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Mycobacterium tuberculosis infection and nontuberculous mycobacteria (NTM) infections exhibit similar clinical symptoms; however, the therapies for these two types of infections are different. Therefore, the rapid and accurate identification of M. tuberculosis and NTM species is very important for the control of tuberculosis and NTM infections. In the present study, a Cas12a/guide RNA (gRNA)-based platform was developed to identify M. tuberculosis and most NTM species. By designing species-specific gRNA probes targeting the rpoB sequence, a Cas12a/gRNA-based platform successfully identified M. tuberculosis and six major NTM species ( Mycobacterium abscessus , Mycobacterium intracellulare , Mycobacterium avium , Mycobacterium kansasii , Mycobacterium gordonae , and Mycobacterium fortuitum ) without cross-reactivity. In a blind assessment, a total of 72 out of 73 clinical Mycobacterium isolates were correctly identified, which is consistent with previous rpoB sequencing results. These results suggest that the Cas12a/gRNA-based platform is a promising tool for the rapid, accurate, and cost-effective identification of both M. tuberculosis and NTM species., (Copyright © 2020 American Society for Microbiology.)

Bedaquiline (BDQ), a diarylquinoline antibiotic that targets ATP synthase, is effective for the treatment of Mycobacterium tuberculosis infections that no longer respond to conventional drugs. While investigating the off-label use of BDQ as salvage therapy, seven of 13 patients with Mycobacterium intracellulare lung disease had an initial microbiological response and then relapsed. Whole-genome comparison of pretreatment and relapse isolates of M. intracellulare uncovered mutations in a previously uncharacterized locus, mmpT5 Preliminary analysis suggested similarities between mmpT5 and the mmpR5 locus, which is associated with low-level BDQ resistance in M. tuberculosis Both genes encode transcriptional regulators and are adjacent to orthologs of the mmpS5-mmpL5 drug efflux operon. However, MmpT5 belongs to the TetR superfamily, whereas MmpR5 is a MarR family protein. Targeted sequencing uncovered nonsynonymous mmpT5 mutations in isolates from all seven relapse cases, including two pretreatment isolates. In contrast, only two relapse patient isolates had nonsynonymous changes in ATP synthase subunit c (atpE), the primary target of BDQ. Susceptibility testing indicated that mmpT5 mutations are associated with modest 2- to 8-fold increases in MICs for BDQ and clofazimine, whereas one atpE mutant exhibited a 50-fold increase in MIC for BDQ. Bedaquiline shows potential for the treatment of M. intracellulare lung disease, but optimization of treatment regimens is required to prevent the emergence of mmpT5 variants and microbiological relapse., (Copyright © 2017 American Society for Microbiology.)

A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Amikacin is a major drug used for the treatment of Mycobacterium avium complex (MAC) disease, but standard laboratory guidelines for susceptibility testing are not available. This study presents in vitro amikacin MICs for 462 consecutive clinical isolates of the MAC using a broth microdilution assay. Approximately 50% of isolates had amikacin MICs of 8 μg/ml, and 86% had MICs of ≤16 μg/ml. Of the eight isolates (1.7%) with MICs of 64 μg/ml, five had an MIC of 32 μg/ml on repeat testing. Ten isolates (2.1%) had an initial amikacin MIC of >64 μg/ml, of which seven (1.5%) had MICs of >64 μg/ml on repeat testing. These seven isolates had a 16S rRNA gene A1408G mutation and included M. avium, Mycobacterium intracellulare, and Mycobacterium chimaera. Clinical data were available for five of these seven isolates, all of which had received prolonged (>6 months) prior therapy, with four that were known to be treated with amikacin. The 16S mutation was not detected in isolates with MICs of ≤64 μg/ml. We recommend primary testing of amikacin against isolates of the MAC and propose MIC guidelines for breakpoints that are identical to the CLSI guidelines for Mycobacterium abscessus: ≤16 μg/ml for susceptible, 32 μg/ml for intermediate, and ≥64 μg/ml for resistant. If considered and approved by the CLSI, this will be only the second drug recommended for primary susceptibility testing against the MAC and should facilitate its use for both intravenous and inhaled drug therapies.

We report the first case of pulmonary disease caused by a strain of Mycobacterium avium complex of presumed veterinary origin in an elderly patient. All serial isolates were identified by multilocus sequence analysis based on rpoB, hsp65, and 16S rRNA fragments. Disease persisted despite macrolide-based combination antibiotic therapy.

Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC.

Early detection of tuberculosis (TB) is essential for infection control. The geneCube (Toyobo) is a novel fully automated gene analyzer that can amplify target DNAs within 60 min. In this study, we evaluated the ability of the geneCube to directly detect Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC) in clinical specimens. The results were then compared with those obtained using conventional culture, microscopy, and the Cobas Amplicor assay (Roche). We examined a total of 516 frozen samples from 69 patients who showed culture-positive infection (73 samples; 39 MTBC, 32 MAC, and 2 mixed infections) and from 354 patients who were culture negative (443 samples). Assays using the geneCube had a sensitivity of 85.4% and a specificity of 99.8% for detection of MTBC and a sensitivity of 85.3% and a specificity of 99.8% for detection of MAC. These results are similar to those obtained using the Amplicor system but were obtained much more rapidly (1 h with the geneCube versus 5.5 h with the Amplicor system). The geneCube thus enables a significant shortening of the assay time with no loss of sensitivity or specificity.

"Pseudomonas andersonii" is a Gram-negative bacillus initially isolated from a granulomatous lung lesion. Novel species status has not been validated for this single strain. We report four additional cases of pulmonary granuloma involving P. andersonii and further characterize the organism. These patients had pulmonary nodules that were surgically resected and which grew P. andersonii on routine culture. Mycobacterium avium complex was concomitantly isolated in two cases, and fungal structures were identified histopathologically in two other cases. The five P. andersonii strains described to date were similar in growth characteristics, biochemical reactions, matrix-assisted laser desorption ionization-time of flight mass spectrometry protein profiles, and susceptibility to antimicrobial agents. Their 16S rRNA genes were 99.9 to 100% identical but less than 95.0% similar to those of all other known bacteria. The gyrA genes of these strains were 99.5 to 100% identical. These shared features illustrate P. andersonii as a unique and distinct bacterium and support the novel species status of the organism.

Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.

The SLOMYCO Sensititre panel and the custom JustOne strip (both from TREK Diagnostic Systems, Cleveland, OH) were evaluated for susceptibility testing of Mycobacterium avium complex isolates against clarithromycin. Seventy-one archived and prospectively collected isolates were tested using both the SLOMYCO panel and the JustOne strip, and the results were compared to those obtained using the BACTEC 460 (BD, Sparks, MD) radiometric method and a broth microdilution reference method. Results obtained by the SLOMYCO panel and the JustOne strip agreed with the BACTEC 460 method for 64/71 isolates (90%). Similarly, concordance with the broth microdilution method was 40/43 isolates (93%) for both test systems. The effect of the source medium on inoculum preparation was evaluated, and there were no differences noted in MICs, regardless of whether the inoculum was prepared from isolates grown in Middlebrook 7H9 medium, on Middlebrook 7H10 agar, or in VersaTREK broth culture bottles (Trek Diagnostics). Clarithromycin susceptibility testing of MAC using the SLOMYCO panel and the JustOne strip methods is easy to set up and simple to read and is readily incorporate into the clinical laboratory. These systems offer advantages over the BACTEC 460 system including the lack of a need for radioactive substrates, sharps, or costly instrumentation.

Members of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food, and other environmental samples and are considered opportunistic pathogens for numerous animal species, mainly birds and pigs, as well as for humans. We have recently demonstrated the usefulness of a PCR-based mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing for the molecular characterization of M. avium subsp. paratuberculosis and M. avium strains exclusively isolated from AIDS patients. In the present study we extended our analysis, based on eight MIRU-VNTR markers, to a strain collection comprehensively comprising the other M. avium subspecies, including M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum, isolated from numerous animal species, HIV-positive and HIV-negative humans, and environmental sources. All strains were fully typeable, with the discriminatory index being 0.885, which is almost equal to that obtained by IS1311 restriction fragment length polymorphism (RFLP) typing as a reference. In contrast to IS1311 RFLP typing, MIRU-VNTR typing was able to further discriminate M. avium subsp. avium strains. MIRU-VNTR alleles strongly associated with or specific for M. avium subspecies were detected in several markers. Moreover, the MIRU-VNTR typing-based results were consistent with a scenario of the independent evolution of M. avium subsp. avium/M. avium subsp. silvaticum and M. avium subsp. paratuberculosis from M. avium subsp. hominissuis, previously proposed on the basis of multilocus sequence analysis. MIRU-VNTR typing therefore appears to be a convenient typing method capable of distinguishing the three main subspecies and strains of the complex and providing new epidemiological knowledge on MAC.

We performed a multicenter prevalence study of nontuberculous mycobacteria (NTM) involving 1,582 patients (mean age, 18.9 years; male/female ratio, 1.06) with cystic fibrosis in France. The overall NTM prevalence (percentage of patients with at least one positive culture) was 6.6% (104/1,582 patients), with prevalences ranging from 3.7% (in the east of France) to 9.6% (in the greater Paris area). Mycobacterium abscessus complex (MABSC; 50 patients) and Mycobacterium avium complex (MAC; 23 patients) species were the most common NTM, and the only ones associated with fulfillment of the American Thoracic Society bacteriological criteria for NTM lung disease. The "new" species, Mycobacterium bolletii and Mycobacterium massiliense, accounted for 40% of MABSC isolates. MABSC species were isolated at all ages, with a prevalence peak between 11 and 15 years of age (5.8%), while MAC species reached their highest prevalence value among patients over 25 years of age (2.2%).

Mycobacterium avium complex (MAC) infections are increasing annually in various countries, including Japan, but the route of transmission and pathophysiology of the infection remain unclear. Currently, a variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of the IS1245-restriction fragment length polymorphism (IS1245-RFLP) typing method and a mycobacterial interspersed repetitive-unit (MIRU)-VNTR typing method reported previously (V. C. Thibault, M. Grayon, M. L. Boschiroli, C. Hubbans, P. Overduin, K. Stevenson, M. C. Gutierrez, P. Supply, and F. Biet, J. Clin. Microbiol. 45:2404-2410, 2007). Seventy clinical isolates identified as M. avium from human immunodeficiency virus-negative patients with MAC infections were used. MATR-VNTR typing using 15 loci distinguished 56 patterns of different allele profiles, yielding a Hunter-Gaston discriminatory index (HGDI) of 0.990. However, IS1245-RFLP and MIRU-VNTR typing yielded HGDIs of 0.960 and 0.949, respectively, indicating that MATR-VNTR has an excellent discriminatory power compared with MIRU-VNTR and IS1245-RFLP typing. Moreover, concomitant use of the MATR-VNTR method and IS1245-RFLP typing increased the HGDI to 0.999. MATR-VNTR typing is inexpensive and easy to perform and could thus be useful in establishing a digital multifacility database that will greatly contribute to the clarification of the transmission route and pathophysiology of M. avium infections.

We report the case of a 3-year-old girl with lymphadenopathy caused by the recently described species Mycobacterium colombiense. M. colombiense is a nonpigmented slow grower that is included in the Mycobacterium avium complex. Partial sequencing of the 16S rRNA gene was used for species identification.

Isolation of Mycobacterium avium complex (MAC) organisms from clinical samples may occur in patients without clinical disease, making the interpretation of results difficult. The clinical relevance of MAC isolates from different types of clinical samples (n = 47) from 39 patients in different sections of a hospital was assessed by comparison with environmental isolates (n = 17) from the hospital. Various methods for identification and typing (commercial probes, phenotypic characteristics, PCR for detection of IS1245 and IS901, sequencing of the hsp65 gene, and pulsed-field gel electrophoresis) were evaluated. The same strain was found in all the environmental isolates, 21 out of 23 (91.3%) of the isolates cultured from urine samples, and 5 out of 19 (26.3%) isolates from respiratory specimens. This strain did not cause disease in the patients. Testing best characterized the strain as M. avium subsp. hominissuis, with the unusual feature that 81.4% of these isolates lacked the IS1245 element. Contamination of certain clinical samples with an environmental strain was the most likely event; therefore, characterization of the environmental mycobacteria present in health care facilities should be performed to discard false-positive isolations in nonsterile samples, mainly urine samples. Molecular techniques applied in this study demonstrated their usefulness for this purpose.

