[Mycobacterium scrofulaceum infection in a pediatric patient from the Galapagos Islands - Ecuador]. / Infección por Mycobacterium scrofulaceum en un niño de las Islas Galápagos - Ecuador.

Mycobacterium scrofulaceum is a slow-growing atypical mycobacteria with a reservoir in the environmen, and occasionally it causes disease in humans. The case of a 10-year-old patient with fever of four months of evolution is presented. Symptoms include bilateral adenopathies of neck, thorax, abdomen and inguinal region, rash, abdominal pain and vomiting; in addition, a positive serology for EBV and a culture of the left inguinal ganglia with growth of M. scrofulaceum were evidenced. Thus, this is the first report of human infection caused by the aforementioned mycobacterium in Ecuador, which it was particularly manifested with an unusual pattern of resistance.

Infección por Mycobacterium scrofulaceum en un niño de las Islas Galápagos - Ecuador / Mycobacterium scrofulaceum infection in a pediatric patient from the Galapagos Islands - Ecuador

Resumen Mycobacterium scrofulaceum es una micobacteria atípica de crecimiento lento que tiene como reservorio el ambiente. De forma ocasional causa enfermedad en humanos. Se presenta el caso de un niño de 10 años de edad con fiebre de cuatro meses de evolución, adenopatías cervicales, torácicas, abdominales e inguinales bilaterales, exantema, dolor abdominal y vómitos; con evidencia concomitante de serología positiva para el VEB y cultivo de los ganglios inguinales izquierdos con crecimiento de M. scrofulaceum. Es la primera comunicación de una infección causada por esta micobacteria en Ecuador, y que particularmente se presentó con un patrón de resistencia inusual.

Mycobacterium scrofulaceum is a slow-growing atypical mycobacteria with a reservoir in the environmen, and occasionally it causes disease in humans. The case of a 10-year-old patient with fever of four months of evolution is presented. Symptoms include bilateral adenopathies of neck, thorax, abdomen and inguinal region, rash, abdominal pain and vomiting; in addition, a positive serology for EBV and a culture of the left inguinal ganglia with growth of M. scrofulaceum were evidenced. Thus, this is the first report of human infection caused by the aforementioned mycobacterium in Ecuador, which it was particularly manifested with an unusual pattern of resistance.

TLR4 and DC-SIGN receptors recognized Mycobacterium scrofulaceum promoting semi-activated phenotype on bone marrow dendritic cells.

Nontuberculous mycobacteria (NTM) are recognized as emerging pathogens and their immune regulatory mechanisms are not well described yet. From them, Mycobacterium avium is known to be a weak activator of dendritic cells (DCs) that impairs the response induced by BCG vaccine. However, whether other NTM such as Mycobacterium scrofulaceum may modulate the activation of DCs, has not been extensively studied. Here, we exposed bone marrow-derived DCs (BMDCs) to M. scrofulaceum and we analyzed the effect on the activation of DCs. We found that M. scrofulaceum has a comparable ability to induce a semi-mature DC phenotype, which was produced by its interaction with DC-SIGN and TLR4 receptors in a synergic effect. BMDCs exposed to M. scrofulaceum showed high expression of PD-L2 and production of IL-10, as well as low levels of co-stimulatory molecules and pro-inflammatory cytokines. In addition to immunophenotype induced on DCs, changes in morphology, re-organization of cytoskeleton and decreased migratory capacity are consistent with a semi-mature phenotype. However, unlike other pathogenic mycobacteria, the DC-semi-mature phenotype induced by M. scrofulaceum was reversed after re-exposure to BCG, suggesting that modulation mechanisms of DC-activation used by M. scrofulaceum are different to other known pathogenic mycobacteria. This is the first report about the immunophenotypic characterization of DC stimulated by M. scrofulaceum.

Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis.

The efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine against pulmonary tuberculosis (TB) varies enormously in different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment, but the precise mechanism has so far not been clarified. Our study demonstrates that prior exposure to live environmental mycobacteria can result in a broad immune response that is recalled rapidly after BCG vaccination and controls the multiplication of the vaccine. In these sensitized mice, BCG elicits only a transient immune response with a low frequency of mycobacterium-specific cells and no protective immunity against TB. In contrast, the efficacy of TB subunit vaccines was unaffected by prior exposure to environmental mycobacteria. Six different isolates from soil and sputum samples from Karonga district in Northern Malawi (a region in which BCG vaccination has no effect against pulmonary TB) were investigated in the mouse model, and two strains of the Mycobacterium avium complex were found to block BCG activity completely.

Skin test reactivity to mycobacterial antigens parallels the phylogenetic structure of their genus.

SETTING: City of Manaus, Amazonas, Brazil. OBJECTIVE: To explore the relationship between positivity to tuberculin and other environmental mycobacteria sensitins, according to a range of criteria and presence of BCG scar. DESIGN: Dual skin testing with tuberculin and four mycobacterial sensitins, and BCG scar recording of 1070 schoolchildren aged 7-14. Four criteria for positivity were used: simple and dominant, with 5 and 10 mm cut-off points. RESULTS: The standardised prevalence of reactions > or = 5 mm for BCG scar negative children was 58.3% for Mycobacterium avium, 54.2% for M. scrofulaceum, 26.8% for M. fortuitum, 17.9% for M. tuberculosis and 7.6% for M. kansasii. Correlations between tuberculin and each sensitin, for BCG scar negative children, were 0.47 for M. avium, 0.53 for M. scrofulaceum, 0.60 for M. kansasii and 0.22 for M. fortuitum (all with P < 0.01). BCG effect was particularly significant for tuberculin (odds ratio = 3.44 for reactions > or = 5 mm, P < 0.001) and influenced the balance between dominant/non-dominant reactions for all sensitins. CONCLUSION: The correlation between tuberculin and each sensitin confirmed the separation of the rapidly (M. fortuitum) and slowly growing mycobacteria (M. tuberculosis, M. avium, M. scrofulaceum and M. kansasii). The influence of BCG on tuberculin reactions was more marked than on other mycobacterial sensitins.

Development of tuberculin reactivity and sensitization to M. scrofulaceum and M. fortuitum in children BCG-vaccinated at birth.

Since the incidence of tuberculosis is steadily declining in Finland and infections by environmental mycobacteria may be increasing, the aim of the present study was to evaluate the development of tuberculin reactivity and sensitization to environmental mycobacteria. Healthy Finnish schoolchildren aged 10.4-12.4 yrs (n=201) were tested with tuberculin purified protein derivative RT23, Mycobacterium scrofulaceum RS95 and M. fortuitum RS20 sensitins. The same children had been previously tested with the same antigens and methods at the age of 4-6 yrs in 1989. Rapid waning of tuberculin reactivity and decrease in sensitization to environmental mycobacteria were observed between 4-6 yrs. Both tuberculin and sensitin skin reaction sizes decreased significantly over the 6-yrs period. The mean tuberculin skin reaction size was 3.2 mm in diameter, which was significantly (p<0.001) smaller than the mean induration size (4.8 mm) at the age of 4-6 yrs. Similarly, the mean skin reaction sizes to M. scrofulaceum and M. fortuitum sensitins were 3.4 and 1.7 mm, respectively, which were significantly (p<0.001) smaller than 6 yrs earlier (mean 4.5 and 3.1 mm). The number of zero reactions to all antigens increased significantly during the follow-up period. Contacts with pets or farm animals were associated with larger reactions. In contrast, children suffering from allergic symptoms had smaller reactions. Contacts with mycobacteria, either with Mycobacterium tuberculosis or environmental mycobacteria, seem to be too rare to maintain tuberculin responsiveness and a high sensitivity to other mycobacteria. Different bacille Calmette-Guérin vaccine products and dosages used, the declining incidence of tuberculosis and geographical factors, which can influence environmental mycobacterial exposure, may explain the disparity between the present and previous Finnish studies.

