Infants hospitalized for Bordetella pertussis infection commonly have respiratory viral coinfections.

BACKGROUND: Whether viral coinfections cause more severe disease than Bordetella pertussis (B. pertussis) alone remains unclear. We compared clinical disease severity and sought clinical and demographic differences between infants with B. pertussis infection alone and those with respiratory viral coinfections. We also analyzed how respiratory infections were distributed during the 2 years study. METHODS: We enrolled 53 infants with pertussis younger than 180 days (median age 58 days, range 17­109 days, 64. 1% boys), hospitalized in the Pediatric Departments at "Sapienza" University Rome and Bambino Gesù Children's Hospital from August 2012 to November 2014. We tested in naso-pharyngeal washings B. pertussis and 14 respiratory viruses with real-time reverse-transcriptase-polymerase chain reaction. Clinical data were obtained from hospital records and demographic characteristics collected using a structured questionnaire. RESULTS: 28/53 infants had B. pertussis alone and 25 viral coinfection: 10 human rhinovirus (9 alone and 1 in coinfection with parainfluenza virus), 3 human coronavirus, 2 respiratory syncytial virus. No differences were observed in clinical disease severity between infants with B. pertussis infection alone and those with coinfections. Infants with B. pertussis alone were younger than infants with coinfections, and less often breastfeed at admission. CONCLUSIONS: In this descriptive study, no associations between clinical severity and pertussis with or without co-infections were found. TRIAL REGISTRATION: Policlinico Umberto I: protocol 213/14, 3085/13.02.2014, retrospectively registered. Bambino Gesù Children's Hospital: protocol n. RF-2010-2317709.

Climate, demographic factors and geographical variations in the incidence of invasive meningococcal disease in Italy.

We investigated the effect of climatic, demographic factors and intra-country geographical variations on the incidence of invasive meningococcal disease (IMD) in Italy. For this purpose, incidence rates of IMD cases reported in Italy between 1994 and 2012 were calculated, and a cluster analysis was performed. A geographical gradient was determined, with lower incidence rates in central and southern Italy, compared to the northern parts, where most clusters were observed. IMD rates were higher in medium-sized towns than in villages. Adults were at lower risk of IMD than children aged ⩽4 years. IMD incidence tended to decrease with increasing monthly mean temperatures (incidence rate ratio 0·94, 95% confidence interval 0·90-0·99). In conclusion, geographical variations in IMD incidence were found, where age and temperature were associated with disease occurrence. Whether geographical variations should be considered in national intervention plans is still a matter for discussion.

Genetic diversity and its impact on disease severity in respiratory syncytial virus subtype-A and -B bronchiolitis before and after pandemic restrictions in Rome.

OBJECTIVES: To scrutinize whether the high circulation of respiratory syncytial virus (RSV) observed in 2021-2022 and 2022-2023 was due to viral diversity, we characterized RSV-A and -B strains causing bronchiolitis in Rome, before and after the COVID-19 pandemic. METHODS: RSV-positive samples, prospectively collected from infants hospitalized for bronchiolitis from 2017-2018 to 2022-2023, were sequenced in the G gene; phylogenetic results and amino acid substitutions were analyzed. Subtype-specific data were compared among seasons. RESULTS: Predominance of RSV-A and -B alternated in the pre-pandemic seasons; RSV-A dominated in 2021-2022 whereas RSV-B was predominant in 2022-2023. RSV-A sequences were ON1 genotype but quite distant from the ancestor; two divergent clades included sequences from pre- and post-pandemic seasons. Nearly all RSV-B were BA10 genotype; a divergent clade included only strains from 2021-2022 to 2022-2023. RSV-A cases had lower need of O2 therapy and of intensive care during 2021-2022 with respect to all other seasons. RSV-B infected infants were more frequently admitted to intensive care units and needed O2 in 2022-2023. CONCLUSIONS: The intense RSV peak in 2021-2022, driven by RSV-A phylogenetically related to pre-pandemic strains is attributable to the immune debt created by pandemic restrictions. The RSV-B genetic divergence observed in post-pandemic strains may have increased the RSV-B specific immune debt, being a possible contributor to bronchiolitis severity in 2022-2023.

Cluster of invasive Neisseria meningitidis infections on a cruise ship, Italy, October 2012.

