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Objectives A seroprevalence study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was conducted in a high-incidence area located in northeastern Italy. Methods All citizens above 10 years of age resident in five municipalities of the Autonomous Province of Trento, with the highest incidence of coronavirus disease 2019 (COVID-19) cases, were invited to participate in the study. Among 6098 participants, 6075 sera and a standardized questionnaire administered face-to-face were collected between 5 May and 15 May 2020 and examined. Symptomatic individuals and their family contacts were tested by RT-PCR. Anti-SARS-CoV-2 antibodies were detected using an Abbott SARS-CoV-2 IgG assay, which was performed on the Abbott Architect i2000SR automated analyser. Seroprevalence was calculated as the proportion of positive results among the total number tested. A multivariable logistic regression model was performed to assess the relationship between seropositive versus seronegative individuals for a set of explanatory variables. Results A total of 1402 participants were positive for IgG antibodies against SARS-CoV-2, with a prevalence of 23.1% (1402/6075). The highest prevalence was found in the age class 40–49 years. Overall, 34.4% (2096/6098) of the participants reported at least one symptom. The ratio between reported cases identified by molecular test and those with seropositive results was 1:3, with a maximum ratio of about 1:7 in the age group 70 years old. The infection fatality rate was 2.5% (35/1402). Among the symptoms, anosmia and ageusia were strongly associated with seropositivity. Conclusions The estimated seroprevalence of 23% was three-fold higher than the number of cases reported in the COVID-19 Integrated Surveillance data in the study area. This may be explained in part by a relatively high number of individuals presenting mild or no illness, especially those of younger age, and people who did not seek medical care or testing, but who may contribute to virus transmission in the community.

BackgroundTyping ofNeisseria meningitidisisolates is crucial for the surveillance of invasive meningococcal disease (IMD). We performed a molecular epidemiology study ofN.meningitidisserogroup B (MenB) causing IMD in Italy between 2014 and 2017 to describe circulating strains belonging to this serogroup, with particular regards to the two factor H-binding protein (FHbp) subfamilies present in the bivalent MenB vaccine.Materials and methodsA total of 109 culture positive and 46 culture negative MenB samples were collected within the National Surveillance System (NSS) of IMD in Italy and molecularly analyzed by conventional methods.ResultsOverall, 71 MenB samples showed the FHbp subfamily A and 83 the subfamily B. The subfamily variants were differently distributed by age. The most frequent variants, A05 and B231, were associated with cc213 and cc162, respectively. All MenB with the FHbp A05 variant displayed the PorA P1.22,14 and 85.7% of them the FetA F5-5. The majority of MenB with the FHbp B231 variant showed the PorA P1.22,14 (65.4%) and 84.6%, the FetA F3-6.ConclusionMenB circulating in Italy were characterized by a remarkable association between clonal complex and FHbp variants, although a high degree of genetic diversity observed over time. A dynamic trend in clonal complexes distribution within MenB was detected. Our results stress the importance of continued meningococcal molecular surveillance to evaluate the potential vaccine coverage of the available MenB vaccines.

Background Neisseria meningitidis can cause sporadic cases and outbreaks of invasive disease, including meningitis and sepsis. The meningococcal serogroup C (MenC) is the second most common serogroup in Italy after MenB. In this study we have estimated the reproductive numbers and the demographic reconstruction on the genomes of invasive N. meningitidis C:P1.5–1,10–8:F3–6:ST–11(cc11) strains isolated in Italy in 2012 – 2017, a period that includes the outbreak in Tuscany. Methods The genomes of N. meningitidis were sequenced using the Illumina MiSeq platform, through the whole genome sequencing (WGS) method and were analyzed by the core genome MLST (cgMLST) approach, using the BIGSdb Genome Comparator tool implemented on the PubMLST website. A Bayesian method was applied to study population dynamics across the entire N. meningitidis dataset. The basic reproduction number R0, which indicates the average number of secondary cases generated by a single primary case, was calculated using a Bayesian method, on the dataset and on the two subsets. The effective reproduction number R(t), defined as the average number of secondary cases per infectious case in a population, made up of susceptible and non-susceptible hosts was studied on the Tuscany dataset, with a Bayesian method. Results An increase in the effective number of the N. meningitidis infections was observed between 2013 and 2016. The estimated R0 parameter was 1.31 (95% HPD: 1.03 – 1.64), 1.22 (95% HPD: 0.90 – 1.64) and 1.4 (95% HPD: 0.91 – 1.9) for the entire dataset, first and second subset, respectively. The BDSKY estimated an initial R(t) of about 2.0 (95% HPD: 0.04 – 5.0), which showed a growing trend at the end of 2014, reaching an average value of 3.22 in 2015, and then declining below 1 from the year 2016. Conclusion Monitoring the effective reproduction number can help to inform future vaccination strategies. The increase in the reproductive number for the Tuscany dataset, was consistent with the amplification event that led to the Tuscany outbreak. Subsequently, the intervention that led to the decline of the cases was followed, suggesting a high effectiveness of the vaccination campaign.

