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Estudo descritivo, qualitativo, que objetivou conhecer os significados da pratica de automedicacao na visao de idosos da UNATI da PUC Goias

Background: The concomitant presence of intestinal parasite infections (IP) and tuberculosis (TB) has relevance. M. tuberculosis immune response is associated with type 1 T helper cell (Th1) while IP is associated with type Th2 cell. However, there are several contradictory reports on cytokine production under coinfection and this could be in association to the mycobacterial antigens used in the studies. Aim: To get insight into the effects of different M. tuberculosis-specific antigens (ESAT-6/CFP-10 and 38 kDa/CFP-10) in generating of appropriate cytokines on peripheral blood mononuclear cells of IPTB co infected patients. Method: ELISA assessed IFN-γ and other 16 cytokines production and plasm IgE. In 18 months, we documented demographic, economic, clinical characteristics and IP frequency in individuals from Brazil. Results: An overall 10/35 (28.5%) were IPTB co infected and 40/76 (52.6%; p = 0.024) asymptomatic intestinal parasite infected community controls (IPCC). Endo-limax nana (40%) and Entamoeba coli (22%), were the most nonpathogenic protozoan identified and Entamoeba histolytica, Giardia intestinalis, Ascaris lumbricoides and Strongyloides stercoralis were the pathogenic species (40%). IgE was higher in IPCC (p = 0.036). Cytokine profiles were significantly biased toward a Th2 type IL-5 (p = 0.001) and IL-13 (p = 0.033), pro-inflammatory GM-CSF (p = 0.019) and borderline lower IL-1β in IPTB, all associated with ESAT-6/CFP-10, while IL-7 was borderline lower, but 38 kDa/CFP-10 associated; as well as IL-8 higher (p < 0.049) vs CC/IPCC. The TB/IPTB IFN-γ levels were similar to both antigens stimuli (p ≥ 0.208). Conclusion: Therefore, coin-cident IPTB coinfection did not exert a significant inhibitory effect in IFN-γ production in response to either of the two antigens, but the partial discrepancy in Th1/Th2 response, is associated with the antigen priming cells.

A herd infected naturally with tuberculosis was investigated by different diagnostic methods. Ninety days after a screening test that identified 21 cows as skin test positive, a Comparative Intradermal Tuberculin Test (CITT) was performed in those 21 cows and in 29 other randomly selected skin test negative cows. Milk samples and nasal swabs were collected prior to the CITT for bacteriological culture and PCR, while blood samples were collected for IFN release and antibody responses to MPB70 and MPB83, at three time points post tuberculin injection. Animals positive by CITT were slaughtered and disease confirmation undertaken. Based on the Kappa test, IFN was comparable to the standard tests (culture, PCR and CITT) at all three sampling points. Results from both antibody ELISAs were similar but were not comparable to the standard tests. T-test analysis of the CITT, IFN and ELISAs demonstrated that their performances were not correlated. There is increasing recognition that individually, available diagnostic tests do not detect all infected cattle. Therefore, a comprehensive strategy for the diagnosis of bovine TB should include test results for the detection of both cellular and humoral immune responses where there may be animals at different stages of infection. Um rebanho bovino naturalmente infectado por tuberculose foi analisado através de diferentes métodos diagnósticos. Um teste intradérmico simples (TIC) identificou 21 animais como positivos. Após 90 dias deste resultado, um teste intradérmico comparativo (TIC) foi aplicado nos 21 animais positivos ao TIS, além de outros 29 animais com resultados prévios negativos escolhidos aleatoriamente. De todos estes animais (50), foram coletadas amostras de leite e secreção nasal para isolamento e identificação de microrganismos por cultura e PCR; amostras de sangue de cada um dos animais foram coletadas para exames de ELISA: produção de Interferon-gama (IFN) e pesquisa de anticorpos frente aos antígenos MPB70 e MPB83. Tais amostras sanguíneas foram coletadas em três diferentes momentos: no dia da execução do TIC e nos dias dia 7 e dia 21 após a execução do TIC. Os animais que foram positivos a este teste foram abatidos; exames de identificação do agente, tais como cultivo e PCR foram realizados post-mortem para confirmação da doença. Baseado na análise Kappa, IFN apresentou resultados estatisticamente comparáveis aos resultados de isolamento e identificação bacteriana por cultura e PCR, além do TIC ao longo de todo o experimento. No entanto, TIC, ELISA e IFN não foram estatisticamente comparáveis. Tais resultados sugeriram que nenhum dos atuais métodos de diagnóstico para tuberculose possibilitou a identificação de todos os animais infectados. Por este motivo, uma estratégia mais abrangente deveria incluir métodos de diagnóstico que pudessem identificar a resposta imune celular e humoral, uma vez que animais de um mesmo rebanho poderiam se encontrar em diferentes estágios da infecção.