Classical methods for identification of Mycobacterium species rely on morphology and biochemical profiles. Speciation of a Mycobacterium isolate using these standard methods is a lengthy process based on subjective data interpretation. In this study, Mycobacterium species were characterized by utilizing matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This technology is designed to provide a characteristic mass spectral fingerprint based on desorbed ions from the cell surface. Thirty-seven strains were analyzed; these represented thirteen species and five subspecies that included the Mycobacterium tuberculosis complex and the M. avium-M. intracellulare complex, as well as rapid- and slow-growing mycobacteria. All 37 strains were analyzed in triplicate, and a database was generated. This method produced species-specific patterns for all but 1 of the 37 isolates and provided reliable differentiation at the strain level. The data suggest that whole-cell MALDI-TOF MS has potential as a rapid and reproducible method for the identification and characterization of Mycobacterium species.

The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated from an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genotypic identity to strain 104. This process was facilitated by the use of a novel two-step approach. In the first step, all 208 strains in the sample were subjected to a high-throughput, large sequence polymorphism (LSP)-based genotyping test, in which DNA from each strain was tested by PCR for the presence or absence of 4 hypervariable genomic regions. Nineteen isolates exhibited an LSP type that resembled that of strain 104. This subset of 19 isolates was then subjected to high-resolution repetitive sequence-based PCR typing, which identified 10 isolates within the subset that were genotypically identical to strain 104. These isolates came from 10 different patients at 5 clinical sites in the western United States, and they were isolated over a 17-year time span. Therefore, the sequenced genome of M. avium strain 104 has been associated with disease in multiple patients in the western United States. Although M. avium is known for its genetic plasticity, these observations also show that strains of the pathogen can be genotypically stable over extended time periods.

The clinical significance and prevalence of Mycobacterium avium and Mycobacterium intracellulare were analyzed in a cohort of 7,472 patients who, from 1999 to 2003, sought care at the University of Texas M.D. Anderson Cancer Center, Houston, and had cultures performed for mycobacteria. Patients were stratified for age, sex, and underlying diseases, and bacteria were identified by 16S rRNA gene sequencing. M. avium was isolated in 62 (0.83%) of 7,472 patients and M. intracellulare in 65 (0.87%). Clinically, only 10 of the 62 (16.2%) patients with M. avium had probable to definite evidence of infection, whereas the majority (83.8%) had weak evidence of infection. Sex and age did not affect the isolation or infection of M. avium. Hematological tumors predisposed to M. avium colonization but not infection. In contrast, 41 of the 65 (63.1%) patients with M. intracellulare had probable to definite infection, a level much higher than those with M. avium (P < 0.001). M. intracellulare was more prevalent in women (1.33% of 3,311) than in men (0.50% of 4,161) (P < 0.001), and underlying diseases had no effect in women. Men with lung cancer had a higher prevalence (1.37%) than men without (0.34%) (4.0-fold; P < 0.001), but it was similar to that in women. A marked age trend for the isolation of M. intracellulare among women was noted: 0.27% (1-fold) for ages of <50 years, 0.85% (3.1-fold) for ages 50 to 59 years, 1.50% (5.6-fold) for ages 60 to 69 years, and 3.74% (13.9-fold) for ages >/=70 years (trend, P < 0.001). The combined rate for women >/=50 was 1.86% (95% confidence interval [1.30 to 2.42%]) (6.9-fold). Together, these results suggest that, among non-AIDS patients, M. intracellulare is more pathogenic and tends to infect women increasingly beyond menopause (age >/=50 years) regardless of underlying disease. The prevalence rate of 1.86% in postmenopausal women suggests the need to further investigate the public health significance of M. intracellulare.

Rapid identification of Mycobacterium avium complex (MAC) is possible by use of AccuProbe (Gen-Probe, San Diego, Calif.). To evaluate the reliability of the MAC AccuProbe for testing 7H9 cultures inoculated with broth from MGIT cultures positive for acid-fast bacilli or growth on a solid medium, we compared probe results to results obtained by sequencing a portion of the 16S rRNA gene. Isolates were sequenced if the MAC probe result was <100,000 relative light units (RLU) or if it was > or =100,000 RLU and the colony morphology was not classic or there were two colony types. For the 1,389 cultures tested in phase 1, conducted to evaluate cutoff values for the MAC probe in testing of 7H9 cultures inoculated with broth from MGIT cultures, the sensitivity and specificity of the MAC AccuProbe were 97.7% and 88.8%, respectively, according to the manufacturer's interpretive criteria (> or =30,000 RLU is positive). If the cutoff for a positive result were 80,000 RLU, the specificity would be 100% and the sensitivity 92.3%. Of the 344 isolates in phase 2, which was conducted to confirm the 80,000-RLU cutoff for a positive result and therefore included only isolates with a MAC probe result of < or =100,000 RLU, 13 of 16 with results of > or =30,000 but <80,000 RLU were identified as mycobacteria other than MAC, including five Mycobacterium tuberculosis complex isolates. These data support the use of 80,000 RLU as the cutoff for a positive result in testing of 7H9 broth cultures with the MAC AccuProbe.

We studied the prevalence and species distribution of nontuberculous mycobacteria (NTM) in relation to age in 385 patients with cystic fibrosis (CF) (mean age +/- standard deviation [range], 12.0 +/- 6.1 [1 to 24] years; sex ratio, 0.53) attending three Parisian centers. The overall prevalence of NTM in sputum was 8.1% (31 out of 385). The following NTM were isolated (n = 33): Mycobacterium abscessus (n = 13, 39.4%), Mycobacterium avium complex (MAC) (n = 7, 21.2%), Mycobacterium gordonae (n = 6, 18.2%), and other (n = 7, 21.2%). Sixteen patients met the American Thoracic Society microbiological criteria for NTM infection, including 11 patients positive for M. abscessus, 4 for MAC, and 1 for MAC and Mycobacterium kansasii. The overall prevalence of NTM was significantly lower in patients under 15 years old than for patients equal to or more than 15 years old (4.8 versus 14.9%, respectively; P = 0.001). M. abscessus was isolated at all ages, while MAC was not recovered before 15 years (prevalence of 0.0 and 5.2% in patients aged 1 to 14 and 15 to 24, respectively; P = 0.001).

