RESUMO
SETTING: The prairie provinces of Canada. OBJECTIVE: To characterize tuberculosis (TB) transmission among the Indigenous and non-Indigenous Canadian-born peoples of the prairie provinces of Canada. DESIGN: A prospective epidemiologic study of consecutively diagnosed adult (age ≥ 14 years) Canadian-born culture-positive pulmonary TB cases on the prairies, hereafter termed "potential transmitters," and the transmission events generated by them. "Transmission events" included new positive tuberculin skin tests (TSTs), TST conversions, and secondary cases among contacts. RESULTS: In the years 2007 and 2008, 222 potential transmitters were diagnosed on the prairies. Of these, the vast majority (198; 89.2%) were Indigenous peoples who resided in either an Indigenous community (135; 68.2%) or a major metropolitan area (44; 22.2%). Over the 4.5-year period between July 1st, 2006 and December 31st 2010, 1085 transmission events occurred in connection with these potential transmitters. Most of these transmission events were attributable to potential transmitters who identified as Indigenous (94.5%). With a few notable exceptions most transmitters and their infected contacts resided in the same community type. In multivariate models positive smear status and a higher number of close contacts were associated with increased transmission; adjusted odds ratios (ORs) and 95% confidence intervals (CIs), 4.30 [1.88, 9.84] and 2.88 [1.31, 6.34], respectively. Among infected contacts, being Indigenous was associated with disease progression; OR and 95% CI, 3.59 [1.27, 10.14] and 6.89 [2.04, 23.25] depending upon Indigenous group, while being an infected casual contact was less likely than being a close contact to be associated with disease progression, 0.66 [0.44, 1.00]. CONCLUSION: In the prairie provinces of Canada and among Canadian-born persons, Indigenous peoples account for the vast majority of cases with the potential to transmit as well as the vast majority of infected contacts. Active case finding and preventative therapy measures need to focus on high-incidence Indigenous communities.
Assuntos
Tuberculose/transmissão , Adolescente , Adulto , Canadá/epidemiologia , Feminino , Humanos , Masculino , Estudos Prospectivos , Tuberculose/epidemiologia , Adulto JovemRESUMO
Mutations in the erm(41) gene of M.abscessus group organisms are associated with differences in inducible macrolide resistance, with current recommendations being to hold rapidly growing isolates for up to 14 days in order to ensure that resistance which develops more slowly can be detected. This study aimed to determine the ideal incubation time for accurate identification of inducible macrolide resistance as well as to determine if there was an association between the time taken to detect inducible resistance in M.abscessus group organisms and their erm(41) sequevar. We amplified and sequenced the erm(41) genes of a total of 104 M.abscessus group isolates and determined their sequevars. The isolates were tested for phenotypic clarithromycin resistance at days 7, 10, 14 and 21, using Trek Diagnostics Sensititre RAPMYCO microbroth dilution plates. Associations between erm(41) gene sequevars and time to detection of resistance were evaluated using Fisher's exact test in R. The samples included in this study fell into 14 sequevars, with the majority of samples falling into Sequevar02 (16), Sequevar06 (15), Sequevar08 (7) and Sequvar 15 (31), and several isolates that were in small clusters, or unique. The majority (82.7%) of samples exhibiting inducible macrolide resistance were interpreted as resistant by day 7. Two isolates in Sequevar02, which has a T28C mutation that is associated with sensitivity, showed intermediate resistance at day 14, though the majority (13) were sensitive at day 14. The majority of isolates with inducible macrolide resistance fell into Sequevars 06,08 and 15, none of which contain the T28C mutation. These sequevars were analyzed to determine if there was any correlation between sequevar and time to detection of resistance. None was found. Based on these findings, we recommend the addition of a day 7 read to the CLSI guidelines to improve turn-around-times for these isolates. It is also recommended that erm(41) gene sequencing be added to routine phenotypic testing for the resolution of cases with difficult-to-interpret phenotypic results.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Macrolídeos/farmacologia , Mycobacterium/genética , Claritromicina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Metiltransferases/genética , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Mycobacterium/isolamento & purificação , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Fatores de TempoRESUMO
The advent and widespread application of next-generation sequencing (NGS) technologies to the study of microbial genomes has led to a substantial increase in the number of studies in which whole genome sequencing (WGS) is applied to the analysis of microbial genomic epidemiology. However, microorganisms such as Mycobacterium tuberculosis (MTB) present unique problems for sequencing and downstream analysis based on their unique physiology and the composition of their genomes. In this study, we compare the quality of sequence data generated using the Nextera and TruSeq isolate preparation kits for library construction prior to Illumina sequencing-by-synthesis. Our results confirm that MTB NGS data quality is highly dependent on the purity of the DNA sample submitted for sequencing and its guanine-cytosine content (or GC-content). Our data additionally demonstrate that the choice of library preparation method plays an important role in mitigating downstream sequencing quality issues. Importantly for MTB, the Illumina TruSeq library preparation kit produces more uniform data quality than the Nextera XT method, regardless of the quality of the input DNA. Furthermore, specific genomic sequence motifs are commonly missed by the Nextera XT method, as are regions of especially high GC-content relative to the rest of the MTB genome. As coverage bias is highly undesirable, this study illustrates the importance of appropriate protocol selection when performing NGS studies in order to ensure that sound inferences can be made regarding mycobacterial genomes.
