Pathological and molecular identification of Mycobacterium avium infection in a loft of domestic pigeons (Columba livia var. domestica) from India.

Mycobacterium avium is a zoonotic pathogen associated with a wide range of pulmonary and extrapulmonary manifestations in a range of host species like humans, animals, and birds. The disease is more common in the avian population, and opportunistic infections have been reported in immune-compromised or debilitated animals and humans. This study reports the pathological and molecular identification of Mycobacterium avium causing avian mycobacteriosis in a loft of domestic pigeons (Columba livia var. domestica). Out of 30 pigeons aged 2-3 years, ten adult racing pigeons revealed a severe chronic and debilitating disease followed by death. The clinical signs included chronic emaciation, dullness, ruffled feathers, lameness, and greenish, watery diarrhea. Post-mortem examination of birds revealed multifocal gray- to yellow-colored raised nodules in the liver parenchyma, spleen, lungs, intestines, bone marrow, and joints. Avian mycobacteriosis was suspected based on the tissue impression smears stained by Ziehl-Neelsen staining. Histopathological examination also revealed multifocal granulomatous lesions in affected organs, which is characteristic of avian mycobacteriosis. The PCR analysis based on 16S rRNA, IS1245, and IS901 regions suggested the presence of Mycobacterium avium infection belonging to either subspecies avium or sylvaticum. This is the first detailed report of avian mycobacteriosis in pigeons from India, warranting a strict surveillance program to identify the carrier status of these microorganisms in the pigeons, which may prove a fatal zoonotic infection in humans.

Mycobacterium Avium in Miniature Schnauzer From Argentina: A Series of Cases.

Environmental mycobacteria such as those from the Mycobacterium avium-intacellulare complex may cause disseminated and severe disease in dogs with genetic predisposition. A series of cases of 4 miniature schnauzers with nonspecific clinical signs and the diagnostic tests are described. Complementary means of diagnosis including complete blood count, biochemical serum analyses and fine needle aspiration cytology staining were performed. The bacteriological culture followed by PCR amplification of 1245 and 901 insertion sequences, allowed the identification of Mycobacterium avium subsp. hominissuis. This environmental Mycobacteria normally do not cause severe disease in dogs or other species, but when CARD-9 gene presents mutations, dogs may become extremely susceptible and disease is fast, disseminated, and fatal. Antibiotic therapy can be applied under veterinary consideration in specific situations, as treatment is usually applied for a long period of time. Although zoonotic risk is low as the Mycobacterium is environmental, contamination of the location may be high, and immunosuppressed animals and humans can develop infection as well. This report may aid clinical veterinarians in the diagnosis, treatment, and prognosis in similar cases of this breed and others with the genetic predisposition.

Nontuberculous mycobacteria in patients with suspected tuberculosis and the genetic diversity of Mycobacterium avium in the extreme south of Brazil.

OBJECTIVE: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. METHODS: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. RESULTS: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. CONCLUSIONS: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.

Nontuberculous mycobacteria in patients with suspected tuberculosis and the genetic diversity of Mycobacterium avium in the extreme south of Brazil / Micobactérias não tuberculosas em pacientes com suspeita de tuberculose e a diversidade genética de Mycobacterium avium no extremo sul do Brasil

ABSTRACT Objective: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. Methods: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. Results: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. Conclusions: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.

RESUMO Objetivo: As micobactérias não tuberculosas (MNT) são um grupo heterogêneo de bactérias amplamente distribuídas na natureza e relacionadas com infecções oportunistas em seres humanos. Os objetivos deste estudo foram identificar MNT em pacientes com suspeita de tuberculose e culturas positivas e avaliar a diversidade genética de cepas identificadas como Mycobacterium avium. Métodos: Foram estudadas amostras pulmonares e extrapulmonares provenientes de 1.248 pacientes. As amostras que apresentaram resultado positivo em cultura e negativo para o complexo M. tuberculosis na identificação molecular foram avaliadas por meio da detecção dos genes hsp65 e rpoB e de sequenciamento de fragmentos conservados desses genes. Todas as cepas identificadas como M. avium foram genotipadas pelo método mycobacterial interspersed repetitive unit-variable-number tandem-repeat com oito loci. Resultados: Das 332 micobactérias isoladas, 25 (7,5%) eram MNT. Dessas 25, 18 (72%) eram M. avium, 5 (20%) eram M. abscessus, 1 (4%) era M. gastri e 1 (4%) era M. kansasii. As 18 cepas de M. avium apresentaram alta diversidade, e apenas duas eram geneticamente relacionadas. Conclusões: Esses resultados mostram a necessidade de considerar a investigação de MNT em pacientes com suspeita de tuberculose ativa e culturas positivas e de avaliar a diversidade genética de cepas de M. avium.

