Publications by authors named "Erica Chimara"

37 Publications

Silent mutations in ribosomal protein genes are associated with high-risk clones of carbapenem-resistant Acinetobacter baumannii prevalent in Brazil.

Infect Genet Evol 2021 Mar 21;88:104686. Epub 2020 Dec 21.

Instituto Adolfo Lutz, São Paulo, SP 01246-902, Brazil; Universidade de São Paulo, São Paulo, SP 01246-903, Brazil. Electronic address:

Objectives: To analyze the relationship of ribosomal protein mutations and clonality of high-risk clones Acinetobacter baumannii.

Methods: Seventy-nine carbapenem-resistant A. baumannii were subjected to whole-genome sequencing (Illumina NextSeq), and codifying sequences of ribosomal proteins were extracted and screened for mutations. MALDI-TOF MS analysis (Bruker Biotyper) and Spectra data from MALDI-TOF was employed to generate a dendrogram based on principal component analysis (PCA) data. Clones were identified by Multilocus sequencing typing (MLST) based on WGS.

Results: Ribosomal RNA protein sequences extracted from the genomes identified mutations that were associated with clonal complexes, but most of them were silent. PCA did not cluster the isolates according to their clonality identified by MLST.

Conclusions: By comparing the nucleotide and amino acid sequences of diversified A. baumannii, and Bruker Biotyper profiles, we showed that silent mutations in ribosomal RNA nucleotides are associated with clonal complexes, but since most of the mutations were silent, MALDI-TOF MS raw data was not a useful tool for typing the high-risk clones of this species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.meegid.2020.104686DOI Listing
March 2021

Genomic and phenotypic characterisation of antimicrobial resistance in carbapenem-resistant Acinetobacter baumannii hyperendemic clones CC1, CC15, CC79 and CC25.

Int J Antimicrob Agents 2020 Dec 9;56(6):106195. Epub 2020 Oct 9.

Centro de Bacteriologia, Instituto Adolfo Lutz, São Paulo, SP, Brazil.

Dissemination of carbapenem-resistant Acinetobacter baumannii (CRAB) is mainly driven by the spread of clonal lineages. High frequencies of CRAB are reported in South America, and clonal complexes CC1, CC15, CC79 and CC25 are predominant. A total of 79 non-redundant CRAB recovered from 26 Brazilian hospitals were selected for antimicrobial susceptibility testing by microdilution and whole-genome sequencing (WGS). Multilocus sequence typing (MLST), acquired antimicrobial resistance genes and phylogeny based on high-quality SNPs were extracted from WGS data. XDR (86.1%), MDR (12.7%) and one PDR isolate from CC15 (1.3%) were identified. Colistin resistance was more frequent in CC25 isolates (P < 0.01). Prevalence of CC79 (n = 22; 27.8%) CC1 (n = 21; 26.6%), CC15 (n = 21; 26.6%) and CC25 (n = 12; 15.2%) was observed. Regarding carbapenem-hydrolysing class D β-lactamases (CHDLs), bla was frequently detected in CC1, CC15 and CC25 isolates, whereas bla was the most frequent CHDL in CC79 isolates [n = 12/22 (54.5%); P < 0.01]. High-quality SNP analysis correlated well with sequence type and revealed that CRAB clones are highly conversed and present some clone-specific resistance determinants. This study provides essential information to understand the antimicrobial resistance patterns of CRAB in Brazilian hospitals, where hyperendemic XDR-CRAB clones are disseminated. Phenotypic and genomic analysis of CRAB recovered from Brazilian hospitals revealed the predominance of XDR phenotype in the majority of international clonal complex CC79, CC1, CC15 and CC25. Dissemination of specific CRAB lineages in Brazil is suggested to be driven by their resistance determinants under antimicrobial selective pressure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijantimicag.2020.106195DOI Listing
December 2020

Pulmonary nontuberculous mycobacterial infections: presumptive diagnosis based on the international microbiological criteria adopted in the state of São Paulo, Brazil, 2011-2014.

J Bras Pneumol 2019 Apr 25;45(2):e20180278. Epub 2019 Apr 25.

. Núcleo de Tuberculose e Micobacterioses, Centro de Bacteriologia, Instituto Adolfo Lutz, São Paulo (SP) Brasil.

Objective: Pulmonary nontuberculous mycobacterial infections are caused by nontuberculous mycobacteria (NTM), the microbiological diagnosis of which involves the isolation and identification of the same species in at least two sputum samples, one BAL fluid sample, or one lung biopsy sample. The objective of the present study was to determine the frequency at which the various NTM species are identified among selected individuals and in potential cases of pulmonary nontuberculous mycobacterial infection.

Methods: This was a retrospective analysis of the data on species isolated from respiratory specimens collected from 2,843 individuals between 2011 and 2014. Potential NTM infection cases were identified on the basis of the international microbiological criteria adopted in the state of São Paulo.

