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1.
Ann Lab Med ; 40(2): 169-173, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31650734

RESUMO

The GENEDIA MTB/NTM Detection Kit (GENEDIA MTB/NTM; Green Cross Medical Science Corp., Chungbuk, Korea) is a multiplex real-time PCR assay used for differential identification of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). While the importance of differential identification of MTB/NTM is recognized, there is limited data on the performance of GENEDIA MTB/NTM assay to date. A total of 687 consecutive sputum specimens were cultured and analyzed with the GENEDIA MTB/NTM and GENEDIA MTB assays. Nineteen specimens (2.8%) were MTBC-positive, and 69 (10.0%) were NTM-positive based on mycobacterial culture. All specimens showed concordant results for MTBC using both assays, with a kappa value of 1.00, overall sensitivity of 63.2% (12/19), and specificity of 100% (668/668). The overall NTM sensitivity and specificity were 23.2% (16/69) and 99.7% (616/618) for GENEDIA MTB/NTM. The association between NTM-positivity using GENEDIA MTB/NTM and the diagnosis of NTM pulmonary disease was not statistically significant. In conclusion, the two real-time PCR assays showed similar diagnostic performance for MTBC detection. However, the sensitivity for NTM detection was lower than that for MTBC detection.


Assuntos
Infecções por Micobactéria não Tuberculosa/diagnóstico , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/genética , Escarro/microbiologia , Tuberculose/diagnóstico , DNA Bacteriano/análise , Humanos , Reação em Cadeia da Polimerase Multiplex , Infecções por Micobactéria não Tuberculosa/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Tuberculose/microbiologia
2.
BMC Genomics ; 20(1): 793, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666009

RESUMO

BACKGROUND: Nontuberculous mycobacteria (NTM) are a major cause of pulmonary and systemic disease in at-risk populations. Gaps in knowledge about transmission patterns, evolution, and pathogenicity during infection have prompted a recent surge in genomic NTM research. Increased availability and affordability of whole genome sequencing (WGS) techniques provide new opportunities to sequence and construct complete bacterial genomes faster and at a lower cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to its slow growth and recalcitrant cell wall. Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA with no additional clean-up steps. RESULTS: Our DNA extraction method was compared to 6 other methods with variations in timing of mechanical disruption and enzymatic digestion of the cell wall, quantity of matrix material, and reagents used in extraction and precipitation. We tested our optimized method on 38 clinical isolates from the M. avium and M. abscessus complexes, which yielded optimal quality and quantity measurements for Oxford Nanopore Technologies sequencing. We also present the efficient completion of circularized M. avium subspecies hominissuis genomes using our extraction technique and the long-read sequencing MinION platform, including the identification of a novel plasmid. CONCLUSIONS: Our optimized extraction protocol and assembly pipeline was both sufficient and efficient for genome closure. We expect that our finely-tuned extraction method will prove to be a valuable tool in long-read sequencing and completion of mycobacterial genomes going forward. Utilization of comprehensive, long-read based approaches will advance the understanding evolution and pathogenicity of NTM infections.


Assuntos
DNA Bacteriano/isolamento & purificação , Genoma Bacteriano , Micobactérias não Tuberculosas/genética , Sequenciamento Completo do Genoma/métodos
3.
BMC Infect Dis ; 19(1): 819, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533664

