Prosthetic hip joint infection by Bacillus Calmette-Guerin therapy following intravesical instillation for bladder cancer identified using whole-genome sequencing: a case report.

BACKGROUND: Joint replacement is an effective intervention and prosthetic joint infection (PJI) is one of the most serious complications of such surgery. Diagnosis of PJI is often complex and requires multiple modalities of investigation. We describe a rare cause of PJI which highlights these challenges and the role of whole-genome sequencing to achieve a rapid microbiological diagnosis to facilitate prompt and appropriate management. CASE PRESENTATION: A 79-year-old man developed chronic hip pain associated with a soft-tissue mass, fluid collection and sinus adjacent to his eight-year-old hip prosthesis. His symptoms started after intravesical Bacillus Calmette-Guerin (BCG) therapy for bladder cancer. Synovasure™ and 16S polymerase chain reaction (PCR) tests were negative, but culture of the periarticular mass and genome sequencing diagnosed BCG infection. He underwent a two-stage joint revision and a prolonged duration of antibiotic therapy which was curative. CONCLUSIONS: BCG PJI after therapeutic exposure can have serious consequences, and awareness of this potential complication, identified from patient history, is essential. In addition, requesting appropriate testing is required, together with recognition that traditional diagnostics may be negative in non-pyogenic PJI. Advanced molecular techniques have a role to enhance the timely management of these infections.

Selection of a new Mycobacterium tuberculosis H37Rv aptamer and its application in the construction of a SWCNT/aptamer/Au-IDE MSPQC H37Rv sensor.

A rapid and accurate detection method for Mycobacterium tuberculosis (M. tuberculosis) is essential for effectively treating tuberculosis. However, current detection methods cannot meet these clinical requirements because the methods are slow or of low specificity. Consequently, a new highly specific ssDNA aptamer against M. tuberculosis reference strain H37Rv was selected by using the whole-cell systematic evolution of ligands by exponential enrichment technique. The selected aptamer was used to construct a fast and highly specific H37Rv sensor. The probe was produced by immobilizing thiol-modified aptamer on an Au interdigital electrode (Au-IDE) of a multichannel series piezoelectric quartz crystal (MSPQC) through Au-S bonding, and then single-walled carbon nanotubes (SWCNTs) were bonded on the aptamer by π-π stacking. SWCNTs were used as a signal indicator because of their considerable difference in conductivity compared with H37Rv. When H37Rv is present, it replaces the SWCNTs because it binds to the aptamer much more strongly than SWCNTs do. The replacement of SWCNTs by H37Rv resulted in a large change in the electrical properties, and this change was detected by the MSPQC. The proposed sensor is highly selective and can distinguish H37Rv from Mycobacterium smegmatis (M. smegmatis) and Bacillus Calmette-Guerin vaccine (BCG). The detection time was 70min and the detection limit was 100cfu/mL. Compared with conventional methods, this new SWCNT/aptamer/Au-IDE MSPQC H37Rv sensor was specific, rapid, and sensitive, and it holds great potential for the early detection of H37Rv in clinical diagnosis.

Sleeping Beauty and the Story of the Bacille Calmette-Guérin Vaccine.

Mycobacterium bovis BCG is the only available vaccine for protection against tuberculosis (TB). While BCG protects children from severe disease, it has little impact on pulmonary disease in adults. A recombinant BCG vaccine BCG ΔureC::hly (strain VPM1002) is in advanced clinical trials and shows promise for improved vaccine safety but little change in efficacy in animal models. A second-generation recombinant BCG vaccine with an additional deletion of the nuoG gene (BCG ΔureC::hly ΔnuoG) shows improved efficacy in a mouse model compared to that of VPM1002. BCG was first used in humans in 1921 and, like Sleeping Beauty pricked by the spinning wheel, we have slept for 100 years, showing a reluctance to invest in clinical development or in biomanufacturing capacity for TB vaccines. The advance of recombinant BCGs should awaken us from our sleep and call us to invest in new-generation TB vaccines and to protect the biomanufacture of our current BCG vaccine.

