Prophylactic effect of mycobacterium bovis BCG vaccination against osteomyelitis in children with Mycobacterium ulcerans disease (Buruli Ulcer)

Mycobacterium ulcerans disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. In 233 consecutive, laboratory-confirmed samples from BU patients in Benin whose Mycobacterium bovis BCG scar status was known, 130 children (<15 years old) and 75 adults had a neonatal BCG vaccination scar. Of 130 children with BCG scars, 10 (7.7%) had osteomyelitis, while 3 of 9 children without BCG scars (33.3%) had osteomyelitis. Our observations support the conclusion that having a BCG vaccination scar provides significant protection against M. ulcerans osteomyelitis in children with BU disease.     

Prophylactic Effect of Mycobacterium bovis BCG Vaccination against Osteomyelitis in Children with Mycobacterium ulcerans Disease (Buruli Ulcer)

Mycobacterium ulcerans disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. In 233 consecutive, laboratory-confirmed samples from BU patients in Benin whose Mycobacterium bovis BCG scar status was known, 130 children (<15 years old) and 75 adults had a neonatal BCG vaccination scar. Of 130 children with BCG scars, 10 (7.7%) had osteomyelitis, while 3 of 9 children without BCG scars (33.3%) had osteomyelitis. Our observations support the conclusion that having a BCG vaccination scar provides significant protection against M. ulcerans osteomyelitis in children with BU disease.     

A booster vaccination with Mycobacterium bovis BCG does not increase the protective effect of the vaccine against experimental Mycobacterium ulcerans infection in mice

Buruli ulcer, caused by Mycobacterium ulcerans, is a necrotizing skin disease emerging particularly in West Africa. M. bovis BCG vaccine offers only short-term protection against experimental footpad infection of C57BL/6 mice with M. ulcerans, and the duration of this protection cannot be prolonged by a booster vaccination.     

Protective efficacy of a DNA vaccine encoding antigen 85A from Mycobacterium bovis BCG against Buruli ulcer

Buruli ulcer, caused by Mycobacterium ulcerans, is characterized by deep and necrotizing skin lesions, mostly on the arms and legs. Together with tuberculosis and leprosy, this mycobacterial disease has become a major health problem in tropical and subtropical regions, particularly in central and western Africa. No specific vaccine is available for Buruli ulcer. There is, however, evidence in the literature that suggests a cross-reactive protective role of the tuberculosis vaccine M. bovis BCG. To identify potential mechanisms for this cross-protection, we identified and characterized the M. ulcerans homologue of the important protective mycobacterial antigen 85 (Ag85A) from BCG. The homologue is well conserved in M. ulcerans, showing 84.1% amino acid sequence identity and 91% conserved residues compared to the sequence from BCG. This antigen was sufficiently conserved to allow cross-reactive protection, as demonstrated by the ability of M. ulcerans- infected mice to exhibit strong cellular immune responses to both BCG and its purified Ag85 complex. To further address the mechanism of cross-reactive protection, we demonstrate here that prior vaccination with either BCG or plasmid DNA encoding BCG Ag85A is capable of significantly reducing the bacterial load in the footpads of M. ulcerans- infected mice, as determined by Ziehl-Neelsen staining and by actual counting of CFU on 7H11 Middlebrook agar. Together, the results reported here support the potential of a cross-protective Ag85-based future vaccine against tuberculosis, Buruli ulcer, and leprosy.     

Dry-reagent-based PCR as a novel tool for laboratory confirmation of clinically diagnosed Mycobacterium ulcerans-associated disease in areas in the tropics where M. ulcerans is endemic

After tuberculosis and leprosy, Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial disease in immunocompetent humans. The disease occurs in tropical countries, with foci in West Africa, Central Africa, and the western Pacific. BU is defined as an infectious disease involving the skin and the subcutaneous adipose tissue characterized by a painless nodule, papule, plaque, or edema, evolving into a painless ulcer with undermined edges and often leading to invalidating sequelae. Due to the fundamental lack of understanding of modes of transmission, disease control in endemic countries is limited to early case detection through improved active surveillance and surgical treatment. The laboratory confirmation of BU is complicated by the absence of a diagnostic "gold standard." Therefore, misclassification and delayed diagnosis of BU may occur frequently, causing a considerable socioeconomic impact in terms of treatment costs due to prolonged hospitalization. In order to respond to the urgent need to develop reliable tools for early case detection and to overcome technical difficulties accompanying the implementation of diagnostic PCR procedures in tropical countries, a dry-reagent-based PCR formulation for the detection of M. ulcerans in diagnostic specimens has been developed at the Bernhard Nocht Institute for Tropical Medicine. Following technical and clinical validation, the assay has been successfully installed and field tested at the Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi, Ghana. Preliminary results show an excellent diagnostic sensitivity of >95%.     

