[Osteitis as a complication of BCG vaccination].

OBJECTIVES: To investigate the incidence and increasing tendency of osteitis after BCG vaccination and, in addition, its clinical features, diagnostic methods and results of treatment. SUBJECTS: 22 cases of Japanese children who received BCG vaccination between 1998 and 2007 and developed osteitis, and were reported in medical journals or meetings. RESULTS AND DISCUSSION: Incidence was very low, 0.2 per 100,000 vaccinations, and an increasing tendency was not seen after 2005, when the vaccination in Japan was limited to below 6 months after birth. However, it might be necessary to follow for much longer period. About 73% of cases of osteitis were seen from 9 to 18 months after receiving the vaccination. The bones of the extremities were commonly affected. Radiography usually showed the defect and cavity formation of the affected bone and often abscess around the lesion. Definitive diagnosis was made by the detection of BCG from the pus or biopsied materials. Recently, multiplex PCR method have been utilized and proved to be a rapid and reliable diagnostic method. Tuberculin reaction was positive, but QFT was negative in all tested cases; QFT will be available for the differential diagnosis of BCG and tuberculous infection. Only 2 patients had multiple lesions, and they had partial interferon-gamma receptor 1 deficiency. Immunodeficiency might have some relationship to the development of osteitis after BCG vaccination. The treatment using INH and RFP was very effective and the outcome was favorable; most of the patients were cured after 6 to 12 months chemotherapy without any complications. However, there is the possibility of defects occurring in the bone and restriction of the articular movement when the diagnosis and treatment are delayed. CONCLUSION: BCG osteitis, although rare, should be considered as a possible complication of the BCG vaccination, and early diagnosis and treatment of this complication is necessary.

[Severe adverse reactions after vaccination with Japanese BCG vaccine: a review].

Japanese BCG vaccine has been admitted by the quality control of World Health Organization (WHO) as the safest BCG vaccine in the world. Even though, BCG, as a live bacterial vaccine, inevitably causes dissemination beyond vaccination site and regional lymph-nodes to various part of the body under certain special conditions. We tried to review the clinical features and immunological status of the cases in which "severe" adverse reactions had developed after vaccination with Japanese freeze-dried BCG vaccine prepared from BCG substrain Tokyo. "Severe" adverse reaction was arbitrarily defined as the adverse reactions of clinical significance developed beyond vaccination site and regional ipsilateral axillary lymph-nodes. By the extensive search of the literatures, 39 cases were identified since 1951 when vaccination with freeze-dried BCG vaccine became compulsory by the Tuberculosis Prevention Law in Japan. Incidence rate was calculated as 0.0182 cases per 100,000 vaccinations. Clinical manifestations of bone and joint were reported in 27 cases (multiple sites: 15 cases, single site: 12 cases), abnormalities in chest X-ray in 13 cases, skin manifestations in 17 cases, diseases in other sites or organs in 8 cases. Most of the cases had lesions in multiple organs. Among these 39 cases, 13 had been diagnosed to have some types of primary immunodeficiency (5 cases: chronic granulomatous disease (CGD); 4 cases: severe combined immunodeficiency (SCID); 4 cases: IFN-gamma receptor 1 deficiency). Further, unidentified defects in cellular immunity were reported in other 6 cases. Death was reported in 6 cases, but in two cases the causes of death were the infections due to different pathogens, namely, pulmonary abscess due to Staphylococcus sp. and bacteremia due to Pseudomonas aeruginosa, respectively, and in only one case death was evidenced as due to disseminated BCG infection by autopsy. All of 6 death cases had some type of immunodeficiency. Apart from fatal cases, outcome of "severe" adverse events were generally favorable and they were successfully treated by anti-tuberculosis therapy with or without surgical treatment for bone and joint lesions. Because the risk of "severe" adverse reactions is high among those of primary immunodeficiency, so it is advisable to avoid BCG vaccination during the first three months after birth when the detection of immunodeficiency is practically impossible.

[Surgical site infection by bacillus Calmette-Guerin (BCG) after radical cystectomy, occurring after intravesical bcg therapy: a case report].

A 51-year-old man received 2 courses of intravesical bacillus Calmette-Guerin (BCG) therapy for carcinoma in situ of the bladder. Two years after the therapy, he underwent left radical nephroureterectomy, cystectomy, urethrectomy and construction of an ileal conduit because of left renal pelvic cancer and severe atrophic bladder. The histopathological diagnosis was carcinoma in situ of the left pelvis and ureter, and epithelioid cell granuloma of left kidney, prostate and bladder. After the operation, he developed extensive surgical site infection (SSI) by BCG, the diagnosis of which was delayed. He recovered from the SSI soon after anti-tuberculosis chemotherapy was begun. We discuss the requirements for more prompt diagnosis of SSI by BCG by analysis of this case.

