Skin reactivity to mycobacterial antigens in children living in an area of Mycobacterium xenopi endemicity

Intradermal skin tests with a 2TU dose of PPD-RT 23 prepared from M. tuberculosis and 0.1 ug/0.1 ml of PPD-RS 631 from M. xenopi were simultaneously carried out in 378 7-year-old children from two localities in North-Bohemian region's capital Ustí n. Lab., a focus of M. xenopi endemicity repeatedly confirmed since its disclosure in 1980 by positive M. xenopi isolations from humans and public water supply network. A further group 157 children serving as controls was from Prague district 4 where no presence of M. xenopi strains was ever recorded. All of these children had received routine immunization at birth with Czech BCG vaccine. The children from the two endemic localities were found to give a positive 6 mm or greater reaction to M. xenopi mycobacterin in 43.3% and 22.3%, to human tuberculin in 12.8% and 12.6%, respectively. The frequency histogram clearly separated a group of reactors with 8-18 mm indurations from a group of nonreactors showing a skin induration of 4-8 mm. The higher reactivity of this exposed child population was also reflected in a larger proportion of reactions greater to M. xenopi PPD than to human tuberculin antigen: the reactions greater by 1-5 mm accounted, respectively, for 25.1% and 20.6%, reactions greater by 6 mm or more for 23.7% and 15.9%. Among a group of children from Prague district 4, 6.4% had medium-sized and 3.8% large-sized reactions to M. xenopi antigen; the proportion of reactions greater to M. xenopi antigen than to human tuberculin accounted for only 5.1%, reactions greater to tuberculin than to sensitin were here in slight predominance. The evidenced skin sensitization to M. xenopi mycobacterin is suggested to result from the different degrees of exposure to infection by environmental mycobacteria.     

The formation of delayed hypersensitivity to mycobacterial antigens

In experiments on guinea pigs and BALB/c mice delayed hypersensitivity to mycobacterial antigens was induced by the sensitization of the animals with live BCG or killed Mycobacterium bovis or M. avium in incomplete Freund's adjuvant. In the study of the dynamics of the development of skin reactivity to tuberculin some advantages of the sensitization of guinea pigs with live mycobacteria were revealed, while after the revaccination of the animals no development of secondary cell-mediated immune response was observed. The immunization of guinea pigs with atypical mycobacteria prior to their sensitization with BCG was found to lead to the development of higher skin reactivity to allergen prepared from atypical mycobacteria than skin reactivity to tuberculin.     

Interpretation of the PPD skin test in BCG-vaccinated children.

Skin testing with 5 tuberculin units (TU) of purified protein derivative (PPD) of tuberculin stabilized with polysorbate (Tween) 80 was done 3 months and 1 year after immunization with bacille Calmette-Guérin (BCG) vaccine in two groups of children: one group vaccinated at birth and another group at age 6 years. Interpretation of the PPD skin test with 5 TU is possible in children 1 year and older vaccinated with BCG at birth: if the diameter of induration is more than 10 to 12 mm the reaction cannot be ascribed to BCG vaccination and is highly suggestive of supervening infection with Mycobacterium tuberculosis or occasionally atypical mycobacteria. In contrast, the interpretation of a PPD test in children vaccinated at age 6 years is extremely difficult.     

Preventive treatment of tuberculin-negative contacts

Five children who had been in close contact with highly infectious tuberculous individuals presented recently to the tuberculosis service of The Montreal Children''s Hospital. Four had developed serious pulmonary tuberculosis and one tuberculous meningitis, all within the three months which followed a post-contact negative tuberculin test.The management of tuberculin-negative children recently exposed to active infectious tuberculosis by repeated skin tests and chest radiography alone is inadequate for their protection. These children are at high risk of developing disease by the time their tuberculin sensitivity has become evident. It is inadvisable to vaccinate them with BCG until three months after their last exposure to the disease. A plea is made for preventive chemotherapy in these cases.     

Specificity of antigenic fractions of tuberculin.

