BCG vaccines for the prevention of tuberculosis in the world]

The BCG vaccines will celebrate the 100th anniversary of their discovery in a decade at the beginning of the next century since Albert Calmette and Camille Guerin had presented it before the Academie des Sciences in 1908. At present tuberculosis kills more people than any other infectious disease about 3 million people a year, including almost 300,000 children under 15, and is producing over 7,000 deaths and over 24,000 new cases every day. Therefore, WHO declared a global health emergency in 1993. More worse, recently multi-drug resistant tubercle bacilli are emerging rapidly making TB patients incurable. Under these situations we need a potent anti-tuberculosis vaccine. So first of all, we must check the century-old BCG before proceeding further. At moment, the BCG vaccines are being used worldwide in the largest quantities in the world, but still most controversial vaccines anywhere. I would like to describe here their success and failure in the combat against the white plague. 1. The Expanded Programme on Immunization (EPI). In 1974, when the EPI was launched by WHO, less than 5% of the world children were immunized against six infectious diseases including tuberculosis. In 1995 statistics, BCG gave the highest vaccination coverage, 87% higher than any other 5 vaccines of EPI for children. The BCG in EPI must have saved a lot of infants as the vaccine, has been proved to be most effective against the blood-born tuberculosis of child type. 2. The efficacy of BCG vaccination against tuberculosis. Results of each 10 of randomized controlled trials (RCT) and Case-control studies (CCS) showed the protective efficacy against tuberculosis as uncertain, unpredictable, as protective efficacy varied from 80% to 0%. More recently, a Meta-analysis of selected papers on BCG field trials which were so far collected. They recalculated vaccine protective effect separately for pulmonary TB and for meningeal/miliary TB in the trials. As the result, it was found that protective effect against pulmonary TB could not be calculated, but protective effect against meningeal and miliary TB was calculated as 86%, 75% respectively, in RCT and CCS, being higher than against pulmonary TB. 3. The duration of BCG efficacy against tuberculosis was confirmed to continue for 15 years after vaccination. The incidence of every form of tuberculosis decreased steeply during the 15 years following vaccination. 4. BCG revaccination. A WHO statement was issued in 1995 mentioning that there is no definitive evidence that repeated BCG vaccination confers additional protection against tuberculosis. Therefore WHO has not recommended to repeat BCG vaccination because of no scientific evidence to support this practice. Multiple BCG revaccinations are not indicated in any persons. 5. Complications with BCG Second IUATLD study (1988) on complications induced by BCG was reviewed, especially following two points: 1-2) Regional suppurative lymphadenitis 3) Generalized lesions: fatal cases 1-2 Several African regions had experienced that the risk of outbreak of suppurative BCG lymphadenitis was low for vaccines with Glaxo and Japanese strains, but much higher for vaccines with Pasteur. This experience in nineteen eighties has led EPI to replace the Pasteur BCG vaccine with less reactogenic BCG, Japanese or Glaxo BCG to solve the outbreak of suppurative adenitis complication. 3 At moment, the only contra-indication of EPI BCG vaccination is symptomatic HIV infection (AIDS), but in the future asymptomatic HIV infection should be placed on alert, because fatal BCG generalized disseminations have already been experienced by HIV positive vaccinees although in a few cases in USA. 6. BCG seed lots for use of vaccination in the world. Nearly 10 seed lots (BCG) are being used in the world at present. However, they are more or less different each other in various characteristics: morphological, biochemical, biophysical, immunological, vaccinological and so on. None of them is the same     

Policy program to minimize spread of infection. Prolonged cough may be a sign of tuberculosis]

