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1.
Afr Health Sci ; 19(1): 1321-1328, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31148957

RESUMEN

Background: Tuberculosis (TB) diagnosis by culture in most resource-limited settings is hampered by high contamination rate varying up to 31%. Reduction of oral microorganism loads by mouth rinse with antiseptic before sputum collection showed a reduction of contamination. Moreover, knowing the characteristic of residual contaminant microorganisms would be an asset to understand contamination issues. Objectives: The aim of this study was to evaluate the effects of mouth rinsing with chlorhexidine on mycobacteria culture contaminations and to characterize morphologically the residual contaminants. Methods: We consecutively included 158 patients in a TB center. Each of them supplied two sputa: The first before mouth rinse, and the second after 60sec of mouth rinsing with chlorhexidine (0.1%). Petroff method and Lowenstein-Jensen media were used for sputum decontamination and inoculation respectively. The contamination rates were compared, and the type of residual contaminants were characterized and compared. Results: The contamination rate did not differ before and after the mouth rinse (respectively 58/150 (39 %) vs 61/150 (41 %), p=0.7). The major residual contaminants were Gram positive spore forming bacteria (94%). Conclusion: Chlorhexidine mouth rinsing before sputum collection did not reduce mycobacterial culture contamination rate. This is probably due to spore forming bacteria, highlighted as major residual contaminants.


Asunto(s)
Antiinfecciosos Locales/farmacología , Antiinfecciosos/farmacología , Clorhexidina/farmacología , Desinfectantes/farmacología , Antisépticos Bucales/farmacología , Mycobacterium tuberculosis/aislamiento & purificación , Esputo/microbiología , Adulto , Antiinfecciosos/administración & dosificación , Antiinfecciosos Locales/administración & dosificación , Burkina Faso , Clorhexidina/administración & dosificación , Desinfectantes/administración & dosificación , Femenino , Humanos , Masculino , Antisépticos Bucales/administración & dosificación , Mycobacterium tuberculosis/crecimiento & desarrollo , Control de Calidad , Esputo/efectos de los fármacos
2.
Sci Rep ; 9(1): 7194, 2019 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-31076625

RESUMEN

The type of commensal microorganisms can influence the efficiency of sputum decontamination for TB diagnosis. A basic characterization of contaminants from LJ contaminated media showed that Gram positive Spore Forming Bacteria (SFB) were the major contaminants. This study aims to identify the species of this contaminants and to evaluate the effectiveness of VCNT at 10 µg of vancomycin to reduce mycobacterial culture contamination mainly linked to SFB. Fifty-three SFB isolated between February 2016 and May 2017 were used. The effectiveness of LJ with VCNT at 10 µg of Vancomycin were evaluated with sputum collected in the same period. SFB had been stored at -20 °C and identified after subculture onto 5% sheep blood Columbia agar and incubated at 37 °C during 24 h. Bacteria cells and isolated colonies were described. API 50CH/B was performed and MALDI-TOF MS was used for external quality control. Thirty- five (66%) isolates representing 4 genera (Bacillus, Paenibacillus, Brevisbacillus and Lysinibacillus) including 10 species were identified. The most important species were Bacillus cereus (30%) and Bacillus licheniformis (21%). Eighteen (34%) isolates were non-reactive Bacillus. The overall contamination rate on LJ with VCNT at 10 µg of vancomycin was statistically lower than which without VCNT (18.7% versus 43.8%) (p = 0.01). The most important SFB identified were B. cereus and B. licheniformis. Almost all identified strains were similar to those currently isolated in fermented traditional food suggesting in part food related contaminants. VCNT containing 10 µg of vancomycin is a good alternative method to reduce mycobacterial culture contamination.

