RESUMO
OBJECTIVES: To evaluate the performance of geneXpert MTB/Rif versus conventional methods (bacilloscopy and culture) in the diagnosis of tuberculosis in a Central Public Health Laboratory (LACEN, Tocantins), Northern Brazil. METHODS: Retrospective study, with information from 1,973 suspected cases of tuberculosis from patients treated from January 2015 to December 2020. RESULTS: From the culture (reference standard), the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the geneXpert MTB/Rif were 100%, 97%, 74%, 100%, and 97%, respectively, against 85%, 98%, 80%, 98%, and 97% of bacilloscopy. CONCLUSIONS: The geneXpert MTB/Rif performed similarly to culture and better than bacilloscopy. Although positive cases with negative culture should be evaluated with caution, its routine use is important for the early detection of tuberculosis.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/genética , Rifampina , Estudos Retrospectivos , Saúde Pública , Sensibilidade e Especificidade , Escarro , Brasil , Tuberculose/diagnósticoRESUMO
ABSTRACT OBJECTIVES To evaluate the performance of geneXpert MTB/Rif versus conventional methods (bacilloscopy and culture) in the diagnosis of tuberculosis in a Central Public Health Laboratory (LACEN, Tocantins), Northern Brazil. METHODS Retrospective study, with information from 1,973 suspected cases of tuberculosis from patients treated from January 2015 to December 2020. RESULTS From the culture (reference standard), the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the geneXpert MTB/Rif were 100%, 97%, 74%, 100%, and 97%, respectively, against 85%, 98%, 80%, 98%, and 97% of bacilloscopy. CONCLUSIONS The geneXpert MTB/Rif performed similarly to culture and better than bacilloscopy. Although positive cases with negative culture should be evaluated with caution, its routine use is important for the early detection of tuberculosis.
Assuntos
Humanos , Masculino , Feminino , Tuberculose , Técnicas de Laboratório Clínico , Mycobacterium tuberculosisRESUMO
CYP21A2 deficiency represents 95% of congenital adrenal hyperplasia (CAH) cases, a group of genetic disorders that affect steroid biosynthesis. The genetic and functional analysis provide critical tools to elucidate complex CAH cases. One of the most accessible tools to infer the pathogenicity of new variants is in silico prediction. Here, we analyzed the performance of in silico prediction tools to categorize missense single nucleotide variants (SNVs) of CYP21A2. SNVs of CYP21A2 characterized in vitro by functional assays were selected to assess the performance of online single and meta predictors. SNVs were tested separately or in combination with the related phenotype (severe or mild CAH form). In total, 103 SNVs of CYP21A2 (90 pathogenic and 13 neutral) were used to test the performance of 13 single-predictors and four meta-predictors. All SNVs associated with the severe phenotypes were well categorized by all tools, with an accuracy of between 0.69 (PredictSNP2) and 0.97 (CADD), and Matthews' correlation coefficient (MCC) between 0.49 (PoredicSNP2) and 0.90 (CADD). However, SNVs related to the mild phenotype had more variation, with the accuracy between 0.47 (S3Ds&GO and MAPP) and 0.88 (CADD), and MCC between 0.18 (MAPP) and 0.71 (CADD). From our analysis, we identified four predictors of CYP21A2 variant pathogenicity with good performance, CADD, ConSurf, DANN, and PolyPhen2. These results can be used for future analysis to infer the impact of uncharacterized SNVs in CYP21A2.
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Genomic-based surveillance on the occurrence of drug resistance and its transmission dynamics has emerged as a powerful tool for the control of tuberculosis (TB). A whole-genome sequencing approach, phenotypic testing and clinical-epidemiological investigation were used to undertake a retrospective population-based study on drug-resistant (DR)-TB in Rio Grande do Sul, the largest state in Southern Brazil. The analysis included 305 resistant Mycobacterium tuberculosis strains sampled statewide from 2011 to 2014, and covered 75.7% of all DR-TB cases identified in this period. Lineage 4 was found to be predominant (99.3%), with high sublineage-level diversity composed mainly of 4.3.4.2 [Latin American and Mediterranean (LAM)/RD174], 4.3.3 (LAM/RD115) and 4.1.2.1 (Haarlem/RD182) sublineages. Genomic diversity was also reflected in resistance of the variants to first-line drugs. A large number of distinct resistance-conferring mutations, including variants that have not been reported previously in any other setting worldwide, and 22 isoniazid-monoresistant strains with mutations described as disputed in the rpoB gene but causing rifampicin resistance generally missed by automated phenotypic tests as BACTEC MGIT. Using a cut-off of five single nucleotide polymorphisms, the estimated recent transmission rate was 55.1%, with 168 strains grouped into 28 genomic clusters. The most worrying fact concerns multi-drug-resistant (MDR) strains, of which 73.4% were clustered. Different resistance profiles and acquisition of novel mutations intraclusters revealed important amplification of resistance in the region. This study described the diversity of M. tuberculosis strains, the basis of drug resistance, and ongoing transmission dynamics across the largest state in Southern Brazil, stressing the urgent need for MDR-TB transmission control state-wide.
