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A Novel Real-Time Simultaneous Amplification and Testing Method to Accurately and Rapidly Diagnose Mycobacterium tuberculosis complex.

The aim of this study was to establish and evaluate a simultaneous amplification and testing method for the M. tuberculosis complex (SAT-TB) in clinical specimens using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB, a 170-bp M. tuberculosis 16s rRNA fragment is reverse-transcribed to DNA using M-MLV with specific primers incorporating the T7 promoter sequence, and undergoes successive cycles of amplification using T7 RNA polymerase.

Using a real-time PCR instrument, hybridization of an internal FAM-DABCYL labeled fluorescent probe can be used to detect RNA amplification. SAT-TB takes less than 1.5 h and the sensitivity for M. tuberculosis H37Rv is 100 CFU/ml. The TB probe has no cross-reactivity with non-tuberculosis mycobacteria or other common respiratory tract pathogens.

In 253 pulmonary tuberculosis (PTB) specimens and 134 non-TB specimens, the SAT-TB correlated with 95.6% (370/387) of the BACTEC MGIT 960 culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of PTB were 67.6%, 100%, 100% and 62.0%, respectively, compared to 61.7%, 100%, 100% and 58.0% for BACTEC MGIT 960 culture. For PTB diagnosis, the sensitivity of the SAT-TB and BACTEC MGIT 960 culture were 97.6% and 95.9% for smear positive specimens, and 39.2% and 30.2% for smear negative specimens.

In conclusion, the SAT-TB is a novel, simple test with high specificity, which may enhance the detection rate of TB. It is therefore a promising tool for rapid diagnosis of M. tuberculosis infection in clinical microbiology laboratories.

Interferon-gamma release assay and Rifampicin therapy for household contacts of tuberculosis.

OBJECTIVES: Longitudinal studies in household contacts to identify subgroups at risk of active tuberculosis are lacking.

METHODS: Household contacts of pulmonary tuberculosis patients were prospectively enrolled to receive chest radiography, sputum studies, and T-SPOT.TB assay at initial visit. Repeat examinations every 6 months for 3 years, and 4-month rifampin preventive therapy for T-SPOT.TB-positive contacts were provided. We investigated factors predicting T-SPOT.TB-positivity and active pulmonary tuberculosis.

RESULTS: 583 contacts were enrolled with a follow-up duration of 20.7 ± 9.4 months. 176 (30.2%) were T-SPOT.TB-positive initially and 32 (18.2%) of them received preventive therapy. Old age, living in the same room/house with the index case, the index case having a high smear grade (3+ ∼ 4+) and pulmonary cavitation were associated with T-SPOT.TB-positivity. Active tuberculosis developed in 9 T-SPOT.TB-positive contacts; risk factors included T-SPOT.TB-positivity without preventive therapy, living in the same room, and the index case being ≤50 years or female. 108 (61.4%) T-SPOT.TB-positive contacts had repeat examinations. Forty-five had T-SPOT.TB reversion and none of them developed active tuberculosis.

CONCLUSION: Household contacts who are T-SPOT.TB-positive and live in the same room as the index case are at risk of active tuberculosis and require preventive therapy and close follow-up.

Diagnostic yield of post-bronchoscopy sputum smear in pulmonary tuberculosis.

Background: The early definitive diagnosis of pulmonary tuberculosis (TB) is important for control of the disease in the community. We performed this study to evaluate the additional gain of post-bronchoscopy sputum in the diagnosis of pulmonary TB.

Methods: Bronchoscopy and bronchoalveolar lavage were performed for 126 patients suspected of pulmonary TB who either had 3 negative sputum smears for acid-fast bacilli or could not expectorate. After bronchoscopy the patients were asked to give sputum samples for 3 consecutive days. All of the obtained specimens were investigated for Mycobacterium tuberculosis by smear and culture.