Results: A total of 50 species were identified using the molecular method PCR-restriction enzyme analysis. Samples collected from 1,014 individuals were analyzed in relation to the microbiological criteria, and 448 (44.18%) had a presumptive diagnosis of pulmonary nontuberculous mycobacterial infection, the species identified most frequently being, in descending order, Mycobacterium kansasii, M. abscessus, M. intracellulare, M. avium, and M. szulgai.

Conclusions: Although various NTM species were identified among the individuals studied, those presumptively identified most frequently on the basis of the microbiological criteria adopted in the state of São Paulo were the ones that are most commonly associated with pulmonary nontuberculous mycobacterial infection worldwide or in specific geographic regions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/1806-3713/e20180278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733752PMC
April 2019

Bedaquiline and linezolid MIC distributions and epidemiological cut-off values for Mycobacterium tuberculosis in the Latin American region.

J Antimicrob Chemother 2019 02;74(2):373-379

Université catholique de Louvain, Institute of Experimental and Clinical Research, Laboratory of Medical Microbiology, Brussels, Belgium.

Objectives: To describe the distributions of bedaquiline and linezolid MIC values for the Mycobacterium tuberculosis WT population and to define the corresponding epidemiological cut-offs (ECOFFs) in three Latin American countries.

Methods: MICs of bedaquiline and linezolid were determined by the resazurin microtitre assay (REMA). In phase 1, interlaboratory reproducibility was assessed using a panel of 10 fully susceptible M. tuberculosis strains. Phase 2 involved MIC determination for 248 clinical isolates from Argentina (n = 58), Brazil (n = 100) and Peru (n = 90) from patients who were treatment-naive for bedaquiline and linezolid. We then determined the ECOFFs for bedaquiline and linezolid by the eyeball method and the ECOFFinder statistical calculator.

Results: Phase 1: REMA MIC values in the three sites were either identical to each other or differed by one 2-fold dilution from the consensus value with the exception of a single value. Phase 2: the bedaquiline MIC range was 0.0039-0.25 mg/L for pan-susceptible and drug-resistant isolates combined. The linezolid MIC range was 0.062-0.5 mg/L for pan-susceptible isolates and 0.031-4 mg/L for drug-resistant isolates. ECOFFs were 0.125 mg/L for bedaquiline and 0.50 mg/L for linezolid.

Conclusions: REMA is reproducible and robust for the determination of bedaquiline and linezolid MIC distributions and ECOFF values when applied in laboratories of medium/low-resource countries. We suggest that WT MIC distributions for both drugs should be used as a monitoring tool to control the possible rapid emergence of resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dky414DOI Listing
February 2019

Nocardial scleritis: A case report and a suggested algorithm for disease management based on a literature review.

Am J Ophthalmol Case Rep 2018 Jun 10;10:1-5. Epub 2018 Jan 10.

Department of Ophthalmology and Visual Sciences, Escola Paulista de Medicina, Universidade Federal de São Paulo, Hospital São Paulo. 821, Botucatu Street, São Paulo, SP, Brazil.

Purpose: To report a case of nocardial scleritis and to propose a logical treatment algorithm based on a literature review.

Observations: It is important to suspect a nocardial infection when evaluating anterior unilateral scleritis accompanied by multiple purulent or necrotic abscesses, especially in male patients with a history of chronic ocular pain and redness, trauma inflicted by organic materials, or recent ophthalmic surgery. A microbiological investigation is essential. In positive cases, a direct smear reveals weakly acid-fast organisms or Gram-positive, thin, beading and branching filaments. Also, the organism (usually) grows on blood agar and Lowenstein-Jensen plates. An infection can generally be fully resolved by debridement of necrotic areas and application of topical amikacin drops accompanied by systemic sulfamethoxazole-trimethoprim.

Conclusions And Significance: Together with the case report described, we review data on a total of 43 eyes with nocardial scleritis. Our proposed algorithm may afford a useful understanding of this sight-threatening disease, facilitating easier and faster diagnosis and management.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajoc.2018.01.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5956651PMC
June 2018

HIV-negative pulmonary disease caused by nontuberculous mycobacteria in Southern Brazil: clinical and microbiological characterization.

J Thorac Dis 2018 Mar;10(3):1903-1911

Ribeirão Preto School of Medicine, University of São Paulo (FMRP-USP), Ribeirão Preto, Brazil.

Background: Nontuberculous mycobacteria (NTM) have been identified with increasing frequency in the clinical practice. The aim of this study was to characterize NTM isolates in respiratory specimens from patients with pulmonary disease and to correlate this with clinical/radiological findings, decision to start treatment and outcomes.

Methods: A cross-sectional descriptive study was performed and included all patients who had at least one NTM isolated in respiratory specimens between 2011 and 2014. NTM culture was performed in liquid medium followed by immunochromatographic identification (anti-MPT64). Species identification was based on nucleic acid amplification followed by restriction analysis of a 441 bp fragment of the hsp65 gene (hsp65 PRA) and patients' records were reviewed.

