Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting

Abstract INTRODUCTION: Nontuberculous mycobacteria (NTM) species, as human pathogens, are increasing in the world, as is the difficulty of accurately identifying them. Differential diagnosis, especially between the M. tuberculosis complex and NTM species, and the characterization of NTM species is important. This study aimed to evaluate the performance of a molecular system based on multiplex real-time PCR with high-resolution melting (HRM) for the identification and differentiation of NTM species of clinical importance of an endemic area for tuberculosis in northeastern Brazil. METHODS: The technical protocol of the molecular system was based on multiplex real-time PCR-HRM, and evaluated the sensitivity and specificity of the detection of NTM species in mycobacterial clinical isolates from the studied region. The gold standard method was specific gene sequencing. RESULTS: The sensitivity and specificity of multiplex real-time PCR-HRM modified for differentiation between NTM and M. tuberculosis were 90% and 100%, respectively. The PCR-HRM sensitivities for the characterization of NTM species (M. kansasii, M. abscesses, M. avium, and M. fortuitum) were 94.59%, 80%, 57.14%, and 54%, respectively. CONCLUSIONS The multiplex real-time PCR-HRM modified assay has the potential to rapidly and efficiently identify nontuberculous mycobacteria of clinical importance, which is crucial for immediate implementation of the appropriate therapy and thus avoiding complications and sequelae in patients.

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples

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.

DEFB1 gene polymorphisms and tuberculosis in a Northeastern Brazilian population

Abstract β-Defensin-1, an antimicrobial peptide encoded by the DEFB1 gene, is known to play an important role in lung mucosal immunity. In our association study we analyzed three DEFB1 functional polymorphisms -52G>A (rs1799946), -44C>G (rs1800972) and -20G>A (rs11362) in 92 tuberculosis patients and 286 healthy controls, both from Northeast Brazil: no association was found between the studied DEFB1 polymorphisms and the disease. However we cannot exclude that this lack of association could be due to the low number of subjects analyzed, as suggested by the low statistical power achieved for the three analyzed SNPs (values between 0.16 and 0.50).

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Osteoarticular tuberculosis in an HIV-positive patient: a case report

The authors report a case of a 38-year-old HIV-positive woman, with subcutaneous nodules on the thoracic region with 3 months of evolution. Clinical, laboratory, and epidemiological features were evaluated and associated with apparent damage to the T11-T12 vertebrae, identification by imaging tests, positivity in a polymerase chain reaction-based test, and reactivity to the Mantoux tuberculin skin test (PPD-RT 23). The patient was diagnosed with osteoarticular tuberculosis and received treatment for a year, and clinical cure was achieved.

Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens / Detecção do complexo Mycobacterium tuberculosis por nested polymerase chain reaction em espécimes pulmonares e extrapulmonares

OBJECTIVE: To compare the performance of nested polymerase chain reaction (NPCR) with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens. METHODS: We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results. RESULTS: Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard), we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively). CONCLUSIONS: Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis. .

OBJETIVO: Comparar o desempenho da técnica nested polymerase chain reaction (NPCR) com aquele de culturas na detecção do complexo Mycobacterium tuberculosis em espécimes pulmonares e extrapulmonares. MÉTODOS: Analisamos 20 e 78 espécimes pulmonares e extrapulmonares, respectivamente, de 67 pacientes hospitalizados com suspeita de tuberculose. Um sistema automatizado foi utilizado na identificação de culturas de Mycobacterium spp., e M. tuberculosis IS6110 foi utilizada como sequência alvo na NPCR. A estatística kappa foi utilizada para verificar a concordância entre os resultados. RESULTADOS: Entre os 67 pacientes, 6 e 5, respectivamente foram diagnosticados com tuberculose pulmonar e extrapulmonar, e a NPCR foi positiva em todos os casos. Entre os 98 espécimes clínicos, a baciloscopia, cultura e NPCR foram positivas em 6,00%, 8,16% e 13,26%, respectivamente. Comparando-se os resultados da NPCR com aqueles da cultura (padrão ouro) nos espécimes pulmonares, a sensibilidade e a especificidade foram 100% e 83%, respectivamente, enquanto essas nos espécimes extrapulmonares foram 83% e 96% respectivamente, com boa concordância entre os testes (kappa, 0,50 e 0,6867, respectivamente). CONCLUSÕES: Embora a NPCR tenha se mostrado uma ferramenta muito útil na detecção do complexo M. tuberculosis, No entanto, os resultados positivos da NPCR devem ser associados à clínica, dados clínicos, epidemiológicos e outros dados laboratoriais devem também ser considerados no diagnóstico e tratamento da tuberculose pulmonar e extrapulmonar. .