Neisseria meningitidis is one of the few commensal bacteria that can even cause large epidemics of invasive meningococcal disease (IMD). N. meningitis serogroup C belonging to the hypervirulent clonal complex 11 (cc11) represents an important public health threat worldwide. We reconstructed the dispersal patterns of hypervirulent meningococcal strains of serogroup C:cc11 by phylogenomic time trees., Neisseria meningitidis is one of the few commensal bacteria that can even cause large epidemics of invasive meningococcal disease (IMD). N. meningitis serogroup C belonging to the hypervirulent clonal complex 11 (cc11) represents an important public health threat worldwide. We reconstructed the dispersal patterns of hypervirulent meningococcal strains of serogroup C:cc11 by phylogenomic time trees. In particular, we focused the attention on the epidemic dynamics of C:P1.5.1,10-8:F3-6;ST-11(cc11) meningococci causing outbreaks, as occurred in the Tuscany region, Italy, in 2015 to 2016. A phylogeographic analysis was performed through a Bayesian method on 103 Italian and 208 foreign meningococcal genomes. The C:P1.5.1,10-8:F3-6;ST-11(cc11) genotype dated back to 1995 (1992 to 1998) in the United Kingdom. Two main clades of the hypervirulent genotype were identified in Italy. The Tuscany outbreak isolates were included in different clusters in a specific subclade which originated in the United Kingdom around 2011 and was introduced in Tuscany in 2013 to 2014. In this work, phylogeographic analysis allowed the identification of multiple introductions of these strains in several European countries and connections with extra-European areas. Whole-genome sequencing (WGS) combined with phylogeography enables us to track the dissemination of meningococci and their transmission. The C:P1.5.1,10-8:F3-6;ST-11(cc11) genotype analysis revealed how a hypervirulent strain may be introduced in previously naïve areas, causing a large and long-lasting outbreak.

In the last decade, the incidence of invasive meningococcal disease (IMD) in Croatia remained stable at approximately 1 case per 100 000 inhabitants, affecting mainly children aged ≤5 years. We report the molecular characterization of meningococci causing IMD occurring from June 2009 to January 2014 in Croatia. Genomic DNA from 50 clinical isolates was analysed for serogroup, multilocus sequence typing and allele type of the two outer membrane protein genes, porA and the iron-regulated fetA. Furthermore, 22 of them were characterized by using whole-genome sequencing to define the meningococcal vaccine four-component meningococcal serogroup B (4CMenB) antigen genes factor H-binding protein (fHbp), Neisseria heparin-binding antigen (nhba) and Neisseria adhesin A (nadA) and the antimicrobial target resistance genes for penicillin (penicillin binding protein 2, penA), ciprofloxacin (DNA gyrase subunit A, gyrA) and rifampicin (β-subunit of RNA polymerase, rpoB). The Etest was used to phenotypically determine the antimicrobial susceptibility of isolated meningococci. The main serogroup/clonal complex combinations were MenB cc41/44, MenC/cc11, MenW/cc174 and MenY/cc23. PorA P1.7-2, FetA F5-5 and F1-5 were the most represented through the serogroups. Meningococci with decreased susceptibility to penicillin (38.9 %) and one strain resistant to ciprofloxacin were identified. Forty-two percent of MenB showed the presence of at least one of the 4CMenB vaccine antigens (fHbp, NHBA, NadA and PorA). Our findings highlight the genetic variability of meningococci causing IMD in Croatia, especially for the serogroup B. Molecular-based characterization of meningococci is crucial to enhance IMD surveillance and to better plan national immunization programmes.