Using INNO-LiPA-MYCOBACTERIA (Lipav1; Innogenetics) and the AccuProbe (Gen-Probe Inc./bioMérieux) techniques, 35 Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAC/MAIS) complex strains were identified between January 2000 and December 2002. Thirty-four of 35 isolates were positive only for the MAIS complex probe by Lipav1 and were further analyzed by INNO-LiPA-MYCOBACTERIA version 2 (Lipav2), hsp65 PCR restriction pattern analysis (PRA), and ribosomal internal transcribed spacer (ITS), hsp65, and 16S rRNA sequences. Lipav2 identified 14 of 34 strains at the species level, including 11 isolates positive for the newly specific MAC sequevar Mac-A probe (MIN-2 probe). Ten of these 11 isolates corresponded to sequevar Mac-A, which was recently defined as Mycobacterium chimerae sp. nov. Among the last 20 of the 34 MAIS isolates, 17 (by hsp65 PRA) and 18 (by hsp65 sequence) were characterized as M. avium. Ten of the 20 were identified as Mac-U sequevar. All these 20 isolates were identified as M. intracellulare by 16S rRNA sequence except one isolate identified as Mycobacterium paraffinicum by 16S rRNA and ITS sequencing. One isolate out of 35 isolates that was positive for M. avium by AccuProbe and that was Mycobacterium genus probe positive and MAIS probe negative by Lipav1 and Lipav2 might be considered a new species. In conclusion, the new INNO-LiPA-MYCOBACTERIA allowed the identification of 40% of the previously unidentified MAIS isolates at the species level. The results of the Lipav2 assay on the MAIS isolates confirm the great heterogeneity of this group and suggest the use of hsp65 or ITS sequencing for precise identification of such isolates.

Commercially available nucleic acid probe- and amplification-based systems for detection and differentiation of mycobacteria are widely used in clinical microbiology laboratories. Here we report two cases of human leprosy in which the COBAS AMPLICOR Mycobacterium intracellulare test led to false- positive results. Correct identification of Mycobacterium leprae was possible only by amplification and comparative sequence analysis of the 16S rRNA gene.

Members of the Mycobacterium avium complex (MAC) are important environmental pathogens that are implicated in several chronic, idiopathic diseases. Diagnosis of MAC-based diseases is compromised by the need to cultivate these fastidious and slowly growing organisms in order to identify which mycobacterial species are present. Detection is particularly difficult when MAC is intracellular or embedded within mammalian tissues. We report on the development of culture-independent, in situ hybridization (ISH) assays for the detection of MAC in culture, sputum, and tissue. This assay includes a highly reliable technique for the permeabilization of mycobacterial cells within culture and tissues. We describe a set of rRNA-based oligonucleotide probes that specifically detect either M. intracellulare, the two M. avium subspecies associated with human disease, or all members of MAC. The results call into question the validity of ISH results derived by the use of other gene loci, such as IS900.

In early 1999, there was an increased incidence of tuberculous lesions in the lymph nodes of slaughtered pigs in the Czech Republic. In part 1 of this study, tuberculous lesions were detected in 140 (62%) tissue samples collected from pigs coming from 15 farms in 15 districts at routine veterinary meat inspections in abattoirs. Mycobacteria were isolated from 37 (16%) tissue samples: 34 Mycobacterium avium subsp. hominissuis isolates and three environmentally derived mycobacteria. In search of infection sources, M. avium subsp. hominissuis was isolated from 38 (79%) samples of peat used as a feed supplement. In part 2 of our study, the head, mesenteric, and inguinal lymph nodes of 117 randomly selected slaughtered pigs from one farm with young piglets fed peat as a supplement were investigated for mycobacterial infection. From 65 (56%) pigs, a total of 76 mycobacterial isolates were identified (56 M. avium subsp. hominissuis isolates, 5 M. avium subsp. avium isolates, 3 M. intracellulare isolates, and 12 environmentally derived mycobacterial isolates). IS1245 restriction fragment length polymorphism (RFLP) types with >20 bands of 45 distinct RFLP types were found in 49 M. avium subsp. hominissuis isolates from pigs (n = 31) and peat (n = 18). Identical RFLP types were found in only four pig isolates. Five randomly selected isolates from pigs and peat were subcultured to six independent clones or colonies. Among the IS1245 RFLP types of 30 clones, identical RFLP types obtained from pigs and peat were identified, which confirmed the hypothesis that peat contaminated with mycobacteria represents a significant source of mycobacterial infection for pigs.

Hemolysin was quantified in 58 isolates of Mycobacterium avium from human, animal, and environmental sources. Human Mav-A and Mav-B isolates were the strongest producers; in contrast, animal and environmental Mav-A isolates and human, animal, and environmental Mav-C organisms were low-level producers. Hemolysin production was not restricted to isolates causing invasive infections.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the gyrB gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63 degrees C. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63 degrees C. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of M. tuberculosis complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 micro l of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 micro l of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.