Crucial requirement for standardization during the development of novel recombinant BCG vaccines: Does the corresponding substrain background matter?

The Bacillus Calmette-Guerin (BCG) vaccine is not a single organism, but consists of substrains that vary in genotypes and phenotypes. Actually, BCG is the common name given to a family of vaccines created in 1921 by the in vitro attenuation of a virulent Mycobacterium bovis in France. Even nearly a century of use, the BCG vaccine lingers generating confusion and debate due to its diversity and failure to protect against tuberculosis (TB). That is probably owing to the enduring lack of standardization during production, distribution and administration procedures. Since the 1940s, substantial sequence modifications among the BCG substrains have been described. To increase the level of complexity, even though that the prolific generation of recombinant BCG vaccines has been promising, the relationships between those candidates used in current clinical trials and their parental substrains are either unsatisfactorily connected or have been never fully delineated. Consequently, the use of the most protective BCG substrain as the background or platform in the development of all recombinant BCG vaccine candidates has not been standardized. In order to schematize and to clarify the subject regarding substrains commonly used to generate those novel vaccines, a sequential emergence of the parental BCG vaccine substrains and their matching recombinant ones, if any, was built. Hence, for a total of 24 BCG substrains currently in circulation worldwide, 9 have been used to sustain one or more genetic modifications, resulting in around 21 novel recombinant BCG vaccines. Although this is a remarkable success, only 2 out of the 21 recombinant BCG substrains harbor a background representative of the most immunogenic group. Systematizing the novel BCG vaccines and their parental strains may facilitate our understanding of protection provided by BCG immunizations.

Ausência de efeito da memória imunológica e antígenos micobacterianos sobre a produção de IgE e citocinas em resposta a Dermatophagoides pteronyssinus / No effect of mycobacterial antigens and immunological memory on the production of cytokines and IgE in response to Dermatophagoides pteronyssinus

Introdução: As doenças respiratórias alérgicas, tais como rinite e asma, afetam elevada proporção da população brasileira. Estima-se que mais de 58 mil pessoas foram afetadas por alguma destas condições no Brasil em 2002-2003. Estudos realizados em humanos e animais sugerem que a exposição ambiental ao Mycobacterium tuberculosis ou imunização com o M. bovis (vacina BCG), podem estar relacionadas à proteção contra doenças alérgicas. Objetivo: Investigar a influência da resposta Th1 a antígenos micobacterianos sobre a modulação da resposta do tipo Th2 ao ácaro Dermatophagoides pteronyssinus (Derp). Métodos: O estudo compreendeu duas fases. Para avaliar o efeito da resposta à revacinação com o BCG sobre a modulação de uma resposta do tipo Th2 ao Derp, foi realizado um estudo de intervenção randomizado com coorte prospectiva, e os voluntários que participaram compuseram a Amostra 1. Para avaliar o efeito da resposta à infecção latente com M. tuberculosis sobre a modulação de uma resposta do tipo Th2 ao Derp, foi feito um estudo de caso-controle e os voluntários que participaram compuseram a Amostra 2. A população foi composta por adultos jovens com idade entre 19 a 33 anos. Todos responderam ao questionáro ISAAC...

Introduction: Allergic respiratory diseases such as asthma and rhinitis, affecting a high proportion of the Brazilian population. More than 58.000 people have been affected by some of these conditions in Brazil in 2002-2003. Studies in humans and animals suggest that environmental exposure to Mycobacterium tuberculosis or immunization with Mycobacterium bovis (BCG), may be related to protection against allergic diseases. Objective: To investigate the influence of Th1 response to mycobacterial antigens on the modulation of Th2-type response to aeroallergen Dermatophagoides pteronyssinus (Derp). Methods: The study comprised two phases. To evaluate the effect of the response to revaccination with BCG on the modulation of a Th2-type response to Derp, we conducted a randomized intervention study with prospective cohort, and the volunteers composed the Sample 1. To evaluate the effect of latent response to infection with M. tuberculosis on the modulation of a Th2-type response to Derp, a study was made of case-control and the volunteers composed the Sample 2. The population consisted of young adults aged 19 to 33 years. All responded to questionnaire ISAAC...

