Challenges and perspectives in vaccination against leishmaniasis

The leishmaniases are a group of diseases caused by protozoa of the genus Leishmania and affect millions of people worldwide. The leishmaniases are transmitted to vertebrate hosts by phlebotomine sand flies. In this review, we focus on several issues that have been poorly addressed in ongoing efforts to develop a vaccine against Leishmania, namely: vaccination with antigens present in sand fly saliva, vaccines based on intracellular Leishmania antigens, and use of recombinant BCG as a vehicle for vaccination. Additionally, we address the differences between L. major and L. braziliensis and the impact that these differences may have on strategies for immunoprophylaxis.     

The current status of zoonotic leishmaniases and approaches to disease control

Leishmaniases are a complex of world-wide diseases with a range of clinical and epidemiological features caused by Leishmania spp. of protozoan parasites. Among 15 well-recognised Leishmania species known to infect humans, 13 have zoonotic nature, which include agents of visceral, cutaneous and mucocutaneous forms of the disease in both the Old and New Worlds. Currently, leishmaniases show a wider geographic distribution and increased global incidence of human disease than previously known. Environmental, demographic and human behavioural factors contribute to the changing landscape of leishmaniasis, which includes increasing risk factors for zoonotic cutaneous leishmaniases and new scenarios associated with the zoonotic visceral leishmaniases. The latter consist of the northward spread of Leishmania infantum transmission in Europe and America, the identification of unusual mammal hosts, and the decline of HIV-Leishmania co-infections in southern Europe following the introduction of the highly active antiretroviral therapy. Few advances have been made in the surveillance and control of the zoonotic leishmaniasis, however a number of tools have been developed for the control of the canine reservoir of L. infantum. These include: (i) several canine vaccine candidates, in particular an FML Leishmania enriched fraction showing good clinical protection, has been registered in Brazil for veterinary use; (ii) a number of insecticide-based preparations have been specifically registered for dog protection against sand fly bites. Laboratory and field studies have shown improved efficacy of these preparations for both individual and mass protection.     

Phlebotomine fauna (Diptera: Psychodidae) and putative vectors of leishmaniases in impacted area by hydroelectric plant, State of Tocantins, Brazil

Background

Although leishmaniases are regarded as serious public health issues in the State of Tocantins, as consequence of the impact of environmental changes, small advances in taxonomic and ecological studies of Phlebotominae fauna are taking place in this state. The present study aimed to improve the knowledge about the sand flies, as well as about the aspects of the bioecology of leishmaniases vectors from Porto Nacional, a city that was directly impacted by the construction of Luís Eduardo Magalhães Hydroelectric Plant (HEP – Lajeado).

Methodology/Principal Findings

Sand flies were collected monthly using CDC light traps and Shannon traps for a period of 40 consecutive months, at different monitoring stations, where 7162 specimens were collected and 48 species were detected. Among the species found, 22 are first records in the state and seven are considered important vectors of leishmaniases. Lutzomyia longipalpis, the vector of American Visceral Leishmaniasis (AVL) showed higher frequency in urban compared to rural areas, and Nyssomyia whitmani, the vector of American Cutaneous Leishmaniasis (ACL), predominated in rural areas. The frequency and habits of sand fly vectors are discussed considering environmental characteristics and climatic factors.

Conclusions/Significance

The construction of dams requires a great amount of labor, therefore attracting people from elsewhere. Increased migration, without adequate structure, leads to bad living conditions in new and unplanned settlements. It also leads to deforestation associated with environmental impacts, which can facilitate the spread of leishmaniases.This study discusses the importance of Lu. longipalpis and Ny. whitmani on the transmission cycles of leishmaniases in Porto Nacional and the record of Bi. flaviscutellata in periurban area of the city.     

Immune response to leishmania: paradox rather than paradigm

The leishmaniases are a group of diseases caused by protozoan parasites of the genus Leishmania. Various Leishmania species can cause human infection, producing a spectrum of clinical manifestations. It is estimated that 350 million people are at risk, with a global yearly incidence of 1-1.5 million for cutaneous and 500,000 for visceral leishmaniasis (VL). VL is a major cause of morbidity and mortality in East Africa and the Indian subcontinent. Coinfection with HIV enhances the risk of the disease. The only control measure currently available in India is case detection and treatment with antimonial drugs, which are expensive, not always available and cannot be self-administered. Newer drugs like oral miltefosine have not become widely available. Vector and reservoir control is difficult due to the elusive nature of the vector and the diversity of the animal reservoir. A detailed knowledge of immune response to the parasite would help in designing prophylactic and therapeutic strategies against this infection.     

