Healed Lesions of Human Cutaneous Leishmaniasis Caused By Leishmania major Do Not Shelter Persistent Residual Parasites

In human cutaneous leishmaniasis (HCL) caused by Leishmania (L.) major, the cutaneous lesions heal spontaneously and induce a Th1-type immunity that confers solid protection against reinfection. The same holds true for the experimental leishmaniasis induced by L. major in C57BL/6 mice where residual parasites persist after spontaneous clinical cure and induce sustainable memory immune responses and resistance to reinfection. Whether residual parasites also persist in scars of cured HCL caused by L. major is still unknown. Cutaneous scars from 53 volunteers with healed HCL caused by L. major were biopsied and the tissue sample homogenates were analyzed for residual parasites by four methods: i) microscope detection of amastigotes, ii) parasite culture by inoculation on biphasic medium, iii) inoculation of tissue exctracts to the footpad of BALB/c mice, an inbred strain highly susceptible to L. major, and iv) amplification of parasite kDNA by a highly sensitive real-time PCR (RT-PCR). Our results show that the scars of healed lesions of HCL caused by L. major do not contain detectable residual parasites, suggesting that this form likely induces a sterile cure at least within the scars. This feature contrasts with other Leishmania species causing chronic, diffuse, or recidivating forms of leishmaniasis where parasites do persist in healed lesions. The possibility that alternative mechanisms to parasite persistence are needed to boost and maintain long-term immunity to L. major, should be taken into consideration in vaccine development against L. major infection

Neighbour-joining tree inferred from the Dps distances calculated for 55 <i>L. major</i> strains isolated from different rodents (40 Tunisian and 15 from other geographic origins) according to the 10 microsatellites analyzed.

<p>Strains isolated among <i>P. obesus</i> (P), <i>M. shawi</i> (M), <i>Tatera sp.</i> (T) and <i>R. opimus</i> (R) were classified into 10 genotypes Lmj01, Lmj02, Lmj14, Lmj15, Lmj17, Lmj21, Lmj37, Lmj39, Lmj65 (as described in<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107043#pone.0107043-AlJawabreh1" target="_blank">[21]</a>) and RdTN from Africa (AF), Middle East (ME) and Central Asia (CA). RdTN indicates the genotype obtained from Tunisian reservoirs. Results are shown as radial tree where the percentages (under 80%) with which a branch is supported in 1000 bootstrap replications are indicated.</p

Descriptive statistics revealing genetic characteristics and variation of the ten microsatellite loci detected in the population of 55 <i>L. major</i> strains isolated from Tunisian rodents and worldwide.

<p>N, number of genotypes; NA, number of allele per locus; Ho, observed heterozygosity; He, expected heterozygosity; Fis, inbreeding coefficient.</p><p>Descriptive statistics revealing genetic characteristics and variation of the ten microsatellite loci detected in the population of 55 <i>L. major</i> strains isolated from Tunisian rodents and worldwide.</p

Comparative genomics of Tunisian Leishmania major isolates causing human cutaneous leishmaniasis with contrasting clinical severity

International audienceZoonotic cutaneous leishmaniasis caused by Leishmania (L.) major parasites affects urban and suburban areas in the center and south of Tunisia where the disease is endemo-epidemic. Several cases were reported in human patients for which infection due to L major induced lesions with a broad range of severity. However, very little is known about the mechanisms underlying this diversity. Our hypothesis is that parasite genomic variability could, in addition to the host immunological background, contribute to the intra-species clinical variability observed in patients and explain the lesion size differences observed in the experimental model. Based on several epidemiological, in vivo and in vitro experiments, we focused on two clinical isolates showing contrasted severity in patients and BALB/c experimental mice model. We used DNA-seq as a high-throughput technology to facilitate the identification of genetic variants with discriminating potential between both isolates. Our results demonstrate that various levels of heterogeneity could be found between both L major isolates in terms of chromosome or gene copy number variation (CNV), and that the intra-species divergence could surprisingly be related to single nucleotide polymorphisms (SNPs) and Insertion/Deletion (InDels) events. Interestingly, we particularly focused here on genes affected by both types of variants and correlated them with the observed gene CNV. Whether these differences are sufficient to explain the severity in patients is obviously still open to debate, but we do believe that additional layers of -omic information is needed to complement the genomic screen in order to draw a more complete map of severity determinants

Theoretical sizes and repetition numbers among the ten-microsatellite loci in Tunisian studied isolates.

<p>*: New allele not previously described.</p><p>Theoretical sizes and repetition numbers among the ten-microsatellite loci in Tunisian studied isolates.</p

Geographical distribution of strains isolated from the Central Tunisia study area.

<p>Panel A shows the square delimited study area in the Governorate of Sidi Bouzid (in gray). Panel B represents the land satellite image of the study area showing the distribution of animal reservoir hosts from which the <i>L. major</i> strains were isolated. Isolates from <i>P. obesus</i> origin were noted P and colored in green, from <i>M. shawi</i> origin were noted Mer and colored in yellow, and from <i>M. nivalis</i> was noted M and colored in red. Spatial data related to the reservoir hosts of these strains were collected using the Global Positioning System (GPS). Satellite imagery: ArcGIS software.</p

Genetic micro-heterogeneity of Leishmania major in emerging foci of zoonotic cutaneous leishmaniasis in Tunisia

International audienceTunisia is endemic for zoonotic cutaneous leishmaniasis (ZCL), a parasitic disease caused by Leishmania (L.) major. ZCL displays a wide clinical polymorphism, with severe forms present more frequently in emerging foci where naive populations are dominant. In this study, we applied the multi-locus microsatellite typing (MLMT) using ten highly informative and discriminative markers to investigate the genetic structure of 35 Tunisian Leishmania (L.) major isolates collected from patients living in five different foci of Central Tunisia (two old and three emerging foci). Phylogenetic reconstructions based on genetic distances showed that nine of the ten tested loci were homogeneous in all isolates with homozygous alleles, whereas one locus (71AT) had a 58/64-bp bi-allelic profile with an allele linked to emerging foci. Promastigote-stage parasites with the 58-bp allele tend to be more resistant to in vitro complement lysis. These results, which stress the geographical dependence of the genetic micro-heterogeneity, may improve our understanding of the ZCL epidemiology and clinical outcome

Negative correlation between expressions of an up-regulated set of miRNAs and their targeted chemokine transcripts.

<p>Expression means of let-7a, miR-25, miR-26a, miR-140, miR-146a and miR-155 at 3 h and miR-23b and miR-132 at 6 h post-infection of three healthy donors (D1, D2 and D3; panel A) is negatively correlated with CCL2, CCL5, CXCL10, CXCL11 and CXCL12 mRNA mean levels at 12 and 24 h post-infection (panel B) in <i>L. major</i>-infected human macrophages. Results were expressed using the 2^−ΔΔCt method.</p