RESUMO

BACKGROUND: Reports on the worldwide ascending trend of pulmonary nontuberculous mycobacteria (NTM) isolation rates and their effective role in respiratory tract infections are compelling. However, as yet, there are no such data relating to Tunisia. METHODS: Here we carried out a retrospective review of mycobacterial cultures originating from Northern Tunisia, which have been processed in the laboratory of mycobacteria of the Institut Pasteur de Tunis, during the time period 2002-2016. All pulmonary NTM (PNTM) isolates available for culture were characterized phenotypically and their taxonomic status was further established based on polymorphisms in rpoB, 16S rRNA, hsp65, and sodA DNA gene sequences. RESULTS: Of the 10,466 specimens collected from HIV-negative Tunisian patients with presumptive clinical pulmonary TB, 60 (0.6%) yielded PNTM isolates. An overall annual PNTM isolation prevalence of 0.2/100,000 was estimated. As far as could be ascertained, this isolation rate accounts amongst the lowest reported hitherto throughout the world. Among the 30 NTM isolates that were available for culture, 27 (90.0%) have been identified to the species level. The most commonly encountered species was Mycobacterium kansasii (23.3%) subtype 1. Strikingly, all M. kansasii cases were male patients originating from Bizerte, an industrialized region particularly known for iron industry. The remaining NTM species were M. fortuitum (16.6%), M. novocastrense (16.6%), M. chelonae (10.0%), M. gordonae (6.6%), M. gadium (6.6%), M. peregrinum (3.3%), M. porcinum (3.3%), and M. flavescens (3.3%). There were no bacteria of the M. avium complex, the most frequently isolated NTM globally, and the main driver of the rise of NTM-lung diseases. CONCLUSIONS: This study uncovered an exceptional low prevalence of PNTM isolation among HIV-negative TB suspects in Northern Tunisia, suggesting a very low burden of NTM pulmonary disease. However, the frequent isolation of M. kansasii subtype 1, the most pathogenic subtype, particularly from the industrialized region of Bizerte, strongly suggests its effective involvement in a typical pulmonary disease.


Assuntos
Infecções por Micobactéria não Tuberculosa/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Tuberculose Pulmonar/diagnóstico , Adulto , Líquido da Lavagem Broncoalveolar/microbiologia , Feminino , Humanos , Masculino , Infecções por Micobactéria não Tuberculosa/epidemiologia , Infecções por Micobactéria não Tuberculosa/microbiologia , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/genética , Filogenia , Prevalência , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Estudos Retrospectivos , Escarro/microbiologia , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/microbiologia , Tunísia/epidemiologia
4.
Ann Agric Environ Med ; 26(3): 511-513, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559813

RESUMO

INTRODUCTION: Mycobacterial diseases of humans and animals can be caused by mycobacteria other than tuberculosis (MOTT). The transmission of the infection primarily occurs via the respiratory or oral routes, but also via a damaged skin barrier. MOTT have high resistance to external factors; therefore, infected, undiagnosed animals can pose a risk for public health. CASE REPORT: The case study describes mycobacterial skin infection in a domestic cat. The correct diagnosis was reached four months after the appearance of the first clinical signs. Those were purulent, granulomatous lesions and fistulas, which could potentially act as a source of the infection for the owners and the veterinarian who cared for the animal. CONCLUSION: Despite using advanced diagnostic techniques, establishing the final cause of the cat's illness was a lengthy process. The skin lesions could contribute to the transmission of the bacteria in the environment. Non-targeted treatments could also cause antimicrobial resistance.


Assuntos
Doenças do Gato/tratamento farmacológico , Doenças do Gato/microbiologia , Infecções por Micobactéria não Tuberculosa/veterinária , Dermatopatias Bacterianas/veterinária , Animais , Antituberculosos/administração & dosagem , Doenças do Gato/diagnóstico , Gatos , Masculino , Infecções por Micobactéria não Tuberculosa/diagnóstico , Infecções por Micobactéria não Tuberculosa/tratamento farmacológico , Infecções por Micobactéria não Tuberculosa/microbiologia , Micobactérias não Tuberculosas/efeitos dos fármacos , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/isolamento & purificação , Dermatopatias Bacterianas/diagnóstico , Dermatopatias Bacterianas/tratamento farmacológico , Dermatopatias Bacterianas/microbiologia
5.
J Dermatol ; 46(10): 917-921, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392741

RESUMO

While the etiology of sarcoidosis remains uncertain, mycobacteria have been suggested as a causative infectious agent. To investigate the causal relationship between mycobacteria and sarcoidosis, we performed a reverse blot hybridization assay (REBA) to identify mycobacteria from the skin samples of nine patients with sarcoidosis. Six of the nine samples were shown to be positive for mycobacteria by REBA, including Mycobacterium tuberculosis and non-tuberculous mycobacteria. This is the first study to identify mycobacteria from the skin samples of sarcoidosis patients using REBA, and our results could strengthen the etiologic association between mycobacteria and sarcoidosis.


Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Sarcoidose/microbiologia , Dermatopatias/microbiologia , Pele/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Sondas de DNA , DNA Bacteriano/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Sonda Molecular , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/genética , Reação em Cadeia da Polimerase , Sarcoidose/patologia , Pele/patologia , Dermatopatias/patologia
6.
Emerg Microbes Infect ; 8(1): 1043-1053, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31287781

RESUMO

The prevalence of nontuberculous mycobacteria (NTM) pulmonary diseases has been increasing worldwide. NTM consist of approximately 200 species and distinguishing between them at the subspecies level is critical to treatment. In this study, we sequenced 63 NTM genomes, 27 of which were newly determined, by hybrid assembly using sequencers from Illumina and Oxford Nanopore Technologies (ONT). This analysis expanded the available genomic data to 175 NTM species and redefined their subgenus classification. We also developed a novel multi-locus sequence typing (MLST) database based on 184 genes from 7547 assemblies and an identification software, mlstverse, which can also be used for detecting other bacteria given a suitable MLST database. This method showed the highest sensitivity and specificity amongst conventional methods and demonstrated the capacity for rapid detection of NTM, 10 min of sequencing of the ONT MinION being sufficient. Application of this methodology could improve disease epidemiology and increase the cure rates of NTM diseases.


Assuntos
Genoma Bacteriano , Infecções por Micobactéria não Tuberculosa/microbiologia , Micobactérias não Tuberculosas/isolamento & purificação , Genômica , Humanos , Tipagem de Sequências Multilocus , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/genética , Filogenia
7.
Int J Food Microbiol ; 306: 108260, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31302488

RESUMO

The aim of this study was to determine the bacteriological quality of bottled water samples obtained from small purification plants located in Mexico City and to identify potentially pathogenic nontuberculous mycobacteria (NTM) species found in these samples. All 111 samples analyzed were positive for aerobic mesophilic bacteria (AMB) and 46 (41.4%) did not comply with Mexico's Official Guidelines. Sixty-nine (62.1%) and 23 (20.7%) water samples were positive for total coliforms (TC) and fecal coliforms (FC), respectively. A total of 81 (72.9%) of the water samples exceeded the maximum allowed limit stipulated in the guideline. Thirty-three (29.7%) of the purified water samples were positive for NTM, being recovered a total of 40 isolates. These NTM isolates were identified using three molecular markers (hsp65, rrs and rpoB genes) which corresponded to the fast-growing mycobacteria M. chelonae (n = 12), M. porcinum (n = 8), M. senegalense (n = 5), M. abscessus (n = 4), M. septicum (n = 4), M. wolinskyi (n = 3), M. mucogenicum (n = 2), M. fortuitum (n = 1) and M. sp. (n = 1). In seven purified water samples, two different NTM species were isolated simultaneously. Overall, these results showed that most of the purified bottled water samples analyzed in this study had unsatisfactory microbiological quality and some harbored NTM associated with illness. Our data could hasten health authorities to intensify efforts in the routine monitoring of activities in the purified bottled water industry in order to supply safe and healthy water to the public.