To compare the performance of the BACTEC 13A (Becton Dickinson, Sparks, Md.), BACTEC MYCO/F LYTIC (Becton Dickinson), BacT/ALERT MB (bioMérieux, Durham, N.C.), and ISOLATOR 10 lysis-centrifugation (Wampole Laboratories, Cranbury, N.J.) systems for detection of mycobacteremia in adults, we inoculated 5-ml aliquots of blood from patients with suspected mycobacteremia into the bottle or tube required for each system. Of 600 sets tested, 85 (14%) yielded Mycobacterium avium complex (MAC) and 9 (2%) yielded other species of mycobacteria. Of 26 complete (three bottles and one tube) adequately filled (5 +/- 1 ml) sets from which MAC was recovered, BACTEC 13A was positive for 19 (73%), BACTEC MYCO/F LYTIC was positive for 21 (81%), BacT/ALERT MB was positive for 22 (85%), and ISOLATOR 10 was positive for 21 (81%). Of the six possible two-way comparisons, the mean times to detection for the recovery of MAC from each bottle in positive adequately paired sets were 15.3 days for BACTEC 13A versus 12.8 days for MYCO/F LYTIC for 33 of 340 pairs, 14.1 days for BACTEC 13A versus 11.6 days for BacT/ALERT MB for 38 of 380 pairs, 12.6 days for BACTEC 13A versus 20.0 days for ISOLATOR 10 for 26 of 261 pairs, 12.8 days for BACTEC MYCO/F LYTIC versus 11.0 days for BacT/ALERT MB for 33 of 340 pairs, 13.2 days for BACTEC MYCO/F LYTIC versus 20.4 days for ISOLATOR 10 for 24 of 230 pairs, and 9.9 days for BacT/ALERT MB versus 19.0 days for ISOLATOR 10 for 24 of 257 pairs. There were no significant differences in yields between the systems. However, the mean time to detection differed significantly among the systems. The time to detection was shortest for BacT/ALERT MB, followed by BACTEC MYCO/F LYTIC and BACTEC 13A and then ISOLATOR 10. Although the numbers were too small for statistical comparison, the time to detection was substantially shorter for MAC than for Mycobacterium tuberculosis complex in the liquid systems. The continuously monitored systems (BACTEC MYCO/F LYTIC and BacT/ALERT MB) were as sensitive and, on balance, faster for the detection of MAC bacteremia than were the heretofore standard manual ISOLATOR 10 and radiometric BACTEC 13A systems.

A molecular analysis of the first Mycobacterium avium complex (MAC) blood isolates from 177 patients from 10 Canadian cities revealed that each cluster of indistinguishable strains consisted of isolates from epidemiologically unrelated patients in the same city or region. This study supports the premise that acquisition of MAC from a common environmental source occasionally occurs.

Broad-range amplification of bacterial DNA from clinical specimens has proved useful for the diagnosis of various bacterial infections, especially during antimicrobial treatment of the patient. Optimal sample processing protocols for diagnostic broad-range bacterial PCR should release DNA from an array of target organisms with equal efficiencies and wash out inhibitory factors from various sample types without introducing bacterial DNA contamination to the amplification reaction. In the present study, two physical cell wall disintegration methods, bead beating and sonication, for enhanced detection of organisms with difficult-to-lyse cell walls were studied. The analytical sensitivities of several commercially available DNA purification kits, which were used with and without additional cell disintegration steps, were compared by using dilution series of model bacteria. Selected purification methods were used to process routine clinical specimens in parallel with the standard phenol-ether DNA extraction, and the results obtained by bacterial PCR and sequencing with the two template preparations were compared. The method with the DNA isolation kit with the lowest detection limits from the bacterial suspensions (Masterpure) did not prove to be superior to the standard method when the two methods were applied to 69 clinical specimens. For another set of 68 clinical specimens, DNA purified with a glass fiber filter column (High Pure) with an additional sonication step yielded results well in accord with those obtained by the standard method. Furthermore, bacterial DNA was detected in four samples that remained PCR negative by the standard method, and three of these contained DNA from gram-positive pathogens. Three samples were positive by the standard method only, indicating the limitations of applying any single method to all samples.

Six isolates of Mycobacterium avium of genotype dnaJ(+) IS901(-) IS1311(+) IS1245(+) and serotypes 6 (n = 1), 6/9, (n = 2), and 9 (n = 3) were obtained within a 5-month period from a human immunodeficiency virus-negative patient treated for tuberculosis. The isolates were identified with PvuII restriction fragment length polymorphism (RFLP) analysis as a single IS1311 RFLP type and six different IS1245 RFLP types. Six separate colonies/clones obtained by subculture from each of the six isolates were tested for MICs of a set of 10 drugs. This report documents the appearance of isolates that are resistant to antimycobacterial drugs as the duration of therapy increases. Because isolates recovered from the patient following longer duration of treatment were more likely to be resistant to more antimycobacterial drugs, we would conclude that there was selection for antimycobacterial drug-resistant isolates. Analyses of all 36 clones identified three IS1311 and 22 IS1245 types forming three clusters. Tests of 105 environmental samples collected in the home and the work place of the patient yielded 16 mycobacterial isolates, of which one M. avium from soil was of genotype dnaJ(+) IS901(+) IS1311(+) IS1245(+) and serotype 2, and the second M. avium from a vacuum cleaner was of genotype dnaJ(+) IS901(-) IS1311(+) IS1245(+) and serotype 9. Overall analyses of the results did not reveal any relation between serotype, RFLP type, and drug susceptibility. Based on the course of the disease in the patient and different serotypes, IS1311 and IS1245 RFLP types of isolates of M. avium we suppose represent polyclonal infection.

Species identification of isolates of the Mycobacterium avium complex (MAC) remains a difficult task. Although M. avium and Mycobacterium intracellulare can be identified with expensive, commercially available probes, many MAC isolates remain unresolved, including those representing Mycobacterium lentiflavum as well as other potentially undefined species. PCR restriction analysis (PRA) of the hsp65 gene has been proposed as a rapid and inexpensive approach. We applied PRA to 278 MAC isolates, including 126 from blood of human immunodeficiency virus (HIV)-infected patients, 59 from sputum of HIV-negative patients with chronic obstructive pulmonary disease, 88 from environmental sources, and 5 pulmonary isolates from a different study. A total of 15 different PRA patterns were observed. For 27 representative isolates, a 441-bp fragment of the hsp65 gene was sequenced; based on 54 polymorphic sites, 18 different alleles were defined, including 12 alleles not previously reported. Species and phylogenetic relationships were more accurately defined by sequencing than by PRA or commercial probe. The distribution of PRA types and, by implication, phylogenetic lineages among blood isolates was significantly different from that for pulmonary and environmental isolates, suggesting that particular lineages have appreciably greater virulence and invasive potential.

