Non-tuberculous mycobacteria: patterns of isolation. A multi-country retrospective survey.

OBJECTIVE: To collect data on non-tuberculous mycobacteria (NTM) isolated from clinical laboratories in different countries to establish: 1) whether the isolation of NTM was increasing, 2) which species were increasing, and 3) whether there was any pattern of geographical distribution. DESIGN: In 1996, the Working Group of the Bacteriology and Immunology Section of the International Union Against Tuberculosis and Lung Disease contacted 50 laboratories in different countries for the necessary information. RESULTS: The number of patients reported with NTM was 36099 from 14 countries. Mycobacterium avium complex, M. gordonae, M. xenopi, M. kansasii and M. fortuitum were the five species most frequently isolated. There was a significant upward trend for M. avium complex and M. xenopi. Pigmented mycobacteria predominated in Belgium, the Czech Republic and the Mediterranean coast of Spain. Non-chromogenic mycobacteria were found to be predominant in the area of the Atlantic coast of Brazil and in Turkey, the United Kingdom, Finland and Denmark. CONCLUSIONS: There was an increase in the number of NTM isolated from clinical samples of patients. Isolation of the most frequent species is constantly changing in most of the geographical areas, and newer species are emerging due to better diagnostic techniques to detect and identify NTM.

Utility of PCR in diagnosing pulmonary tuberculosis.

At present, the rapid diagnosis of pulmonary tuberculosis rests with microscopy. However, this technique is insensitive and many cases of pulmonary tuberculosis cannot be initially confirmed. Nucleic acid amplification techniques are extremely sensitive, but when they are applied to tuberculosis diagnosis, they have given variable results. Investigators at six centers in Europe compared a standardized PCR system (Amplicor; Roche) against conventional culture methods. Defined clinical information was collected. Discrepant samples were retested, and inhibition assays and backup amplification with a separate primer pair were performed. Mycobacterium tuberculosis complex organisms were recovered from 654 (9.1%) of 7,194 samples and 293 (7.8%) of 3,738 patients. Four hundred fifty-two of the M. tuberculosis isolates from 204 patients were smear positive and culture positive. Among the culture-positive specimens, PCR had a sensitivity of 91.4% for smear-positive specimens and 60.9% for smear-negative specimens, with a specificity of 96.1%. Analysis of 254 PCR-positive, culture-negative specimens with discrepant results revealed that 130 were from patients with recently diagnosed tuberculosis and 94 represented a presumed laboratory error. Similar analysis of 118 PCR-negative, culture-positive specimens demonstrated that 27 discrepancies were due to presumed uneven aliquot distribution and 11 were due to presumed laboratory error; PCR inhibitors were detected in 8 specimens. Amplicor enables laboratories with little previous experience with nucleic acid amplification to perform PCR. Disease in more than 60% of the patients with tuberculosis with smear-negative, culture-positive specimens can be diagnosed at the time of admission, and potentially all patients with smear-positive specimens can immediately be confirmed as being infected with M. tuberculosis, leading to improved clinical management.

DNA fingerprinting and phenotyping of Mycobacterium tuberculosis isolates from human immunodeficiency virus (HIV)-seropositive and HIV-seronegative patients in Tanzania.

With the purpose of determining whether the risk of infection with a particular clone of Mycobacterium tuberculosis is influenced by the human immunodeficiency virus (HIV) status of the host, we analyzed and compared 68 mycobacterial isolates obtained from HIV-seropositive patients with tuberculosis (TB) in Dar es Salaam, Tanzania, with 66 mycobacterial isolates obtained from HIV-seronegative patients with TB in the same geographical region by using both DNA fingerprinting and classical phenotyping methods. One hundred one different IS6110 fingerprinting patterns were observed in the 134 isolates. The level of diversity of the DNA fingerprints observed in the HIV-seropositive group was comparable to the level of the diversity observed in the HIV-seronegative group. Resistance to a single anti-TB drug was found in 8.8% of the tested isolates, and 3.2% of the isolates were resistant to more than one anti-TB drug. The drug susceptibility profiles were not significantly difference between the two groups of isolates compared in the present study. Phenotypic characteristics which classify M. tuberculosis strains as belonging to the Asian subgroup correlated with a low IS6110 copy number per isolate. However, the occurrence of Asian subgroup strains was not associated with the HIV status of the patients. The results of the study suggested an equal risk of infection with a defined M. tuberculosis clone for HIV-seropositive and HIV-seronegative individuals.