[Specificity of the Mantoux-reaction to the new tuberculin RT23 in relation to that of previously used PPD-RIVM; a comparative study in hospital staff]. / Specificiteit van de Mantoux-reactie op het nieuwe tuberculine RT23 ten opzichte van de reactie op het oude PPD-RIVM; vergelijkend onderzoek onder ziekenhuispersoneel.

OBJECTIVE: In the Netherlands, an increase in prevalence of tuberculin positivity has been noted since the replacement of the Dutch PPD-RIVM preparation by RT23. This investigation was carried out to assess the specificity of RT23 compared with PPD-RIVM. DESIGN: Comparative. METHOD: In 90 hospital workers simultaneous tuberculin tests were performed in both forearms with RT23 (Statens Serum Institut, Copenhagen, Denmark; lot number 13751; 2 tuberculin units) and PPD-RIVM (Rijksinstituut voor Volksgezondheid en Milieu, Bilthoven, the Netherlands; lot number 76B; 1 IU), respectively. Sensitivity to atypical mycobacteria (Mycobacterium scrophulaceum, M. avium and M. kansasii) was assessed where differences were > 3 mm. RESULTS: In two of 73 non-vaccinated subjects, reactions to RT23 were 11 and 15 mm larger than to PPD-RIVM; both showed sensitivity to M. scrophulaceum and M. avium. Five of nine BCG vaccinated subjects showed differences of five to eight mm; in four of these, the reaction to RT23 was larger. Repeat testing in one showed no cross-reactions. The specificity of RT23 compared with PPD-RIVM was 96% (cut-off at 10 mm diameter). CONCLUSIONS: In a low tuberculosis risk population, tuberculin testing with RT23 yields more positive results than PPD-RIVM. This is probably due to stronger cross-reactivity with atypical mycobacteria of RT23. Decisions on isoniazide prophylaxis should take into account an increased false-positivity rate with RT23.

Prevalence of allergy in children in relation to prior BCG vaccination and infection with atypical mycobacteria.

By influence on the Th1/Th2 cell balance, infectious agents may affect the development of atopic allergy. In this study, we investigated whether previous BCG vaccination or infection with atypical mycobacteria might be related to the development of atopic disease. The study, which involved skin testing with mycobacteria and answers to a questionnaire for more than 6000 children in Sweden, revealed a low prevalence of allergy among BCG-vaccinated children who were immigrants or adopted from other countries. Vaccinated children born in Sweden, however, did not have significantly lower allergy prevalence than age-matched, unvaccinated children. Furthermore, the overall frequencies of skin-test reactivity to the atypical mycobacteria M. avium and M. scrofulaceum were higher rather than lower in allergic than in nonallergic children. By contrast, there was a tendency toward a lower frequency of more strongly positive skin reactions (> or = 10 mm) to mycobacteria in allergic than in nonallergic children. These findings do not support the hypothesis that early mycobacterial infections have a suppressive effect on the development of atopic disease. Earlier findings of an apparent association between atopy and lack of previous mycobacterial infection may possibly be explained by a relatively decreased ability of atopic patients to mount strong Th1 cell-mediated immune responses.

Species-specific B-cell epitope on the C-terminal region of the alpha antigen from Mycobacterium scrofulaceum.

In the amino acid (AA) sequences of alpha antigen from mycobacteria, C-terminal regions were variable among a variety of mycobacterial species though the N-terminal regions were relatively conserved. These regions may possess some species-specific antigenic determinants of the alpha antigen from Mycobacterium scrofulaceum (S-alpha). AAs288-300 of S-alpha fused to beta-galactosidase was reactive with the antisera raised against S-alpha. The same fused peptide did not react with the antisera raised against the alpha antigen from Mycobacterium avium (A-alpha) and Mycobacterium bovis BCG (B-alpha). B-cell epitope mapping then was performed focusing on the C-terminal region of S-alpha using the synthetic peptides. Their reactivities with antisera raised against the alpha antigens of three different mycobacterial species were assessed by ELISA. AAs279-286 were a cross-reactive common immunodominant region among three mycobacterial species. This region may be one of the cross-reactive common epitopes in mycobacterial species. And AAs291-300 were reactive only with the antisera raised against S-alpha. This region may possess a species-specific epitope.

