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Ante-mortem diagnosis of bovine tuberculosis (bTB) is based mainly on the tuberculin skin test (TST) and the ɣ-IFN release assay (IGRA). Some infected animals escape screening tests, thus, limit herd sanitation. Previous reports have suggested a predominant pattern of multi-organ lesions attributable to Mycobacterium bovis (the causative agent of bTB) bacteraemia. A case-control study was conducted to investigate blood PCR as an alternative tool for improving ante-mortem detection of TST false-negative bovines. Cases comprised 70 TST false-negative bovines (cases), which were serology positive, and controls included 81 TST positive bovines; all of them confirmed as infected with M. bovis. Detection of the IS6110 target through touchdown blood-PCR (IS6110 TD-PCR) was performed. The positivity of the blood-PCR was 27.2% in the control group. This performance was similar to the 15% obtained among cases (p = 0.134). Most cases identified by the IS6110 TD-PCR exhibited focalized lesions (p = 0.002). Results demonstrated that blood-PCR could detect TST false-negative cattle, even if they are negative for IGRA. Considering that cases exhibited humoral response to M. bovis, further studies conducted in a pre-serological stage could provide evidence about the real contribution of the technique in herds.
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Molecular techniques have revolutionized tuberculosis (TB) diagnosis by offering a faster and more sensitive approach, detecting Mycobacterium tuberculosis (Mtb) DNA directly from samples. Single-tube nested PCR (STNPCR) combines two PCR reactions with separate oligonucleotide sets in a single tube. Moreover, colorimetric methods in PCR products have been studied for pathogen detection. Thus, this study aimed to establish a novel system based on colorimetric STNPCR for Mtb detection using microtiter plates with IS6110-amplified fragments. The results showed a general colorimetric STNPCR detection limit of 1 pg/µl. Its general sensitivity and specificity were 76.62 and 60.53%, respectively, with kappa index agreement of 0.166.
A total of 318 biological samples (urine, plasma, peripheral blood mononuclear cells, pleural fluid and sputum) from pulmonary/extrapulmonary TB and non-TB patients were used in this study. The colorimetric STNPCR assay using IS6110 as the target gene was developed and optimized for Mtb detection based on similar validated systems. Cut-off values based on receiver operator characteristic curve analysis were defined to determine the sensitivity and specificity for each sample type. The technique's performance was assessed according to kappa index calculations and interpretation.
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Colorimetria , DNA Bacteriano , Mycobacterium tuberculosis , Reação em Cadeia da Polimerase , Tuberculose , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Colorimetria/métodos , Reação em Cadeia da Polimerase/métodos , Humanos , Tuberculose/diagnóstico , Tuberculose/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/análise , Sensibilidade e Especificidade , Limite de DetecçãoRESUMO
INTRODUCTION: Mycobacterium tuberculosis genotyping has impacted evolutionary studies worldwide. Nonetheless, its application and the knowledge generated depend on the genetic marker evaluated and the detection technologies that have evolved over the years. Here we describe the timeline of main genotypic methods related to M. tuberculosis in Latin America and the main findings obtained. METHODOLOGY: Systematic searches through the PubMed database were performed from 1993 to May 2021. A total of 345 articles met the inclusion criteria and were selected. RESULTS: Spacer oligonucleotide typing (spoligotyping) was the most widely used method in Latin America, with decreasing use in parallel with increasing use of mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) and whole genome sequencing (WGS). Among the countries, Brazil, Mexico, and Argentina had the most publications, and a considerable part of the articles were in collaboration with Latin American or non-Latin American institutions; a small proportion of studies needed partnerships to perform the genotypic methods. The genotypic methods allowed the identification of M. tuberculosis genotypes with greater capacity for clonal expansion and revealed the predominance of the Euro-American lineage in Latin America. There was a notable presence of the Beijing family in Peru and Colombia. CONCLUSIONS: The data obtained demonstrated the importance of expanding collaborative networks of tuberculosis (TB) research groups to countries with low productivity in this area, the commitment of the few Latin American countries to advance TB research, as well as the inestimable value of building a Latin America database, considering ease of population mobility between countries.