Assuntos
Água Potável/microbiologia , Enterobacteriaceae/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Purificação da Água , Qualidade da Água , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Humanos , Incidência , México , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/genética
8.
Eur J Clin Microbiol Infect Dis ; 38(10): 1873-1881, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31313101

RESUMO

The diagnosis of mycobacterial infections has been dramatically improved by the introduction of molecular methods aimed to reduce the time to diagnosis as compared with culture. The broad range pan-mycobacterial PCR can detect all the mycobacterial species directly from clinical specimens. We aimed to evaluate its usefulness and its clinical added value for the diagnosis of nontuberculous mycobacterial (NTM) infections. We performed a retrospective study (2003-2013) including 952 samples taken from 639 patients with clinical suspicion of NTM infection. The performance of smear microscopy, PCR and culture was established using clinical data to investigate discrepant results. We also compared the time to microbial diagnosis between the direct PCR and culture. The sensitivity, specificity, positive and negative predictive values of the PCR were 61.6% (53.5-69.1), 99.1% (98.2-99.6), 92.8% (85.8-96.5) and 93.4% (91.6-94.9), respectively, when considering all specimens. When considering smear-positive specimens and smear-negative specimens, the sensitivity was 81.6% and 40%, respectively. The sensitivity for pulmonary and extra-pulmonary smear-positive specimens was 85.2% versus 72.7%. The median time to identification at species level was 35 days (SD, 17.67) for culture and 6 days (SD, 2.67) for the PCR (when positive), which represents a 29-day shorter time to results (p < 0.0001). The 16S rRNA gene pan-mycobacterial PCR displays a substantial benefit in terms of time to diagnose NTM infections when compared with culture. Despite an excellent specificity, its sensitivity is yet limited in particular for smear-negative specimens, which might be improved by relying onto real-time PCRs.


Assuntos
Genes de RNAr , Técnicas de Diagnóstico Molecular/métodos , Infecções por Micobactéria não Tuberculosa/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Humanos , Microscopia/métodos , Micobactérias não Tuberculosas/genética , Valor Preditivo dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Fatores de Tempo
10.
Emerg Infect Dis ; 25(5): 849-855, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31002056

RESUMO

We investigated a cluster of Mycobacterium fortuitum and M. goodii prosthetic joint surgical site infections occurring during 2010-2014. Cases were defined as culture-positive nontuberculous mycobacteria surgical site infections that had occurred within 1 year of joint replacement surgery performed on or after October 1, 2010. We identified 9 cases by case finding, chart review, interviews, surgical observations, matched case-control study, pulsed-field gel electrophoresis of isolates, and environmental investigation; 6 cases were diagnosed >90 days after surgery. Cases were associated with a surgical instrument vendor representative being in the operating room during surgery; other potential sources were ruled out. A tenth case occurred during 2016. This cluster of infections associated with a vendor reinforces that all personnel entering the operating suite should follow infection control guidelines; samples for mycobacterial culture should be collected early; and postoperative surveillance for <90 days can miss surgical site infections caused by slow-growing organisms requiring specialized cultures, like mycobacteria.


Assuntos
Artrite Infecciosa/epidemiologia , Artrite Infecciosa/microbiologia , Prótese Articular/efeitos adversos , Infecções por Micobactéria não Tuberculosa/epidemiologia , Infecções por Micobactéria não Tuberculosa/microbiologia , Micobactérias não Tuberculosas , Infecções Relacionadas à Prótese/epidemiologia , Infecções Relacionadas à Prótese/microbiologia , Idoso , Artrite Infecciosa/diagnóstico , Artrite Infecciosa/história , Estudos de Casos e Controles , Infecção Hospitalar , Surtos de Doenças , Microbiologia Ambiental , Feminino , História do Século XXI , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Infecções por Micobactéria não Tuberculosa/diagnóstico , Infecções por Micobactéria não Tuberculosa/história , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/isolamento & purificação , Oregon/epidemiologia , Infecções Relacionadas à Prótese/diagnóstico , Infecções Relacionadas à Prótese/história , Infecção da Ferida Cirúrgica
11.
J Water Health ; 17(2): 350-356, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30942784