A correlação entre a resistência à pirazinamida (PZA) e a resistência a outros fármacos antituberculose de primeira linha foi investigada em 395 cepas de Mycobacterium tuberculosis provenientes de espécimes clínicos, que representavam 14% do total de isolados de M. tuberculosis no período entre 2003 e 2008 no laboratório de um hospital universitário de referência para tuberculose. Uma alta correlação foi encontrada entre resistência a PZA e multirresistência, assim como entre resistência a PZA e resistência a rifampicina, isoniazida e etambutol (p < 0,01 para todos). Esses resultados enfatizam a importância da realização do teste de sensibilidade a PZA antes de prescrever a droga para o tratamento de tuberculose resistente e multirresistente.

In 2005 and 2006, 8,121 clinical specimens submitted to the Mycobacteriology Laboratory of the Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, in the city of Rio de Janeiro, Brazil, were inoculated on Lowenstein-Jensen medium containing glycerol and pyruvate. There were 79 mycobacteria isolates that presented growth only on pyruvate-containing medium, and those isolates were selected for the presumptive identification of Mycobacterium bovis. The selected isolates were screened with biochemical tests, PCR amplification (with the specific primers Rv0577 and Rv1510), and pyrazinamide susceptibility tests. All of the strains isolated showed specific phenotypical and genotypical patterns characteristic of M. tuberculosis, and no M. bovis strains were detected.

OBJETIVO: Identificar micobactérias não tuberculosas (MNT) isoladas de sítios estéreis em pacientes internados no Hospital Universitário Clementino Fraga Filho, Rio de Janeiro (RJ) entre 2001 e 2006. MÉTODOS: Durante o período do estudo, 34 isolados de MNT de sítios estéreis de 14 pacientes, a maioria HIV positivos, foram submetidos a identificação fenotípica e hsp65 PCR-restriction enzyme analysis (PRA, análise por enzimas de restrição por PCR do gene hsp65). RESULTADOS: A maioria dos isolados foi identificada como Mycobacterium avium, seguida por M. monacense, M. kansasii e M. abscessus em menores proporções. CONCLUSÕES: A combinação de PRA, um método relativamente simples e de baixo custo, com algumas características fenotípicas pode fornecer a identificação correta de MNT na rotina de laboratórios clínicos.

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p < 0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.

An epidemic of infections after video-assisted surgery (1,051 possible cases) caused by rapidly growing mycobacteria (RGM) and involving 63 hospitals in the state of Rio de Janeiro, Brazil, occurred between August 2006 and July 2007. One hundred ninety-seven cases were confirmed by positive acid-fast staining and/or culture techniques. Thirty-eight hospitals had cases confirmed by mycobacterial culture, with a total of 148 available isolates recovered from 146 patients. Most ( n = 144; 97.2%) isolates presented a PRA- hsp65 restriction pattern suggestive of Mycobacterium bolletii or Mycobacterium massiliense . Seventy-four of these isolates were further identified by hsp65 or rpoB partial sequencing, confirming the species identification as M. massiliense . Epidemic isolates showed susceptibility to amikacin (MIC at which 90% of the tested isolates are inhibited [MIC 90 ], 8 μg/ml) and clarithromycin (MIC 90 , 0.25 μg/ml) but resistance to ciprofloxacin (MIC 90 , ≥32 μg/ml), cefoxitin (MIC 90 , 128 μg/ml), and doxycycline (MIC 90 , ≥64 μg/ml). Representative epidemic M. massiliense isolates that were randomly selected, including at least one isolate from each hospital where confirmed cases were detected, belonged to a single clone, as indicated by the analysis of pulsed-field gel electrophoresis (PFGE) patterns. They also had the same PFGE pattern as that previously observed in two outbreaks that occurred in other Brazilian cities; we designated this clone BRA100. All five BRA100 M. massiliense isolates tested presented consistent tolerance to 2% glutaraldehyde. This is the largest epidemic of postsurgical infections caused by RGM reported in the literature to date in Brazil.