Assuntos
Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mycobacterium tuberculosis , Manejo de Espécimes/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
BACKGROUND: Outcome of Mycobacterium tuberculosis (Mtb) infection is affected by virulence of the infecting strain of Mtb, host environment, co-morbidities, and the genetic composition of the host, specifically the presence or absence of genes involved in immune responses/regulation. It is hypothesized that specific killer immunoglobulin-like receptor (KIR) genes may be associated with Mtb infection and clinical outcome. This cross-sectional study examined the KIR gene frequencies, profiles, and haplotypes of individuals with active tuberculosis, latent tuberculosis infection, compared to TB and HIV negative healthy controls. RESULTS: Analysis of KIR gene frequencies revealed differences among disease status groups, suggesting that enrichment or depletion of specific KIR genes may direct the disease outcome. Mtb infected individuals were more likely to have a centromeric-AA haplotype compared to controls. CONCLUSION: The differences in KIR gene frequencies and haplotypes may result in differential cytokine expression, contributing to different disease outcomes, and suggest a genetic influence on Mtb susceptibility and pathogenesis.
Assuntos
Frequência do Gene , Haplótipos , Interações Hospedeiro-Patógeno/genética , Receptores KIR/genética , Tuberculose/epidemiologia , Tuberculose/genética , Adolescente , Adulto , Idoso , Canadá/epidemiologia , Estudos Transversais , Feminino , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Avaliação de Resultados da Assistência ao Paciente , Tuberculose/imunologia , Tuberculose/microbiologia , Adulto JovemRESUMO
The critical concentration (CC) for ethambutol testing on the Bactec MGIT 960 M. tuberculosis susceptibility testing has been questioned in recent publications. In this study, we correlate susceptibility results from the Bactec 460, MGIT 960 and embB gene sequencing to determine if the Bactec MGIT 960 adequately detects ethambutol resistance. We discovered discrepancies between the methods that highlight a need to re-evaluate ethambutol susceptibility testing recommendations, namely by considering lowering currently recommended CC on the MGIT 960. Further studies on the clinical significance of low-level ethambutol resistance are also required.
Assuntos
Antituberculosos/farmacologia , Etambutol/farmacologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , HumanosRESUMO
Killer immunoglobulin-like receptors (KIR) on natural killer (NK) cells interact with other immune cells to monitor the immune system and combat infectious diseases, such as tuberculosis (TB). The balance of activating and inhibiting KIR interactions helps determine the NK cell response. In order to examine the enrichment or depletion of KIRs as well as to explore the association between TB status and inhibitory/stimulatory KIR haplotypes, we performed KIR genotyping on samples from 93 Canadian First Nations (Dene, Cree, and Ojibwa) individuals from Manitoba with active, latent, or no TB infection, and 75 uninfected Caucasian controls. There were significant differences in KIR genes between Caucasians and First Nations samples and also between the First Nations ethnocultural groups (Dene, Cree, and Ojibwa). When analyzing ethnicity and tuberculosis status in the study population, it appears that the KIR profile and centromeric haplotype are more predictive than the presence or absence of individual genes. Specifically, the decreased presence of haplotype B centromeric genes and increased presence of centromeric-AA haplotypes in First Nations may contribute to an inhibitory immune profile, explaining the high rates of TB in this population.