Cytokine response to antigen stimulation of whole blood from patients with Mycobacterium ulcerans disease compared to that from patients with tuberculosis

Mycobacterium ulcerans disease (Buruli ulcer) is a skin-ulcerating infection common in some parts of the tropics. We have investigated cytokine secretion after stimulation of whole blood from Buruli ulcer (BU) patients in a region of endemicity in Ghana with M. ulcerans sonicate or culture filtrate antigens to investigate the development of the response over time and its specificity by comparison with the response to Mycobacterium tuberculosis sonicate in human immunodeficiency virus-negative tuberculosis patients. Significant gamma interferon (IFN-gamma) production in response to whole-blood stimulation with M. ulcerans sonicate was detected in patients with ulcers, which was higher than that in patients with nodules but similar to subjects with healed BU. The mean IFN-gamma response in household contacts of BU patients was not significantly different from that in healthy control subjects from an area of nonendemicity. Results in patients with untreated, smear-positive pulmonary tuberculosis and tuberculosis patients on treatment for more than 2 weeks showed that BU patients responded better to M. ulcerans antigens than tuberculosis patients. In contrast, interleukin-10 results were higher in patients with active M. ulcerans disease than in those with healed lesions, but the pattern of response was similar to that seen in tuberculosis. A similar pattern of cytokine secretion was found using M. tuberculosis sonicate as an antigen. Neither of the two culture filtrate antigens of M. ulcerans appeared to be more specific than M. ulcerans sonicate. In the early stages of M. ulcerans disease there was a mixed Th1 and Th2 cytokine response, but the Th1 response emerged as the dominant type.     

Prospects for vaccine development against Buruli disease

Buruli disease, caused by Mycobacterium ulcerans, is an emerging infectious disease in tropical areas, particularly West Africa, which can cause deep necrotizing skin lesions, called Buruli ulcer. Buruli disease affects all age groups but about 50% of the cases are diagnosed in children. There is no evidence that Buruli disease is transmitted by direct person-to-person contact and it is very likely that contaminated water of rivers, swamps and lakes serves as the wildlife reservoir of M. ulcerans. This review briefly discusses the epidemiology, microbiology, pathology and treatment of the disease. It describes in detail the current knowledge of the immune response and focuses on the studies that have dealt with vaccination. Finally, experimental approaches for future immunoprophylaxis are discussed.     

Evidence for an intramacrophage growth phase of Mycobacterium ulcerans

Mycobacterium ulcerans is the etiologic agent of Buruli ulcer (BU), an emerging tropical skin disease. Virulent M. ulcerans secretes mycolactone, a cytotoxic exotoxin with a key pathogenic role. M. ulcerans in biopsy specimens has been described as an extracellular bacillus. In vitro assays have suggested a mycolactone-induced inhibition of M. ulcerans uptake by macrophages in which its proliferation has not been demonstrated. Therefore, and uniquely for a mycobacterium, M. ulcerans has been classified as an extracellular pathogen. In specimens from patients and in mouse footpad lesions, extracellular bacilli were concentrated in central necrotic acellular areas; however, we found bacilli within macrophages in surrounding inflammatory infiltrates. We demonstrated that mycolactone-producing M. ulcerans isolates are efficiently phagocytosed by murine macrophages, indicating that the extracellular location of M. ulcerans is not a result of inhibition of phagocytosis. Additionally, we found that M. ulcerans multiplies inside cultured mouse macrophages when low multiplicities of infection are used to prevent early mycolactone-associated cytotoxicity. Following the proliferation phase within macrophages, M. ulcerans induces the lysis of the infected host cells, becoming extracellular. Our data show that M. ulcerans, like M. tuberculosis, is an intracellular parasite with phases of intramacrophage and extracellular multiplication. The occurrence of an intramacrophage phase is in accordance with the development of cell-mediated and delayed-type hypersensitivity responses in BU patients.     