Firefly luciferase assay of adenosine triphosphate as a tool of quantitation of the viability of BCG vaccines.

The purpose of the present study was to compare conventional colony forming unit (CFU) enumeration with a bioluminescent measurement in order to quantitate living bacteria in BCG vaccine. Forty batches of BCG vaccine quantitated with respect to weight (10 each of concentration 120 mg/vial, 30 mg/vial, 1.5 mg/vial and 0.75 mg/vial, respectively) were tested. Adenosine triphosphate (ATP) was extracted using the boiling Tris-EDTA extraction method preceded by treatment with the ATP-hydrolysing enzyme apyrase, and the liberated ATP was assayed using a LKB 1251 luminometer. The sensitivity of the assay was 10(-11) M ATP which corresponded to 5 x 10(5) CFU/ml. A proportional relation between CFU and ATP content per vial was found up to 30 mg/vial, but for 120 mg/vial the CFU method seemed unreliable in contrast to the bioluminescence method. The measurements error for the logarithm (base 10) of ATP was 0.051 and this method was therefore more exact than the CFU method (0.097). Because the bioluminescence method is sensitive, reliable, time-saving and less expensive it seems preferable to CFU enumeration in the quality control of BCG vaccines.

Serovars of Mycobacterium avium complex isolated from patients in Denmark.

Danish isolates of Mycobacterium avium complex were serotyped by the use of seroagglutination. The most prevalent serovars among patients with AIDS (n = 89) were 4 and 6, while among non-AIDS patients the most prevalent serovars were 1, 6, and 4, with no major differences between those in patients with pulmonary disease (n = 65) and those in patients with lymph node infection (n = 58). The results suggest a Scandinavian distribution of serovars with a predominance of serovar 6 and fail to demonstrate any selective protection against different serovars by Mycobacterium bovis BCG vaccination.

Identification of immunodominant antigens during infection with Mycobacterium tuberculosis.

T lymphocytes isolated from mice infected with Mycobacterium tuberculosis response vigorously to proteins secreted by the bacilli and these antigens may be of importance in the generation of protective immunity against the disease. In this study, short-term culture filtrate (ST-CF), which constitutes a complex mixture of secreted proteins, was fractionated by a modified preparative SDS-PAGE technique. The ability of each fraction to be recognized by T cells isolated from infected mice was evaluated by quantifying proliferation and IFN-gamma production in cell cultures. Two molecular mass regions 4-11 and 26-35 kDa were found to possess marked stimulatory properties. Four potent single antigens were mapped within the stimulatory regions. These purified antigens stimulated T cells isolated from mice at the height of a tuberculous infection to produce large amounts of IFN-gamma. Two of these stimulatory antigens belonged to the antigen 85 complex.

Results of the third immunology of leprosy/immunology of tuberculosis antimycobacterial monoclonal antibody workshop.

An international workshop was sponsored by the World Health organization to screen new antimycobacterial monoclonal antibodies and to identify antibodies which could be recommended as standard reagents giving consistent results under differing assay conditions. Fifty-eight antibodies were submitted to the workshop by eight independent laboratories. Nineteen of the antibodies recognized antigens distinct from those identified in earlier workshops, defining at least 10 new protein antigens. Monoclonal antibodies characterized in the workshop provide a set of convenient reagents for further characterization of mycobacterial antigens.

Detection of mycobacteria from blood and bone marrow: a decade of experience.

This study reports our experience with methods used at our department from 1981 through 1990 for detection of mycobacteria in blood and bone marrow specimens. Direct inoculation on Lowenstein Jensen media was replaced by Isolator lysis-centrifugation followed by inoculation on conventional solid media, and the Bactec 12B and Bactec 13A systems. A total of 3033 specimens were analyzed. A total of 137 mycobacterial isolates were obtained from 42 patients, all HIV-positive except one. Mycobacteremia caused by M. avium-intracellulare (83%), M. tuberculosis, M. scrofulaceum and M. kansasii was found. Of 680 blood specimens tested by the last three methods, 7.6% were found to be positive by at least one method and revealed recovery rates of 6.8% for the Isolator-solid media system, 3.4% for the Isolator-12B system and 6.9% for the 13A system (all isolates MOTT). Mean detection times for 21 cultures found positive by all three methods were 23.6, 23.3 and 17.7 days for the Isolator-solid media, Isolator-12B and 13A systems, respectively, with a significantly shorter detection time for the 13A system. Low degree (less than 1 cfu/ml) mycobacteremia (MOTT) caused delay in the Isolator-solid media and the 13A systems and no detection in the Isolator-12B system. Antituberculous therapy significantly prolonged the detection times for MOTT in the 13A system in contrast to the other systems.

