Immunoblot Analysis for Serodiagnosis of Tuberculosis Using a 45/47-Kilodalton Antigen Complex of Mycobacterium tuberculosis

Transcription

1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1997, p Vol. 4, No X/97/$ Copyright 1997, American Society for Microbiology Immunoblot Analysis for Serodiagnosis of Tuberculosis Using a 45/47-Kilodalton Antigen Complex of Mycobacterium tuberculosis SERGE DIAGBOUGA, 1 FRANCIS FUMOUX, 1 ALAIN ZOUBGA, 2 PAUL THOMAS SANOU, 3 AND GILLES MARCHAL 4 * Centre MURAZ, Organisation de Coordination et de Coopération pour la lutte contre les Grandes Endémies, 1 Centre Régional de Lutte Anti-Tuberculeuse, 2 and Service MST/SIDA, Direction Provinciale de la Santé, 3 Bobo-Dioulasso, Burkina Faso, and Unité de Physiopathologie de l Infection, Institut Pasteur, Paris, France 4 Received 28 October 1996/Accepted 26 February 1997 We evaluated the immunoglobulin G (IgG) antibody response to the 45/47-kDa secreted protein of Mycobacterium tuberculosis by immunoblot assay, to assess its potential value for serological diagnosis. Control subjects consisted of healthy volunteers with negative or positive tuberculin skin tests. Most (>98%) scored negative in an immunoblot test when the sera were analyzed at a 1:400 dilution. Approximately 40% of sera (diluted 1 in 400) from tuberculous patients (positive smears) recognized the antigen complex. The sensitivity of the test for patients suffering from extrapulmonary tuberculosis was similar to that for patients suffering from pulmonary tuberculosis but who had negative smears. The frequency of positive reactions among the patients suffering from other pulmonary diseases was similar to that among the control subjects. In tuberculous patients infected with human immunodeficiency virus, the sensitivity of the immunoblot test was significantly lower. Thus, this test based on an antigen complex used in an immunoblot assay to detect the presence of IgG antibody has a specificity of 98% and a sensitivity of 40%. The simultaneous use of different purified antigens, selected at the same high specificity level, may improve the sensitivity of such an assay. * Corresponding author. Mailing address: Unité de Physiopathologie de l Infection, Institut Pasteur, 25, rue du Dr. Roux, Paris, France. Phone: (33) Fax: (33) Present address: INSERM U 399, Faculté de Médecine de la Timone, Marseille Cédex 05, France. Despite the availability of effective drugs, tuberculosis remains a major public health problem in most developing countries. Furthermore, its incidence is increasing in some industrialized countries (16). Current recommendations for the control of tuberculosis emphasize case detection so as to allow treatment of patients and thereby limit transmission of the bacilli (15). There is no simple, straightforward diagnostic test for active tuberculosis. Clinical symptoms are suggestive but often nonspecific, and the mycobacteriological techniques available to confirm diagnosis are far from satisfactory (8). Direct sputum smears are rapid but poorly sensitive, with only 40% of truly active pulmonary tuberculosis being confirmed even with optimal staining and satisfactory microscopic examination (1, 4, 5). Culture techniques are time-consuming and provide a definitive diagnosis only 4 weeks after the first clinical examination. Moreover, it is difficult to obtain clinical specimens from patients with less-extensive pulmonary disease and from patients with extrapulmonary tuberculosis (6). PCR and other methods for detecting Mycobacterium tuberculosis DNA sequences are expensive and are unsuitable for routine diagnostic testing, especially in developing countries (15). Serological techniques, in contrast, are simple, inexpensive, suitable, and applicable as rapid diagnostic tests. However, the specificity of serological techniques and the level of human antibody response to M. tuberculosis are low (6, 8). Immunization by environmental mycobacteria or by other bacteria present during bacterial disease may be responsible for this low specificity (8). Attempts to improve the sensitivity of serological assays often result in decreased specificity, for example if nonpurified or only partly purified antigens are used (6). Recent reports have demonstrated the presence in sera of antibodies reactive to purified M. tuberculosis antigens or to particular epitopes (2, 3, 9). The development of a serological assay for tuberculosis may require combining several antigens, chosen for their capacity to bind specific antibodies in sera from tuberculous patients. Ideally, this association of specific antigens would react poorly or not at all with sera from healthy persons previously infected with M. tuberculosis, i.e., tuberculin-positive healthy persons. We investigated the frequency and the level of antibody response to the purified 45/47-kDa antigen complex, also called APA, in a population living in a tropical zone where tuberculosis is endemic. This