Pretreatment time to detection of Mycobacterium tuberculosis in liquid culture is associated with relapse after therapy.
Ontology highlight
ABSTRACT:
SUBMITTER: Bark CM
PROVIDER: S-EPMC3264180 | biostudies-literature | 2012 Feb
REPOSITORIES: biostudies-literature
Publications
Pretreatment time to detection of Mycobacterium tuberculosis in liquid culture is associated with relapse after therapy.
Bark Charles M CM Thiel Bonnie A BA Johnson John L JL
Journal of clinical microbiology 20111123 2
Similar Datasets
Project description:Mycobacterium tuberculosis culture, a critical technique for routine diagnosis of tuberculosis, takes more than two weeks. Here, step-by-step improvements in the protocol including a new medium, microaerophlic atmosphere or ascorbic-acid supplement and autofluorescence detection dramatically shortened this delay. In the best case, primary culture and rifampicin susceptibility testing were achieved in 72 hours when specimens were inoculated directly on the medium supplemented by antibiotic at the beginning of the culture.
| S-EPMC3937792 | biostudies-literature
Project description:Mycobacterium tuberculosis strains of the Beijing genotype were first identified in China and neighboring countries and have attracted special attention due to their global emergence and association with drug resistance. To further analyze the spread and special characteristics of Beijing genotype strains, accurate, rapid and sensitive methods that overcome the drawbacks of the classical methods such as IS6110 DNA fingerprinting or spoligotyping for the identification of strains of this genotype are needed. Based on the nucleotide sequences of M. tuberculosis SAWC0780 and H37Rv, primers and fluorogenic 5' nuclease (TaqMan) probes for real-time PCR assays specific for Beijing and non-Beijing strains, respectively, were designed. The detection limits for the real-time PCR assays were about 5 and 10 copies of chromosomal DNA, respectively. In mixtures of Beijing and non-Beijing DNA, a multiplex assay was able to detect (i) one copy of Beijing DNA in approximately 1,000 copies of non-Beijing DNA and (ii) one copy of non-Beijing DNA in approximately 2,000 copies of Beijing DNA. In a blinded analysis of a collection of 103 multidrug-resistant strains isolated in Germany in 2001, all 62 Beijing and all 41 non-Beijing strains were correctly identified. In conclusion, the real-time assay allows for the rapid and specific detection of Beijing and non-Beijing strains. The major advantages of this test in comparison to other methods used for the identification of Beijing strains are its simplicity and sensitivity and the fact that amplification and detection occur within one reaction tube.
| S-EPMC1392668 | biostudies-literature
Project description:BACKGROUND:Current laboratory methods for monitoring the response to therapy for tuberculosis (TB) rely on mycobacterial culture. Their clinical usefulness is therefore limited by the slow growth rate of Mycobacterium tuberculosis. Rapid methods to reliably quantify the response to anti-TB drugs are desirable. METHODS:We developed 2 real-time PCR assays that use hydrolysis probes to target DNA of the IS6110 insertion element and mRNA for antigen 85B. The nucleic acids are extracted directly from concentrated sputum samples decontaminated with sodium hydroxide and N-acetyl-L-cysteine. We prospectively compared these assays with results obtained by sputum mycobacterial culture for patients receiving anti-TB therapy. RESULTS:Sixty-five patients with newly diagnosed TB and receiving a standardized first-line anti-TB drug regimen were evaluated at week 2 and at months 1, 2, and 4 after therapy initiation. Both the DNA PCR assay (98.5% positive) and the mRNA reverse-transcription PCR (RT-PCR) assay (95.4% positive) were better than standard Ziehl-Neelsen staining techniques (83.1%) for detecting M. tuberculosis in culture-positive sputum samples. The overall agreement between culture and mRNA RT-PCR results for all 286 sputum samples was 87.1%, and compared with culture, the mRNA RT-PCR assay's diagnostic sensitivity and specificity were 85.2% and 88.6%, respectively. For monitoring efficacy of therapy, mRNA RT-PCR results paralleled those of culture at the follow-up time points. CONCLUSIONS:The continued presence of viable M. tuberculosis according to culture and results obtained by RT-PCR analysis of antigen 85B mRNA correlated clinically with resistance to anti-TB drugs, whereas the DNA PCR assay showed a high false-positive rate. This mRNA RT-PCR assay may allow rapid monitoring of the response to anti-TB therapy.
