaxone and azithromycin, 10.3% were penicillin G intermediate (range, 8% in 2008-16.7% in 2010), and <1% were ciprofloxacin, rifampin, or penicillin G resistant. Of the penicillin G intermediate or resistant isolates, 63% contained mutations in the penA gene associated with reduced susceptibility to penicillin G. All ciprofloxacin-resistant isolates contained mutations in the gyrA gene associated with reduced susceptibility. CONCLUSIONS:. Resistance of N meningitidis to antimicrobials used for empirical treatment of meningitis in the United States has not been detected, and resistance to penicillin G and chemoprophylaxis agents remains uncommon. Therapeutic agent recommendations remain valid. Although periodic surveillance is warranted to monitor trends in susceptibility, routine clinical testing may be of little use.

Neisseria meningitidis serogroup B (MnB) is a leading cause of bacterial meningitidis; however, as opposed to the disease state, MnB is most commonly associated with asymptomatic carriage in the nasopharyngeal cavity. Two vaccines are now licensed for the prevention of MnB disease; an additional possible benefit of these vaccines could be to indirectly protect against disease by disrupting nasopharyngeal carriage, e.g. herd protection. To investigate this potential, accurate diagnostic approaches are required to characterize MnB carriage isolates. In contrast to invasive meningococcal disease (IMD) isolates that can readily be serogrouped, carriage isolates often lack capsule expression making standard phenotypic assays untenable for strain characterization. Several antibody based methods were evaluated for their ability to serogroup isolates and compared to two genotyping methods (real-time PCR (rt-PCR) and whole genome sequencing (WGS) to identify which approach might most accurately ascertain the polysaccharide group associated with carriage isolates. WGS and rt-PCR were in agreement for 99% of IMD isolates, including those carrying coding sequences for MnB, MnC, MnW, and MnY, and the phenotypic methods correctly identified serogroups for between 69 and 98% of IMD isolates. In contrast, only 47% of carriage isolates were groupable by genotypic methods due to mutations within the capsule operon, and of the isolates identified by genotypic methods, ≤43% were serogroupable by any of the phenotypic methods tested. These observations highlight the difficulties in serogrouping and capsular genogrouping meningococcal carriage isolates. Based on our findings, WGS is the most suitable approach for the characterization of meningococcal carriage isolates.

BACKGROUND: Surveillance for acute flaccid paralysis with laboratory confirmation has been a key strategy in the global polio eradication initiative, and the laboratory platform established for polio testing has been expanded in many countries to include surveillance for cases of febrile rash illness to identify measles and rubella cases. Vaccine-preventable disease surveillance is essential to detect outbreaks, define disease burden, guide vaccination strategies and assess immunization impact. Vaccines now exist to prevent Japanese encephalitis (JE) and some etiologies of bacterial meningitis. METHODS: We evaluated the feasibility of expanding polio-measles surveillance and laboratory networks to detect bacterial meningitis and JE, using surveillance for acute meningitis-encephalitis syndrome in Bangladesh and China and acute encephalitis syndrome in India. We developed nine syndromic surveillance performance indicators based on international surveillance guidelines and calculated scores using supervisory visit reports, annual reports, and case-based surveillance data. RESULTS: Scores, variable by country and targeted disease, were highest for the presence of national guidelines, sustainability, training, availability of JE laboratory resources, and effectiveness of using polio-measles networks for JE surveillance. Scores for effectiveness of building on polio-measles networks for bacterial meningitis surveillance and specimen referral were the lowest, because of differences in specimens and techniques. CONCLUSIONS: Polio-measles surveillance and laboratory networks provided useful infrastructure for establishing syndromic surveillance and building capacity for JE diagnosis, but were less applicable for bacterial meningitis. Laboratory-supported surveillance for vaccine-preventable bacterial diseases will require substantial technical and financial support to enhance local diagnostic capacity.

