**4.** *Leishmania* **parasite evolution, genetics and genome analyses – Consequences and prospects**

For many years *Leishmania* parasites have been considered to replicate clonally, without genetic exchange. Indeed, Tibayrenc proposed that clonal evolution in micropathogens be defined as restrained recombination on an evolutionary scale, with genetic exchange scarce enough to not break the prevalent pattern of clonal population structure (Reviewed in [141,142]). The two main manifestations of clonal evolution are strong linkage disequilibrium (LD) and wide‐ spread genetic clustering ("near-clading"). These authors hypothesized that this pattern is not mainly due to natural selection, but would originate chiefly from in-built genetic properties of pathogens, that would allow like for other microorganisms (viruses, bacteria, protozoan parasites) to keep a balance between clonality and recombination, which would help escape from recombinational load. This way, to face evolutionary challenges, pathogens would be equipped with "clonality/sexuality machinery" that would function as alternative allelic systems [141,142]. However, an accumulation of molecular evidence indicates that there are inter-specific [115,137,143–146] and intra-specific [132,138] hybrids among natural popula‐ tions. Genetic exchange was finally demonstrated experimentally in 2009 [99]. In fact, double drug resistant *Leishmania* major hybrids were produced by co-infecting *Phlebotomus duboscqi* (a natural *L. major* vector) sand flies with two strains carrying different drug resistance markers. The nuclear genotypes were consistent with a Mendelian transmission leading to a heterozy‐ gous first generation progeny [99]. The anticipated continuity of these studies was to co-infect sand flies with transgenic *Leishmania* carrying two different markers that are fluorescent, in an attempt to visualize the recombination events microscopically [147]. In 2011, for the first time, using a fluorescent protein detection system to observe yellow hybrid promastigotes in *Phlebotomus perniciosus* and *Lutzomyia longipalpis* midguts, *L. donovani* hybrids were observed, 2 days post bloodmeal, and the morphological stages involved were found to be short procyclic promastigotes [100]. However, the parasites could not be recovered and propagated to confirm their hybrid genotypes [100]. Recently, the analysis of the mating competency of *L. major* strains have been expanded to include pairwise matings of multiple isolates bearing independent drug markers [148]. Also, the timing of the appearance of hybrids and their developmental stage associations within both natural (*Phlebotomus duboscqi*) and unnatural (*Lutzomyia longipalpis*) sand fly vectors was followed. Genotype analysis of a large number of progeny clones showed a chromosomal inheritance of both parental alleles at 4–6 unlinked nuclear loci, consistent with a meiotic process, and a uniparental inheritance of kinetoplast DNA [148]. A low frequency of nuclear loci showed only one parental allele, suggesting loss of heterozygos‐ ity, most likely arising from aneuploidy, which is common in *Leishmania*. In the natural vector, when comparing the timing of hybrid formation and the presence of developmental stages, the authors suggested that nectomonad promastigotes are the most likely mating competent forms, with hybrids emerging before the first appearance of metacyclic promastigotes [148].

MLMT analysis showed that recombination events are much more frequent in *Leishmania* than previously thought. Indeed, MLMT analysis of Bolivian and Peruvian *L. braziliensis* showed frequent sexual crosses of individuals from the same strain (inbreeding) [138]. The substantial heterozygote deficiency and extreme inbreeding found in this study is not consistent with a strictly clonal reproduction. The authors came to the conclusion that *Leishmania* parasites may alternate between clonal and sexual modes of reproduction, occurring most probably in the vector [138]. Sexual fusion may frequently take place between genetically related parasites or even within the same strain with occasional recombination events between individuals of different genotypes.

Also, *L. braziliensis*/*L. peruviana* hybrids were found to be quite common in a Peruvian focus where both species can occur sympatrically [137]. In the Old World, natural *L. infantum*/*L. major* hybrids were experimentally transmitted by *Ph. papatasi,* usually only competent to transmit *L. major* [149]. This suggests that hybrids may circulate using this sand fly vector and spread into new foci throughout the broad range of *Ph. papatasi* distribution.

The fact that *Leishmania* can undergo genetic exchange is potentially of profound epidemio‐ logical significance since this could facilitate the emergence and spread of new genotypes and phenotypic traits. Also, hybrid offspring might show a strong selective advantage relative to the parental strains. In [149], the authors have shown that natural hybrids could have enhanced transmission potential and a positively affected fitness.