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Mycobacterium tuberculosis , Tuberculose , Humanos , América Latina/epidemiologia , Genótipo , Polimorfismo de Fragmento de Restrição , Técnicas de Tipagem Bacteriana/métodos , Tuberculose/epidemiologia , Tuberculose/microbiologia , Mycobacterium tuberculosis/genética , Repetições MinissatélitesRESUMO
Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.
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Humanos , Polimorfismo de Fragmento de Restrição/genética , Repetições Minissatélites/genética , Mycobacterium tuberculosis/genética , Brasil/epidemiologia , Técnicas de Tipagem Bacteriana , Epidemiologia Molecular , Sequenciamento Completo do Genoma , Genótipo , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
BACKGROUND: Since the implementation of the Xpert MTB/RIF in Sao Paulo, Brazil, numerous Mycobacterium tuberculosis isolates presenting "rifampicin-resistant genotype with rifampicin-susceptible phenotype" were observed. OBJECTIVE: To evaluate the prevalence, rpoB mutations and transmission of M. tuberculosis resistant to rifampicin on Xpert MTB/RIF but susceptible on BACTEC MGIT system, in Sao Paulo state. METHODS: Patients' isolates with this pattern of rifampicin discordance, collected from 2014 to 2017, had their rpoB predominant rifampicin-resistance-determining region sequenced and were genotyped by IS6110 restriction fragment-length polymorphism. FINDINGS: The prevalence of rifampicin-discordant M. tuberculosis with genotypic resistance was 55.1% (156/283). Among the sequenced and genotyped isolates, 75.5% (111/147) were in clusters, largely associated with the type of rpoB mutation. Most isolates (98.6%; 72/73) harbouring the predominant mutation, His445Asn, were pooled into the two largest clusters, SP2ga (42/72; 58.3%) and SP5o (12/72; 16.7%). Ranking second, isolates carrying the silent mutation Phe433Phe were mostly (92.3%; 24/26) gathered into four groups of the family SP25. CONCLUSION: These findings suggest that this unusual high rifampicin discrepancy proportion was greatly influenced by few actively circulating clusters. Further studies on many of the rpoB mutations identified in our setting are needed to elucidate their association with phenotypic rifampicin resistance.
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Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Epidemias/estatística & dados numéricos , Mutação , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibióticos Antituberculose/farmacologia , Brasil/epidemiologia , Estudos Transversais , RNA Polimerases Dirigidas por DNA/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Fenótipo , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto JovemRESUMO
Tuberculosis (TB) has a high incidence, prevalence and mortality in the world. Due to its high level of transmission and long-term pharmacological treatment, it is important to have sensitive and specific diagnostic tests. Recently, the PureLyse® system, which is a novel DNA extraction method, was proposed to be an important tool for molecular diagnosis of TB. Here, we compare the PureLyse® system followed by an IS6110 nested PCR (PureLyse® - IS6110 nested PCR) with the Xpert® MTB/RIF test for Mycobacterium tuberculosis complex (MTBC) identification in 40 clinical samples. Among the 40 samples, 26 samples were positive and 14 negative for the Xpert® MTB/RIF test as well as for the PureLyse® - IS6110 nested PCR. According to the Xpert® MTB/RIF test, positive samples presented different bacillary concentrations from "High" to "Very low" and rifampin resistance was observed in 5 samples. The concordance of both molecular methods makes the PureLyse® - IS6110 nested PCR suitable for MTBC detection in patients for low-income resources.