Cross-reactive immune responses against Mycobacterium bovis BCG in mice infected with non-tuberculous mycobacteria belonging to the MAIS-Group.

Two bacillus Calmette-Guérin (BCG)-susceptible mouse strains, BALB/c and C57BL/6, were infected intravenously with Mycobacterium intracellulare, M. avium or M. scrofulaceum and monitored during 3 months for mycobacterial replication and antibody and Th1-type cytokine production in response to cytoplasmic and secreted antigens from M. bovis BCG. Whereas initial colony-forming unit (CFU) counts of M. intracellulare and M. avium were higher in lungs than in spleen, the opposite was observed for M. scrofulaceum. Mycobacterium intracellulare was the most virulent species and its replication could not be controlled in either mouse strain. It also induced the strongest antibody response. Mycobacterium avium was eliminated in both mouse strains and M. scrofulaceum finally was eliminated in C57BL/6 but multiplied in spleen from BALB/c mice. Significant sustained interleukin-2 and interferon-gamma production towards BCG antigens was only found in M. scrofulaceum infection. As in BCG-vaccination, M. scrofulaceum-infected C57BL/6 mice demonstrated a higher response towards whole BCG culture filtrate, BCG extract and purified antigen 85 complex (Ag85) from BCG than did BALB/c mice. The data suggest that the presence of M. scrofulaceum in the environment may possibly interfere in genetically predisposed subjects with BCG vaccine and its protective efficacy against M. tuberculosis.

Implications of simultaneous tests for tuberculin and non-tuberculous mycobacteria antigen in the elderly.

It is well established that old people living in institutions are more susceptible to tuberculous reactivation and exogenic infection than the general population. Therefore, annual screening with a tuberculin (T) skin test is suggested. Unfortunately, the specificity and sensitivity of this test is limited because of cross-reactivity with non-tuberculous mycobacteria (NTM) and anergy. We therefore investigated the possibility that a simultaneous test with NTM antigen might be helpful in the interpretation of the T test in the elderly. We tested 220 inhabitants of a geriatric institution in Israel simultaneously with T and Mycobacterium scrofulaceum sensitin (S). Ninety-six subjects reacted to the T test, and 64 of them reacted to S as well. In this last group, 38 reacted more to T, 22 more to S, and 4 reacted equally to both antigens. Of the 124 subjects who did not react to T, 24 did react to S and 123 were retested with T. Twenty-two of the latter group had a positive booster effect, but 9 of the positive booster responders initially reacted to S. The difference between the proportions of the positive booster effect in the responders and the non-responders to S was significant. We conclude that in an area where infection with NTM is present a simultaneous S test may clarify the meanings of initial and booster-positive responses to T in the elderly, as well as exclude anergy when the T test is negative.

Investigation into typical and atypical tuberculin sensitivity in the Royal Netherlands Army, resulting in a more rational indication for isoniazid prophylaxis.

SETTING: The Department of Military Health Services (DMGZ) vaccination clinic, Utrecht, The Netherlands. OBJECTIVES: To improve upon PPD skin testing procedure by introducing a double Mantoux test. DESIGN: During the first part of the study, from 1986-1988, army recruits were double-tested simultaneously with PPD and Mycobacterium scrofulacaeum sensitin. During the second part of the study, from 1989 to 1993, recruits reacting to PPD, with an induration in the range of 10-15 mm, underwent a second skin test with M. scrofulaceum sensitin. The total study population consisted of 237,692 non-BCG-vaccinated recruits. RESULTS: From 1986-1993 and average of 0.45% persons reacted with indurations > or = 10 mm to PPD. An average of 7.76% if army recruits reacted with indurations > or = 10 mm to M. scrofulaceum sensitin during the first part of the study. Using a modified ITSC (International Tuberculosis Surveillance Centre) model, 48% of the persons reacting to PPD with indurations in the range 10 mm and 15 mm were classified as false-positive. A total of 16% with indurations > or = 10 mm to PPD were classified as false positive. False-positive persons were then excluded from INH chemoprophlaxis. CONCLUSIONS: In areas with a high prevalence of non-tuberculous mycobacteria infection the use of double skin testing might be useful in differentiating between indurations due to tubercle bacilli and those due to infection with non-tuberculous mycobacteria.