New high-throughput sequencing technologies have opened the door for population genome analyses and genome-wide association studies. Genome of the *L. major* species was the first to be fully sequenced [150] followed by *L. infantum* and *L. braziliensis* [151]. Comparison of the three genomes revealed conservation of synteny and identified only 200 genes having a differential distribution between the three species. Such genes may encode for proteins implicated in host-pathogen interactions and parasite survival in the macrophage [151]. The species *L. mexicana* and *L. donovani* were subsequently sequenced [152,153] and the reference genomes for *L. major*, *L. infantum*, and *L. braziliensis* were refined [152]. This has allowed the identification of a remarkably low number of genes or paralog groups unique to each of the species *L. mexicana, L. major, L. infantum*, and *L. braziliensis* (2, 14, 19, and 67, respectively). Besides, *L. major* and *L. infantum* were found to have a surprisingly low number of predicted heterozygous SNPs compared with *L. braziliensis* and *L. mexicana*. Chromosome copy number also varied significantly between species, with nine supernumerary chromosomes in *L. infantum*, four in *L. mexicana*, two in *L. braziliensis*, and one in *L. major*. The authors also showed that gene duplication events occur more frequently on disomic chromosomes [152]. In addition to sequencing of an *L. donovani* reference genome, a recent study also included sequence analysis of a set of 16 related clinical lines, isolated from VL patients in Nepal and India, which also differ in their *in vitro* drug response [153]. Sequence comparisons with other *Leishmania* species and analysis of single-nucleotide diversity showed evidence of selection acting on different surface- and transport-related genes, including genes associated with drug resistance. Extensive variation in chromosome copy number between the analyzed lines was also shown. In association to drug resistance, they also showed structural variation, including gene dosage and copy number variation of a circular episome, present in all lines [153].

their hybrid genotypes [100]. Recently, the analysis of the mating competency of *L. major* strains have been expanded to include pairwise matings of multiple isolates bearing independent drug markers [148]. Also, the timing of the appearance of hybrids and their developmental stage associations within both natural (*Phlebotomus duboscqi*) and unnatural (*Lutzomyia longipalpis*) sand fly vectors was followed. Genotype analysis of a large number of progeny clones showed a chromosomal inheritance of both parental alleles at 4–6 unlinked nuclear loci, consistent with a meiotic process, and a uniparental inheritance of kinetoplast DNA [148]. A low frequency of nuclear loci showed only one parental allele, suggesting loss of heterozygos‐ ity, most likely arising from aneuploidy, which is common in *Leishmania*. In the natural vector, when comparing the timing of hybrid formation and the presence of developmental stages, the authors suggested that nectomonad promastigotes are the most likely mating competent forms, with hybrids emerging before the first appearance of metacyclic promastigotes [148]. MLMT analysis showed that recombination events are much more frequent in *Leishmania* than previously thought. Indeed, MLMT analysis of Bolivian and Peruvian *L. braziliensis* showed frequent sexual crosses of individuals from the same strain (inbreeding) [138]. The substantial heterozygote deficiency and extreme inbreeding found in this study is not consistent with a strictly clonal reproduction. The authors came to the conclusion that *Leishmania* parasites may alternate between clonal and sexual modes of reproduction, occurring most probably in the vector [138]. Sexual fusion may frequently take place between genetically related parasites or even within the same strain with occasional recombination events between individuals of

86 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

Also, *L. braziliensis*/*L. peruviana* hybrids were found to be quite common in a Peruvian focus where both species can occur sympatrically [137]. In the Old World, natural *L. infantum*/*L. major* hybrids were experimentally transmitted by *Ph. papatasi,* usually only competent to transmit *L. major* [149]. This suggests that hybrids may circulate using this sand fly vector and

The fact that *Leishmania* can undergo genetic exchange is potentially of profound epidemio‐ logical significance since this could facilitate the emergence and spread of new genotypes and phenotypic traits. Also, hybrid offspring might show a strong selective advantage relative to the parental strains. In [149], the authors have shown that natural hybrids could have enhanced

New high-throughput sequencing technologies have opened the door for population genome analyses and genome-wide association studies. Genome of the *L. major* species was the first to be fully sequenced [150] followed by *L. infantum* and *L. braziliensis* [151]. Comparison of the three genomes revealed conservation of synteny and identified only 200 genes having a differential distribution between the three species. Such genes may encode for proteins implicated in host-pathogen interactions and parasite survival in the macrophage [151]. The species *L. mexicana* and *L. donovani* were subsequently sequenced [152,153] and the reference genomes for *L. major*, *L. infantum*, and *L. braziliensis* were refined [152]. This has allowed the identification of a remarkably low number of genes or paralog groups unique to each of the species *L. mexicana, L. major, L. infantum*, and *L. braziliensis* (2, 14, 19, and 67, respectively). Besides, *L. major* and *L. infantum* were found to have a surprisingly low number of predicted

spread into new foci throughout the broad range of *Ph. papatasi* distribution.

transmission potential and a positively affected fitness.

different genotypes.

