The Mycobacterium avium complex.

Mycobacterium avium complex (MAC) disease emerged early in the epidemic of AIDS as one of the common opportunistic infections afflicting human immunodeficiency virus-infected patients. However, only over the past few years has a consensus developed about its significance to the morbidity and mortality of AIDS. M. avium was well known to mycobacteriologists decades before AIDS, and the MAC was known to cause disease, albeit uncommon, in humans and animals. The early interest in the MAC provided a basis for an explosion of studies over the past 10 years largely in response to the role of the MAC in AIDS opportunistic infection. Molecular techniques have been applied to the epidemiology of MAC disease as well as to a better understanding of the genetics of antimicrobial resistance. The interaction of the MAC with the immune system is complex, and putative MAC virulence factors appear to have a direct effect on the components of cellular immunity, including the regulation of cytokine expression and function. There now is compelling evidence that disseminated MAC disease in humans contributes to both a decrease in the quality of life and survival. Disseminated disease most commonly develops late in the course of AIDS as the CD4 cells are depleted below a critical threshold, but new therapies for prophylaxis and treatment offer considerable promise. These new therapeutic modalities are likely to be useful in the treatment of other forms of MAC disease in patients without AIDS. The laboratory diagnosis of MAC disease has focused on the detection of mycobacteria in the blood and tissues, and although the existing methods are largely adequate, there is need for improvement. Indeed, the successful treatment of MAC disease clearly will require an early and rapid detection of the MAC in clinical specimens long before the establishment of the characteristic overwhelming infection of bone marrow, liver, spleen, and other tissue. Also, a standard method of susceptibility testing is of increasing interest and importance as new effective antimicrobial agents are identified and evaluated. Antimicrobial resistance has already emerged as an important problem, and methods for circumventing resistance that use combination therapies are now being studied.     

Identification of mycobacteria from culture by using the Gen-Probe Rapid Diagnostic System for Mycobacterium avium complex and Mycobacterium tuberculosis complex.

Commercially available kits (Mycobacterium avium Complex Rapid Diagnostic System and Mycobacterium tuberculosis Complex Rapid Diagnostic System; Gen-Probe, Inc., San Diego, Calif.) utilizing nucleic acid hybridization for the rapid identification of members of the M. avium-M. intracellulare complex and M. tuberculosis complex were evaluated by using 339 clinical and American Type Culture Collection (Rockville, Md.) isolates. The tests, which can be performed in approximately 2 h, use specific [125I]DNA probes complementary to the rRNAs of M. avium, M. intracellulare, and M. tuberculosis complex, the latter of which includes M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti. The M. avium-M. intracellulare probes correctly identified 99 of 114 M. avium-M. intracellulare isolates, with 7 false-negatives and 8 false-positives, for a sensitivity of 93.4% and a specificity of 96.6%. After repeat testing, 110 of 114 were correctly identified, with 4 false-negatives and no false-positives, for a sensitivity of 96.5% and a specificity of 100%. The M. tuberculosis complex probe correctly identified 99 of 102 M. tuberculosis isolates, with 1 false-negative and 2 false-positives, for a sensitivity of 99% and a specificity of 99.2%. After repeat testing, 100 of 102 isolates were correctly identified, with no false-negatives and 2 false-positives, for a sensitivity of 100% and a specificity of 99.2%. Overall, there were 15 discrepant M. avium-M. intracellulare results, with 4 such results after repeat testing, and 3 discrepant M. tuberculosis complex results, with 2 such results after repeat testing. The Gen-Probe kits are highly sensitive and specific for use in identifying M. avium-M. intracellulare complex and M. tuberculosis complex isolates and will be useful in the clinical laboratory which can use the present radionuclide-containing kits cost effectively.     

Identification of Mycobacterium avium complex strains and some similar species by high-performance liquid chromatography.

Strains of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium xenopi, and Mycobacterium gordonae were identified by high-performance liquid chromatography (HPLC) analysis of mycolic acids as bromophenacyl esters. HPLC criteria were used to develop a flow chart identification scheme, which was evaluated in our laboratory with a set of 234 strains representing five species and a hitherto undescribed species. Correct identifications of M. gordonae and M. xenopi were easily made. Flow chart differentiation of M. avium, M. intracellulare, and M. scrofulaceum was done with 97.9, 97.5, and 89.2% accuracies, respectively. Independent evaluation of the flow chart at a separate laboratory demonstrated an overall identification accuracy of 97% for M. avium complex. Strains that have been described biochemically as being intermediate between M. avium-M. intracellulare and M. scrofulaceum were identified as one or the other of these known species. Strains which were negative with the species-specific radioactive probe for M. avium complex but which were positive with the nonradioactive SNAP X probe were usually identified as M. intracellulare and M. scrofulaceum but rarely as M. avium.     