Summary Objectives To describe a spatio-temporal cluster of invasive meningococcal disease (IMD) due to serogroup C meningococci, occurred in a restricted area of Tuscany between January and October 2015, and the results of whole genome sequencing (WGS). Methods Surveillance activities and public health measures were implemented in the Region. Bacterial isolates from IMD cases were characterized by the National Reference Laboratory of the Istituto Superiore di Sanita (ISS), and WGS was performed on available strains. The kSNP software was used to identify core genome SNPs. Results Overall, 28 IMD cases due to meningococcus C were identified up to 31st October, 2015. Of them, 26 were due to meningococcus C:P1.5-1,10-8: F3-6:ST-11 (cc11) and 2 to C:P1.5-1,10-8: F3-6:ST-2780 (cc11). WGS of 13 meningococci isolated during the outbreak occurred in Tuscany in 2015 showed higher similarity when compared with those of 47 C: P1.5-1,10-8: F3-6:ST-11 (cc11) invasive strains from sporadic cases previously detected in Italy. Conclusions A highly aggressive meningococcal C strain was involved in the cluster of severe IMD occurred in Tuscany, a Region with high vaccine coverage among children. Whether this was due to low herd immunity related to the short duration of vaccine protection needs further investigation.

PorB is a pan-Neisserial major outer membrane protein with a trimeric β-barrel structure. Each monomer presents eight periplasmic turns and eight surface exposed loop regions with sequence variability. PorB induces activation of host cell responses via a TLR2-dependent mechanism likely mediated by electrostatic interactions between TLR2 and PorB surface exposed loops. Variability in the loop amino acid sequence is known to influence cell responses to PorB in vitro, particularly for the residues in L5 and L7. In this work, the sequence of the porB gene and the electrostatic surface charges of PorB from 35 invasive meningococcal isolates belonging to the main clonal complexes identified in Italy and from five carriage genomes available on the website http://pubmlst.org/neisseria/ were examined. Analysis of the porB encoding regions from the invasive meningococci has identified four new alleles and a potential association between porB alleles, serogroup, and clonal complexes. Through computer-based modeling and analysis of the electrostatic surface charges of PorB from these strains, loop charge segregation between PorB from invasive serogroups B and C was observed. Specifically, loops 1, 4, and 7 were negatively charged and L2 and L8 were mostly neutral in serogroup B isolates, while an overall homogeneous positive surface charge was present in PorB from invasive serogroup C strains. A higher PorB sequence variability was observed among carriage genomes, and a general prevalence of negative loop surface charges. The surface charge differences in PorB from serogroups B and C invasive and carriage strains may, in part, influence the outcomes of Neisseriae interactions with host cells. © 2016 IUBMB Life, 68(6):488-495, 2016.

Meningococcal disease incidence in Italy remains quite low in the overall population except for infants. Within a study on carriage isolates among high school students we aimed to define: i) the prevalence of carriage isolates, ii) the phenotypic and iii) the molecular features of meningococci by Whole Genome Sequencing (WGS). A total of 1697 pharyngeal samples from undergraduate students (age range 14–19 years) were collected from 2012 to 2013 from six larger cities in Italy. One hundred and twenty culture positive meningococci (7%) were analyzed. Carriage isolates were sent to the National Reference Laboratory for invasive meningococcal disease (IMD) for PCR-based serogroup identification, Multilocus Sequence Typing, PorA and FetA typing. Moreover, factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA) were typed. Core genome MLST (cgMLST) was performed on a subsample of 75 carriage isolates. Capsule null locus (cnl) predominated (47%), followed by serogroup B (27%). The antimicrobial susceptibility profile revealed an high prevalence of reduced susceptibility to penicillin G (54%) and a full susceptibility to ceftriaxone, ciprofloxacin and rifampicin. Carriage isolates presented a high genetic diversity: the clonal complexes (ccs) cc1136, cc198 and cc41/44, were the predominant. An high heterogeneity was also observed for PorA and FetA types. The fhbp and nhba genes were identified in all the carriage isolates; only 5% of the carriage isolates presented the nadA gene. The core genome MLST analysis revealed that the majority of the cnl isolates clustered in a distinct group. The evidence gathered during this study provides the estimate of carriage isolates in high school students in a non-epidemic period in Italy that was lower than expected. Moreover, the highest proportion of carriage isolates were cnl and, overall, they were molecular heterogeneous.

The aim of this study was to evaluate the antimicrobial susceptibilities of 866 Neisseria meningitidis invasive strains during 11 years of surveillance in Italy. Two and six strains were resistant to ciprofloxacin and rifampin, respectively. Forty-five percent were penicillin intermediate, associated with hypervirulent serogroup C clonal complex 11. All of the strains were susceptible to cephalosporins.