In a worldwide epidemiological perspective, Sweden is well favoured with an annual tuberculosis incidence of approximately six cases per 100,000 of the population. Neither the impact of the HIV pandemic nor the occurrence of multiresistant strains of Mycobacterium tuberculosis has yet become a major problem in the care of tuberculosis patients in Sweden. Only a few per cent of HIV patients have developed tuberculosis, and during the period, 1991-94, only one per cent of M. tuberculosis isolates in Sweden were resistant to such antimycobacterials as isoniazid and rifampicin. However, the epidemiological situation in the neighbouring Baltic states is a matter for concern. Bovine tuberculosis has been eradicated in Sweden, the last case having been diagnosed in 1978. Although the reported efficacy of BCG (bacillus Calmette-Guérin) tuberculosis vaccine varies according to the population studied, protective rates of 70-85 per cent have been reported for Sweden and other west European countries. Re-vaccination of tuberculin-negative individuals has not been shown to yield added protection. The aim of a national programme for protection against tuberculosis is to preserve our favourable epidemiological situation by early detection of new cases, effective contact tracing, and BCG vaccination of children in population groups at risk. The primary means of achieving this is the education of health care personnel to retain tuberculosis as a differential diagnosis. Moreover, national guidelines for contact tracing must be duly observed, and immigrants from high prevalence areas need to be screened for tuberculosis. Registration of all cases of tuberculosis should be maintained at regional and national levels, and follow-up must be meticulous until a successful outcome of treatment is accomplished. Recommendations for dealing with tuberculosis should be made available and duly implemented at all hospitals caring for tuberculosis patients, in order to avoid nosocomial transmission. Although BCG vaccination at birth was formerly general in Sweden, since 1975 only children considered to be at risk have been vaccinated. Thus, non-vaccinated young adults are now entering the health care sector as students or employees, and should be offered BCG vaccination. Moreover, the epidemiological situation both in Sweden and in neighbouring countries needs to be monitored carefully in order that recommendations concerning BCG vaccination and other preventive measures can be modified if necessary.     

Detection of bacillus Calmette-Guerin in the blood by the polymerase chain reaction method of treated bladder cancer patients

PURPOSE: Following intravesical bacillus Calmette-Guerin (BCG) instillation, we attempted to detect BCG in the blood using the polymerase chain reaction (PCR) method and correlate these findings with the occurrence of major complications due to this treatment. MATERIALS AND METHODS: Intravesical BCG immunotherapy was given to 22 consecutive patients with superficial bladder tumors. In 2 patients the BCG instillation had to be discontinued due to serious side effects of therapy. Blood samples (252 aliquots) were obtained from 126 BCG courses in 22 cases, and 2 additional samples (4 aliquots) were obtained from 1 patient 1 and 3 months after cessation of therapy. All blood samples were analyzed by the PCR technique for detection of deoxyribonucleic acid tuberculosis Mycobacterium tuberculosis. RESULTS: Of the 126 blood samples 9 (7.1%) were PCR positive for M. tuberculosis. These 9 positive samples belonged to 3 patients, all of whom were among those 4 patients who had major clinical side effects. CONCLUSIONS: We demonstrated that rapid and sensitive detection of mycobacteremia by PCR correlated with the clinical course of these patients. We also demonstrated that PCR can be used to monitor BCG in the blood after antituberculous therapy. The early, fast and accurate diagnosis of BCG in the blood by PCR may alter the serious clinical course of these patients by initiation of specific treatment early. However, further extensive studies are needed to validate these results.     

Tuberculosis in BCG-vaccinated and unvaccinated young Swedish Men. A comparative study

Tuberculosis causing exemption from military service was studied in 47,791 tuberculin-tested military recruits between 1941 and 1946. A total of 46.6% were tuberculin negative, and two-thirds were BCG vaccinated. An analysis was made of 304 cases of tuberculosis discovered from 6 months after the test up to the end of 1960. It has been calculated that 69% of "expected cases" of tuberculosis were in the BCG-vaccinated men during the 5-year period after the test, and 20% during the following period; 46% of "expected cases" of post-primary pulmonary tuberculosis and 83% of "expected" deaths were prevented. The differences on which these percentages are based were statistically significant. The efficacy of BCG vaccination was lower in areas with widespread cattle tuberculosis than in areas where cattle tuberculosis is less frequent; this is explained by the influence of previously acquired immunity.     

