Sara Thulin Hedberg

Prevalence and sequence variations of the genes encoding the five antigens included in the novel 5CVMB vaccine covering group B meningococcal disease

During the recent years, projects are in progress for designing broad-range non-capsular-based meningococcal vaccines, covering also serogroup B isolates. We have examined three genes encoding antigens (NadA, GNA1030 and GNA2091) included in a novel vaccine, i.e. the 5 Component Vaccine against Meningococcus B (5CVMB), in terms of gene prevalence and sequence variations. These data were combined with the results from a similar study, examining the two additional antigens included in the 5CVMB (fHbp and GNA2132). nadA and fHbp v. 1 were present in 38% (n=36), respectively 71% (n=67) of the isolates, whereas gna2132, gna1030 and gna2091 were present in all the Neisseria meningitidis isolates tested (n=95). The level of amino acid conservation was relatively high in GNA1030 (93%), GNA2091 (92%), and within the main variants of NadA and fHbp. GNA2132 (54% of the amino acids conserved) appeared to be the most diversified antigen. Consequently, the theoretical coverage of the 5CVMB antigens and the feasibility to use these in a broad-range meningococcal vaccine is appealing.

Antibiotic Susceptibility and Characteristics of Neisseria meningitidis Isolates from the African Meningitis Belt, 2000 to 2006: Phenotypic and Genotypic Perspectives

ABSTRACT Up-to-date information regarding the antibiotic susceptibility of Neisseria meningitidis strains from African countries is highly limited. Our aim was to comprehensively describe the antibiotic susceptibilities of a selection of N. meningitidis isolates recovered between 2000 and 2006 from 18 African countries, mainly those within the meningitis belt. Susceptibilities to 11 antibiotics were determined using Etest for 137 N. meningitidis isolates (stringently selected from 693 available isolates). The isolates were also characterized by serogrouping, multilocus sequence typing, genosubtyping, and penA allele identification. All N. meningitidis isolates were susceptible to ceftriaxone, chloramphenicol, and ciprofloxacin. No isolate produced β-lactamase. Only three isolates (2%) displayed reduced susceptibility to penicillin G. The two isolates with the highest penicillin G MICs were the only isolates showing reduced susceptibility to ampicillin and cefuroxime. One of these isolates was also resistant to penicillin V. One percent of isolates displayed reduced susceptibility to rifampin, while 52% of the isolates were resistant to tetracycline, 74% were resistant to erythromycin, and 94% were resistant to sulfadiazine. The MICs of rifampin and tetracycline seemed to be associated with the serogroup of the isolates. In total, 18 sequence types (STs), 10 genosubtypes, and 8 different penA alleles were identified; the most common were ST-7, P1.20,9,35-1, and penA4, respectively. A high level of correlation was found between ST, genosubtype, and penA allele. In conclusion, N. meningitidis isolates from the African meningitis belt remain highly susceptible to the antibiotics used. Regarding β-lactam antibiotics, rare isolates showed a reduced susceptibility to penicillins, but the expanded-spectrum cephalosporins are not affected at present.