Infection with Mycobacterium bovis in a patient with AIDS: a late complication of BCG vaccination.

We present a 28-year-old HIV-infected man with a 2-year delayed complication of BCG immunization. When immunized the man was healthy, with an unknown HIV status, but 2 years later he was diagnosed with AIDS because of a Pneumocystis carinii pneumonia. He was successfully treated and discharged in a state of good health. A few months later he presented with an enlarged lymph node and Mycobacterium bovis, BCG strain, was cultured. No sign of dissemination was found. We discuss the indications for BCG vaccination in adults, especially in areas and in populations with a high prevalence of HIV.

Proteins released from Mycobacterium tuberculosis during growth.

Proteins secreted from Mycobacterium tuberculosis during growth are believed to be important for protective immunity against tuberculosis. We have investigated the growth of M. tuberculosis in an enriched liquid medium. The release of isocitrate dehydrogenase from the bacilli served as a marker of autolysis and was observed during the late logarithmic growth phase. The release of proteins during the culture period was investigated by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three major groups of proteins, which differed markedly with respect to profile of release and location in intact bacilli, were defined. A short-term filtrate devoid of autolytic products was defined and found to be composed of 33 major components. Five proteins were identified by monoclonal antibodies. Pronounced superoxide dismutase activity was detected in the filtrate. The enzyme was purified and identified as a dominating component of short-term filtrate.

Affinity purification, biological characterization and serological evaluation of defined antigens from Mycobacterium tuberculosis.

Nine mycobacterial antigens have been purified by affinity chromatography using monoclonal antibodies (m. abs) as immunosorbent. The importance of the individual antigens have been evaluated by different immunological methods.

Quantitative and qualitative studies on the major extracellular antigen of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG.

The Mycobacterium tuberculosis antigen 85 is a biologically important antigen. Tuberculosis patients may have strong antibodies against it, and their peripheral blood mononuclear cells respond to it with gamma-interferon production and lymphocyte proliferation. Antigen 85 is actively secreted into the culture medium during culture in vitro and is known to bind human fibronectin. A double-antibody enzyme-linked immunosorbent assay (ELISA) for quantification of antigen 85 is described. A mouse monoclonal antibody, HYT27, was used as capture antibody in the assay. HYT27 was characterized in crossed immunoelectrophoresis and found to bind all three components of the antigen 85 complex. By radioimmunoassay, HYT27 was found to bind equally well to antigens 85A and 85B. In the ELISA assay, a rabbit anti-antigen 85 antiserum was used in the second antibody layer. The specificity of the assay was tested using several different antigen preparations. The purified BCG 85A and 85B components were compared, and there was a 10 times lower sensitivity for antigen 85A due to weaker rabbit antibodies toward this component. The purified components MPT44 and MPT59 from M. tuberculosis H37Rv were compared with the components of BCG and found to correspond to BCG 85A and 85B, respectively. Mycobacterium kansasii and Mycobacterium avium both contained partially identical antigens. Small amounts of antigen 85 were detected in Mycobacterium leprae sonicates. Detecting antigen 85 by sensitive methods may be of great value in the early diagnosis of mycobacterial disease.

Plasmid profiles of mycobacterium avium/intracellulare isolated from patients with AIDS or cervical lymphadenitis and from environmental samples.

Plasmid profile analysis was performed on Mycobacterium avium/M. intracellulare isolates from patients with AIDS, from children with cervical lymphadenitis, and from environmental sources. The frequency of plasmid-containing strains was found to be, respectively, 5/16 (31%), 4/15 (27%) and 1/15 (7%). The data indicate that plasmids occur more frequently among clinical isolates than among environmental isolates, and support a possible pathogenic role of the plasmids.

Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG.