Mycobacterium avium complex: Advances in therapy

DisseminatedMycobacterium avium complex (MAC) is one of the most common opportunistic infections in AIDS patients and is increasingly recognized as a significant pathogen in chronic pulmonary disease in nonimmunocompromised patients. Important progress in therapy has occurred over the last several years. In AIDS patients, multidrug therapy has been shown to be beneficial in terms of reducing circulating bacteremia and improving clinical symptoms. Clarithromycin and azithromycin, two broad-spectrum antimicrobials with minimal activity againstMycobacterium tuberculosis, have emerged as potent, well tolerated agents pivotal to treatment regimens. In AIDS patients, rifabutin prophylaxis reduced the frequency of MAC bacteremia by 50 % in two placebo controlled trials. Despite these advances, there remains a need for determining the optimal combination regimens for therapy, and more effective drugs for prophylaxis which are beneficial both in terms of survival and functional capacity of patients.     

Mycobacterium avium complex, an emerging pathogen in Massachusetts.

We report a study of 1,953 patients whose laboratory records from 1972 through 1983 at the Massachusetts Mycobacteria Reference Laboratory indicated the isolation of Mycobacterium avium complex (MAC) organisms. At least one clinical specimen from each patient during this period exhibited the organism. The incidence of isolation of MAC has increased fivefold since 1972, with a doubling of the number of patients with positive MAC specimens from normally sterile sites occurring since 1980. A concomitant increase of more than fourfold in other nontuberculous mycobacteria has occurred. Most isolates came from high-density population centers. Communities whose drinking water comes from a distant rather than a local source were more likely to have patients with MAC.     

Gen-Probe Rapid Diagnostic System for the Mycobacterium avium complex does not distinguish between Mycobacterium avium and Mycobacterium paratuberculosis.

Three reference and 16 field strains of Mycobacterium paratuberculosis were tested with the Gen-Probe Mycobacterium avium complex DNA probe (Gen-Probe Inc., San Diego, Calif.). All reference strains and 12 of 16 field strains gave positive hybridization results with the probe. This study shows that the M. avium complex probe does not distinguish between M. avium and M. paratuberculosis and indicates heterogeneity in the 16S rRNA gene of M. paratuberculosis.     

Genetic diversity among Mycobacterium avium complex AccuProbe-positive isolates.

The partial 32-kDa-protein gene sequences of 22 Mycobacterium avium complex (MAC) clinical isolates that were positive by the AccuProbe MAC probe only (not by the M. avium or M. intracellulare probe) were determined. The obtained nucleotide sequences were compared with the published sequences for M. tuberculosis, M. avium, and M. intracellulare by a sequence analysis program. There was a wide range of genetic diversity among the strains studied. Most of them (16 of 22) had sequences similar but not identical to those of M. avium and M. intracellulare. These strains were considered to be true MAC strains. Five strains had sequences in the category of the novel MAIX sequence, which was very different from the sequences of other mycobacteria analyzed thus far. In addition to these strains, one isolate had a sequence that differed greatly from the reference sequences. These results support previous findings showing that the MAC probably contains several additional species. Our results also suggest that the MAC AccuProbe may react with strains that do not belong to the MAC.     

Serovars of Mycobacterium avium complex isolated from patients in Sweden.

The serovars of clinical isolates of Mycobacterium avium complex from 24 acquired immunodeficiency syndrome (AIDS) and 140 non-AIDS patients in Sweden were studied by using type-specific antisera. A wide distribution of serovars was seen. Serovar 6 was predominant in both groups of patients, isolated from 33 and 16% of the AIDS and non-AIDS patients, respectively. The results indicate geographical as well as disease-related differences in the distribution of M. avium complex serovars of clinical importance.     

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.     

Mycobacterium avium complex pseudobacteriuria from a hospital water supply.

From July 1983 through November 1985, organisms belonging to Mycobacterium avium complex were isolated from the urine of 29 patients. Strains recovered from the urine of nine patients from July 1983 through August 1984 were serotyped. Eight of the nine samples belonged to serovar 4. M. avium complex was isolated from the urine of 21 patients during the period from November 1984 through November 1985. While the possibility of a point source contamination was investigated, M. avium complex was recovered from the phenol red solution used for processing urine specimens in the mycobacteriology laboratory and the deionized tap water of that laboratory that is used to make the reagent. M. avium complex serovar 4 was subsequently recovered from the tap water of the laboratory and four hospital wards. During the year following the installation of a microbiological filter for the mycobacteriology laboratory deionized tap water, 2 urine isolates were recovered, compared to 26 the previous year. This study demonstrates the importance of filtration devices at tap water sites that are used to make laboratory reagents and the value of serotyping as a marker for the detection of a specific source of M. avium complex contamination.     

