Usefulness of the polymerase chain reaction dot-blot assay, used with Ziehl-Neelsen staining, for the rapid and conveni­ent diagnosis of pulmonary tuberculosis in human immuno­deficiency virus-seropositive and -seronegative individuals

There are scarce data regarding the value of molecular tests, when used in parallel with classical tools, for the diagnosis of tuberculosis (TB) under field conditions, especially in regions with a high burden of TB-human immunodeficiency virus (HIV) co-infection. We evaluated the usefulness of the polymerase chain reaction dot-blot assay (PCR) used in parallel with Ziehl-Neelsen staining (ZN) for pulmonary tuberculosis (PTB) diagnosis, in a TB-HIV reference hospital. All sputum samples from 277 patients were tested by ZN, culture, and PCR. Performances were assessed individually, in parallel, for HIV status, history of anti-TB treatment, and in different simulated TB prevalence rates. Overall, the PTB prevalence was 46% (128/277); in HIV-seropositive (HIV+) individuals, PTB prevalence was 54% (40/74); the ZN technique had a lower sensitivity (SE) in the HIV+ group than in the HIV-seronegative (HIV–) group (43% vs. 68%; Fisher test, P<0.05); and the SE of PCR was not affected by HIV status (Fisher test; P=0.46). ZN, in parallel with PCR, presented the following results: i) among all PTB suspects, SE of 90%, specificity (SP) of 84%, likelihood ratio (LR)+ of 5.65 and LR– of 0.12; ii) in HIV– subjects: SE of 92%, LR– of 0.10; iii) in not previously treated cases: SE of 90%, LR– of 0.11; iv) in TB, prevalence rates of 5-20%; negative predictive values (NPV) of 98-99%. ZN used in parallel with PCR showed an improvement in SE, LR–, and NPV, and may offer a novel approach in ruling out PTB cases, especially in not previously treated HIV– individuals, attended in hospitals in developing nations

A hipermetioninemia reduz os níveis de fosfolipídios, colesterol e gangliosídios em córtex cerebral de ratos

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Cost-effectiveness analysis of PCR for the rapid diagnosis of pulmonary tuberculosis

<p>Abstract</p> <p>Background</p> <p>Tuberculosis is one of the most prominent health problems in the world, causing 1.75 million deaths each year. Rapid clinical diagnosis is important in patients who have co-morbidities such as Human Immunodeficiency Virus (HIV) infection. Direct microscopy has low sensitivity and culture takes 3 to 6 weeks <abbrgrp><abbr bid="B1">1</abbr><abbr bid="B2">2</abbr><abbr bid="B3">3</abbr></abbrgrp>. Therefore, new tools for TB diagnosis are necessary, especially in health settings with a high prevalence of HIV/TB co-infection.</p> <p>Methods</p> <p>In a public reference TB/HIV hospital in Brazil, we compared the cost-effectiveness of diagnostic strategies for diagnosis of pulmonary TB: Acid fast bacilli smear microscopy by Ziehl-Neelsen staining (AFB smear) plus culture and AFB smear plus colorimetric test (PCR dot-blot).</p> <p>From May 2003 to May 2004, sputum was collected consecutively from PTB suspects attending the Parthenon Reference Hospital. Sputum samples were examined by AFB smear, culture, and PCR dot-blot. The gold standard was a positive culture combined with the definition of clinical PTB. Cost analysis included health services and patient costs.</p> <p>Results</p> <p>The AFB smear plus PCR dot-blot require the lowest laboratory investment for equipment (US$20,000). The total screening costs are 3.8 times for AFB smear plus culture versus for AFB smear plus PCR dot blot costs (US$ 5,635,760 versus US$1,498, 660). Costs per correctly diagnosed case were US$ 50,773 and US$13,749 for AFB smear plus culture and AFB smear plus PCR dot-blot, respectively. AFB smear plus PCR dot-blot was more cost-effective than AFB smear plus culture, when the cost of treating all correctly diagnosed cases was considered. The cost of returning patients, which are not treated due to a negative result, to the health service, was higher in AFB smear plus culture than for AFB smear plus PCR dot-blot, US$ 374,778,045 and US$ 110,849,055, respectively.</p> <p>Conclusion</p> <p>AFB smear associated with PCR dot-blot associated has the potential to be a cost-effective tool in the fight against PTB for patients attended in the TB/HIV reference hospital.</p

PCR colorimetric dot-blot assay and clinical pretest probability for diagnosis of Pulmonary Tuberculosis in Smear-Negative patients

