Cutaneous and visceral Leishmaniasis

Discover our DNA extraction for sensitive diagnosis of Leishmaniasis

Illustration of Leishmania cells

Disease profile

Leishmaniasis is a Neglected Tropical Disease (NTD) caused by hemoflagellated protozoans of the family Leishmania. The pathogens are transmitted through bites of two sandfly genera (Americas: Lutzomyia; Asia, Africa, Europe: Phlebotomus). Canids such as dogs as well as rodents serve as natural reservoirs. Leishmaniasis is endemic in almost 100 countries in Asia, Africa, South & Central America and Southern Europe, where about 10 million people are infected. More than 25,000 people die from Leishmaniasis every year.

First illustrations and descriptions of humans having deformations and lesions typical for cutaneous leishmaniasis date back about 3000 years. In the late 19th century, the Russian surgeon Peter Borovsky was the first who correctly connected the lesions with a new protozoan parasite. 

Leishmania is one of three parasite genera belonging to the order Trypanosomatida, who cause diseases of major concern in humans (the other two conditions are Chagas disease and Sleeping sickness). There are about 20 Leishmania species known to cause Leishmaniasis in humans. The most important ones are L. major, L. infantum, and L. braziliensis. The replication of Leishmania takes place in macrophages (mononuclear phagocytes), which attack the protozoa after injection by the sandfly. In the human body the protozoa transform from the promastigote stage into the amastigote stage. The latter is actively infecting new macrophages.

Leishmaniasis can distinguished in two major forms: cutaneous leishmaniasis (CL) and visceral leishmaniasis. CL is characterized by skin ulcers developing around the site of the sandfly bite. If the ulcers are affecting the nose and mouth region, it is called mucocutaneous leishmaniasis. Ulcers can grow significantly and resembling leprosy lesions, and can ultimately destroy large skin areas. These lesions usually heal spontaneously after several weeks or months and the patient seem to be healed.

However, the protozoa are still inside the body and invade liver and spleen. This causes fever, anemia, and organ damage - the typical symptoms of visceral leishmaniasis (VL), also known as Kala-azar or Black fever. VL has an extremely high mortality rate if left untreated. And even if the patient was treated correctly or healed spontaneous, the disease can recur in a secondary form called Post Kala Azar, which causes various severe skin conditions with hypopigmented areas, ulcers or plaques. 

Read more on Wikipedia

 

Molecular diagnosis

Leishmaniasis is traditionally detected through microscopic visualization of the amastigote stage from blood, organ aspirates, or skin lesions. Immunological detection is possible through ELISA or direct agglutination tests, However, the most sensitive approaches to detect Leishmania are molecular. The sample types are identical with the above-mentioned: whole blood to detect visceral leishmaniasis and fine needle aspirates, skin punches or slit skin smears to detect cutaneous leishmaniasis.

Molecular diagnosis of Leishmaniasis requires a field-friendly extraction of DNA from whole blood and skin samples so that it can be applied in low-ressource setting laboratories or even literally in the field at the point-of-care. Amplification and detection of Leishmania DNA itself is usually performed using real-time PCR, RPA, RAA or LAMP.

 

Applicable Xpedite Diagnostics products

SwiftX DNA Kit for rapid extraction of DNA and RNA

SwiftX™ DNA (25 extractions)

For magnetic bead-based capture of Leishmania cells and rapid extraction of Leishmania DNA.

Validated for whole blood, skin punch biopsies, slit skin smears and fine needle aspirates.

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User protocols

Extraction of Leishmania DNA from skin biopsies

Unpublished protocol by Kobialka et al. from the Institute for Animal Hygiene and Public Veterinary Services (Leipzig, Germany)

  • 250mg glass beads (1mm diameter) were placed in a 1.5mL microtube
  • 300µL Buffer DL, 10µL Proteinase K (20 mg/mL), and 30µL Beads A were added
  • 10 milligram of a skin biopsy was added
  • mixture was vortexed for 10 seconds at 2000rpm
  • mixture was incubated for 15 minutes 65°C, and was briefly vortexed every 5 minutes
  • mixture was incubated 5 minutes at 95°C
  • microtube was removed from heat block and placed in a magnetic rack for 1 minute
  • supernatant was used for real-time PCR and real-time RPA detection

 

Magnetic capturing of Leishmania in lymph node aspirates followed by DNA extraction

National Reference Center for Leishmaniasis in Palermo (Italy) (see Application Note 2022-03 below)

  • 400µL Buffer EN and 30µL Beads A were mixed
  • 200µL lymph node aspirate was added and mixed
  • mixture was incubated for 3 minutes for binding of cells to Beads A
  • tubes were placed in a magnetic rack for 1 minute for bead separation
  • supernatant was removed
  • Beads A were washed with 500µL Buffer EN
  • Beads A with Leishmania & leukocytes were resuspended in 100µL Buffer DL
  • mixture was incubated for 10 minutes at 95°C
  • tubes were removed from heat block and placed in a magnetic rack for 1 minute
  • supernatant was used for real-time PCR detection

 

Magnetic capturing of Leishmania in leukocytes from whole blood followed by DNA extraction

Mondal et al. (2016) Parasites & Vectors (see below link to the publication)

  • 1500µL Buffer EN and 30µL Beads A was mixed
  • 500µL whole blood was added and mixed
  • mixture was incubated for 3 minutes for binding of leukocytes to Beads A
  • tubes were placed in a magnetic rack for 1 minute for bead separation
  • supernatant was removed
  • Beads A were washed twice with 500µL Buffer EN
  • Beads A with Leishmania & leukocytes were resuspended in 100µL Buffer DL
  • mixture was incubated for 10 minutes at 95°C
  • tubes were removed from heat block and placed in a magnetic rack for 1 minute
  • 5µL supernatant was used for real-time RPA detection