104 research outputs found
A commercial line probe assay for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis
BACKGROUND: Mycobacterium tuberculosis is a leading cause of death worldwide. In multi-drug resistant tuberculosis (MDR-TB) infectiousness is frequently prolonged, jeopardizing efforts to control TB. The conventional tuberculosis drug susceptibility tests are sensitive and specific, but they are not rapid. The INNO-LiPA Rif. TB (® )(LiPA) is a commercial line probe assay designed to rapidly detect rifampicin resistance, a marker of MDR-TB. Although LiPA has shown promising results, its overall accuracy has not been systematically evaluated. METHODS: We did a systematic review and meta-analysis to evaluate the accuracy of LiPA for the detection of rifampicin-resistant tuberculosis among culture isolates and clinical specimens. We searched Medline, Embase, Web of Science, BIOSIS, and Google Scholar, and contacted authors, experts and the manufacturer. Fifteen studies met our inclusion criteria. Of these, 11 studies used culture isolates, one used clinical specimens, and three used both. We used a summary receiver operating characteristic (SROC) curve and Q* index to perform meta-analysis and summarize diagnostic accuracy. RESULTS: Twelve of 14 studies that applied LiPA to isolates had sensitivity greater than 95%, and 12 of 14 had specificity of 100%. The four studies that applied LiPA directly to clinical specimens had 100% specificity, and sensitivity that ranged between 80% and 100%. The SROC curve had an area of 0.99 and Q* of 0.97. CONCLUSION: LiPA is a highly sensitive and specific test for the detection of rifampicin resistance in culture isolates. The test appears to have relatively lower sensitivity when used directly on clinical specimens. More evidence is needed before LiPA can be used to detect MDR-TB among populations at risk in clinical practice
GATE : a simulation toolkit for PET and SPECT
Monte Carlo simulation is an essential tool in emission tomography that can
assist in the design of new medical imaging devices, the optimization of
acquisition protocols, and the development or assessment of image
reconstruction algorithms and correction techniques. GATE, the Geant4
Application for Tomographic Emission, encapsulates the Geant4 libraries to
achieve a modular, versatile, scripted simulation toolkit adapted to the field
of nuclear medicine. In particular, GATE allows the description of
time-dependent phenomena such as source or detector movement, and source decay
kinetics. This feature makes it possible to simulate time curves under
realistic acquisition conditions and to test dynamic reconstruction algorithms.
A public release of GATE licensed under the GNU Lesser General Public License
can be downloaded at the address http://www-lphe.epfl.ch/GATE/
Bead Array Direct rRNA Capture Assay (rCapA) for Amplification Free Speciation of Mycobacterium Cultures
Mycobacterium cultures, from patients suspected of tuberculosis or nontuberculous mycobacteria (NTM) infection, need to be identified. It is most critical to identify cultures belonging to the Mycobacterium tuberculosis complex, but also important to recognize clinically irrelevant or important NTM to allow appropriate patient management. Identification of M. tuberculosis can be achieved by a simple and cheap lateral flow assay, but identification of other Mycobacterium spp. generally requires more complex molecular methods. Here we demonstrate that a paramagnetic liquid bead array method can be used to capture mycobacterial rRNA in crude lysates of positive cultures and use a robust reader to identify the species in a direct and sensitive manner. We developed an array composed of paramagnetic beads coupled to oligonucleotides to capture 16 rRNA from eight specific Mycobacterium species and a single secondary biotinilated reporter probe to allow the captured rRNA to be detected. A ninth less specific bead and its associated reporter probe, designed to capture 23S rRNA from mycobacteria and related genera, is included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genera. Using this rRNA capture assay (rCapA) with the array developed we were already able to confirm the presence of members of the M. tuberculosis complex and to discriminate a range of NTM species. This approach is not based on DNA amplification and therefore does not require precautions to avoid amplicon contamination. Moreover, the new generation of stable and cost effective liquid bead readers provides the necessary multiplexing potential to develop a robust and highly discriminatory assay
Rapid Molecular Detection of Rifampicin Resistance Facilitates Early Diagnosis and Treatment of Multi-Drug Resistant Tuberculosis: Case Control Study