Assuntos
Haplótipos , Células Matadoras Naturais/imunologia , Tuberculose Latente/etnologia , Tuberculose Latente/genética , Receptores KIR/genética , Tuberculose Pulmonar/etnologia , Tuberculose Pulmonar/genética , Adolescente , Adulto , Idoso , Centrômero , Estudos de Coortes , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Indígenas Norte-Americanos , Inuíte , Células Matadoras Naturais/microbiologia , Tuberculose Latente/imunologia , Tuberculose Latente/microbiologia , Desequilíbrio de Ligação , Masculino , Manitoba/epidemiologia , Pessoa de Meia-Idade , Receptores KIR/classificação , Receptores KIR/imunologia , Telômero , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , População BrancaRESUMO
Macrolides are an important tool in the treatment of Mycobacterium avium complex infections. Here, we evaluate the use of 23S rRNA gene sequencing for the rapid detection of macrolide resistance. Routine sequencing of the 23S rRNA gene is highly specific for macrolide resistance but lacks in sensitivity.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Macrolídeos/farmacologia , Técnicas de Diagnóstico Molecular/métodos , Complexo Mycobacterium avium/efeitos dos fármacos , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Complexo Mycobacterium avium/genética , RNA Ribossômico 23S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodosRESUMO
SETTING: Nationwide Canadian public health surveillance. OBJECTIVE: Description of demographic features and disease characteristics of drug-resistant tuberculosis (TB) in Canada over a 12 year period. DESIGN: Continuous surveillance of all cases of culture-confirmed TB in Canada. Demographic and microbiologic features were analyzed and comparisons between drug-susceptible, multidrug-resistant (MDR), and drug-resistant not-MDR were made. Cases of extensively drug resistant TB are described. RESULTS: 15,993 cases of culture-confirmed TB were reported during the study period. There were 5 cases of XDR-TB, 177 cases of MDR-TB, and 1,234 cases of first-line drug resistance not-MDR. The majority of drug-resistant cases were reported in foreign-born individuals, with drug-resistant cases diagnosed earlier post-arrival in Canada compared to drug-susceptible cases. In MDR-TB isolates, there was a high rate of drug-resistance to other first- and second-line drugs, making reliable empiric therapeutic recommendations for MDR-TB difficult. There was a statistically significant association between both MDR and drug-resistance not-MDR, and the risk of a negative treatment outcome (defined as treatment failure, absconded, or treatment ongoing >3 yrs). CONCLUSION: Drug-resistance complicates TB management even in developed nations with well-established TB control programs. The predominantly international origin of drug-resistant cases highlights the need for global strategies to combat TB.
Assuntos
Antituberculosos/uso terapêutico , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Adolescente , Adulto , Antituberculosos/farmacologia , Canadá/epidemiologia , Criança , Pré-Escolar , Demografia , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Fatores de Tempo , Resultado do Tratamento , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/microbiologia , Organização Mundial da Saúde , Adulto JovemRESUMO
Nontuberculosis mycobacteria (NTM) are an important cause of human disease and infections. Though less notorious than tuberculosis, these infections are clinically significant and have been associated with outbreaks in various settings. To accommodate outbreak investigations for the numerous species of NTM, we evaluated a DiversiLab repetitive-sequence-based PCR (rep-PCR) kit for genotyping of mycobacteria. This kit was used to genotype both rapidly and slowly growing mycobacteria and was compared with other PCR-based genotyping methods, including random amplified polymorphic DNA (RAPD) analysis, hsp65 gene sequencing, and mycobacterial interspersed repetitive unit - variable number of tandem repeat (MIRU-VNTR) analysis. Compared with RAPD analysis, rep-PCR achieved better reproducibility in testing. When compared with hsp65 gene sequencing and MIRU-VNTR for Mycobacterium avium , rep-PCR provided results that agreed with these less discriminatory genotyping methods but provided a higher level of discrimination for situations such as outbreak investigations. We also evaluated the kit for its ability to identify closely related rapidly growing NTM. While rep-PCR was informative in some cases, a much larger library of isolates would be necessary to truly evaluate it as an identification tool. Overall, rep-PCR was able to provide improved reproducibility over RAPD and a discriminatory genotyping method for the isolates evaluated in this study.