antigen complex has been purified for its ability to interact mainly with antibodies present in the sera of guinea pigs immunized with living M. tuberculosis (10, 14). This antigen complex was used in immunoblot assays to detect the immunoglobulin G (IgG) antibody response in pulmonary and extrapulmonary tuberculosis. It had a sensitivity of 40% regardless of the clinical form of tuberculosis when the specificity level was set at 98%. MATERIALS AND METHODS Study population. (i) Healthy subjects. Control serum samples (111 samples) were obtained from 20 tuberculin-negative and from 91 tuberculin-positive volunteers in February 1994 in Bobo-Dioulasso, Burkina Faso. Tuberculin (10 IU; 0.1 ml per injection) was injected intradermally into the volar surface of the left forearm, and 72 h later the transverse diameter of palpable induration at the site of injection was measured. The healthy individuals were about 20 years old (range, 17 to 22 years) and were attending college. Few had been vaccinated against M. tuberculosis BCG, and most were tuberculin positive, presumably as a consequence of exposure to the bacilli in their environment. A blood sample (10 ml) was collected from each subject on the day of tuberculin injection. The sera were frozen at 20 C until immunoblotting was performed. Subjects were excluded from the control group if they presented symptoms of clinical tuberculosis 334

2 VOL. 4, 1997 TUBERCULOSIS ASSAY USING 45/47-kDa ANTIGEN COMPLEX 335 or malaria. All individuals were human immunodeficiency virus (HIV) seronegative. (ii) Tuberculous patients with or without HIV type 1 (HIV-1) infection. Between October 1993 and October 1995, 127 patients with pulmonary or extrapulmonary tuberculosis were recruited from the tuberculosis clinic of the Centre Régional de Lutte Anti-Tuberculeuse, Bobo-Dioulasso, Burkina Faso. Only patients whose sputum samples were positive in direct smear tests or in culture were judged to be authentic pulmonary tuberculous patients. Extrapulmonary tuberculosis diagnosis was performed by analysis of various data (radiographic signs and clinical symptoms). Blood samples from all tuberculous patients were drawn before or at the beginning (less than 2 weeks after the start) of antituberculous treatment. The tuberculous patients were later classified according to HIV status. The mean age of the tuberculous patients was 35 years (range, 24 to 56 years). (iii) HIV-1-seropositive patients without tuberculosis. Between August 1994 and September 1995, 43 HIV-1-seropositive patients without tuberculosis were recruited at the Sexually Transmitted Disease Treatment Centre (outpatient clinics for dermatology and venereal diseases, including AIDS), Bobo-Dioulasso, Burkina Faso. These patients did not suffer from clinical tuberculosis as assessed by physical examination. They were tuberculin skin tested and classified as HIV-1 tuberculin-positive and HIV-1 tuberculin-negative patients. Their mean age was 32 years (range, 22 to 55 years). (iv) Nontuberculous pulmonary disease patients. Twenty-nine serum samples were obtained from patients with pulmonary diseases other than tuberculosis who were receiving consultation at the Centre Régional de Lutte Anti-Tuberculeuse. They were HIV seronegative, and their mean age was 37 years (range, 24 to 60 years). Patients with HIV-1 infection did not receive antiretroviral treatment. All the subjects were informed about the objectives of the study and only subjects from whom informed consent was obtained were included. The protocol was approved by the National AIDS Control Committee, Ministry of Health, Burkina Faso. HIV antibody assay. HIV-1 antibody tests were performed in the Centre MURAZ laboratory, by using an enzyme-linked immunosorbent assay (ELISA) (GENELAVIA mixt) confirmed by Western blotting (NEW LAV Blot 1) according to World Health Organization criteria (18). HIV serological kits were purchased from Sanofi Diagnostic Pasteur, Marnes la Coquette, France. Preparation of 45/47-kDa antigen. Antigen was prepared by the Unité de Physiopathologie de l Infection, Institut Pasteur, Paris, France. The methods for preparation and purification have been published elsewhere (14). Briefly, M. tuberculosis H37 Rv (a virulent strain) was cultured in round flasks containing 130 ml of synthetic Sauton medium. The culture medium was harvested after 14 days and filtered through gauze and a m-pore-size filter. The medium was intensively washed at 4 C with 4% butanol in deionized water on a PM10 Amicon membrane and concentrated to 20-fold. The concentrated medium containing molecules with molecular masses of more than 10 kda was freeze-dried. Each major fraction eluted from a series of high-pressure liquid chromatography columns (gel filtration, DEAE, and reverse-phase chromatography columns) was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto two nitrocellulose sheets. The blots were probed with antibodies raised in