Assuntos
Antibióticos Antituberculose/uso terapêutico , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antituberculosos/uso terapêutico , Brasil/epidemiologia , Perfilação da Expressão Gênica , Genoma Bacteriano/genética , Humanos , Isoniazida/uso terapêutico , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único/genética , Estudos Retrospectivos , Rifampina/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
PURPOSE: The establishment of candidate genetic determinants associated with tuberculosis (TB) is a challenge, considering the divergent frequencies among populations. The objective of this study was to evaluate the association between MIF - 794 CATT 5-8 polymorphism and susceptibility to TB. METHODS: Case-control study. Patients > 18 years, with pulmonary TB were included. The control group consisted of blood donors and household contacts, not relatives, healthy and > 18 years. MIF - 794 CATT 5-8 were genotyped using sequencing of PCR and capillary electrophoresis. RESULTS: 126 patients and 119 controls were included. The genotype 5/5 was more frequent among cases (15.1%) than in controls (5.9%) (p = 0.019). Cases had more frequently the allele 5 (29.4%) as compared with controls (19.3%) (p = 0.010). Prevalence of 7/X + 8/X genotypes was not different between cases and controls (p = 0.821). There was no difference between patients with alleles 7 and 8 and those with alleles 5 and 6 (p = 0.608). CONCLUSIONS: The genotype 5/5 and the allele 5 of MIF - 794 CATT 5-8 were more frequent among TB patients than in controls.
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Predisposição Genética para Doença , Fatores Inibidores da Migração de Macrófagos , Tuberculose , Estudos de Casos e Controles , Frequência do Gene , Genótipo , Humanos , Oxirredutases Intramoleculares , Fatores Inibidores da Migração de Macrófagos/genética , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Tuberculose/genéticaRESUMO
We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.
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Programas de Rastreamento/métodos , Mycobacterium tuberculosis/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Manejo de Espécimes/métodos , Tuberculose Pulmonar/diagnóstico , Brasil , DNA Bacteriano/isolamento & purificação , Humanos , Limite de Detecção , Programas de Rastreamento/economia , Programas de Rastreamento/instrumentação , Mycobacterium tuberculosis/genética , Estudo de Prova de Conceito , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/economia , Escarro/microbiologia , Tuberculose Pulmonar/microbiologiaRESUMO
Abstract Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.
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Humanos , Tuberculose Pulmonar , Mycobacterium tuberculosis , Sonicação , Escarro , Brasil , DNA , DNA Bacteriano/genética , Sensibilidade e Especificidade , Mycobacterium tuberculosis/genéticaRESUMO
Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14â¯CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.
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Mycobacterium tuberculosis , Tuberculose Pulmonar , Brasil , DNA , DNA Bacteriano/genética , Humanos , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , Sonicação , EscarroRESUMO
PURPOSE: The establishment of candidate genes associated with susceptibility to TB is a challenge especially due to divergent frequencies among different populations. The objective of this study was to evaluate the association between macrophage migration inhibitory factor (MIF) -173 G>C single nucleotide polymorphism (SNP) and susceptibility to pulmonary TB in a population of southern Brazil. METHODS: Case-control study. Patients > 18 years old, diagnosed with pulmonary TB were included. The control group consisted of blood donors and household contacts, not relatives, healthy and > 18 years old. MIF -173 G>C SNPs were genotyped using real-time PCR using a TaqMan SNP Genotyping assay. RESULTS: 174 patients and 166 controls were included. There were no statistically significant differences between cases and controls regarding genotype prevalence (p>0.05). Comparing patients with normal genotype (GG) with those with at least one C allele, there was also no statistically significant difference (p = 0.135). Also, there was no statistically significant difference comparing the homozygous for the mutation (CC) with the other patients (GG and CG) (p = 0.864). CONCLUSIONS: We did not find association between MIF -173 G>C polymorphism and susceptibility to pulmonary TB.
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Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Polimorfismo de Nucleotídeo Único , Tuberculose Pulmonar/genética , Adolescente , Adulto , Idoso , Brasil , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tuberculose Pulmonar/patologiaRESUMO
BACKGROUND: Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES: To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS: Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS: The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS: qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.