BACKGROUND: The incidence of meningococcal disease is currently at historic lows in the United States; however, incidence remains highest among infants aged <1 year. With routine use of Haemophilus influenzae type b and pneumococcal vaccines in infants and children in the United States, Neisseria meningitidis remains an important cause of bacterial meningitis in young children. METHODS: Data were collected from active, population- and laboratory-based surveillance for N meningitidis conducted through Active Bacterial Core surveillance during 2006 through 2012. Expanded data collection forms were completed for infant cases identified in the surveillance area during 2006 through 2010. RESULTS: An estimated 113 cases of culture-confirmed meningococcal disease occurred annually among infants aged <1 year in the United States from 2006 through 2012, for an overall incidence of 2.74 per 100 000 infants. Among these cases, an estimated 6 deaths occurred. Serogroup B was responsible for 64%, serogroup C for 12%, and serogroup Y for 16% of infant cases. Based on the expanded data collection forms, a high proportion of infant cases (36/58, 62%) had a smoker in the household and the socioeconomic status of the census tracts where infant meningococcal cases resided was lower compared with the other Active Bacterial Core surveillance areas and the United States as a whole. CONCLUSIONS: The burden of meningococcal disease remains highest in young infants and serogroup B predominates. Vaccines that provide long-term protection early in life have the potential to reduce the burden of meningococcal disease, especially if they provide protection against serogroup B meningococcal disease.

BACKGROUND: Meningococcal conjugate vaccines (MenACWY) against serogroups A, C, W and Y are recommended for routine use in adolescents aged 11-18 years. Impact of these vaccines on meningococcal population structure in the US remained to be evaluated. METHODS: Meningococcal isolates from 2006-10 (post-MenACWY) collected through Active Bacterial Core surveillance (ABCs) were characterized; serogroup distribution and molecular features of these isolates were compared to previously published data on ABCs isolates from 2000-05 (pre-MenACWY). p values were generated using chi-squared statistics and exact methods. RESULTS: There was a significant change (p<0.05) in serogroup distribution among all age groups between the two periods. A small proportion of isolates has shown evidence of capsular switching in both periods. Between the two periods, significant changes were observed in the distribution of PorA, FetA, and strain genotypes among vaccine and non-vaccine serogroups. CONCLUSIONS: The population structure of U.S. meningococcal isolates is dynamic; some changes occurred over time but the basic structure remained. Vaccine-induced serogroup replacement was not observed, although a small proportion of isolates had undergone capsule switching possibly driven by non-vaccine mediated selection. Changes in the distribution of molecular features are likely due to horizontal gene transfer and changes in serogroup distribution.

In 2010, Burkina Faso became the first country to introduce meningococcal serogroup A conjugate vaccine (PsA-TT). During 2012, Burkina Faso reported increases in Neisseria meningitidis serogroup W, raising questions about whether these cases were a natural increase in disease or resulted from serogroup replacement after PsA-TT introduction. We analyzed national surveillance data to describe the epidemiology of serogroup W and genotyped 61 serogroup W isolates. In 2012, a total of 5,807 meningitis cases were reported through enhanced surveillance, of which 2,353 (41%) were laboratory confirmed. The predominant organism identified was N. meningitidis serogroup W (62%), and all serogroup W isolates characterized belonged to clonal complex 11. Although additional years of data are needed before we can understand the epidemiology of serogroup W after PsA-TT introduction, these data suggest that serogroup W will remain a major cause of sporadic disease and has epidemic potential, underscoring the need to maintain high-quality case-based meningitis surveillance after PsA-TT introduction.

BACKGROUND: Meningococcocal meningitis represents an important cause of mortality and morbidity in sub-Saharan countries. Confirmatory bacteriological or molecular diagnosis is essential for patient management/treatment and meningitis surveillance, but many laboratory tests are expensive and rarely available for low-income countries. A rapid diagnostic test (RDT) represents a valuable alternative to improve case management and surveillance. METHODS: A dipstick RDT developed in early 2000s that detects Neisseria meningitidis serogroups A, C, W and Y but for which a new conjugated antibody (L4-8) for the detection of serogroup A replaced the original K15-2 was assessed in the field by trained staff from health centres and district hospitals in Niger. The results were compared to those obtained in the reference laboratory and the sensitivity and specificity of RDTs were determined using conventional and real-time PCR assays as a gold standard. RESULTS: RDT results from field staff and the reference laboratory obtained for 2095 cerebrospinal fluid (CSF) specimens presented a strong concordance of 94% with Cohen's kappa coefficient of 0.88. The observed concordance between RDTs operated by staff from the reference laboratory vs combination of conventional and real-time PCR assays was 89% with Cohen's kappa coefficient of 0.76 indicating very good agreement. The theoretical overall sensitivity for RDT was 91.5% and the specificity 84.6%. CONCLUSIONS: RDT has proven to be relatively sensitive and specific for the detection of meningococcal serogroups A/C/Y/W. We confirmed that these RDTs can be reliably operated by trained but non-specialised staff in basic health facilities.