The genus Leishmania in Italy

Seventy-four Leishmania isolates collected in Italy from six different Regions where leishmaniases are endemic, have been typed. Parasites have been isolated from: man (VL and CL), dog, black rat (Rattus rattus), fox (Vulpes vulpes) and geckoes (Tarentola mauritanica and Cyrtodactylus kotschyi). The isolates have been characterized by starch-gel electrophoresis for 9-16 enzymes whose mobility was compared with that of international reference strains for L. infantum, L. tropica, L. major, L. donovani, L. aethiopica and L. tarentolae. The results obtained have shown that the genus Leishmania in Italy is represented by five zymodemes which may be grouped into two taxa: L. infantum s.l. (L. infantum s.st., L. infantum NH130 variant, L. infantum NH140 variant and L. infantum GOT, MDH, NH variant), agent of mammalian leishmaniases (including human leishmaniases), and L. tarentolae, parasite of geckoes. At the moment, the absence of L. tropica in Italy as agent of CL has been revealed. Through the analysis of epidemiological data obtained from the foci where Leishmania parasites were isolated two zymodemes only, L. infantum s.st. and L. infantum NH140 variant, show to be widely distributed. However, L. infantum s.st. appears to be prevalent in Thyrrenean foci which are characterized by VL cases and by high density of Phlebotomus perniciosus, and L. infantum NH140 variant is present in Adriatic areas where CL is diffuse and P. perfiliewi is the probable vector.     

Microsatellite analysis reveals genetic structure of Leishmania tropica

The current rapid spread of leishmaniases caused by Leishmania tropica and the complexity of its clinical spectrum call for this parasite's epidemiological and evolutionary investigation. Evaluation of its population structure by isoenzyme electrophoresis and previous molecular biological analysis has proved difficult. In this study, we used 21 microsatellite loci to type 117 strains from different African and Asian locations. Eighty-one different genotypes were found. A genetic bottleneck supported by a gradient in the number of alleles and consistent with the geographical structure of the Middle East suggests an African origin of this species. A Bayesian approach identified 10 genetic clusters that correlated predominantly with geographical origin. The strains in the 'Asia' cluster form a very heterogeneous sub-population, with a varied but inter-related genotype that is geographically very widely dispersed and consistent with anthroponotic transmission of the parasite. The other nine clusters were more homogenous. The propagation of L. tropica appears to be predominantly clonal. In Africa and the Middle East, anthroponotic and zoonotic systems of distribution may contribute to the development of overlapping, genetically distinct populations of L. tropica.     

Risk factors in the spread of leishmaniases: towards integrated monitoring?

Environmental changes, immune status and treatment failure constitute the three major risk factors for the (re-)emergence and spread of leishmaniases. Except for Leishmania-HIV co-infection, these risk factors are not systematically monitored and their interaction is poorly studied and understood. Recently, the multidisciplinary network Leish-Med was launched to document this issue around the Mediterranean and to promote transborder control strategies.     

Leishmania proteins derived from recombinant DNA: current status and next steps

The parasite Leishmania is a major cause of disease worldwide. In the past 15 years, many groups have analysed DNA-derived proteins from Leishmania. Large amounts of data obtained by these groups can be collated to direct future research into Leishmania and to find novel immunological mechanisms and information about its metabolism. International coordination will increase both the basic knowledge about Leishmania and the capacity to apply this knowledge to combat leishmaniases.     

Leishmaniasis: complexity at the host-pathogen interface

Leishmania is a genus of protozoan parasites that are transmitted by the bite of phlebotomine sandflies and give rise to a range of diseases (collectively known as leishmaniases) that affect over 150 million people worldwide. Cellular immune mechanisms have a major role in the control of infections with all Leishmania spp. However, as discussed in this Review, recent evidence suggests that each host-pathogen combination evokes different solutions to the problems of parasite establishment, survival and persistence. Understanding the extent of this diversity will be increasingly important in ensuring the development of broadly applicable vaccines, drugs and immunotherapeutic interventions.     