Infection with Mycobacterium ulcerans induces persistent inflammatory responses in mice

Buruli ulcer (BU) is a devastating, necrotizing, tropical skin disease caused by infections with Mycobacterium ulcerans. In contrast to other mycobacterioses, BU has been associated with minimal or absent inflammation. However, here we show that in the mouse M. ulcerans induces persistent inflammatory responses with virulence-dependent patterns. Mycolactone-positive, cytotoxic strains are virulent for mice and multiply progressively, inducing both early and persistent acute inflammatory responses. The cytotoxicity of these strains leads to progressive destruction of the inflammatory infiltrates by postapoptotic secondary necrosis, generating necrotic acellular areas with extracellular bacilli released by the lysis of infected phagocytes. The necrotic areas, always surrounded by acute inflammatory infiltrates, expand through the progressive invasion of healthy tissues around the initial necrotic lesions by bacteria and by newly recruited acute inflammatory cells. Our observations show that the lack of inflammatory infiltrates in the extensive areas of necrosis seen in advanced infections results from the destruction of continuously produced inflammatory infiltrates and not from M. ulcerans-induced local or systemic immunosuppression. Whether this is the mechanism behind the predominance of minimal or absent inflammatory responses in BU biopsies remains to be elucidated.     

The effect of neonatal BCG vaccination on atopy and asthma at age 7 to 14 years: an historical cohort study in a community with a very low prevalence of tuberculosis infection and a high prevalence of atopic disease

BACKGROUND: There are conflicting reports on the effect of BCG vaccination on the subsequent development of atopy and asthma. There are no data on the effects of neonatal BCG vaccination on cytokine responses of lymphocytes that are exposed in vitro to allergens. OBJECTIVES: We sought to test the hypothesis that neonatal BCG vaccination or, alternatively, evidence of an immunologic memory of this vaccination is associated with a reduced prevalence of allergic sensitization, asthma, eczema, and hay fever during childhood. METHODS: An historical cohort study was conducted among 7- to 14-year-old children who were born in 2 districts in Sydney, Australia, and whose mothers were born in southeast Asia. One district had routinely administered BCG vaccination to infants born to overseas-born mothers and the other had not. Eligible subjects were identified from birth registers. Consenting subjects completed questionnaires, performed spirometric and airway hyperresponsiveness testing, and had allergen skin prick testing and tuberculin skin testing. Blood was collected to measure total serum IgE levels and for in vitro lymphocyte culture in the presence of an extract of house dust mite, the dominant allergen in this region, and purified protein derivative of Mycobacterium tuberculosis (tuberculin). IL-4, IL-5, IL-10, and IFN-gamma were measured in the culture supernatant. RESULTS: The cohort included 309 BCG-vaccinated subjects and 442 non-BCG-vaccinated subjects. BCG-vaccinated subjects did not have a lower rate of allergic sensitization than nonvaccinated subjects. However, among the subgroup of subjects with a family history of rhinitis or eczema, BCG vaccination was associated with a lower prevalence of current asthma (defined as recent wheezing plus airway hyperresponsiveness; relative risk, 0.46; 95% CI, 0.22-0.95). BCG vaccination was also associated with lower levels of allergen-stimulated IL-10 production in vitro. Among the BCG-vaccinated subjects, the 44 (14.3%) who had tuberculin skin test reaction sizes of 5 mm or greater and the 31 (18.3%) who demonstrated an in vitro IFN-gamma response to purified protein derivative of M tuberculosis did not have lower rates of allergic sensitization and, overall, did not have a lower prevalence of allergic disease than tuberculin skin test or IFN-gamma nonreactors. CONCLUSION: We conclude that neonatal BCG vaccination has an effect on T-cell allergen responsiveness 7 to 14 years after vaccination and that among a subgroup of subjects with an inherited predisposition to allergic disease, this is associated with clinically relevant beneficial effects. The findings of this study encourage the view that external influences on the immune system in the neonatal period have consequences that extend into later childhood and influence the expression of asthma. Genetic factors are likely to modify the effect of those external factors.     