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Testes Diagnósticos de Rotina/instrumentação , Testes Diagnósticos de Rotina/métodos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Rifampina/farmacologia , Tuberculose/diagnóstico , Antibióticos Antituberculose/farmacologia , Proteínas de Bactérias/genética , Técnicas Bacteriológicas/instrumentação , Técnicas Bacteriológicas/métodos , DNA Bacteriano/análise , Farmacorresistência Bacteriana , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose/microbiologiaRESUMO
Abstract INTRODUCTION: This study evaluated the performance of the IS6110-TaqMan® assay in different types of biological samples and tissues for laboratory diagnosis of extrapulmonary tuberculosis. METHODS: 143 biological samples and tissues from patients with suspected extrapulmonary tuberculosis from the health services of Recife/Pernambuco/Brazil were evaluated with the IS6110-TaqMan® assay. RESULTS: The sensitivities of the IS6110-TaqMan® assay calculated for blood, urine, both blood and urine samples, tissue biopsies, extrapulmonary body fluid samples, and all samples from patients calculated together were 55.9%, 33.3%, 68.8%, 43.8%, 29.6%, and 73.7%, respectively, and the specificities were 80%, 100%, 78.6%, 100%, 100%, and 84.2%, respectively. CONCLUSIONS The accuracy of qPCR was high in various clinical sample types. The analysis of more than one type of clinical sample collected from the same patient with extrapulmonary tuberculosis enhances the diagnostic power of the IS6110-TaqMan® assay when compared with the use of only one clinical sample.
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Humanos , Tuberculose/diagnóstico , DNA Bacteriano/análise , Mycobacterium tuberculosis/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/genética , Método Duplo-Cego , Estudos Prospectivos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase em Tempo Real , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
BACKGROUND In high tuberculosis (TB) burden countries, there are few data on the performance of new molecular commercialised assays developed locally. OBJECTIVE To evaluate the performance of a new molecular commercialised assay for TB diagnosis (Detect-TB) in three laboratories. METHODS A total of 302 sputum samples from an equal number of patients with presumptive diagnosis of pulmonary tuberculosis (PTB) were submitted for routine smear microscopy, culture, and Detect-TB assay at three different sites in Brazil (the cities of Caxias do Sul, São Paulo and Canoas). FINDINGS Seventy four (24.7%) TB cases were diagnosed (65 bacteriologically confirmed). When compared to smear microscopy/culture results, the overall sensitivity and specificity of Detect-TB assay was 84.6% (CI 95%; 73.7-91.6) and 93.1% (CI 95%; 89.1-95.8), respectively. When compared to bacteriological and clinical diagnostic criteria, the sensitivity and specificity of Detect-TB assay was 74.3% (CI 95%; 63.3-82.9) and 92.9% (CI 95%; 88.7-95.6), respectively. Among the three sites - Caxias do Sul, São Paulo and Canoas - the sensitivity and specificity were respectively 94.7% and 97.8%; 71.4% and 93.9%, 82.1% and 88.9%. MAIN CONCLUSIONS These findings suggest that the Detect-TB assay could be applied routinely in reference laboratories across different regions in Brazil.
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Humanos , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/genética , Brasil , DNA Bacteriano , Reações Falso-NegativasRESUMO
Abstract Objective The diagnosis of extrapulmonary tuberculosis is still a challenge because of its pauci-bacillary nature. The aim of the study was to evaluate the role of a multiplex PCR assay in the diagnosis of extrapulmonary tuberculosis and to compare the efficiency of two targets, IS6110 and MPB64 to detect Mycobacterium tuberculosis. Methods 150 extrapulmonary samples (61 pus/aspirate, 46 tissue, 32 body fluids, and 11 urine) from clinically suspected cases of tuberculosis were included in the study. All the samples were subjected to direct fluorescent microscopy, TB culture (BacT/ALERT 3D, biomerieux, Durham, North Carolina, USA) and a Multiplexed Tandem PCR targeting two mycobacterial DNA sequences, IS6110 and MPB64. Master-Mix reagents and primers were prepared by AusDiagnostics Pvt. Ltd (Alexandria, New South Wales, Australia). The performance of the assay was assessed using a composite gold standard, which included clinical characteristics, microbiology smear as well as culture, histopathology, cytology, radiology, and response to antitubercular therapy. Results 20.3%, 23.6%, and 45.3% of specimens were positive by smear, culture, and PCR, respectively. The sensitivity and specificity of the multiplex PCR was 91.9% and 88.4%, respectively, using the composite gold standard. Positive and negative predictive values of the PCR were estimated as 85.1% and 93.8%, respectively. Higher positivity was observed with target IS6110 (44.6%) as compared to target MPB64 (18.9%). The sensitivities of IS6110 and MPB64 individual targets were 90.3% and 64.5%, respectively, and specificities were 88.4% and 97.7%, respectively. Conclusion PCR can play an important role in rapid and accurate diagnosis of extrapulmonary tuberculosis. IS6110 alone is an effective target in our part of the country.
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Humanos , Tuberculose/diagnóstico , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/genética , Antígenos de Bactérias/genética , DNA Bacteriano/análise , Amplificação de Genes , Técnicas Bacteriológicas/métodos , Sensibilidade e Especificidade , Meios de CulturaRESUMO
Abstract: INTRODUCTION Characterization of Mycobacterium tuberculosis (MTB) isolates by DNA fingerprinting has contributed to tuberculosis (TB) control. The aim of this study was to determine the genetic diversity of MTB isolates from Tehran province in Iran. METHODS MTB isolates from 60 Iranian and 10 Afghan TB patients were fingerprinted by standard IS6110-restriction fragment length polymorphism (RFLP) analysis and spoligotyping. RESULTS The copy number of IS6110 ranged from 10-24 per isolate. The isolates were classified into 22 clusters showing ≥ 80% similarity by RFLP analysis. Fourteen multidrug-resistant (MDR) isolates were grouped into 4 IS6110-RFLP clusters, with 10 isolates [71% (95% CI: 45-89%)] in 1 cluster, suggesting a possible epidemiological linkage. Eighteen Iranian isolates showed ≥ 80% similarity with Afghan isolates. There were no strains with identical fingerprints. Spoligotyping of 70 isolates produced 23 distinct patterns. Sixty (85.7%) isolates were grouped into 13 clusters, while the remaining 10 isolates (14.2%) were not clustered. Ural (formerly Haarlem4) (n = 22, 31.4%) was the most common family followed by Central Asian strain (CAS) (n = 18, 25.7%) and T (n = 9, 12.8%) families. Only 1strain was characterized as having the Beijing genotype. Among 60 Iranian and 10 Afghan MTB isolates, 25% (95% CI: 16-37) and 70% (95% CI: 39-89) were categorized as Ural lineage, respectively. CONCLUSIONS A higher prevalence of Ural family MTB isolates among Afghan patients than among Iranian patients suggests the possible transmission of this lineage following the immigration of Afghans to Iran.
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Humanos , Tuberculose/microbiologia , Variação Genética/genética , DNA Bacteriano/genética , Técnicas de Tipagem Bacteriana/métodos , Mycobacterium tuberculosis/genética , Polimorfismo de Fragmento de Restrição , Análise por Conglomerados , Impressões Digitais de DNA , Epidemiologia Molecular , Genótipo , Irã (Geográfico) , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
OBJECTIVE: The diagnosis of extrapulmonary tuberculosis is still a challenge because of its pauci-bacillary nature. The aim of the study was to evaluate the role of a multiplex PCR assay in the diagnosis of extrapulmonary tuberculosis and to compare the efficiency of two targets, IS6110 and MPB64 to detect Mycobacterium tuberculosis. METHODS: 150 extrapulmonary samples (61 pus/aspirate, 46 tissue, 32 body fluids, and 11 urine) from clinically suspected cases of tuberculosis were included in the study. All the samples were subjected to direct fluorescent microscopy, TB culture (BacT/ALERT 3D, biomerieux, Durham, North Carolina, USA) and a Multiplexed Tandem PCR targeting two mycobacterial DNA sequences, IS6110 and MPB64. Master-Mix reagents and primers were prepared by AusDiagnostics Pvt. Ltd (Alexandria, New South Wales, Australia). The performance of the assay was assessed using a composite gold standard, which included clinical characteristics, microbiology smear as well as culture, histopathology, cytology, radiology, and response to antitubercular therapy. RESULTS: 20.3%, 23.6%, and 45.3% of specimens were positive by smear, culture, and PCR, respectively. The sensitivity and specificity of the multiplex PCR was 91.9% and 88.4%, respectively, using the composite gold standard. Positive and negative predictive values of the PCR were estimated as 85.1% and 93.8%, respectively. Higher positivity was observed with target IS6110 (44.6%) as compared to target MPB64 (18.9%). The sensitivities of IS6110 and MPB64 individual targets were 90.3% and 64.5%, respectively, and specificities were 88.4% and 97.7%, respectively. CONCLUSION: PCR can play an important role in rapid and accurate diagnosis of extrapulmonary tuberculosis. IS6110 alone is an effective target in our part of the country.
