Molecular Epidemiology of Sporadic and Outbreak-Related Salmonella Typhi Isolates in the Brazilian North Region: A Retrospective Analysis from 1995 to 2013.

Typhoidal salmonellosis is a global public health problem occurring in developing endemic regions. In Brazil, cases are mostly registered in the North and Northeast regions. Molecular characterization of the strains is important to understand the epidemiology of disease infections and to design control strategies. The present study retrospectively evaluates the genotyping features of sporadic and outbreak-related Salmonella Typhi isolates from the Brazilian North region. Bacterial isolates were recovered from blood and a rectal swab of patients in the states of Acre and Pará, Brazilian North region, in the period of 1995 to 2013, and were submitted to genotyping by applying Multilocus sequence typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE) reference methods. MLST genotyping revealed the presence of epidemic clones ST1 and ST2, and 20 pulsotypes were identified by PFGE, including four distinct clusters (A-D), and six subclusters (A1-D1) with indistinguishable strains in different periods and locations. To conclude, the obtained data demonstrates the temporal stability, adaptation, and transmission of outbreak-related and sporadic S. Typhi strains over time, contributing to the transmission chain in the region.

Clinical and laboratory data, radiological structured report findings and quantitative evaluation of lung involvement on baseline chest CT in COVID-19 patients to predict prognosis.

OBJECTIVE: To evaluate by means of regression models the relationships between baseline clinical and laboratory data and lung involvement on baseline chest CT and to quantify the thoracic disease using an artificial intelligence tool and a visual scoring system to predict prognosis in patients with COVID-19 pneumonia. MATERIALS AND METHODS: This study included 103 (41 women and 62 men; 68.8 years of mean age-range, 29-93 years) with suspicious COVID-19 viral infection evaluated by reverse transcription real-time fluorescence polymerase chain reaction (RT-PCR) test. All patients underwent CT examinations at the time of admission in addition to clinical and laboratory findings recording. All chest CT examinations were reviewed using a structured report. Moreover, using an artificial intelligence tool we performed an automatic segmentation on CT images based on Hounsfield unit to calculate residual healthy lung parenchyma, ground-glass opacities (GGO), consolidations and emphysema volumes for both right and left lungs. Two expert radiologists, in consensus, attributed at the CT pulmonary disease involvement a severity score using a scale of 5 levels; the score was attributed for GGO and consolidation for each lung, and then, an overall radiological severity visual score was obtained summing the single score. Univariate and multivariate regression analysis was performed. RESULTS: Symptoms and comorbidities did not show differences statistically significant in terms of patient outcome. Instead, SpO2 was significantly lower in patients hospitalized in critical conditions or died while age, HS CRP, leukocyte count, neutrophils, LDH, d-dimer, troponin, creatinine and azotemia, ALT, AST and bilirubin values were significantly higher. GGO and consolidations were the main CT patterns (a variable combination of GGO and consolidations was found in 87.8% of patients). CT COVID-19 disease was prevalently bilateral (77.6%) with peripheral distribution (74.5%) and multiple lobes localizations (52.0%). Consolidation, emphysema and residual healthy lung parenchyma volumes showed statistically significant differences in the three groups of patients based on outcome (patients discharged at home, patients hospitalized in stable conditions and patient hospitalized in critical conditions or died) while GGO volume did not affect the patient's outcome. Moreover, the overall radiological severity visual score (cutoff ≥ 8) was a predictor of patient outcome. The highest value of R-squared (R2 = 0.93) was obtained by the model that combines clinical/laboratory findings at CT volumes. The highest accuracy was obtained by clinical/laboratory and CT findings model with a sensitivity, specificity and accuracy, respectively, of 88%, 78% and 81% to predict discharged/stable patients versus critical/died patients. CONCLUSION: In conclusion, both CT visual score and computerized software-based quantification of the consolidation, emphysema and residual healthy lung parenchyma on chest CT images were independent predictors of outcome in patients with COVID-19 pneumonia.

Molecular epidemiology of mycobacteria among herds in Marajó Island, Brazil, reveals strains genetically related and potential zoonotic risk of clinical relevance.