guinea pigs immunized either with living or with heat-killed mycobacteria. Cross-reactive antigens that stained in both immunoblots were eliminated. Major antigens (the 45/47-kDa antigen complex) interacting with antibodies raised after immunization with only living bacteria were identified and purified (14). This antigen preparation was stable during storage in the lyophilized state. Immunoblot analysis. The Phast System (Pharmacia, Uppsala, Sweden) was used for immunoblot analysis at local room temperature (22 C). The 45/47-kDa antigen complex (0.25 g) was separated by electrophoresis on 12.5% polyacrylamide PhastGels in the presence of SDS, in parallel with molecular weight markers (Pharmacia, Uppsala, Sweden). Separated components were then transferred onto a nitrocellulose sheet (pore size, 0.22 m) in a transblot unit (217 Multiphor 2; LKB, Uppsala, Sweden). Nitrocellulose sheets and Whatman paper were soaked in transfer buffer, and the minigel was encompassed in a sandwich; 45 min after the transfer, the nitrocellulose sheet was stained with Ponceau red and destained in methanol-acetic acid. The position of the 45/47-kDa antigen complex was checked by comparison with the positions of molecular weight markers. The nitrocellulose sheet was cut into eight strips and washed with sodium phosphate buffer (PBS). The strips were blocked with nonfat dry milk (5%) in PBS for 4 h with gentle agitation and then washed twice for 5 min in T-PBS (PBS 0.1% Tween 20). They were incubated overnight with gentle agitation at room temperature in the presence of sera diluted in PBS containing Tween 20 (0.5%) and nonfat dry milk (5%). The strips were washed three times with T-PBS and then incubated for 4 h with 1 ml of the second antibody [alkaline phosphatase-conjugated AffinePure F(ab ) 2 fragment goat anti-human IgG (H L); Jackson ImmunoResearch Laboratories, Inc., Baltimore, Md.] diluted 1:2,000 in T-PBS containing nonfat dry milk. The washing procedure was repeated and followed by the addition of 500 l of substrate containing bromochloroindolyl phosphate-nitroblue tetrazolium (BCIP/NBT) (Sigma, Saint Quentin Fallavier, France). The development time was limited to 15 min, allowing a purple coloration to appear on positive strips. The results were interpreted FIG. 1. Immunoblot analyses of various sera with the 45/47-kDa antigen complex. The proteins were separated by SDS-polyacrylamide gel electrophoresis (PhastGel homogeneous 12.5% polyacrylamide gel; Pharmacia) and transferred onto nitrocellulose. The strips were incubated in sera (diluted 1:200) from tuberculin-negative (lanes 1) or tuberculin-positive (lanes 2) healthy subjects and from tuberculosis patients (lanes 3). visually by two independent investigators. All the dilutions for each serum were tested in duplicate. Data analysis. Sigma Plot/PC software (Jandel Scientific) was used for statistical data analysis. Sensitivity and specificity values were calculated by using the Mann-Whitney U test. A P value of less than 0.05 was considered to be statistically significant. RESULTS Development of the immunoblot test. Serum samples from seven pulmonary tuberculos patients with positive sputum samples and from five healthy subjects were used for preliminary experiments. The sera were diluted 1:200 in the development buffer. The 45/47-kDa antigen complex was labeled by five of the tuberculous patient samples but not by four of the healthy subject samples. The test was then repeated randomly with these 12 serum samples after various periods of time to ensure reproducibility (r 0.99; P ). These sera were used thereafter as positive and negative controls and were included in each experimental run. Representative results are reported in Fig. 1. Control group. The control group gave low-intensity, tuberculin-induced inflammatory reactions. However, the distribution of the induration diameters was bimodal, allowing classification into reactors (diameter, 2 mm) and nonreactors (diameter, 1 mm) (Fig. 2). Among 20 serum samples collected from nonreactors (16 subjects) and weak reactors (four subjects showing 1-mm diameters of induration), 4 and 3 labeled the 45/47-kDa antigen complex when diluted 1:50 and 1:200, respectively. No serum diluted 1:400 labeled the antigen complex. Among 91 serum samples from subject reactors, 56 and 50 labeled the antigen complex when diluted 1:50 and 1:200, respectively. Only two of them labeled the antigen complex at a dilution of 1:400 (Fig. 3 and Table 1). The diameters of the indurations of the tuberculin reactions were relatively high for these two subjects (7 and 9 mm). Pulmonary tuberculosis patients without HIV infection. (i) Patients with positive smears. Serum samples from 59 patients with bacteriologically confirmed tuberculosis were tested. The 45/47-kDa antigen complex was labeled by 48 serum samples diluted 1:50, 46 serum samples diluted 1:200, and 23 serum samples diluted 1:400 (Fig. 3 and Table 1).