Assuntos
Antígenos de Bactérias/genética , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/genética , Tuberculose/diagnóstico , Técnicas Bacteriológicas/métodos , Meios de Cultura , DNA Bacteriano/análise , Amplificação de Genes , Humanos , Sensibilidade e EspecificidadeRESUMO
The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3%, 100%, 100% and 89.7%, respectively whereas accuracy was 90.7%. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method.
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Mycobacterium tuberculosis/genética , Tuberculose/diagnóstico , DNA Bacteriano/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase , Segurança , Manejo de EspécimesRESUMO
The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3%, 100%, 100% and 89.7%, respectively whereas accuracy was 90.7%. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method
La posibilidad de obtener ADN a partir de frotis es una valiosa alternativa para remediar la falta de muestras cuando estas son totalmente utilizadas para la baciloscopia; esta opción soluciona, además, el problema de bioseguridad asociado a la posibilidad de accidente al transportar frascos que contienen muestras clínicas potencialmente infectivas. Por lo tanto, el propósito de este estudio fue utilizar para el diagnóstico de la tuberculosis la secuencia de inserción IS6110 para amplificación del ADN a partir de frotis que resultaron positivos por baciloscopia. Del análisis de 52 baciloscopias positivas surge que la sensibilidad, la especificidad, el valor predictivo positivo y el valor predictivo negativo de esta técnica fueron, respectivamente, del 52,3%, del 100%, del 100% y del 89,7%; y la precisión fue del 90,7%. La PCR IS6110 para el diagnóstico de tuberculosis, desarrollada con ADN extraído de frotis positivos, es un método rápido, simple, específico y seguro
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Tuberculose/diagnóstico , Reação em Cadeia da Polimerase/métodos , Contenção de Riscos Biológicos/métodosRESUMO
The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75 percent) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.
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Adulto , Feminino , Humanos , Masculino , Adulto Jovem , DNA Bacteriano , Variação Genética , Repetições Minissatélites , Mycobacterium tuberculosis , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , Genótipo , Índia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Sequências Repetitivas de Ácido NucleicoRESUMO
The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8 percent and 52 percent in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29 percent and 26.9 percent in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease.
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Humanos , Sangue , Genoma Bacteriano , Técnicas In Vitro , Mycobacterium tuberculosis , Reação em Cadeia da Polimerase , Urina , Técnicas e Procedimentos Diagnósticos , Métodos , PacientesRESUMO
Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2 percent (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85 percent and 98 percent, and 94 percent and 100 percent, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.
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Humanos , Mycobacterium tuberculosis , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Escarro , Tuberculose Pulmonar , Colorimetria , DNA Bacteriano , Mycobacterium tuberculosis , Sondas de Oligonucleotídeos , Kit de Reagentes para Diagnóstico , Sensibilidade e EspecificidadeRESUMO
The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8% and 52% in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29% and 26.9% in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease.