BackgroundIn 2015-2016, a cross-sectional carriage survey was performed in Tuscany Region, Italy, during an outbreak of invasive meningococcal disease due to Neisseria meningitidis serogroup C clonal complex 11 (MenC:cc11). This study aims to evaluate the genomic profile of meningococcal carriage isolates collected during the survey.MethodsWhole-genome sequencing (WGS) was performed using Illumina MiSeq on 85 cultivated meningococcal carriage isolates received at the Dept. of Infectious Disease, National Institute of Health (Istituto Superiore di Sanità, ISS), as National Reference Laboratory (NRL) for Invasive Meningococcal Disease (IMD). De novo assembled genomes were scanned by the BIGSdb platform to assign: the genotypic profiles, the cgMLST, the vaccine antigen variants and allele types of antimicrobial resistance associated genes, together with denitrification pathway loci.ResultsCapsule null and non-groupable meningococci accounted for 52.9% and 10.6%, respectively. Among the remaining carriage isolates, serogroup B was the predominant (71.0%). Serogroup C meningococci were culture negative and unavailable for WGS. Overall, 64 genotypic profiles were identified and, based on cgMLST, isolates clustered according to clonal complexes. Eight isolates (9.4%) harbored at least one gene encoding a 4CMenB vaccine antigen. Mutated penA alleles were found in more than 82%. Finally, complete aniA and norB coding sequences were detected among 71.8% of carriage isolates.ConclusionsMeningococcal carriage isolates collected during the MenC:cc11 outbreak were characterized by an extensive genetic diversity. The lack of outbreak-related isolates among carriage might be attributable to the high transmissibility with low duration of colonization of MenC:cc11 meningococci.

Summary Background A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe. Methods We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain. Findings 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63–90, range of point estimates 73–87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries. Interpretation MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future. Funding Novartis Vaccines and Diagnostics.

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.

Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, Pen I ) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the Pen I phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work ( http://neisseria.org/nm/typing/penA ). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

Meningococcal invasive disease is a life-threatening infection that affects mostly children and adolescents. The present study was performed during 2003–2005 to compare the phenotypic characteristics of meningococcal isolates from these two main groups at risk with those of isolates from other age groups to assess whether strategies for treatment and prevention implemented elsewhere can also be applied in Italy. The results showed that serogroup C meningococci were predominant, and that a dramatic increase in the circulation of strains with decreased susceptibility to penicillin was associated mainly with a prevalent phenotype C:2b:P1.5,2, which belongs to the hyper-virulent ST8/A4 cluster.

In Italy, the incidence of invasive meningococcal disease (IMD) has remained stable since 2007 (around 0.3 cases/100,000 inhabitants). However, as reported for other European countries, an increase of serogroup Y Neisseria meningitidis has been observed. In this study we report IMD cases from 2007 to 2013 in Italy and investigate the clinical and epidemiological features of cases affected by serogroup Y. Molecular characteristics of serogroup Y strains are also described. During the study period, the proportion of IMD cases due to serogroup Y increased, ranging from 2% in 2007 to 17% in 2013 (odds ratio (OR): 8.8), whereby the five to 14 years age group was mostly affected (p lpxL1 gene; however, no associations among lpxL1 mutations, ST and age group were identified. Overall, these findings generate scientific evidence for the use of the quadrivalent meningococcal conjugate vaccine in the five to 14 years age group.

Gonorrhea may become untreatable due to the spread of resistant or multidrug-resistant strains. Cefixime-resistant gonococci belonging to sequence type 1407 have been described worldwide. We report the genome sequence of Neisseria gonorrhoeae strain G2891, a multidrug-resistant isolate of sequence type 1407, collected in Italy in 2013.

We report pertussis cases in 4 infants less than 6 months admitted with symptoms compatible with pertussis to the intensive care unit of the Universita Cattolica del Sacro Cuore in Rome, April 2014. Realtime PCR confirmed pertussis diagnosis for the 4 infants, 2 of them were cousins, and for the household contacts of 1 of them. Analysis of pertussis toxin, its promoter and pertactin was also performed. First of all, this report emphasizes the need to investigate household contact of infants with pertussis; secondly, to evaluate the selective vaccination of household members of newborns as an effective program to reduce pertussis in infants.

In this study, we set up a real-time reverse transcriptase PCR assay to measure the relative amounts of brkA transcripts in 50 Bordetella isolates. The results suggested that brkA expression is strain dependent and its level may play a role in determining the serum resistance or susceptibility phenotype. Pertussis immunocompetent sera were unable to kill Bordetella parapertussis via complement deposition.