Wide geographic variations of sensitivity to MOTT sensitins in Greece.

Several studies have suggested that large bodies of water are a main source of infection with mycobacteria other than tuberculosis (MOTT). If this is correct, there should be a gradient in the infection rate with MOTT between mountainous and seaside areas. To test this hypothesis, we performed skin testing with tuberculin and sensitins in 19,470 Greek Armed Forces recruits. Initially, several MOTT sensitins were used, but when it became clear that the Mycobacterium scrofulaceum sensitin was the most appropriate, the study was continued with it alone in 17,403 recruits. Finally, in order to evaluate the geographical distribution of sensitivity to sensitins, we studied the results of 8,507 of these recruits living in or near their birthplace. They were divided into three geophysical areas: seaside 3,389 recruits; mountains 2,692 recruits; and inland plains 2,426 recruits. MOTT sensitivity rates were 4.1% in mountainous areas and 7.1% in seaside areas. All small Aegean islands had high MOTT rates (above 8%). In inland plains, high MOTT rates (above 8%) were observed among those living near big rivers. This geographical distribution of MOTT sensitivity supports the theory that large bodies of water are a main source of infection with MOTT.

Determination of humoral immunoglobulins M and G directed against mycobacterial antigen 60 failed to diagnose primary tuberculosis and mycobacterial adenitis in children.

The serodiagnosis of primary tuberculosis (TB) and mycobacterial adenitis in children was tried using the Anda-Tb tests (Anda Biologicals, France) that measure immunoglobulins (Ig) M and G directed against mycobacterial antigen 60 (A60) by enzyme-linked immunosorbent assay. The 188 cases studied included 81 healthy or mycobacteria-unrelated diseased children with no reaction to tuberculin skin test (STN); 9 recent BCG vaccination (BCG); 35 asymptomatic (AsTB), 29 symptomatic (STB) primary TB and 11 adenitis caused by atypical mycobacteria from the group avium-intracellulare-scrofulaceum (MAIS) tested before treatment; and 23 past primary TB tested at different times after completion of specific treatment (past TB). The individual IgM and IgG levels largely overlapped whatever the clinical status of the children. Setting the normal upper limit at the 95th percentile of the STN values, which by definition gives 95% specificity, the highest IgM sensitivity was found in past TB (35%), AsTB showing 23%, STB 17%, and MAIS 18% sensitivity. IgG sensitivity was also the highest in past TB (26%) and was equal to 6, 14, and 9% in AsTB, STB, and MAIS, respectively. Positive and negative predictive values and the test efficiency (63 and 62% for IgM and IgG, respectively) were far too low. Combining positivity for IgM and/or IgG did not improve the results. In our study, the anti-A60 IgM and IgG measurements using the Anda-Tb tests in primary TB and mycobacterial adenitis in children did not prove of any diagnostic help.

Comparative skin testing with PPD tuberculin, Mycobacterium avium and M. scrofulaceum sensitin in schoolchildren in Saudi Arabia.

SETTING: Jizan region, Saudi Arabia. OBJECTIVE: To test methodology for studies of tuberculin sensitivity and effectiveness of BCG vaccination programme. DESIGN: School children (n = 315) aged 6-12 years simultaneously tested intradermally with PPD tuberculin RT23, 2TU and 0.1 microgram Mycobacterium avium or M. scrofulaceum sensitin. Presence of BCG scar as evidence of previous BCG vaccination. Cervical lymph nodes palpated. RESULTS: BCG coverage was 75%. The prevalences of positive reactions (induration > or = 6 mm) were higher among children with than without scar. Prevalences of positive reactions to PPD and M. scrofulaceum sensitin were higher among girls than boys. In children with scar, the mean size of positive reactions to PPD was larger among girls than boys, and the prevalence of positive reactions was smaller in this group among children with than without palpable cervical lymph nodes. The sizes of reactions both to M. avium and to M. scrofulaceum sensitin were correlated to the size of the tuberculin reactions. Previous admission to hospital was reported less often by children with than without scar. CONCLUSIONS: The technique applied and training of staff was satisfactory. In further studies, however, we will apply three simultaneous tests to each person.