Real‐time PCR detection of five prevalent bacteria causing acute meningitis

Acute bacterial meningitis is life-threatening (1–3) with an incidence of four to six cases per 100 000 adults in developed countries (4–7). Correct and rapid aetiological diagnosis is crucial for early optimal therapy. Neisseria meningitidis and Streptococcus pneumoniae are responsible for about 80% of the cases of acute bacterial meningitis in countries with general vaccination against Haemophilus influenzae type b (4). In addition, several other bacteria can cause meningitis, such as Streptococcus agalactiae (Group B streptococci; GBS), Listeria monocytogenes and H. influenzae. More rarely, Escherichia coli, Klebsiella pneumoniae, staphylococci, salmonella, enterococci and others are causes. Molecular methods offer rapid diagnosis of acute bacterial meningitis (8, 9). A limitation has been that cultured bacteria normally are needed for antibiotic susceptibility testing. Lately several molecular methods have, however, been developed to identify antibiotic resistance, such as sequencing of the penA gene in N. meningitidis (10, 11). The aims of the present study were to develop a real-time PCR assay for rapid and specific detection of five prevalent bacteria causing acute meningitis, i.e. N. meningitidis, H. influenzae, S. pneumoniae, S. agalactiae and L. monocytogenes, as well as general species designation using partial 16S rRNA gene sequencing. The method was developed and optimized using five different reference strains, i.e. N. meningitidis MC58 (12), S. pneumoniae CCUG 36696, H. influenzae ATCC 10211, S. agalactiae CCUG 4208 and L. monocytogenes CCUG 51681. As a positive preparation control a Staphylococcus haemolyticus isolate, CCUG 55899, was included. The PCR method was tested using bacterial isolates of relevant species (n = 416, see Table 1). For evaluation of the method, 18 clinical culture-positive frozen cerebrospinal fluid (CSF) samples collected between 1999 and 2008 were examined, i.e. N. meningitidis n = 3, S. pneumoniae n = 7, H. influenzae n = 1, S. agalactiae n = 2 and other species n = 5 (E. coli n = 1, S. aureus n = 1, coagulase-negative staphylococci n = 3). All fresh consecutive culture-negative CSF samples referred to the laboratory during November 2007 to September 2008, with a clinical query of acute bacterial meningitis were also included (n = 82). Isolation of DNA from the bacterial isolates was performed using the MagNA Pure LC ⁄Compact (Roche Diagnostics GmbH, Mannheim, Germany) or QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA from the clinical CSF samples was prepared using MagNA Pure Compact. To improve lysis a prepreparation step with 100 units Mutanolysin (Sigma-Aldrich, St Louis, MO, USA) was added to 200 ll of a sample, and incubated at 37 C for 30 min. Segments of highly species-associated genes of N. meningitidis (ctrA), S. pneumoniae (lytA), H. influenzae (p6), S. agalactiae (cfb) and L. monocytogenes (hly), as well as the 16S rRNA gene (universal region 2-3), were used as targets (see Table 2). The genes were amplified using the LightCycler 2.0 system (Roche Diagnostics GmbH) with probe detection [95 C 10 min, 45 cycles: 95 C 5 s, 60 C 20 s (single fluorescence measurement), 72 C 8 s]. Each PCR mixture (20 ll) contained 2 ll of LightCycler Faststart Reaction Mix Hybridisation Probes (Roche Diagnostics GmbH), MgCl2, primers, probes (concentrations listed in Table 2) and 5 ll of DNA template. As positive PCR controls for the target specific PCRs, DNA preparations (100–1000 bacterial genome ⁄PCR) of the five different reference strains were used. A negative preparation control (containing only kit reagents) was used for each reaction mix. For the 16S rRNA gene PCR four controls were used; one positive (S. haemolyticus) and one negative preparation control (see above), as well as two PCR controls of sterile H2O and DNA preparation of negative CSF. All fresh consecutive culturenegative samples were tested for inhibition by diluting at a ratio of 1:10 and by adding S. pneumoniae DNA (1 ll added to 4 ll sample). APMIS 117: 856–86

Multicenter Study for Defining the Breakpoint for Rifampin Resistance in Neisseria meningitidis by rpoB Sequencing

ABSTRACT Identification of clinical isolates of Neisseria meningitidis that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number (n = 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of ≥0.25 mg/liter (n = 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of <0.25 mg/liter were also examined (n = 231). Typing of isolates was performed, and a 660-bp DNA fragment of the rpoB gene was sequenced. Sequences differing by at least one nucleotide were defined as unique rpoB alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of >1 mg/liter possessed rpoB alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of ≤1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of ≤1 mg/liter. The rpoB allele sequence and isolate data have been incorporated into the PubMLST Neisseria database (http://pubmlst.org/neisseria/ ). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates.

Genome-Based Characterization of Emergent Invasive Neisseria meningitidis Serogroup Y Isolates in Sweden from 1995 to 2012

ABSTRACT Invasive meningococcal disease (IMD) caused by Neisseria meningitidis serogroup Y has increased in Europe, especially in Scandinavia. In Sweden, serogroup Y is now the dominating serogroup, and in 2012, the serogroup Y disease incidence was 0.46/100,000 population. We previously showed that a strain type belonging to sequence type 23 was responsible for the increased prevalence of this serogroup in Sweden. The objective of this study was to investigate the serogroup Y emergence by whole-genome sequencing and compare the meningococcal population structure of Swedish invasive serogroup Y strains to those of other countries with different IMD incidence. Whole-genome sequencing was performed on invasive serogroup Y isolates from 1995 to 2012 in Sweden (n = 186). These isolates were compared to a collection of serogroup Y isolates from England, Wales, and Northern Ireland from 2010 to 2012 (n = 143), which had relatively low serogroup Y incidence, and two isolates obtained in 1999 in the United States, where serogroup Y remains one of the major causes of IMD. The meningococcal population structures were similar in the investigated regions; however, different strain types were prevalent in each geographic region. A number of genes known or hypothesized to have an impact on meningococcal virulence were shown to be associated with different strain types and subtypes. The reasons for the IMD increase are multifactorial and are influenced by increased virulence, host adaptive immunity, and transmission. Future genome-wide association studies are needed to reveal additional genes associated with serogroup Y meningococcal disease, and this work would benefit from a complete serogroup Y meningococcal reference genome.