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

Introduction This study evaluated the performance of an in-house nested-PCR system for the detection of the Mycobacterium tuberculosis complex in pleural fluid, blood and urine samples from pleural effusion tuberculosis patients by health services physicians in Pernambuco, Brazil. Methods A prospective double-blind study with 37 hospitalized patients of both sexes, aged over 15, was used to investigate the diagnosis of pleural effusion. The criteria used to define the cases included the demonstration of bacillus in biological samples by smear or culture or by a granulomatous finding in the histopathological examination, associated with an evident response to specific treatments to each clinical situation. Pleural fluid, blood and urine samples were collected and subjected to routine tests and the nested PCR technique to assess for M. tuberculosis amplification. Results In total, 37 pleural effusion patients took part in the study, of whom 19 (51.3%) had tubercular etiologies and 18 (48.7%) had etiologies from other causes. When the pleural fluid, blood and/or urine sample in-house nested-PCR sensitivities were evaluated simultaneously, the results were positive regardless of the biological specimen (the sensitivity was 84.2%); however, when the blood and/or urine samples were analyzed together, the sensitivity was 72.2%. When the pleural fluid samples were evaluated alone, the sensitivity was only 33.3%. Conclusions The performance of the diagnostic pleural tuberculosis nested-PCR was directly related to the diversity of the samples collected from the same patient. Additionally, this study may identify a need to prioritize non-invasive blood and urine collection for this diagnosis. .

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Introduction The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) Results Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. Conclusions Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques. .

Bone tuberculosis: a case report on child

The authors report a case of a 12-year-old child with a complaint of pain and deformity in the lower thoracic region that had lasted for two years. Clinical, epidemiological and laboratory characteristics associated with images of apparent damage in the T9-T10 and T11-T12 vertebrae taken by radiography of the thoracic spine and nuclear magnetic resonance in addition to the positivity of the molecular test based on the polymerase chain reaction, led to tuberculous spondylitis being diagnosed and specific therapy was started. Culture of vertebral biopsy was positive for Mycobacterium tuberculosis after thirty days.

Avaliação da associação de polimorfismos de base única em região promotora de genes das citocinas fator de necrose tumoral (TNF) e Interleucina-10 (IL-10) com a susceptibilidade ou resistência a tuberculose pulmonar / Evaluation of the association of single nucleotide polymorphisms in the promoter region of genes of cytokines tumor necrosis factor alpha (TNF) and interleukin-10 (IL-10) with susceptibility or resistance to pulmonary tuberculosis

