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

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

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

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

High Frequency of Enterocytozoon bieneusi Genotype WL12 Occurrence among Immunocompromised Patients with Intestinal Microsporidiosis

International audienceMicrosporidiosis is an emerging opportunistic infection causing severe digestive disorders in immunocompromised patients. The aim of this study was to investigate the prevalence of intestinal microsporidia carriage among immunocompromised patients hospitalized at a major hospital complex in the Tunis capital area, Tunisia (North Africa), and perform molecular epidemiology and population structure analyses of Enterocytozoon bieneusi, which is an emerging fungal pathogen. We screened 250 stool samples for the presence of intestinal microsporidia from 171 patients, including 81 organ transplant recipients, 73 Human Immunodeficiency Virus (HIV)-positive patients, and 17 patients with unspecified immunodeficiency. Using a nested PCR-based diagnostic approach for the detection of E. bieneusi and Encephalitozoon spp., we identified 18 microsporidia-positive patients out of 171 (10.5%), among which 17 were infected with E. bieneusi. Microsporidia-positive cases displayed chronic diarrhea (17 out of 18), which was associated more with HIV rather than with immunosuppression other than HIV (12 out of 73 versus 6 out of 98, respectively, p = 0.02) and correlated with extended hospital stays compared to microsporidia-negative cases (60 versus 19 days on average, respectively; p = 0.001). Strikingly, internal transcribed spacer (ITS)-based genotyping of E. bieneusi strains revealed high-frequency occurrence of ITS sequences that were identical (n = 10) or similar (with one single polymorphic site, n = 3) to rare genotype WL12. Minimum-spanning tree analyses segregated the 17 E. bieneusi infection cases into four distinct genotypic clusters and confirmed the high prevalence of genotype WL12 in our patient population. Phylogenetic analyses allowed the mapping of all 17 E. bieneusi strains to zoonotic group 1 (subgroups 1a and 1b/1c), indicating loose host specificity and raising public health concern. Our study suggests a probable common source of E. bieneusi genotype WL12 transmission and prompts the implementation of a wider epidemiological investigation