Bordetella pertussis, the causative agent of whooping cough, is internalized by several cell types, including epithelial cells, monocytes, and neutrophils. Although its ability to survive intracellularly is still debated, it has been proven that cell-mediated immunity (CMI) plays a pivotal role in protection. In this study we aimed to clarify the interaction ofB. pertussiswith human monocyte-derived dendritic cells (MDDC), evaluating the ability of the bacterium to enter MDDC, to survive intracellularly, to interfere with the maturation process and functional activities, and to influence the host immune responses. The results obtained showed thatB. pertussishad a low capability to be internalized by—and to survive in—MDDC. Upon contact with the bacteria, immature MDDC were induced to undergo phenotypic maturation and acquired antigen-presenting-cell functions. Despite the high levels of interleukin-10 (IL-10) and the barely detectable levels of IL-12 induced byB. pertussis, the bacterium induced maturation of MDDC and T helper 1 (Th1) polarized effector cells. Gene expression analysis of the IL-12 cytokine family clearly demonstrated thatB. pertussisinduced high levels of the p40 and p19 subunits of IL-23 yet failed to induce the expression of the p35 subunit of IL-12. Overall our findings show thatB. pertussis, even if it survives only briefly in MDDC, promotes the synthesis of IL-23, a newly discovered Th1 polarizing cytokine. A Th1-oriented immune response is thus allowed, relevant in the induction of an adequate CMI response, and typical of protection induced by natural infection or vaccination with whole-cell vaccines.

To the Editor: Invasive meningococcal disease is usually defined by the occurrence of meningitis or septicemia. Pericarditis might occur during the course of invasive infection. This clinical picture, defined as disseminated meningococcal disease with pericarditis (1) or secondary meningococcal pericarditis, was reported in 1918 (2). In 1939, primary or isolated meningococcal pericarditis (1,3) was described. In this form of pericarditis, pericardial or blood cultures are positive for Neisseria meningitidis but there is no meningeal involvement or clinical meningococcemia (4). Since its description, several cases of primary meningococcal pericarditis have been reported (5). Although its pathogenesis remains largely undefined, it has been hypothesized that the onset of primary pericarditis occurs after a transient bacteremia or as a consequence of involvement of the lower respiratory tract (4). Blaser et al. reported that serogroup C meningococci are usually associated with this disease, especially in adults. However, serogroups B, W, and Y have also been identified (4). We report a case-patient with primary meningococcal pericarditis caused by a serogroup C strain of N. meningitidis. The patient was a 32-year-old man who lived in Sardinia, Italy. He had no predisposing factors, such as immunodeficiency or other chronic disorders. Disease onset occurred on August 29, 2015. Clinical manifestations were fever (temperature 38°C), hypotension, epigastralgia, arthralgia, asthenia, chest pain, and reduced vesicular murmur. The left ventricle was widely hypokinetic, and a light ST increase was observed. A blood culture was positive for N. meningitidis. The patient was given piperacillin/tazobactam (4.5 g 3×/d) and metronidazole (500 mg 3×/d) for 4 days. After 4 days, treatment with ceftriaxone (2 g 2×/d) for 4 days was started. Because of persistent fever (38.8°C), levofloxacin (500 mg 2×/d) for 23 days was also started on day 7. On day 10, ceftriaxone was replaced with piperacillin/tazobactam (4.5 g 4×/d) for 21 days. A major bilateral pleural effusion was detected on the left side. On day 11, the fever had resolved. The outcome was favorable for this patient. Drug resistance of the strain was determined by using the MIC Test Strip Method (Liofilchem, Abruzzi, Italy). Breakpoints used were those recommended by the European Committee on Antimicrobial Susceptibility Testing version 5.0 (http://www.eucast.org/). The strain was susceptible to cefotaxime, ceftriaxone, ciprofloxacin, penicillin G, and rifampin. Serogroup was determined by using slide agglutination with commercial antisera (Remel Europe, Ltd., Dartford, UK) and confirmed by PCR (6). Whole-genome sequencing was conducted to obtain molecular data and enable comparison with other meningococci of the same serogroup that were isolated in Italy. Multilocus sequence typing (MLST) and typing of porA and fetA genes and Bexsero (meningcoccal group B vaccine) antigen genes (http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm431374.htm) were conducted as described (http://neisseria.org/). Whole-genome sequence was analyzed by using the BIGSdb Genome Comparator Tool (http://pubmlst.org/neisseria/). Genomes of meningococci belonging to the same finetype were compared by using the core genome MLST (cgMLST) approach. The N. meningitidis strain of serogroup C was susceptible to all antimicrobial drugs tested. Although serogroup C was associated with 53 (41%) of 132 invasive meningococcal disease cases in Italy in 2015 (http://www.iss.it/binary/mabi/cont/Report_MBI_20151223_v4.pdf), this serogroup has not been detected in Sardinia since 2010. Molecular analyses showed that the strain belonged to the hypervirulent clonal complex (cc) 11, sequence type (ST) 11. The complete finetype was C:5–1,10–8:F3–6:ST-11(cc11). This finetype has been reported in the United States and several countries in Europe (7), including Italy, and is responsible for several disease outbreaks. In Italy, this finetype represents 61% (70/115) of all serogroup C strains collected during 2012–2015. Two outbreaks caused by this strain were reported in Italy in 2007 (8) and in 2012 (9). The N. meningitidis factor H binding protein and heparin binding protein alleles were 1.13 and 20, respectively. The N. meningitidis adhesin A variant had an insertion sequence that disrupted this gene, as described for ET-15 meningococci (10). On the basis of results of cgMLST, the strain was determined to be related to strains responsible for an outbreak in Italy in 2015. In summary, we report a case of meningococcal pericarditis caused by a strain of N. meningitidis. This strain belongs to hyperinvasive clonal complex cc11 and was identified as C:P1.5–1,10–8:F3–6:ST-11(cc11), an emerging strain in Italy and worldwide. Timely diagnosis and complete molecular characterization of this strain, which causes a rare form of invasive disease (4), is needed for appropriate management of patients with this disease.