Results: From 14,394 cultures in 4 years, 590 (4.10%) grew NTM and 305 (51.7%) isolates were characterized till species level, representing 290 patients including those with and without human immunodeficiency virus (HIV) infection. Two hundred and eleven non-HIV patients had NTM isolated from respiratory specimens, 49 (23.2%) had criteria for active disease based on the American Thoracic Society (ATS) 2007. The majority was men above 51 years old and M. intracellulare was detected in 59.2% (29/49), followed by M. avium 14.3% (7/49), and M. abscessus 12.2% (6/49).

Conclusions: Old age, nodular and nodular/bronchiectasis radiographic pattern, previous tuberculosis (TB) treatment and M. intracellulare were more frequent among NTM-disease patients compared to those only colonized. Positive culture and maintenance of clinical symptoms (poor outcome) was a rule when M. abscessus caused NTM-disease. Positive acid-fast smear in respiratory specimen is a strong predictor of disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21037/jtd.2018.03.66DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906231PMC
March 2018

Growth characteristics of liquid cultures increase the reliability of presumptive identification of Mycobacterium tuberculosis complex.

J Med Microbiol 2018 Jun 23;67(6):828-833. Epub 2018 Apr 23.

Núcleo de Tuberculose e Micobacterioses, Centro de Bacteriologia, Instituto Adolfo Lutz - Av. Dr. Arnaldo, 351 - 9° andar - 01246-902 - São Paulo - SP, Brazil.

We evaluated the microscopic and macroscopic characteristics of mycobacteria growth indicator tube (MGIT) cultures for the presumptive identification of the Mycobacterium tuberculosis complex (MTBC) and assessed the reliability of this strategy for correctly directing isolates to drug susceptibility testing (DST) or species identification. A total of 1526 isolates of mycobacteria received at the Instituto Adolfo Lutz were prospectively subjected to presumptive identification by the observation of growth characteristics along with cord formation detection via microscopy. The presumptive identification showed a sensitivity, specificity and accuracy of 98.8, 92.5 and 97.9 %, respectively. Macroscopic analysis of MTBC isolates that would have been erroneously classified as non-tuberculous mycobacteria based solely on microscopic morphology enabled us to direct them rapidly to DST, representing a substantial gain to patients. In conclusion, the growth characteristics of mycobacteria in MGIT, when considered along with cord formation, increased the reliability of the presumptive identification, which has a great impact on the laboratory budget and turnaround times.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jmm.0.000734DOI Listing
June 2018

Use of an immunochromatographic assay for rapid identification of complex clinical isolates in routine diagnosis.

J Med Microbiol 2018 May;67(5):683-686

Tuberculosis and Mycobacteriosis Core, Center of Bacteriology, Adolfo Lutz Institute, Av. Dr. Arnaldo 351, CEP: 01246-902, São Paulo, SP, Brazil.

Accurate identification of complex (MTBC) isolates is essential for tuberculosis (TB) control, especially in a high-burden country such as Brazil. Conventional identification methods are laborious and time-consuming, while rapid molecular methods are expensive and require skilled personnel and appropriate physical laboratory infrastructure. Immunochromatographic assays (ICAs) have been shown to provide a rapid and reliable TB diagnosis at a low cost. The use of the SD Bioline TB Ag MPT64 ICA (MPT64 assay) for rapid identification of MTBC clinical isolates in the routine diagnosis of a large-volume reference TB laboratory was evaluated. We analysed 375 isolates on solid and liquid media concurrently with conventional phenotypic methods, the PRA-65 molecular technique and the MPT64 assay. The sensitivity, specificity and accuracy of the ICA were 97.7, 100 and 98.1 %, respectively. The MPT64 assay yielded rapid and accurate results, enabling the treatment to be initiated early and also impacting on TB control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jmm.0.000726DOI Listing
May 2018

Genetic correlates of clarithromycin susceptibility among isolates of the Mycobacterium abscessus group and the potential clinical applicability of a PCR-based analysis of erm(41).

J Antimicrob Chemother 2018 04;73(4):862-866

Tuberculosis and Mycobacteriosis Laboratory, Bacteriology Center, Instituto Adolfo Lutz, São Paulo, Brazil.

Objectives: To define the genetic basis of clarithromycin resistance among isolates of the Mycobacterium abscessus group (MAG).

Methods: We analysed 133 isolates identified as MAG. Species identification was confirmed by sequencing the rpoB gene. Clarithromycin susceptibility testing was performed according to CLSI recommendations, with an extended 14 day incubation. Known resistance genotypes of erm(41) and rrl were identified by sequencing; the presence of deletions in erm(41) was detected by PCR.