3.
Infect Drug Resist ; 11: 1617-1625, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30319278

RESUMEN

Objective: Nigeria ranks fourth among the high tuberculosis (TB) burden countries. This study describes the prevalence of drug resistance and the genetic diversity of Mycobacterium tuberculosis in Abuja's Federal Capital Territory. Materials and methods: Two hundred and seventy-eight consecutive sputum samples were collected from adults with presumptive TB during 2013-2014. DNA was extracted from Löwenstein-Jensen cultures and analyzed for the identification of nontuberculous mycobacteria species, detection of drug resistance with line probe assays, and high-throughput spacer oligonucleotide typing (spoligotyping) using microbead-based hybridization. Results: Two hundred and two cultures were positive for M. tuberculosis complex, 24 negative, 38 contaminated, and 15 positive for nontuberculous mycobacteria. Five (2.5%) M. tuberculosis complex isolates were resistant to rifampicin (RIF) and isoniazid (multidrug resistant), nine (4.5%) to RIF alone, and 15 (7.4%) to isoniazid alone; two RIF-resistant isolates were also resistant to fluoroquinolones and ethambutol, and one multidrug resistant isolate was also resistant to ethambutol. Among the 180 isolates with spoligotyping results, 164 (91.1%) were classified as lineage 4 (Euro-American), 13 (7.2%) as lineage 5 (West African 1), two (1.1%) as lineage 2 (East Asia), and one (0.6%) as lineage 6 (West African 2). One hundred and fifty-six (86.7%) isolates were grouped in 17 clusters (2-108 isolates/cluster), of which 108 (60.0%) were grouped as L4.6.2/Cameroon (spoligotype international type 61). Conclusion: The description of drug resistance prevalence and genetic diversity of M. tuberculosis in this study may be useful for improving TB control in Nigeria.

4.
Mem Inst Oswaldo Cruz ; 112(11): 769-774, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29091137

RESUMEN

BACKGROUND: The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB. METHOD: We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible. FINDINGS: Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. MAIN CONCLUSIONS: Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.


Asunto(s)
Técnicas Bacteriológicas/métodos , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Brasil , Genotipo , Humanos , Técnicas de Diagnóstico Molecular , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tuberculosis Resistente a Múltiples Medicamentos/microbiología
5.
Mem. Inst. Oswaldo Cruz ; 112(11): 769-774, Nov. 2017. tab
Artículo en Inglés | LILACS | ID: biblio-894852

RESUMEN

BACKGROUND The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB. METHOD We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible. FINDINGS Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. MAIN CONCLUSIONS Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.


Asunto(s)
Humanos , Técnicas Bacteriológicas/métodos , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Mycobacterium tuberculosis/genética , Brasil , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Patología Molecular , Genotipo
6.
Am J Trop Med Hyg ; 97(3): 806-809, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28722603

RESUMEN

We evaluated Tuberculosis-Spoligo-Rifampicin-Isoniazid Typing (TB-SPRINT), a microbead-based method for spoligotyping and detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis. For that, 67 M. tuberculosis complex strains were retrospectively selected. Membrane-based spoligotyping, restriction fragment length polymorphism, DNA sequencing/pyrosequencing of rpoB, katG, and inhA promoter, TB-SPRINT, and SNP typing were performed. Concordance between spoligotyping methods was 99.6% (2,785/2,795 spoligotype data points). For most of the discordant cases, the same lineage was assigned with both methods. Concordance between phenotypic drug susceptibility testing and TB-SPRINT for detecting rifampicin and isoniazid resistance was 98.4% (63/64) and 93.8% (60/64), respectively. Concordance between DNA sequencing/pyrosequencing and TB-SPRINT for detecting mutations in rpoB, katG, and inhA were 98.4% (60/61), 100% (64/64), and 96.9% (62/64), respectively. In conclusion, TB-SPRINT is a rapid and easy-to-perform assay for genotyping and detecting drug resistance in a single tube; therefore, it may be a useful tool to improve epidemiological surveillance.


Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Genotipo , Pruebas de Sensibilidad Microbiana/métodos , Tipificación Molecular/métodos , Sensibilidad y Especificidad
7.
Infect Genet Evol ; 45: 461-473, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27746295

RESUMEN

Two geographically distant M. tuberculosis sublineages, Tur from Turkey and T3-Osaka from Japan, exhibit partially identical genotypic signatures (identical 12-loci MIRU-VNTR profiles, distinct spoligotyping patterns). We investigated T3-Osaka and Tur sublineages characteristics and potential genetic relatedness, first using MIRU-VNTR locus analysis on 21 and 25 samples of each sublineage respectively, and second comparing Whole Genome Sequences of 8 new samples to public data from 45 samples uncovering human tuberculosis diversity. We then tried to date their Most Recent Common Ancestor (MRCA) using three calibrations of SNP accumulation rate (long-term=0.03SNP/genome/year, derived from a tuberculosis ancestor of around 70,000years old; intermediate=0.2SNP/genome/year derived from a Peruvian mummy; short-term=0.5SNP/genome/year). To disentangle between these scenarios, we confronted the corresponding divergence times with major human history events and knowledge on human genetic divergence. We identified relatively high intrasublineage diversity for both T3-Osaka and Tur. We definitively proved their monophyly; the corresponding super-sublineage (referred to as "T3-Osa-Tur") shares a common ancestor with T3-Ethiopia and Ural sublineages but is only remotely related to other Euro-American sublineages such as X, LAM, Haarlem and S. The evolutionary scenario based on long-term evolution rate being valid until T3-Osa-Tur MRCA was not supported by Japanese fossil data. The evolutionary scenario relying on short-term evolution rate since T3-Osa-Tur MRCA was contradicted by human history and potential traces of past epidemics. T3-Osaka and Tur sublineages were found likely to have diverged between 800y and 2000years ago, potentially at the time of Mongol Empire. Altogether, this study definitively proves a strong genetic link between Turkish and Japanese tuberculosis. It provides a first hypothesis for calibrating TB Euro-American lineage molecular clock; additional studies are needed to reliably date events corresponding to intermediate depths in tuberculosis phylogeny.


Asunto(s)
Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Tuberculosis/epidemiología , Tuberculosis/microbiología , ADN Bacteriano/análisis , ADN Bacteriano/genética , Epidemias/historia , Epidemias/estadística & datos numéricos , Evolución Molecular , Genotipo , Historia del Siglo XVI , Historia del Siglo XVII , Historia del Siglo XVIII , Historia Antigua , Historia Medieval , Humanos , Japón , Repeticiones de Minisatélite/genética , Epidemiología Molecular , Tipificación Molecular , Filogenia , Polimorfismo de Nucleótido Simple/genética , Tuberculosis/historia , Turquia
8.
BMC Infect Dis ; 15: 306, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-26231661

RESUMEN

BACKGROUND: We aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil. METHODS: One hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions). RESULTS: Fifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB. CONCLUSIONS: Although our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20-30 % of the isolates we analyzed might be MDR-TB transmission cases.


Asunto(s)
Variación Genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Alelos , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Brasil/epidemiología , Análisis por Conglomerados , ADN Bacteriano/análisis , ARN Polimerasas Dirigidas por ADN , Genotipo , Humanos , Isoniazida/uso terapéutico , Repeticiones de Minisatélite/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/aislamiento & purificación , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología
9.
J Clin Microbiol ; 52(7): 2410-5, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24759720

RESUMEN

A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. pneumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n = 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n = 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.


Asunto(s)
Legionella pneumophila/clasificación , Legionella pneumophila/genética , Microesferas , Tipificación Molecular/métodos , Automatización de Laboratorios , Francia , Ensayos Analíticos de Alto Rendimiento , Humanos , Epidemiología Molecular/métodos
10.
J Clin Microbiol ; 51(11): 3527-34, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23966495

RESUMEN

As a follow-up of the "spoligoriftyping" development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.


Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Tipificación Molecular/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Monitoreo Epidemiológico , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Epidemiología Molecular/métodos , Mutación Missense , Mycobacterium tuberculosis/genética , Medicina de Precisión/métodos , Rifampin/farmacología , Sensibilidad y Especificidad
11.
J Clin Microbiol ; 50(10): 3172-9, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22814456

RESUMEN

We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.


Asunto(s)
Tipificación Molecular/métodos , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Hibridación de Ácido Nucleico/métodos , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/farmacología , Bulgaria , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana , Genotipo , Alemania , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Microesferas , Mycobacterium tuberculosis/efectos de los fármacos , Nigeria , Oligonucleótidos/genética , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico
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