Purified protein derivative from Mycobacterium bovis was separated into 6 fractions by electrophoresis at 1500 V/40 mA in pH 4.2 acetic acid-pyridine buffer. Further purification of one of the fractions on Sephadex G 25 column and by acid hydrolysis yielded antigen "PS" eliciting tuberculin reaction only in animals vaccinated with M. bovis BCG but not in those sensitized with M. avium and some fast-growing mycobacteria. The specificity of antigen "PS" was confirmed in vitro: the antigen induced blastic transformation only in lymphocytes of guinea pigs sensitized with M. bovis BCG.     

Purified Protoplasmic Peptides of Mycobacteria: Isolation of Species-Specific Peptides from Protoplasm of Mycobacteria

Infections with mycobacteria other than tubercle bacilli are responsible for a variable percentage of cross-reactions to tuberculin. Two major suggestions for circumventing this problem have been made: the first, development of a quantitative tuberculin test, is based on the fact that most cross-reactions are smaller than those caused by true tuberculous infections; the second, preparation of purified skin test antigens from other mycobacteria, is based on the hope that greater specificity will be displayed by homologous sensitin. Effort so far has been focused on the culture filtrates as the source of antigen. This article describes the preparation of low molecular weight purified protoplasmic peptides (PPP) of specificity and sensitivity superior to purified protein derivatives.     

Purified Protoplasmic Peptides of Mycobacteria: In Vivo and In Vitro Comparison of the Species Specificity of Purified Protoplasmic Peptides and Purified Protein Derivatives of Mycobacterial Culture Filtrates

The specificity of purified protein derivatives (PPD) prepared from the culture filtrates of Mycobacterium tuberculosis (PPD), M. kansasii (PPD-Y), M. intracellulare (PPD-B), and M. scrofulaceum (PPD-G) were compared to comparable protoplasmic extracts (PPP) of the same organisms by gel diffusion and delayed hypersensitivity reactions in sensitized guinea pigs. PPD and, to a lesser degree, PPD-Y demonstrated specificities sufficient to enable identification of homologously sensitized guinea pigs in the above group of four mycobacteria. PPD-B and PPD-G did not always elicit the largest reaction in homologously sensitized animals. The PPP sensitins from M. tuberculosis and M. kansasii produced as good skin reactions at 24 and at 48 hr as did their PPD counterparts. The PPP from M. scrofulaceum and M. intracellulare were more specific and more reactive than corresponding PPD, regardless of the time of comparison. Although based on different immunological mechanisms, the specificity of these two groups of sensitins, as demonstrated by delayed hypersensitivity, correlated well with serological comparisons in the gel diffusion test. The low degree of specificity of PPD-B and PPD-G in contrast to that of corresponding PPP was reflected in the precipitin bands in agar gel.     

Interspecies transfer by "immune" RNA of lymphocyte proliferative response to specific antigen

Ribonucleic acid (RNA) extracted from the lymph nodes of BCG sensitized cattle transferred tuberculin sensitivity to normal guinea pig lymphocytes as indicated by increased incorporation in vitro of ³H-thymidine in response to Purified Protein Derivative (PPD). The RNA treated lymphocytes were unresponsive to a nonspecific antigen, histoplasmin. Ribonuclease treatment of the RNA abolished its ability to transfer tuberculin reactivity and RNA extracted from the lymph nodes of normal cattle was also ineffective.     

Immunogenicity of Cell Walls from Various Mycobacteria Against Airborne Tuberculosis in Mice

Protective potency of oil-treated cell walls of various mycobacteria against airborne infection of mice with a few cells of Mycobacterium tuberculosis H37Rv was compared with that of viable BCG. Although less potent than BCG cell walls, the cell walls of atypical mycobacteria of Runyon's groups I to IV protected against challenge by aerosol to some degree. Protection afforded by cell walls of H37Rv and of the avirulent mutants H37Ra and Washington II was comparable to that provided by BCG cell walls. However, cell walls of a highly virulent strain of M. bovis (Bovinus I) provided the best protection yet achieved. Present evidence suggests that protective substances are shared by all mycobacteria but in differing amounts; the relationship between virulence and immunogenicity has yet to be clarified.     