Sixty-nine blood samples from 47 patients infected with human immunodeficiency virus were analyzed by using PCR to detect Mycobacterium avium. The sensitivity can be up to 95.7%, depending on the detection method used and the number of blood samples analyzed from each patient. The procedure can be helpful in the diagnosis of mycobacterial disease.

We evaluated the sensitivity of a DNA amplification test for the detection of Mycobacterium avium in blood samples using different blood components and different DNA extraction methods. M. avium-inoculated blood samples were processed to obtain separate blood components: peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNCs), and whole-blood sodium dodecyl sulfate (SDS)-lysate pellets. The sensitivity for the detection of the lowest mycobacterial load (1 CFU/ml) was significantly greater (P < 0.01) with DNA extracted from SDS-lysate pellets than with DNA extracted from PBMCs or PMNCs. Subsequently, DNA extraction methods based on guanidine NaOH, and proteinase were compared. The sensitivity of the guanidine-based method was significantly greater (P < 0.01) than those of the others.

We evaluated the ability of the AccuProbe (Gen-Probe, San Diego, Calif.) to detect Mycobacterium gordonae and Mycobacterium avium complex directly in liquid medium flagged positive by the MB/BacT (Organon Teknika Corp., Durham, N.C.). Seventy-one bottles from clinical specimens containing M. gordonae and 34 containing M. avium, confirmed by culture, were tested by direct AccuProbe assay for both organisms after additional incubation for > or = 48 h and centrifugation at 4,500 x g for 15 min. Relative light unit (RLU) values were analyzed using the manufacturer's recommended cutoff of 30,000 RLU and a lower cutoff of 10,000 RLU. Using the 30,000 RLU cutoff, 55 of 71 (77.5%) specimens containing M. gordonae yielded positive results, whereas 28 of 34 (82.3%) M. avium complex specimens were correctly identified by direct probe. No specimens shown by culture to contain either M. gordonae or M. avium complex tested positive with the probe for the opposite organism (100% specificity). When the cutoff was lowered to 10,000 RLU, 67 of 71 M. gordonae (94.4%) and 32 of 34 M. avium complex (94.1%) specimens were correctly identified. This difference was significant for M. gordonae (P = 0.004) but not for M. avium complex (P = 0.26) compared to detection using the recommended RLU cutoff. Specificity was 100% for specimens containing M. gordonae that were tested with the M. avium complex probe using the 10,000 RLU cutoff, whereas specificity for specimens containing M. avium complex tested with the M. gordonae probe was 97%. Using a lower RLU cutoff for determining a positive result using the M. gordonae or M. avium complex probes when testing instrument-positive MB/BacT bottles directly will improve sensitivity without substantially compromising specificity.

To characterize changes in serum cytokine levels in human immunodeficiency virus type 1 (HIV-1)-infected persons with Mycobacterium avium complex (MAC) bacteremia, the levels of IL-1alpha (interleukin-1alpha), IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), soluble type II TNF receptor (sTNF-RII), and transforming growth factor beta (TGF-beta) in serum were measured in two cohorts of HIV-1-infected persons with MAC bacteremia. The first cohort was part of a MAC prophylaxis study. Patients with bacteremia were matched with controls without bacteremia. Elevated IL-6, IL-10, TNF-alpha, sTNF-RII, and TGF-beta levels were noted at baseline for all subjects, a result consistent with advanced HIV-1 disease. IL-1alpha was not detected. No differences in cytokine levels in serum were noted at baseline and at the time of bacteremia between patients with MAC and controls. In the second cohort, subjects had serum samples collected at the time of MAC bacteremia and thereafter while on macrolide therapy. Serum samples at time of bacteremia were collected from HIV-1-infected persons at a time when neither highly active antiretroviral therapy (HAART) nor MAC prophylaxis was used routinely. MAC treatment resulted in decreased levels of IL-6 and TNF-alpha in serum, which were evident for IL-6 by 4 to 6 weeks and for TNF-alpha by 8 to 16 weeks. Thus, antibiotic treatment for MAC results in decreased levels of IL-6 and TNF-alpha in serum in HIV-1-infected persons who are not on HAART.

Polyclonal infection by Mycobacterium avium was detected by hsp65 PCR-restriction enzyme analysis (PRA) in a bone marrow isolate from an AIDS patient. Two M. avium strains, differing in colony morphology, PRA HaeIII digestion pattern, insertion element (IS) 1245 amplification, and restriction fragment length polymorphism fingerprints with IS1245 and IS1311 probes, were isolated.

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.

The LiPA MYCOBACTERIA (Innogenetics NV, Ghent, Belgium) assay was used to identify mycobacterial isolates using culture fluid from positive BACTEC 12B bottles. The LiPA method involves reverse hybridization of a biotinylated mycobacterial PCR fragment, a 16 to 23S rRNA spacer region, to oligonucleotide probes arranged in lines on a membrane strip, with detection via biotin-streptavidin coupling by a colorimetric system. This system identifies Mycobacterium species and differentiates M. tuberculosis complex, M. avium-M. intracellulare complex, and the following mycobacterial species: M. avium, M. intracellulare, M. kansasii, M. chelonae group, M. gordonae, M. xenopi, and M. scrofulaceum. The mycobacteria were identified in the laboratory by a series of tests, including the Roche AMPLICOR Mycobacterium tuberculosis (MTB) test, the Gen-Probe ACCUPROBE, and a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 65-kDa heat shock protein gene. The LiPA MYCOBACTERIA assay detected 60 mycobacterium isolates from 59 patients. There was complete agreement between LiPA and the laboratory identification tests for 26 M. tuberculosis complex, 9 M. avium, 3 M. intracellulare complex, 3 M. kansasii, 4 M. gordonae, and 5 M. chelonae group (all were M. abscessus) isolates. Three patient samples were LiPA positive for M. avium-M. intracellulare complex, and all were identified as M. intracellulare by the PCR-RFLP analysis. Seven additional mycobacterial species were LiPA positive for Mycobacterium spp. (six were M. fortuitum, and one was M. szulgai). The LiPA MYCOBACTERIA assay was easy to perform, and the interpretation of the positive bands was clear-cut. Following PCR amplification and gel electrophoresis, the LiPA assay was completed within 3 h.