Sequence analysis of the penA gene, encoding penicillin-binding protein 2 (PBP2), in 30 penicillin-intermediate (PenI) Neisseria meningitidis strains showed altered gene sequences due to the translocation of exogenous DNA blocks derived from commensal neisseriae, which are known to have PBP2 proteins with decreased affinity for the antibiotic. In order to obtain a rapid and reproducible method for predicting the PenI phenotype, a real-time PCR assay was set up with primers and probes designed on the basis of the penA gene. The A→G mutation at codon 566, in the transpeptidase domain of the penA gene (which is present in the whole sample of 30 PenI strains and in all the 41 sequences of PenI meningococci isolated worldwide and has been deposited in the sequence databank), was chosen as a marker of penA translocations. Two hybridization probes were designed to distinguish the wild-type penA gene in penicillin-susceptible (PenS) meningococci from the mutated penA gene at codon 566 in PenI strains. Thermal analysis of probe hybridization revealed a melting temperature difference of at least 6°C between PenI and PenS strains. This real-time PCR protocol characterizes the penicillin phenotype of N. meningitidis in a few hours without DNA sequencing and is useful for rapid screening of the penicillin-intermediate genotype among meningococcal isolates.

The relative value of antibodies and/or T-cell immune responses toBordetella pertussisantigens in the immunity induced by acellular pertussis (aP) vaccines is still an open issue, probably due to the incomplete knowledge on the mechanisms of protective immunity to pertussis. The relevance of T-cell immune responses in protection from pertussis has been demonstrated in murine and human models of infection; thus, in this study, the ability of different vaccine preparations of three component (pertussis toxin, filamentous hemagglutinin, and pertactin) aP vaccines to induce T-cell responses was investigated in mice. All vaccine preparations examined passed the immunogenicity control test, based on antibody titer assessment, according to European Pharmacopoeia standards, and protected mice fromB. pertussisintranasal challenge, but not all preparations were able to prime T cells to pertussis toxin, the specificB. pertussisantigen. In particular, one vaccine preparation was unable to induce proliferation and gamma interferon (IFN-γ) production while the other two gave borderline results. The evaluation of T-cell responses to pertussis toxin antigen may provide information on the protective immunity induced by aP vaccines in animal models. Considering the critical role of the axis interleukin-12-IFN-γ for protection from pertussis, our results suggest that testing the induction of a key protective cytokine such as IFN-γ could be an additional tool for the evaluation of the immune response induced by aP vaccines.

The molecular characteristics of 14 B:NT:P1.14 Neisseria meningitidis isolates, collected from 2007 through 2010 in Italy, have been investigated. The B:NT:P1.14 phenotype has only more recently been identified in our country, mainly associated with clonal complex CC162, which is rare in Italy.

Meningococcal serogroup C strains, in particular those belonging to the ST-11 clonal complex, are known to cause invasive diseases worldwide. We report the genome sequence of a Neisseria meningitidis strain linked to a cluster of cases of invasive meningococcal disease on a cruise ship that was described in 2012.

The meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or “relative potency” (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified.