Rapid identification of Leishmania species from formalin-fixed biopsy samples by polymorphism-specific polymerase chain reaction

The precise identification and classification of Leishmania species is important for public health surveillance since different species cause different clinical features of the disease. A highly specific polymerase chain reaction (PCR) panel was developed to enable the identification of the five major Leishmania species that cause New World cutaneous leishmaniases. The primers used for this panel were designed to distinguish the polymorphism in sequences of commonly amplified DNA bands of the parasites produced by arbitrarily primed PCR. These polymorphism-specific PCR diagnoses were performed with formalin-fixed biopsy specimens of the leishmanial lesions from four patients in Ecuador and one hamster skin lesion, and these lesions were determined to be caused by Leishmania (Viannia) panamensis, L. (Leishmania) mexicana, and L. (L.) amazonensis. The PCR panel may offer an important and practical approach to the standardized identification of Leishmania species in field examinations.     

Parasite identification in the surveillance of imported leishmaniasis cases in Italy

An accurate Leishmania classification was defined since 1980s by the use of isoenzyme analysis. To date, this procedure still represents the reference identification technique, despite the increasing use of molecular approaches. Studies and surveillance methods on leishmaniases are strongly conditioned by the knowledge and mapping of all the parameters characterizing each nosogeographical entity. On this respect, the identification of parasites from all the actors of the natural life cycle plays a key role. With the increasing population movements and climate changes, novel risk factors could be identified associated to Leishmania geographical distribution and spreading: a) the introduction into Italy of new populations of L. infantum from other countries; b) the introduction of new Leishmania species that may find a suitable milieau to support their life cycle in our country. The objective of this report is to present the surveillance activity on imported leishmaniases by the Leishmania Identification Reference Centre, ISS. Two different methodologies were routinely applied: a) isoenzyme electrophoretic analysis, which requires parasite culture, and b) a number of molecular techniques, used for both diagnosis and parasite identification, differently applied according to the geographical origin of the suspected leishmaniasis case. When possible, both types of methodologies were applied. From 1986 to June 2002, 38 imported cases of leishmaniases were identified: 9 visceral (VL) and 29 cutaneous (CL) cases, of which 22 from the Old and 7 from the New World. Pathology, Leishmania species/zymodeme and geographical origin features are reported in the paper. Seven out of 9 VL patients were HIV positive, of whom 5 detected in the period 1993-1995. This high importation rate can be associated to the general increase in Mediterranean Leishmania/HIV coinfections in that period. Following HAART treatment, VL imported cases became occasional; no introduction of new L. infantum populations has been detected. On the other hand, our findings show an increase of CL imported cases from different areas of the Old and New Worlds. This phenomenon, however, is so far limited to new Leishmania species that could hardly be introduced in our country, because of their strict biological requirements (i.e. vectors and/or natural reservoir hosts). Since June 2002, 10 further suspected imported cases were recorded. For these patients--whose Leishmania identification is still in course--the origin/visited geographical areas were only slight different from the previous. However, the characteristics of the patients are changing: there are more immigrants that occasionally visited their place of origin, and Italian military staff.     

Kinetoplastida: new therapeutic strategies

New formulations and therapeutic switching of the established drugs, amphotericin B and paromomycin, together with the discovery of miltefosine, have significantly improved the opportunities for treatment of visceral leishmaniasis (VL) chemotherapy. However, for human African trypanosomiasis (HAT), Chagas disease and cutaneous leishmaniases there has been limited progress. For HAT, a novel diamidine, parfuramidine, is in phase III clinical trial for early-stage disease, but for the treatment of late-stage disease there are no new drugs and combinations of eflornithine with melarsoprol or nifurtimox have been the focus of clinical studies. For Chagas disease, different classes of compounds that have validated biochemical targets, sterol biosynthesis methylases and cysteine proteases, are in various stages of development. The genome sequences that are now available for the pathogens that cause the leishmaniases and trypanosomiases, and new methods for rapid validation of targets, are part of the solution to discover new drugs. The integration of medicinal chemistry, pharmacokinetics, project planning and interaction with the pharma/biotech sector are essential if progress is to be made. Although there are financial constraints, the appearance of new funding sources and not-for-profit product development partnerships offers hope for drug development.     