Description of a novel adhesin of Mycobacterium avium subsp. paratuberculosis.

The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the µM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding.

Characterization of a Mycobacterium avium subsp. avium operon associated with virulence and drug detoxification.

The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo.

Shared Mycobacterium avium genotypes observed among unlinked clinical and environmental isolates.

Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this assumption, a high-resolution PCR-based genotyping approach, large-sequence polymorphism (LSP)-mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR), was selected and used to analyze clinical and environmental isolates of M. avium from geographically diverse sources. Among 127 clinical isolates from seven locations in North America, South America, and Europe, 42 genotypes were observed. Among 12 of these genotypes, matches were seen in isolates from apparently unlinked patients in two or more geographic locations. Six of the 12 were also observed in environmental isolates. A subset of these isolates was further analyzed by alternative strain genotyping methods, pulsed-field gel electrophoresis and MIRU-VNTR, which confirmed the existence of geographically dispersed strain genotypes. These results suggest that caution should be exercised in interpreting high-resolution genotypic matches as evidence for an acquisition event.

First description of natural and experimental conjugation between Mycobacteria mediated by a linear plasmid.

BACKGROUND: In a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. The transfer mechanism of this insertion sequence to M. kansasii was investigated here. METHODOLOGY/PRINCIPAL FINDINGS: A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. The linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau. CONCLUSIONS/SIGNIFICANCE: Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.

Difference in virulence of Mycobacterium avium isolates sharing indistinguishable DNA fingerprint determined in murine model of lung infection.

BACKGROUND: Opportunistic Mycobacterium avium typically causes disease in immunocompromised patients and in some groups of apparently healthy individuals. The high virulence of some bacterial lineages increases the disease risk. High-resolution molecular genotyping studies of M. avium clinical isolates demonstrated that some genotype patterns were more prevalent than others, suggesting that close genetic relatedness of these successful isolates sharing a similar genotype could determine similar biological properties associated with high virulence. METHODS AND FINDINGS: In this study, we aimed to compare the virulence and pathogenic properties of two epidemiologically unrelated M. avium isolates sharing an indistinguishable DNA fingerprint in a well-characterized model of pulmonary infection in mice, resistant or susceptible to mycobacteria. The mice, C57BL/6 wild- type or IFN-gamma gene disrupted (GKO), respectively, were intratracheally infected with two isolates, H27 (human blood isolate) and P104 (pig lymph node isolate), and the lungs were examined for bacterial loads, histopathology and cytokine gene expression. The obtained data demonstrated significant differences in the virulence properties of these strains. Although the H27 strain grew significantly faster than P104 in the early stage of infection, this bacterium induced protective immunity that started to reduce bacterial numbers in the wild-type mice, whereas the P104 strain established a chronic infection. In the GKO mice, both strains were capable of causing a chronic infection, associated with higher bacterial burdens and severe lung pathology, in a similar manner. CONCLUSIONS/SIGNIFICANCE: The results demonstrated that the studied isolates differed in the pathogenic properties although were indistinguishable by actually widely used genotyping techniques demonstrating that the genotype similarity does not predict similarity in virulence of M. avium isolates.

Natural occurrence of horizontal transfer of Mycobacterium avium- specific insertion sequence IS1245 to Mycobacterium kansasii.

Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. The insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome.

Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Parotid mycobacteriosis is frequently caused by Mycobacterium tuberculosis in advanced AIDS.