Immune response to infection with Mycobacterium ulcerans

Mycobacterium ulcerans is a slow-growing, acid-fast bacillus that causes chronic necrotizing skin ulcers known as Buruli ulcers. Previously reported information on immunity to this mycobacterium is limited. We examined immune responses to M. ulcerans and M. bovis BCG in patients with M. ulcerans disease and in 20 healthy control subjects (10 tuberculin test positive and 10 tuberculin test negative). Cell-mediated immunity was assessed by stimulating peripheral blood mononuclear cells (PBMC) with whole mycobacteria and then measuring PBMC proliferation and the production of gamma interferon (IFN-gamma). Humoral immunity was assessed by immunoblotting. PBMC from all subjects showed significantly greater proliferation and IFN-gamma production in response to stimulation with living mycobacteria compared with killed cells. However, PBMC from subjects with past or current M. ulcerans disease showed significantly reduced proliferation and production of IFN-gamma in response to stimulation with live M. ulcerans or M. bovis than PBMC from healthy, tuberculin test-positive subjects (P < 0.001) and showed results in these assays comparable to those of tuberculin test-negative subjects (P > 0.2). Serum from 9 of 11 patients with M. ulcerans disease, but no control subject, contained antibodies to M. ulcerans. The results indicate that patients with M. ulcerans infection mount an immune response to M. ulcerans as evidenced by antibody production, but they demonstrate profound systemic T-cell anergy to mycobacterial antigens. These findings may explain some of the distinct clinical and pathological features of M. ulcerans-induced disease.     

The contribution of skin autograft in open air in the treatment of the Buruli ulcer

The Buruli ulcer (BU) rages in a lot of tropical and subtropical countries. In Democratic Republic of Congo, some BU cases were reported between the 50s and 70s. This disease offers resistance to the usual chemotherapy. The only alternative for the treatment remains the surgical one (trimming, wound dressing, graft). The overarching aim of this study, conducted in the province of Lower Congo, is to put forward a surgical therapeutic approach adapted to the precarious conditions met on the ground in our rural areas. The evidence of a Mycobacterium ulcerans infection has been bacteriological and histopathological. The slight skin autograft or "of Davis", in open air or with dressing was performed in 37 patients. After three months this skin autograft in open air has experienced 78.5% of complete healing against 70% for the skin autograft with dressing. The exposure of skin graftings to open air favors an hyperoxygenation which also fights against the Mycobacterium ulcerans, for this latter develops better in an hypoxical area.     

The protective effect of the Mycobacterium bovis BCG vaccine is increased by coadministration with the Mycobacterium tuberculosis 72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected guinea pigs

A tuberculosis vaccine candidate consisting of a 72-kDa polyprotein or fusion protein based upon the Mtb32 and Mtb39 antigens of Mycobacterium tuberculosis and designated Mtb72F was tested for its protective capacity as a potential adjunct to the Mycobacterium bovis BCG vaccine in the mouse and guinea pig models of this disease. Formulation of recombinant Mtb72F (rMtb72F) in an AS02A adjuvant enhanced the Th1 response to BCG in mice but did not further reduce the bacterial load in the lungs after aerosol challenge infection. In the more stringent guinea pig disease model, rMtb72F delivered by coadministration with BCG vaccination significantly improved the survival of these animals compared to BCG alone, with some animals still alive and healthy in their appearance at >100 weeks post-aerosol challenge. A similar trend was observed with guinea pigs in which BCG vaccination was boosted by DNA vaccination, although this increase was not statistically significant due to excellent protection conferred by BCG alone. Histological examination of the lungs of test animals indicated that while BCG controls eventually died from overwhelming lung consolidation, the majority of guinea pigs receiving BCG mixed with rMtb72F or boosted twice with Mtb72F DNA had mostly clear lungs with minimal granulomatous lesions. Lesions were still prominent in guinea pigs receiving BCG and the Mtb72F DNA boost, but there was considerable evidence of lesion healing and airway remodeling and reestablishment. These data support the hypothesis that the coadministration or boosting of BCG vaccination with Mtb72F may limit the lung consolidation seen with BCG alone and may promote lesion resolution and healing. Collectively, these data suggest that enhancing BCG is a valid vaccination strategy for tuberculosis that is worthy of clinical evaluation.     

Variability in 3' end of 16S rRNA sequence of Mycobacterium ulcerans is related to geographic origin of isolates.

Mycobacterium ulcerans causes extensive ulcers (Buruli ulcers) in the skin of humans. Analysis of the 3'-terminal region of the 16S rRNA gene sequence of 17 strains of M. ulcerans from Africa, the Americas, and Australia revealed three subgroups corresponding to the continent of origin, and some variable phenotypic characteristics. This sequence is useful for the rapid detection of M. ulcerans and discriminates M. marinum and M. shinshuense from M. ulcerans.     