This study evaluated the biodiversity of 28 clinical and 24 environmental Mycobacterium isolates from Rio de Janeiro, Brazil, by using hsp65 sequences, with the aim of contributing to a better understanding of the genetic diversity and usefulness of this marker. An extensive phylogenetic analysis was performed. The nucleotide diversity was similar between clinical (0.06508) and environmental (0.06221) isolates.

Summary Objectives Rapidly growing mycobacteria (RGM) have emerged as important pathogens in clinical settings, associated with esthetic procedures and postsurgical infections, pulmonary infections among cystic fibrosis patients, and other structural pulmonary diseases. Microorganisms belonging to Mycobacterium abscessus – Mycobacterium chelonae and to Mycobacterium fortuitum groups have frequently been associated with outbreaks and various epidemics. In the present study, RGM strains were characterized in order to investigate molecular markers based on proteomic analysis. Methods Multilocus enzyme electrophoresis (MLEE) was used for species identification and clonal analysis of RGM recovered from postsurgical wound infections during an epidemic. The study included 30 M. abscessus subsp. bolletii clinical isolates, most belonging to the BRA100 clone (epidemic in Rio de Janeiro city), as well as 16 RGM ATCC reference strains. Results Molecular typing allowed the detection of diversity in the studied population and revealed species-specific isoenzymatic patterns. Additionally, the clonal relationship among M. abscessus subsp. bolletii outbreak isolates, as examined using MLEE, was markedly consistent. Conclusions Isoenzymatic characterization was found to be a useful molecular tool to identify RGM species and to determine the relatedness among closely related M. abscessus subsp. bolletii isolates. This may be considered a powerful approach for epidemiological studies on RGM.

This study aimed to assess drug resistance in Mycobacterium tuberculosis strains isolated from sputum samples. To that end, sputum samples were collected from 263 patients suspected of having tuberculosis. All subjects lived in the Complexo de Manguinhos, which is located in the city of Rio de Janeiro, Brazil. Cultures testing positive between October of 2000 and December of 2002 were tested to determine strain susceptibility to isoniazid, rifampicin, streptomycin, ETHionamide, and ETHambutol. Of the 75 patients diagnosed with tuberculosis, resistance to at least one of the drugs was found in 16 (21.4%). Of those 16 patients, 8 (50%) were new cases, and 8 (50%) had previously been treated. Multidrug-resistant tuberculosis was identified in 8 (10.6%) of the 75 patients, being associated with previous treatment in 6 (8%). The incidence of multidrug-resistant tuberculosis might have been underestimated, since M. tuberculosis was not isolated from all of the samples testing positive for acid-fast bacilli. However, at least, our findings shed some light on the problem.

Forty-four cows from five herds infected with tuberculosis (TB) and without paratuberculosis (PTB), and 21 cows from a herd without either infection were studied. The cattle presented concordant results in both the skin test and gamma-interferon assay for TB and two commercial ELISAs for PTB. Animals were divided according to TB test results into Group A with 28 TB-infected animals, Group B with 16 TB-negative animals from infected herds, and Group C with 21 TB-negative cows from a tuberculosis-free herd (which were used as controls). Twenty of 28 animals from Group A (71.4%), 6/16 from Group B (37.5%) and none from Group C were reactive to PTB ELISAs, suggesting that these commercial kits were unable to distinguish between PTB and TB. It is proposed that natural occurring TB strongly interferes in the diagnosis of PTB and that commercial ELISAs cannot be considered reliable tools in the diagnosis of paratuberculosis in tuberculosis-infected herds.

