Viability-PCR for the selective detection of and in live bacteria-containing products.

To exert their beneficial effects, microorganisms used in live bacteria-containing products must be viable and present in certain amounts. In this study, we developed a viability assay based on quantitative PCR coupled with propidium monoazide for the identification and enumeration of viable and . In order to optimize the protocol, the thermal inactivation conditions for the two target microorganisms and the PMA concentration inhibiting DNA amplification from the dead cells while allowing it from the live cells were first determined.

Live biotherapeutic products and their regulatory framework in Italy and Europe.

In Italy and Europe, live microorganisms-containing products meant to be used by vulnerable or sick people for preventing or curing a disease are defined as live biotherapeutic products and are regulated as biological drugs. As such, they must undergo extensive quality, safety and efficacy testing and evaluation before receiving a marketing authorization. This review describes the regulatory framework of live biotherapeutic products with special focus on the European Pharmacopoeia monograph 3053 that set mandatory requirements for this kind of medicines, including verification of the number of live microorganisms and absence of certain contamination indicator microorganisms.

Development of a new, simple, rapid ultra-high-performance liquid chromatography (UHPLC) method for the quantification of 2-phenoxyethanol in vaccines for human use.

A new, simple and rapid method for the quantitative determination of the antimicrobial preservative 2-phenoxyethanol, based on reverse phase ultra-high-performance liquid chromatography has been developed. The validation was performed according the ICH Q2 guideline "Validation of Analytical Procedures". The desired chromatographic separation was achieved on a Waters Symmetry C18 (150 × 4.

Optimization of the monocyte activation test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine.

Pyrogen content is a key quality feature that must be checked in all injectable products, including vaccines. Four tests are currently available in the European Pharmacopoeia to monitor pyrogen/endotoxin presence: the rabbit pyrogen test (RPT), the bacterial endotoxin test, the recombinant factor C test, and the monocyte activation test (MAT). Here, we explored the possibility to replace the RPT with the MAT in the quality control of a vaccine against tick-borne encephalitis virus (TBEV).

Tetanus-diphtheria vaccination in adults: the long-term persistence of antibodies is not dependent on polyclonal B-cell activation and the defective response to diphtheria toxoid re-vaccination is associated to HLADRB1∗01.

Cellular and humoral immune responses to tetanus-diphtheria vaccine (Td) were assessed in human leukocyte antigen (HLA)-typed Italian military personnel who received multiple concomitant vaccines. Td-specific antibodies and T-lymphocytes were measured in individuals with one (group-1) and more than one (group-2) Td boosters. A third group (group-3), who received several vaccines, but not Td, was studied to verify the hypothesis of the polyclonal B-cell activation as mechanism for antibody persistence.

Anti-polysaccharide and anti-diphtheria protective antibodies after 13-valent pneumococcal conjugate vaccination in rheumatoid arthritis patients under immunosuppressive therapy.

Immunogenicity of 13-valent pneumococcal polysaccharide (PnPS) conjugate vaccine (PCV13) was evaluated in 38 rheumatoid arthritis patients under immunosuppressive treatment and 20 healthy controls (HC). Antibodies to all PnPS and diphtheria-toxin analogue conjugate protein were measured pre- (T0), 1 (T1), 6 (T2), 12 (T3) months post-immunization. Patients and HC had similar response to individual PnPS.

Adherence and invasive properties of Corynebacterium diphtheriae strains correlates with the predicted membrane-associated and secreted proteome.

Background: Non-toxigenic Corynebacterium diphtheriae strains are emerging as a major cause of severe pharyngitis and tonsillitis as well as invasive diseases such as endocarditis, septic arthritis, splenic abscesses and osteomyelitis. C. diphtheriae strains have been reported to vary in their ability to adhere and invade different cell lines.

Tetanus in Italy 2001-2010: a continuing threat in older adults.

Despite being a completely preventable disease, tetanus cases continue to occur in Italy and notification and hospitalization rates have been reported to be higher with respect to European and other industrialized countries. We examined statutory notification, hospitalization, mortality and seroprevalence data to describe tetanus epidemiology in Italy from 2001 to 2010. A total of 594 tetanus cases were notified, with an average annual incidence of 1.

Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human.

The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study.

Identification and molecular discrimination of toxigenic and nontoxigenic diphtheria Corynebacterium strains by combined real-time polymerase chain reaction assays.

With the recognition of several diphtheria outbreaks and the emergence of nontoxigenic corynebacteria strains, there has been renewed interest in the development of laboratory diagnostic methods. Previously reported polymerase chain reaction (PCR) assays can have low diagnostic sensitivity or give species misidentifications among clinical isolates. The aim of the present study was the development of combined real-time PCR assays, based on the tox and rpoB genes, for the detection and differentiation of toxigenic and nontoxigenic corynebacteria.

Improved specificity of a multiplex immunoassay for quantitation of anti-diphtheria toxin antibodies with the use of diphtheria toxoid.

A nonspecific binding of antibodies to diphtheria toxin, especially in adult serum samples, was observed in our diphtheria-tetanus-pertussis multiplex immunoassay (DTaP4 MIA). This can be significantly reduced by the use of diphtheria toxoid, achieving a good correlation with the Vero cell neutralization test and the toxin binding inhibition assay.

External quality assessment for the determination of diphtheria antitoxin in human serum.

Accurate determination of diphtheria toxin antibodies is of value in determining the rates of immunity within broad populations or the immune status of individuals who may be at risk of infection, by assessing responses to vaccination and immunization schedule efficacy. Here we report the results of an external quality assessment (EQA) study for diphtheria serology, performed within the dedicated surveillance network DIPNET. Twelve national laboratories from 11 European countries participated by testing a standard panel of 150 sera using their current routine method: Vero cell neutralization test (NT), double-antigen enzyme-linked immunosorbent assay (ELISA; DAE), dual double-antigen time-resolved fluorescence immunoassay (dDA-DELFIA), passive hemagglutination assay (PHA), toxin binding inhibition assay (ToBI), and in-house or commercial ELISAs.

Isolation of Corynebacterium tuscaniae sp. nov. from blood cultures of a patient with endocarditis.

A strain of an unknown coryneform bacterium was repeatedly isolated in pure culture from the blood of a patient affected by endocarditis. Comparative 16S rRNA gene sequence analysis revealed that this isolate represented a new subline within the genus Corynebacterium. This new taxon can be identified by the presence of corynomycolic acids and its enzymatic activities and fermentation of sugars.

Experimental model of infection with non-toxigenic strains of Corynebacterium diphtheriae and development of septic arthritis.

Corynebacterium diphtheriae is a well-known cause of localized respiratory tract infections. However, this micro-organism can also be associated with invasive infections, such as endocarditis, septic arthritis and osteomyelitis. Invasive infections are often caused by non-toxigenic strains.

Association of group A streptococcal emm types with virulence traits and macrolide-resistance genes is independent of the source of isolation.

Streptococcus pyogenes (group A streptococci; GAS) recovered from paediatric pharyngitis (101 isolates) and asymptomatic children (79 isolates) in the same geographical area and period, as well as isolates collected during an enhanced national surveillance programme for GAS invasive diseases (79 isolates), were screened for the incidence of the streptococcal pyrogenic exotoxin (spe) genes speA and speC, as well as the macrolide-resistance genes erm(B), erm(A) subclass erm(TR) and mef(A), and typed by emm sequencing. The speA gene was detected with comparable incidence among throat isolates (13.9 % of asymptomatic children and 16.

Internalization of non-toxigenic Corynebacterium diphtheriae by cultured human respiratory epithelial cells.

Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, and diphtheria is one of the most worried diseases, this microorganism can be associated also with invasive infections such as endocarditis, septic arthritis, and osteomyelitis. Invasive infections are usually caused by non-toxigenic C. diphtheriae strains.

Inhibition of nitric oxide synthase exacerbates group B streptococcus sepsis and arthritis in mice.

The role of nitric oxide in group B Streptococcus (GBS) infection was evaluated by inhibiting its production with aminoguanidine (AG). AG-treated mice displayed higher mortality rates and more frequent and severe arthritis than controls. Worsening of arthritis correlated with a higher number of GBS cells in the joints and local interleukin-1 beta production.