Mycobacterium bovis is the main causative agent of bovine tuberculosis (bTB) being among the animal-adapted Mycobacterium tuberculosis complex. Herds can also be infected with non-tuberculous mycobacteria (NTM) causing a negative effect on the economy and on animal and human health through zoonotic infections. Molecular tools are required for mycobacteria identification; thus, it is laborious to determine the epidemiological information of mycobacteria among herds. We aimed to describe the mycobacterial pathogens associated with cases of suspected bTB lesions in cattle/buffaloes slaughtered for consumption and to investigate bTB transmission. We evaluated 74 lesion samples from 48 animals (27 bovine/21 buffaloes) from 16 mapped farms. Positives samples from nested-PCR were cultured in Lowenstein-Jensen (LJ), 2% pyruvate (LJ + P), and 2% glycerol (LJ + G) media, followed by Ziehl-Neelsen (ZN) staining technique and partial gene sequencing (hsp65, rpoB, and 16S-rRNA). Spoligotyping and 24-MIRU-VNTR were performed. The LJ + P increased the chance of obtaining bacilli. The respiratory tract and the oral cavity were the most important infection route. In addition, the calcified part of the lesions suggested chronic bTB. Spoligotypes of M. bovis (SIT986/SB0885) differed from others found in South America, and the MIRU-VNTR 24 loci suggested that bTB was associated to highly related strains. The NTM species found are of clinical importance in humans.

Molecular Identification, Genotyping, Phenotyping, and Antifungal Susceptibilities of Medically Important Trichosporon, Apiotrichum, and Cutaneotrichosporon Species.

Recently, Trichosporon taxonomy has been reevaluated and new genera of the Trichosporonaceae family have been described. Here, 26 clinical isolates were submitted for identification via sequencing of the intergenic space 1 (IGS1) region, genotyping, and investigation of virulence factors. Antifungal susceptibility was determined using the CLSI broth microdilution method for fluconazole (FLC), itraconazole (ITC), and amphotericin B (AMB). Of these, 24 isolates were identified, including 12 T. asahii, 4 T. inkin, 3 T. faecale, 1 T. coremiiforme, 1 T. japonicum, 2 Cutaneotrichosporon dermatis (formerly T. dermatis), and 1 Apiotrichum mycotoxinivorans (formerly T. mycotoxinivorans). Species-level identification of 2 isolates was not successful; they were described as Trichosporon sp. We observed optimal colonial development at 35-40 °C. Lipase was the major extracellular enzyme produced (100%); caseinase was not produced (0%). Biofilms were produced by all isolates (classified as low). High AMB minimum inhibitory concentration (MIC) was observed, with all strains resistant. Fluconazole was the most active drug among the antifungals tested. However, high MICs for FLC were observed in C. dermatis and A. mycotoxinivorans species, which also showed resistance to ITC and AMB. This study, conducted in the Northern region of Brazil, identified 5 Trichosporon species along with C. dermatis and A. mycotoxinivorans and demonstrated their pathogenic potential through their ability to produce important virulence factors. This may contribute to our understanding of the epidemiology and factors related to the pathogeneses of species in the Trichosporonaceae family.

Clinical aspects in patients with pulmonary infection caused by mycobacteria of the Mycobacterium abscessus complex, in the Brazilian Amazon.

OBJECTIVE: To describe the clinical manifestations of patients with pulmonary infection caused by mycobacteria of the Mycobacterium abscessus complex (MABSC), and to compare these manifestations with those of patients infected with other nontuberculous mycobacteria (NTM). METHODS: This was a retrospective cohort study involving 43 patients divided into two groups: the MABSC group, consisting of patients with pulmonary infection caused by MABSC (n = 17); and the NTM group, consisting of patients with pulmonary infection caused by NTM other than MABSC (n = 26). Patients were previously treated with a regimen of rifampin, isoniazid, pyrazinamide, and ethambutol before the diagnosis of NTM was confirmed by two culture-positive sputum samples. The nucleotide sequences of the hsp65, 16S rRNA, and/or rpoB genes were analyzed to identify the mycobacteria. Data were collected on demographic, clinical, and radiological characteristics, as well as on treatment responses and outcomes. RESULTS: Loss of appetite was the only clinical manifestation that was significantly more common in the MABSC group than in the NTM group (p = 0.0306). The chance of having to use a second treatment regimen was almost 12 times higher in the MABSC group than in the NTM group. Treatment success was significantly higher in the NTM group than in the MABSC group (83.2% vs. 17.6%; p < 0.0001). The chance of recurrence was approximately 37 times higher in the MABSC group than in the NTM group. CONCLUSIONS: In the study sample, treatment response of pulmonary disease caused by MABSC was less favorable than that of pulmonary disease caused by other NTM.

