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Find video protocols related to scientific articles indexed in Pubmed.
Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.
J. Clin. Microbiol.
PUBLISHED: 06-04-2014
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Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus.
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Prospective cross-sectional evaluation of the small membrane filtration method for diagnosis of pulmonary tuberculosis.
J. Clin. Microbiol.
PUBLISHED: 05-07-2014
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Smear microscopy has suboptimal sensitivity, and there is a need to improve its performance since it is commonly used to diagnose tuberculosis (TB). We prospectively evaluated the diagnostic accuracy of the small membrane filtration (SMF) method, an approach that uses a vacuum manifold and is designed to concentrate bacilli onto a filter that can be examined microscopically. We enrolled hospitalized adults suspected to have pulmonary TB in Kampala, Uganda. We obtained a clinical history and three spontaneously expectorated sputum specimens for smear microscopy (direct, concentrated, and SMF), MGIT (mycobacterial growth indicator tube) 960 and Lowenstein-Jensen (LJ) cultures, and Xpert MTB/RIF testing. We performed per-specimen (primary) and per-patient analyses. From October 2012 to June 2013, we enrolled 212 patients (579 sputum specimens). The participants were mostly female (63.2%), and 81.6% were HIV infected; their median CD4 cell count was 47 cells/?l. Overall, 19.0%, 20.4%, 27.1%, 25.2%, and 25.9% of specimens tested positive by direct smear, concentrated smear, MGIT culture, LJ culture, and Xpert test, respectively. In the per-specimen analysis, the sensitivity of the SMF method (48.5%; 95% confidence interval [CI], 37.4 to 59.6) was lower than those of direct smear (60.9%; 51.4 to 70.5 [P = 0.0001]) and concentrated smear (63.3%; 53.6 to 73.1 [P < 0.0001]). Subgroup analyses showed that SMF performed poorly in specimens having a low volume or low bacterial load. The SMF method performed poorly compared to standard smear techniques and was sensitive to sample preparation techniques. The optimal laboratory SMF protocol may require striking a fine balance between sample dilution and filtration failure rate.
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Importance of cough and M. tuberculosis strain type as risks for increased transmission within households.
PLoS ONE
PUBLISHED: 01-01-2014
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The degree to which tuberculosis (TB) is transmitted between persons is variable. Identifying the factors that contribute to transmission could provide new opportunities for TB control. Transmission is influenced by host, bacterial and environmental factors. However, distinguishing their individual effects is problematic because measures of disease severity are tightly correlated, and assessing the virulence of Mycobacterium tuberculosis isolates is complicated by epidemiological and clinical confounders.
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Discordance of tuberculin skin test and interferon gamma release assay in recently exposed household contacts of pulmonary TB cases in Brazil.
PLoS ONE
PUBLISHED: 01-01-2014
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Interferon-gamma (IFN-?) release assays (IGRAs) such as the Quantiferon Gold In-tube test are in vitro assays that measure IFN-? release from T cells in response to M. tuberculosis (Mtb)-specific antigens. Unlike the tuberculin skin test (TST), IGRA is specific and able to distinguish Mtb-infection from BCG vaccination. In this study we evaluated the concordance between TST and IGRA and the efficacy of IGRA in diagnosing new Mtb infection in household contacts (HHC) of pulmonary tuberculosis (PTB) cases. A total of 357 HHC of TB cases in Vitória, Brazil were studied. A TST was performed within 2 weeks following enrollment of the HHC and if negative a second TST was performed at 8-12 weeks. HHC were categorized as initially TST positive (TST+), persistently TST negative (TST-), or TST converters (TSTc), the latter representative of new infection. IGRA was performed at 8-12 weeks following enrollment and the test results were positive in 82% of TST+, 48% of TSTc, and 12% of TST-, indicating poor concordance between the two test results among HHC in each category. Evaluating CXCL10 levels in a subset of IGRA supernatants or lowering the IGRA cutoff value to define a positive test increased agreement between TST and IGRA test results. However, ROC curves demonstrated that this resulted in a trade-off between sensitivity and specificity of IGRA with respect to TST. Together, the findings suggest that until the basis for the discordance between TST and IGRA is fully understood, it may be necessary to utilize both tests to diagnose new Mtb infection in recently exposed HHC. Operationally, in IGRA negative HHC, it may be useful to employ a lower cutoff value for IGRA to allow closer monitoring for potential conversion.
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Multilocus sequence typing scheme for the Mycobacterium abscessus complex.
Res. Microbiol.
PUBLISHED: 09-17-2013
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We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576 bp): the M. abscessus sensu stricto group (44 STs), the "M. massiliense" group (31 STs) and the "M. bolletii" group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients. Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world.