Induced sensitization to nickel in guinea pigs immunized with mycobacteria by injection of purified protein derivative with nickel

Nickel has been reported to be one of the most common causes of allergic contact dermatitis. Despite the fact that nickel is a frequent sensitizer in humans, establishing animal models for nickel allergy has met with considerable difficulties. In clinical cases, allergic contact hypersensitivity to nickel develops much more readily in inflamed skin than normal skin. In this study, we tried to induce nickel sensitization when inflammation has been evoked in guinea pigs immunized with mycobacteria followed by co-administration of a mycobacterial component with nickel. We first examined the delayed-type hypersensitivity (DTH) reaction of mycobacterial components such as the cell wall, cell membrane, 70S ribosomal fraction, cytoplasm, tuberculin purified protein derivative (PPD), RNA and DNA from Mycobacterium bovis BCG in guinea pigs immunized with live M. bovis BCG or heat killed M. tuberculosis. When PPD was used, the hypersensitivity reaction was strongest. Next, we tested whether PPD with nickel could induce nickel sensitivity in guinea pigs immunized with mycobacteria. Strong sensitization to nickel was achieved by injecting PPD with nickel. However, if too large an amount of PPD or nickel salts was used, sensitization to nickel decreased. In this way, sensitization of nickel developed much more easily in guinea pigs immunized with mycobacteria by injection of an appropriate amount of nickel at the inflammation site induced by a suitable amount of PPD.     

Comparative Study of 14C-Labeled Purified Protein Derivative from Various Mycobacteria: II. Skin Cross-Reactivity in Sensitized Guinea Pigs

A study on skin cross-reactivity between stabilized ¹⁴C-labeled mycobacterial antigens, namely tuberculin purified protein derivative (PPD; from Mycobacterium tuberculosis), PPD-A (M. avium), PPD-Y (M. kansasii), PPD-G (M. scrofulaceum), PPD-B (M. intracellulare), and PPD-F (M. fortuitum), has been carried out in groups of guinea pigs sensitized with one of the following heat-killed mycobacteria: M. tuberculosis, M. avium, M. kansasii, M. scrofulaceum, M. intracellulare, or M. fortuitum. For each type of sensitization, the average response for the corresponding PPD antigen was higher than the average response for any of the other antigens. However, the responses to the heterologous PPD antigens were not necessarily significantly different among themselves, and the significant differences of the heterologous PPD antigens were distributed differently according to the type of sensitization. Therefore, ¹⁴C-PPD antigens skin cross-reacted in guinea pigs essentially in the same manner as reported by others for nonradioactive PPD antigens.     

Induction of delayed-type hypersensitivity and antitumor immunity by systemic BCG

Both delayed-type hypersensitivity (DTH) and antitumor resistance induced in mice by intravenous (i.v.) and local injection of highly immunogenic irradiated Meth A cells were potentiated by prior systemic BCG infection. DTH and antitumor immunity were not elicited by i.v. injection of poorly immunogenic irradiated mastocytoma cells, P 815 (MA), but were induced by the local injection of these cells when animals were systemically infected with BCG. The level of the potentiated response corresponded with the dose of immunogen up to an optimum, beyond which additional immunogen was suppressive. At all dose levels the subcutaneous (s.c.) route of immunogen inoculation was more effective than the i.v. route. Significant DTH was first detected 7 days after the local administration of immunogen and was correlated with antitumor immunity. Systemically administered BCG grew mainly in the liver and spleen until the development of maximal tuberculin sensitivity when the number of organisms decreased. However, the small number of mycobacteria that reached the peripheral lymph nodes remained constant after maximal tuberculin sensitivity but failed to augment the cell proliferation that occurred in these lymph nodes as a result of the local inoculation of irradiated tumor cells. Autoradiographs of such nodes revealed proliferation in the thymus-dependent areas whereas nodes from mice immunized with immunogen alone manifested B- as well as T-cell activity. Local immunization in both BCG-infected and uninfected hosts was also associated with a proliferative response in the red pulp of the spleen but the BCG-infected hosts differed conspicuously by virtue of the presence of tubercles and depletion of lymphoid cells from the periarteriolar sheath. Immunity generated by the local administration of immunogen in systemically infected mice was tumor specific and could be adoptively transferred with spleen cells.     