Background Following the introduction of meningococcal serogroup C conjugate vaccine in Italy in 2005, changes in the epidemiology of Invasive Meningococcal Disease (IMD) were expected. The study aims were to describe the epidemiological trend and to characterize the isolates collected during the period 2008/09-2012/13 by multilocus sequence typing (MLST). Data on laboratory confirmed meningococcal diseases from National Surveillance System of IMD were reported. Methods Poisson regression models were used to estimate the incidence rate over time. Serogrouping and MLST were performed following published methods. Results The incidence rate of laboratory confirmed meningococcal disease decreased from 0.33 per 100,000 population in 2008/09 to 0.25 per 100,000 population in 2012/13. The serogroup B incidence rate was significantly higher (p

Bordetella pertussis, the causative agent of whooping cough, possesses an array of virulence factors, including adenylate cyclase toxin (ACT), relevant in the establishment of infection. Here we better define the impact of cyclic AMP (cAMP) intoxication due to the action of ACT on dendritic cell (DC)-driven immune response, by infecting monocyte-derived DC (MDDC) with an ACT-deficientB. pertussismutant (ACT−18HS19) or its parental strain (WT18323). Both strains induced MDDC maturation and antigen-presenting cell functions; however, only ACT−18HS19 infected MDDC-induced production of interleukin-12 (IL-12) p70. Gene expression analysis of the IL-12 cytokine family subunits revealed that both strains induced high levels of p40 (protein chain communal to IL-12 p70 and IL-23) as well as p19, a subunit of IL-23. Conversely only ACT−18HS19 infection induced consistent transcription of IL-12 p35, a subunit of IL-12 p70. Addition of the cAMP analogousd-butyril-cAMP (d-cAMP) abolished IL-12 p70 production and IL-12 p35 expression in ACT−18HS19-infected MDDC. ACT−18HS19 infection induced the expression of the transcription factors interferon regulatory factor 1 (IRF-1) and IRF-8 and of beta interferon, involved in IL-12 p35 regulation, and the expression of these genes was inhibited byd-cAMP addition and in WT18323-infected MDDC. The concomitant expression of IL-12 p70 and IL-23 allowed ACT−18HS19 to trigger a more pronounced T helper 1 polarization compared to WT18323. The present study suggests that ACT-dependent cAMP induction leads to the inhibition of pathways ultimately leading to IL-12 p35 production, thus representing a mechanism forB. pertussisto escape the host immune response.

A rise in invasive diseases due to Neisseria meningitidis C:2b:P1.5 with decreased penicillin susceptibility occurred in Italy during the last 2 years. Real-time PCR identified the Pen i phenotype, and the penA sequence revealed the mosaicism of the gene. Molecular analyses assigned the isolates to a single emergent clone of the hypervirulent A4 cluster.

This report describes the C-to-B capsular switching in four Neisseria meningitidis strains belonging to the electrophoretic type 37 (ET-37) complex. In particular, one strain belonged to the new sequence type 1860, which was first detected in the year 2000 in Italy and is now frequently isolated. The presence of switched serogroup B strains deserves special attention if they prove as able to spread as their serogroup C progenitors belonging to the hypervirulent ET-37 complex.

The availability of new polysaccharide-protein conjugate vaccines against Neisseria meningitidis serogroup C prompted European National Health authorities to carefully monitor isolate characteristics. In Italy, during 1999–2001, the average incidence was 0.4 cases per 100,000 inhabitants. Serogroup B was predominant and accounted for 75% of the isolates, followed by serogroup C with 24%. Serogroup C was isolated almost twice as frequently in cases of septicemia than in cases of meningitis, and the most common phenotypes were C: 2a:P1.5 and C:2b:P1.5. Among serogroup B meningococci, the trend of predominant phenotypes has changed from year to year, with a recent increase in the frequency of B:15:P1.4. Only a few meningococci had decreased susceptibility to penicillin, and, in the penA gene, all of these strains had exogenous DNA blocks deriving from the DNA of commensal Neisseria flavescens, Neisseria cinerea, and Neisseria perflava/sicca. Fluorescent amplified fragment–length polymorphism analysis revealed the nonclonal nature of the strains with decreased susceptibility to penicillin. Infections caused by Neisseria meningitidis are still an important public health problem because of the severity and highly contagious nature of the disease. The level of interest related to the issue can be inferred from the number of reports published over the years and from

In this study, phagocytosis of Bordetella pertussis was assessed using a human monocyte-derived macrophage line (THP-1) and immune sera from children who had received primary vaccination during the Italian clinical trial on the efficacy of two acellular three-component (PT-FHA-PRN) and one whole-cell pertussis vaccines. The results demonstrate that phagocytosis of opsonized bacteria with specific immune sera is not significantly enhanced compared with that of non-opsonized bacteria or bacteria opsonized with non-immune sera. A similar result was obtained also using B. pertussis strains showing variants of the pertactin antigen suggesting that those variations do not reduce the capability of the bacterium to invade the monocytes.

