Direcció estratègica i lideratge, febrer 2013

Recurs d'aprenentatge de la Universitat Oberta de Catalunya.Recurso de aprendizaje de la "Universitat Oberta de Catalunya".Learning material of the "Universitat Oberta de Catalunya"

Probability of the spectrum of rpoB mutations in the INH-resistant mutant and the wildtype parent being identical.

<p> <i>The probability of hypothesis h0_b (the spectrum of rpoB-mutations of the INH-resistant mutant is not different than that of the wildtype parent) determined by X² for all INH-resistant strains for both methods. For all of the tested strains the statistical power of the X² test was reduced due to one or more mutations occurring less than five times. NA: not available, mutants were not obtained for these strains.</i></p><p>*<i>: since both 2001-1669 and 2001-1670 are resistant to INH (via katG-S315T), the spectrum was compared to the only susceptible LAM strain, 9900098. </i><b><i>Bold</i></b><i>: p_a<0.05, therefore hypothesis is rejected.</i></p

Proportion of rpoB-S531L and other mutations in rpoB acquired by INH-resistant M. tuberculosis strains compared to their susceptible parent strains.

<p>Results for all INH-resistant strains are grouped, as well as the results for are wildtype strains. Mutation distribution obtained for the INH-resistant group and the wildtype group and rpoB mutation distribution obtained for the two different experimental methods are compared. The red bars depict the proportion of rpoB-S531L mutations that were detected. The blue bars represent all other rpoB-mutations, whereas for the X² test depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029108#pone-0029108-t004" target="_blank">tables 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029108#pone-0029108-t005" target="_blank">5</a> only the four most prevalent mutations were taken into account. Numbers in the bar graphs represent the number of samples carrying the specific mutation as a percentage of the total amount of rpoB mutants and as absolute numbers (in brackets). The p-values above the bar graphs represent the probability that the distributions compared are similar.</p

Probability of an equal distribution of the four targeted rpoB mutations in various M. tuberculosis strains.

<p> <i>The probability (p_a) of hypothesis h0_a (the targeted rpoB-mutations (S531L, H526D, H526Y and S522L) have an equal chance of occurring) was determined by X² for each strain for both method 1 and method 2. For all tested strains, except in the first experiment with MTB72, the statistical power of the X² test was reduced due to one or more mutations occurring less than five times. NA: not available, mutants were not obtained for these strains. </i><b><i>Bold</i></b><i>: p_a<0.05, therefore hypothesis is rejected.</i></p

Spectrum of spontaneous rpoB-mutations obtained by method 2 (single colonies from 10-ml cultures).

<p> <i>Results for MTB72 (first row) and strains H103, 2001–2184 and 2001–2185 were obtained in the first experiment, where we assessed the influence of pre-existing INH resistance on the spectrum of mutations. </i><i>Results for MTB72 (second row) and 9900098, 2001-1669 and 2001-1670 were obtained in the second experiment, where we determined the role of the LAM genotype on the spectrum of rpoB-mutations. </i><i>Results for MTB72 (third row) and 9500592, 2002-1640, 2002-1585 and 17583 were obtained in the third experiment, where we determined the role of the Beijing genotype on the spectrum of rpoB-mutations. ins: insertion, indel: combined insertion/deletion, Δ: deletion.</i></p

Mutation rates (×10⁻⁸/cell division), determined with 8 µg/ml rifampicin, for the M. tuberculosis strains used in this study.

<p>Description of the strains can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029108#pone-0029108-t001" target="_blank">Table 1</a>. Red bars indicate replicates from a second, independent experiment.</p

Description of M. tuberculosis strains used in this study.

<p><i>Strains are identified by the name given by either the RIVM (numerical codes) or the KIT (letter+number). DST: drug susceptibility profile, H: isoniazid, R: rifampicin, S: susceptible, R: resistant. The genotype of the strains indicated in the table is determined by spoligotyping, mutations in parentheses are characteristic genotypic mutations identified by MLPA and confirmed by sequencing (ogt, mutT2, mutT4, Ag85C). Mutations in katG confer resistance to isoniazid; deletions were initially picked up by two different PCR reactions, amplifying either the region that covers the drug resistance mutations at codon 315 or the genotypic mutation at codon 463. The notation in this table indicates that the PCR fragment in question was absent and that therefore the region was deleted in the specific strain</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029108#pone.0029108-Bergval1" target="_blank">[2]</a>.</p

Epidemiological links between tuberculosis cases identified twice as efficiently by whole genome sequencing than conventional molecular typing: A population-based study

<div><p>Background</p><p>Patients with <i>Mycobacterium tuberculosis</i> isolates sharing identical DNA fingerprint patterns can be epidemiologically linked. However, municipal health services in the Netherlands are able to confirm an epidemiological link in only around 23% of the patients with isolates clustered by the conventional variable number of tandem repeat (VNTR) genotyping. This research aims to investigate whether whole genome sequencing (WGS) is a more reliable predictor of epidemiological links between tuberculosis patients than VNTR genotyping.</p><p>Methods</p><p>VNTR genotyping and WGS were performed in parallel on all <i>Mycobacterium tuberculosis</i> complex isolates received at the Netherlands National Institute for Public Health and the Environment in 2016. Isolates were clustered by VNTR when they shared identical 24-loci VNTR patterns; isolates were assigned to a WGS cluster when the pair-wise genetic distance was ≤ 12 single nucleotide polymorphisms (SNPs). Cluster investigation was performed by municipal health services on all isolates clustered by VNTR in 2016. The proportion of epidemiological links identified among patients clustered by either method was calculated.</p><p>Results</p><p>In total, 535 isolates were genotyped, of which 25% (134/535) were clustered by VNTR and 14% (76/535) by WGS; the concordance between both typing methods was 86%. The proportion of epidemiological links among WGS clustered cases (57%) was twice as common than among VNTR clustered cases (31%).</p><p>Conclusion</p><p>When WGS was applied, the number of clustered isolates was halved, while all epidemiologically linked cases remained clustered. WGS is therefore a more reliable tool to predict epidemiological links between tuberculosis cases than VNTR genotyping and will allow more efficient transmission tracing, as epidemiological investigations based on false clustering can be avoided.</p></div

Correlation between genetic distances in SNPs and events for which an epidemiological link was confirmed (red) or not (green) of all 134 VNTR clustered isolates in 2016.

<p>The frequencies on the y-axis represent the number of events (n-1) within VNTR clusters rather than the number of isolates. The dashed line indicates the threshold of ≤ 12 SNPs used to rule in transmission in this study.</p

Venn diagram of VNTR and WGS typing of 535 <i>M</i>. <i>tuberculosis</i> complex isolates from the Netherlands and confirmed epidemiological links in cluster investigation.

<p>* Isolates with unique VNTR profiles in 2016 were not investigated for epidemiological links. ** Epidemiological link information is based on geographical proximity, as cluster investigation was not conducted for isolates with different VNTR profiles.</p