Identification of two subpopulations of Bacillus Calmette-Guérin (BCG) Tokyo172 substrain with different RD16 regions.

Two types of colonies with different morphologies (smooth: S and rough: R) formed when Bacillus Calmette-Guérin (BCG) Tokyo172 substrain was cultured on Middlebrook 7H10 agar medium, and their genotypes were analyzed by multiplex PCR on five RD regions and SenX3-RegX3. In most cases these two colony types had different genotypes, i.e., S colonies showed a characteristic 22 bp deletion in Rv3405c of the RD16 region (type I), and R colonies did not have this deletion (type II) similar to many other BCG substrains. Thus, there was a strong relationship between colony morphology and genotype. Both genotypes were found in every Tokyo172 preparation tested, including the seed lot for production, the origin of seed lot from the 1960s and ATCC BCG Japan. Type I was always in the majority. It was suggested that types I and II constituted independent subpopulations within the Tokyo172 substrain. Type I was shown to have a growth advantage over type II both on culture media and in mice organs.

Bacillus calmette-guerin Tokyo172 substrain for superficial bladder cancer: characterization and antitumor effect.

PURPOSE: We investigated the preparation of bacillus Calmette-Guerin (BCG) Tokyo172 substrain (Japan BCG Laboratory, Ltd., Tokyo, Japan) on the characteristics of bacilli and antitumor activity in a mouse model in comparison with a preparation of the Connaught substrain (Aventis Pasteur, Ltd., Toronto, Ontario, Canada). MATERIALS AND METHODS: Lyophilized BCG preparations of Tokyo172 and Connaught for superficial bladder cancer were tested. The number of bacilli and cfu per dose, dispersion, size and attachment to murine bladder tumor cells were determined after reconstitution. Antitumor activity was assessed by intradermal injection of tumor cells with various doses of either BCG preparation into the flanks of syngeneic mice, followed by the observation of tumor suppression and survival in mice. RESULTS: Each dose of Tokyo172 had about half the bacilli in a dose of Connaught but the cfu content was about 13-fold higher for Tokyo172 than for Connaught. After reconstitution Tokyo172 bacilli were better dispersed with fewer aggregates than Connaught bacilli. Tokyo172 bacilli were about half as long as Connaught bacilli and Tokyo172 bacilli showed better attachment to tumor cells in vitro. In mice Tokyo172 achieved similar tumor suppression at a lower dose than Connaught. CONCLUSIONS: High viability, good dispersion and efficient binding to tumor cells by BCG bacilli in the Tokyo172 preparation seem to be the main reasons for the lower clinical dose of this preparation compared with the Connaught preparation (18 vs 81 mg dry weight).

Modified multiplex PCR for identification of Bacillus Calmette-Guérin substrain Tokyo among clinical isolates.

When an adverse reaction occurs and a mycobacterial species is isolated from a person vaccinated with Bacillus Calmette-Guérin (BCG) or a patient receiving BCG immunotherapy, it is essential to identify whether the isolate is BCG or another mycobacterial species. However, differentiation of BCG from other members of Mycobacterium tuberculosis complex has been very difficult. Using several specific primer-pairs, Bedwell et al. [Bedwell J, Kairo SK, Behr MA, Bygraves JA. Identification of substrains of BCG vaccine using multiplex PCR. Vaccine 2001; 19: 2146-51] recently reported that they could distinguish BCG substrains. We modified their method to improve differentiation of Tokyo 172 from other members of the M. tuberculosis complex, and examined whether this modified method could be applied to clinical isolates. Our method clearly identified BCG substrain (BCG Tokyo 172) among clinical isolates and easily distinguished between M. tuberculosis and wild-type Mycobacterium bovis.

The BCG scar after percutaneous multiple puncture vaccination may help establish the nationalities of unidentified cadavers.

BCG vaccination using the multiple puncture device (the Heaf gun) has been used in Japan and in the United Kingdom. The appearance of the BCG scars therefore differs from that caused by the conventional intradermal BCG vaccination, which is used throughout the world. The aim of this study was to investigate the usefulness of examining BCG scars to identify the nationalities of unidentified cadavers. We investigated the BCG vaccination program not only in Japan, but also in other countries, along with the relation between the BCG scar and nationality. The results showed that the countries which domestically make and use the Heaf gun are Japan (where it is the only method that has been in use since 1968), the United Kingdom (where it has been used, in part, since 1982), and South Africa (where it has been used, in part, since 1972). In addition, for the past 10 years, the Japanese Heaf gun method has been partially applied in the Republic of Korea and in Brazil. The Heaf gun scar can be clearly distinguished from the intradermal scar, and is visible throughout a person's life when good technique is used to administer the vaccination. If the Heaf gun scar is found on the left upper arm of an unidentified Asian cadaver, it is sure to be that of a Japanese. The findings of this present study indicate that the Heaf gun scar can be examined to identify the nationalities of unidentified cadavers.