3 336 DIAGBOUGA ET AL. CLIN. DIAGN. LAB. IMMUNOL. TABLE 1. Proportions of positive serum samples in each group a Group No. of subjects No. (%) of positive serum samples Control groups Tuberculin-negative 20 0 (0) Tuberculin-positive 91 2 (2) Tuberculous patients with: Positive smears (39) Negative smears 7 4 (57) Extrapulmonary tuberculosis 15 2 (13) Patients with nontuberculous pulmonary disease 29 2 (7) FIG. 2. Distribution of tuberculin (10 IU) reactions of 111 healthy students. To be useful for diagnosis, a serological test should differentiate between healthy persons, including those previously infected with M. tuberculosis, and patients developing active tuberculosis. By our method, the dilution giving the best discrimination between tuberculous and control subjects was 1:400. At this dilution, the immunoblot test showed a high specificity ( 98% [range, 93 to 100%]) but the sensitivity was moderate (40% [range, 27 to 53%]). (ii) Patients with negative smears. Sera from seven patients with pulmonary tuberculosis whose sputum samples were negative in each of three different smear tests were analyzed; four of these serum samples were positive in the immunoblot assay when diluted 1:400 (Table 1). FIG. 3. Percentages of serum samples labeling the 45/47-kDa antigen complex in the immunoblot test. Tuberculin, tuberculin-positive; Tuberculin, tuberculin-negative. HIV-1-seropositive patients Without tuberculosis 43 2 (5) With tuberculosis 46 1 (2) a Sera were diluted 1:400 and tested in the immunoblot assay against the 45/47-kDa antigen complex. Extrapulmonary tuberculosis patients without HIV infection. Sera from 15 patients, suffering from pleurisy (n 5), lymph node tuberculosis (n 1), osteoarticular tuberculosis (n 2), and genitourinary tuberculosis (n 7), were tested; two serum samples labeled the 45/47-kDa antigen complex at a 1:400 dilution (Table 1). Thus, among the 22 tuberculous patients, 6 had sera positive at a 1:400 dilution. The sensitivity of the assay, around 27% (range, 12 to 50%), was similar to that for patients suffering from active, smear-positive pulmonary tuberculosis. Analysis of a larger number of serum samples from extrapulmonary tuberculosis patients would allow a better statistical evaluation of the test. Nontuberculous pulmonary disease patients without HIV infection. Sera from 29 patients, hospitalized for nontuberculous pulmonary diseases, were examined. Two of the serum samples labeled the antigen complex when diluted 1:400 (Table 1). HIV-1-seropositive patients. (i) Patients without clinical tuberculosis. Sera from HIV-1-seropositive patients without clinical tuberculosis were tested, including 18 serum samples from tuberculin-positive subjects and 25 from tuberculin-negative subjects. One serum sample from each group of patients was reactive with the antigen complex when diluted 1:400 (Table 1). (ii) Patients with clinical tuberculosis. Sera diluted 1:400 from 32 bacteriologically confirmed pulmonary tuberculosis HIV-1-infected patients did not label the antigen complex. Only one of three serum samples from pulmonary tuberculosis patients without microscopic confirmation was able to label the antigen complex. None of the 11 extrapulmonary tuberculosispositive HIV-1 patients (9 pleurisy patients, 1 patient with lymph node localization, and 1 patient with pericarditis) was reactive (Table 1). Thus, among 46 serum samples from HIV-1-seropositive patients suffering from tuberculosis, only 1 contained significant levels of IgG against the 45/47-kDa antigen complex. These results illustrate the serological unresponsiveness of HIV-1-infected persons. DISCUSSION The control of tuberculosis in a population is based on the detection of cases of overt tuberculosis and their cure by chemotherapy. Unfortunately, the diagnosis of active cases is dif-

4 VOL. 4, 1997 TUBERCULOSIS ASSAY USING 45/47-kDa ANTIGEN COMPLEX 337 ficult. Although sputum is easily available for examination from patients with advanced pulmonary tuberculosis, it is difficult to obtain specimens from some patients, especially children and patients with closed lesions. Furthermore, accurate microscopic examination of smears is not easy and interpretation can vary. Sound laboratory practice and a high level of expertise are required. Furthermore, this approach could be dangerous because of the possible risks of contamination. The development of a straightforward, low-cost, and widely available diagnostic test would be of great value to the control of tuberculosis (6, 8, 15). To meet all these requirements, any ELISA or immunoblot test would require good specificity and sensitivity, and thus its value