Leishmania spp.: Leishmaniosis in humans and dogs – Molecular diagnosis (Real-time PCR); Species identification (PCR and sequencing); Antibodies.

Information 27-02-2018.

Leishmaniosis is a group of clinical manifestations produced by a protozoan parasite of the genus Leishmania, which is transmitted to humans by the bite of infected female phlebotomine. Leishmaniasis is the third most important vector-borne disease in the world. Its geographical distribution is conditioned by the distribution of its vectors and its sensitivity to cold, and includes the Mediterranean basin, Southeast Asia, East and North Africa, and Central and South America, being endemic in 88 countries. In spite of the advances obtained by the programs of elimination of the disease in the main endemic countries, the burden of this disease has increased in the last decade due to the migration of humans and infected dogs, the new geographical distribution of the insect by the global warming, coinfection with immunosuppressive diseases and hygienic-sanitary conditions. The World Health Organization estimates that between 700,000 and one million new cases occur each year and between 20,000 and 30,000 deaths. Leishmaniosis is mainly a canine zoonosis, so its study and diagnosis is important in both veterinary and human medicine.

Fifty three species of Leishmania have been described, classified in five subgenera and complexes: Leishmania, Viannia, Sauroleishmania, the complex L. enrietti, and Paraleishmania. Among them, 31 species are parasites of mammals and 20 species, included in the genera Leishmania and Viannia, are pathogenic for humans.

Leishmania spp. causes four main clinical forms, depending on the location of the parasites in the tissues: cutaneous, diffuse cutaneous, mucocutaneous, and visceral leishmaniosis. The health status of the host and its genetics influence the form of the disease, but the main determinant of the tropism and the pathology of the infection is the infecting Leishmania species.

The different clinical pictures are caused by different species of Leishmania in the Old and New World. The subgenus Viannia (L. braziliensis, L. guyanensis, L. panamensis, L. naiffi, L. shawi, L. lainsoni, and L. peruviana) and the L. mexicana complex (L. mexicana, L. amazonensis, L. pifanoi, L. garnhami, and L. venezuelensis) cause cutaneous leishmaniosis in the New World, while L. major, L. tropica, and L. aethiopica are the species responsible for cutaneous leishmaniasis in the Old World. Among them, infections caused by L. aethiopica and L. amazonensis can also give rise to diffuse cutaneous leishmaniosis. Some of these species, mainly L. braziliensis, and to a lesser extent L. guyanensis, L. panamensis, and L. amazonensis, can migrate to nasopharyngeal tissues and cause mucocutaneous leishmaniosis. The most severe form of the disease, visceral leishmaniosis, is caused by two species of the L.donovani complex: L. donovani and L. infantum, also called L.archivaldi and L. chagasi respectively in the New World. Also, some cases of visceral leishmaniosis caused by the infection of L. tropica and L. amazonensis have been reported.

The biological cycle of Leishmania essentially comprises the alternative passage from a vertebrate host to another invertebrate, and viceversa, with two main morphological forms, the intracellular or amastigote that is localized in the cells of the mononuclear phagocytic system of the vertebrate host, and the extracellular form or Promastigote located in the intestinal tract of arthropod vectors, sandflies. Sandflies are small Diptera of 2-3 mm, of which at least 78 species are proven vectors of leishmaniosis. In the New World, the species involved in the transmission of infection belong to the genus Lutzomyia, Nyssomyia, Psychodopygus, and Verrucarum, while in the Old World, they are mostly included in the genera Phlebotomus and Paraphlebotomus. The passage of the parasite to the vector takes place when the females of the sandflies ingest blood from the vertebrate host to feed and develop their eggs. In the digestive tract of the vector, Leishmania multiplies under the amastigote form, rapidly transforming to the promastigote form, which actively multiplies in its stomach and intestine. After a paramastigote phase, the metacyclic promastigotes, the infecting form, are located in the buccal region or trunk of the vector, from where they will pass to the vertebrate host. Although these dipterans are the accepted vectors of the disease, the dipterans Forcipomyia spp. and some species of ticks such as Rhipicephalus sanguineus have been described as potential vectors of Leishmania.

Most leishmaniosis are zoonoses in which different wild and domestic animal species, fundamentally canids and different types of rodents, act as a reservoir of the parasite for its subsequent spread to humans through sandflies. However, in some forms of leishmaniosis, man can also act as the sole vertebrate host. The cycle in the vertebrate host begins when the sandfly inoculates the infecting or metacyclic promastigotes through its bite. The promastigotes are captured by the macrophages of the dermis and, in the cytoplasm, they are included in a parasitophore vacuole where they are transformed to amastigote form. Inside the macrophages, the amastigotes can be destroyed by the oxygenated metabolites and the lysosomal hydrolases, if not, the parasites are actively divided until the cell explodes. Depending on the form of leishmaniosis, free parasites can be picked up by another macrophage in the area, repeating the multiplicative process, disseminate directly through the skin or the skin circulation to reach the mucous membranes, or be carried by the bloodstream and lymphatic with macrophages, localizing in the lymphatic organs, mainly bone marrow, liver and spleen. In humans, when the invasion of Leishmania spp. is limited to the macrophages of the area of ​​the bite, presents the clinical picture of cutaneous leishmaniosis, also known as Oriental sore. Cutaneous leishmaniosis is the most frequent form of the disease, and it produces localized infections in the exposed areas of the body with one or more skin lesions, especially ulcers, which leave scars for life. In most cases it can resolve spontaneously, although the duration of the process is prolonged (months or years). If, on the other hand, a weak immune response occurs and the isolation of the primary lesion by a lymphocytic barrier does not take place, the parasite spreads over the entire cutaneous surface producing diffuse cutaneous leishmaniosis, characterized by the formation of plaques, papules and/or nodules mainly on the face and limbs. The metastasis of the parasites towards the mucous membranes causes mucocutaneous leishmaniosis or sputum, which causes ulceration and erosion of the soft tissue and cartilage of the affected areas, mainly the nasal area but also the pharynx, larynx and upper lip, which can be highly deformative. Finally, the visceralization of the parasite is known as visceral leishmaniosis or Kala-azar. This clinical form usually affects children, people in a state of malnutrition and patients with some type of immunosuppression and, if untreated, is fatal in more than 95% of cases. It is characterized by irregular episodes of fever, weight loss, hepatosplenomegaly and anemia. The main organs affected are the spleen, liver, bone marrow and lymphatic tissue, but there may be involvement of other areas such as the lungs and intestinal mucosa. In the affected organs, small granulomas are formed that damage the viscera, causing hyperplasia and hypertrophy thereof, congestion of the blood vessels, necrosis of the tissues and alterations of their normal functioning. In addition, the important humoral response results in the appearance of circulating immune complexes that sometimes deposit in the kidneys causing glomerulonephritis.