Results: The 133 MAG isolates included 82 M. abscessus, 27 Mycobacterium massiliense and 24 Mycobacterium bolletii. After the 3 day incubation, only five isolates demonstrated clarithromycin resistance (R); after 14 days of extended incubation, an additional 92 exhibited inducible resistance (IR), with the remaining being susceptible (S). The distribution of susceptibility phenotypes varied among the species. Among M. abscessus isolates, 11% were S, 84% IR and 5% R; among M. bolletii isolates, 96% were IR and 4% R; and among M. massiliense isolates 100% were S. Sequencing of rrl identified only a single isolate with the A2058G mutation. Deletions in erm(41) were present in 30 susceptible isolates; among the remaining 103 isolates, 97 were R or IR (sensitivity, 83%; specificity, 100%; positive predictive value, 100%; negative predictive value, 94%). Among the six susceptible isolates without deletions, all carried the erm(41) T28C point mutation.

Conclusions: A significant proportion of MAG isolates demonstrate inducible resistance to clarithromycin that is only detectable with an extended 14 day incubation. Further, the majority of clarithromycin-susceptible MAG isolates have characteristic deletions in erm(41) that can rapidly and reliably be detected by a simple PCR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkx476DOI Listing
April 2018

Genomic epidemiology of a national outbreak of post-surgical wound infections in Brazil.

Microb Genom 2017 05 3;3(5):e000111. Epub 2017 May 3.

1​Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

An epidemic of post-surgical wound infections, caused by a non-tuberculous mycobacterium, has been on-going in Brazil. It has been unclear whether one or multiple lineages are responsible and whether their wide geographical distribution across Brazil is due to spread from a single point source or is the result of human-mediated transmission. 188 isolates, collected from nine Brazilian states, were whole genome sequenced and analysed using phylogenetic and comparative genomic approaches. The isolates from Brazil formed a single clade, which was estimated to have emerged in 2003. We observed temporal and geographic structure within the lineage that enabled us to infer the movement of sub-lineages across Brazil. The genome size of the Brazilian lineage was reduced relative to most strains in the three subspecies of and contained a novel plasmid, pMAB02, in addition to the previously described pMAB01 plasmid. One lineage, which emerged just prior to the initial outbreak, is responsible for the epidemic of post-surgical wound infections in Brazil. Phylogenetic analysis indicates that multiple transmission events led to its spread. The presence of a novel plasmid and the reduced genome size suggest that the lineage has undergone adaptation to the surgical niche.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mgen.0.000111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562415PMC
May 2017

Epidemiological and laboratorial profile of patients with isolation of nontuberculous mycobacteria.

Int J Mycobacteriol 2017 Jul-Sep;6(3):239-245

Instituto Adolfo Lutz, Núcleo de Tuberculose e Micobacterioses, IAL Central São Paulo, SP Av. Dr. Arnaldo, 355 Cerqueira César, São Paulo, Brazil.

Background: An increase in NTM diseases in the international scenario has been observed in recent years.

Aims: To analyze the epidemiological and laboratory profiles of patients with isolation of nontuberculous mycobacteria (NTM) over one decade. A retrospective analysis of records of a mycobacterial reference laboratory found 135 cases with isolation of NTM.

Methods: Clinical and epidemiological data were collected from the records of government health clinics and from the State notification system (TBWEB). The cases were geocoded by location based on the street address in the Mercator Transverse Universal projection, Datum SAD/69 and MapInfo software.

Results: Most patients were male (66.7%), older than 50 years (40%) and had only completed elementary schooling (38.5%). Associated health problems were found in 71.8% of the subjects, with 43.7% being HIV positive and 25.9% having had tuberculosis in the past. Hospitals were the most able institutions to diagnose cases (45.2%). Sputum was the most common material tested (63.0%) with the bacilloscopy being positive in 33.3% of cases. The most common mycobacteria species in the region were Mycobacterium avium and M. abscessus/M. massiliense/M. bolletii. When the regional reference municipality was analyzed, M. avium and M. fortuitum were the most common species isolated in the urban area.

Conclusions: In the study region, mycobacteriosis most affected adult males with low schooling. Most patients presented comorbidities in particular co-infection with the HIV virus. M. avium is the most prevalent species in the region with the M. abscessus/M. massiliense/M. bolletii species being the main cause of nosocomial infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4103/ijmy.ijmy_87_17DOI Listing
April 2018

Characterization of -Like Strains by Comparative Genomics.

Front Microbiol 2017 8;8:789. Epub 2017 May 8.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São PauloSão Paulo, Brazil.