An automated cultivation system for mycobacteria, the MGIT 960 system (MGIT system), was compared in the clinical routine with two variants of Löwenstein-Jensen (L-J) medium. A total of 152 isolates were recovered from 2,015 specimens: 139 (91%) with the MGIT system and 127 (84%) with L-J media (P = 0.05). These included 68 isolates of Mycobacterium tuberculosis, of which 88% grew in the MGIT system and 93% grew in L-J media (P = 0.389), and 84 isolates of mycobacteria other than M. tuberculosis (MOTT), of which 94% grew in the MGIT system and 76% grew in L-J media (P = 0.003). More M. avium complex isolates were detected in the MGIT system (n = 65) than in L-J media (n = 50) (P = 0.001). Growth in the MGIT system was detected in 2 weeks for 78% of the isolates, whereas growth was detected in the two L-J media for 17 and 25% of the isolates, respectively. The mean times to detection of M. tuberculosis were 12 days in the MGIT system and 20 days in L-J media, and for M. avium complex the mean times to detection were 8 and 22 to 25 days, respectively. The contamination rates were similar (8.7 to 8.9%) in all media. A commercial amplification system (COBAS AMPLICOR) was evaluated for its ability to rapidly identify M. tuberculosis, M. avium, and M. intracellulare directly from 393 samples in MGIT system broth. A correct PCR result, as evaluated by culture or clinical data, was obtained for 96% of the samples, with inhibition being detected for 2% of the samples. Of the 89 results positive for M. tuberculosis, 91% were regarded as true positive, 8% were regarded as inconclusive, and 2% were considered false positive. For results positive for M. avium and M. intracellulare, 97 and 79%, respectively, were regarded as true positive. Increased rapidity and enhanced isolation of MOTT were obtained with the MGIT system. COBAS AMPLICOR was suitable for rapid identification of these three common pathogens from MGIT system broth.

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.

Organisms in the Mycobacterium avium complex (MAC; M. avium, M. intracellulare, and "nonspecific or X" MAC) are emerging pathogens among individual organisms of which significant genetic variability is displayed. The objective of the present study was to evaluate various molecular methods for the rapid and definitive identification of MAC species. Isolates were obtained from both human immunodeficiency virus (HIV)-positive patients and HIV-negative patients with and without known predisposing conditions. The isolates were initially hybridized with nucleic acid probes complementary to the rRNA of the respective mycobacterial species (AccuProbe Culture Confirmation kits for M. avium, M. intracellulare, and MAC species; Gen-Probe). Isolates were also examined by PCR and in some cases by Southern blot hybridization for the insertion element IS1245. Two other techniques included a PCR assay that amplifies the mig gene, a putative virulence factor for MAC, and hsp65 gene amplification and sequencing. This study led to the following observations. Eighty-five percent of the isolates from HIV-positive patients were M. avium and 86% of the isolates from HIV-negative patients were M. intracellulare. Fifteen of the M. avium isolates did not contain IS1245 and 7% of the M. intracellulare isolates were found to carry IS1245. All of the M. avium strains were mig positive, and all of the M. intracellulare strains were mig negative.

In the present study we compared the clinical presentations of patients with a clinical diagnosis of AIDS and disseminated Mycobacterium genavense infection (n = 12) with those of patients with AIDS and disseminated M. avium complex (MAC) infection (n = 24). Abdominal pain was seen more frequently in the group of patients infected with M. genavense than in patients infected with MAC (P = 0. 003). Analysis of microbiological data revealed that stool specimens from patients infected with M. genavense were more often smear positive than stool specimens from patients infected with MAC (P = 0. 00002). However, M. genavense could be cultured on solid media from only 15.4% of the stool specimens, whereas MAC could be cultured from 71.4% of the specimens. Bone marrow and liver biopsy specimens yielded growth of M. genavense within a reasonably short time, allowing species identification by DNA technology. Microbiological data clearly demonstrated the importance of acidic liquid medium for primary culture, the avoidance of pretreatment and the use of additives in culture, and the necessity for prolonged incubation if M. genavense is suspected. Susceptibility testing showed that M. genavense is sensitive to rifamycins, fluoroquinolones, and macrolides, whereas it is resistant to isoniazid. Susceptibility to ethambutol and clofazimine could not be evaluated. The mean survival times of patients in the two groups were similar.

The BACTEC MGIT 960 instrument is a fully automated system that exploits the fluorescence of an oxygen sensor to detect growth of mycobacteria in culture. Its performance was compared to those of the radiometric BACTEC 460 instrument and egg-based Lowenstein-Jensen medium. An identical volume of sample was inoculated in different media, and incubation was carried out for 6 weeks with the automatic systems and for 8 weeks on solid media. A total of 2,567 specimens obtained from 1,631 patients were cultured in parallel. Mycobacteria belonging to nine different taxa were isolated by at least one of the culture systems, with 75% of them being represented by Mycobacterium tuberculosis complex. The best yield was obtained with the BACTEC 460 system, with 201 isolates, in comparison with 190 isolates with the BACTEC MGIT 960 system and 168 isolates with Lowenstein-Jensen medium. A similar but not significant difference was obtained when the most-represented organisms, the M. tuberculosis complex, Mycobacterium xenopi, and the Mycobacterium avium complex, were analyzed separately and when combinations of a solid medium with the BACTEC MGIT 960 system and with the BACTEC 460 system were considered. The shortest times to detection were obtained with the BACTEC MGIT 960 system (13.3 days); 1.5 days earlier than that with the BACTEC 460 system (14.8 days) and 12 days earlier than that with Lowenstein-Jensen medium (25.6 days). The BACTEC MGIT 960 system had a contamination rate of 10.0%, intermediate between those of the radiometric system (3.7%) and the egg-based medium (17.0%). We conclude, therefore, that the BACTEC MGIT 960 system is a fully automated, nonradiometric instrument that is suitable for the detection of growth of tuberculous and other mycobacterial species and that is characterized by detection times that are even shorter than that of the "gold standard," the BACTEC 460 system. The contamination rate was higher than that for the radiometric BACTEC 460 system and needs to be improved.