Assuntos
Técnicas de Tipagem Bacteriana/normas , Micobactérias não Tuberculosas/genética , Reação em Cadeia da Polimerase , Técnica de Amplificação ao Acaso de DNA Polimórfico , Análise por Conglomerados , Genótipo , Micobactérias não Tuberculosas/classificação , Reprodutibilidade dos TestesRESUMO
The purpose of this study was to establish a standardized protocol for second-line antimicrobial susceptibility testing of Mycobacterium tuberculosis using the Bactec MGIT 960 system in Canadian laboratories. Four Canadian public health laboratories compared the susceptibility testing results of 9 second-line antimicrobials between the Bactec 460 and Bactec MGIT 960 systems. Based on the data generated, we have established that the Bactec MGIT 960 system provides results comparable to those obtained with the previous Bactec 460 method. The critical concentrations established for the testing of the antimicrobials used are as follows: amikacin, 1 µg/ml; capreomycin, 2.5 µg/ml; ethionamide, 5 µg/ml; kanamycin, 2.5 µg/ml; linezolid, 1 µg/ml; moxifloxacin, 0.25 µg/ml; ofloxacin, 2 µg/ml; p-aminosalicylic acid, 4 µg/ml; rifabutin, 0.5 µg/ml.
Assuntos
Antituberculosos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Mycobacterium tuberculosis/efeitos dos fármacos , Canadá , HumanosRESUMO
Rifampin is a key component of standard short-course first-line therapy against Mycobacterium tuberculosis (MTB). Rifampin monoresistant MTB, previously a rare phenomenon, is now being reported at increasing rates worldwide. We report a mutation in the rpoB region leading to low level rifampin monoresistance in a cluster of HIV-positive patients. All rifampin monoresistant isolates identified from 2004 to 2006 underwent susceptibility confirmation, sequencing of rpoB and genotyping. Three patients were found to have a previously undocumented 3 base pair insertion at codon 525 in the rpoB region. The earliest initial case was infected with fully susceptible MTB. Disease relapse occurred 7 months later with a genotypically identical MTB isolate, showing acquired rifampin monoresistance. MTB isolates from 2 subsequent patients showed primary rifampin monoresistance with an identical genotype to the index case. Patients with rifampin monoresistant MTB tend to have poorer outcomes than those with fully susceptible strains. Risk factors for the development of rifampin monoresistance include co-morbid HIV infection and previously treated tuberculosis. HIV infection has been associated with malabsorption of anti-tuberculous medications leading to sub-therapeutic levels of administered drugs. These factors may have played a role in the development of this previously undocumented mutation.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Infecções por HIV/genética , Mutação , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Tuberculose Pulmonar/genética , Adulto , RNA Polimerases Dirigidas por DNA , Feminino , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência de DNA , Resultado do Tratamento , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/tratamento farmacológicoRESUMO
The current gold standard for Mycobacterium tuberculosis complex (MTBC) genotyping is insertion sequence (IS) 6110 restriction fragment length polymorphism (RFLP) as it provides the highest discriminatory power of all available MTBC genotyping methods. However, RFLP is labour intensive and the interpretation of data from this method can be susceptible to errors. In 2001 a rapid, reproducible variable number of tandem repeat (VNTR) based typing method using 12 mycobacterial interspersed repetitive units (MIRU) was developed. Despite this advancement, this method lacked the discriminatory power of IS6110-RFLP. More recently a set of 24 MIRU-VNTR loci was reported to have greater discriminatory power than the original 12 locus system and may exceed that of RFLP when combined with spoligotyping. We compared the 24 locus method to the 12 locus method in order to improve surveillance of tuberculosis in Canada. A random sample of 650 MTBC isolates from British Columbia, Saskatchewan, Manitoba and Quebec Canada was genotyped using the 24 MIRU loci. Comparison of the data for the 12 and 24 MIRU loci showed an increase of the Hunter-Gaston discriminatory index (HGDI) from 0.895 (12 loci) to 0.920 (24 loci). The implementation of the 24 locus MIRU-VNTR methods offers improvement in discriminatory power over the traditional 12 locus method. For long-term surveillance of MTBC within Canada, the use of 24 MIRU-VNTR loci will provide rapid, highly discriminatory molecular epidemiology information.