Isolates of the - complex are subdivided into four clusters (CHI to CHIV) in the INNO-LiPA® spp DNA strip assay. A considerable phenotypic variability was observed among isolates of the CHII cluster. In this study, we examined the diversity of 26 CHII cluster isolates by phenotypic analysis, drug susceptibility testing, whole genome sequencing and single-gene analysis. Pairwise genome comparisons were performed using several approaches, including average nucleotide identity (ANI) and genome-to-genome distance (GGD) among others. Based on ANI and GGD the isolates were identified as (14 isolates), (2 isolates) and (1 isolate). The remaining 9 isolates were subdivided into three novel putative genomospecies. Phenotypic analyses including drug susceptibility testing, as well as whole genome comparison by TETRA and delta differences, were not helpful in separating the groups revealed by ANI and GGD. The analysis of standard four conserved genomic regions showed that alone and the concatenated sequences clearly distinguished the taxonomic groups delimited by whole genome analyses. In conclusion, the CHII INNO-LiPa is not a homogeneous cluster; on the contrary, it is composed of closely related different species belonging to the complex and also several unidentified isolates. The detection of these isolates, putatively novel species, indicates a wider inner variability than the presently known in this complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2017.00789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5420552PMC
May 2017

Pseudooutbreak of rapidly growing mycobacteria due to Mycobacterium abscessus subsp bolletii in a digestive and respiratory endoscopy unit caused by the same clone as that of a countrywide outbreak.

Am J Infect Control 2016 11 11;44(11):e221-e226. Epub 2016 Aug 11.

Hospital das Clínicas, University of São Paulo, São Paulo, Brazil.

Background: The nontuberculous mycobacteria (NTM) are widely spread. In Brazil, 2,520 cases of rapidly growing mycobacteria (RGM) infections after medical procedures were reported, with 5.4% of cases related to nonsurgical invasive procedures and with an occurrence of 1 clone (BRA100) of Mycobacterium abscessus subsp bolletii.

Objective: To describe a pseudooutbreak of M abscessus subsp bolletii in an endoscopy and bronchoscopy unit.

Methods: The alert for a pseudooutbreak was given when 3 patients, in the same week, had a positive bronchoalveolar lavage culture for M abscessus subsp bolletii. The patients had no symptoms/signs of mycobacterial infection; thus, contamination of bronchoscopes was suspected. Samples for culturing were collected from bronchoscopes, digestive endoscopes, automated disinfection machines, and the water supply. Clinical samples were identified by polymerase chain reaction restriction-enzyme analysis (PRA) of the hsp65 gene and their pulsed-field gel electrophoresis pattern was compared with environmental samples.

Results: The investigation demonstrated a contamination of bronchoscopes, digestive endoscopes, and disinfection machines. Molecular typing demonstrated that all strains belonged to the same clone (MAB01), identical to clone BRA100.

Discussion: Cross-transmission due to poor disinfection as well as resistance to glutaraldehyde may play roles in the spread of MAB01 M abscessus subsp bolletii, which may have a unique resistance to the environment and adaption to human hosts. However the water supply may have played a role. Attention is needed to ensure the quality of water used to rinse disinfected equipment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajic.2016.06.019DOI Listing
November 2016

Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting.

Mem Inst Oswaldo Cruz 2016 Sep 1;111(9):545-50. Epub 2016 Sep 1.

Instituto Adolfo Lutz, Centro de Bacteriologia, Núcleo de Tuberculose e Micobacterioses, São Paulo, SP, Brasil.

Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/0074-02760160048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027861PMC
September 2016

Resazurin Microtiter Assay for Clarithromycin Susceptibility Testing of Clinical Isolates of Mycobacterium abscessus Group.

J Clin Lab Anal 2016 Sep 12;30(5):751-5. Epub 2016 May 12.

Nucleo de Tuberculose e Micobacterioses, Instituto Adolfo Lutz, Sao Paulo, Brazil.

Background: Mycobacterium abscessus group has heterogeneous susceptibility pattern among species. The species is most common cause of nosocomial infections. Macrolides minimum Inhibitory concentration (MIC) determination is essential for the treatment.

Methods: Thirty-six strains were randomly selected for performing Resazurin Microtiter Assay (REMA) for clarithromycin testing in comparison to MIC test according to Clinical and Laboratory Standards Institute (2011) recommendation. REMA has been used for detection of drug resistance in M. tuberculosis. Extended incubation was performed to detect induced resistance.

Results: Thirty microliters of resazurin (0.01%) was added after visually taking MIC reading. Resistance was observed in 11.1% of M. bolletti and 4.8% of M. abscessus strains; and induced resistance was detected in 77.8% and 95.2% of M. bolletti and M. abscessus strains, respectively. All strains of M. massiliense were susceptible. The samples presented same MIC value both by visual reading and through resazurin.

Conclusion: The present study showed 100% concordance between both readings, with REMA providing easier to read and report results benefit. This change in reading can also reflect on the MIC determination and report, improving the test.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcla.21933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6806693PMC
September 2016

Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae--Mycobacterium abscessus group.

Int J Syst Evol Microbiol 2015 Dec;65(12):4403-4409

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results,demonstrated that they share characteristics with M. chelonae–M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/ijsem.0.000590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772522PMC
December 2015

Mycobacterium franklinii sp. nov., a species closely related to members of the Mycobacterium chelonae-Mycobacterium abscessus group.