Double repetitive element (DRE) PCR amplification is a simple Mycobacterium tuberculosis typing method, however amplification failure or poor resolution of bands commit its efficacy. In order to verify if whether or not these features could be minimized by improving DNA extraction procedures or Taq polymerise quality, DRE-PCR was performed on 24 M. tuberculosis DNA samples extracted by heat-shock, mechanical and enzymatic methods applying conventional and hot start Taq pol. We demonstrated that when dealing with the Mycobacterium tuberculosis DRE-PCR typing method, Taq pol of better quality might be more important to improve amplification than the DNA extraction method.Amplificação de duplo elemento repetido (DRE) por PCR é um método simples para tipagem de Mycobacterium tuberculosis, entretanto falha ou a baixa resolução das bandas na amplificação compromete a eficiência do método. Com o objetivo de verificar se estes problemas podem ou não ser minimizados pela utilização de diferentes procedimentos de extração de DNA ou de qualidades de Taq polimerase, DRE-PCR foi ensaiado em 24 amostras de DNA de M. tuberculosis extraídos pelos métodos de choque-térmico, - mecânico e enzimático utilizando Taq polimerase convencional e hot start Taq pol. Foi demonstrado que a qualidade da Taq pol utilizada talvez seja mais importante para uma melhor amplificação que o método de extração de DNA empregado.

Forty-four cows from five herds infected with tuberculosis (TB) and without paratuberculosis (PTB), and 21 cows from a herd without either infection were studied. The cattle presented concordant results in both the skin test and gamma-interferon assay for TB and two commercial ELISAs for PTB. Animals were divided according to TB test results into Group A with 28 TB-infected animals, Group B with 16 TB-negative animals from infected herds, and Group C with 21 TB-negative cows from a tuberculosis-free herd (which were used as controls). Twenty of 28 animals from Group A (71.4%), 6/16 from Group B (37.5%) and none from Group C were reactive to PTB ELISAs, suggesting that these commercial kits were unable to distinguish between PTB and TB. It is proposed that natural occurring TB strongly interferes in the diagnosis of PTB and that commercial ELISAs cannot be considered reliable tools in the diagnosis of paratuberculosis in tuberculosis-infected herds.Quarenta e quatro animais provenientes de cinco rebanhos infectados com tuberculose e livres de paratuberculose, e 21 animais provenientes de rebanhos livres de ambas as infecções foram analisados. Todos os animais foram testados para tuberculose pelo teste intradérmico de PPD-bovino e pelo ensaio comercial de Interferon-gama (IFN-gama). Para o diagnóstico de paratuberculose, dois ELISAs comerciais foram usados neste estudo. Os animais foram divididos em três grupos, de acordo com os resultados obtidos pelos testes diagnósticos para tuberculose. O grupo A foi composto por 28 animais com resultados positivos para tuberculose; o grupo B foi formado por 16 animais provenientes de rebanhos infectados com tuberculose, porém com resultados negativos nos testes diagnósticos para esta infecção. O grupo C foi composto por 21 animais provenientes de propriedades livres de ambas as doenças e que foram utilizados como grupo controle deste estudo. Vinte dos 28 animais do grupo A (71,4%), 6/16 do grupo B (37,5%) e nenhum animal do grupo C foram reativos aos ELISAs para diagnóstico de paratuberculose, o que demonstrou que os kits comerciais utilizados não são capazes de diferenciar entre ambas as doenças. Os resultados aqui obtidos sugerem que a ocorrência natural de tuberculose pode interferir no diagnóstico da paratuberculose e os ELISAs comerciais disponíveis não são seguros para utilização em rebanhos onde há tuberculose.

