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

Population Pharmacokinetics of Intravenous Salbutamol in Children with Refractory Status Asthmaticus

Background: Intravenous salbutamol is used to treat children with refractory status asthmaticus, however insufficient pharmacokinetic data are available to guide initial and subsequent dosing recommendations for its intravenous use. The pharmacologic activity of salbutamol resides predominantly in the (R)-enantiomer, with little or no activity and even concerns of adverse reactions attributed to the (S)-enantiomer. Objective: Our aim was to develop a population pharmacokinetic model to characterize the pharmacokinetic profile for intravenous salbutamol in children with status asthmaticus admitted to the pediatric intensive care unit (PICU), and to use this model to study the effect of different dosing schemes with and without a loading dose. Methods: From 19 children (median age 4.9 years [range 9 months–15.3 years], median weight 18 kg [range 7.8–70 kg]) treated with continuous intravenous salbutamol at the PICU, plasma samples for R- and S-salbutamol concentrations (111 samples), as well as asthma scores, were collected prospectively at the same time points. Possible adverse reactions and patients’ clinical data (age, sex, weight, drug doses, liver and kidney function) were recorded. With these data, a population pharmacokinetic

Evolution of pathogenicity and sexual reproduction in eight Candida genomes

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.publishe

Summary of the MLPA probes designed and used in this study^a.

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

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

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. ^aStrain-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

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

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