BACKGROUND: Recombinant vaccines containing factor H-binding protein (fHBP) have been developed for the purpose of protection from invasive meningococcal serogroup B disease. Neisseria meningitidis fHBP sequences can be divided into 2 genetically and immunologically distinct subfamilies (A and B); thus, cross protection is conferred within but not between subfamilies. A comprehensive understanding of fHBP epidemiology is required to accurately assess the potential vaccine impact when considering different vaccination implementation strategies. METHODS: Systematically collected invasive meningococcal serogroup B isolates from England, Wales, Northern Ireland, the United States, Norway, France and the Czech Republic were previously characterized for fHBP sequence. This study expanded the evaluation with additional meningococcal serogroup B disease isolates from Spain (n = 346) and Germany (n = 205). This expanded set (n = 1841), collected over a 6-year period (2001 to 2006), was evaluated for fHBP sequence and fHBP subfamily relative to patient age. RESULTS: All 1841 isolates contained fhbp. fHBP sequences from Spain and Germany fell within the previously described subfamilies, with 69% of isolates belonging to subfamily B and 31% to subfamily A; prevalent sequence variants were also similar. Stratification of data by age indicated that disease in infants <1 year of age was caused by a significantly higher proportion of isolates with fHBP subfamily A variants than that seen in adolescents and young adults 11-25 years (47.7% versus 19.5%, P < 0.0001, respectively). CONCLUSIONS: These observations highlight a difference in epidemiology of fHBP subfamilies in different age groups, with fHBP subfamily A strains causing more disease in vulnerable populations, such as infants, than in adolescents.

High molecular weight (Hmw) proteins 1 and 2, type IV pilin protein (PilA), outer-membrane protein P5 (OmpP5), Haemophilus protein D (Hpd) and Haemophilus adhesive protein (Hap) are surface proteins involved in the adherence of non-typeable Haemophilus influenzae. One hundred clinical isolates were evaluated for the presence of the genes encoding these proteins by PCR and for their adherence capacity (AC) to Detroit 562 nasopharyngeal cells (D562). The majority of isolates were from blood (77/100); other sites were also represented. Confluent D562 monolayers (1.2x10(5) cells per well) were inoculated with standardized minimal infective doses (m.o.i.) of 10(2), 10(3) or 10(4) c.f.u. per well. The AC was categorized as low (<10 %) or high (≥10 %) depending on the percentage of c.f.u. adhering per well. All the isolates evaluated showed adherence: 69/100 (69 %) demonstrated high adherence, while 31/100 (31 %) showed low adherence. Of all the genes evaluated, hmw1A and/or hmw2A were detected in 69/100 (69 %) of isolates. The presence of hmw1A and/or hmw2A was associated with increased adherence to D562 cells (P≤0.001). Dot immunoblots were performed to detect protein expression using mAbs 3D6, AD6 and 10C5. Among the high-adherence isolates (n = 69), 72 % reacted with 3D6 and 21 % with 10C5. Our data indicate that the absence of Hmw1 and/or Hmw2 was associated with decreased adherence to D562 cells.

BACKGROUND: College students living in residential halls are at increased risk of meningococcal disease. Unlike for serogroups prevented by quadrivalent meningococcal vaccines, public health response to outbreaks of serogroup B meningococcal disease is limited by lack of a US licensed vaccine. METHODS: In March 2010 we investigated a prolonged outbreak of serogroup B disease associated with a university. In addition to case ascertainment, molecular typing of isolates was performed to characterize the outbreak. We conducted a matched case-control study to examine risk factors for serogroup B disease. Five controls per case, matched by college year, were randomly selected. Participants completed a risk factor questionnaire. Data were analyzed using conditional logistic regression. RESULTS: Between January 2008 and November 2010, we identified 13 meningococcal disease cases (seven confirmed, four probable, and two suspected) among university students (ten) or university-linked persons (three). One student died. Ten cases were determined to be serogroup B. Isolates from six confirmed cases had an indistinguishable pulse-field gel electrophoresis pattern and belonged to sequence type ST-269, clonal complex 269. Factors significantly associated with disease were Greek Society membership (matched odds ratio [mOR] 15.0; p=0.03), >1 kissing partner (mOR 13.7; p=0.03) and attending bars (mOR 8.1; p=0.04). CONCLUSIONS: The outbreak was associated with a novel serogroup B strain (CC269) and risk factors indicative of increased social mixing. Control measures were appropriate but limited by lack of vaccine. Understanding serogroup B transmission in college and other settings will help inform use of serogroup B vaccines currently under consideration for licensure.