To describe some aspects of tuberculosis in a low-income community (the Complexo de Manguinhos, in Rio de Janeiro, Brazil), a retrospective study was carried out. Of the 290 cases reported in the 2000-2002 period, 75.8% were new cases. The annual incidence rates were 157/100,000 (2000), 205/100,000 (2001), and 145/100,000 (2002). Although there was a tendency toward a decrease in the number of cases over the period studied, the difference was not significant, suggesting that tuberculosis continues to be endemic in the area. Therefore, despite the existence of local public health care services, more efficient strategies should be implemented in order to increase the effectiveness of tuberculosis control programs in the area.

There are no reliable means for detecting subclinical mycobacterial infections. The recent sequencing of several mycobacterial genomes has now afforded new opportunities for the development of pathogen-specific diagnostic tests, critical in the context of leprosy and tuberculosis control. In the present study, we applied a multi-parametric flow cytometric analysis that allowed the investigation of T-cell functions in order to define immunological markers that measure previous exposure to mycobacteria. We compared the in vivo response to PPD, the gold standard skin test reagent for measuring previous exposure to Mycobacterium tuberculosis, with in vitro parameters of leukocyte activation in five PPD positive and five PPD negative healthy volunteers. PPD-stimulated peripheral leukocytes expressing CD4, CD69, cutaneous lymphocyte-associated antigen (CLA) and intracellular IFN-gamma were enumerated in whole blood and compared with the size of in vivo PPD-induced induration and IFN-gamma production levels as measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells. The reactivity to the tuberculin skin test (TST) was associated with markedly increased frequencies of PPD-responsive activated (CD69+) and IFN-gamma-producing CD4+T cells. Detection of PPD-specific IFN-gamma producing leukocytes was restricted to CD4+T cells and a subset of these cells was shown to express the skin homing molecule CLA. Multiple linear regression modeling of responses to PPD showed the highest association between skin test indurations and frequencies of PPD-responsive IFN-gamma-producing CD4+CD69+ T cells. Our data show that the in vitro enumeration of antigen-specific IFN-gamma-producing CD4+ T cells can provide an alternative to the in vivo tuberculin test for the detection of latent Mycobacterium tuberculosis infection. Moreover, the measurement of these immunological parameters can be useful for the screening of new specific antigens defined by the genome sequence allowing selection of the best candidates for new diagnostics (including new skin tests), and vaccines for leprosy and tuberculosis.

BACKGROUND: \ud \ud The use of liquid medium (MGIT960) for tuberculosis (TB) diagnosis was recommended by WHO in 2007. However, there has been no evaluation of its effectiveness on clinically important outcomes.\ud \ud METHODS AND FINDINGS: \ud \ud A pragmatic trial was carried out in a tertiary hospital and a secondary health care unit in Rio de Janeiro City, Brazil. Participants were 16 years or older, suspected of having TB. They were excluded if only cerebral spinal fluid or blood specimens were available for analysis. MGIT960 technique was compared with the Lowenstein-Jensen (LJ) method for laboratory diagnosis of active TB. Primary outcome was the proportion of patients who had their initial medical management changed within 2 months after randomisation. Secondary outcomes were: mean time for changing the procedure, patient satisfaction with the overall treatment and adverse events. Data were analysed by intention-to-treat. Between April 2008 and September 2011, 693 patients were enrolled (348 to MGIT, 345 to LJ). Smear and culture results were positive for 10% and 15.7% of participants, respectively. Patients in the MGIT arm had their initial medical management changed more frequently than those in the LJ group (10.1% MGIT vs 3.8% LJ, RR 2.67 95% CI 1.44-.96, p = 0.002, NNT 16, 95% CI 10-39). Mean time for changing the initial procedure was greater in LJ group at both sites: 20.0 and 29.6 days in MGIT group and 52.2 and 64.3 in LJ group (MD 33.5, 95% CI 30.6-36.4, p = 0.0001). No other important differences were observed.\ud \ud CONCLUSIONS: \ud \ud This study suggests that opting for the MGIT960 system for TB diagnosis provides a promising case management model for improving the quality of care and control of TB.\ud \ud TRIAL REGISTRATION: \ud \ud Controlled-Trials.com ISRCTN79888843.

