Evolutionary history and spread of the Mycobacterium tuberculosis Latin American and Mediterranean (L4.3/LAM) sublineage, Tunisia.

BACKGROUND: To infer the origin and spread of the Mycobacterium tuberculosis Latin American and Mediterranean (L4.3/LAM) sublineage in a Mediterranean country, Tunisia, where it predominates. METHODS: We combined Bayesian (STRUCTURE) and maximum likelihood (MIGRAINE) estimation approaches based on a global 24-loci mycobacterial interspersed repetitive units-variable numbers of tandem repeats (MIRU-VNTR24) genotyping dataset consisting of 1573 L4.3/LAM clinical strains from four continents, including 252 isolates originating from Tunisia. RESULTS: Phylogenetic analyses coupled with Bayesian estimations suggested that the most predominant L4.3/LAM subpopulation in Tunisia (65.07%), which is dominated by a single clonal complex, TUN4.3_CC1 (94.51%), has evolved from an ancestral pool that is restricted to Europe and Africa, contrasting with the remaining L4.3/LAM subpopulations whose ancestry was traced all over the word. Maximum likelihood analysis revealed that TUN4.3_CC1 has been undergoing a demographic expansion since 131 years ago (CI95%: 90.7-205), thus explaining its preponderance relative to the second most predominant CC, TUN4.3_CC2, whose population was found under contraction. CONCLUSIONS: The preponderance of L4.3/LAM in Tunisia stems from a 130-year expansion process of a locally evolved clone.

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

Microsporidiosis 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.

Successful expansion of Mycobacterium tuberculosis Latin American and Mediterranean sublineage (L4.3/LAM) in Tunisia mainly driven by a single, long-established clonal complex.

OBJECTIVES: To explore the evolutionary history of Mycobacterium tuberculosis Latin American and Mediterranean (L4.3/LAM) sublineage in Tunisia, where it predominates. METHODS: High-resolution genotyping of 252 L4.3/LAM clinical strains was undertaken, and whole-genome sequencing was performed on 31 representative isolates. RESULTS: Genotyping data coupled with Bayesian analyses split the Tunisian L4.3/LAM strain collection into two divergent entities (65.07% vs 34.92%): a major subpopulation, dominated by a single clonal complex (CC), TUN4.3_CC1 (94.51%); and a minor subpopulation, dominated by TUN4.3_CC2 (42.04%). TUN4.3_CC1 is clearly thriving in Tunisia, accounting for 61.5% of the L4.3/LAM sublineage. TUN4.3_CC1 displayed higher mean allelic richness compared with TUN4.3_CC2 and predominated throughout the entire region, indicating a long-established history. The very low proportion of drug resistance among TUN4.3_CC1 isolates is indicative of their intrinsic ability to spread successfully in the host population. Genomic analyses further confirmed the clear genetic separation between the two main CCs (pairwise fixation index 0.56), and suggested the relatively ancient origin of TUN4.3_CC1. Consistent with its successful expansion, TUN4.3_CC1 showed reduced mean pairwise genetic distance between genomes. CONCLUSIONS: These findings link the successful expansion of L4.3/LAM in Tunisia to a single long-established clone.

Automated IS6110-based fingerprinting of Mycobacterium tuberculosis: Reaching unprecedented discriminatory power and versatility.

BACKGROUND: Several technical hurdles and limitations have restricted the use of IS6110 restriction fragment length polymorphism (IS6110 RFLP), the most effective typing method for detecting recent tuberculosis (TB) transmission events. This has prompted us to conceive an alternative modality, IS6110-5'3'FP, a plasmid-based cloning approach coupled to a single PCR amplification of differentially labeled 5' and 3' IS6110 polymorphic ends and their automated fractionation on a capillary sequencer. The potential of IS6110-5'3'FP to be used as an alternative to IS6110 RFLP has been previously demonstrated, yet further technical improvements are still required for optimal discriminatory power and versatility. OBJECTIVES: Here we introduced critical amendments to the original IS6110-5'3'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. METHODS: IS6110-5'3'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 E. coli 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. RESULTS: In doing so, the overall turnaround time of IS6110-5'3'FP was reduced to 4 hours. The new protocol allowed detecting almost all 5' and 3' IS6110 polymorphic fragments of any given strain, including IS6110 high-copy number Beijing strains. IS6110-5'3'FP proved much more discriminative than 24-loci MIRU-VNTR, particularly with strains of the M. tuberculosis lineage 4. CONCLUSIONS: The IS6110-5'3'FP protocol described herein reached the optimal discriminatory potential of IS6110 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.