RESUMO

Microbiological control of hospital waters as one of the main sources of nontuberculous mycobacteria (NTM) is important for the prevention of NTM-associated illness. This study aimed to investigate the prevalence of NTM in the hospital water systems of Tehran, Iran. A total of 218 samples from different hospital waters (i.e., tap water and medical devices such as humidifying cup of oxygen manometer, dialysis devices, nebulizers, and dental units) were included in this study. Phenotypic and molecular tests were used to identify the isolated organisms to species level. Of 218, 85 (39.0%) samples at 37 °C and 87 (40.0%) samples at 25 °C were identified as NTM. Using hsp65-sequencing method, Mycobacterium lentiflavum was the most frequently encountered, followed by M. gordonae and M. paragordonae. No significant difference was seen in frequency and species in mycobacteria isolated at 37 °C and 25 °C temperatures. Humidifying cup of oxygen manometer had the most contaminated water among the investigated water distribution systems in hospitals. Isolation of NTM from hospital water sources is a serious public health problem in Iran and merits further attention by health authorities. Establishment of microbiological monitoring systems for hospital waters and expanding the number of facilitated laboratories are strongly recommended.


Assuntos
Micobactérias não Tuberculosas/genética , Microbiologia da Água , Hospitais , Humanos , Irã (Geográfico)/epidemiologia , Infecções por Micobactéria não Tuberculosa/epidemiologia , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/isolamento & purificação
12.
Vet Dermatol ; 30(3): 262-e80, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30883992

RESUMO

BACKGROUND: Cutaneous disseminated mycobacteriosis is rare in dogs. To the best of the authors' knowledge, the slowly growing mycobacterial species Mycobacterium nebraskense has not been described before in this species. OBJECTIVE: Description of clinical features, laboratory analyses and treatment regimen of this unusual case. ANIMAL: A 9-year-old female-spayed West Highland white terrier dog presented with progressive nodules and ulcerations on both sides of the thorax and the rostral aspect of the chest. METHODS AND MATERIALS: Investigations involved histopathological examination of skin biopsies (including special stains for fungi, bacteria and mycobacteria), standard and mycobacterial culture (including susceptibility testing), 16S/23S rRNA sequencing and BLAST similarity searching. RESULTS: Ziehl-Neelsen staining of decontaminated biopsy material revealed acid-fast bacteria morphologically consistent with mycobacteria. Treatment with clarithromycin and marbofloxacin achieved partial resolution. A change in the treatment regimen to pradofloxacin and azithromycin resulted in rapid deterioration of skin lesions. Final healing occurred with the addition of prednisolone at an anti-inflammatory dose. The results of mycobacterial culture and susceptibility testing were received 10 and 12 months, respectively, after the first presentation of the dog. Therapy was stopped after 16 months without recurrence of skin lesions. CONCLUSIONS AND CLINICAL IMPORTANCE: This case is noteworthy for the description of a new mycobacterial species contributing to disseminated panniculitis in a dog and for the difficulties experienced in the lengthy empirical treatment of slowly growing nontuberculous mycobacterial infections. The addition of prednisolone to induce complete healing raises the question of whether the mycobacterial infection was primary or whether it occurred secondarily to an ongoing sterile panniculitis.


Assuntos
Infecções por Micobactéria não Tuberculosa/veterinária , Dermatopatias Bacterianas/veterinária , Pele/patologia , Animais , Antibacterianos/uso terapêutico , Cães , Feminino , Fluoroquinolonas/uso terapêutico , Infecções por Micobactéria não Tuberculosa/diagnóstico , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/isolamento & purificação , RNA Ribossômico 16S/genética , Pele/microbiologia , Dermatopatias Bacterianas/sangue , Suíça
13.
Tuberculosis (Edinb) ; 114: 17-23, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711153