Paratuberculosis is an important enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease is officially considered exotic in Brazil, but recent serological surveys and the isolation of the agent suggest it may occur in our herds. The aim of this study was to evaluate three different formulations of Herrold's egg yolk agar with mycobactin J (HEYM) and four faecal culture protocols considering their ability for Map growth as well as cost and ease of application. Three formulations of HEYM were inoculated with two suspensions of Map. Spiked faeces and naturally contaminated faecal samples were treated by the four faecal culture protocols. Centrifugation protocol and HEYM recommended by OIE showed the best results on the recovery of Map.

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65(PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.

Paratuberculosis (Johne's disease) is a chronic enteritis in cattle caused by infection with Mycobacterium avium subsp. paratuberculosis (Map). In Brazil, few reports describe the isolation of the organism. The gold standard test for Map is its isolation from tissues or feces. Moreover, bacterial growth is slow and test results are available only after three to five months of incubation Furthermore, shedding of bacilli at levels detectable by fecal culture is irregular and does not occur during the early stages of infection, which compromises the sensitivity of this methodology. The most common immunological tests to identify Map infection are complement fixation test (CFT), agar gel immunodiffusion (AGID) and enzyme-linked immunoassay (ELISA). In Brazil, commercial ELISA kits are imported and too expensive to be used as part of diagnostic laboratorial routine. Apart from that, their use has not yet been approved in the country. The aim of the present study was to improve an original assay PPA-ELISA protocol established by our group, and to determine sensitivity and specificity of the modified test. In a first step, we introduced modifications that minimized plate-to-plate and between-well variations, thus making the test more stable and reliable. In the second part of this study, a panel of 106 sera samples was tested by this modified PPA-ELISA protocol in order to estimate its sensitivity and specificity. The new assay presented overall sensitivity of 76.9% and specificity of 70.0%. Our study demonstrated that this assay could be recommended as a valuable tool for diagnosis and control of paratuberculosis in Brazil and other developing countries.Paratuberculose (doença de Johne) se caracteriza por uma enterite crônica de ruminantes, causada por Mycobacterium avium subesp. paratuberculosis (Map). O padrão ouro diagnóstico da infecção é a cultura e isolamento do microrganismo a partir de tecidos ou fezes. No entanto, seu crescimento demora de três a cinco meses e a excreção de Map em níveis detectáveis em cultura é irregular e não ocorre durante os primeiros estágios da infecção, o que diminui seus níveis de sensibilidade. No Brasil, existem poucos relatos de seu isolamento. Os testes imunológicos mais comuns para a identificação da infecção são: Fixação de complemento, imunodifusão em gel de agarose e ensaio imunoenzimático do tipo ELISA. No Brasil, kits comerciais ELISA de diagnóstico são importados e seu uso não está ainda aprovado. O objetivo deste estudo é melhorar um ensaio ELISA-PPA in-house previamente desenvolvido (10) e determinar sua sensibilidade e especificidade. Na primeira etapa deste estudo, as alterações do protocolo original contribuíram para diminuir as variações encontradas entre poços na mesma placa e entre placas em experimentos diferentes, o que demonstrou uma maior estabilidade e reprodutibilidade do teste. Na segunda etapa, um painel de 106 amostras de soros foi testado pelo protocolo modificado do ELISA-PPA para estimar seus índices de sensibilidade e especificidade. O ensaio apresentou sensibilidade de 76,9% e especificidade de 70,0%. Nosso estudo demonstrou que este ensaio pode ser recomendado como uma valiosa ferramenta para diagnóstico e controle da paratuberculose no Brasil e em outros países em desenvolvimento.

In view of the fact that the World Health Organization has recommended the use of the mycobacteria growth indicator tube (MGIT) 960 system for the diagnosis of tuberculosis and that there is as yet no evidence regarding the clinical impact of its use in health care systems, we conducted a pragmatic clinical trial to evaluate the clinical performance and cost-effectiveness of the use of MGIT 960 at two health care facilities in the city of Rio de Janeiro, Brazil, where the incidence of tuberculosis is high. Here, we summarize the methodology and preliminary results of the trial. (ISRCTN.org Identifier: ISRCTN79888843 [http://isrctn.org/]) In view of the fact that the World Health Organization has recommended the use of the mycobacteria growth indicator tube (MGIT) 960 system for the diagnosis of tuberculosis and that there is as yet no evidence regarding the clinical impact of its use in health care systems, we conducted a pragmatic clinical trial to evaluate the clinical performance and cost-effectiveness of the use of MGIT 960 at two health care facilities in the city of Rio de Janeiro, Brazil, where the incidence of tuberculosis is high. Here, we summarize the methodology and preliminary results of the trial. (ISRCTN.org Identifier: ISRCTN79888843 [http://isrctn.org/]).