In the Mediterranean Basin, L.infantum is the agent responsible for the cutaneous and visceral form of leishmaniosis in humans, with the cases being declared essentially visceral, most of them in patients infected with HIV. L. infantum is associated with a zoonotic cycle where dogs are considered the main hosts of the parasite and the main reservoir of canine and human infection. It should be added that L. infantum can also cause cutaneous leishmaniosis in cats and horses, with feline leishmaniosis being an increasingly recognized disease in endemic areas. Canine leishmaniosis is endemic in the Mediterranean basin, with the average seroprevalence of L. infantum in southern Europe in domestic dogs of up to 25%. In dogs, L.infantum infection has an incubation period that varies from months to years and the clinical picture includes a spectrum that ranges from subclinical infection to canine leishmaniosis, a severe, chronic, systemic, and even fatal disease. Resistance is associated with the development of cellular immunity, and most infected dogs do not get sick. The most frequent clinical manifestations are lymphoadenomegaly, skin alterations, anorexia, eye injuries, lameness, weakness, weight loss, hair loss, epistaxis, anemia, and renal dysfunction.

 The laboratory diagnosis of leishmaniasis is based, generally, on the joint application of direct and indirect diagnostic methods, varying according to the clinical picture. The direct diagnosis is made mainly by observing the parasite under a microscope after staining with the dyes usually used in hematology. Also, culture, inoculation in laboratory animals, xenodiagnosis, and detection of antigens by Western blot, or DNA by PCR can be performed. Among the indirect methods, the intradermoreaction of Montenegro and the serological diagnosis of visceral leishmaniosis stand out. Currently, the detection of the DNA of the parasite by PCR is one of the most tested techniques due to its great sensitivity and speed. Serological diagnosis is also a useful method in visceral leishmaniosis in immunocompetent patients, but not in immunosuppressed patients. L. infantum, the endemic species in the Mediterranean area, presents an important enzymatic polymorphism and, in the laboratory, the identification of the different zymodemas constitutes a useful taxonomic tool that contributes to the study of the epidemiology of the pathogen. In humans, different zymodemas can be considered dermotropes, viscerotropes, or causes of both forms of leishmaniosis, and also, be more associated with immunocompetent or immunosuppressed individuals. In addition, the comparison of species by PCR-RFLP allows to differentiate strains of L. infantum, constituting a useful method to compare two strains of different origins, or two samples of the same subject to differentiate between relapse or reinfection.

Tests carried out in IVAMI:

• Molecular detection (real-time PCR) of Leishmania spp. by amplifying the AAP3 gene in humans and animals.

• Identification of Leishmania spp. at the species level (PCR and sequencing).

• Detection of antibodies against L. infantum in humans and dogs.

Recommended sample:

• For the diagnosis of tegumentary leishmaniosis, the recommended samples are those obtained from active lesions of the skin or mucous membranes. In ulcerated lesions, the sample must be taken at the edges of the lesion or at the bottom of the ulcer, after removing the upper necrotic area. Sampling can be done by scraping the lesion, needle aspiration, needle biopsy, or punching.

• For the diagnosis of visceral leishmaniosis, the sampling method most used for its ease and sensitivity is the bone marrow aspirate. The parasite can also be found occasionally in peripheral blood leukocytes.

• For serological diagnosis in immunocompetent individuals should be sent serum or plasma (2 mL), or whole peripheral blood drawn with EDTA (5 mL) for the subsequent separation of plasma in our laboratory.

Conservation and shipment of the sample:

• Refrigerated (preferred) for less than 2 days.

• Frozen: more than 2 days.

• In the case of sending whole blood extracted with EDTA for serological diagnosis, the sample should not be frozen.

Delivery of results:

• Molecular detection (real-time PCR) of Leishmania spp. by amplification of the AAP3 gene: 24 to 48 hours.

• Identification of Leishmania spp. at the species level (PCR and sequencing): 48 to 72 hours.

• Detection of antibodies against L. infantum: 48 to 72 hours.

Cost of the test:

• Molecular detection (real-time PCR) of Leishmania spp. by amplifying the AAP3 gene: consult ivami@ivami.com

• Identification of Leishmania spp. (PCR and sequencing):: consult ivami@ivami.com

• Detection of antibodies against L. infantum: consult ivami@ivami.com