Oral recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing HIV-1 antigens as a freeze-dried vaccine induces long-term, HIV-specific mucosal and systemic immunity.

Induction of HIV-1-specific immune responses was evaluated using a recombinant BCG (rBCG) vector-based vaccine expressing HIV-1 Env V3 peptide (rBCG-pSOV3J1). rBCG-pSOV3J1 was manufactured as a freeze-dried preparation based on good laboratory practice guidelines. Guinea pigs were immunized with the freeze-dried rBCG vaccine by oral administration to test the effectiveness of what is generally considered the most convenient and practical route for vaccination. While delayed-type hypersensitivity (DTH) skin reactions to purified protein derivative were not detected in any of the animals receiving oral rBCG-pSOV3J1, HIV-1 V3J1 antigen-specific DTH responses were detected in all of the immunized guinea pigs 1.5 years after immunization. In addition, significant proliferative responses against HIV-1 V3J1 antigen were measured in peripheral blood mononuclear cells and splenocytes from all animals receiving oral rBCG. Interestingly, intestinal intraepithelial lymphocytes from the animals also exhibited high levels of proliferative activity against HIV-1 V3J1 antigen. These results suggest that oral vaccination of guinea pigs with freeze-dried rBCG-pSOV3J1 induces high levels of functional T cells specific for HIV-1 antigens in both mucosal and systemic compartments and suggest that this approach has potential for use as a vaccine against HIV-1.

Immunological protection induced by bacillus Calmette-Guérin treatment in a murine bladder tumor model.

BACKGROUND: It has been previously reported that MBT-2 tumor growth is completely inhibited when mice are inoculated with bacillus Calmette-Guérin (BCG). In this study it was examined whether or not vaccination with a mixture of BCG and MBT-2 cells also induces immunological protection against murine bladder tumors. METHODS: Seven hundred thousand MBT-2 cells and 1 mg of BCG (Tokyo 172 strain) per mouse were injected subcutaneously into female C3H/HeN mice. Four and eight weeks after vaccination with this mixture, animals were reinoculated with MBT-2 cells alone or MBT-2 cells cocultured with BCG. RESULTS: Animals vaccinated with a mixture of BCG and MBT-2 cells showed MBT-2 tumor growth but completely rejected the MBT-2 cells cocultured with BCG. MBT-2 cells cocultured with BCG developed into tumors when they were inoculated into the control animals. Splenocytes prepared from vaccinated animals showed specific cytocidal activity against MBT-2 cells precultured with BCG. CONCLUSIONS: The results suggest that a mixture of BCG and MBT-2 cells induces antitumor immunological protection against BCG- or MBT-2-associated antigens presented on MBT-2 cells precultured with BCG.

Surface antigen expression on bladder tumor cells induced by bacillus Calmette-Guérin (BCG): A role of BCG internalization into tumor cells.

BACKGROUND: The antitumor mechanisms of bacillus Calmette-Guérin (BCG) against bladder cancer is still unclear. We previously reported that BCG was internalized by and survived within murine bladder tumor cells (MBT-2) for at least 40 days. In the present study, we investigated the effect of BCG on the surface antigen expression of bladder tumor cells and the characteristics of these cells as antigen-presenting cells in vitro. METHODS: Surface antigen (major histocompatibility complex (MHC) Class II, CD1, CD80 and intercellular adhesion molecule-1 (ICAM-1)) expression on BCG-treated murine (MBT-2) and human (T-24, J82) bladder tumor cells were analyzed using flow cytometry. The production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) from murine lymphocytes sensitized with BCG or BCG-treated tumor cells were also investigated. RESULTS: The expressions of MHC Class II, CD1, CD80 and ICAM-1 were augmented in all of the bladder tumor cell lines used; however, they were augmented to varying degrees among the cell lines that were treated with live BCG. Heat-killed BCG had little or no effect. When murine lymph node cells sensitized with BCG or BCG-treated MBT-2 cells were cocultured with BCG-treated MBT-2 cells, significant amounts of IL-2 and IFN-gamma were produced in the culture medium. CONCLUSIONS: BCG induced the augmented expression of surface antigens, such as MHC Class II, CD1, CD80 and ICAM-1, of bladder tumor cells. Furthermore, BCG-treated MBT-2 cells could stimulate BCG-sensitized lymphocytes to produce IL-2 and IFN-gamma. These results strongly suggest that bladder tumor cells gained the characteristics and functions of antigen-presenting cells (APC).