| S-EPMC2874158 | biostudies-literature
Project description:BackgroundHeteroresistant Mycobacterium tuberculosis infections (defined as concomitant infection with drug-resistant and drug-susceptible strains) may explain the higher risk of poor tuberculosis treatment outcomes observed among patients with mixed-strain M. tuberculosis infections. We investigated the clinical effect of mixed-strain infections while controlling for pretreatment heteroresistance in a population-based sample of patients with tuberculosis starting first-line tuberculosis therapy in Botswana.MethodsWe performed 24-locus mycobacterial interspersed repetitive unit-variable number tandem-repeat analysis and targeted deep sequencing on baseline primary cultured isolates to detect mixed infections and heteroresistance, respectively. Drug-sensitive, micro-heteroresistant, macro-heteroresistant, and fixed-resistant infections were defined as infections in which the frequency of resistance was <0.1%, 0.1%-4%, 5%-94%, and ≥95%, respectively, in resistance-conferring domains of the inhA promoter, the katG gene, and the rpoB gene.ResultsOf the 260 patients with tuberculosis included in the study, 25 (9.6%) had mixed infections and 30 (11.5%) had poor treatment outcomes. Micro-heteroresistance, macro-heteroresistance, and fixed resistance were found among 11 (4.2%), 2 (0.8%), and 11 (4.2%), respectively, for isoniazid and 21 (8.1%), 0 (0%), and 10 (3.8%), respectively, for rifampicin. In multivariable analysis, mixed infections but not heteroresistant infections independently predicted poor treatment outcomes.ConclusionsAmong patients starting first-line tuberculosis therapy in Botswana, mixed infections were associated with poor tuberculosis treatment outcomes, independent of heteroresistance.
| S-EPMC6217728 | biostudies-literature
Project description:A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.
| S-EPMC3255615 | biostudies-literature
Project description:BackgroundTuberculosis Trials Consortium Study 28, was a double blind, randomized, placebo-controlled, phase 2 clinical trial examining smear positive pulmonary Mycobacterium tuberculosis. Over the course of intensive phase therapy, patients from African sites had substantially delayed and lower rates of culture conversion to negative in liquid media compared to non-African patients. We explored potential explanations of this finding.MethodsIn TBTC Study 28, protocol-correct patients (n = 328) provided spot sputum specimens for M. tuberculosis culture in liquid media, at baseline and weeks 2, 4, 6 and 8 of study therapy. We compared sputum culture conversion for African and non-African patients stratified by four baseline measures of disease severity: AFB smear quantification, extent of disease on chest radiograph, cavity size and the number of days to detection of M. tuberculosis in liquid media using the Kaplan-Meier product-limit method. We evaluated specimen processing and culture procedures used at 29 study laboratories serving 27 sites.ResultsAfrican TB patients had more extensive disease at enrollment than non-African patients. However, African patients with the least disease by the 4 measures of disease severity had conversion rates on liquid media that were substantially lower than conversion rates in non-African patients with the greatest extent of disease. HIV infection, smoking and diabetes did not explain delayed conversion in Africa. Some inter-site variation in laboratory processing and culture procedures within accepted practice for clinical diagnostic laboratories was found.ConclusionsCompared with patients from non-African sites, African patients being treated for TB had delayed sputum culture conversion and lower sputum conversion rates in liquid media that were not explained by baseline severity of disease, HIV status, age, smoking, diabetes or race. Further investigation is warranted into whether modest variation in laboratory processes substantially influences the efficacy outcomes of phase 2 TB treatment trials or if other factors (e.g., nutrition, host response) are involved.Trial registrationClinicalTrials.gov NCT00144417.
| S-EPMC3073969 | biostudies-literature
Project description:Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.
