Pertussis diagnosis in Belgium: results of the National Reference Centre for Bordetella anno 2015.

In 2015, the Belgian National Reference Centre for Bordetella analyzed 4110 respiratory samples by qPCR and 4877 serum samples by serology. Whereas about 50% of respiratory samples were from infants and children below the age of five, serum samples were distributed among all age categories. A total of 394 (9·6%) cases was diagnosed as positive for Bordetella pertussis by qPCR and 844 (17·3%) cases were diagnosed as acute infection by serology (anti-pertussis toxin (PT) IgG > 125 IU/ml). Another 1042 (21·4%) sera had anti-PT IgG between 55 and 125 IU/ml reflecting a vaccination or pertussis infection during the last 1-2 years. Seventy per cent of the pertussis cases diagnosed by qPRC were in infants and children younger than 14 years old, whereas the highest number of sera with anti-PT levels >125 IU/ml was in the age group of 10-14 years old. Based on the limited data of the last vaccination (reported for only 15% of the samples), recent booster vaccination in the teenager group may have contributed only minimally to these elevated anti-PT levels. The highest number of sera with anti-PT titers between 55 and 125 IU/ml was found in the age category 50-59 years old. It is clear that pertussis continues to be a problem in Belgium and that other vaccination strategies (maternal vaccination, cocoon vaccination) and ultimately better vaccines will be needed to control this highly infectious respiratory disease.

Investigation of inflammatory and allergic responses to common mold species: Results from in vitro experiments, from a mouse model of asthma, and from a group of asthmatic patients.

Most studies on molds focus on Alternaria alternata and Aspergillus fumigatus. Here, we report on inflammatory and allergenic properties of more typical indoor species Aspergillus versicolor, P. chrysogenum, C. cladosporioïdes, and C. sphaerospermum that were compared to A. alternata and A. fumigatus. In a mouse model, after intranasal instillation, A. alternaria, A. versicolor, and C. sphaerospermum induced the early recruitment of neutrophils and the strong expression of inflammatory markers in the bronchoalveolar lavages fluids. A. fumigatus also induced the early accumulation of neutrophils but with lower levels of inflammatory markers. Chronic treatment induced variable response according to species: P. chrysogenum and A. fumigatus appeared strong pro-allergenic inducers compared to A. alternata and C. sphaerospermum while A. versicolor and C. cladosporioides induced a mixed pro-allergenic/pro-inflammatory response. In mold-sensitized asthmatics, mold-specific Immunoglobulin E (IgE) were detected with an in-house dot-blot assay. A. fumigatus and A. alternata were the most frequent sensitizers. Altogether, P. chrysogenum, P. brevicompactum, C. sphaerospermum, and C. cladosporïoides were the "major sensitizer" (defined as the strongest response against a single mold species) for almost 30% of the asthmatics. These results show that, not only A. alternata and A. fumigatus, but also indoor species have strong inflammatory and allergic properties and a harmful potency.

Synthesis and anti-tubercular activity of N(2)-arylbenzo[g]isoquinoline-5,10-dione-3-iminium bromides.

Tuberculosis has remained a challenge for medicinal chemists worldwide. In the framework of a collaborative program to identify and evaluate novel antitubercular candidate compounds, the biological properties of benzo[g]isoquinoline-5,10-diones have been found to be very promising. In this paper we have further expanded the library by incorporation of an amidinium moiety into the benzo[g]isoquinoline-5,10-dione scaffold. The presence of this functional group also increased the solubility of the quinones in polar solvents. To this purpose N(2)-arylbenzo[g]isoquinoline-5,10-dione-3-iminium bromides were synthesized in a straightforward way by means of a reaction of anilines with 2-(bromomethyl)-3-(cyanomethyl)-1,4-dimethoxynaphthalene. Following the biological evaluation, N(2)-(4-chlorophenyl)-5,10-dioxobenzo[g]isoquinoline-3(2H)-iminium bromide (MIC = 1.16 µM, CC50 = 28.51 µM, SI = 24.58) was selected as the most promising representative. Apart from the nano-molar anti-mycobacterial activity, the compound was able to target intracellular residing Mycobacterium tuberculosis and the susceptibility of a multi-drug-resistant strain towards the compound was confirmed.