<p>Abstract</p> <p>Background</p> <p>Smear-negative pulmonary tuberculosis (SNPTB) accounts for 30% of Pulmonary Tuberculosis (PTB) cases reported annually in developing nations. Polymerase chain reaction (PCR) may provide an alternative for the rapid detection of <it>Mycobacterium tuberculosis </it>(MTB); however little data are available regarding the clinical utility of PCR in SNPTB, in a setting with a high burden of TB/HIV co-infection.</p> <p>Methods</p> <p>To evaluate the performance of the PCR dot-blot in parallel with pretest probability (Clinical Suspicion) in patients suspected of having SNPTB, a prospective study of 213 individuals with clinical and radiological suspicion of SNPTB was carried out from May 2003 to May 2004, in a TB/HIV reference hospital. Respiratory specialists estimated the pretest probability of active disease into high, intermediate, low categories. Expectorated sputum was examined by direct microscopy (Ziehl-Neelsen staining), culture (Lowenstein Jensen) and PCR dot-blot. Gold standard was based on culture positivity combined with the clinical definition of PTB.</p> <p>Results</p> <p>In smear-negative and HIV subjects, active PTB was diagnosed in 28.4% (43/151) and 42.2% (19/45), respectively. In the high, intermediate and low pretest probability categories active PTB was diagnosed in 67.4% (31/46), 24% (6/25), 7.5% (6/80), respectively. PCR had sensitivity of 65% (CI 95%: 50%–78%) and specificity of 83% (CI 95%: 75%–89%). There was no difference in the sensitivity of PCR in relation to HIV status. PCR sensitivity and specificity among non-previously TB treated and those treated in the past were, respectively: 69%, 43%, 85% and 80%. The high pretest probability, when used as a diagnostic test, had sensitivity of 72% (CI 95%:57%–84%) and specificity of 86% (CI 95%:78%–92%). Using the PCR dot-blot in parallel with high pretest probability as a diagnostic test, sensitivity, specificity, positive and negative predictive values were: 90%, 71%, 75%, and 88%, respectively. Among non-previously TB treated and HIV subjects, this approach had sensitivity, specificity, positive and negative predictive values of 91%, 79%, 81%, 90%, and 90%, 65%, 72%, 88%, respectively.</p> <p>Conclusion</p> <p>PCR dot-blot associated with a high clinical suspicion may provide an important contribution to the diagnosis of SNPTB mainly in patients that have not been previously treated attended at a TB/HIV reference hospital.</p