Clinical aspects in patients with pulmonary infection caused by mycobacteria of the Mycobacterium abscessus complex, in the Brazilian Amazon / Aspectos clínicos em pacientes com infecção pulmonar por micobactérias do complexo Mycobacterium abscessus na Amazônia brasileira

ABSTRACT Objective: To describe the clinical manifestations of patients with pulmonary infection caused by mycobacteria of the Mycobacterium abscessus complex (MABSC), and to compare these manifestations with those of patients infected with other nontuberculous mycobacteria (NTM). Methods: This was a retrospective cohort study involving 43 patients divided into two groups: the MABSC group, consisting of patients with pulmonary infection caused by MABSC (n = 17); and the NTM group, consisting of patients with pulmonary infection caused by NTM other than MABSC (n = 26). Patients were previously treated with a regimen of rifampin, isoniazid, pyrazinamide, and ethambutol before the diagnosis of NTM was confirmed by two culture-positive sputum samples. The nucleotide sequences of the hsp65, 16S rRNA, and/or rpoB genes were analyzed to identify the mycobacteria. Data were collected on demographic, clinical, and radiological characteristics, as well as on treatment responses and outcomes. Results: Loss of appetite was the only clinical manifestation that was significantly more common in the MABSC group than in the NTM group (p = 0.0306). The chance of having to use a second treatment regimen was almost 12 times higher in the MABSC group than in the NTM group. Treatment success was significantly higher in the NTM group than in the MABSC group (83.2% vs. 17.6%; p < 0.0001). The chance of recurrence was approximately 37 times higher in the MABSC group than in the NTM group. Conclusions: In the study sample, treatment response of pulmonary disease caused by MABSC was less favorable than that of pulmonary disease caused by other NTM.

RESUMO Objetivo: Descrever as manifestações clínicas de pacientes com infecção pulmonar por micobactérias do complexo Mycobacterium abscessus (CMA) e compará-las com as daqueles infectados com outras micobactérias não tuberculosas (MNT). Métodos: Estudo de coorte retrospectivo envolvendo 43 pacientes divididos em dois grupos: grupo CMA, com pacientes com infecção pulmonar por CMA (n = 17); e grupo MNT, com pacientes com infecção pulmonar por MNT que não CMA (n = 26). Os pacientes foram previamente tratados com o esquema rifampicina, isoniazida, pirazinamida e etambutol antes de o diagnóstico de MNT ser confirmado com culturas positivas em duas amostras de escarro diferentes. As sequências nucleotídicas dos genes hsp65, RNAr 16S e/ou rpoB foram analisadas para a identificação das micobactérias. Foram coletadas características demográficas, clínicas e radiológicas, assim como respostas terapêuticas e desfechos. Resultados: A única manifestação clínica significativamente mais frequente no grupo CMA que no grupo MNT foi hiporexia (p = 0,0306). A chance de haver a necessidade de utilização de um segundo esquema terapêutico foi quase 12 vezes maior no grupo CMA que no grupo MNT. O sucesso terapêutico foi significativamente maior no grupo MNT que no grupo CMA (83,2% vs. 17,6%; p < 0,0001). A chance de recidiva no grupo CMA foi aproximadamente 37 vezes maior que no grupo MNT. Conclusões: Na amostra estudada, a resposta terapêutica da doença pulmonar causada por CMA evoluiu de forma menos favorável do que naquela causada pelas demais MNT.

A case of pulmonary infection due to Mycobacterium paraffinicum from the Amazon Region.

M. paraffinicum, a slow-growing scotochromogenic mycobacterium that uses paraffinic hydrocarbons other than methane, i.e. inorganic carbon sources, was originally isolated from soil samples, but only in 2010 definitely achieved the species status. We have described here the first report of pulmonary disease due to M. paraffinicum in Amazon Region.

A fatal case of pulmonary infection by Mycobacterium colombiense in Para State, Amazon Region, Brazil.