We describe a cluster of four cases of invasive meningococcal disease that occurred on a cruise ship sailing along the Italian coast in October 2012. All four cases were hospitalised with severe illness and one of them died. This report illustrates the importance of rapid implementation of emergency control measures such as administration of prophylaxis to all crew members and passengers to prevent the spread of the disease in such a close environment.

Diversity of Neisseria meningitidis invasive isolates in Italy in the period 2008-2010.

In the period 2008-2010, 309 Neisseria meningitidis, isolated in Italy within the National Surveillance of the Invasive Meningococcal Diseases, have been tested for their phenotypic and genotypic characteristics. The main results obtained are: (a) an increase of the strains of serogroup B and a decrease of serogroup C; (b) a phenotypic and genotypic variability of the ST-41/44 clonal complex, the most frequently isolated among serogroup B strains; (c) a decrease of ST-8 clonal complex among serogroup C meningococci whereas strains belonging to ST-11 clonal complex are nowadays the most frequently isolated.

Azithromycin-resistant Neisseria gonorrhoeae strains recently isolated in Italy.

OBJECTIVES: The aim of this study was to characterize 22 azithromycin-resistant Neisseria gonorrhoeae isolates, collected in Italy from January 2007 through June 2008, during a study of the prevalence of antibiotic resistance. METHODS: MICs of azithromycin, ciprofloxacin, ceftriaxone, penicillin and tetracycline were determined by the Etest method. Azithromycin-resistant strains (MIC > or = 1 mg/L) were genetically analysed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and PFGE. RESULTS: A total of 22 azithromycin-resistant isolates were found among the 219 collected. Five of the 22 isolates showed high-level azithromycin resistance (MICs of 128 or 256 mg/L). Sixteen of the 22 were isolated from men who have sex with men. Among the 14 sequence types (STs) found by NG-MAST, 5 STs, containing clusters of two, three or four strains, were homogeneous with respect to epidemiology and/or antibiotic susceptibility. PFGE divided the 22 strains into two main groups that were possibly related. CONCLUSIONS: This is the first report of gonococci with high-level resistance to azithromycin circulating in Italy. Correlation between NG-MAST results and epidemiological data for some of the analysed strains and patients could be established. This study represents a reference point for future surveillance in Italy and suggests the need to add azithromycin to the antibiotic susceptibility panel for gonococci.

Characterisation of Neisseria meningitidis C strains causing two clusters in the north of Italy in 2007 and 2008.

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.

An outbreak of severe invasive meningococcal disease due to a capsular switched Neisseria meningitidis hypervirulent strain B:cc11.

OBJECTIVES: The aim was to investigate an outbreak of invasive meningococcal disease (IMD) in Southern Sardinia. METHODS: Epidemiological and microbiological investigations were performed. The latter included antimicrobial susceptibility testing and whole-genome sequencing (WGS). RESULTS: Seven individuals with severe IMD were found to be infected with serogroup B (MenB) Neisseria meningitidis in the first quarter of 2018. Five of the seven cases (five males; mean age 19 years; range 18-21 years; CFR 40%) were due to a unique strain B:P1.5-1,10-8:F3-6:ST-11(cc11), probably switched from the hypervirulent C-cc11, as confirmed by WGS. All five patients had attended the same nightclub in the 2 weeks prior to symptom onset. Public health measures, including chemoprophylaxis of contacts and active immunization against MenB, were implemented. CONCLUSIONS: We observed five IMD cases due to the same switched MenB strain. The hypervirulent B:P1.5-1,10-8:F3-6:ST-11(cc11) strain, probably switched from C-cc11, is of concern due to the observed high virulence and case fatality rates. All the patients shared the same place of probable exposure. The molecular characterization of the invasive strain allowed the outbreak to be confirmed, which was then controlled through timely public health action.

Characterisation of invasive meningococcal isolates from Italian children and adolescents.

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.

Genome-based study of a spatio-temporal cluster of invasive meningococcal disease due to Neisseria meningitidis serogroup C, clonal complex 11.

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 Sanità (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.

Changing epidemiology of Infant Meningococcal Disease after the introduction of meningococcal serogroup C vaccine in Italy, 2006-2014.