Phase transitions in phospholipid monolayers at the air-water interface: a fluorescence study

A study was made of the specific sensitivity to tuberculin in the animals vaccinated with BCG in case of their additional sensitization with various atypical mycobacteria. The mentioned experimental study appeared to be necessary for the purpose of a more proper treatment of the epidemiological date referred to the sensitivity of man to tuberculin and sensitins against the background of mass BCG vaccination. Preliminary BCG vaccination of the animals with their subsequent infection with atypical mycobacteria altered the allergic response to the antigens from the mycobacteria increasing the response reactions not only to tuberculin, but also to sensitins from mycobacteria of the I--III groups by Runyon's classification, closely connected in antigenic respect with mycobacteria tuberculosis. Skin reactions to sensitins from the saprophytic mycobacteria which had in their composition much less common antigens with mycobacteria tuberculosis, remained at the low level. Sensitization with atypical mycobacteria of animals preliminarily vaccinated with BCG failed to cause significant influence on the production of immunity to the subsequent virulent infection with tuberculosis.     

Tuberculin tests in school leavers in canton Berne , Neuenburg and Wallis 1992/1993

To determine the prevalence of tuberculosis infection in Switzerland, standardized tuberculin tests using 2 units of tuberculin Berna PPD RT 23, administered by specially trained personnel, were performed on school leavers in 3 Swiss cantons in 1992/1993. Of the 7036 school leavers, averaging 15 years of age, only 294 (4.18%) were not BCG-vaccinated. Non-vaccinated persons had tuberculin test indurations > 15 mm in 2.04% (6663 BCG vaccinated persons in 1.14%). Calculations of potentially influential factors using stepwise ordinal polychotomous regression showed that tuberculin test indurations are significantly larger after BCG vaccination, as well as with increasing age at immigration from high prevalence tuberculosis countries. Indurations were smaller with increasing time passed since BCG vaccination, as well as in females. Pets at home did not significantly influence the size of tuberculin reactions. Theoretically the positive predictive value of tuberculin tests in Switzerland is small because of the low tuberculosis prevalence. From our data the maximal prevalence of infection in 15-year-olds is estimated at 0.91% (2.48% in the non-vaccinated) in Swiss and 2.54% (9.77% in the non-vaccinated) in foreign born school children. These rates, higher than extrapolated from previous studies, are comparable to data from other industrialized countries. They do not warrant a change in BCG vaccination policy in Switzerland, which since 1987 requires BCG vaccination in children immigrating from countries with high tuberculosis prevalence only.     

Using the outcome-based quality improvement model and OASIS to improve HMO patients'' outcomes. Outcome Assessment and Information Set

In Japan, BCG vaccination, which covers more than 90% of infants, has been given according to the national immunization policy. Moreover, first-grade children in elementary school are screened with tuberculin skin test, and those who show negative reaction in the Japanese standard, i.e. size of erythema less than 10 mm, are re-vaccinated with BCG according to the Tuberculosis Prevention Law. However, since the incidence of tuberculosis among children below age 14 is as low as 1.5/100,000 in Japan, it is time to reconsider the BCG vaccination policy. As the first step to assess the efficiency of the present program, we observed the occurrence of Koch's phenomenon after BCG vaccination in elementary school children in Chiba City in 1995 and 1996, and we introduced the two-step tuberculin test to elementary school children in 1997. Among 180 BCG vaccinated children in 1995 and 1996, 168 (93.3%) had been vaccinated by 4-year of age. We could follow local reaction of BCG re-vaccination and observed Koch's phenomenon in 117 (69.6%, 95% C.I. of 62.7-76.6%). Among 92 tuberculin negative children in 1997, 85 (92.4%) had been vaccinated by 4-year of age. In the two-step tuberculin test program of 85 initial negative-reactors, 63 (74.1%, 95% C.I. of 64.8-83.4%) turned to positive by the second test. Those results suggest that more than 69% of tuberculin-negative school children who were vaccinated previously maintained immunity with BCG. Our studies raised a problem of the current BCG re-vaccination policy that depends on the result of tuberculin test. Due to the discrepancy between tuberculin allergy and immunity in tuberculosis, many school children may be given BCG vaccination unnecessarily. Taking into consideration the incidence of tuberculosis in children, discontinuation of BCG re-vaccination policy at elementary school entrance should be considered.     