Multi-drug resistant tuberculosis (MDR-TB) is a major public health concern since diagnosis is often delayed, increasing the risk of spread to the community and health care workers. Treatment is prolonged, and the total cost of treating a single case is high. Diagnosis has traditionally relied upon clinical suspicion, based on risk factors and culture with sensitivity testing, a process that can take weeks or months. Rapid diagnostic molecular techniques have the potential to shorten the time to commencing appropriate therapy, but have not been put to the test under field conditions.This retrospective case-control study aimed to identify risk factors for MDR-TB, and analyse the impact of testing for rifampicin resistance using RNA polymerase B (rpoB) mutations as a surrogate for MDR-TB. Forty two MDR-TB cases and 84 fully sensitive TB controls were matched by date of diagnosis; and factors including demographics, clinical presentation, microbiology findings, management and outcome were analysed using their medical records. Conventionally recognised risk factors for MDR-TB were absent in almost half (43%) of the cases, and 15% of cases were asymptomatic. A significant number of MDR-TB cases were identified in new entrants to the country. Using rpoB mutation testing, the time to diagnosis of MDR-TB was dramatically shortened by a median of 6 weeks, allowing patients to be commenced on appropriate therapy a median of 51days earlier than those diagnosed by conventional culture and sensitivity testing.MDR-TB is frequently an unexpected finding, may be asymptomatic, and is particularly prevalent among TB infected new entrants to the country. Molecular resistance testing of all acid fast bacilli positive specimens has the potential to rapidly identify MDR-TB patients and commence them on appropriate therapy significantly earlier than by conventional methods
Molecular analysis of Mycobacterium isolates from extrapulmonary specimens obtained from patients in Mexico
<p>Abstract</p> <p>Background</p> <p>Little information is available on the molecular epidemiology in Mexico of <it>Mycobacterium </it>species infecting extrapulmonary sites in humans. This study used molecular methods to determine the <it>Mycobacterium </it>species present in tissues and body fluids in specimens obtained from patients in Mexico with extrapulmonary disease.</p> <p>Methods</p> <p>Bacterial or tissue specimens from patients with clinical or histological diagnosis of extrapulmonary tuberculosis were studied. DNA extracts from 30 bacterial cultures grown in Löwenstein Jensen medium and 42 paraffin-embedded tissues were prepared. Bacteria were cultured from urine, cerebrospinal fluid, pericardial fluid, gastric aspirate, or synovial fluid samples. Tissues samples were from lymph nodes, skin, brain, vagina, and peritoneum. The DNA extracts were analyzed by PCR and by line probe assay (INNO-LiPA MYCOBACTERIA v2. Innogenetics NV, Gent, Belgium) in order to identify the <it>Mycobacterium </it>species present. DNA samples positive for <it>M. tuberculosis </it>complex were further analyzed by PCR and line probe assay (INNO-LiPA Rif.TB, Innogenetics NV, Gent, Belgium) to detect mutations in the <it>rpo</it>B gene associated with rifampicin resistance.</p> <p>Results</p> <p>Of the 72 DNA extracts, 26 (36.1%) and 23 (31.9%) tested positive for <it>Mycobacterium species </it>by PCR or line probe assay, respectively. In tissues, <it>M. tuberculosis </it>complex and <it>M. genus </it>were found in lymph nodes, and <it>M. genus </it>was found in brain and vagina specimens. In body fluids, <it>M. tuberculosis </it>complex was found in synovial fluid. <it>M. gordonae</it>, <it>M. smegmatis</it>, <it>M. kansasii</it>, <it>M. genus</it>, <it>M. fortuitum/M. peregrinum </it>complex and <it>M. tuberculosis </it>complex were found in urine. <it>M. chelonae/M. abscessus </it>was found in pericardial fluid and <it>M. kansasii </it>was found in gastric aspirate. Two of <it>M. tuberculosis </it>complex isolates were also PCR and LiPA positive for the <it>rpo</it>B gene. These two isolates were from lymph nodes and were sensitive to rifampicin.</p> <p>Conclusion</p> <p>1) We describe the <it>Mycobacterium </it>species diversity in specimens derived from extrapulmonary sites in symptomatic patients in Mexico; 2) Nontuberculous mycobacteria were found in a considerable number of patients; 3) Genotypic rifampicin resistance in <it>M. tuberculosis </it>complex infections in lymph nodes was not found.</p
Drug resistance mutations and heteroresistance detected using the GenoType MTBDRplus assay and their implication for treatment outcomes in patients from Mumbai, India