Systemic suppression of interferon-gamma responses in Buruli ulcer patients resolves after surgical excision of the lesions caused by the extracellular pathogen Mycobacterium ulcerans

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial infection in immunocompetent humans besides tuberculosis and leprosy. We have compared by ex vivo enzyme-linked immunospot analysis interferon-gamma (IFN-gamma) responses in peripheral blood mononuclear cells (PBMC) from BU patients, household contacts, and individuals living in an adjacent M. ulcerans nonendemic region. PBMC were stimulated with purified protein derivative (PPD) and nonmycobacterial antigens such as reconstituted influenza virus particles and isopentenyl-pyrophosphate. With all three antigens, the number of IFN-gamma spot-forming units was reduced significantly in BU patients compared with the controls from a nonendemic area. This demonstrates for the first time that M. ulcerans infection-associated systemic reduction in IFN-gamma responses is not confined to stimulation with live or dead mycobacteria and their products but extends to other antigens. Interleukin (IL)-12 secretion by PPD-stimulated PBMC was not reduced in BU patients, indicating that reduction in IFN-gamma responses was not caused by diminished IL-12 production. Several months after surgical excision of BU lesions, IFN-gamma responses of BU patients against all antigens used for stimulation recovered significantly, indicating that the measured systemic immunosuppression was not the consequence of a genetic defect in T cell function predisposing for BU but is rather related to the presence of M. ulcerans bacteria.     

Early BCG vaccination and reduction in atopy in Guinea-Bissau

BACKGROUND: It has been proposed that certain viral and bacterial infections in early childhood may prevent allergic sensitization, by inducing Th1-type immune responses. This has led to speculation that mycobacterial vaccines might, through their Th1-stimulating properties, also protect against atopy. OBJECTIVE: To investigate whether the prevalence of atopy is lower in children who have been vaccinated with BCG in infancy than in children who have not been vaccinated. METHODS: We measured skin test reactivity to three allergens (Dermatophagoides pteronyssinus, D. farinae and cockroach) in 400 children, aged 3-14 years, as part of a follow-up study to examine the immune sequelae of measles in an urban area of Bissau, the capital of Guinea-Bissau in west Africa. Information on childhood vaccinations, including BCG in infancy, was available from child records. Of these children, 271 had been vaccinated with BCG (according to records) and 53 had not been vaccinated (no record and no BCG scar). Atopy was defined in two ways, according to the presence of any allergen reaction > or = 2 mm and any reaction > or = 3 mm. RESULTS: Of the children who had received BCG vaccine, 57 (21%) were atopic (any reaction > or = 2 mm), compared with 21 (40%) of the unvaccinated children [odds ratio, after controlling for potential confounding factors, 0.19 (95% CI 0.06-0.59)]. When atopy was defined using the 3-mm criterion, the reduction in atopy associated with BCG was greater the earlier the age at vaccination, and the largest reduction was seen in children vaccinated in the first week of life. CONCLUSION: BCG vaccination given early in infancy may prevent the development of atopy in African children.     

Effect of Mycobacterium bovis BCG vaccination upon Mycobacterium lepraemurium infection.

Mice were infected with 10(8) Mycobacterium lepraemurium in the footpad (unsuppressed mice), and some of these animals were concurrently given 10(9) heat-killed M. lepraemurium intravenously (suppressed mice). These groups of mice were preimmunized with 10(7) viable organisms of Mycobacterium bovis BCG by several routes. BCG inhibited the proliferation of M. lepraemurium in the unsuppressed mice, but not in the suppressed mice. In effect, the intravenous administration of heat-killed M. lepraemurium suppressed the immunity to M. lepraemurium that BCG vaccination had engendered. BCG did not protect normal mice against intravenous infection with M. lepraemurium. It appears that normal mice against intravenous infection with M. lepraemurium. It appears that the inhibitory effect of BCG vaccination upon M. lepraemurium infection is due to cross-reactive immunity rather than to nonspecific immunity or immunopotentiation. Thus, the route of BCG vaccination was immaterial, and vaccination 12 weeks before M. lepraemurium infection was as beneficial as vaccination 4 weeks before infection. Moreover, spleen cells from M. lepraemurium-immunized mice conferred adoptive immunity to BCG. The implications of this study for the use of BCG as a prophylactic and therapeutic agent in human leprosy are discussed.     