Application of heat killed Mycobacterium bovis-BCG into the lung inhibits the development of allergen-induced Th2 responses.

We have previously reported that an infection of the lung with BCG-inhibited ovalbumin (OVA)-induced airway eosinophilia. In the current study, we investigated if the intranasal application of heat killed (HK)-BCG or purified protein derivative (PPD) from Mycobacterium tuberculosis had the same effect. For this purpose we treated mice intranasally with either live BCG, HK-BCG or PPD and analyzed if the mice developed airway eosinophilia after immunization and intranasal challenge with OVA. Our results clearly showed that an intranasal vaccination with live and HK-BCG but not PPD, given 4 or 8 weeks prior to allergen airway challenge, resulted in a strong suppression of airway eosinophilia. The inhibition of airway eosinophilia correlated with reduced levels of IL-5 production by T cells from the lymph node of the lungs and a strong reduction in Th2 cell numbers present in the airways of OVA-challenged mice. Furthermore, HK-BCG-induced suppression of airway eosinophilia was strongly reduced in IFN-gamma deficient mice. HK-BCG in contrast to live BCG may also be a promising candidate for a prospective asthma vaccine in humans since negative side effects due to mycobacterial infection can be ruled out.

How can immunology contribute to the control of tuberculosis?

Tuberculosis poses a significant threat to mankind. Multidrug-resistant strains are on the rise, and Mycobacterium tuberculosis infection is often associated with human immunodeficiency virus infection. Satisfactory control of tuberculosis can only be achieved using a highly efficacious vaccine. Tuberculosis is particularly challenging for the immune system. The intracellular location of the pathogen shields it from antibodies, and a variety of T-cell subpopulations must be activated to challenge the bacterium's resistance to antibacterial defence mechanisms. A clear understanding of the immune responses that control the pathogen will be important for achieving optimal immunity, and information provided by functional genome analysis of M. tuberculosis will be vital in the design of a future vaccine.

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens.

Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovis BCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10) as or at an even higher level (TB10.4) than ESAT-6. The non-ESAT-6 family member, TB7.3, for comparison, was recognized at a much lower level. CFP10 was found to distinguish TB patients from BCG-vaccinated donors and is, together with ESAT-6, an interesting candidate for the diagnosis of TB. The striking immunodominance of antigens within the ESAT-6 family is discussed, and hypotheses are presented to explain this targeting of the immune response during TB infection.

Immunogenicity and safety of Mycobacterium tuberculosis culture filtrate proteins in non-human primates.

The development of improved vaccines is considered a high priority in the effort to control tuberculosis (TB) world wide. Results from several studies performed in relevant animal models have demonstrated that Mycobacterium tuberculosis secreted antigens may represent major components of improved TB vaccines. To characterize further the M. tuberculosis secreted antigens as they relate to specific features important for vaccine development, rhesus macaques were immunized with either one of two different preparations containing M. tuberculosis culture filtrate (CF) proteins. These preparations differed in relative protein content and in the presence or absence of lipoarabinomannan. Animals received a total of three monthly intramuscular injections consisting of CF proteins resuspended in RIBI adjuvant and were tested for development of specific antibody and cellular proliferative responses. In addition, all animals were constantly monitored for local and systemic reactions as well as for the development of DTH reactions to intradermal tuberculin injection. Results from this study show that the two CF preparations are relatively safe and immunogenic in non-human primates. These two CF preparations differed in their ability to induce specific antibody responses, but were comparable in their ability to induce specific cellular proliferative responses. Induction of both humoral and cellular responses occurred even in presence of pre-existing antibodies directed against M. tuberculosis antigens. However, these responses appeared to be short-lived. Only one of the four animals produced interferon-gamma (IFN-gamma) in response to immunization with CF proteins. No DTH reaction to intradermal tuberculin injection was observed in any immunized animal. Although it is clear that additional studies are required to design strategies for the improvement of the immunogenicity of CF proteins, our observations support the currently accepted view that secreted protein-based preparations may represent promising vaccine candidates for TB.