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Increased sensitivity in diagnosis of tuberculosis in HIV-positive patients through the small-membrane-filter method of microscopy.
J. Clin. Microbiol.
PUBLISHED: 06-26-2013
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The sensitivity of microscopy for the diagnosis of tuberculosis (TB) is around 50% but decreases by about 15% in patients with suspected TB who are coinfected with HIV. Here, we compared the accuracies of three microscopy methods for processing sputum smears (concentration by centrifugation with or without N-acetyl-L-cysteine [NALC] and concentration by filtration on a polycarbonate membrane) to that of culture on Ogawa-Kudoh medium as the gold standard method. Sputum samples were obtained from 432 patients with suspected pulmonary TB, of whom 60% were infected with HIV. Analysis was performed using the first specimen. Compared to the gold standard culture, the small-membrane-filter (SMF) method was the most sensitive microscopic method. In HIV-infected TB patients, the sensitivity of the SMF method was significantly higher than those for centrifugation of sputum samples with or without NALC treatment (61.9%, 47.6%, and 45.2%, respectively; P = 0.001). Similarly, in TB patients without HIV infection, the sensitivity of the SMF method was significantly higher than those for centrifugation of sputum samples with or without NALC treatment (81.8%, 63.6%, and 57.5%, respectively; P = 0.001). In the two study groups, TB patients with or without HIV, no significant differences between the specificities of the three methods were observed. Handling of the second sputum sample similarly by centrifugation with or without NALC and by the SMF method increased positivities by 13%, 11%, and 4%, respectively. The overall agreement between microscopy and culture was above 90% for all groups. Microscopic evaluation of the sputum samples treated with NALC compared to those not treated with NALC did not show any increase in sensitivity. Altogether, the sensitivity of the SMF method is higher than those of the other two microscopic methods studied without a loss of specificity.
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Extrapulmonary tuberculosis: Mycobacterium tuberculosis strains and host risk factors in a large urban setting in Brazil.
PLoS ONE
PUBLISHED: 01-01-2013
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Factors related to the development of extrapulmonary forms of tuberculosis (EPTB) are still poorly understood, particularly in high-endemic countries like Brazil. The objective of the paper is to determine host and Mycobacterium tuberculosis (MTB) strain-related factors associated with the development of EPTB in Espírito Santo state, Brazil.
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Evaluation of oral antiseptic rinsing before sputum collection to reduce contamination of mycobacterial cultures.
J. Clin. Microbiol.
PUBLISHED: 06-15-2011
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To assess whether rinsing with oral antiseptics before sputum collection would reduce contamination of mycobacterial cultures, 120 patients with suspected tuberculosis were randomly assigned to rinse with chlorhexidine or cetylpyridinium mouthwash before collection. The culture contamination rate was significantly lower after rinsing with chlorhexidine before collection, especially for cultures grown in MGIT medium.
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Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains.
J. Clin. Microbiol.
PUBLISHED: 11-24-2010
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Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.
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Household members and health care workers as supervisors of tuberculosis treatment.
Rev Saude Publica
PUBLISHED: 03-27-2010
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To compare tuberculosis cure rates among patients supervised by household members or health care workers.
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Saline nebulization before gastric lavage in the diagnosis of pulmonary tuberculosis in children and adolescents.
J. Trop. Pediatr.
PUBLISHED: 03-05-2010
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The main objective is to assess whether nebulization before gastric lavage (GL) improves its sensitivity for the diagnosis of childhood tuberculosis (TB). Children and adolescents suspected of pulmonary TB were randomly assigned (1 : 2) to nebulization with hypertonic saline 30 min before GL (Neb group; n = 36) or GL without prior nebulization (controls; n = 68). The proportion of positive GL smears was greater in Neb group than in the control group; however, no statistical significance was observed (36.3% vs. 22.2%; p = 0.4). Inhalation of nebulized hypertonic saline before GL did not improve TB diagnosis in this study. Nevertheless, the validation of our data will require large longitudinal studies.
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III Brazilian Thoracic Association Guidelines on tuberculosis.
J Bras Pneumol
PUBLISHED: 08-20-2009
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New scientific articles about tuberculosis (TB) are published daily worldwide. However, it is difficult for health care workers, overloaded with work, to stay abreast of the latest research findings and to discern which information can and should be used in their daily practice on assisting TB patients. The purpose of the III Brazilian Thoracic Association (BTA) Guidelines on TB is to critically review the most recent national and international scientific information on TB, presenting an updated text with the most current and useful tools against TB to health care workers in our country. The III BTA Guidelines on TB have been developed by the BTA Committee on TB and the TB Work Group, based on the text of the II BTA Guidelines on TB (2004). We reviewed the following databases: LILACS (SciELO) and PubMed (Medline). The level of evidence of the cited articles was determined, and 24 recommendations on TB have been evaluated, discussed by all of the members of the BTA Committee on TB and of the TB Work Group, and highlighted. The first version of the present Guidelines was posted on the BTA website and was available for public consultation for three weeks. Comments and critiques were evaluated. The level of scientific evidence of each reference was evaluated before its acceptance for use in the final text.