BACKGROUND: Tuberculosis is one of the leading infectious diseases in the world, with more than 2 million new cases annually. It is one of the main causes of death of human immunodeficiency virus (HIV)-positive patients, involving multiple organs and particularly the lungs. Nevertheless there are few consistent studies about tuberculosis involving the parotid of HIV patients. The objective of this work was to describe the histological and immunohistochemical characteristics of 10 cases of mycobacteriosis involving the parotid of autopsied patients with advanced acquired immunodeficiency syndrome (AIDS), including identification of the Mycobacterium species. METHODS: Detection of 'M. tuberculosis complex' was performed by polymerase chain reaction (PCR) and ligase chain reaction (LCR) and Mycobacterium avium by PCR. RESULTS: All cases showed involvement of intraparotid lymph nodes, but the glandular parenchyma was affected in only three cases. Most of the cases (80%) presented a chronic non-caseating granulomatous inflammation, and in two cases predominated foamy macrophages, full of bacteria, and no granuloma formation. In areas of mycobacteriosis, macrophages predominated followed by TCD8, B and TCD4 lymphocytes. All cases were infected by Mycobacterium genus and 'M. tuberculosis complex' was detected in five cases by LCR and in eight by PCR, while M. avium was positive in one case only, which was also positive for M. tuberculosis. CONCLUSIONS: Parotid mycobacteriosis in advanced AIDS is characterized by intraparotid lymph node non-caseating inflammatory granulomatous lesion, caused mainly by M. tuberculosis.

Mycobacterium avium restriction fragment length polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains.

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 AIDS inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Identification of Mycobacterium avium genotypes with distinctive traits by combination of IS1245-based restriction fragment length polymorphism and restriction analysis of hsp65.

One-hundred eight Mycobacterium avium isolates from pigs, humans, birds, and bovines were typed by the IS1245-based restriction fragment length polymorphism (RFLP) method and PCR-restriction enzyme analysis (PRA) of hsp65. Nine clusters of isolates showing more than 80% similarity in their RFLP profiles were detected. The largest cluster (cluster B) included 32 of 79 pig isolates (40.5%), 3 of 25 human isolates (12%), and 1 of 2 bovine isolates, comprising 33% of all isolates. The second largest cluster (cluster A) included 18 pig isolates (22.8%) and 6 human isolates (24%). Six smaller clusters included six pig isolates (clusters C and D), four and two human isolates (clusters E and F, respectively), two pig isolates (cluster I), and two pig isolates plus one bovine isolate and the avian purified protein derivative strain (cluster H). Cluster G represented the "bird-type" profile and included the bird isolate in this series, one pig isolate, plus reference strain R13. PRA revealed four allelic variants. Seventy-seven isolates were identified as M. avium PRA variant I, 24 were identified as M. avium PRA variant II, 6 were identified as M. avium PRA variant III, and 1 was identified as M. avium PRA variant IV. Except for three isolates from cluster B, each of the RFLP clusters was associated with a single PRA pattern. Isolates with unique (nonclustered) RFLP profiles were distributed between PRA variants I and II, and there was one unique isolate of PRA variant IV. These observations are consistent with divergent evolution within M. avium, resulting in the emergence of distinct lineages with particular competence to infect animals and humans.

Profiles of the mRNA expression by macrophages infected with mycobacterium leprae and mycobacterium avium complex

In the present study, we examined profiles of the interaction of Mycobacterium leprae and Mycobacterium avium complex (MAC) with murine peritoneal macrophages (M phi s) in terms of up-regulation of M phi expression of proinflammatory and immunosuppressing cytolines (CKs) after infection. First, the reverse transcription polymerase chain reaction (RT-PCR) assay revealed that both MAC and M. leprae infections up-regulated M phi mRNA expression IL-12, TNF-alpha, IL-10, and transformating growth factor-beta (TGF-beta), except that M. leprae-infected M phi s showed no increase in the IL-12 mRNA expression. Second, the RT-PCR assay also showed some differences between M. leprae- and MAC-infected M phi s with respect to the modes of IL-10 and inducible nitric oxide synthase (iNOS) mRNA expression. That is MAC, but not M. leprae, infection caused a prolonged increase in the expression of IL-10 and iNOS mRNAs. Third, a ribonuclease protection assay revealed that M phi s co-infected with MAC and M. leprae showed the Il-12, TNF-alpha and IL-10 mRNA expression in an intermediate mode of those of M phi s infected with either M. leprae or MAC alone. This implies that the CK expression of M. leprae-infected M phi s may be modified by co-infection with MAC. These findings may suggest differential interactions of M. leprae and MAC organisms with murine peritoneal M phi s in terms of the activation of signal transduction pathways for expression of some kinds of immunoregulatory cytokines and immunoprotective enzymes.