Bordetella pertussis seroprevalence in Belgian adults aged 20-39 years, 2012.

SUMMARY The last report on pertussis seroprevalence in Belgium concerned samples collected during 1993-1994. In the context of the Eupert-Labnet WP6 seroprevalence study (comparing sera from 16 European member states), 1500 anonymized leftover diagnostic samples were collected randomly during the second semester of 2012 by the clinical chemistry laboratories of six participating Belgian centres, distributed equally between Flanders, Wallonia and Brussels Capital Region. As suggested by the WP6 organizers, a total of 750 samples (125/centre) were selected from subjects in the 20-29 years age group and 750 samples (125/centre) from subjects in the 30-39 years age group. Anti-PT IgG levels were measured using Virion-Serion ELISA and analysed using predefined cut-off levels. Sixty-one (4%) sera were indicative of an infection in the past 2 years (between 50 and 100 IU/ml) and another 61 (4%) sera had anti-PT IgG antibodies reflecting acute infection (>100 IU/ml). These results highlight the presence of a Bordetella pertussis reservoir in the adult 'healthy' Belgian population.

[Hooping cough in adults, think about it!]. / Coqueluche chez l'adulte, pensez-y!

We report the case of a 47 year old patient who had been suffering from persistent cough for more than three weeks. Patient coughed predominantly during night time, without fever. The amoxicillin-clavulanic acid initially prescribed was not effective. A series of complementary investigations were performed before serology finally identified Bordetella pertussis infection after two months of symptoms which improved slowly without evident benefit of macrolide treatment. The diagnosis of whooping cough was also established for the wife of the patient with fast resolution of the symptoms after rapid unset of treatment with macrolides.

Immunogenicity and protective efficacy of a tuberculosis DNA vaccine.

Tuberculosis is the most widespread and lethal infectious disease affecting humans. Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell-mediated immune responses and conferred significant protection against challenge with live M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M. tuberculosis, leading to the development of a more effective vaccine.

Assessment of IgA anti-PT and IgG anti-ACT reflex testing to improve Bordetella pertussis serodiagnosis in recently vaccinated subjects.

OBJECTIVES: Quantifying IgG antibodies to pertussis toxin (PT) is the most specific and sensitive method for the serodiagnosis of a Bordetella pertussis infection. Since PT is a component of acellular pertussis vaccines, anti-PT IgG is also induced by vaccination, precluding pertussis serodiagnosis based exclusively on anti-PT IgG in recently vaccinated subjects. Here, we aim to identify additional B. pertussis-specific serological markers that can discriminate between infection and recent vaccination. METHODS: The clinical usefulness of measuring IgA directed to the vaccine antigen PT and IgG directed to non-vaccine antigens (Fim2/3, LPS, ACT, CatACT) was evaluated in nine well characterized subject groups, aged 10-89 years (n = 390). Serum anti-PT IgG levels (>125 IU/mL) served as an indicator for a recent B. pertussis infection. Comparing symptomatic pertussis-infected subjects (n = 140) with recently vaccinated, non-infected subjects (n = 100) revealed the optimal cut-off, accuracy, sensitivity and specificity for each single parameter. RESULTS: For pertussis diagnosis in recently vaccinated subjects, the measurement of anti-PT IgA (cut-off 15 IU/mL) and anti-ACT IgG (cut-off 15 U/mL) resulted in accuracies of 95% (91.5-97.1) and 87.5% (82.7-91.1), sensitivities of 92.9% (87.4-96.0) and 83.6% (76.5-88.8) and specificities of 98% (93.0-99.4) and 93% (86.3-96.6), respectively. Comparing anti-PT IgA levels between the youngest (10-19 years, n = 38) and oldest (70-89 years, n = 17) age groups revealed an age-dependent increase in antibody levels in pertussis-infected subjects (p < 0.0001). CONCLUSIONS: Reflex testing of anti-PT IgA and anti-ACT IgG improves pertussis serodiagnosis in recently vaccinated symptomatic subjects with elevated anti-PT IgG levels. Furthermore, both markers can discriminate between vaccination and recent infection in pertussis serosurveillance studies.