Polimorfismos de base única (SNPs) em regiões promotoras de genes das citocinas TNF-alfa e IL-10 influenciam a resposta imunológica e podem estar envolvidos na resistência ou susceptibilidade a tuberculose. Este trabalho objetivou avaliar a associação entre os SNPs funcionais dos genes das citocinas TNF-alfa (-308G/A) e IL-10 (-1082A/G) com a resistência ou susceptibilidade a tuberculose pulmonar ativa em pacientes oriundos do estado de Pernambuco. Participaram 282 indivíduos, distribuídos em grupos de estudo de acordo com achados clínicos, radiológicos e exames laboratoriais. O grupo caso foi formado por 71 pacientes com tuberculose pulmonar ativa; controle 1: 53 pacientes sintomáticos respiratórios com tuberculose latente; controle 2: 57 pacientes sintomáticos respiratórios portadores de infecções pulmonares inespecíficas e o controle 3 por 101 indivíduos clinicamente saudáveis, de serviços públicos de saúde. Foram coletados 5-10mL de escarro e 4,5mL de sangue periférico, realizada extração de DNA genômico e determinação do polimorfismo genético através de qPCR. A análise polimórfica comparativa entre os grupos de estudo demonstrou que o alelo mutante -1082G [p<0.0001; OR=3.90 [2.45- 6.59] e os genótipos -1082GA [p<0.0001; OR=2.90 [1.42- 5.95] e -1082GG [p<0.0001; OR=15.50 [5.18- 1.62] estava associado com o risco de desenvolvimento da tuberculose pulmonar quando o grupo caso foi comparado com o controle 3. O genótipo heterozigoto -308AG [p=0.005; OR=0.374 [0.17–0.76] apresentou associação estatística significante entre o grupo caso e o controle 3

Não houve diferença estatística significante na análise de associação dos SNP´s -308G/A e -1082A/G na comparação entre o grupo caso e os controles 1 e 2. Portanto, portadores do alelo mutante -1082G, equivalente a níveis protéicos aumentados de IL-10 podem estar influenciando a resposta imune contra o M. tuberculosis. Entretanto, portadores do genótipo heterozigoto -308GA, demonstrou um fator de proteção ao desenvolvimento da doença. O estudo demonstrou que os SNPs apresentaram um importante papel na susceptibilidade ou resistência a tuberculose pulmonar na população pernambucana estudada

Evaluation of a nested-pcr for Mycobacterium tuberculosis detection in blood and urine samples

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.

Diferenciação de micobactérias por PCR multiplex / Differentiation of micobacteria by multiplex PCR

O trabalho visou à otimização de um método baseado na reação em cadeia da polimerase multiplex - para diferenciação de micobactérias de interesse para a saúde pública. A PCR Multiplex baseou-se na amplificação simultânea do genehsp65, presente em todo gênero Mycobacterium, do gene dnaJ, presente apenas em Mycobacterium tuberculosis e Mycobacterium avium e da sequência de inserção IS6110 presente no complexo Mycobacterium tuberculosis, gerando amplicons de 165pb, 365pb e 541pb, respectivamente. O limite de detecção foi de 1fg para o alvo hsp65, 100pg para o dnaJ e 0,1fg para o IS6110. A PCR multiplex detectou até 100pg de DNA de Mycobacterium tuberculosis. O sistema demonstrou ser específico e sensível na detecção de Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium e Mycobacterium smegmatis. Os resultados obtidos utilizando cepas de referência demonstraram que a PCR multiplex pode ser uma ferramenta rápida, sensível e específica na diferenciação de micobactérias.

This study aimed to optimize a method based on the polymerase chain reaction - multiplex PCR - for differentiation of mycobacteria species of interest for public health. The multiplex PCR was based on simultaneous amplification of the hsp65 gene, which is present in all species of the Mycobacterium genus, the dnaJ gene, which is present only in Mycobacterium tuberculosis and Mycobacterium avium and the IS6110 insertion sequence, which is present in the Mycobacterium tuberculosis complex, generating amplicons of 165 bp, 365 bp and 541 bp, respectively. The detection limit was 1 fg for the hsp65 target, 100 pg for dnaJ and 0.1 fg for IS6110. The multiplex PCR detected down to 100 pg of DNA of Mycobacterium tuberculosis. The system was shown to be specific and sensitive for detection of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. The results obtained using reference strains of mycobacteria showed that multiplex PCR may be a fast, sensitive and specific tool for differentiation of mycobacteria.