Bordetella holmesii, a human pathogen, can confound the diagnosis of respiratory illness caused by Bordetella pertussis. We present the draft genome sequences of two B. holmesii isolates, one from blood, F627, and one from the nasopharynx, H558. Interestingly, important virulence genes that are present in B. pertussis are not found in B. holmesii.

BACKGROUND: Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard. METHODS AND FINDINGS: CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously. RESULTS: Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF. CONCLUSION: Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.

Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate (M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the M16917 genome sequence, which led to the introduction of an approximately 2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.

BACKGROUND: The serogroup A conjugate meningococcal vaccine, MenAfriVac, was introduced in mass vaccination campaigns in December 2010 in Burkina Faso, Mali and Niger. In the coming years, vaccination will be extended to other African countries at risk of epidemics. To document the molecular characteristics of disease-causing meningococcal strains circulating in the meningitis belt of Africa before vaccine introduction, the World Health Organization Collaborating Centers on Meningococci in Europe and United States established a common strain collection of 773 isolates from cases of invasive meningococcal disease collected between 2004 and 2010 from 13 sub-Saharan countries. METHODOLOGY: All isolates were characterized by multilocus sequence typing, and 487 (62%) were also analyzed for genetic variation in the surface antigens PorA and FetA. Antibiotic susceptibility was tested for part of the collection. PRINCIPAL FINDINGS: Only 19 sequence types (STs) belonging to 6 clonal complexes were revealed. ST-5 clonal complex dominated with 578 (74.8%) isolates. All ST-5 complex isolates were remarkably homogeneous in their PorA (P1.20,9) and FetA (F3-1) and characterized the serogroup A strains which have been responsible for most epidemics during this time period. Sixty-eight (8.8%) of the 773 isolates belonged to the ST-11 clonal complex which was mainly represented by serogroup W135, while an additional 38 (4.9%) W135 isolates belonged to the ST-175 complex. Forty-eight (6.2%) serogroup X isolates from West Africa belonged to the ST-181 complex, while serogroup X cases in Kenya and Uganda were caused by an unrelated clone, ST-5403. Serogroup X, ST-181, emerged in Burkina Faso before vaccine introduction. CONCLUSIONS: In the seven years preceding introduction of a new serogroup A conjugate vaccine, serogroup A of the ST-5 clonal complex was identified as the predominant disease-causing strain.

BACKGROUND: Since the introduction of effective vaccines, the incidence of invasive Haemophilus influenzae type b (Hib) disease among children <5 years of age has decreased by 99% in the United States. In response to a limited vaccine supply that began in 2007, Hib booster doses were deferred for 18 months. METHODS: We reviewed national passive and active surveillance (demographic and serotype) and vaccination status data for invasive H. influenzae disease in children aged <5 years before (1998-2007) and during (2008-2009) the vaccine shortage years to assess the impact of the vaccine deferral on Hib disease. We estimated the average annual number of Hib cases misclassified as unknown (not completed or missing) serotype. RESULTS: From 1998 to 2007 and 2008 to 2009, the annual average incidence of Hib disease per 100,000 population was 0.2 and 0.18, respectively; no significant difference in incidence was found by age group, gender, or race. Among Hib cases in both time periods, most were unvaccinated or too young to have received Hib vaccine. During 2001 to 2009, there were <53 Hib cases per year, with an estimated 6 to 12 Hib cases misclassified as unknown serotype. CONCLUSIONS: The booster deferral did not have a significant impact on the burden of invasive Hib disease in children <5 years of age. Continued surveillance and serotype data are important to monitor changes in Hib incidence, especially during vaccine deferrals. Hib booster deferral is a reasonable short-term approach to a Hib vaccine shortage.

