10 research outputs found
Combined species identification, genotyping, and drug resistance detection of mycobacterium tuberculosis cultures by mlpa on a bead-based array
The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management
Classifying recurrent Mycobacterium tuberculosis cases in Georgia using MIRU-VNTR typing.
INTRODUCTION:Recurrent tuberculosis (TB) is one of the main challenges in TB control. Genotyping based on Mycobacterial Interspersed Repetitive Units-Variable Tandem Repeats (MIRU-VNTR) has been widely used to differentiate between relapse and reinfection, which are the two main causes of recurrent TB. There is a lack of data regarding the causes of TB recurrence in Georgia, and while differentiating between relapse and reinfection plays a key role in defining appropriate interventions, the required genotyping methodologies have not been implemented. The objective of this study was to implement MIRU-VNTR genotyping at the National Center for Tuberculosis and Lung Diseases (NCTBLD) and differentiate between relapse and reinfection in multidrug resistant (MDR-) TB patients from Tbilisi, Georgia. METHODS:Recurrent MDR tuberculosis cases from 2014-2016 diagnosed at NCTLD were included in the study when bacterial samples from both episodes were available. Genotyping based on the MIRU-VNTR 24 loci was implemented and used for differentiating between relapse and reinfection. Paired samples showing the same MIRU-VNTR pattern or one locus difference were classified as relapse, while two and more loci differences were treated as reinfection. Exact logistic regression was used to identify predictors of recurrence. RESULTS:Thirty two MDR-TB patients (64 samples) were included and MIRU-VNTR 24 typing was performed on the corresponding paired samples. Of the 32 patients, 25 (83.3%) were identified as relapse while 5 (16.7%) were due to re-infection. Patients with a history of incarceration were significantly associated with TB reinfection (p< 0.05). CONCLUSION:Recurrent TB in MDR patients in Georgia are mainly caused by relapse, raising concerns on the efficacy of the TB control program. An association between incarceration and reinfection likely reflects high levels of ongoing TB transmission in prisons, indicating the need for better TB infection control measures in these settings. Our results add to the rationale for implementing genotypic surveillance of TB more broadly to support TB control in Georgia
High percentage of mixed infections in a high MDR-TB country identified from lung surgery samples
Trabajo presentado en Applied Bioinformatics and Public Health Microbiology Cambridge, UK, 5-7 June 201
The relative transmission fitness of multidrug-resistant Mycobacterium tuberculosis in a drug resistance hotspot
Geographical hotspots with high frequency of multi-drug resistant tuberculosis (MDR-TB) have been observed in several locations, such as the country of Georgia. Here, the authors analyse genomic sequences from tuberculosis bacteria isolated from Georgia to show that the transmission fitness of MDR-TB strains is heterogeneous, and highly drug-resistant and transmissible isolates contribute to the emergence and maintenance of MDR-TB hotspots
Algorithm applied to all strains analysed for species identification of <i>M. tuberculosis</i> complex and non-tuberculous mycobacteria.
<p>MLPA markers are framed and final NTM species, MTBC lineages or sublineages are shown in bold. The species identification of a sample always starts with the MTBC 16SrRNA marker. As an example the call for the Beijing lineage K1 is highlighted with bold arrows. The following markers are present or absent in a strain belonging to the Beijing K1 lineage: MTBC 16S rRNA (present), TbD1 (present), RD750 (absent), pks15/1–7 (absent), RD105 (present), fbpB-238 (present), muT2-58 (present), acs-1551 (absent), RD131 (present). * as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043240#pone.0043240-Comas2" target="_blank">[25]</a>.</p
Reproducibility of the MLPA assay.
<p>DNA from <i>M. tuberculosis</i> strains 16, 27, 42 and 75 was analysed by MLPA in duplicate and in three experimental replicates. Mean Median Fluorescence Intensity (MFI) values with standard error of the mean (SEM) are shown. Each bar represents one experiment. The dashed line at MFI 150 indicates the set threshold.</p
Summary of the MLPA probes designed and used in this study<sup>a</sup>.
a<p>only probes that were functional in this study are shown. Probes are named after the gene and specific codon, nucleotide position (bold), or region they target. Probes are either targeting the mutation (mut) or the wild type (wt) sequence or the presence or absence of an RD. Bacterial DNA sequences are targeted with the left oligo (capital letters), spanning oligo (bold), right oligo (italics), iii =  inosine. xTAG sequences are not shown. RD  =  region of difference.</p
Overview of the bead-based Multiplex Ligation-dependent Probe Amplification (MLPA) assay.
<p>(a) MLPA oligo design. MLPA oligos were designed to test for (1) single nucleotide polymorphism, the absence (2) or presence (3) of a region of difference (RD), (4) species-specific sequences (b) Hybridisation of MLPA oligos to target DNA. Sequence-specific sequences hybridise to target DNA (DNA1 and DNA2). Each probe consists of a target-specific sequence (grey bars), a unique xTAG (orange bar), forward and reverse primer binding sequences (red and green bars). The MLPA oligos perfectly match to the sequence of DNA1 that harbours a SNP but not to DNA2. (c) Ligation of hybridised oligos. Only oligos that are hybridised directly adjacent to each other are ligated. (d) Amplification of ligated oligos by PCR. All ligated oligos are amplified in a PCR reaction using a single Cy3-labelled forward primer and unlabelled reverse primer. (e) Hybridisation of MLPA products to beads. Amplified probes hybridise to their anti-xTAG coupled to an individual bead species. (f) Analysis of bead-probe complexes on the MAGPIX. A red light emitting diode (LED) and a CCD camera identify first the individual bead species before green LEDs excite the reporter molecules on the probes. The signal is translated into Median Fluorescence Intensity (MFI). For DNA1 a reporter signal is detected on the bead species indicating the presence of the SNP, thus a mutation, in the respective DNA.</p
Dot plot of MLPA probe-specific MFI values of strains analysed.
<p>Median fluorescence intensity (MFI) values are indicated for each MLPA probe for every mycobacterial strain tested. The threshold used to call the presence or absence of a maker, MFI of 150, is indicated with a horizontal dashed line. Non-functional MLPA probes are indicated to the right side of the plot separated with a vertical dashed line. False positives or false negatives are highlighted in red. Brackets indicate whether a MLPA probe targets the wildtype sequence (wt), SNP (mut), the presence (P) or absence (A) of an RD, or a species-specific sequence (S).</p
Validation of MLPA probes on 88 previously characterised mycobacterial strains.
<p>The MLPA was performed on 79 <i>M. tuberculosis</i> isolates (strains 1–79), nine non-tuberculosis mycobacteria (strains 80–88) and one species unrelated to mycobacteria (strain 89). Species identification was determined on the basis of the presence or absence of MLPA markers following calls mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043240#pone-0043240-g002" target="_blank">Figure 2</a>. Results obtained by MLPA were compared to results obtained from testing the same strain by other molecular methods. <sup>a</sup>Strain-specific drug resistance profiles and genotypic information obtained by other molecular methods is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043240#pone.0043240.s001" target="_blank">Table S1</a>. The presence or absence of an MLPA product is indicated with a black square or a white square, respectively. The confirmation of the MLPA result by other molecular methods is indicated with a green dot; conflicting results between MLPA and other molecular methods are indicated with a red cross. ND =  Analysis for this marker was not done. MTB4 is defined as <i>M. tuberculosis</i> group 4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043240#pone.0043240-Hershberg1" target="_blank">[26]</a> but not X family, LAM or Haarlem.</p