Assuntos
Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Repetições Minissatélites , Mycobacterium tuberculosis/genética , Tuberculose/genética , Canadá/epidemiologia , Genótipo , Humanos , Epidemiologia Molecular , Mycobacterium tuberculosis/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Reprodutibilidade dos Testes , Tuberculose/epidemiologiaRESUMO
During susceptibility testing of 743 isolates of Mycobacterium tuberculosis to pyrazinamide (PZA) using the Bactec 960 system, 57 (7.7%) isolates showed PZA resistance. Repeat testing of resistant isolates with the Bactec 460 reference method confirmed 33 (4.4%) of these isolates as resistant, and 24 (3.2%) were susceptible. Erroneous results for resistance with the Bactec 960 were confirmed by testing the 24 discordant isolates for pyrazinamidase and mutations in the pncA gene.
Assuntos
Antituberculosos/farmacologia , Erros de Diagnóstico , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , HumanosRESUMO
We present a report of a diabetic patient with an infection of his left thumb and thenar eminence. Standard cultures of drainage and tissue biopsy were unrevealing. The infection progressed despite empiric antibacterial agent therapy and multiple debridements. Two intraoperative tissue biopsies revealed a yellow-pigmented, rapidly growing Mycobacterial nontuberculous species. The organism was identified as Mycobacterium monacense, a newly described species. The patient was cured with a 6-week course of clarithromycin and levofloxacin.
Assuntos
Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas , Dermatopatias Bacterianas/diagnóstico , Dermatopatias Bacterianas/microbiologia , Mãos , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Mycobacterium não Tuberculosas/terapia , Dermatopatias Bacterianas/terapiaAssuntos
Bacteriemia/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/classificação , DNA Espaçador Ribossômico/genética , Humanos , Malaui , Micobactérias não Tuberculosas/genética , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Especificidade da Espécie , Tanzânia , Tailândia , Estados UnidosRESUMO
A case of presumed bacillus Calmette-Guérin (BCG) cystitis in an elderly female patient following direct intravesical BCG instillation treatment for papillary transitional cell carcinoma is reported. The organism cultured from urine samples was eventually identified as a rifampin-resistant Mycobacterium bovis BCG isolate. Because the patient had received rifampin monotherapy during the course of treatment for presumed BCG disease, the clinical picture favoured acquired rifampin resistance. Sequencing of the target gene for rifampin (rpoB) confirmed a known mutation responsible for conferring high levels of resistance to both rifampin and rifabutin (Ser531Tyr). To the authors' knowledge, this is the first reported case of M bovis BCG disease in a non-HIV patient where the organism had acquired drug resistance to rifampin, and the second reported case of M bovis BCG that had acquired drug resistance. The present case demonstrates the necessity to re-evaluate appropriate guidelines for the effective treatment of BCG disease.
RESUMO
Mycobacterium peregrinum consists of two taxa: types I and II. We evaluated 43 clinical type II strains from throughout the United States. They were responsible for soft-tissue and bone infections, catheter-related infections, and possible pneumonitis. By carbohydrate utilization, they were indistinguishable from type I strains, being D-mannitol and trehalose positive. However, they had a distinct susceptibility pattern that included intermediate ciprofloxacin MICs but low clarithromycin and doxycycline MICs of < or =1 microg/ml. These features were also shared by reference isolates of Mycobacterium senegalense from African bovine cases of "farcy." By 16S rRNA gene sequencing, the type II isolates shared 100% sequence identity with M. senegalense. Partial sequencing of the type II hsp65 gene (441 bp) revealed four sequevars showing > or =98.4% identity with each other and > or =98.6% identity with the sequence of five bovine strains of M. senegalense. There was < or =97.1% identity with M. peregrinum type I isolates and other Mycobacterium fortuitum group species. Sequencing of additional gene targets including the 16S-23S rDNA internal transcribed spacer region and the rpoB gene (partial sequence) revealed a similar phylogenetic grouping. DNA-DNA hybridization showed 76 to 99% relatedness between the bovine and human strains. These studies demonstrate that type II isolates are not isolates of M. peregrinum but represent human strains of M. senegalense. This study is the first to demonstrate this species as a human pathogen. Representative human M. senegalense strains include ATCC 35755 and newly submitted strains ATCC BAA-849, ATCC BAA-850, and ATCC BAA-851.