Role of interleukin-18 in experimental group B streptococcal arthritis.

Objective: To assess the role of interleukin-18 (IL-18) in the evolution of septic arthritis induced by group B streptococci (GBS) in mice.

Methods: CD1 mice were inoculated intravenously with 8 x 10(6) colony-forming units (CFU) of type IV GBS (strain 1/82), and administered intraperitoneally 1 hour before infection with anti-IL-18 monoclonal antibodies (0.25 mg/mouse).

Multiplex PCR assay for direct identification of group B streptococcal alpha-protein-like protein genes.

We developed a group B streptococcus multiplex PCR assay which allows, by direct analysis of the amplicon size, determination of the surface protein antigen genes of alpha-C protein, epsilon protein, Rib, Alp2, Alp3, and Alp4. The multiplex PCR assay offers a rapid and simple method of subtyping Streptococcus agalactiae based on surface protein genes.

Simple and rapid technique for monitoring the quality of meningococcal polysaccharides by high performance size-exclusion chromatography.

The molecular size of meningococcal polysaccharides is an important physico-chemical parameter which correlates with immunogenicity. This paper describes the experimental conditions for high-performance size-exclusion chromatography on a PL Aquagel-OH 60 column to follow changes in the size distribution and therefore in the distribution coefficient (K(D)) of the meningococcal polysaccharides of groups A, C, Y and W-135 used to formulate anti-Neisseria meningitidis vaccines. The experimental conditions were also found to be suitable for a rapid monitoring of the quality (no group A polysaccharide depolymerization) of the tetravalent meningococcal polysaccharide vaccine.

Molecular epidemiology and characteristics of Corynebacterium diphtheriae and Corynebacterium ulcerans strains isolated in Italy during the 1990s.

Five cases of diphtheria were reported in Italy between January 1990 and June 2001. Three cases were confirmed microbiologically by the isolation of toxigenic Corynebacterium diphtheriae (two cases) and Corynebacterium ulcerans (one case). Over the same period, 11 cases of non-toxigenic C.

Role of macrophages in experimental group B streptococcal arthritis.

Septic arthritis is a clinical manifestation of group B Streptococcus (GBS) infection in both neonates and adults. Because macrophages are known to participate in tissue injury, the role of this cell population in GBS-induced arthritis was investigated. Mice were rendered monocytopenic by administration of etoposide, a drug that selectively depletes the monocyte/macrophage population and then injected with GBS (1 x 10(7) colony-forming units per mouse).

Regulatory role of interleukin-10 in experimental group B streptococcal arthritis.

Intravenous inoculation of CD-1 mice with 10(7) CFU of type IV group B Streptococcus (GBS) results in a high incidence of diffuse septic arthritis, associated with high levels of systemic and local production of interleukin-1beta (IL-1beta) and IL-6. In this study, the role of the anti-inflammatory cytokine IL-10 in the evolution of GBS systemic infection and arthritis was evaluated. IL-10 production was evident in sera and joints of GBS-infected mice.

Immunogenicity of anti-Haemophilus influenzae type b CRM197 conjugate following mucosal vaccination with oligodeoxynucleotide containing immunostimulatory sequences as adjuvant.

Most vaccines are delivered by injection. Mucosal vaccination would increase compliance and decrease the risk of spread of infectious diseases due to a reduction of mucosal colonization and of contaminated syringes. However, most vaccines are unable to induce immune responses when administered mucosally, and require the use of strong adjuvant or effective delivery systems.

The beneficial effect of interleukin-12 on arthritis induced by group B streptococci is mediated by interferon-gamma and interleukin-10 production.

Objective: To assess the effect of interleukin-12 (IL-12) administration on the evolution of systemic infection and septic arthritis induced by group B streptococci (GBS) in mice.

Methods: CD1 mice were inoculated intravenously with arthritogenic strain 1/82 of type IV GBS. Exogenous murine IL-12 was administered intraperitoneally 18 hours or 5 days after infection with 1 x 10(7) GBS, at doses ranging from 0.