BACKGROUND: An affordable, highly immunogenic Neisseria meningitidis serogroup A meningococcal conjugate vaccine (PsA-TT) was licensed for use in sub-Saharan Africa in 2009. In 2010, Burkina Faso became the first country to implement a national prevention campaign, vaccinating 11.4 million people aged 1-29 years. We analysed national surveillance data around PsA-TT introduction to investigate the early effect of the vaccine on meningitis incidence and epidemics. METHODS: We examined national population-based meningitis surveillance data from Burkina Faso using two sources, one with cases and deaths aggregated at the district level from 1997 to 2011, and the other enhanced with results of cerebrospinal fluid examination and laboratory testing from 2007 to 2011. We compared mortality rates and incidence of suspected meningitis, probable meningococcal meningitis by age, and serogroup-specific meningococcal disease before and during the first year after PsA-TT implementation. We assessed the risk of meningitis disease and death between years. FINDINGS: During the 14 year period before PsA-TT introduction, Burkina Faso had 148 603 cases of suspected meningitis with 17 965 deaths, and 174 district-level epidemics. After vaccine introduction, there was a 71% decline in risk of meningitis (hazard ratio 0.29, 95% CI 0.28-0.30, p<0.0001) and a 64% decline in risk of fatal meningitis (0.36, 0.33-0.40, p<0.0001). We identified a statistically significant decline in risk of probable meningococcal meningitis across the age group targeted for vaccination (62%, cumulative incidence ratio [CIR] 0.38, 95% CI 0.31-0.45, p<0.0001), and among children aged less than 1 year (54%, 0.46, 0.24-0.86, p=0.02) and people aged 30 years and older (55%, 0.45, 0.22-0.91, p=0.003) who were ineligible for vaccination. No cases of serogroup A meningococcal meningitis occurred among vaccinated individuals, and epidemics were eliminated. The incidence of laboratory-confirmed serogroup A N meningitidis dropped significantly to 0.01 per 100,000 individuals per year, representing a 99.8% reduction in the risk of meningococcal A meningitis (CIR 0.002, 95% CI 0.0004-0.02, p<0.0001). INTERPRETATION: Early evidence suggests the conjugate vaccine has substantially reduced the rate of meningitis in people in the target age group, and in the general population because of high coverage and herd immunity. These data suggest that fully implementing the PsA-TT vaccine could end epidemic meningitis of serogroup A in sub-Saharan Africa. FUNDING: None.

A new Group A meningococcal (Men A) conjugate vaccine, MenAfriVac, was prequalified by the World Health Organization (WHO) in June 2010. Because Burkina Faso has repeatedly suffered meningitis epidemics due to Group A Neisseria meningitidis special efforts were made to conduct a country-wide campaign with the new vaccine in late 2010 and before the onset of the next epidemic meningococcal disease season beginning in January 2011. In the ensuing five months (July-November 2010) the following challenges were successfully managed: (1) doing a large safety study and registering the new vaccine in Burkina Faso; (2) developing a comprehensive communication plan; (3) strengthening the surveillance system with particular attention to improving the capacity for real-time polymerase chain reaction (PCR) testing of spinal fluid specimens; (4) improving cold chain capacity and waste disposal; (5) developing and funding a sound campaign strategy; and (6) ensuring effective collaboration across all partners. Each of these issues required specific strategies that were managed through a WHO-led consortium that included all major partners (Ministry of Health/Burkina Faso, Serum Institute of India Ltd., UNICEF, Global Alliance for Vaccines and Immunization, Meningitis Vaccine Project, CDC/Atlanta, and the Norwegian Institute of Public Health/Oslo). Biweekly teleconferences that were led by WHO ensured that problems were identified in a timely fashion. The new meningococcal A conjugate vaccine was introduced on December 6, 2010, in a national ceremony led by His Excellency Blaise Compaore, the President of Burkina Faso. The ensuing 10-day national campaign was hugely successful, and over 11.4 million Burkinabes between the ages of 1 and 29 years (100% of target population) were vaccinated. African national immunization programs are capable of achieving very high coverage for a vaccine desired by the public, introduced in a well-organized campaign, and supported at the highest political level. The Burkina Faso success augurs well for further rollout of the Men A conjugate vaccine in meningitis belt countries.

We report seven cases of Haemophilus haemolyticus invasive disease detected in the United States, which were previously misidentified as non-typeable Haemophilus influenzae (Hi). All cases had different symptoms and presentations. Our study suggests that a testing scheme that includes reliable PCR assays and standard microbiological methods should be used in order to improve H. haemolyticus identification.