[Humoral immune response in humans and animals infected and immunized with Mycobacteria of the MAIS complex]. / Gumoralnyi immunnyi otvet u zarazhennykh i immunizirovannykh mikobakteriiami kompleksa MAIS liudei i zhivotnykh.

Hyperimmunization of mice and rabbits with surface (whole cells) and complex (ultrasound disintegrants) with the MAIS complex Mycobacterium antigens induced a humoral immune response which was largely aimed against cross-reacting than "unique" Mycobacterium antigens. Infection of BALB/c mice and rabbits with M. avium induced a humoral immune response which was chiefly against cross-antigenic Mycobacterium determinants. A series of consecutive immunochemical procedures were used to isolate from M. avium antiserum the antibody reacting with the antigenic determinants 18, 20, 24, 27, 39, 68, 94, kDa. Mab to the antigenic determinant 79 kDa of M. avium, which belong to the IgG class and unreacting with antigens of other Mycobacterium types were obtained. The antibodies isolated from hyperimmune serum and Mabs were used in the competitive EIA to determine antimycobacterial antibodies in man, mice, and rabbits. With Mab, positive results were obtained in 75% of M. avium-infected mice, 63.6% of rabbits and 63.6% of patients with M. avium infection (with 5.0% false positive results in humans). With antiserum, 83.3% of positive results were obtained in mice (with 10.0% of false positive results), 81.8 in rabbits (with 25% of false positive results) and 90.9% in M. avium-infected persons (with 30% of false positive results).

Protective response in guinea pigs exposed to Mycobacterium aviumintracellulare/M. scrofulaceum, BCG & south Indian isolates of M. tuberculosis.

The protective immunity resulting from exposure to nontuberculous mycobacteria (NTM), BCG and virulent mycobacteria in different sequences was studied in the guinea pig model employing strains prevalent in the south Indian BCG trial area and time kinetics to observe the immuno-modulation. The findings suggest that during the early course of challenge infection in guinea pigs there was no interference with the immunity due to BCG, by prior exposure to NTM. In the animals sensitised with M. avium intracellulare before immunisation, the challenge infection was localised and confined to the site of inoculation, and only a few organisms reached the spleen. However, at the later stages of the infection, as seen by the spleen viable counts at 12 wk, it appeared that the barrier at the localised site of infection may not be intact in the animals with prior exposure to NTM, and a few organisms disseminate to the spleen.

Sensitivity to sensitins and tuberculin in Swedish children. Part 5: A study of school children in an inland rural area.

SETTING: Since 1986 we have tested approximately 7000 non-BCG-vaccinated Swedish children with tuberculin and with Mycobacterium avium or M. scrofulaceum sensitin. In Göteborg, an urban coastal area, 25% of 8-9-year-old school children reacted to M. avium sensitin and 32% of M. scrofulaceum sensitin using a 6 mm cut-off. OBJECTIVE: To evaluate if geographical factors have an influence on the prevalence of children reacting to tuberculin and sensitins. DESIGN: 2000 schoolchildren of the same age as those in Göteborg but living in an inland rural area were Mantoux-tested in a similar way (simultaneously on separate arms with PPD RT23 and either of the two sensitins). RESULTS: 15% reacted to PPD RT23, 9.7% to M. avium and 13.9% to M. scrofulaceum sensitin. None of the children with a PPD RT23 reaction > = 6 mm showed signs of tuberculosis or had any known exposure. A reaction to PPD RT23 was usually combined with a still larger sensitin reaction. CONCLUSION: There was a considerable geographic difference in sensitin reactivity, low in an inland rural area compared to a coastal urban area. Children reacting to PPD RT23 had probably not been infected by M. tuberculosis but more likely by atypical mycobacteria. The reactions should be interpreted as cross-reactions.