Automated IS<i>6110</i>-based fingerprinting of <i>Mycobacterium tuberculosis</i>: Reaching unprecedented discriminatory power and versatility

<div><p>Background</p><p>Several technical hurdles and limitations have restricted the use of IS<i>6110</i> restriction fragment length polymorphism (IS<i>6110</i> RFLP), the most effective typing method for detecting recent tuberculosis (TB) transmission events. This has prompted us to conceive an alternative modality, IS<i>6110</i>-5’3’FP, a plasmid-based cloning approach coupled to a single PCR amplification of differentially labeled 5’ and 3’ IS<i>6110</i> polymorphic ends and their automated fractionation on a capillary sequencer. The potential of IS<i>6110</i>-5’3’FP to be used as an alternative to IS<i>6110</i> RFLP has been previously demonstrated, yet further technical improvements are still required for optimal discriminatory power and versatility.</p><p>Objectives</p><p>Here we introduced critical amendments to the original IS<i>6110</i>-5’3’FP protocol and compared its performance to that of 24-loci multiple interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR), the current standard method for TB transmission analyses.</p><p>Methods</p><p>IS<i>6110</i>-5’3’FP protocol modifications involved: (i) the generation of smaller-sized polymorphic fragments for efficient cloning and PCR amplification, (ii) omission of the plasmid amplification step in <i>E</i>. <i>coli</i> for shorter turnaround times, (iii) the use of more stable fluorophores for increased sensitivity, (iv) automated subtraction of background fluorescent signals, and (v) the automated conversion of fluorescent peaks into binary data.</p><p>Results</p><p>In doing so, the overall turnaround time of IS<i>6110</i>-5’3’FP was reduced to 4 hours. The new protocol allowed detecting almost all 5’ and 3’ IS<i>6110</i> polymorphic fragments of any given strain, including IS<i>6110</i> high-copy number Beijing strains. IS<i>6110</i>-5’3’FP proved much more discriminative than 24-loci MIRU-VNTR, particularly with strains of the <i>M</i>. <i>tuberculosis</i> lineage 4.</p><p>Conclusions</p><p>The IS<i>6110</i>-5’3’FP protocol described herein reached the optimal discriminatory potential of IS<i>6110</i> fingerprinting and proved more accurate than 24-loci MIRU-VNTR in estimating recent TB transmission. The method, which is highly cost-effective, was rendered versatile enough to prompt its evaluation as an automatized solution for a TB integrated molecular surveillance.</p></div

Box plot showing the sizes of IS<i>6110</i> polymorphic amplicons generated by IS<i>6110</i>-5’3’FP using the 11-banded laboratory reference strain genomic DNA digested either by <i>BstU</i>I or <i>Hinc</i>II.

<p>The IS<i>6110</i>-5’3’FP products were fractionated without being diluted on an ABI PRISM 3100 capillary DNA sequencer (Applied Biosystems Inc., CA, USA). The boxes show the 25% to 75% interquartile range.</p

Assessment of the discriminatory power of IS<i>6110</i>-5’3’FP and 24-loci MIRU-VNTR.

<p><i>M</i>. <i>tuberculosis</i> strain collections belonging to Haarlem (A), LAM (B), and Beijing (C) genotypes were used.</p

Comparative cost estimates (USD).

<p>Comparative cost estimates (USD).</p

Overview of IS<i>6110</i>-5’3’FP original protocol and its simplified highly performing version.

<p>(A). The original IS<i>6110</i>-5’3’FP protocol as described in Thabet et al. (2014) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197913#pone.0197913.ref011" target="_blank">11</a>]. (B). The new protocol of IS<i>6110</i>-5’3’FP. In this new protocol version, aside from using the frequently cutting <i>BstU</i>I enzyme instead of <i>Hinc</i>II, there is no need for plasmid library amplification in <i>E</i>. <i>coli</i>, a modification that considerably shortens the method turnaround. Moreover, amplification in <i>E</i>. <i>coli</i> could result in the loss of some IS<i>6110</i>-containing plasmid most likely because of clone instability. Therefore, omission of this step increases the sensitivity of the method.</p

Comparison of the discriminatory power of IS<i>6110</i>-5’3’FP and 24-loci MIRU-VNTR.

<p>Comparison of the discriminatory power of IS<i>6110</i>-5’3’FP and 24-loci MIRU-VNTR.</p

Number of IS<i>6110</i>-5’3’FP-generated peaks relative to IS<i>6110</i> copies as determined from IS<i>6110</i> RFLP profiles.

<p>Number of IS<i>6110</i>-5’3’FP-generated peaks relative to IS<i>6110</i> copies as determined from IS<i>6110</i> RFLP profiles.</p

Participant selection from the parent micronutrient trial for the sputum culture conversion analysis.

<p>Participant selection from the parent micronutrient trial for the sputum culture conversion analysis.</p

Baseline characteristics of participants with drug sensitive smear-positive pulmonary tuberculosis.^#

#<p>Continuous data expressed as median (IQR).</p><p>NA: Not applicable. Data was complete, therefore not imputed.</p

A participatory scenario method to explore the future of marine social-ecological systems

Anticipating future changes in marine social‐ecological systems (MSES) several decades into the future is essential in the context of accelerating global change. This is challenging in situations where actors do not share common understandings, practices, or visions about the future. We introduce a dedicated scenario method for the development of MSES scenarios in a participatory context. The objective is to allow different actors to jointly develop scenarios which contain their multiple visions of the future. The method starts from four perspectives: “fisheries management,” “ecosystem,” “ocean climate,” and “global context and governance” for which current status and recent trends are summarized. Contrasted scenarios about possible futures are elaborated for each of the four single perspectives before being integrated into multiple‐perspective scenarios. Selected scenarios are then developed into storylines. Focusing on individual perspectives until near the end allows actors with diverse cultures, interests and horizons to confront their own notions of the future. We illustrate the method with the exploration of the futures of the Barents Sea MSES by 2050. We emphasize the following lessons learned: first, many actors are not familiar with scenario building and attention must be paid to explaining the purpose, methodology, and benefits of scenarios exercises. Second, although the Barents Sea MSES is relatively well understood, uncertainties about its future are significant. Third, it is important to focus on unlikely events. Fourth, all perspectives should be treated equally. Fifth, as MSES are continuously changing, we can only be prepared for future changes if we collectively keep preparing