This study evaluated the oxacillin susceptibilities of 152 coagulase-negative staphylococcal (CoNS) strains of 12 species by disk diffusion; agar dilution; E-test; the slide latex agglutination test (Slidex MRSA Detection test; bioMérieux S/A, Paris, France); the agar screening test with 1, 2, 4, or 6 μg of oxacillin per ml and incubation for 24 or 48 h; and detection of the mecA gene by PCR. The results revealed that the agar screening test with 4 μg of oxacillin per ml and incubation for 48 h was superior to any single phenotype-based susceptibility assay, presenting a sensitivity and a specificity of 100% each. For the different methods evaluated, the sensitivities and specificities were as follows: for disk diffusion, 94.2 and 91.8%, respectively; for the agar dilution test 100 and 73.5%, respectively; for E-test, 100 and 71.4%, respectively; and for the slide latex agglutination test, 97.1 and 98%, respectively. A good correlation was observed between oxacillin susceptibility testing results and PCR results for Staphylococcus epidermidis , S. haemolyticus , S. hominis subsp. hominis , and all mecA -positive strains. However, at least 60% of the mecA -negative isolates of the species S. saprophyticus , S. cohnii subsp. urealyticum , S. lugdunensis , and S. sciuri were erroneously classified as oxacillin resistant by the agar dilution test. Conversely, the slide latex agglutination test presented a high sensitivity (97.1%) and a high specificity (98%) for all CoNS species. Our results demonstrated the accuracy of the agar screening test with 4 μg of oxacillin per ml and incubation for 48 h and the slide latex agglutination test for the appropriate detection of the oxacillin susceptibilities of CoNS isolates. Both assays are technically simple and can be easier to perform in routine laboratories than PCR.

The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.

The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95% and 94%, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries.

Techniques to improve the sensitivity of smear microscopy would facilitate early tuberculosis (TB) diagnosis and disease control, especially in low-income countries where the positive predictive value is high. C 18 -carboxypropylbetaine (CB-18) is a zwitterionic detergent that helps to compensate for the innate buoyancy of mycobacteria, potentially enhancing recovery by centrifugation. Previous data suggest that CB-18 may increase the sensitivity of smear, culture, and molecular amplification diagnostic testing. The goal of the present study was to evaluate if the sensitivity of the smear technique using light microscopy could be improved by treating respiratory samples with CB-18. In the first phase, respiratory specimens were collected consecutively from patients with suspected pulmonary tuberculosis in a tertiary-care hospital in Rio de Janeiro, Brazil (236 specimens were analyzed). After protocol modifications, another 120 respiratory specimens were evaluated. The standard technique was N -acetyl- l -cysteine with sodium hydroxide (NALC-NaOH) treatment, smear concentration with centrifugation, and Ziehl-Neelsen staining. Culture on Löwenstein-Jensen slants was performed on all specimens for use as the “gold standard.” No specimens from patients undergoing active TB treatment were included. The initial protocol for CB-18 processing resulted in a sensitivity of 59.6% and specificity of 96.8% compared to standard processing with a sensitivity of 66.0% and specificity of 96.8%. Using the modified protocol, the sensitivity of CB-18 increased to 71.4% with a specificity of 97.0% versus standard processing with a sensitivity of 61.9% and a specificity of 99.0%. The diagnostic yield of acid-fast bacillus smear with CB-18 in the absence of fluorescence microscopy and PCR compared to standard processing with NALC-NaOH was not significantly different, although the power to detect a difference by the modified assay was low.