Success of MIRU-VNTR typing in correlation with smear microscopy.

<p>MIRU-VNTR typing results of all 79 samples taken from 26 TB patients in correlation with smear microscopy result. Smear microscopy grades are coded as follows: 3+ (>10 bacilli/field); 2+ (1–10 bacilli/field); 1+ (10–99 bacilli/100 fields); 2AFB (7–9 bacilli/100 fields), and 1 AFB (3–6 bacilli/100 fields).</p

Prospective Genotyping of <i>Mycobacterium tuberculosis</i> from Fresh Clinical Samples

<div><p>Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of <i>Mycobacterium tuberculosis</i> directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (<i>p</i> = 0.0003) and for 53 follow-up samples (<i>p</i> = 0.002). For 20 of the 26 patients for whom complete or even incomplete <i>M. tuberculosis</i> isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of <i>M. tuberculosis</i> can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample.</p></div

MIRU-VNTR typing results of all 79 samples, taken from 26 patients, depending on initiation of TB treatment and result of smear microscopy.

<p>Missing alleles are coded as <i>na</i> (i.e., non amplified).</p><p>MIRU-VNTR typing results of all 79 samples, taken from 26 patients, depending on initiation of TB treatment and result of smear microscopy.</p

Allelic diversities of 24 MIRU-VNTR loci of Slovenian <i>M. tuberculosis</i> isolates (<i>n</i> = 919) from a retrospective population-based study and proportion of missing allele per locus in our prospective study from direct sputum samples (<i>n</i> = 79).

a<p>Variant alleles specifically observed for locus 0580. These “s” alleles consist of <i>n</i>×77-bp repeat units (taken into account for allele coding), in contrast to classical alleles of <i>n</i>×77-bp repeat units (taken into account for allele coding), plus a single 53-bp repeat unit (not taken into account for allele coding).</p><p>Allelic diversities of 24 MIRU-VNTR loci of Slovenian <i>M. tuberculosis</i> isolates (<i>n</i> = 919) from a retrospective population-based study and proportion of missing allele per locus in our prospective study from direct sputum samples (<i>n</i> = 79).</p

Admixed Phylogenetic Distribution of Drug Resistant <em>Mycobacterium tuberculosis</em> in Saudi Arabia

<div><h3>Background</h3><p>The phylogeographical structure of <em>Mycobacterium tuberculosis</em> is generally bimodal in low tuberculosis (TB) incidence countries, where genetic lineages of the isolates generally differ with little strain clustering between autochthonous and foreign-born TB patients. However, less is known on this structure in Saudi Arabia—the most important hub of human migration as it hosts a total population of expatriates and pilgrims from all over the world which is equal to that of its citizens.</p> <h3>Methodology</h3><p>We explored the mycobacterial phylogenetic structure and strain molecular clustering in Saudi Arabia by genotyping 322 drug-resistant clinical isolates collected over a 12-month period in a national drug surveillance survey, using 24 locus-based MIRU-VNTR typing and spoligotyping.</p> <h3>Principal Findings</h3><p>In contrast to the cosmopolitan population of the country, almost all the known phylogeographic lineages of <em>M. tuberculosis</em> complex (with noticeable exception of <em>Mycobacterium africanum</em>/West-African 1 and 2) were detected, with Delhi/CAS (21.1%), EAI (11.2%), Beijing (11.2%) and main branches of the Euro-American super-lineage such as Ghana (14.9%), Haarlem (10.6%) and Cameroon (7.8%) being represented. Statistically significant associations of strain lineages were observed with poly-drug resistance and multi drug resistance especially among previously treated cases (p value of < = 0.001 for both types of resistance), with relative over-representation of Beijing strains in the latter category. However, there was no significant difference among Saudi and non-Saudi TB patients regarding distribution of phylogenetic lineages (p = 0.311). Moreover, 59.5% (22/37) of the strain molecular clusters were shared between the Saudi born and immigrant TB patients.</p> <h3>Conclusions</h3><p>Specific distribution of <em>M. tuberculosis</em> phylogeographic lineages is not observed between the autochthonous and foreign-born populations. These observations might reflect both socially favored ongoing TB transmission between the two population groups, and historically deep-rooted, prolonged contacts and trade relations of the peninsula with other world regions. More vigorous surveillance and strict adherence to tuberculosis control policies are urgently needed in the country.</p> </div

Distributions of <i>M. tuberculosis</i> complex strain lineages among Saudi and non-Saudi cases.

<p>Lineages identified as described in text were segregated as per “Saudi” and “Non-Saudi” nationalities. Bar diagrams show the proportion (in percentage) of each lineage in each of these two groups. The lineages TUR, NEW-I and URAL were clubbed under the label “Others” as the corresponding isolates are very few in number.</p

Splits Graph of the 17 Concatenated Sequences of the Six Housekeeping Genes

<p>The nodes represent strains and are depicted as small red (smooth tubercle bacilli) or blue (MTBC members) squares. The scale bar represents Hamming distance. Numbers at the edges represent the percent bootstrap support of the splits obtained after 1,000 replicates. The fit was 61.7%. Note that the branching order of MTBC strains is weakly supported, and it should therefore not be seen as contradicting previous evolutionary hypotheses based on deletion patterns [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010005#ppat-0010005-b16" target="_blank">16</a>].</p

Demographical summary of the study samples.

1<p>Eastern Saudi Arabia.</p>2<p>Central Saudi Arabia.</p>3<p>Western Saudi Arabia.</p>4<p>Southern Saudi Arabia.</p>5<p>Bangladesh, India, Nepal, Pakistan, Sri Lanka,</p>6<p>China, Indonesia, Malaysia, Philippines.</p>7<p>Yemen.</p>8<p>Algeria, Chad, Egypt, Eritrea, Ethiopia, Nigeria, Senegal, Somalia, Sudan.</p

Drug resistant pattern of the enrolled isolates.

1<p>Newly diagnosed cases.</p>2<p>Patient received >1 month of anti TB drug therapy.</p>3<p>Multidrug resistant tuberculosis; resistant to at least isoniazid and rifampicin.</p>4<p>Pan-resistance was defined here as resistance to all four first-line drugs.</p

Phylogenetic Position of the Tubercle Bacilli within the Genus <i>Mycobacterium</i>

<p>The blue triangle corresponds to tubercle bacilli sequences that are identical or differing by a single nucleotide. The sequences of the genus <i>Mycobacterium</i> that matched most closely to those of <i>M. tuberculosis</i> were retrieved from the BIBI database (<a href="http://pbil.univ-lyon.fr/bibi/" target="_blank">http://pbil.univ-lyon.fr/bibi/</a>) and aligned with those obtained for 17 smooth and MTBC strains. The unrooted neighbor-joining tree is based on 1,325 aligned nucleotide positions of the 16S rRNA gene. The scale gives the pairwise distances after Jukes-Cantor correction. Bootstrap support values higher than 90% are indicated at the nodes.</p