A total of 1,830 specimens (75.7% respiratory and 24.3% nonrespiratory) were cultured in parallel with the MB/BacT and BACTEC 460 TB systems and on Lowenstein-Jensen (LJ) medium. Mycobacteria were identified from 173 (6.5%) specimens. The most common species recovered were Mycobacterium tuberculosis complex (65. 9%), Mycobacterium avium complex (22.5%), and Mycobacterium chelonae (9.2%). The recovery rates by individual systems were 96.5, 99.4, and 95.9% for MB/BacT, BACTEC 460 TB, and LJ medium, respectively, for all mycobacteria; the recovery rates were 99.1, 100, and 98.2%, respectively, for M. tuberculosis complex alone. The difference among the recovery rates for all mycobacteria and those for individual species was not significant. The BACTEC 460 TB system detected M. tuberculosis isolates more rapidly than the MB/BacT system (8 versus 11.8 days for smear-positive specimens [P < 0.01] and 18 versus 21 days for smear-negative specimens [P < 0.05]), whereas the MB/BacT system more rapidly detected the nontuberculous mycobacteria (17.1 versus 12.7 days [P < 0.01]). These results indicate that the nonradiometric MB/BacT system is a rapid, sensitive, and efficient method for the recovery of M. tuberculosis and nontuberculous mycobacteria from both pulmonary and extrapulmonary clinical specimens.

We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with AseI. The LRF patterns of Mycobacterium avium isolates recovered from potable water in three homes, two commercial buildings, one reservoir, and eight hospitals had varying degrees of relatedness to 19 clinical isolates recovered from 17 patients. The high number of M. avium isolates recovered from hospital water and their close relationship with clinical isolates suggests the potential threat of nosocomial spread. This study supports the possibility that potable water is a source for the acquisition of M. avium infections.

We evaluated the BACTEC MGIT 960 system, which is a fully automated, noninvasive system for the growth and detection of mycobacteria with a capacity to incubate and continuously monitor 960 7-ml culture tubes. We studied 3,330 specimens, 2,210 respiratory and 1,120 nonrespiratory specimens, collected from 2,346 patients treated at six sites. Processed specimens were inoculated into the BACTEC MGIT 960 and BACTEC 460 TB systems, as well as onto Lowenstein-Jensen slants and Middlebrook 7H11/7H11 selective plates. From all culture systems, a total of 362 isolates of mycobacteria were recovered; these were recovered from 353 specimens collected from 247 patients. The greatest number of isolates of mycobacteria (289, or 80% of the 362 isolates) was recovered with the BACTEC MGIT 960, followed by the BACTEC 460 TB (271, or 75%) and solid media (250, or 69%). From all culture systems a total of 132 isolates of Mycobacterium tuberculosis complex were recovered. The greatest number of isolates of M. tuberculosis complex was recovered when liquid medium was combined with conventional solid media; the number recovered with BACTEC 460 TB plus solid media was 128 (97%), that recovered with BACTEC MGIT 960 plus solid media was 121 (92%), that recovered with BACTEC 460 TB was 119 (90%) and that recovered with all solid media combined was 105 (79%). The recovery with BACTEC MGIT 960 alone was 102 (77%). The mean times to detection (TTD) for M. tuberculosis complex were 14.4 days for BACTEC MGIT, 15.2 days for BACTEC 460 TB, and 24.1 days for solid media. The numbers of isolates of Mycobacterium avium complex (MAC) recovered were 172 (100%) for all systems, 147 (85%) for BACTEC MGIT 960, 123 (72%) for BACTEC 460 TB, and 106 (62%) for all solid media combined. The TTD for MAC in each system were 10.0 days for BACTEC MGIT 960, 10.4 days for BACTEC 460 TB, and 25.9 days for solid media. Breakthrough contamination rates (percentages of isolates) for each of the systems were 8.1% for BACTEC MGIT 960, 4.9% for BACTEC 460 TB, and 21.1% for all solid media combined.

In the present study restriction fragment length polymorphism analyses with the recently described insertion sequence IS1245 as a probe was performed with clinical Mycobacterium avium complex strains cultured in Denmark during a 2-year period. The overall aim of the study was to disclose potential routes of transmission of these microorganisms. As a first step, the genetic diversity among isolates from AIDS patients and non-human immunodeficiency virus (HIV)-infected patients was described. In addition, a number of isolates from nonhuman sources cultured during the same period were analyzed and compared to the human isolates. A total of 203 isolates from AIDS patients (n = 90), non-HIV-infected patients (n = 91), and nonhuman sources (n = 22) were analyzed. The presence of IS1245 was restricted to Mycobacterium avium subsp. avium isolates. The majority of human isolates had large numbers of IS1245 copies, while nonhuman isolates could be divided into a high-copy-number group and a low-copy-number group. Groups of identical strains were found to be geographically widespread, comprising strains from AIDS patients as well as strains from non-HIV-infected patients. Samples of peat (to be used as potting soil) and veterinary samples were found to contain viable M. avium isolates belonging to genotypes also found in humans.

The use of the COBAS AMPLICOR System (Roche Molecular Diagnostics, Basel, Switzerland), the only automated system for PCR testing, was evaluated for a rapid identification of mycobacteria with positive BACTEC 12B cultures. Two hundred ninety-six specimens with a growth index of >/=30 were analyzed for the presence of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare. Compared to traditional methods and provided that samples with PCR inhibition are retested at a 1:10 dilution, the sensitivity and specificity of the COBAS AMPLICOR System with BACTEC 12B cultures were 100 and 98%, respectively. The COBAS AMPLICOR method is rapid and reliable for identifying the most common mycobacteria in cultures.

Isolates of the Mycobacterium avium complex were examined for hemolysin expression. Only invasive isolates of M. avium were observed to be hemolytic (P < 0.001), with activity the greatest for isolates of serovars 4 and 8. Thus, M. avium hemolysin appears to represent a virulence factor necessary for invasive disease.