Avaliação da técnica de PCR in house no diagnóstico de tuberculose pulmonar

Objetivos: Avaliar o desempenho e o custo-efetividade de duas técnicas de PCR (Polymerase Chain Reaction) in house: PCR dot-blot e o PCR utilizando a eletroforese em gel de agarose (PCR-AG) no diagnóstico de Tuberculose Pulmonar em pacientes infectados ou não pelo HIV . Material e Métodos: Um estudo prospectivo foi conduzido (de Maio de 2003 a Maio de 2004) em um Hospital de Referência para TB/HIV. Escarros de 277 indivíduos com suspeita de Tuberculose Pulmonar (PTB) foram testados pelas técnicas de microscopia direta pela coloração de Ziehl-Neelsen (ZN), cultura e pelas metodologias de PCR in house (PCR dot-blot e PCR-AG). A acurácia dos testes foi avaliada de acordo com o status de HIV, com o status de tratamento prévio ou não, e com a estimativa de probabilidade pré-teste feita pelos pneumologistas. O padrão ouro utilizado foi a cultura positiva combinada com a definição clínica de PTB (usando sintomas, fatores de risco e Raios-X). O custo efetividade foi expresso por: a) custo por correto caso diagnosticado de Tuberculose; b) custo por correto caso diagnosticado de Tuberculose e tratado incluindo tratamento de casos falsamente diagnosticados; c) custo por casos incorretamente diagnosticados e não tratados. Resultados: Prevalência de PTB foi 46,2% (128/277); em HIV soropositivo (HIV+) foi de 54% (40/74). Em pacientes com baciloscopias negativas foi de 28,4% (43/151) e nos grupos com probabilidades pré-teste alta, intermediária e baixa foi de 67,4% (31/46); 24% (6/25) e 7,5% (6/80). A sensibilidade e a especificidade do PCR dot-blot foi 74% (CI 95%; 66,1%-81,2%) e 85% (CI 95%; 78,8%-90,3%); do PCR-AG foi 43% (CI 95%; 34,5%-51,6%) e 76% (CI 95%; 69,2%-82,8%), respectivamente. Sensibilidades do PCR dot-blot (72% vs 75%; p=0,46) e PCR-AG (42% vs 43%; p=0,54) foi similar para HIV+ and HIV seronegativo (HIV-). Em relação ao efeito da suspeita clínica no desempenho do PCR em pacientes com PTB e com a baciloscopia negativa, o PCR dot-blot combinado com alta probabilidade pré-teste de TB mostrou uma sensibilidade, Valor Preditivo Negativo (VPN) e Razão de Verossimilhança negativa (RV-) de 93%, 96% e 0,1, respectivamente. Usando a estratégia de associar ZN a Cultura, a PCR-AG e PCR dot-blot. A ZN associada à Cultura mostrou o melhor desempenho com sensibilidade de 94% e VPN de 99%, e RV- de 0,07, seguida da ZN associado ao PCR dot-blot com sensibilidade de 90%, com VPN de 99% e uma RV- de 0,12. Não houve diferença significativa no desempenho do PCR em relação ao status de HIV. Na análise de custo-efetividade os custos por correto caso diagnosticado de Tuberculose foram U$1.462,00 , U$1.296,00 e U$1.136,00; os custos por correto caso diagnosticado de Tuberculose e tratado, incluindo tratamento de casos falsamente diagnosticados, foram de US$1.608,00 para a estratégia do ZN associado ao PCR-dotblot e de US$2.359,00 para a estratégia de ZN associado a Cultura. Os custos por casos não diagnosticados que retornam aos serviços de saúde foram de US$3.735,00 para a estratégia de ZN associado ao PCR-dot-blot e de US$2.329,00 para a estratégia de ZN associada à Cultura. Conclusão: Este estudo mostra que as técnicas de PCR in house podem oferecer uma melhora para o diagnóstico de exclusão de TB em pacientes com suspeita de TB atendidos em hospitais com alta prevalência de TB e HIV.Objective: To evaluate the performance and cost-effectiveness of two in house PCR (Polymerase Chain Reaction) techniques: PCR dot-blot methodology (PCR dot-blot) and PCR agarose gel electrophoresis (PCR-AG) for the diagnosis of Pulmonary Tuberculosis (PTB) in patients with or without HIV. Methods: A prospective study was conducted (from May 2003 to May 2004) in a TB/HIV reference hospital. First sputum from 277 PTB patients suspects was tested by Ziehl-Neelsen direct microscopy staining (ZN), culture and in house PCR assays (PCR dot-blot and PCR-AG). The accuracy of tests was evaluated in according to HIV status, previous treatment and the estimative of pretest probability (PP) of PTB performed by chest physicians. Gold standard was the culture positive combined with the definition of clinical PTB (based on symptoms, risk factors and chest X- Ray). The cost effectiveness was expressed as: a) cost per correctly diagnosed case of PTB; b) cost per case correctly diagnosed and treated, including treatment of falsely diagnosed cases; c) cost per case incorrectly diagnosed and non treated. Results: The prevalence of PTB was 46$1,462, U$1,296 and U$1,136 for ZN plus culture, ZN plus PCR-AG and ZN plus PCR dot-blot, respectively. The cost per case correctly diagnosed and treated, including treatment of falsely diagnosed cases, was US$1,608 for ZN plus PCR dot-blot and US$2,359 for ZN plus culture. Cost of the return of all false negatives to health service was US$3,735 for ZN plus PCR dot-blot and US$2,329 for ZN plus Culture. Conclusion: This study shows that in house PCR may offer an improvement for ruling out TB diagnosis for PTB suspects assisted at hospitals with a high prevalence of TB and HIV

Detecção de heterozigotos para MPS II (doença de Hunter) através da biologia molecular

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Detecção de heterozigotos para MPS II (doença de Hunter) através da biologia molecular

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Smear plus detect-TB for a sensitive diagnosis of pulmonary tuberculosis : a cost-effectiveness analysis in an incarcerated population

Background: Prison conditions can favor the spread of tuberculosis (TB). This study aimed to evaluate in a Brazilian prison: the performance and accuracy of smear, culture and Detect-TB; performance of smear plus culture and smear plus Detect-TB, according to different TB prevalence rates; and the cost-effectiveness of these procedures for pulmonary tuberculosis (PTB) diagnosis. Methods: This paper describes a cost-effectiveness study. A decision analytic model was developed to estimate the costs and cost-effectiveness of five routine diagnostic procedures for diagnosis of PTB using sputum specimens: a) Smear alone, b) Culture alone, c) Detect-TB alone, d) Smear plus culture and e) Smear plus Detect-TB. The cost-effectiveness ratio of costs were evaluated per correctly diagnosed TB case and all procedures costs were attributed based on the procedure costs adopted by the Brazilian Public Health System. Results: A total of 294 spontaneous sputum specimens from patients suspected of having TB were analyzed. The sensibility and specificity were calculated to be 47% and 100% for smear; 93% and 100%, for culture; 74% and 95%, for Detect-TB; 96% and 100%, for smear plus culture; and 86% and 95%, for smear plus Detect-TB. The negative and positive predictive values for smear plus Detect-TB, according to different TB prevalence rates, ranged from 83 to 99% and 48 to 96%, respectively. In a cost-effectiveness analysis, smear was both less costly and less effective than the other strategies. Culture and smear plus culture were more effective but more costly than the other strategies. Smear plus Detect-TB was the most cost-effective method. Conclusions: The Detect-TB evinced to be sensitive and effective for the PTB diagnosis when applied with smear microscopy. Diagnostic methods should be improved to increase TB case detection. To support rational decisions about the implementation of such techniques, cost-effectiveness studies are essential, including in prisons, which are known for health care assessment problems