Use of Mycobacterium avium subsp. paratuberculosis specific coding sequences for serodiagnosis of bovine paratuberculosis.

In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) was screened for specific coding sequences that could be very valuable in the design of a sensitive and specific Map detection serological assay. Eighty-seven Map-specific sequences were retained. Among these, three candidate antigens have been analysed for their serodiagnostic potential. These antigens were selected on the basis of their putative immunogenicity as predicted by in silico analysis. The antigens were cloned in Escherichia coli, expressed, and purified before testing in an antibody detection ELISA test, using a well characterized panel of 18 and 48 sera from Map infected and uninfected cattle, respectively. Two of these antigens, antigen 6 and MAP1637c, yielded in our conditions a sensitivity of 72% and 82%, respectively, for a specificity of 98%. It is particularly noticeable that, when probed with the same serum panel, the most widely used European paratuberculosis commercial seroassay (Pourquier test) yielded a sensitivity of 72% for a specificity of only 92%.

Aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 11 European countries.

OBJECTIVES: To describe the role of bacteria (including bacterial resistance), viruses (including those recently described) and mixed bacterial-viral infections in adults presenting to primary care with lower respiratory tract infection (LRTI). METHODS: In all, 3104 adults with LRTI were enrolled, of whom 141 (4.5%) had community-acquired pneumonia (CAP), and 2985 matched controls in a prospective study in 16 primary care networks in Europe, and followed patients up at 28-35 days. We detected Streptococcus pneumoniae and Haemophilus influenzae and assessed susceptibility, atypical bacteria and viruses. RESULTS: A potential pathogen was detected in 1844 (59%) (in 350 (11%) bacterial pathogens only, in 1190 (38%) viral pathogens only, and in 304 (10%) both bacterial and viral pathogens). The most common bacterial pathogens isolated were S. pneumoniae (5.5% overall, 9.2% in CAP patients) and H. influenzae (5.4% overall, 14.2% in CAP patients). Less than 1% of S. pneumoniae were highly resistant to penicillin and 12.6% of H. influenzae were ß-lactamase positive. The most common viral pathogens detected were human rhinovirus (20.1%), influenza viruses (9.9%), and human coronavirus (7.4%). Influenza virus, human parainfluenza viruses and human respiratory syncytial virus as well as human rhinovirus, human coronavirus and human metapneumovirus were detected significantly more frequently in LRTI patients than in controls. CONCLUSIONS: A bacterial pathogen is identified in approximately one in five adult patients with LRTI in primary care, and a viral pathogen in just under half, with mixed infections in one in ten. Penicillin-resistant pneumococci and ß-lactamase-producing H. influenzae are uncommon. These new findings support a restrictive approach to antibiotic prescribing for LRTI and the use of first-line, narrow-spectrum agents in primary care.

Inhibitory effect of interferon on the growth of spontaneous mammary tumors in mice.

Partially purified mouse interferon type I (Mu IFN-beta), crude mouse interferon type II (Mu IFN-gamma) serum, and the interferon inducer polyinosinic acid-polycytidylic acid (poly I:C) were evaluated for their effects on the growth of spontaneously occurring mammary carcinomas in BALB/c mice. As soon as their tumors had reached a palpable size (diameter: 3-5 mm), the mice received three ip injections of Mu IFN-beta, Mu IFN-gamma, or poly I:C per week for 6 weeks. A significant (fourfold to fivefold) reduction in tumor size was achieved with Mu IFN-beta (at 10(6) U/mouse), Mu IFN-gamma (at 10(3) U/mouse), and poly I:C (at 1 mg/mouse). A similar reduction in tumor size was noted when the mice were treated with cyclophosphamide (2.5 mg/mouse, three ip injections per wk for 6 wk). Treatment with Mu IFN-beta combined with Mu IFN-gamma resulted in a greater tumor growth-inhibitory effect than treatment with either Mu IFN-beta or Mu IFN-gamma alone. In addition to inhibiting the growth of primary tumors, interferon also reduced the incidence of lung metastases. However, complete tumor regression was not observed in any mouse while under interferon therapy.

Genome Sequences of Four Strains of Mycobacterium avium subsp. hominissuis, Isolated from Swine and Humans, Differing in Virulence in a Murine Intranasal Infection Model.

