**Acknowledgements**

**5. Conclusion**

88 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

parasites.

achieve its public health potential.

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

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

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.

Research on *Leishmania* Molecular diagnostics in our laboratory has received support from the Ministry of Higher Education and Scientific Research and Technology in Tunisia (BSP46, LR00SP04 & LR11IPT04) and from different international programs: EU–Avicenne (CT920013), STD3 (CT930253), INCO-DC (CT970256); TDR–RSG (ID890266), TDR–RTG (920781), TDR–PAG (A30380); MERC–NIAID–NIH (NO1AI45183); IAEA (TUN06–12; CRP15111); CRDF (TN1–7009–TP–09); AUF (PCSI 6319PS011).