Results of the BCG vaccination in Hungary since 1929: evaluation of preventive and immunotherapeutic effectiveness

BACKGROUND: The BCG (Bacille Calmette-Guérin), a living attenuated bacterial vaccine with a characteristic residual virulence, has been used to prevent tuberculosis since 1921 (in Hungary non-systematically since 1929) and applied for immunostimulation in neoplasia since the 1960s. MEASURES: Considering the grave tuberculosis epidemiological situation in Hungary, the BCG revaccination became compulsory up to 20 years old tuberculin negatives since 1959. The Pasteur P1173P2 BCG strain has been used for vaccine manufacturing with improved quality control methods according to the requirements of the WHO. With in systematic BCG primo and revaccination policy 8.1 million BCG vaccination from 1959 to 1983 then further 3.1 million between 1984 and 1996 have been performed. RESULTS: Linear regression analysis demonstrates that the decrease of the TB incidence in children was 3-5 times more rapid (annual average decrease was 25.5%) than in adult since 1959. Multiple regression analysis indicates that the BCG is the strongest explanatory variable decreasing children TB incidence among other antituberculosis measures. The BCG vaccination efficacy ins demonstrated by 2 x 2 table analysis. The systematic BCG vaccination, the living and persisting BCG in the macrophages, confers acquired resistance against virulent TB infections. The immunostimulation in neoplasia has been applied with concentrated BCG developed in Hungary since 1979. The adverse reactions are at accepted frequency. The number of BCG vaccinated subjects was estimated at 1.5 billion from 1948 to 1974 in the world. The yearly number of BCG vaccination in the WHOI-EPI System is estimated 50-100 million. CONCLUSION: The efficacy of the BCG vaccination can only be ensured if the vaccine is manufactured and controlled with standardized methods, and applied in a systematic vaccination programme. The effectiveness has to be evaluated in statistically valid biostatistical models.     

Tuberculosis and its control programme in Japan

The author analyses the factors which brought about the rapid decline of tuberculosis in Japan during the past 30 years. Among the modern measures combating tuberculosis, chemotherapy and the extensive use of BCG vaccination are at the paramount place. As a result of intensive control the problem of tuberculosis has become smaller and smaller and a turning point is reached. The future direction of tuberculosis control is determined by chemoprophylaxis of high risk groups and by symptomatic casefinding combined with selective mass miniature radiography for high risk groups using high technical standards. By means of intensive initial chemotherapy the duration of treatment will be shortened. In 1974 the policy of BCG vaccination was changed; primary vaccination is provided for children in the age of 0 to 3 years, and revaccination at entrance to primary school and in leavers from middle school for tuberculin non-reactors. If the annual risk of infection will continue to decline the primary vaccination age it intended to be raised up to school entrance.     

Bacillus Calmette-Guérin infection after vaccination of human immunodeficiency virus-infected children

The use of Mycobacterium bovis/Bacillus Calmette-Guérin (BCG) to vaccinate against tuberculosis remains controversial. The development of tuberculosis in human immunodeficiency virus (HIV)-infected children demands specific evaluation of the risk/benefit ratio of BCG vaccination in this situation. In our institution 9 of 68 HIV-infected children vaccinated with BCG before the diagnosis of HIV infection was suspected developed vaccine-related complications: 7 of these children had a large satellite adenopathy with or without skin fistulae, whereas the other 2 had disseminated BCG infection beyond the satellite ganglion (involvement of the spleen and mesenteric and mediastinal lymph nodes in one case and the liver and lungs in the other). The children were vaccinated soon after birth; no particular problems were observed at that time, but complications appeared 3 to 35 months later. All but one of these children had a rapidly progressive form of HIV disease. The possibility of delayed local or disseminated BCG infection must be considered in analysis of the risk/benefit ratio of vaccination of HIV-infected children. The prognosis of HIV infection must be taken into account, even if the child is asymptomatic when vaccination is being considered.     