RESUMO
El objetivo de esta investigación fue la detección molecular de micobacterias del complejo tuberculosis en el tejido periodontal afectado de un grupo de pacientes VIH+ y VIH- con tuberculosis pulmonar. Se incluyeron 20 pacientes, 10 VIH+ y 10 VIH- con diagnóstico microbiológico convencional de tuberculosis, provenientes de la consulta de Tisiología, y de Sala de VIH, del Hospital Simón Bolívar, Caracas. De cada uno de los pacientes fue obtenido el consentimiento informado previo al inicio de la investigación, se le realizó una minuciosa historia clínica y un examen clínico bucal, con el fin de establecer la condición periodontal de cada uno de los sujetos participantes en este estudio. Se tomaron muestras de tejido periodontal afectado de cada uno de los pacientes con indicación de cirugía periodontal. Resultados: 50% (10/20) de los pacientes con TBC VIH- presentaron gingivitis crónica localizada y el 30% (3/20) de los pacientes con TBC VIH+ presentaron gingivitis crónica generalizada. El grupo restante mostró gingivitis localizada y cuadros sugerentes de periodontitis. Por medio de la reacción en cadena de la polimerasa (PCR) en 60% (12/20) de las muestras de tejido periodontal se detectó la secuencia IS6110, que está presente en micobacterias del complejo tuberculosis. La amplificación de blancos moleculares es una metodología sensible para la detección de este grupo de microorganismos en enfermedad periodontal y pudiera ser utilizada en el diagnóstico diferencial de lesión de la cavidad bucal
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Humanos , Masculino , Feminino , Boca/patologia , Doenças da Boca/microbiologia , Doenças da Boca/patologia , Mycobacterium tuberculosis , Reação em Cadeia da Polimerase/métodosRESUMO
The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8% and 52% in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29% and 26.9% in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease.
RESUMO
La tuberculosis (TBC) es una causa importante de morbimortalidad en nuestro país. Las herramientas de diagnóstico no son 100% eficientes y rápidas para ayudar al clínico en su decisión médica. Nuevas pruebas, como la reacción en cadena de la polimerasa (PCR), son necesarias. Evaluar la utilidad clínica de una PCR para IS6110, en muestras clínicas de pacientes con sospecha de tuberculosis pulmonar o extrapulmonar. Sesenta y una muestras de 46 pacientes del Hospital General del Este, Dr. Domingo Luciani de Caracas, Venezuela, fueron procesadas para la detección del M. Tuberculosis. Se practicó baciloscopia, cultivo en LJ y PCR e hibridación. Los pacientes se clasificaron como TBC positivos si tenían evidencia clínica o radiológica, y positivas algunas de estas pruebas: PPD, ZN, cultivo, biopsia positiva o respuesta favorable al tratamiento. Los TBC negativos con evidencia clínica o radiológica positiva y todas las pruebas negativas. La sensibilidad de la PCR sola fue del 75% y la especificidad del 61%. La PCR e hibridación juntas mostraron sensibilidad del 90% y especificidad del 57%. Los valores predictivos positivos y negativos fueron del 62% y 88%, respectivamente
Tuberculosis (TBC) remains a leading cause of morbidity and mortality in Venezuela. The diagnos tic tools used are not 100% specific, sensitive, efficient or fast enough to help clinicians take the right clinical decision. New tests, like polymerase chain reaction (PCR), are necessary. We evaluated the clinical usefulness of an IS6110 in-house PCR in clinical samples from patients suspected of having pulmonary or extrapulmonary TBC. Sixty one samples from 46 patients were processed for detection of M. tuberculosis by ZN stained smear examination, LJ medium culture, and PCR and hybridization assay. The patients were classified as TBC positive when they had clinical or radiological evidence plus any positive of the following tests: PPD, ZN, LJ culture, biopsy or anti-TBC treatment response and TBC negative when they had clinical or radiological evidence but all the applied tests used were negative. PCR sensibility was 75% and specificity was 61%, using PCR alone, but when PCR and hybridization were evaluated together the sensibility was 90% and specificity was 57%. The predictive positive and negative values were 62% and 88% respectively