BACKGROUND: In Italy, the incidence of Invasive Meningococcal Disease (IMD) was around 0.28 per 100,000 over the last years. Since the risk IMD is usually high among infants aged less than 1 year, we decided to evaluate the trend of IMD cases reported between 2006 and 2014 in this age group. In particular, the study aim was to describe the main characteristics of IMD cases in infants following the introduction of MCC vaccine (2005) and to estimate the number of cases which are potentially preventable through early vaccination. METHODS: The National Surveillance System of Bacterial Meningitis was established in 1994 and in 2007 was extended to all invasive bacterial diseases. Clinical data and isolates and/or clinical samples are collected from hospitalized patients throughout the country. IMD cases are reported by clinicians to the local health authorities, and samples are sent to the Reference Laboratory at the Istituto Superiore di Sanità for further characterization and storage at -80°C. In particular, serogroup identification is obtained by agglutination with commercial antisera or by multiplex PCR. RESULTS: The annual incidence for infants <1 year old remained rather stable of 3.6 per 100,000, with several upward and downward oscillations and a peak in 2010. The incidence of IMD among infants was more than 10 times higher than the overall rate of IMD observed in Italy. Finally, serogroup B was more frequently detected among infants aged <1 year, accounting for 65% of the total (p<0.01). CONCLUSIONS: During the study period, IMD incidence reported among infants aged less than one year old was 10 times higher than the overall rate, and serogroup B was the most commonly detected over time. The long-term impact of meningococcal C conjugate vaccine and the effect of the introduction of meningococcal B vaccination among infants need to be evaluated.

Draft Genome Sequence of Neisseria gonorrhoeae Sequence Type 1407, a Multidrug-Resistant Clinical Isolate.

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.

Draft Genome Sequence of a Bordetella pertussis Strain with the Virulence-Associated Allelic Variant ptxP3, Isolated in Italy.

Despite a universal immunization program, pertussis has persisted and resurged, and is of particular concern for infants in terms of morbidity and mortality. Here, we report the genome sequence of a Bordetella pertussis strain with the virulence-associated allelic variant ptxP3, isolated from a 45-day-old infant.

Polymerase chain reaction for the identification of Bordetella pertussis and Bordetella parapertussis.

The polymerase chain reaction (PCR) for the detection of Bordetella pertussis and Bordetella parapertussis DNA in clinical samples was well documented by recent studies. Different regions in Bordetella pertussis DNA have been successfully used as targets for this method by various authors. In this work we report the usefulness of the PCR assay also for speciating Bordetellae isolates in those cases where the biochemical and serological tests gave inconclusive results.

Polymerase chain reaction for the detection of Bordetella pertussis in clinical nasopharyngeal aspirates.

A PCR procedure for the detection of Bordetella pertussis in nasopharyngeal aspirates (NPAs) was developed with primers derived from the pertussis toxin promoter region. The amplification resulted in a 191-bp PCR product specific for B. pertussis. A total of 681 NPAs collected from children with cough lasting >7 days was evaluated by PCR and culture; 104 aspirates were positive by PCR and 93 by culture. Sixteen cases were positive only by PCR and five culture positive aspirates were negative by PCR. An internal control was included in the assay to monitor the performance of the PCR and to identify possible inhibitory components in clinical samples. The PCR method was more efficient than culture in detecting B. pertussis in samples collected late in the disease, in antibiotic-treated children and in patients with mild disease.

Determination of pyrethroid pesticide residues in fatty materials by solid-matrix dispersion partition, followed by mini-column size-exclusion chromatography.

The method studied uses a combination of a solid-matrix dispersion partition (SMDP) followed by high-performance size-exclusion chromatography on a minicolumn (HPmSEC) of 7.8 mm I.D. for the separation of pyrethroid (PYR) residues from fatty material. The solid-matrix dispersion extraction is carried out by absorbing a fat solution onto an Extrelut-3 cartridge (filled with a macroporous diatomaceous material) and extracting the PYR residues with acetonitrile. Up to 1 g of fatty material can be extracted with 15 ml acetonitrile. The small amount (mean +/- S.D. = 12.4 +/- 5.9 mg) of fatty material which is eluted into the acetonitrile is further removed by HPmSEC. PYR pesticide residues are collected in a 2-ml fraction between 7 and 9 ml, the column being washed up to 24 ml. The two techniques used in series allow a better removal of fat, a greater input of sample and a lower consumption of solvent compared to the sole SEC on macrocolumns, and a lower limit of determination compared to the sole SEC on minicolumns. Recoveries of 9 PYR out of the 14 investigated residues from soya oil were in the range 66-83% at spiking levels ranging 0.49-2.57 mg/kg, while for 6 PYR residues tested at spiking levels in the range 0.13-0.53 mg/kg the recoveries were in the range 80-111%. Recovery of fluvalinate and permethrin could not be calculated due to interferences from soya oil, while lambda-cyhalothrin, esfenvalerate and tralomethrin gave low recovery. The final extract contains small amount (mean +/- S.D. = 2.4 +/- 0.9 mg) of lipid residue and is not completely free from interferences.