This paper announces the genome sequences of four strains of Mycobacterium avium subsp. hominissuis, isolated from cases of lymphadenopathy in swine and humans, differing in virulence in a murine intranasal infection model.

A Mycobacterium tuberculosis gene cluster encoding proteins of a phosphate transporter homologous to the Escherichia coli Pst system.

We report the cloning and sequencing of three M. tuberculosis genes encoding proteins homologous to E. coli PstA, PstC and PstB. They are tentatively called pstA-2, pstC-1 and pstB. They encode proteins of 302, 336 and 275 amino acids, respectively. In E. coli, PstB is the ATP binding component and PstA/PstC are the two hydrophobic subunits of a phosphate permease belonging to the family of ABC (ATP-binding cassette) transporters. In mycobacteria, PstS-1, the phosphate binding subunit (Andersen and Hansen, 1989), is encoded by a gene directly surrounded by pstB, pstC-1 and pstA-2 within a potential operon (pstB, pstS-1, pstC-1, pstA-2). Phosphate uptake by whole, suspension grown, M. bovis BCG cells was measured and could be inhibited by a monoclonal antibody directed against the PstS-1 subunit, suggesting that these genes encode subunits of a functional mycobacterial phosphate permease.

Identification of a second Mycobacterium tuberculosis gene cluster encoding proteins of an ABC phosphate transporter.

Following the identification of a M. tuberculosis phosphate transporter belonging to the superfamily of ABC transporters, we report on the cloning and sequencing of two additional genes, called pstS-3 and pstC-2, encoding proteins homologous to PstS and PstC of Escherichia coli, respectively. Together with the previously isolated M. tuberculosis gene similar to the E. coli pstA, these are included in a cluster encoding a second putative phosphate transport system. We demonstrate that pstS-3 encodes the previously described Ag 88, a 40 kDa M. bovis BCG culture filtrate antigen (immunodominant in H-2b haplotype type mice). Finally, a signature motif identifying integral transmembrane proteins of prokaryotic phosphate binding-dependent permeases is proposed.

Vaccinia expression of Mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of M. tuberculosis Ag85.

There is increasing evidence to implicate a role for CD8(+) T cells in protective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins can be used both as tools to dissect CD8(+) T-cell responses and, in attenuated form, as candidate vaccines capable of inducing a balanced CD4(+)/CD8(+) T-cell response. A panel of rVV was constructed to express four immunodominant secreted proteins of MTB: 85A, 85B and 85C and ESAT-6. A parallel group of rVV was constructed to include the heterologous eukaryotic tissue plasminogen activator (tPA) signal sequence to assess if this would enhance expression and immunogenicity. Clear expression was obtained for 85A, 85B and ESAT-6 and the addition of tPA resulted in N-glycosylation and a 4-10-fold increase in expression. Female C57BL/6 mice were immunised using the rVV-Ag85 constructs, and interleukin-2 and gamma-interferon were assayed using a co-culture of immune splenocytes and recall antigen. There was a marked increase in cytokine production in mice immunised with the tPA-containing constructs. We report the first data demonstrating enhanced immunogenicity of rVV using a tPA signal sequence, which has significant implications for future vaccine design.

An isoelectric focusing method for the study of the humoral response against the antigen 85 complex of Mycobacterium bovis BCG in the different forms of leprosy.

Isoelectric focusing was used to separate the three components of the antigen 85 complex of Mycobacterium bovis BCG. Antibody responses of leprosy patients against each Mycobacterium bovis BCG. Antibody responses of leprosy patients against each component were quantitated by densitometric analysis of immunoblot assays. The 85A component was recognized by 40% (8/20) of the lepromin positive and negative healthy subjects, by 76% (19/25) of the tuberculoid and by 96% (24/25) of the lepromatous leprosy sera. In contrast, the 85B component was not stained by the control sera, nor by the tuberculoid leprosy sera but by 64% (16/25) of the lepromatous leprosy sera. The results suggest that antigen 85B contains one or several epitopes that are specifically recognized by sera of lepromatous leprosy patients only.

Vaccination of guinea pigs with DNA encoding Ag85A by gene gun bombardment.