Surveillance of invasive Neisseria meningitidis with a serogroup Y update, Sweden 2010 to 2012

An increase of invasive meningococcal disease caused by Neisseria meningitidis serogroup Y has been noted in Sweden since 2005, and to a lower extent throughout Europe. The present study describes the epidemiology of invasive N. meningitidis isolates in Sweden in the period between 2010 and 2012, with a focus on serogroup Y. We also aimed to find an optimal molecular typing scheme for both surveillance and outbreak investigations. All invasive N. meningitidis isolates in Sweden during the study period (n=208) were genetically characterised. Serogroup Y predominated with 22/57, 31/61 and 44/90 of all invasive isolates (incidence 0.23, 0.33 and 0.46 per 100,000 population) in 2010, 2011 and 2012 respectively. In each of these years, 15/22, 22/31 and 19/44 of serogroup Y isolates were genetically clonal (Y: P1.5–2,10–1,36–2: F4–1: ST-23(cc23), ‘porB allele 3–36, fHbp allele 25 and penA allele 22). Our findings further support those of others that currently recommended FetA typing could be replaced by FHbp. Moreover, in line with a previous study that we conducted, the current results indicate that highly variable multilocus variable-number tandem repeat analysis (HV-MLVA) can be used as a first-hand rapid method for small outbreak investigations.

Evaluation of the BD MAX™ Enteric Parasite Panel for clinical diagnostics

ABSTRACT We compared the performance of the BD Max enteric parasite panel to routine microscopy and an in-house PCR for the detection of Giardia intestinalis, Entamoeba histolytica, and Cryptosporidium spp. The enteric parasite panel showed good specificity for all targets and good sensitivity for E. histolytica and Cryptosporidium spp. Sensitivity for G. intestinalis with the BD Max enteric parasite panel was equivalent to that with microscopy.

Target gene sequencing to define the susceptibility of Neisseria meningitidis to ciprofloxacin

ABSTRACT Meningococcal gyrA gene sequence data, MICs, and mouse infection were used to define the ciprofloxacin breakpoint for Neisseria meningitidis. Residue T91 or D95 of GyrA was altered in all meningococcal isolates with MICs of ≥0.064 μg/ml but not among isolates with MICs of ≤0.032 μg/ml. Experimental infection of ciprofloxacin-treated mice showed slower bacterial clearance when GyrA was altered. These data suggest a MIC of ≥0.064 μg/ml as the ciprofloxacin breakpoint for meningococci and argue for the molecular detection of ciprofloxacin resistance.

Evaluation of molecular typing methods for identification of outbreak‐associated Neisseria meningitidis isolates

It is essential in an outbreak investigation that strain characterization of Neisseria meningitidis is performed in a rapid and accurate manner. This study evaluated two new molecular typing methods, multiple‐locus variable number tandem repeat analysis (MLVA) and repetitive sequence‐based PCR (rep‐PCR) (DiversiLab; bioMérieux) and compared them with current recommended methodologies. This retrospective study included 36 invasive N. meningitidis serogroup C isolates collected in Sweden 2001 through 2009 and previously subjected to outbreak investigation. All strains were typed with highly variable‐MLVA (HV‐MLVA) and rep‐PCR. The isolates were further characterized by multilocus sequence typing (MLST) and sequencing of the fetA, fHbp, penA, porA and porB genes. The results showed that HV‐MLVA had the highest index of diversity (0.99) and rep‐PCR had the highest congruence (40%) with the currently recommended typing methods. The HV‐MLVA correlated best to the spatiotemporal connections and had the overall highest Adjusted Wallace coefficients, suggesting that HV‐MLVA can predict the results of the other typing methods in the study. We therefore suggest that after initial confirmation of species, serogroup and genosubtype, HV‐MLVA should be used as the most discriminatory method for first hand investigation of N. meningitidis serogroup C isolates.

Antibiotic susceptibility of invasive Neisseria meningitidis isolates from 1995 to 2008 in Sweden : the meningococcal population remains susceptible

Abstract The susceptibility to 7 antibiotics was determined for all Swedish invasive Neisseria meningitidis isolates from 1995 to 2008 (N = 717). In general, these remain highly susceptible to the antibiotics recommended for use. Accordingly, penicillin G remains effective for the treatment of invasive meningococcal disease and ciprofloxacin appropriate for prophylaxis.