Fibronectin (FN)-binding antigens are prominent components of short-term culture supernatants of Mycobacterium tuberculosis. In 3-day-old supernatants, a 30-kilodalton (kDa) protein was identified as the major FN-binding molecule. In 21-day-old supernatants, FN bound to a double protein band of 30 and 31 kDa, as well as to a group of antigens of larger molecular mass (57 to 60 kDa). FN-binding molecules in this size range, but not of 30 to 31 kDa, were also found in sonicates. We showed that the 31- and 30-kDa FN-binding bands correspond to components A and B of the BCG85 complex, previously shown to be abundant in culture supernatants of Mycobacterium bovis BCG. Thus, a polyclonal antibody to the BCG85 complex bound to the 30- and 31-kDa antigens and inhibited binding of FN to them on immunoblots of the culture filtrates. Similarly, FN bound to the purified components of the BCG85 complex, and this binding was blocked by the antibody. A monoclonal antibody, HYT27, also bound both to the BCG85 components A and B and to the 30- and 31-kDa FN-binding molecules of M. tuberculosis, but it did not block the binding of FN. Related molecules appear to be present on the surface of BCG and to mediate the binding of BCG to FN-coated plastic surfaces, since this binding could also be blocked by the polyclonal anti-BCG85 antibody and by the purified components of BCG85, particularly component A, but not by monoclonal antibody HYT27. The binding of these mycobacterial antigens to FN appears to be of very high affinity, and we suggest that this property of major secreted antigens of M. tuberculosis indicates an important role in mycobacterial disease and in the binding of BCG to tumor cells during immunotherapy of bladder cancer.

The antigens of Mycobacterium tuberculosis, H37Rv, studied by crossed immunoelectrophoresis. Comparison with a reference system for Mycobacterium bovis, BCG.

On the basis of a previously developed reference system for Mycobacterium bovis, BCG, in crossed immunoelectrophoresis (CIE), antigens of Mycobacterium tuberculosis were identified in an analogous system. A majority of the numbered lines in the BCG system were also present and identified in the M. tuberculosis system. The corresponding antigens in the two systems were identified by dual dilution in CIE, and using monospecific antisera and monoclonal antibodies. Some of the antigens were specifically identified by the demonstration of enzyme activity and by means of hydroxyapatite, concanavalin A (Con A), EDTA, and blue-Sepharose. Three antigens (nos 10, 78, and 81), which were found in high concentrations in M. tuberculosis culture fluid, were not identified or were present in low concentrations in BCG culture fluid. The high percentage of corresponding antigens confirms that there is a very close taxonomic relationship between BCG and M. tuberculosis. Corresponding antigens in BCG and M. tuberculosis did not differ in electrophoretic mobility in the antigenic preparations studied.

Allergenic and blastogenic reactivity of three antigens from Mycobacterium tuberculosis in sensitized guinea pigs.

Three antigens from a culture filtrate of Mycobacterium tuberculosis H37Rv were purified by affinity chromatography, using monoclonal antibodies. The molecular weights of the purified antigens are 17,000 to 19,000, 32,000 to 33,000, and 39,000, respectively, and by their migration patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, they all appeared to be single-chain polypeptides. Western blot and enzyme-linked immunosorbent assay analyses indicated that the antigens are non-cross-reactive. All antigens generated an intermediate to strong skin reaction when tested in guinea pigs previously immunized with a live M. bovis BCG vaccine or with an oil emulsion preparation of phenol-or heat-killed M. tuberculosis. Lymphocytes isolated from peripheral blood or lymph nodes of similarly immunized guinea pigs could be stimulated by purified protein derivative and the purified antigens. Qualitative differences in stimulatory capacity between the preparations were demonstrated. The antigens may prove useful in further studies of the immunology and pathogenesis of tuberculosis.

Interspecies reactivity of five monoclonal antibodies to Mycobacterium tuberculosis as examined by immunoblotting and enzyme-linked immunosorbent assay.

Five different murine monoclonal antibodies (MAbs) to Mycobacterium tuberculosis were examined for degree of cross-reactivity with other mycobacterial species by enzyme-linked immunosorbent assay and immunoblotting. One MAb reacted solely with M. tuberculosis and M. bovis BCG. Two of the MAbs reacted with all mycobacterial species examined, whereas two MAbs demonstrated a limited reactivity pattern. The epitopes are located on molecules susceptible to protease treatment, and two of these molecules possess concanavalin A-binding moieties. Two of the antigens defined by these five MAbs are present in tuberculin purified protein derivative.

Production and partial characterization of monoclonal hybridoma antibodies to Mycobacterium tuberculosis.

The production and partial characterization of monoclonal hybridoma antibodies against Mycobacterium tuberculosis is described. 30 clones have been produced. Their reactivity towards M. tuberculosis antigens have been tested in immunoblotting from SDS-PAGE, in crossed immunoelectrophoresis and in ELISA. The Mab's separated into four reactivity groups, one of which may be relevant in assays for antigen specific for M. tuberculosis.