Expression of the HGF/SF receptor, c-met, and its ligand in human colorectal cancers

To characterize the clinical features of childhood tuberculosis, we analyzed the symptoms, signs, and laboratory findings of the 89 children with tuberculosis admitted to the Yokohama City University Hospital from 1975 to 1994. Compared with the numbers of patients admitted from 1975 to 1979, those of patients of the past 5 years (from 1990 to 1994) were reduced by half. Of the 89 subjects, 56.2% were below 3 years of age and 24.7% were under 1 year of age. 51.7% had primary complex and 20.2% had serious tuberculosis (tuberculous meningitis 14.6%, miliary tuberculosis 3.4%, and bone and joint tuberculosis 2.2%). Tuberculous children below 3 years of age consisted of primary complex (60.0%) and serious tuberculosis (32.0%). The majority (86.0%) of tuberculous children below 3 years of age had not received BCG vaccination. In 55 (61.8%) of 89 subjects, the sources of tuberculosis were clarified. Of these subjects, 83.6% were infected in the family. The rate of BCG inocluation tended to decrease with decreasing age, especially that of children below 3 years of age was 14.0%. Of the 89 subjects, only 16.9% proved to be smear-positive. Taken together, in order to eliminate tuberculous children below 3 years of age, the following is necessary; (1) BCG inoculation in early infancy, (2) early diagnosis of index cases with adult tuberculosis, and (3) prompt and appropriate family contact examination.     

Specific differentiation between Mycobacterium bovis BCG and virulent strains of the Mycobacterium tuberculosis complex

A PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system, named SenX3-RegX3, was developed and was shown to be suitable for identifying Mycobacterium bovis BCG. The senX3-regX3 IR contains a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). All tested BCG strains exclusively contained 77-bp MIRUs within the senX3-regX3 IR, whereas all non-BCG M. tuberculosis complex strains contained a 53-bp MIRU, in addition to the 77-bp MIRUs. All 148 strains analyzed so far could be divided into eight different groups according to the copy numbers of the 77-bp MIRU and to the presence or absence of the 53-bp MIRU. BCG strains contained either one, two, or three 77-bp MIRUs. The other strains contained one to five 77-bp MIRUs invariably followed by a 53-bp MIRU. The consistent absence of the 53-bp MIRU in BCG strains and its presence in virulent strains allowed us to develop an enzyme-linked immunosorbent assay using specific capture oligonucleotide probes to distinguish between BCG and other M. tuberculosis complex strains.     

Tuberculosis in children

Newly registered active tuberculosis in children is under 300 persons a year in Japan, now. In order to lessen further and exterminate childhood tuberculosis in near future, physician have to make effort to find the infected children by good-timing thorough surveillance when he find an adult tuberculosis patient. Chemoprophylaxis could prevent the infected children from progressing to active disease. BCG vaccination in infantile period is still important in Japan, especially to protect young children from disseminated tuberculosis. Pulmonary tuberculosis in children is successfully treated with 6 months standard multi-drugs therapy.     

Transfer to the leukocytes of neonates by means of the plasma of patients with tuberculosis and of healthy persons of the ability to depress migration under the influence of antigens of mycobacterium tuberculosis in vitro

A study was made of the effect of the blood plasma of patients suffering from tuberculosis and of healthy individuals on the in vitro migration in the presence of old tuberculin and the BCG vaccine of the leukocytes of neonates sensitive and insensitive to these antigens. Plasma of tuberculosis patients and more rarely of healthy persons created favourable conditions for depression of migration with old tuberculin and the BCG vaccine of the neonatal leukocytes insensitive to it on autoplasma, i.e. realized the transfer of the cellulo-mediated reactivity. In some cases the neonatal leukocyte migration by tuberculin on autoplasma was absent on the plasma of patients with tuberculosis of the lungs. A conclusion was drawn that the blood plasma of neonates suffering from tuberculosis and of adult healthy individuals contained quantitatively and qualitatively different factors determining different sensitivity of their leukocytes to the Mycobacteria antigens.     

Effectiveness of mass neonatal BCG vaccination in the prevention of pulmonary tuberculosis: a case-control study in Nagpur, India

SETTING: Government Medical College, Nagpur, India, a tertiary care hospital. OBJECTIVE: To estimate the effectiveness of mass neonatal BCG vaccination in the prevention of pulmonary tuberculosis in Nagpur, India. DESIGN: A hospital-based pair-matched case-control study with a case of 1:3, including 144 cases of pulmonary tuberculosis and 432 controls. RESULTS: The overall vaccine effectiveness estimated in the present study was 60% (95% Confidence Interval [CI] 43%-72%). The protective effect was more in males in the age group 21-30 years. The prevented fraction was 39% (95% CI 24%-52%). CONCLUSION: The moderate effectiveness demonstrated in this study needs to be substantiated for other forms of tuberculosis by undertaking community-based case-control studies, before attempting to justify the use of mass neonatal BCG vaccination strategy as a part of the national programme.     