Assuntos
Técnicas de Tipagem Bacteriana , Doenças dos Bovinos/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/genética , Animais , Bovinos , DNA Bacteriano/análise , DNA Espaçador Ribossômico/análise , Genes de RNAr , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Micobactérias não Tuberculosas/metabolismo , Micobactérias não Tuberculosas/patogenicidade , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNARESUMO
BACKGROUND: In the field of clinical mycobacteriology, Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating/chemical fixation) may not consistently kill MTB organisms. METHODS: An inclusive viability study (n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks. RESULTS: Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80 degrees C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again (n = 18), resulting in 0% viability. CONCLUSIONS: This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures.
Assuntos
Técnicas de Laboratório Clínico/normas , Contenção de Riscos Biológicos/normas , Viabilidade Microbiana , Mycobacterium tuberculosis , Contenção de Riscos Biológicos/métodos , Impressões Digitais de DNA , DNA Bacteriano/isolamento & purificação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Saúde Ocupacional , Polimorfismo de Fragmento de Restrição , Medição de RiscoRESUMO
Continuous monitoring systems for the detection of Mycobacterium tuberculosis are reported to have higher contamination rates than traditional radiometric technologies. Multiple decontamination methods have recently been reported in an attempt to optimize contamination rates for these systems. In this study, several decontamination methods for sputum were evaluated using viable colony count and flow cytometry. The decontamination protocols evaluated include N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH), modified Petroffs's method, and the Yamane procedure. Several parameters of the NALC-NaOH method were analyzed including final NaOH concentrations of 0.5-3%, NaOH exposure times of 0-30 min, and variations in resuspension media for the resultant pellet. All decontamination methods were performed on pooled and sterilized sputum seeded separately with either a mixture of common contaminating bacteria or M. tuberculosis H37Ra. Viability of organisms following decontamination was assessed by both colony counts and flow cytometric analysis. Flow cytometry viability assays utilized a combination of viability dyes and reference beads to determine viable organism concentrations. The results indicated that no decontamination method was clearly superior, however a concentration of 1-2% NaOH and an increase in the time of NaOH exposure to 30 min will effectively kill contaminating bacteria without significantly affecting the viability of M. tuberculosis H37Ra. While flow cytometry viability analysis did not directly correspond to viable colony counts, it was a useful tool for rapid viability analysis M. tuberculosis.
Assuntos
Citometria de Fluxo/métodos , Mycobacterium tuberculosis/isolamento & purificação , Hidróxido de Sódio/farmacologia , Escarro/microbiologia , Análise de Variância , Técnicas Bacteriológicas , Contagem de Colônia Microbiana , Humanos , Probabilidade , Sensibilidade e Especificidade , Manejo de Espécimes , Tuberculose Pulmonar/diagnósticoRESUMO
Sequencing of the 16S ribosomal DNA (rDNA) for identification of nontuberculous mycobacteria (NTM) has contributed to the establishment of more than 35 new species during the last decade. Increasingly, NTM are accepted as potential or proven pathogens. We identified, by 16S rDNA sequence analysis, slowly growing NTM isolates negative by AccuProbe (GenProbe, San Diego, CA) that previously were identified by using conventional biochemical techniques, to determine the accuracy of reporting AccuProbe-negative NTM prior to sequence-based identification. Of 82 strains, 30 were deemed novel. An attempt was made to determine the clinical importance of previously misidentified novel species. Clinical cases are described for a number of strains previously identified as Mycobacterium terrae complex, Mycobacterium scrofulaceum, and Mycobacterium avium complex. As sequence-based identification methods become more commonplace in clinical microbiology laboratories, there is a need to understand the significance of previously undescribed species, which often mimic and subsequently are identified as well-established species.