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Population pharmacokinetics of linezolid in adults with pulmonary tuberculosis.
Antimicrob. Agents Chemother.
PUBLISHED: 06-29-2009
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Nineteen adults with pulmonary tuberculosis received linezolid (600 mg) once or twice daily in an early bactericidal activity trial. A one-compartment population model produced median values for the absorption rate constant, volume of distribution, and elimination rate constant of 1.5 h(-1), 29.6 liters, and 0.25 h(-1) (once daily) and 2.7 h(-1), 32.1 liters, and 0.15 h(-1) (twice daily). Linezolid administered twice daily produced higher values for free drug area under the concentration-time curve (AUC)/MIC and time above MIC. Both regimens achieved free AUC/MIC ratios > 100. Median times above the MIC for free drug were 100% (twice daily) and 63% (once daily).
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Correlations of mutations in katG, oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America.
BMC Microbiol.
PUBLISHED: 02-19-2009
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Mutations associated with resistance to rifampin or streptomycin have been reported for W/Beijing and Latin American Mediterranean (LAM) strain families of Mycobacterium tuberculosis. A few studies with limited sample sizes have separately evaluated mutations in katG, ahpC and inhA genes that are associated with isoniazid (INH) resistance. Increasing prevalence of INH resistance, especially in high tuberculosis (TB) prevalent countries is worsening the burden of TB control programs, since similar transmission rates are noted for INH susceptible and resistant M. tuberculosis strains.
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Mycobacterium tuberculosis DNA fingerprint clusters and its relationship with RD(Rio) genotype in Brazil.
Tuberculosis (Edinb)
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Mycobacterium tuberculosis (Mtb) strains designated as RD(Rio) are responsible for a large cluster of new cases of tuberculosis (TB) in Rio de Janeiro. They were previously shown to be associated with severe manifestations of TB. Here, we used three genotyping methods (IS6110 RFLP, spoligotyping, and multiplex PCR) to characterize RD(Rio) and non-RD(Rio) strains from the metropolitan area of Vitória, State of Espirito Santo in southeast Brazil to determine strain diversity and transmission patterns. Strains with identical IS6110 RFLP patterns were considered to belong to a cluster indicative of recent transmission. Between 2000 and 2010, we identified 5470 new TB patients and genotyped 981 Mtb strains. Of these, 376 (38%) were RD(Rio). By RFLP, 180 (48%) of 376 RD(Rio) strains and 235 (40%) of 593 non-RD(Rio) strains belonged to RFLP cluster pattern groups (p = 0.023). Simpsons diversity index based on RFLP patterns was 0.96 for RD(Rio) and 0.98 for non-RD(Rio) strains. Thus, although RD(Rio) strains appear to be comprised of a fewer number of RFLP genotypes, they represent a heterogeneous group. While TB cases caused by RD(Rio) appear more likely to be due to recent transmission than cases caused by non-RD(Rio) strains, the difference is small. These observations suggest that factors other than inherent biological characteristic of RD(Rio) lineages are more important in determining recent transmission, and that public health measures to interrupt new transmissions need to be emphasized for TB control in Vitória.
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The small membrane filter method of microscopy to diagnose pulmonary tuberculosis.
J. Clin. Microbiol.
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We modified microscopy for acid-fast bacilli to diagnose tuberculosis (TB) using small membrane filters (SMFs) after special processing and prefiltration. With the first specimen obtained from each of 335 persons suspected of having TB, the sensitivity of the new SMF method using fluorescence microscopy (FM) was 89% (95% confidence interval [CI]: 80%, 95%). This was significantly better (P = 0.0001) than the sensitivity of routine FM of centrifuged specimens of 60% (95% CI: 49%, 71%) or that of direct sputum smears of 56% (95% CI: 40%, 72%).
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Evaluation of processing methods to equitably aliquot sputa for mycobacterial testing.
J. Clin. Microbiol.
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We compared bacillary loads after splitting sputum specimens by chemical (N-acetyl-l-cysteine [NALC]) and mechanical homogenization by vortexing with sterile glass beads. NALC and vortexing with glass beads were equally effective at homogenizing sputum specimens, resulting in an equal distribution of tubercle bacilli in the aliquots.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.