Gene replacement by homologous recombination in Mycobacterium bovis BCG.

Gene replacement by homologous recombination is a powerful tool for fundamental studies of gene function, as well as allowing specific attenuation of pathogens, but has proved difficult to achieve for Mycobacterium tuberculosis. We have used a plasmid-based test system to demonstrate the occurrence of homologous recombination in the tuberculosis vaccine strain Mycobacterium bovis BCG, and we have successfully replaced a target gene in BCG by homologous recombination, using a shuttle plasmid. Specific inactivation of selected genes will facilitate study of virulence factors and drug resistance as well as allowing rational attenuation of M. tuberculosis for the production of new vaccines.

The T cell response to secreted antigens of Mycobacterium tuberculosis.

Recent information from several laboratories points to proteins secreted from live Mycobacterium tuberculosis as being involved in protective immunity. We have studied protein release from M. tuberculosis during growth and have defined 3 different groups of proteins: excreted proteins, secreted proteins of the outer cell wall and cytoplasmic proteins released at late culture timepoints. These findings have lead to the definition of a short-term culture filtrate (ST-CF) enriched in excreted/secreted proteins and with a minimal content of autolytic products. ST-CF was tested as antigen in experimental vaccines against tuberculosis. A vaccine based on the adjuvant dimethyldioctadecylammonium chloride (DDA) was constructed and demonstrated to induce a potent cell mediated immune response of the Th-1 type. The vaccine was tested in parallel with a BCG standard vaccine and both vaccines induced a highly significant protection of the same magnitude. Molecules within the Ag85 complex and a 6-kDA secreted protein were mapped as the major antigenic targets for long-lived T cells involved in protective immunity against M. tuberculosis.

Immunotherapy of tuberculosis with Mycobacterium vaccae NCTC 11659.

The history of immunotherapy for tuberculosis is briefly reviewed, and the early appreciation of the importance of secreted antigens, common mycobacterial antigens and stress proteins is noted. The methods by which Mycobacterium vaccae strain NCTC 11659 was selected for special attention, and results of some of the pilot studies of its use as an immunotherapeutic for tuberculosis are reviewed. The results suggested that immunotherapy with M. vaccae may be an important step forward in the treatment and eventual control of tuberculosis. Used in combination with modern short course chemotherapy, treatment failures and deaths during treatment can be significantly reduced. Preliminary data suggests that shortened courses of chemotherapy may be possible when combined with immunotherapy, and such treatment may also be effective in patients co-infected with HIV. Studies at several centers show that M. vaccae may have an important part to play in the treatment of multi-drug resistant tuberculosis, especially when resistance is of the primary type. The mechanism by which M. vaccae achieves these results may be through adrenal endocrine influences on immunity, but remains speculative.

The Mycobacterium bovis 32-kilodalton protein antigen induces human cytotoxic T-cell responses.

The 30-kDa protein (P32) is a mycobacterial secreted antigen which is homologous in Mycobacterium bovis and M. tuberculosis. In vitro, P32 induced T-cell proliferation. M. tuberculosis- or P32-stimulated T-cell lines lysed macrophages pulsed with P32 or M. tuberculosis, respectively. We conclude that P32 stimulates cytotoxic T cells specifically.

[Test-systems of polymerase chain reaction for determining the tuberculosis pathogen]. / Test-sistemy na osnove polimeraznoi tsepnoi reaktsii dla identifikatsii vozbuditelia tuberkulëza.