| S-EPMC4390653 | biostudies-literature
Project description:Very fast amplification of DNA in small volumes can be continuously monitored with a rapid cycler that incorporates fluorimetric detection. Primers were designed to amplify a 157-bp fragment of the rpoB gene spanning codons 526 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Mycobacterium tuberculosis. Most mutations associated with resistance to rifampin (RMP) and isoniazid (INH) in clinical isolates occur in these codons. Two pairs of hybridization probes were synthesized; one in each pair was 3' labeled with fluorescein and hybridized upstream of the codon with the mutation; the other two probes were 5' labeled with LightCycler-Red 640. Each pair of probes recognized adjacent sequences in the amplicon. After DNA amplification was finished by using a LightCycler, the temperature at which the Red 640 probe melted from the product was determined in a 3-min melt program. Twenty M. tuberculosis clinical isolates susceptible to streptomycin, INH, RMP, and ethambutol and 36 antibiotic-resistant clinical M. tuberculosis isolates (16 resistant to RMP, 16 to INH, and 4 to both antimicrobial agents) were amplified, and the presence of mutations was determined using single-strand conformation polymorphism analysis, the LiQor automated sequencer, and the LightCycler system. Concordant results were obtained in all cases. Within 30 min, the LightCycler method correctly genotyped all the strains without the need of any post-PCR sample manipulation. Overall, this pilot study demonstrated that real-time PCR coupled to fluorescence detection is the fastest available method for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.
| S-EPMC87353 | biostudies-literature
Project description:Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35 degrees C (63.27 to 65.42 degrees C); M. kansasii, 59.20 degrees C (58.07 to 60.33 degrees C); M. avium, 57.82 degrees C (57.05 to 58.60 degrees C); M. intracellulare, 54.46 degrees C (53.69 to 55.23 degrees C); M. marinum, 58.91 degrees C (58.28 to 59.55 degrees C); rapidly growing mycobacteria, 53.09 degrees C (50.97 to 55.20 degrees C) or 43.19 degrees C (42.19 to 44.49 degrees C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.
| S-EPMC262464 | biostudies-literature
Project description:BackgroundApproximately 5% of patients with drug-susceptible tuberculosis have a relapse after 6 months of first-line therapy, as do approximately 20% of patients after 4 months of short-course therapy. We postulated that by analyzing pretreatment isolates of Mycobacterium tuberculosis obtained from patients who subsequently had a relapse or were cured, we could determine any correlations between the minimum inhibitory concentration (MIC) of a drug below the standard resistance breakpoint and the relapse risk after treatment.MethodsUsing data from the Tuberculosis Trials Consortium Study 22 (development cohort), we assessed relapse and cure isolates to determine the MIC values of isoniazid and rifampin that were below the standard resistance breakpoint (0.1 μg per milliliter for isoniazid and 1.0 μg per milliliter for rifampin). We combined this analysis with clinical, radiologic, and laboratory data to generate predictive relapse models, which we validated by analyzing data from the DMID 01-009 study (validation cohort).ResultsIn the development cohort, the mean (±SD) MIC of isoniazid below the breakpoint was 0.0334±0.0085 μg per milliliter in the relapse group and 0.0286±0.0092 μg per milliliter in the cure group, which represented a higher value in the relapse group by a factor of 1.17 (P=0.02). The corresponding MIC values of rifampin were 0.0695±0.0276 and 0.0453±0.0223 μg per milliliter, respectively, which represented a higher value in the relapse group by a factor of 1.53 (P<0.001). Higher MIC values remained associated with relapse in a multivariable analysis that included other significant between-group differences. In an analysis of receiver-operating-characteristic curves of relapse based on these MIC values, the area under the curve (AUC) was 0.779. In the development cohort, the AUC in a multivariable model that included MIC values was 0.875. In the validation cohort, the MIC values either alone or combined with other patient characteristics were also predictive of relapse, with AUC values of 0.964 and 0.929, respectively. The use of a model score for the MIC values of isoniazid and rifampin to achieve 75.0% sensitivity in cross-validation analysis predicted relapse with a specificity of 76.5% in the development cohort and a sensitivity of 70.0% and a specificity of 100% in the validation cohort.ConclusionsIn pretreatment isolates of M. tuberculosis with decrements of MIC values of isoniazid or rifampin below standard resistance breakpoints, higher MIC values were associated with a greater risk of relapse than lower MIC values. (Funded by the National Institute of Allergy and Infectious Diseases.).
| S-EPMC6317071 | biostudies-literature