RESUMO

The purpose of the present study was to create a real-time PCR test system allowing simultaneous detection of nontuberculous mycobacteria (NTM) and Mycobacterium tuberculosis complex (MTBC) both in culture and sputum. NTM cultures (18 strains, 18 species), MTBC cultures (16 strains, 2 species) and non-mycobacterial microorganisms from the collection of the Central Research TB Institute (CTRI) were used for the preliminary evaluation of the test system. 301 NTM cultures from patients with mycobacteriosis were used to assess the sensitivity of the developed test system. Clinical respiratory samples (sputum) from 104 patients with mycobacteriosis, 3627 patients with tuberculosis and 118 patients with other lung diseases were used for diagnostic sensitivity and specificity testing. The specificity and sensitivity of the assay for MTBC was found to be 100% both in culture and sputum samples; for NTM, the specificity was 100% in culture and sputum, the sensitivity reached 100% in culture and 73.1% in sputum samples. Positive predictive value (PPV) and negative predictive value (NPV) of the assay for culture were both 100%, for clinical material 100% and 80.8%, respectively. The limit of detection at the probability of detection 95% (LoD95%) was estimated to be 16 cfu/ml for M. tuberculosis H37RV and 1200 cfu/ml for M. avium.


Assuntos
Infecções por Micobactéria não Tuberculosa/diagnóstico , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Tuberculose/diagnóstico , Técnicas de Tipagem Bacteriana/métodos , DNA Bacteriano/isolamento & purificação , Diagnóstico Diferencial , Humanos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/genética , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Escarro/microbiologia
15.
Lett Appl Microbiol ; 68(3): 219-225, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30636048

RESUMO

Quantitated Mycobacterium tuberculosis (M.tb) H37Rv DNA was used to analyse the sensitivity and the specificity was assessed using DNA isolated from the reference strain H37Rv, 12 nontuberculous mycobacterium (NTM) species and five nonmycobacterium species. Furthermore, performance of the assay was evaluated on the sputum samples and compared with smear microscopy, culture and PCR. mpt64 (also called mpb64 or Rv1980c) loop-mediated isothermal amplification (LAMP) successfully detected 1 pg DNA within 40 min and successfully rejected NTMs and other bacterial species tested. It specifically detected all the 119 confirmed TB cases and 100 of the 104 control cases. The resulting sensitivity and specificity of LAMP assay was found to be 100% (95% CI: 96·79-100%) and 96·15% (95% CI; 90·44-98·94%) respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Loop-mediated isothermal amplification (LAMP) is a technique for isothermal DNA amplification suitable for cost-limited settings as it prevents the use of sophisticated instruments. Using mpt64 antigenic protein gene, we developed a LAMP assay especially for organisms of the M. tuberculosis complex. mpt64 LAMP assay showed 100% sensitivity and detected all the bacteriologically and clinically positive TB cases not detected by smear, culture or PCR methods.


Assuntos
Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Estudos de Casos e Controles , DNA Bacteriano/genética , Feminino , Humanos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Tuberculose Pulmonar/microbiologia
16.
Microb Drug Resist ; 25(2): 264-270, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30256172

RESUMO

The prevalence of nontuberculous mycobacteria (NTM) has increased in tuberculosis (TB)-suspected clinical samples. These bacteria are now recognized as important emerging pathogens, which affect both immunocompetent and immunocompromised individuals. The aim of this study was to evaluate the frequency of NTM in clinical samples and to efficacy of genomic loci as targets for detection of NTM species. This cross-sectional study was performed on 8166 clinical samples to determine the presence of NTM species from April 2013 to December 2015. The phenotypic methods were applied for preliminary NTM identification. The PCR-RFLP assay of heat shock protein-65 (hsp-65) gene and multilocus sequence analysis based on 16S-23S internal transcribes spacer (ITS), rpoB, and 16S rRNA genes were applied for species identification. In a total of 520 isolates from TB-suspected cases, 61 samples (11.7%) were identified as NTM. Overall, Mycobacterium (M.) fortuitum (63.9%) was the most frequently encountered species, followed by M. kansasii (9.8%), M. simiae (9.8%), M. abscessus (6.7%), M. gordonae (4.9%), M. flavescens (3.3%), and M. setense (1.6%). Moreover, sequencing of 16S rRNA and rpoB genes could identify all NTM species. In conclusion, we showed that the samples were infected by six NTM species, and M. fortuitum was the most frequent NTM strain. Based on the findings, 16S rRNA and rpoB genes were superior to ITS gene in identification of NTM species.