Two polymerase chain reaction-based test systems were used to identify Mycobacteria tuberculosis by using a clinical material for practical health purposes. The former test system strictly specifically enables M. tuberculosis to be detected. The latter also allows one to differentiate a BCG vaccine strain from the M. tuberculosis. The test systems were tested by using 11 clinical sputum samples, three of them were taken from patients with fibrocavernous tuberculosis, two from those with focal tuberculosis and one from that with infiltrative one, 5 from those with non-specific pulmonary diseases (chronic bronchitis and asthma). In addition, one serum sample from a patient with fibrocavernous tuberculosis was examined. All tests obtained from patients with tuberculosis were positive, whereas those from patients with non-specific pulmonary diseases were negative. This suggests that differentiation of BCG vaccine strain from M. tuberculosis can be used both in model experiments and in the study of clinical material lysates.

Rapid electroelution of two-dimensionally separated protein mixtures: its use in in vitro assays of T cell activities.

A recently developed electroelution method for separated mixtures of proteins and its application in vaccine research were investigated. The method combines the high resolution power of two-dimensional gel electrophoresis with the advantage of direct probing of separated proteins with viable cells. An electroelution time of only 30 min was sufficient for complete protein transfer, as shown by Coomassie Brilliant Blue and silver staining. Inclusion of sodium dodecyl sulfate (SDS) into the electrophoresis buffer for the second dimension considerably improved the separation capacity. Furthermore, because of the low concentration of SDS (0.03%) no deleterious effects on the cells were seen. It was shown that T lymphocytes from cattle vaccinated with dead M. bovis BCG responded to numerous mycobacterial protein antigens, whereas unvaccinated control animals showed no, or very weak, responses. A comparison of T cell proliferation profiles obtained with different protein separations demonstrated the reproducibility of the method.

Protective efficacy of different cell-wall fractions of Mycobacterium tuberculosis.

Immunization with various cell-wall fractions of M. tuberculosis H37Ra, progressively depleted of lipids (cell-wall-insoluble fraction; CWIF), soluble proteins (cell-wall core; CWC), mycolic acids and arabinogalactans (cell-wall-protein-peptidoglycan complex; CW-PPC) elicited significant levels of both humoral and cell-mediated immune response. Mice immunized with these fractions, when challenged with an LD50 dose of M. tuberculosis H37Rv, exhibited significant protection as revealed by high survival rates and decreased bacterial load in lungs, liver and spleen, as compared to nonimmunized animals.

Development of BCG as a live recombinant vector system: potential use as an HIV vaccine.

Bacille Calmette-Guèrin (BCG), a live attenuated tubercle bacillus, is currently the most widely used vaccine in the world. Because of its unique characteristics, including low toxicity, adjuvant potential, and long-lasting immunity, BCG represents a novel vaccine vehicle with which to deliver protective antigens of multiple pathogens. We have developed episomal and integrative expression vectors employing regulatory sequences of major BCG heat shock proteins for stable maintenance and expression of foreign antigens in BCG vaccine strains (22). Shuttle plasmids capable of autonomous replication in Escherichia coli and BCG were constructed with a DNA cassette containing a minimal replicon derived from the Mycobacterium fortuitum plasmid pAL5000. Efficient and stable chromosomal integration of recombinant plasmids into BCG was achieved using a DNA segment containing the mycobacteriophage L5 attachment site and integrase coding sequence. Using the BCG hsp60 and hsp70 stress gene promoters, we were able to express Escherchia coli beta-galactosidase to levels in excess of 10% of total cell protein. The major antigens of HIV-1 gag, pol, and env were also stably expressed using our vector systems. The recombinant BCG elicited long-lasting humoral and cellular immune responses to these antigens in mice. Antibody responses to beta-galactosidase using as few as 200 colony-forming units were detected 6 weeks after immunization, and titers (1:30,000) were sustained for more than 10 weeks. Cellular immune responses, of both cytotoxic T cell (CTL) and helper T lymphocytes, were detected to beta-galactosidase. CTL responses were also induced to the HIV-1 envelope protein. Thus, we have demonstrated stable recombinant antigen expression, processing, and presentation using our recombinant BCG vector system. This live recombinant vector system shows promise as a universally applicable and safe vaccine vehicle for protection against various infectious diseases.