Mycobacterium avium complex (MAC) is a heterogeneous group of species found in several environmental sources and that exhibit variable degrees of pathogenicity. Among the MAC members, Mycobacterium colombiense has been related to pulmonary disease and disseminated infection in HIV-infected patients in Colombia. Lymphadenopathy cases have also been reported. We have described a fatal case of M. colombiense pulmonary disease in a Brazilian patient without evidence of HIV infection or other known causes of immunosuppression.

Molecular identification of nontuberculous mycobacteria isolates in a Brazilian mycobacteria reference laboratory.

This study utilized the hsp65 polymerase chain reaction restriction analysis (PRA) method in the identification of nontuberculous mycobacteria (NTMs) isolated in a Brazilian mycobacteria laboratory. NTM isolates from clinical specimens collected from 192 patients were characterized using the hsp65 PRA method and analyzed using both 16S rRNA and hsp65 gene sequencing. Only 30% of the NTM strains were correctly identified through PRA, though the suggested inclusion of an additional restriction enzyme could increase the resolution to roughly 90%. A total of 17 NTM strains were not identified to species level and may represent a new taxonomic entity classified as belonging to the Mycobacterium simiae complex. This study demonstrates the applicability of hsp65 PRA in the identification of several NTM strains in a reference laboratory, though the results suggest that some modifications to the original PRA method could increase its resolution substantially.

Caracterização fenotípica e genotípica de Serratia marcescens provenientes de Unidade Neonatal de Referência em Belém, Pará, Brasil / Phenotypic and genotypic characterization of Serratia marcescens from a Neonatal Unit in Belém, Pará State, Brazil

A Serratia marcescens tem sido relatada como importante agente de infecções relacionadas à saúde, destacando-se por apresentar elevado nível de resistência intrínseca aos antimicrobianos usados em neonatologia, além de persistir por longos períodos no ambiente hospitalar. Neste trabalho foram avaliadas, por métodos fenotípicos e moleculares, S. marcescens recuperadas a partir de colonização do trato gastrointestinal ou sepse tardia em neonatos internados em Unidade Neonatal em Belém. A identificação das S. marcescens e o teste de sensibilidade foram realizados por meio de sistema automatizado Vitek (BioMérieux); a suscetibilidade ao ertapenem foi avaliada com auxílio de disco contendo 10 μg da droga (Oxoide). A genotipagem foi feita por ERIC-PCR usando os primers ERIC1 (5'-TGAATCCCCAGGAGCTTACAT-3')e ERIC2 (5'-AAGTAAGTGACTGGGGTGAGCG-3'). Foram obtidas 22 cepas de S. marcescens, sendo 15 recuperadas de hemoculturas, e sete de vigilância (swab retal); todas apresentaram resistência a: ampicilina, ampicilina-sulbactam, gentamicina e cefalotina. Não foi observada resistência a: ciprofloxacina, imipenem, meropenem e ertapenem. Quanto aos demais antibióticos avaliados, o perfil de suscetibilidade foi variável. Foram obtidos 11 padrões de amplificação por ERIC-PCR, dois foram compartilhados por 14 isolados. Foi possível observar um padrão polimórfico característico para as cepas provenientes de colonização gastrointestinal, exceto em dois casos, que apresentaram padrões genotípicos relacionadas a casos de sepse. Os dados obtidos neste trabalho confirmam o elevado índice de resistência da S. marcescens aos antimicrobianos; no entanto, todos os isolados apresentaram sensibilidade à ciprofloxacina e aos carbapenêmicos. A tipagem por meio de antibiograma e ERIC-PCR sugere dispersão de clones associados à colonização ou sepse entre alas na Unidade Neonatal do hospital estudado.