depends upon the selection and use of appropriate antigens (12, 17). The 45/47-kDa antigen complex was purified from BCG and M. tuberculosis culture filtrate according to its ability to interact only with antibodies raised in guinea pigs immunized with live M. tuberculosis. Antibodies present in the sera of guinea pigs immunized with dead bacteria contain no or only a very low level of antibodies directed against the complex (14). In preliminary tests, specific antibodies were detected in sera from patients suffering from active pulmonary tuberculosis but not in sera from patients suffering from other infectious diseases. Consequently we investigated the suitability of the 45/47-kDa antigen complex for serodiagnosis. Various fractions purified or partly purified from mycobacteria have previously been proposed for use in diagnostic tests. In most cases, although the difference between controls and patients was statistically significant, the cutoff between positive and negative test results was not established clearly. For our test, we investigated sera from healthy subjects and from tuberculous patients living in the same area under similar or nearly similar socioeconomical conditions in order to evaluate the sensitivity of the assay at the best specificity level. The induration diameters of positive tuberculin reactions observed in control healthy individuals were small and similar to those observed in a cohort of 49 patients with active pulmonary tuberculosis (data not shown). We have no good explanation for these weak reactions in positive subjects despite technically satisfactory tuberculin injection and the standardized evaluation of reactivity: two readers recorded similar results in a blind assay. However, the responses fell into two groups: positive and negative tuberculin reaction. The sera from tuberculin-negative subjects were generally less reactive than sera from tuberculin-positive patients, when different dilutions were assayed on the antigen complex (Fig. 3). The chosen cutoff dilution (1:400) allowed the discrimination of subjects with a high level of specific antibodies from other subjects. The specificity and sensitivity of the immunoblot test using this cutoff were 98 and 40%, respectively, for HIV-negative subjects. Among sera from patients suffering from nontuberculous pulmonary diseases, the frequency of positive results was similar to that in the control subjects. Despite the moderate sensitivity, this test may help physicians choose to start chemotherapy for patients in whom tuberculosis is suspected but whose smear results are negative (or when microscopic examination is not possible, for example, in cases of renal or osteoarticular tuberculosis). HIV-1-seropositive subjects suffering from bacteriologically proven tuberculosis developed antibody titers lower than those of tuberculous HIV-1-seronegative subjects. Recent studies have reported a reduced sensitivity of tuberculosis serodiagnosis in patients with AIDS in Uganda and in Zaire (7, 13). The antigens used were the 30-kDa (85B) antigen and the A60 antigen of M. tuberculosis in an ELISA. Our results with the 45/47-kDa antigen complex in an immunoblot assay were similar in terms of decreased levels of IgG antibody response in HIV-seropositive subjects. The reasons for this are beyond the scope of this work, but it may be of interest to compare the IgG antibody response of HIV-seropositive subjects to protein antigens from mycobacteria, which are facultative intracellular bacteria, with that to protein antigens from extracellular bacteria. A previous study reported no difference between the results of HIV and HIV subjects living in Spain who were tested for antibody responses to mycobacterial sulfolipid (11). This suggests that reactivity varies with the chemical nature of the antigens or that the disease process may differ among distant geographical regions. In summary, the 45/47-kDa complex antigen is recognized as an antigen by the sera of at least 80% of humans. The specific antibody titer differs between healthy subjects (without disease) who have been previously infected by M. tuberculosis and tuberculous patients (pulmonary and extrapulmonary cases). Thus, the antigen complex may be helpful as an element of a serological test. The comparison of assays using the 30-kDa (85A), 19-kDa, and A60 antigens or other purified molecules under very similar conditions and the use of a well-defined library of sera obtained from healthy tuberculin-positive individuals would allow optimal conditions to be defined for each antigen and help to determine the cutoff values. 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