Leishmania species: Comparative ultrastructure of experimental nodules and diffuse human cutaneous lesions in American leishmaniases

Experimental nodules of American leishmaniases were obtained by inoculating 0.1–1 × 10⁵ amastigotes into the dorsum of the hindpaws of golden hamsters and of C57B1/6J mice. The amastigotes were obtained by biopsy of lesions in six human cases of cutaneous leishmaniases and were serially maintained in golden hamsters and in a fetal calf serum-containing medium. Human nodules were obtained by biopsy from several patients with diffuse cutaneous leishmaniases, always prior to treatment. Within the same host species, no ultrastructural differences were seen in the tissue response to isolates of Leishmania mexicana, L. brasiliensis, or L. garnhami, nor were there differences between the host species in response to a particular isolate of the genus Leishmania. The typical inflammatory response was a macrophage granuloma with abundant polymorphonuclear neutrophils, some eosinophils, and plasma cells. Simple human cutaneous leishmanial lesions, as well as experimental nodules in regression, show many fibroblasts, much collagen fiber, but very few parasites. In typical lesions, parasites occurred within macrophage phagolysosomes, within distended lacunar cells, and in the intercellular spaces. Leishmaniae strongly adhered to parasitophorous vacuoles by a site of their plasma membrane directly opposite the flagellum, and the host cell cytoplasm close to the adherence site became highly vacuolated. In most cases the intra- and extracellular parasites show normal morphology, which suggest the inability of phagocytic cells to attack them.     

A profile of research on the parasitic trypanosomatids and the diseases they cause

The parasitic trypanosomatids cause lethal and debilitating diseases, the leishmaniases, Chagas disease, and the African trypanosomiases, with major impacts on human and animal health. Sustained research has borne fruit by assisting efforts to reduce the burden of disease and by improving our understanding of fundamental molecular and cell biology. But where has the research primarily been conducted, and which research areas have received the most attention? These questions are addressed below using publication and citation data from the past few decades.     

Interaction of Leishmania with the host macrophage

The leishmaniases are a group of diseases with a spectrum of clinical manifestations ranging from self-healing cutaneous ulcers to severe visceral disease and even death. In mammals, the macrophage is the main host for the Leishmania amastigote. However, the macrophage is also the immune effector cell that, upon activation, is able to kill intracellular organisms. Therefore, understanding the parasite mechanisms which allow establishment of infection, and the host immune mechanisms that are responsible for parasite recognition and killing should lead to the development of new drugs and vaccines.     

Roe deer (Capreolus capreolus) are a novel potential reservoir for human visceral leishmaniasis in the Emilia-Romagna region of northeastern Italy

Leishmaniasis is a complex human disease caused by intracellular parasites of the genus Leishmania, predominantly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused exclusively by Leishmania infantum, responsible for the human and canine visceral leishmaniases (HVL and CVL, respectively). Within the Emilia-Romagna region, two different foci are active in the municipalities of Pianoro and Valsamoggia (both in the province of Bologna). Recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different, suggesting that there is an animal reservoir other than dogs for human visceral leishmaniasis in the Emilia-Romagna region. In this work, we analyzed specimens from wild animals collected during hunts or surveillance of regional parks near active foci of human visceral leishmaniasis for L. infantum infection in the province of Bologna. Out of 70 individuals analyzed, 17 (24%) were positive for L. infantum. The infection prevalence in hedgehogs (Erinaceus europaeus), roe deer (Capreolus capreolus), badgers (Meles meles), and bank voles (Myodes glareolus) was 80, 33, 25, and 11%, respectively. To distinguish the two strains of L. infantum we have developed a nested PCR protocol optimized for animal tissues. Our results demonstrated that most (over 90%) of L. infantum infections in roe deer were due to the strain circulating in humans in the Emilia-Romagna region.     

Immunobiology of experimental cutaneous leishmaniasis

The study of the murine model of infection with Leishmania major is providing important insights into the understanding of the complex interactions between the host and intracellular pathogens. Using this model system, basic research is actively leading to the identification of host factors promoting or circumventing the development of immunity to L. major. Here, Geneviève Milon, Giuseppe Del Giudice and Jacques A. Louis review recent results related to the characterization of immunological host factors determining resistance and susceptibility to this parasite, and try to identify areas where further research is required for a better understanding of the complex events triggered by intracellular parasites within their hosts. Extrapolation to the human leishmaniases of the rapid advances made in this murine model of infection, should pave the way to the rational design of future immunoprophylactic and immunotherapeutic measures.     

Microplate method for obtaining Leishmania clonal populations

The various clinical expressions observed in human leishmaniases result from complex host-parasite relationships in which the biodiversity of the parasite is a determining factor. Because Leishmania strains isolated from humans are composed of heterogeneous populations, it is crucial to use clonal lineages for studies on the characterization of these parasites. Presently, techniques used for cloning Leishmania spp. parasites are time-consuming and show poor efficiency. Here, a method developed in 96-well microplates is described, which allows one to rapidly obtain numerous clones of Leishmania in the most versatile and efficient way. The technique may be useful for cloning various protozoa as well as Leishmania spp.