Serratia marcescens has been reported as an important agent of health care-related infections and has been highlighted for presenting a high level of intrinsic resistance to antimicrobials used in neonatology, besides persisting in hospital environments for long periods. In this work, S. marcescens was recovered from colonies in the gastrointestinal tract or late sepsis in newborn infants hospitalized in a Neonatal Unit in Belém. The identification of S. marcescens and the sensitivity test was carried out using a Vitek (BioMérieux) automated system; susceptibility to ertapenem was assessed using e-test strips (Oxoid). Genotyping was executed by ERIC-PCR using the primers ERIC1 (5’-TGAATCCCCAGGAGCTTACAT-3’) and ERIC2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’). Twenty-two strains of S. marcescens were recovered: 15 from hemocultures and seven from surveillance (rectal swab culture). All presented resistance to ampicillin, ampicillin-sulbactam, gentamicin and cephalothin. There were no indications of resistance to ciprofloxacin, imipenem, meropenemor ertapenem. The susceptibility profiles varied for other antibiotics. Eleven amplification patterns by ERIC-PCR were obtained, and two were shared by 14 isolates. It was possible to observe a characteristic polymorphic pattern in the strains from gastrointestinal colonization, except for two cases, which presented genotypic patterns related to cases of sepsis. The data obtained in this work confirm the high level of resistance of S. marcescens against antimicrobials; however, all isolates displayed sensitivity to ciprofloxacin and carbapenemics. Antibiogram and ERIC-PCR typing suggest a dispersion of clones associated with colonization or sepsis among the wards of the Neonatal Unit in the surveyed hospital.

Desenvolvimento de PCR multiplex para detecção e diferenciação de categorias de Escherichia coli diarreiogênicas / Development of multiplex PCR to detect and differentiate the categories of diarrheagenic Escherichia coli

INTRODUÇÃO: As Escherichia coli diarreiogênicas (DEC) são consideradas importante causa de diarréia nos países em desenvolvimento. Para sua correta identificação, esses microrganismos devem ser diferenciados dos membros não patogênicos da microbiota intestinal. As DEC podem ser classificadas em seis categorias, de acordo com seu mecanismo de patogenicidade. MATERIAIS E MÉTODOS: Foram desenvolvidos e avaliados dois sistemas de PCR multiplex para a detecção de E. coli enteropatogênicas (EPEC), enterotoxigênica (ETEC), enteroinvasora (EIEC) e produtora da toxina de Shiga (STEC). RESULTADOS: Quatro categorias diarreiogênicas foram detectadas entre os isolados de E. coli obtidos de indivíduos infectados pelo HIV, com EIEC e EPEC representando os patótipos mais frequentes. Duas STEC stx1 e eae positivas foram identificadas, sendo este o primeiro relato de isolamento dessa categoria em indivíduos infectados pelo HIV no estado do Pará. A PCR multiplex mostrou-se como técnica eficiente, rápida e reprodutível para detecção dos isolados de DEC. Os dois sistemas de PCR multiplex apresentaram resultados 100 por cento concordantes com os encontrados na PCR simples. CONCLUSÃO: Devido sua simplicidade, economia e eficiência torna-se possível a aplicação dos protocolos multiplex para agilizar o diagnóstico molecular das categorias de DEC, o que promoverá a elaboração de novos projetos de pesquisa e darão suporte as atividades de vigilância epidemiológica desenvolvidos pelos institutos de saúde pública...

INTRODUCTION: Diarrheagenic Escherichia coli (DEC) are considered an important cause of diarrhea in developing countries. The correct identification of these microorganisms depends on their differentiation from non-pathogenic members of the intestinal microbiota. DEC can be classified into one of six categories according to their mechanism of pathogenicity. MATERIALS AND METHODS: Two multiplex PCR systems used to detect enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC) and Shiga Toxin-producing (STEC) E. coli were evaluated and described. RESULTS: Four categories of DEC were detected among isolates of E. coli obtained from individuals infected with HIV. EIEC and EPEC were among the most prevalent pathotypes. Furthermore, two STEC strains that were both stx1-and eae-positive were identified. This is the first report of this kind of isolation in individuals infected with HIV in the State of Pará. Multiplex PCR proved to be an efficient, fast and reproducible technique for detection of DEC isolates. Both multiplex PCR systems described here produced results 100 per cent similar to those obtained from individual PCR reactions. CONCLUSION: Given their simplicity, cost and efficiency, it is possible to use these protocols to expedite the molecular diagnosis of the distinct categories of DEC. In addition to facilitating the development of new research projects, these findings could support the epidemiological surveillance undertaken by public health agencies and institutes...