Int J Syst Evol Microbiol 2015 Jul 9;65(7):2148-2153. Epub 2015 Apr 9.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

Two isolates from water, D16Q19 and D16R27, were shown to be highly similar in their 16S rRNA, 16S-23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences to 'Mycobacterium franklinii' DSM 45524, described in 2011 but with the name not validly published. They are all nonpigmented rapid growers and are related phenotypically and genetically to the Mycobacterium chelonae-Mycobacterium abscessus group. Extensive characterization by phenotypic analysis, biochemical tests, drug susceptibility testing, PCR restriction enzyme analysis of the hsp65 gene and ITS, DNA sequencing of housekeeping genes and DNA-DNA hybridization demonstrated that 'M. franklinii' DSM 45524, D16Q19 and D16R27 belong to a single species that is separated from other members of the M. chelonae-M. abscessus group. On the basis of these results we propose the formal recognition of Mycobacterium franklinii sp. nov. Strain DSM 45524(T) ( = ATCC BAA-2149(T)) is the type strain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/ijs.0.000234DOI Listing
July 2015

Mycobacterium chelonae cutaneous infection in a patient with mixed connective tissue disease.

An Bras Dermatol 2015 Jan-Feb;90(1):104-7

Pontifícia Universidade Católica de Campinas, Campinas, SP, Brazil.

Around 50 mycobacteria species cause human disease. Immunosuppressive states predispose to non-tuberculous mycobaterium infection, such as Mycobacterium chelonae: AFB, non-tuberculous, fast growth of low virulence and uncommon as a human pathogen. It may compromise the skin and soft tissues, lungs, lymph nodes and there is also a disseminated presentation. The diagnosis involves AFB identification and culture on Agar and Lowenstein-Jensen medium base. A 41-year-old female with MCTD (LES predominance) is reported, presenting painless nodules in the right forearm. She denied local trauma. Immunosuppressed with prednisone and cyclophosphamide for 24 months. Lesion biopsy has demonstrated positive bacilloscopy (Ziehl-Neelsen stain) and M.chelonae in culture (Lowenstein-Jensen medium base), therefore clarithromycin treatment has been started (best therapy choice in the literature).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/abd1806-4841.20152276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323705PMC
July 2015

Differentiation between Nocardia spp. and Mycobacterium spp.: Critical aspects for bacteriological diagnosis.

Rev Inst Med Trop Sao Paulo 2014 Sep-Oct;56(5):397-401

Tuberculosis and Mycobacteriosis Branch, Instituto Adolfo Lutz, São Paulo, SP, Brazil.

New methodologies were developed for the identification of Nocardia but the initial diagnosis still requires a fast and accurate method, mainly due to the similarity to Mycobacterium, both clinical and bacteriologically. Growth on Löwenstein-Jensen (LJ) medium, presence of acid-fast bacilli through Ziehl-Neelsen staining, and colony morphology can be confusing aspects between Nocardia and Mycobacterium. This study describes the occurrence of Nocardia spp. in a mycobacterial-reference laboratory, observing the main difficulties in differentiating Nocardia spp. from Mycobacterium spp., and correlating isolates with nocardiosis cases. Laboratory records for the period between 2008 and 2012 were analyzed, and the isolates identified as Nocardia sp. or as non-acid-fast filamentous bacilli were selected. Epidemiological and bacteriological data were analyzed as well. Thirty-three isolates identified as Nocardia sp. and 22 as non-acid-fast bacilli were selected for this study, and represented 0.12% of isolates during the study period. The presumptive identification was based on macroscopic and microscopic morphology, resistance to lysozyme and restriction profiles using the PRA-hsp65 method. Nocardia spp. can grow on media for mycobacteria isolation (LJ and BBL MGIT™) and microscopy and colony morphology are very similar to some mycobacteria species. Seventeen patients (54.8%) were reported and treated for tuberculosis, but presented signs and symptoms of nocardiosis. It was concluded that the occurrence of Nocardia sp. during the study period was 0.12%. Isolates with characteristics of filamentous bacilli, forming aerial hyphae, with colonies that may be pigmented, rough and without the BstEII digestion pattern in PRA-hsp65 method are suggestive of Nocardia spp. For a mycobacterial routine laboratory, a flow for the presumptive identification of Nocardia is essential, allowing the use of more accurate techniques for the correct identification, proper treatment and better quality of life for patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172110PMC
http://dx.doi.org/10.1590/s0036-46652014000500005DOI Listing
April 2015

Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.

J Clin Microbiol 2014 Aug 4;52(8):2881-91. Epub 2014 Jun 4.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JCM.00688-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136145PMC
August 2014

Viability of stressed Mycobacterium tuberculosis and association with multidrug resistance.

Braz J Microbiol 2013 30;44(2):465-8. Epub 2013 Oct 30.

Instituto Adolfo Lutz, São Paulo, SP, Brazil.