Genetic diversity of mycobacterium avium recovered from AIDS patients in the caribbean as studied by a consensus IS1245-RFLP method and pulsed-field gel electrophoresis.

A total of 45 strains of Mycobacterium avium from 31 human immunodeficiency virus (HIV)-positive patients in the French Caribbean islands and Guiana were subjected to DNA fingerprinting using a recently described consensus IS1245 restriction fragment length polymorphism (RFLP) method, and pulsed-field gel electrophoresis (PFGE). The IS1245-RFLP resulted in three distinct clusters composed of three 27-banded isolates from two patients (cluster A), nine two-banded isolates from six patients (cluster B), and three 20-banded isolates from three patients (cluster C). PFGE results obtained after XbaI and DraI digestions gave similar clustering results irrespective of the enzyme used, and confirmed the molecular clonality for high IS1245 copy number isolates (clusters A and C). However, PFGE further discriminated the low IS1245 copy number cluster B into two distinct subclusters: subcluster I containing six isolates from four patients during the same time period from a single hospital in Guadeloupe, and subcluster IV composed of four isolates from two patients, out of which three isolates were from a single patient (patient 19). Interestingly, in the latter case, PFGE grouped together all three isolates from patient 19 despite the fact that IS1245 fingerprinting permitted grouping only two of the three isolates (the remaining unclustered isolate contained two additional bands of 3.5 and 5 kbp, and was initially considered as evidence of polyclonal infection). A combined numerical analysis of the IS1245-RFLP and PFGE results corroborated the existence of four instead of three clusters. A comparison of IS1245-RFLP versus PFGE results suggested that the standardized RFLP procedure is compatible with PFGE only for M. avium isolates containing > or = 5 copies of IS1245. Consequently, the typing results for low IS1245 copy number isolates (31% of isolates in this study) should be reconsidered for secondary typing using PFGE. Lastly, the absence of a predominant genotype of M. avium infecting HIV-positive patients over a 5-year period in this tropical region argues in favor of a lack of a privileged ecological niche for M. avium, and instead suggests that microepidemics of M. avium may prevail during limited periods of time.

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe.

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated 'A', 'B', 'C' and 'E') were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern 'A', was found in 46 isolates (75%). The second, pattern 'E', included 8 isolates (13%), while the third, pattern 'B', included 6 isolates (10%). Pattern 'C' was found for only one isolate. All of the deer isolates were classified as pattern 'A', while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern 'A', was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns 'B', 'C' and 'E', were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.

Identification of two novel Mycobacterium avium allelic variants in pig and human isolates from Brazil by PCR-restriction enzyme analysis.

Mycobacterium avium complex (MAC) is composed of environmental mycobacteria found widely in soil, water, and aerosols that can cause disease in animals and humans, especially disseminated infections in AIDS patients. MAC consists of two closely related species, M. avium and M. intracellulare, and may also include other, less-defined groups. The precise differentiation of MAC species is a fundamental step in epidemiological studies and for the evaluation of possible reservoirs for MAC infection in humans and animals. In this study, which included 111 pig and 26 clinical MAC isolates, two novel allelic M. avium PCR-restriction enzyme analysis (PRA) variants were identified, differing from the M. avium PRA prototype in the HaeIII digestion pattern. Mutations in HaeIII sites were confirmed by DNA sequencing. Identification of these isolates as M. avium was confirmed by PCR with DT1-DT6 and IS1245 primers, nucleic acid hybridization with the AccuProbe system, 16S ribosomal DNA sequencing, and biochemical tests. The characterization of M. avium PRA variants can be useful in the elucidation of factors involved in mycobacterial virulence and routes of infection and also has diagnostic significance, since they can be misidentified as M. simiae II and M. kansasii I if the PRA method is used in the clinical laboratory for identification of mycobacteria.