Identificação genotípica de membros do complexo Mycobacterium avium isolados de infecções pulmonares no Estado do Pará, Brasil / Genotypic identification of members of the Mycobacterium avium complex isolated from pulmonary infections in Pará State, Brazil

INTRODUÇÃO: O complexo Mycobacterium avium (MAC) compreende micobactérias de crescimento lento, naturalmente encontradas no meio ambiente, capazes de causar infecções em diversas espécies de seres vivos, incluindo aves, suínos e humanos. Tais infecções podem ser assintomáticas, clinicamente significantes e, em alguns casos, fatais. No entanto, existe a necessidade de se disponibilizarem técnicas que ofereçam uma identificação conclusiva de bactérias estreitamente relacionadas. As técnicas de biologia molecular se apresentam como ferramentas promissoras para uma identificação mais precisa. MATERIAIS E MÉTODOS: Neste trabalho foram avaliados os marcadores moleculares RNAr 16S, hsp65 e rpoB aplicados à distinção de membros do MAC isolados no Laboratório de Micobactérias do Instituto Evandro Chagas. RESULTADOS: Amostras de MAC colhidas em 15 pacientes foram previamente avaliadas pelo método de análise de polimorfismo de fragmentos de restrição do gene hsp65 (PRA-hsp65), que forneceu três diferentes perfis: (I) BstEII: 235/115/100, HaeIII: 145/130/60; (II) BstEII: 235/210, HaeIII: 130/105; e (III) BstEII: 235/210, HaeIII:145/130. Árvores filogenéticas foram construídas após análise do RNAr 16S, em que as amostras foram distribuídas em dois grupos, semelhante ao encontrado na análise de hsp65. Entretanto, os resultados de rpoB foram discordantes daqueles das outras árvores, devido à modificação de topologia. CONCLUSÃO: Os achados deste estudo sugerem que diferentes forças evolucionárias podem estar atuando sobre o gene rpoB, e, desta forma, é necessário que se tenha precaução ao estabelecer esse alvo para fins taxonômicos. Adicionalmente, recomenda-se que mais de um marcador, incluindo o RNAr 16S, seja avaliado para a identificação micobacteriana.

INTRODUCTION: The Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria naturally found in the environment capable of causing infections in a wide variety of living species, including birds, swines and humans. These infections can be asymptomatic, clinically significant and, in some cases, fatal. There is a demand for techniques that are capable of conclusively identifying closely related bacteria. Molecular biological techniques are promising tools for a more precise identification. MATERIAL AND METHODS: In this study, we evaluated the ability of 16S rRNA, hsp65 and rpoB molecular markers to distinguish between members of the MAC isolated in the Mycobacteria Laboratory at the Instituto Evandro Chagas. RESULTS: MAC samples collected from 15 patients were previously evaluated using an hsp65 gene restriction fragment length polymorphism analytical method (RFLP-hsp65), and they showed three different profiles: (I) BstEII: 235/115/100, HaeIII: 145/130/60; (II) BstEII: 235/210, HaeIII: 130/105; and (III) BstEII: 235/210, HaeIII: 145/130. We constructed phylogenetic trees using 16S rRNA analysis in which the samples were distributed into two groups, similarly to those found in the hsp65 analysis. However, the results from the rpoB analysis disagreed with those of the other trees due to changes in topology. CONCLUSION: The findings from this study warrant that various evolutionary forces may be acting on the rpoB gene. Thus, it is necessary to be cautious when using this target for taxonomic analyses. Additionally, we recommend that multiple markers (including16S rRNA) be evaluated when identifying mycobacteria.

Molecular identification of rapidly growing mycobacteria isolates from pulmonary specimens of patients in the State of Pará, Amazon region, Brazil.

We isolated 44 strains of rapidly growing mycobacteria (RGM) from 19 patients with pulmonary infections assisted at the Instituto Evandro Chagas (Pará, Brazil) from 2004 to 2007. Identification at the species level was performed by PCR restriction fragment length polymorphism analysis (PRA) of a 441 bp hsp65 fragment and partial 16S rRNA, hsp65, and rpoB gene sequencing. Genotyping by PRA yielded 3 digestion patterns: one identical to Mycobacterium abscessus type I (group I); another to M. abscessus type II, Mycobacterium bolletii, and Mycobacterium massiliense (group II); and a third typical for Mycobacterium fortuitum type I (group III). When comparing analysis of the 3 genes, more discrimination was obtained by rpoB gene sequence, which allowed good distinction between group I, II, and III strains and subclassification of group II strains in SG IIa (M. bolletii) and SG IIb (M. massiliense). In this study, we show that the description of new RGM species requires the establishment of standardized procedures for RGM identification and the alert of the clinician about their involvement in pulmonary disease and the necessity of treatment for control and cure.