Naturally occurring antibodies against Mycobacterium avium complex

Serum obtained from 57 healthy individuals and patients admitted to the hospital owing to diverse pathological causes, as well as serum from seven patients with AIDS and disseminated Mycobacterium avium complex (MAC) infection, were studied to determine the prevalence of IgG and IgM antibodies against surface proteins of M. avium complex. Immunodot assay to detect serum positivity against MAC and Western Blot technique in order to determine the MAC antigens eliciting antibody production were performed. Sera from 89 percent and 81 percent of the non-AIDS population had IgG and IgM antibodies against MAC antigens, respectively. In contrast, 43 percent and 71 percent of the AIDS population had IgG and IgM antibodies against MAC antigens, respectively, in the serum. To define further the antigens recognized by these naturally occurring antibodies, the serum of 14 non-AIDS and four acquired immunodeficiency syndrome (AIDS) patients were studied. Multiple antigens of MAC, with molecular weight ranging from 10 to 95 kilodaltons were recognized by IgG and IgM antibodies present in the sera. The IgM type antibodies were shown to react mainly against 10 kilodalton and 31 kilodalton antigenic protein, while the IgG type antibodies were produced mainly against the 10, 31, and 65 kilodalton proteins. Although the pattern of reaction was consistent between non-AIDS and AIDS populations, IgM antibodies were not detected against the 10 kilodalton protein nor were IgG antibodies detected against the 31 kilodalton protein in the AIDS population.     

Rapid identification of Mycobacterium avium complex in culture using DNA probes.

A commercial DNA probe procedure for identification of Mycobacterium avium complex isolates was evaluated for accuracy and applicability for use in the clinical laboratory. The test (Gen-Probe Rapid Diagnostic System for Mycobacterium Avium Complex; Gen-Probe Corp., San Diego, Calif.) uses hybridization in solution of two 125I-labeled cDNA probes. One probe is complementary to rRNA from M. avium, and the other is complementary to rRNA from M. intracellulare. Results are expressed as absolute percent hybridization, with values greater than or equal to 10% considered positive. The procedure accurately identified all 134 M. avium complex isolates and concomitantly identified them to species level. There were no false-positives with 66 other mycobacteria, including 22 M. tuberculosis and 18 M. kansasii isolates, or with 8 Nocardia isolates. The mean percent hybridization (+/- the standard deviation) of M. avium probe-positive isolates (94 isolates) was 48.0 +/- 9.9 (range, 11.5 to 72.7); for M. intracellulare (40 isolates), it was 45.7 +/- 8.8 (range, 22.7 to 60.7). Among the 74 non-M. avium complex isolates, the percent hybridization range was 1.0 to 4.2, except for a single value of 9.7 which was less than 5 when the test was repeated. Four M. avium complex isolates reacted positively with both probes on initial testing, and three were confirmed. On repeat testing of subcultures, each reacted with only one probe, suggesting the presence of a mixed culture. The procedure can be completed in as little as 2 h and could easily be performed in most clinical laboratories.     

Comprehensive approach to identification of serovars of Mycobacterium avium complex.

Serotyping of nontuberculous mycobacteria, especially those of the Mycobacterium avium complex, provides important epidemiological information, particularly in tracing origins of infections. Seroagglutination with whole cells and polyclonal rabbit antibodies was the original way of identifying serovars and is still commonly used. The discovery of the glycolipid nature of the typing antigens allows differentiation of serovars on the basis of thin-layer chromatography of whole antigens and gas chromatography-mass spectrometry of the characteristic sugars of the oligosaccharide haptens of these antigens. In particular, the generation of monoclonal antibodies to the glycolipid antigens allows facile differentiation of serovars through enzyme-linked immunosorbent assay. All of these protocols were applied in developing a comprehensive approach to the typing of members of the M. avium complex.     

Differential responses of bovine macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically similar organisms; however, they differ in their virulence for cattle. M. avium subsp. paratuberculosis causes a chronic intestinal infection leading to a chronic wasting disease termed paratuberculosis or Johne's disease, whereas M. avium subsp. avium causes only a transient infection. We compared the response of bovine monocyte-derived macrophages to ingestion of M. avium subsp. paratuberculosis and M. avium subsp. avium organisms by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-8 (IL-8), IL-10, IL-12, and granulocyte-monocyte colony-stimulating factor (GM-CSF). Unlike M. avium subsp. paratuberculosis, macrophages were able to kill approximately half of the M. avium subsp. avium organisms after 96 h of incubation. This difference in killing efficiency was not related to differences in nitric oxide or superoxide production. Compared to macrophages activated with IFN-gamma and lipopolysaccharide, macrophages incubated with M. avium subsp. paratuberculosis showed greater expression of IL-10 and GM-CSF (all time points) and IL-8 (72 h) and less expression of IL-12 (72 h), IFN-gamma (6 h), and TNF-alpha (6 h). When cytokine expression by macrophages incubated with M. avium subsp. paratuberculosis was compared to those of macrophages incubated with M. avium subsp. avium, M. avium subsp. paratuberculosis-infected cells showed greater expression of IL-10 (6 and 24 h) and less expression of TNF-alpha (6 h). Therefore, the combination of inherent resistance to intracellular degradation and suppression of macrophage activation through oversecretion of IL-10 may contribute to the virulence of M. avium subsp. paratuberculosis in cattle.     

Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified.