Incremento da baciloscopia no diagnóstico de tuberculose pulmonar em pessoas privadas de liberdade / Increment of the microscopy in the diagnosis of pulmonary tuberculosis in inmates

Objetivo: Avaliar um procedimento de fácil execução e baixo custo para incrementar o diagnóstico da tuberculose entre pessoas privadas de liberdade sem riscos de contaminação para profissionais de laboratório. Métodos: Amostras de escarro foram analisadas por baciloscopia após tratamento com hipoclorito de sódio e sedimentação espontânea em comparação à baciloscopia direta convencional, cultura pelo método Ogawa-Kudoh e o teste molecular rápido pelo sistema Xpert®MTB/RIF. Para as análises estatísticas foram empregados os programas Open Epi e SPSS. Resultados: De 436 amostras de escarro submetidas ao cultivo 71 foram positivas (verdadeiros positivos) e dessas 50 foram positivas pela baciloscopia direta convencional e 67 pela baciloscopia do escarro processado, o que corresponde a um incremento de 29% na positividade. Conclusão: O procedimento proposto preserva as vantagens e aumenta a sensibilidade da baciloscopia direta convencional. A implementação dessa técnica para diagnóstico entre grupos vulneráveis em locais de acesso e recursos limitados poderá aumentar a identificação de casos de tuberculose pulmonar.

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

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.

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.

Microscopic detection of Mycobacterium tuberculosis in direct or processed sputum smears.

INTRODUCTION: Microscopic identification of active pulmonary tuberculosis (PTB) from direct smears of sputum (DS) is widely used for detection, but has limited sensitivity. Here, we assessed the yield of acid-fast bacilli (AFB) detection in processed sputum smears (PSS). METHODS: Sputum samples were simultaneously analyzed by direct sputum smearing and after chemical treatment and spontaneous sedimentation. RESULTS: Of the 1,719 samples analyzed, 16.4% were positive for AFB in conventional DS and 21.4% in PSS, corresponding to a 30% increase in detection. CONCLUSIONS: Increased sensitivity from analyzing PSS and better safety protocols will contribute to improved detection and control of the disease.

Microscopic detection of Mycobacterium tuberculosis in direct or processed sputum smears

Abstract INTRODUCTION: Microscopic identification of active pulmonary tuberculosis (PTB) from direct smears of sputum (DS) is widely used for detection, but has limited sensitivity. Here, we assessed the yield of acid-fast bacilli (AFB) detection in processed sputum smears (PSS). METHODS: Sputum samples were simultaneously analyzed by direct sputum smearing and after chemical treatment and spontaneous sedimentation. RESULTS: Of the 1,719 samples analyzed, 16.4% were positive for AFB in conventional DS and 21.4% in PSS, corresponding to a 30% increase in detection. CONCLUSIONS: Increased sensitivity from analyzing PSS and better safety protocols will contribute to improved detection and control of the disease.

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

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.

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.

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.

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.

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. .

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.

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.

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.

Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients.

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 nPCR amplified 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 was 26.15% 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 specific technique, 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.

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.