Assuntos
Infecções por Micobactéria não Tuberculosa/epidemiologia , Infecções por Micobactéria não Tuberculosa/microbiologia , Micobactérias não Tuberculosas/genética , Adulto , Idoso , Estudos Transversais , Feminino , Variação Genética , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Prevalência , RNA Ribossômico 16S/genética
17.
OMICS ; 23(1): 1-16, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30207826

RESUMO

Nontuberculous mycobacterial (NTM) species present a major challenge for global health with serious clinical manifestations ranging from pulmonary to skin infections. Multiomics research and its applications toward clinical microbial proteogenomics offer veritable potentials in this context. For example, the Mycobacterium abscessus, a highly pathogenic NTM, causes bronchopulmonary infection and chronic pulmonary disease. The rough variant of the M. abscessus UC22 strain is extremely virulent and causes lung upper lobe fibrocavitary disease. Although several whole-genome next-generation sequencing studies have characterized the genes in the smooth variant of M. abscessus, a reference genome sequence for the rough variant was generated only recently and calls for further clinical applications. We carried out whole-genome sequencing and proteomic analysis for a clinical isolate of M. abscessus UC22 strain obtained from a pulmonary tuberculosis patient. We identified 5506 single-nucleotide variations (SNVs), 63 insertions, and 76 deletions compared with the reference genome. Using a high-resolution LC-MS/MS-based approach (liquid chromatography tandem mass spectrometry), we obtained protein coding evidence for 3601 proteins, representing 71% of the total predicted genes in this genome. Application of proteogenomic approach further revealed seven novel protein-coding genes and enabled refinement of six computationally derived gene models. We also identified 30 variant peptides corresponding to 16 SNVs known to be associated with drug resistance. These new observations offer promise for clinical applications of microbial proteogenomics and next-generation sequencing, and provide a resource for future global health applications for NTM species.


Assuntos
Mycobacterium abscessus/genética , Micobactérias não Tuberculosas/genética , Resistência Microbiana a Medicamentos/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Proteogenômica/métodos , Proteômica/métodos , Tuberculose Pulmonar/microbiologia
18.
Clin Chem ; 65(2): 333-341, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30523201

RESUMO

BACKGROUND: Nontuberculous mycobacteria (NTM) species are a rising threat, especially to patients living with pulmonary comorbidities. Current point-of-care diagnostics fail to adequately identify and differentiate NTM species from Mycobacterium tuberculosis (Mtb). Definitive culture- and molecular-based testing can take weeks to months and requires sending samples out to specialized diagnostic laboratories. METHODS: In this proof-of-concept study, we developed an assay based on PCR amplification of 16S ribosomal RNA (rRNA) rrs genes by using universal mycobacterial primers and interrogation of the amplified fragments with a panel of binary deoxyribozyme (BiDz) sensors to enable species-level identification of NTM (BiDz-NTMST). Each BiDz sensor consists of 2 subunits of an RNA-cleaving deoxyribozyme, which form an active deoxyribozyme catalytic core only in the presence of the complimentary target sequence. The target-activated BiDz catalyzes cleavage of a reporter substrate, thus triggering either fluorescent or colorimetric (visually observed) signal depending on the substrate used. The panel included BiDz sensors for differentiation of 6 clinically relevant NTM species (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium kansasii, and Mycobacterium gordonae) and Mtb. RESULTS: Using the fluorescent BiDz-NTMST assay, we successfully identified the species of 38 clinical isolates. In addition, a subset of strains was tested with visual BiDz sensors, providing proof-of-concept for species typing of NTM by the naked eye. CONCLUSIONS: The BiDz-NTMST assay is a novel platform for rapid identification of NTM species. This method is highly specific and significantly faster than current tools and is easily adaptable for onsite diagnostic laboratories in hospitals or clinical laboratories.


