Leishmaniasis, caused by protozoan parasites of the Leishmania genus, represents an important health problem in many regions of the world. Lack of effective point-of-care (POC) diagnostic tests applicable in resources-limited endemic areas is a critical barrier to effective treatment and control of leishmaniasis. The development of the loop-mediated isothermal amplification (LAMP) assay has provided a new tool towards the development of a POC diagnostic test based on the amplification of pathogen DNA. LAMP does not require a thermocycler, is relatively inexpensive, and is simple to perform with high amplification sensitivity and specificity. In this review, we discuss the current technical developments, applications, diagnostic performance, challenges, and future of LAMP for molecular diagnosis and surveillance of Leishmania parasites. Studies employing the LAMP assay to diagnose human leishmaniasis have reported sensitivities of 80% to 100% and specificities of 94% to 100%. These observations suggest that LAMP offers a good molecular POC technique for the diagnosis of leishmaniasis and is also readily applicable to screening at-risk populations and vector sand flies for Leishmania infection in endemic areas.

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97–99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes. Leishmania promastigotes are able to undergo genetic exchange during their growth and development in the sand fly vector, however, it is still not known if they have a true sexual cycle involving meiosis. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of 44 experimental hybrids generated between different strains of L. major, L. infantum, and L. tropica. In almost every case the number of chromosomes and the allele contributions from each parent matched the inheritance patterns expected under meiosis. Rare instances of hybrid chromosomes that did not fit Mendelian expectations were observed, including gain or loss of somy, and loss of heterozygosity. Strong evidence for a meiotic-like process was also obtained from the genome wide patterns of recombination observed in the offspring generated from backcrosses involving an experimental or natural hybrid, consistent with crossing over occurring between homologous chromosomes during meiosis. The frequency and position of the recombination breakpoints observed on each chromosome allowed us to construct the first physical and genetic maps in Leishmania. The results demonstrate that forward genetic approaches are possible for positional cloning of important genes.

Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64°C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per μl. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas.

Leishmania major and an uncharacterized species have been reported from human patients in a cutaneous leishmaniasis (CL) outbreak area in Ghana. Reports from the area indicate the presence of anthropophilic Sergentomyia species that were found with Leishmania DNA. In this study, we analyzed the Leishmania DNA positive sand fly pools by PCR-RFLP and ITS1 gene sequencing. The trypanosome was determined using the SSU rRNA gene sequence. We observed DNA of L. major, L. tropica and Trypanosoma species to be associated with the sand fly infections. This study provides the first detection of L. tropica DNA and Trypanosoma species as well as the confirmation of L. major DNA within Sergentomyia sand flies in Ghana and suggests that S. ingrami and S. hamoni are possible vectors of CL in the study area. The detection of L. tropica DNA in this CL focus is a novel finding in Ghana as well as West Africa. In addition, the unexpected infection of Trypanosoma DNA within S. africana africana indicates that more attention is necessary when identifying parasitic organisms by PCR within sand fly vectors in Ghana and other areas where leishmaniasis is endemic. Cutaneous leishmaniasis (CL) is one of the world's most neglected diseases transmitted by female sand flies and affecting mostly developing countries with about 1.2 million cases every year. In most African countries, the disease is typically caused by one of two species of Leishmania parasite: L. major or L. tropica. Clinical symptoms of both infections are similar, producing ulcerative and nodular lesions. Notwithstanding their similarity, lesions caused by L. major self-heal and bestow immunity to re-infection and therapy, if applied, is often by antimonials administered intra-lesionally, whereas the treatment of CL caused by L. tropica is difficult. Differentiating between these agents in any CL focus is important. Following the outbreak of CL in Ghana, we focused on the sand fly species present in the area in order to detect Leishmania DNA in potential vectors. Our study provides evidence on the occurrence of L. tropica and L. major DNA, and the detection of Trypanosoma DNA in Sergentomyia sand flies in Ghana. These findings have considerable implications in determining the epidemiology and dynamics of the disease. Significantly, our study supports the possibility of Sergentomyia sand flies as the vectors of CL in Ghana other than Phlebotomus, which contains all currently known vectors for Leishmania in the Old World.