A DNA vaccine encoding Ag85A from Mycobacterium tuberculosis was administered to guinea pigs by epidermal gene gun bombardment and its protective efficacy was determined. Vaccination with Ag85A DNA twice significantly reduced the severity of pulmonary pathology and number of pulmonary colony-forming units (CFU) (p<0.01). When immunogenic synthetic Ag85A peptide was used as a booster, lung pathology was improved significantly and pulmonary CFU were reduced dramatically. Neither Ag85A DNA nor BCG Tokyo protected the guinea pigs from hematogenous spread of tubercle bacilli to the spleen because splenic granulomas without central necrosis were recognized. When the vaccinated guinea pigs were followed up for 7 months, the pulmonary lesions became fibrotic in guinea pigs vaccinated with Ag85A DNA plus Ag85A peptide, or BCG Tokyo, and no tubercle bacilli were detected. The protective efficacy of the tuberculosis Ag85A DNA vaccine was improved significantly by peptide boosting. It is concluded that dosage and peptide boosting are important in the induction of higher protective efficacy by a tuberculosis DNA vaccine.

Influence of in vivo immunosuppressive drugs on production of lymphokines.

The influence in vivo of immunosuppressive drugs (cyclosporine, azathioprine, and corticosteroids) on the production of various lymphokines (alpha and gamma interferon, interleukin 2), both in organ transplant recipients and in normal volunteers taking 100 mg hydrocortisone orally has been studied. To avoid interference with the rejection process or viral infection, patients were studied in a steady state with low maintenance immunosuppression consisting of prednisolone combined with azathioprine or with cyclosporine. In patients treated with both drug regimens, significant depression of production of the three lymphokines was found. Normal volunteers challenged with 100 mg hydrocortisone showed inhibition of production of interleukin 2 and alpha and gamma interferon in 4 hr, a time corresponding to the nadir of T cell lymphopenia, affecting the OKT4 subset preferentially. The percentage of OKT8 cells remained unchanged. Percentages of large granular lymphocytes increased, but their absolute number was not significantly modified. Changes in lymphocyte markers were fully reversible after 24 hr, but interleukin 2 production remained markedly depressed, showing that the redistribution patterns induced by corticosteroids on lymphocyte subsets may be dissociated from functional consequences.

Influence of in vivo interferon treatment on mitogen responsiveness of T-and B-cells is dependent on the mouse genotype.

Intravenous inoculation of moderate doses of viral interferon (up to 10(4) I.U./mouse) induced subsequent modification of the mitogen responsiveness of T-and B-lymphocytes from the spleen. The modulation, however, was dependent on the mouse genotype. Whereas BALB/c mice demonstrated enhanced T-cell responses (especially to PHA), C57B1/6 mice were generally suppressed in their T-cell response by the same treatment. B-cell proliferation to LPS showed a moderate enhancement in both strains, probably due to activation of accessory cells.

Suppression of CTL responses in vitro by large granular T cells.

The generation of large granular T cells (LGC) that display cytotoxicity similar to activated natural killer (NK) cells was described previously by the authors. To determine whether LGC can regulate the generation of allospecific CTL (C57BL/6, anti-DBA/2), we used primed responding T cells (Thy-1.2) and (Thy-1.1) to perform co-cultures. The cytolytic activities and the phenotype of the harvested T cells were examined after a co-culture period (3-6 days). We observed that the survival of fresh responding T cells was impaired in the co-cultures. Activated (4 days) responding T cells can survive in similar coculture conditions. This led to the conclusion that LGC can inactivate the CTL generation in fresh T-cell populations and that fresh and activated responding cells have different susceptibilities to LGC interactions.

DNA vaccines against tuberculosis.

DNA plasmids encoding Mycobacterium tuberculosis antigen 85 (Ag85) were tested as vaccines in animal models. Ag85 DNA induced relevant immune responses (i.e. T helper (Th) cells, Th1 cytokines and cytotoxic T lymphocytes) and was protective in mouse and guinea pig models of mycobacterial disease. Therefore, DNA vaccination holds promise as an effective means of preventing tuberculosis in humans. Furthermore, this technique is amenable to identifying the protective antigens of M. tuberculosis.