Study of systemic fever reaction of the delayed type hypersensitivity

The possibility of elaborating a model of fever reaction to a simple protein antigen, ovalbumin, was investigated. Administration of the antigen in adjuvants into the foot-pads or intravenously proved unsatisfactory and did not sensitize the animal to induction of a fever reaction to a challenging dose of antigen. Sensitization by the simultaneous intravenous administration of ovalbumin together with living BCG vaccine yielded positive results. The fever response to ovalbumin is specific, since rabbits sensitized with BCG vaccine only did not respond to the administration of ovalbumin. The degree of fever reactions to tuberculin and ovalbumin in the individual rabbits was more or less proportional. The given model is reproducible and is useful for experimental studies. Further experiments will be necessary, however, for detailed characterization and for analysis of the mechanism (antibody-mediated or delayed type hypersensitivity).     

Comparative Study of 14C-Labeled Purified Protein Derivative from Various Mycobacteria: I. Preparation of 14C-Labeled Purified Protein Derivative Antigens and Their Adsorption to Glass

Biologically active (14)C-labeled purified protein derivative ((14)C-PPD) has been prepared from the culture filtrates of seven species of mycobacteria, namely Mycobacterium tuberculosis Johnston strain (PPD), M. bovis BCG (PPD-BCG), M. avium (PPD-A), M. kansasii (PPD-Y), M. intracellulare (PPD-B), M. scrofulaceum (PPD-G), and M. fortuitum (PPD-F). These mycobacteria were grown in a culture medium containing a mixture of (14)C-labeled amino acids. The yield and specific radioactivity of the PPD, of the nucleic acid, of the bacterial cells, and of the CO(2) developed during growth have been determined for each of the seven species of mycobacteria. Although the yields of (14)C-PPD antigens differed greatly for the different species of mycobacteria tested, their specific radioactivities were similar. The (14)C-PPD antigens have been used as a means to measure their adsorption to glass. When glass ampoules containing dilute solutions (0.001 mg of PPD per ml) of these PPD antigens (PPD, PPD-BCG, PPD-A, PPD-Y, PPD-G, PPD-B, and PPD-F) were stored for 12 months at 5 C, it was found that they all adsorbed equally well to glass surfaces. In fact, regardless of the origin of the PPD, a loss due to adsorption of about 90% occurred during the first month of storage, and thereafter the PPD content remained practically constant for the rest of the duration of the storage period. The addition of 0.0005% Tween 80 to the PPD solutions effectively reduced the adsorption to glass of most PPD antigens. However, adsorption of PPD-BCG was not quite so effectively prevented, even when the Tween 80 concentration was increased from 0.0005 to 0.0005%.     

The Tuberculin Skin Test (TST) Is Affected by Recent BCG Vaccination but Not by Exposure to Non-Tuberculosis Mycobacteria (NTM) during Early Life

The tuberculin skin test (TST) is widely used in TB clinics to aid Mycobacterium tuberculosis (M.tb) diagnosis, but the definition and the significance of a positive test in very young children is still unclear. This study compared the TST in Gambian children at 4½ months of age who either received BCG vaccination at birth (Group 1) or were BCG naïve (Group 2) in order to examine the role of BCG vaccination and/or exposure to environmental mycobacteria in TST reactivity at this age. Nearly half of the BCG vaccinated children had a positive TST (≥5 mm) whereas all the BCG naïve children were non-reactive, confirming that recent BCG vaccination affects TST reactivity. The BCG naïve children demonstrated in vitro PPD responses in peripheral blood in the absence of TST reactivity, supporting exposure to and priming by environmental mycobacterial antigens. Group 2 were then vaccinated at 4½ months of age and a repeat TST was performed at 20–28 months of age. Positive reactivity (≥5 mm) was evident in 11.1% and 12.5% infants from Group 1 and Group 2 respectively suggesting that the timing of BCG vaccination had little effect by this age. We further assessed for immune correlates in peripheral blood at 4½ months of age. Mycobacterial specific IFNγ responses were greater in TST responders than in non-responders, although the size of induration did not correlate with IFNγ. However the IFNγ: IL-10 ratio positively correlated with TST induration suggesting that the relationship between PPD induced IFNγ and IL-10 in the peripheral blood may be important in controlling TST reactivity. Collectively these data provide further insights into how the TST is regulated in early life, and how a positive response might be interpreted.     