Results: Pulmonary TB was confirmed in 56 patients. Among all confirmed cases, the sensitivity of bronchoalveolar lavage smear was 57.1% (32 of 56), sensitivity of post-bronchoscopy smear was 76.7% (43 of 56), and the yield of a combination of the 2 methods was 83.9% (47 of 56). Results of post-bronchoscopy sputum smears were not significantly related to sex, age, human immunodeficiency virus (HIV) infection, presence of cavitary lesions on chest X-ray, or the ability to expectorate before bronchoscopy (p > 0.05).

Conclusion: Evaluation of post-bronchoscopy sputum smears is helpful for earlier diagnosis of pulmonary TB and is an inexpensive and accessible assay.

Levels of Evidence in Endoscopic Ultrasonography: A Systematic Review.

BACKGROUND AND AIMS: A prolific trend currently designates endoscopic ultrasonography (EUS) literature. We aimed to record all EUS-studies published during the past decade and evaluate them in terms of scientific quality, creating a stratification based on levels of evidence (LE).

METHODS: All articles on EUS published between January 2001 and December 2010 were retrieved using a Pubmed and Cochrane Library search. Inclusion criteria were: original research papers (randomized controlled trials-RCTs, prospective and retrospective studies), meta-analyses, reviews and surveys pertinent to gastrointestinal EUS. Levels of evidence (LE) were assessed using the North of England evidence-based guidelines.

RESULTS: Overall, 1,832 eligible articles were reviewed. The majority (46.1%) of reports comprised retrospective descriptive studies (LE III). Expert reviews and committee reports (LE IV) accounted for 28.9%, well-designed quasi-experimental studies (LE IIb) for 20.1%, RCTs (LE Ib) for 2.4%, prospective controlled trials (LE IIa) for 1.4%, and meta-analyses (LE Ia) for 1.1% of the total. High LE (Ia-Ib) were assigned to loco-regional staging of luminal gastrointestinal cancers; mediastinal staging of lung cancer; diagnostic work-up of solid pancreatic tumors, suspected biliary obstruction and choledocholithiasis; celiac plexus neurolysis; and pancreatic pseudocysts drainage. Intermediate to low LE (IIa-IV) were assigned to submucosal tumors, pancreatic cysts, chronic pancreatitis and novel therapeutic applications (pancreato-biliary drainage, tumor ablation).

CONCLUSIONS: Diagnostic and staging EUS has matured and has proven its clinical impact on patient management. Therapeutic or interventional EUS is still evolving and more quality research and data are needed to establish EUS as the best next intervention to perform once firm evidence is available.

New aspects of photodynamic therapy for central type early stage lung cancer.

BACKGROUND: and Objective Photodynamic therapy (PDT) has come to be considered as the first choice of treatment for central type early stage lung cancer (CELC). Recent advances in the ability to diagnose CELC, and in photosensitizers, as well as sophisticated clinical management, may improve the therapeutic outcome and expand the indications of PDT.

MATERIALS AND METHODS: We made the search for papers on PDT for lung cancer to select the most relevant articles. Based on this review and our recent data, we discussed the best available evidence for the diagnosis, the definition of indications, photosensitizers, and clinical management with regard to PDT.

RESULTS: To obtain complete response (CR) by PDT, the selection of the indications is extremely important, including the extent of the tumor on the bronchial surface and the depth of invasion in the bronchial wall. The development of autofluorescence bronchoscopy (AFB) and endobronchial ultrasonography (EBUS) have had a large impact on diagnostic bronchoscopy for CELC. CELCs less than 1 cm in diameter showed a favorable cure rate by PDT, thus this is a good indication for PDT. The relatively newer photosensitizer NPe6, which has a stronger antitumor effect than Photofrin, showed similar treatment outcome even for large tumors >1.0 cm in diameter. Furthermore, comprehensive management including photodynamic diagnosis before and after PDT should be effective to minimize the possibility of local recurrence after PDT.

CONCLUSION: The present guidelines of PDT for CELC were established based on the data obtained from studies in the 1980's. We postulate that comprehensive diagnosis and the new generation of photosensitizers may increase the CR rate and expand the indications of PDT for larger tumors.

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