Genomic research on *Leishmania* is taking promising directions, mainly upon sequencing of the main pathogenic species [150–153] and also the non pathogenic *L. tarentolae* [154] which will enable to answer key questions on population genetics and ultimately unravel many important aspects related to drug resistance and virulence, which are especially relevant for control of the disease.

Novel genomics technologies are expected to bring more powerful tools to characterize the pathogens and particularly the infectious stages of *Leishmania* parasites. It will be particularly useful to fully characterize the parasites within the lesions/hosts in their microenvironment. While so far expression profiling relied mainly on microarray analysis which revealed only a limited number of differentially expressed genes across developmental stages [155], or species [156]. RNA sequencing technology seems very promising to highlight transcriptional events that are associated to parasite life cycle, infection or pathology. Previous studies have dem‐ onstrated a correlation between gene expression and gene copy number [157,158]. It was further hypothesized that "Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression in response to environmental conditions in the host, providing a genetic basis for disease tropism" [152]. Other studies have also suggested that *Leishmania* parasites do not respond dynamically to host immune pressure, and that any influence of varying transcript levels on virulence and pathogenicity of the different *Leishma‐ nia* species is likely to result from the differential expression of conserved genes between species and/or the expression of a small number of genes that are differentially distributed between species [159].

Genome-wide multilocus genotyping in malaria research through novel sequencing technol‐ ogies has allowed the identification of almost 47000 single nucleotide polymorphisms (SNPs) across the *Plasmodium* genome [160]. This allowed development of microarray–based plat‐ forms for screening more than 3000 SNPs that were successfully applied for population genetic analyses and genome-wide association studies in *P. falciparum* [161,162]. Similar studies still need to be developed for *Leishmania*.

### **5. Conclusion**

Epidemiological, taxonomic and population genetic studies of *Leishmania* require good sampling methods and appropriate molecular markers that allow discrimination at different levels. Answering key epidemiological questions requires new or improved tools that allow discrimination of *Leishmania* parasites at different levels. The MLEE, considered as gold standard technique, needs cultured parasites and lacks discriminatory power. PCR assays are likely to replace isoenzyme analysis since they enable direct detection and identification of different *Leishmania* species in human and animal samples and also in infected sand flies. Many of the PCR assays described in the literature have proven useful in numerous field studies. However, they still need to be standardized and validated as diagnostic PCR assays and comparisons of the sensitivity and specificity parameters of the different approaches need also validation under routine conditions. In general, more than one assay is necessary to obtain fully satisfactory analysis of field samples. Given emergence context and changing ecoepidemiological trends, multiple tools will be needed to fully investigate the transmitted parasites.

At the strain level differentiation, MLMT has potential for being a gold standard, because on its principle it is expected to be reproducible and brings possibility of data storage and exchange. However, microsatellite markers are largely species-specific in *Leishmania* and different marker sets have to be used according to species. Such databases do not exist yet and data generation will need standardization. It may also require access to automated sequencers and good knowledge of population genetics programs. On the other hand MLST appears potentially as more powerful for phylogenetic and evolutionary studies although less dis‐ criminatory than MLMT. It is most probably this technique that will advantageously replace MLEE in the future. Some results showed that the same targets could be used across the *Leishmania* genus, which will enable comparisons of distances between the species but also of the degree of genetic diversity within species [163]. Here also it will require access to automated sequencers and adequate analytical programs. Cost of both approaches are relatively high and inherent limitations will be most likely overcome by the next generation sequencing ap‐ proaches expected to gain momentum in a near future. *Leishmania* population genomics still needs to be developed and made accessible to researchers in disease endemic countries to best achieve its public health potential.

Parasite knowledge is so far built on strains obtained *in vitro*. Increasing interest in *Leishma‐ nia* parasite analyses will likely identify novel genotypes or organisms, a challenge for our current knowledge on parasite taxonomy and assays to identify and characterize parasites. Improving ways to enhance knowledge on parasites within samples remains a priority.

In spite of the increasing potential of sophisticated technologies and techniques, some disease endemic areas still need simple assays for eco-epidemiological investigations or diagnosis as well as capacity building in this highly relevant area to disease control.