Nineteen cases of Buruli ulcer diagnosed in Japan from 1980 to 2010

The etiology, clinical manifestations, and treatment of 19 sporadic cases of Buruli ulcer (BU) in Japan are described. The cases originated in different regions of Honshu Island, with no evidence of patient contact with an aquatic environment. The majority (73.7%) of cases occurred in females, with an average age of 39.1 years for females and 56.8 years for males. All patients developed ulcers on exposed areas of the skin (e.g., face, extremities). Most ulcers were <5 cm in diameter (category I), except in one severe progressive case (category II). Pain was absent in 10 of the 19 cases. Fourteen ulcers were surgically excised, and nine patients needed skin grafting. All cases were treated with various antibiotic regimens, with no reported recurrences as of March 2011. Mycobacterium ulcerans-specific IS2404 was detected in all cases. Ten isolates had identical 16S rRNA gene sequences, which were similar to those of M. ulcerans. However, the rpoB gene showed a closer resemblance to Mycobacterium marinum or Mycobacterium pseudoshottsii. PCR identified pMUM001 in all isolates but failed to detect one marker. DNA-DNA hybridization misidentified all isolates as M. marinum. The drug susceptibility profile of the isolates also differed from that of M. ulcerans. Sequence analysis revealed "Mycobacterium ulcerans subsp. shinshuense" as the etiologic agent of BU in Japan. Clinical manifestations were comparable to those of M. ulcerans but differed as follows: (i) cases were not concentrated in a particular area; (ii) there was no suspected connection to an aquatic environment; (iii) drug susceptibility was different; and (iv) bacteriological features were different.     

Vaccination of neonatal calves with Mycobacterium bovis BCG induces protection against intranasal challenge with virulent M. bovis

Vaccination of neonates with Mycobacterium bovis bacillus Calmette-Guerin (BCG) may be a strategy that overcomes reduced vaccine efficacy associated with exposure to environmental mycobacteria in humans and cattle. Preliminary comparisons indicated that 2-week-old calves produced an immune response to vaccination at least as intense as that observed in adults. Subsequently, five gnotobiotic hysterotomy derived calves aged 1 day were inoculated with BCG and 3 months later were challenged intranasally with virulent M. bovis. The number of tissues with lesions and the pathological extent of these lesions was reduced significantly in vaccinates. Furthermore, lesions were evident in the lung or associated chest lymph nodes of four of five controls but none of five vaccinates. BCG vaccination reduced significantly the level of bacterial colonization. However, lesions in the head associated lymph nodes were observed in three of five BCG-vaccinated cattle. Levels of interferon gamma (IFN-gamma) detected by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISPOT) in individual vaccinated animals at challenge did not correlate with subsequent resistance and in general immune responses post-challenge were lower in vaccinated calves. Low IL-10 responses were evident but IL-4 was not detected. Responses to ESAT-6 and/or CFP-10 were evident in four of four control calves that had lesions. Two of the BCG vaccinates with lesions did not produce a response to ESAT-6 and CFP-10, indicating that these antigens did not distinguish vaccinated immune animals from vaccinated animals with lesions. Overall, vaccination of neonatal calves with BCG induced significant protection against disease and has potential as a strategy for the reduction of the incidence of bovine tuberculosis.     

Study of immune response in Warao children from communities with high tuberculosis prevalence

The present study was carried out in a Warao childhood population with extremely high tuberculosis (TB) rate of 3190/100,000 in 0-15 years old children. One hundred seven serum and saliva samples were tested, 32 from patients with active TB (27 positive and 5 negative for the tuberculin skin test, TST) and 75 apparently healthy contact children (45 positive and 30 negative for the TST). The innate, immunoglobulin and cellular responses were studied. The results showed that both, patients and controls, had a high percentage of children with increased levels of complement C3 and C4 components. A high percentage of children with increased total serum IgA and IgG (13.8% and 79.3% respectively) was observed in children with TB in comparison to control children (0% and 69.2%). A high percentage of control children had increased levels of IgM and sIgA (69.2% and 56.16%, respectively) in comparison to patients (48.3% and 31.25%). Both groups showed children with increased levels of IgE. The results concerning to the cellular immune response to PPD and the BCG vaccination status showed that there was a correlation between an increase in PPD reactivity and age. The PPD reactivity in children less than 7 years old was similar and also independent of the BCG vaccination status. A significant number of children without or with scars (46.8% and 27.6%, respectively) showed induration values of 0 mm to tuberculin skin test. The Candida reactivity showed a high percentage of children (80%) with anergy status. In conclusion, an increase in the levels of complement components C3 and C4 and hypergammaglobulinemia was observed in Warao children, and these results were independent from PPD reactivity and BCG vaccination. The isotype results showed that the decrease in sIgA could be and active disease marker, while the increase in IgM levels could represent a marker of recent disease.