This study investigated biological characteristics of recovered stressed M. tuberculosis isolates that failed to grow in differential culture media for phenotypic identification and in culture media containing anti-tuberculosis drugs for drug-susceptibility testing, despite of having grown in primary culture. It represents an improvement in the diagnosis of MDR tuberculosis and tuberculosis control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/S1517-83822013000200019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3833144PMC
January 2015

The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study.

Eur Respir J 2013 Dec 18;42(6):1604-13. Epub 2013 Apr 18.

A full list of the authors' affiliations can be found in the Acknowledgements.

A significant knowledge gap exists concerning the geographical distribution of nontuberculous mycobacteria (NTM) isolation worldwide. To provide a snapshot of NTM species distribution, global partners in the NTM-Network European Trials Group (NET) framework (www.ntm-net.org), a branch of the Tuberculosis Network European Trials Group (TB-NET), provided identification results of the total number of patients in 2008 in whom NTM were isolated from pulmonary samples. From these data, we visualised the relative distribution of the different NTM found per continent and per country. We received species identification data for 20 182 patients, from 62 laboratories in 30 countries across six continents. 91 different NTM species were isolated. Mycobacterium avium complex (MAC) bacteria predominated in most countries, followed by M. gordonae and M. xenopi. Important differences in geographical distribution of MAC species as well as M. xenopi, M. kansasii and rapid-growing mycobacteria were observed. This snapshot demonstrates that the species distribution among NTM isolates from pulmonary specimens in the year 2008 differed by continent and differed by country within these continents. These differences in species distribution may partly determine the frequency and manifestations of pulmonary NTM disease in each geographical location.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1183/09031936.00149212DOI Listing
December 2013

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil.

Mem Inst Oswaldo Cruz 2012 Dec;107(8):969-77

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil.

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/s0074-02762012000800002DOI Listing
December 2012

Diversity of pulsed-field gel electrophoresis patterns of Mycobacterium abscessus type 2 clinical isolates.

J Clin Microbiol 2011 Jan 17;49(1):62-8. Epub 2010 Nov 17.

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, São Paulo, SP, Brazil.

An epidemic of infections by rapidly growing mycobacteria related to surgical procedures between 2004 and 2008 in Brazil was caused by a unique strain showing the Mycobacterium abscessus type 2 pattern when it was analyzed by the molecular method of PCR-restriction enzyme analysis of the hsp65 gene (PRA-hsp65). In order to investigate the diversity of M. abscessus type 2 clinical isolates and to assess whether this epidemic strain was present in specimens from nonsurgical patients, we studied 52 isolates from 38 patients showing this characteristic PRA-hsp65 pattern obtained between 2005 and 2009. All isolates were identified by sequencing of region V of the rpoB gene and typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. Seven isolates obtained from sputum, bronchoalveolar lavage fluid, and urine in three different Brazilian states showed rpoB sequences 100% similar to the rpoB sequence of epidemic strain INCQS 594 and PFGE patterns highly related to the patterns of isolates, evidencing the presence of the epidemic strain in isolates from patients not associated with the surgical epidemic. The remaining isolates showed diverse rpoB sequences, with the highest similarities being to the corresponding sequences of M. massiliense(T) CIP 108297 (21 isolates), M. bolletii(T) CIP 108541 (19 isolates), or M. abscessus(T) ATCC 19977 (5 isolates). Two additional clusters could be detected by PFGE. PFGE showed 100% typeability and reproducibility and discriminatory powers, calculated by Simpson's index of diversity, of 0.978 (DraI) and 0.986 (AseI), confirming its suitability for the discrimination of M. abscessus type 2 isolates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JCM.01665-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020437PMC
January 2011

Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil.

Future Microbiol 2010 Jun;5(6):971-80

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862 3 degrees Andar, 04023-062 - São Paulo, SP, Brazil.

Aim: Our aim is to investigate if the clusters of postsurgical mycobacterial infections, reported between 2004 and 2008 in seven geographically distant states in Brazil, were caused by a single mycobacterial strain.

Materials & Methods: Available information from 929 surgical patients was obtained from local health authorities. A total of 152 isolates from surgical patients were identified by PCR restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene. Isolates were typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. A total of 15 isolates not related to surgical cases were analyzed for comparison.

Results: All isolates were identified as Mycobacterium abscessus ssp. massiliense. Isolates from surgical patients and one sputum isolate grouped in a single PFGE cluster, composed of two closely related patterns, with one band difference. A total of 14 other isolates unrelated to surgical cases showed distinctive PFGE patterns.

Conclusion: A particular strain of M. abscessus ssp. massiliense was associated with a prolonged epidemic of postsurgical infections in seven Brazilian states, suggesting that this strain may be distributed in Brazilian territory and better adapted to cause surgical-site infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2217/fmb.10.49DOI Listing
June 2010

Cord factor detection and macroscopic evaluation of mycobacterial colonies: an efficient combined screening test for the presumptive identification of Mycobacterium tuberculosis complex on solid media.