PCR detecting the protein D (hpd) and fuculose kinase (fucK) genes showed high sensitivity and specificity for identifying Haemophilus influenzae and differentiating it from H. haemolyticus. Phylogenetic analysis using the 16S rRNA gene demonstrated two distinct groups for H. influenzae and H. haemolyticus.

Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect Nm ctrA, Hi hpd and Sp lytA, and serogroup-specific genes in the cap locus for Nm serogroups A, B, C, W135, X and Y. However, the assay sensitivity for serogroups B, W135 and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays B-synD, W-synG, and Y-synF showed 100% specificity for detecting Nm species, with high sensitivity [B-synD, 99%(75/76); W-synG, 97%(38/39); and Y-synF, 100%(66/66)]. The lower limit of detection (LLD) was 9, 43 and 10 copies/reaction for B-synD, W-synG, and Y-synF assays, respectively, a significant improvement compared to the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of: (i) Nm ctrA, Hi hpd and Sp lytA (NHS); (ii) Nm serogroups A, W135 and X (AWX), and (iii) Nm serogroups B, C and Y (BCY). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for singleplex. Pairwise comparison of real-time PCR between multiplex and singleplex showed that cycle threshold values of the multiplex were similar to those for singleplex. There were no substantial differences in the sensitivity and specificity between these multiplex and singleplex real-time PCR assays.

Alexander Langmuir, founder of the CDC Epidemic Intelligence Service (EIS), was quoted in the early 1960s instructing incoming EIS officers that the only need for the laboratory in an outbreak investigation was to "prove their conclusions were right." Understandably, this was not well received by the CDC Laboratory Branch. However, Langmuir's point was not to denigrate the laboratory but to emphasize the power of an investigation based on a solid clinical case definition and established field epidemiologic principles. In truth, in 1960, when CDC assumed responsibility for publishing MMWR, the laboratory provided little added value in many investigations, except to confirm "what the etiologic agent wasn't." Existing diagnostic laboratory procedures for infectious and noninfectious diseases of public health importance were reasonably reliable but basic and laborious. For diagnosis of many diseases and conditions, no laboratory procedures existed. Since 1961, advances in molecular sciences, analytical chemistry, and technology have revolutionized the public health laboratory investigative capacity, capability, and specificity and have emphasized the importance of more independent laboratory research. The term "molecular epidemiology" is widely applied, and the number of diseases for which laboratory diagnoses are available today is substantially larger. This article describes the principles and practices of the state-of-the-art public health laboratory in 1961 and provides examples of scientific, technologic, and strategic advances since that time that characterize the still evolving public health laboratory of the 21st century. | | Browsing through MMWR, volume 10, week 1, January 13, 1961, provides insight into the public health laboratory of 1961 and the topics of most interest and visibility at that time. Subsequently, progress and contributions made by the public health laboratories are provided in a more detailed account by using several illnesses and conditions of public health importance as examples. They span both infectious and noninfectious arenas. Some were listed in the first MMWR summary, but some were not under consideration in 1961 or were yet to be discovered.

BACKGROUND: With the introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines, there has been a dramatic reduction of Hib disease in young children and the epidemiological trends of invasive H. influenzae have shifted. METHODS: Data were collected from active surveillance for invasive H. influenzae disease conducted through Active Bacterial Core surveillance sites during 1989-2008. RESULTS: During 1999-2008, the estimated mean annual incidence of H. influenzae infection was 1.62 cases per 100,000 population; 15.3% of cases were fatal. Incidence was higher among adults aged ≥65 years, compared with other age groups. The largest burden of disease among children aged <5 years was in infants aged <1 year; many of these cases occurred during the first month of life in preterm or low-birth weight infants. An estimated 10% of the total burden of disease among children aged <5 years occurred in American Indian and Alaska Native children. During 1989-2008, 7559 cases of H. influenzae disease were reported from Active Bacterial Core surveillance sites. Small increases in the incidence of serotypes a, e, and f were observed during 1989-2008. The largest of these increases was in serotype f and was primarily among adults aged ≥18 years. CONCLUSIONS: Since the introduction of Hib conjugate vaccines, the incidence of invasive disease caused by H. influenzae in the United States has decreased dramatically; however, a considerable burden of non-Hib disease is still present in the oldest and youngest age groups. There is no evidence of substantial replacement disease with non-b serotypes in young children in the United States.