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos / Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients

Objetivo: Avaliar o desempenho da técnica nested PCR (nPCR) para detectar o complexo Mycobacterium tuberculosis em amostras de sangue de pacientes com suspeita de TB para sua possível utilização como uma ferramenta auxiliar no diagnóstico laboratorial da doença em crianças. Métodos: Detecção do complexo M. tuberculosis em amostras de sangue usando como alvo a sequência de inserção IS6110 do DNA genômico do bacilo. Foram avaliados 120 pacientes, menores de 15 anos de idade, de ambos os sexos, provenientes de hospitais públicos do Recife (PE), no período entre janeiro de 2003 e agosto de 2005. O diagnóstico de TB foi realizado pelo médico assistente do serviço de saúde de acordo com os critérios da Sociedade Torácica Americana. A nPCR amplificou um fragmento de 123 pb com oligonucleotídeos externos (IS1/IS2) e, na reação subsequente, com oligonucleotídeos internos (IS3/IS4), gerando um amplicon de 81 pb. Resultados: A TB ativa ou latente esteve presente em 65 pacientes, foi descartada em 28 suspeitos e 27 não tinham a doença (controles). A sensibilidade da nPCR foi de 26,15%, sendo significativamente maior na forma extrapulmonar (55,56%) em relação à pulmonar (18,18%), e a especificidade foi de 92,73%. Conclusões: Diante das dificuldades diagnósticas da TB infantil e do baixo número de casos estudados,a nPCR em sangue demonstrou ser uma técnica rápida e específica, mas com baixa sensibilidade. Para saber a suareal utilidade no diagnóstico de formas paucibacilares, sobretudo as extrapulmonares, novas pesquisas devem ser desenvolvidas com uma casuística maior de crianças e com outros espécimes biológicos além do sangue.

Objective: To evaluate the performance of nested PCR (nPCR) in detecting the Mycobacterium tuberculosis complex in blood samples of patients suspected of having TB, in order to determine its potential for use as an auxiliary tool in the laboratory diagnosis of TB in children. Methods: Detection of the M. tuberculosis complex in blood samples using as a target the insertion sequence IS6110 of the genomic DNA of the bacillus. Blood samples of 120 patients were evaluated. All of the patients were under 15 years of age at the time of their treatment at public hospitals in the city of Recife, Brazil (between January of 2003 and August of 2005). Attending physicians at the hospitals diagnosed TB based on the criteria recommended by the American Thoracic Society. The nPCRamplified a 123-bp fragment with outer oligonucleotides (IS1/IS2) and, in the subsequent reaction, using inner oligonucleotides (IS3/IS4), generating an 81-bp amplicon. Results: Active or latent TB was found in 65 patients,TB was ruled out in 28 suspected cases, and 27 patients were TB-free (controls). The sensitivity of nPCR was26.15% and was significantly higher for the extrapulmonary form of the disease (55.56%) than for the pulmonary form (18.18%). The specificity was 92.73%. Conclusions: Despite the difficulties in diagnosing TB in children and the low number of cases evaluated in the present study, nPCR in blood samples proved to be a rapid and specifictechnique, albeit one with low sensitivity. In order to establish its true usefulness in the diagnosis of paucibacillary forms, especially extrapulmonary TB, further studies need to be carried out with a larger sample of children and analyzing biological specimens other than blood.

Espondilite tuberculosa em adolescente / Tuberculous spondylitis in teenager

Relata-se o caso de uma adolescente com tuberculose osteoarticular em coluna lombossacral, uma localização incomum. O seu diagnóstico permanece um desafio por apresentar sintomas gerais inespecíficos e lesões ósseas que podem ser confundidas com outras afecções. A doença é degenerativa e de prognóstico reservado. São discutidos aspectos clínicos, laboratoriais e de imagem, incluindo tomografia computadorizada e ressonância magnética. A reação em cadeia da polimerase, usando o marcador IS 6110 para M. tuberculosis, foi positiva, sugerindo fortemente a presença do patógeno. Este ensaio é particularmente indicado quando se exige um diagnóstico de tuberculose rápido e sensível.