Selective clean-up applicable to aqueous acetone extracts for the determination of carbendazim and thiabendazole in fruits and vegetables by high-performance liquid chromatography with UV detection.

Fungicide residues in vegetables (benomyl, carbendazim, thiabendazole) are analyzed through a clean-up procedure that uses a portion of the aqueous acetone extract prepared for multiresidue methodology. A portion of the aqueous acetone extract (equivalent to 5 g of vegetables) is loaded onto an Extrelut-20 cartridge (the cartridge is filled with a coarse, large-pore diatomaceous material). Then, acetone is partially removed by an upward stream of nitrogen at 2l/min for 30 min. Benzimidazolic fungicides are recovered by percolating the cartridge with 100 ml of 0.1 M phosphoric acid solution, which also serves to convert benomyl to carbendazim. The percolating acid solution is drained on-line through a strong cation-exchange (SCX) solid-phase extraction cartridge with the aid of a slight vacuum. Benzimidazolic fungicides are retained on the SCX cartridge. The phosphoric acid solution is discarded together with the washings of the SCX cartridge, i.e., water followed by methanol-water (75:25), that remove unwanted coextractives. Finally, benzimidazolic fungicides are recovered by eluting the SCX cartridge with methanol-ammonium formate buffer (75:25). The final extract is then analyzed by reversed-phase HPLC with UV detection. Recoveries from crops such as apples, lettuce, strawberries and citrus fruits are generally greater than 80% and no interferences were observed. The clean-up is simple and straightforward, requires only disposable items, water solutions and a few milliliters of solvent and a minimum number of manipulations, and does not require concentration steps or electrical equipment.

Typing of Neisseria meningitidis isolates from patients with invasive disease by molecular analysis of porin genes.

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism assay (PCR-RFLP) for porA and porB genes of Neisseria meningitidis was applied to type 64 strains isolated from cases of invasive disease in Italy. The technique was also successfully used on non-viable strains and on cerebrospinal fluid samples from patients with meningococcal meningitis diagnosed by PCR. RFLP patterns were obtained for all strains, including those nontypeable or nonsubtypeable by the serological methods. The results confirmed the usefulness of the PCR-RFLP for porA and porB genes of Neisseria meningitidis in differentiating strains belonging to the same serological type and provided discriminative patterns in the increased proportion of non serotypeable/serosubtypeable strains circulating in Italy in recent years.

Characterization of Bordetella pertussis strains of recent isolation.

During the clinical trial conducted in Italy to evaluate the efficacy of new acellular pertussis vaccines, the most favorable conditions for the recovery and characterization of Bordetella pertussis strains, isolated from children with cough, were adopted. The nasopharyngeal aspirates were collected and sent to the laboratory in refrigerated conditions within 24 hours. Charcoal agar selective and non selective plates were used, and most of the isolates were recovered after 3-4 days of incubation. Confirmation of all suspected colonies included the use of biochemical tests and specific agglutination reaction with B. pertussis antiserum. Serotyping of fimbriae, susceptibility to erythromycin and DNA fingerprinting by Pulsed Field Gel Electrophoresis (PFGE), were performed to characterize B. pertussis isolates and to determine relatedness among different strains. Serotype 1,3 was the most represented in the bacterial population examined. A predominant pulsetype (PTA) characterized most of the isolates accounting for 71.4% of the strains examined. Eight subclones (23.5%) and three unrelated pulsetypes were also found. No resistant strains to erythromycin were detected.

Draft Genome Sequence of C:P1.5-1,10-8:F3-6:ST-11 Meningococcal Clinical Isolate Associated with a Cluster on a Cruise Ship.

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.