Mutations in the rpoB gene affecting two amino acids were found in eight rifampicin-resistant Neisseria meningitidis group B and C strains isolated in Italy. The Asp542-->Val substitution, documented for the first time in N. meningitidis, was found in four of the isolates; the His552-->Tyr or Asn substitution in the other four resistant strains. Mutations in the mtr gene did not seem to be involved in the resistance since the same mutations occurred in both resistant and susceptible strains. Two different clusters were identified among these resistant strains, without any correlation with the specific mutations detected in the rpoB gene.

To the Editor: Neisseria meningitidis serogroup X was first described in the 1960s and has been found to be responsible of rare cases of invasive meningococcal diseases, in particular, meningitis, in North America, Europe, Australia, Africa, and the People’s Republic of China (1–3). This serogroup has recently emerged in Africa as an increasing cause of meningitis; unfortunately, it is not covered by current vaccine programs. Serogroup X outbreaks have been reported in Niger, Ghana, and Kenya (4–6). In particular, in Niger during January–June 2006, N. meningitidis serogroup X represented 51% of confirmed cases of meningitis (4). To investigate the population structure of serogroup X meningococci isolated during recent decades in Africa, Europe, and North America, Gagneux et al. (1) compared the molecular characteristics among them. That study highlighted a low genetic variability between African serogroup X strains, which contrasts with higher genetic variability among isolates from Europe and the United States (1). We describe a case of invasive meningococcal disease caused by a serogroup X N. meningitidis strain isolated in Italy. The patient was a 55-year-old Italian woman with no immune deficiency. The onset of disease started quickly with high fever (39°C) on June 1, 2009. No contacts with persons coming from abroad were reported. This case was diagnosed on the basis of clinical signs and symptoms and results of laboratory confirmatory tests, including blood culture. The patient received ceftriaxone (2 g/day) for 7 days with a favorable outcome. The strain was susceptible to penicillin G, rifampin, ciprofloxacin, and ceftriaxone, as determined by Etest method (bioMerieux, Florence, Italy). The breakpoints were those recommended by the Clinical and Laboratory Standards Institute (7). Serogroup was determined by serum agglutination, and serotype/subtype, NT:P1.15, 19 were determined by standard whole-cell ELISA with monoclonal antibodies (obtained from the National Institute for Biological Standards and Control, South Mimms, UK) (8). PorA variable regions, FetA, and multilocus sequence typing analyses were performed according to standard procedures from the Neisseria Multi Locus Sequence Typing Web site (http://pubmlst.org/neisseria). The isolate from Italy had the pattern PorA VR1–19, VR2–15, and VR3–36; F5–5 and sequence type (ST)-2888. The same ST was already described in Greece in 2002 but in a noninvasive strain (http://pubmlst.org/neisseria). The pattern obtained by pulsed-field gel electrophoresis (9), using the rare-cutting enzyme NheI, (data not shown), was identical to patterns found among meningococci X strains isolated in United Kingdom and belonging to ST-750, clonal group X-II (1). In particular, ST-2888 resembles, except for gdh gene sequence, ST-2317, which was found among the X meningococci isolated in the United Kingdom in 2002 with phenotype X:4:P1.7 (http://pubmlst.org/neisseria). Our data document a rare case of invasive meningococcal meningitis in Italy, caused by N. meningitidis serogroup X ST-2888. Future surveillance data may be able to determine epidemiologic influences, likely emanating from nearby countries, on the spread of such a strain into Italy.

Two clusters of invasive meningococcal disease in the north of Italy both due to serogroup C/ST-11 clonal complex are here described. The objective of the investigation was to analyse the phenotype and the genotype of meningococci involved in the two clusters which were of national relevance due to the fatal outcome of the majority of cases (six of the total of 10 cases). All the strains were C:2a:P1.5 ST-11/ET-37 clonal complex. Two pulsed field gel electrophoresis (PFGE) and variable number tandem repeats (VNTR) profiles were identified, one for each cluster. VNTRs were different from those detected in Italy for C/ST-11 strains isolated from sporadic cases in the same period. This laboratory surveillance report highlights the importance and the crucial role of molecular characterisation to confirm the relatedness among meningococci responsible for clusters of cases.