J Bras Pneumol 2009 Dec;35(12):1212-6

Instituto Adolfo Lutz, São Paulo, Brazil.

Objective: The rapid differentiation between Mycobacterium tuberculosis and nontuberculous mycobacteria is fundamental for patients co-infected with tuberculosis and HIV. To that end, we use two methods in our laboratory: detection of cord factor and PCR-restriction enzyme analysis (PRA). The objective of this study was to evaluate the accuracy of a screening test on solid medium as a rapid method for the presumptive identification of M. tuberculosis complex, considering costs and turnover time.

Methods: A total of 152 strains were submitted to a combined screening test, consisting of the detection of cord factor under microscopy (Ziehl-Neelsen staining) and evaluation of the macroscopic aspect of colonies, as well as to PRA, which was used as the gold standard. Costs were estimated by calculating the price of all of the materials needed for each test.

Results: The overall accuracy of cord factor detection alone was 95.4% (95% CI: 90.7-98.1%), and that of the combined screening test was 99.3% (95% CI: 96.4-100%). Cord factor detection costs US$ 0.25, whereas the PRA costs US$ 7.00. Results from cord factor detection are ready in 2 days, whereas PRA requires 4 days to yield results.

Conclusions: The presumptive identification of M. tuberculosis using the macroscopic evaluation of colonies combined with cord factor detection under microscopy is a simple, rapid and inexpensive test. We recommend the combined screening test to rapidly identify M. tuberculosis in resource-poor settings and in less well-equipped laboratories while awaiting a definite identification by molecular or biochemical methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/s1806-37132009001200008DOI Listing
December 2009

Nontuberculous mycobacteria isolated in São José do Rio Preto, Brazil between 1996 and 2005.

J Bras Pneumol 2008 Nov;34(11):950-5

Instituto Adolfo Lutz, São José do Rio Preto, SP, Brasil.

Objective: To study the incidence of nontuberculous mycobacteria and the range of species isolated between 1996 and 2005 at a regional branch of the Adolfo Lutz Institute-located in the city of São José do Rio Preto, Brazil-and to show the importance of laboratory testing.

Methods: Mycobacteria were isolated from pulmonary and extrapulmonary specimens and identified through phenotyping and molecular methods (polymerase chain reaction-restriction enzyme analysis).

Results: We isolated 317 nontuberculous mycobacterium strains: Mycobacterium avium complex, 182 (57.4%); M. gordonae, 33 (10.4%); M. fortuitum, 25 (7.9%); M. chelonae, 8 (2.5%); M. terrae complex, 8(2.5%); M.kansasii, 7 (2.2%); and less frequent species, 54 (17%). During this period, 72 cases (33.3%) were characterized as mycobacteriosis, according to bacteriological criteria established by the American Thoracic Society in 2007. Of those 72 cases, 56 were attributed to M.avium complex. Of those 56, 29 (51.8%) were characterized as disseminated disease. Six cases were attributed to M. fortuitum, 3 to M. gordonae, 2 to M. chelonae, 1 to M. abscessus, 1 to M. kansasii, 1 to M. intracellulare, 1 to M. malmoense and 1 to Mycobacterium ssp.

Conclusions: These results show the importance of the bacteriological diagnosis, since identification of the species enables early and appropriate treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/s1806-37132008001100010DOI Listing
November 2008

Descriptive study of the frequency of nontuberculous mycobacteria in the Baixada Santista region of the state of São Paulo, Brazil.

J Bras Pneumol 2008 Aug;34(8):590-4

Adolfo Lutz Institute, Santos Regional Laboratory, Santos, Brazil.

Objective: The present study aims at describing the frequency of nontuberculous mycobacteria (NTM) species identified through laboratory testing of samples collected from non-sterile sites (sputum), as well as its frequency in HIV-infected and non-HIV-infected individuals in the Baixada Santista region of the state of São Paulo, Brazil, in the period from 2000 to 2005.

Methods: Retrospective analysis of sputum smear microscopy results and culture was conducted based on the records on file at the Instituto Adolfo Lutz-Santos, the regional tuberculosis laboratory.

Results: We analyzed 194 NTM strains isolated from 125 individuals, of whom 73 (58.4%) were HIV-negative and 52 (41.6%) were HIV-positive. Thirteen different species were identified: Mycobacterium kansasii; M. avium complex; M. fortuitum; M. peregrinum; M. gordonae; M. terrae; M. nonchromogenicum; M. intracellulare; M. flavescens; M. bohemicum; M. chelonae; M. shimoidei; and M. lentiflavum. In 19.2% of the cases, the bacteriological diagnosis was confirmed by isolation of the same species in at least two consecutive samples.

Conclusions: Our results show the importance of including systematic identification of NTM in the laboratory routine, and that its integration into the clinical routine could improve the characterization of the disease, thereby informing the planning of effective control measures in specific populations, such as individuals presenting tuberculosis/HIV co-infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/s1806-37132008000800008DOI Listing
August 2008
-->