Assuntos
DNA Catalítico/metabolismo , Micobactérias não Tuberculosas/genética , Colorimetria , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Infecções por Micobactéria não Tuberculosa/diagnóstico , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo
19.
Enferm Infecc Microbiol Clin ; 37(3): 160-166, 2019 03.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-29871765

RESUMO

INTRODUCTION: The American Thoracic Society and the Infectious Diseases Society of America recommend that clinically significant non-tuberculous mycobacteria (NTM) should be identified to the species level in order to determine their clinical significance. The aim of this study was to evaluate identification of rapidly growing NTM (RGM) isolated from clinical samples by using MALDI-TOF MS and a commercial molecular system. The results were compared with identification using a reference method. METHODS: We included 46 clinical isolates of RGM and identified them using the commercial molecular system GenoType® CM/AS (Hain, Lifescience, Germany), MALDI-TOF MS (Bruker) and, as reference method, partial rpoß gene sequencing followed by BLAST and phylogenetic analysis with the 1093 sequences available in the GeneBank. RESULTS: The degree of agreement between GenoType® and MALDI-TOF MS and the reference method, partial rpoß sequencing, was 27/43 (62.8%) and 38/43 cases (88.3%) respectively. For all the samples correctly classified by GenoType®, we obtained the same result with MALDI-TOF MS (27/27). However, MALDI-TOF MS also correctly identified 68.75% (11/16) of the samples that GenoType® had misclassified (p=0.005). CONCLUSIONS: MALDI-TOF MS classified significantly better than GenoType®. When a MALDI-TOF MS score >1.85 was achieved, MALDI-TOF MS and partial rpoß gene sequencing were equivalent. GenoType® was not able to distinguish between species belonging to the M. fortuitum complex. MALDI-TOF MS methodology is simple, rapid and associated with lower consumable costs than GenoType®. The partial rpoß sequencing methods with BLAST and phylogenetic analysis were not able to identify some RGM unequivocally. Therefore, sequencing of additional regions would be indicated in these cases.


Assuntos
Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Técnicas Bacteriológicas/métodos , Sequência de Bases , DNA Bacteriano/análise , Genótipo , Humanos , Micobactérias não Tuberculosas/fisiologia , Filogenia
20.
J Clin Microbiol ; 57(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30333128

RESUMO

The sustained increase in the incidence of nontuberculous mycobacterial (NTM) infection and the difficulty in distinguishing these infections from tuberculosis constitute an urgent need for NTM species-level identification. The MeltPro Myco assay is the first diagnostic system that identifies 19 clinically relevant mycobacteria in a single reaction based on multicolor melting curve analysis run on a real-time PCR platform. The assay was comprehensively evaluated regarding its analytical and clinical performances. The MeltPro Myco assay accurately identified 51 reference mycobacterial strains to the species/genus level and showed no cross-reactivity with 16 nonmycobacterial strains. The limit of detection was 300 bacilli/ml, and 1% of the minor species was detected in the case of mixed infections. Clinical studies using 1,163 isolates collected from five geographically distinct health care units showed that the MeltPro Myco assay correctly identified 1,159 (99.7%) samples. Further testing with 94 smear-positive sputum samples showed that all samples were correctly identified. Additionally, the entire assay can be performed within 3 h. The results of this study confirmed the efficacy of this assay in the reliable identification of mycobacteria, suggesting that it might potentially be used as a screening tool in regions endemic for tuberculosis.


Assuntos
Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Infecções por Micobactéria não Tuberculosa/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Coinfecção/diagnóstico , Coinfecção/microbiologia , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Humanos , Técnicas Microbiológicas/normas , Técnicas de Diagnóstico Molecular/normas , Infecções por Micobactéria não Tuberculosa/microbiologia , Micobactérias não Tuberculosas/genética , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Escarro/microbiologia , Fatores de Tempo , Tuberculose/diagnóstico , Tuberculose/microbiologia
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