<p>Abstract</p> <p>Background</p> <p>Only 5% of the estimated global multidrug resistant TB (MDRTB) load is currently detected. Endemic Mumbai with increasing MDR would benefit from the introduction of molecular methods to detect resistance.</p> <p>Methods</p> <p>The GenoType MTBDR<it>plus </it>assay was used to determine mutations associated with isoniazid and rifampicin resistance and their correlation with treatment outcomes. It was performed on a convenience sample comprising 88 onset and 67 fifth month isolates for which phenotypic drug susceptibility testing (DST) was determined by the Buddemeyer technique for an earlier study. Simultaneous presence of wild type and mutant bands was referred to as "mixed patterns" (heteroresistance).</p> <p>Results</p> <p>Phenotypically 41 isolates were sensitive; 11 isoniazid, 2 rifampicin, 2 pyrazinamide and 5 ethambutol monoresistant; 16 polyresistant and 78 MDR. The agreement between both methods was excellent (kappa = 0.72-0.92). Of 22 rifampicin resistant onset isolates, the predominant <it>rpoB </it>mutations were the singular lack of WT8 (n = 8) and mixed D516V patterns (n = 9). Of the 64 rifampicin resistant fifth month isolates, the most frequent mutations were in WT8 (n = 31) with a further 9 showing the S531L mutation. Mixed patterns were seen in 22 (34%) isolates, most frequently for the D516V mutation (n = 21). Of the 22 onset and 35 fifth month <it>katG </it>mutants, 13 and 12 respectively showed the S315T1 mutation with loss of the WT. Mixed patterns involving both S315T1 and S315T2 were seen in 9 and 23 isolates respectively. Seventeen of 23 and 23/35 <it>inhA </it>mutant onset and fifth month isolates showed mixed A16G profiles. Additionally, 10 fifth month isolates lacked WT2. Five onset and 6 fifth month isolates had both <it>katG </it>and <it>inhA </it>mutations. An association was noted between only <it>katG </it>but not only <it>inhA </it>resistance and poor outcome (<it>p </it>= 0.037); and additional resistance to ethambutol (<it>p </it>= 0.0033). More fifth month than onset isolates had mixed profiles for at least 1 gene (<it>p </it>= 0.000001).</p> <p>Conclusions</p> <p>The use of the assay to rapidly diagnose MDR could guide simultaneous first- and second-line DST, and reduce the delay in administering appropriate regimens. Furthermore, detection of heteroresistance could prevent inaccurate "cured" treatment outcomes documented through smear microscopy and permit more sensitive detection of neonascent resistance.</p
In-Depth Molecular Characterization of Mycobacterium tuberculosis from New Delhi – Predominance of Drug Resistant Isolates of the ‘Modern’ (TbD1−) Type
BACKGROUND: India has the highest estimated burden of tuberculosis in the world, accounting for 21% of all tuberculosis cases world-wide. However, due to lack of systematic analysis using multiple markers the available information on the genomic diversity of Mycobacterium tuberculosis in India is limited. METHODOLOGY/PRINCIPAL FINDINGS: Thus, 65 M. tuberculosis isolates from New Delhi, India were analyzed by spoligotyping, MIRU-VNTR, large deletion PCR typing and single nucleotide polymorphism analysis (SNP). The Central Asian (CAS) 1 _DELHI sub-lineage was the most prevalent sub-lineage comprising 46.2% (n = 30) of all isolates, with shared-type (ST) 26 being the most dominant genotype comprising 24.6% (n = 16) of all isolates. Other sub-lineages observed were: East-African Indian (EAI)-5 (9.2%, n = 6), EAI6_BGD1 (6.2%, n = 4), EAI3_IND, CAS and T1 with 6.2% each (n = 4 each), Beijing (4.6%, n = 3), CAS2 (3.1%, n = 2), and X1 and X2 with 1 isolate each. Genotyping results from five isolates (7.7%) did not match any existing spoligopatterns, and one isolate, ST124, belonged to an undefined lineage. Twenty-six percent of the isolates belonged to the TbD1+ PGG1 genogroup. SNP analysis of the pncA gene revealed a CAS-lineage specific silent mutation, S65S, which was observed for all CAS-lineage isolates (except two ST26 isolates) and in 1 orphan. Mutations in the pncA gene, conferring resistance to pyrazinamide, were observed in 15.4% of all isolates. Collectively, mutations in the rpoB gene, the katG gene and in both rpoB and katG genes, conferring resistance to rifampicin and isoniazid, respectively, were more frequent in CAS1_DELHI isolates compared to non-CAS_DELHI isolates (OR: 3.1, CI95% [1.11, 8.70], P = 0.045). The increased frequency of drug-resistance could not be linked to the patients' history of previous anti-tuberculosis treatment (OR: 1.156, CI95% [0.40, 3.36], P = 0.79). Fifty-six percent of all new tuberculosis patients had mutations in either the katG gene or the rpoB gene, or in both katG and rpoB genes. CONCLUSION: CAS1_DELHI isolates circulating in New Delhi, India have a high frequency of mutations in the rpoB and katG genes. A silent mutation (S65S) in the pncA gene can be used as a putative genetic marker for CAS-lineage isolates
Unsustainability of recommended fertilization rates for coffee monoculture due to high N2O emissions
A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis
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