Risk of complications during endovesical treatment with BCG for superficial tumor of the bladder

OBJECTIVES: BCG therapy is the reference adjuvant treatment for multiple and voluminous or recurrent superficial bladder cancer and can cause specific complications. We assessed the frequency and therapeutic modalities involved associated with such complications in a personal retrospective series of patients. PATIENTS AND METHODS: BCG therapy was given to 148 patients who were followed for a mean 40 months. RESULTS: Forty-six percent of the patients developed a follicular reaction in the bladder wall: 14 developed complications requiring anti-tuberculosis treatment. The frequency of BCG therapy complications was higher in patients who had had tuberculosis previously (50%) than those who did not (13.8%). Complications were more frequent after early treatment. In the patients who developed BCGitis with fever, a 3-month regimen of rifampicin and isoniazine appeared to be sufficient. DISCUSSION: The frequency of BCG therapy complications (bladder anomalies excluded) was 7.8% with only 2.8% major complications. The development of a follicular reaction of the bladder wall does not appear to have any prognostic value. Special surveillance is needed in patients with a past history of tuberculosis. CONCLUSION: Adjuvant BCG therapy requires careful follow-up because of the risk of BCGitis which can be effectively treated with a 3-month double-antibiotic regimen.     

Clinical manifestations and epidemiology of childhood tuberculosis in Stockholm 1976-95

81 cases of tuberculosis infection (17) and disease (64), seen between 1977 and 1995 at St G?ran's Children's hospital, Stockholm, Sweden are reviewed. The incidence of tuberculosis disease increased from 1 to 6/10(5) children/y. The increase was due to immigration from high-prevalence countries, with an incidence of 20/10(5) in a partly segregated suburb. Most of the children were foreign-born. Of the 31 0-4-y-old cases, 19 were born in Sweden, and 7 had received BCG vaccination. For Swedish-born children with Swedish-born parents, the incidence of tuberculosis disease remained stable at < 0.5. 50 patients were symptomatic when first seen (60% pulmonary tuberculosis, 8% military tuberculosis, 25%, cervical adenitis, 15% other extrapulmonary tuberculosis). There was 1 death, and in 2 children complicated tuberculosis courses. Side effects of drug therapy were seen in 5% of the children. In conclusion, tuberculosis remains an important differential diagnosis in children of immigrants from high-prevalence countries for at least 5 y after settlement in Sweden. The practice of delaying BCG vaccination of them until 6 months of age can be disputed.     

Serodiagnosis of tuberculosis by detection of antituberculous glycolipid antigen (TBGL antigen) antibodies in serum using enzyme-linked immunosorbent assay: clinical evaluation of anti-TBGL antibodies assay kit

Kyowa Medex Co., Ltd. developed the kit for the sero-diagnosis of tuberculosis, which detects IgG antibodies against tuberculous glycolipids antigen containing cord factor (TBGL antigen) prepared from M. tuberculosis using the enzyme-linked immunosorbent assay technique. We evaluated the kit using clinical specimens and the results are as follows: 1) In total, 34 out of 39 cases (87.2%) with active pulmonary tuberculosis showed positive anti-TBGL antibody. 2) Patients with cavity, patients with extensive lesions and patients excreting large amount of acid fast bacilli tended to show high positivity rates. 3) The antibody titers increased in 7 out of 11 cases after starting the antituberculous chemotherapy. 4) The use of the antibody is unsuitable for the determination of the activity of tuberculosis since the antibody titers only slightly decreased even after chemotherapy for two years. 5) Two out of four nontuberculous mycobacteriosis cases showed high antibody titers 6) All three AIDS patients with tuberculosis showed low antibody titers. 7) The antibody was negative in almost all healthy controls showing a positive PPD skin test after vaccination with BCG, and it was therefore assumed that the antibody titer is not increased by BCG vaccination. 8) The antibody titers of the staff members working in the tuberculosis wards were not high compared with those of staff members working in the other wards.     

Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria

Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.