Comparative testing of skin reactions to PPD mycobacterins from Mycobacterium tuberculosis and Mycobacterium scrofulaceum in school-age children

Comparative skin tests with 2 TU PPD-RT 23 with Tween 80 (prepared from M. tuberculosis) and 5 TU PPD-RS 95 with Tween 80 (prepared from M. scrofulaceum) were intradermally given to a total of 1,140 7-year-old children in two towns of Karviná district (340 and 255 children) and in Teplice (267 children) and Prague (278 children). In the two groups of Karviná district children the percentages of small-sized reactions (6-9 mm) to PPD-RT 23 were 13.6 and 22.3% compared to 7.1% in Teplice and 5.7% in Prague. The prevalence of small reactions to the PPD-RS 95 test in district of Karviná was 14.4 and 16.9%, in Teplice 4.5% and in Prague 6.8%. In the two towns of Karviná district the percentages of children whose reaction to PPD-RS 95 was larger than to PPD-RT 23 were 56.4 and 42.8%, in Teplice 24% and in Prague 24%. The hypothesis is advanced that the higher degree of skin hypersensitivity to the M. scrofulaceum mycobacterin which was found among the Karviná district children tested is due to sensitization with environmental mycobacteria which are common in this area.     

Interactions between heterochronic cells in the formation of delayed-type hypersensitivity.

The effect of ageing on lymphocyte and macrophage functional activity in the induction of tuberculin sensitivity was studied in experiments on 440 CBA mice of three age groups. The capacity of old animals to generate delayed-type hypersensitivity following administration of BCG cells was found to be diminished and their lymphocytes caused decreased hypersensitivity on transplantation. The capacity of recipients to reproduce delayed-type hypersensitivity after thymocyte and bone marrow cell transplantation was influenced by the age of transplanted thymocytes. This capacity was markedly suppressed on the recipients of old cells. The antigen presentation function of macrophage did not after significantly as a function of age.     

Molecular cloning and sequencing of the merozoite surface antigen 2 gene from Plasmodium falciparum strain FCC-1/HN and expression of the gene in mycobacteria

Strain bacillus Calmette-Guerin (BCG) of Mycobacterium bovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-1/HN Plasmodium falciparum genome, sequenced, and expressed in M. bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa. This first report of expression of the full-length P. falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.     

Tuberculosis Screening by Tuberculosis Skin Test or QuantiFERON?-TB Gold In-Tube Assay among an Immigrant Population with a High Prevalence of Tuberculosis and BCG Vaccination

Rationale

Each year 1 million persons acquire permanent U.S. residency visas after tuberculosis (TB) screening. Most applicants undergo a 2-stage screening with tuberculin skin test (TST) followed by CXR only if TST-positive at > 5 mm. Due to cross reaction with bacillus Calmette-Guérin (BCG), TST may yield false positive results in BCG-vaccinated persons. Interferon gamma release assays exclude antigens found in BCG. In Vietnam, like most high TB-prevalence countries, there is universal BCG vaccination at birth.

Objectives

1. Compare the sensitivity of QuantiFERON ®-TB Gold In-Tube Assay (QFT) and TST for culture-positive pulmonary TB. 2. Compare the age-specific and overall prevalence of positive TST and QFT among applicants with normal and abnormal CXR.

Methods

We obtained TST and QFT results on 996 applicants with abnormal CXR, of whom 132 had TB, and 479 with normal CXR.

Results

The sensitivity for tuberculosis was 86.4% for QFT; 89.4%, 81.1%, and 52.3% for TST at 5, 10, and 15 mm. The estimated prevalence of positive results at age 15–19 years was 22% and 42% for QFT and TST at 10 mm, respectively. The prevalence increased thereafter by 0.7% year of age for TST and 2.1% for QFT, the latter being more consistent with the increase in TB among applicants.

Conclusions

During 2-stage screening, QFT is as sensitive as TST in detecting TB with fewer requiring CXR and being diagnosed with LTBI. These data support the use of QFT over TST in this population.