Leishmania donovani - Laboratory Diagnosis

Laboratory Diagnosis of Leishmania donovani

The laboratory diagnosis of Leishmania donovani includes:

Specimen

• biopsy samples from the spleen, bone marrow, lymph node, liver, and peripheral blood smear

• spleen aspiration – 98% sensitivity but risky as seen rupture can be fatal

• bone marrow aspiration (collected from the sternum, iliac crest) – 85% sensitivity

• lymph node aspiration – 60% sensitivity (useful for African leishmaniasis but not for Indian leishmaniasis)

• peripheral blood smears – low sensitivity

Microscopy

• Leishmania donovani amastigotes, also known as LD bodies) can be demonstrated in biopsy smears

• stains such as Leishman stain, Giemsa stain, Brown-Hopps stain, or Wright stain can be used

• LD bodies can be found within macrophages as well as freely in the host circulatory system which had recently escaped after rupturing from macrophages

• observation of LD bodies in spleen aspiration, bone marrow aspiration, and lymph node aspiration also confirms infection by Leishmania donovani

• in cases of stained peripheral blood smears, LD bodies can be microscopically demonstrated inside neutrophils and circulating monocytes

Culture

Leishmania donovani can be cultured in laboratory animals as well as in-vitro culture.

In-vitro culture

• media used includes NNN, Schneider Drosophila medium or any biphasic medium

• tissue samples and aspirates are inoculated into the water of condensation or into the fluid of a liquid medium

• incubated at 22° C - 26° C for 1-4 weeks

• at the end of each week, a drop of culture is microscopically examined for promastigotes

• motile promastigotes can be observed in positive cultures

• has a sensitivity of 75%

Laboratory animal inoculation

• laboratory animals such as Chinese and golden hamsters are inoculated intraperitoneally by Leishmania donovani culture

• if the animal dies, its spleen smear is stained and visualized under a microscope

• if the animal lives, they are killed after six months to visualize its stained spleen smear under a microscope

Serodiagnosis

Serodiagnosis is mostly useful to diagnose the early stages of visceral leishmaniasis caused by Leishmania donovani.

Complement Fixation Test (CFT)

• Complement Fixation Test (CFT) is used to detect serum antibodies in cases of visceral leishmaniasis

• Mycobacterium tuberculosis antigen WKK (Witebsky Klingenstein, Kuhn) and an antigen from Kedrowsky acid-fast bacillus are used

Indirect Immuno-fluorescent (IFA)

• Indirect Immuno-fluorescent (IFA) uses cultured Leishmania donovani promastigotes as antigen

• the disadvantage includes cross-reactivity with sera from leprosy, schistosomiasis, malaria, cutaneous leishmaniasis, and Chagas disease

Enzyme-Linked Immunosorbent Assay (ELISA)

• Enzyme-Linked Immunosorbent Assay (ELISA) uses cultured Leishmania donovani promastigotes as antigen

• the disadvantage includes cross-reactivity with sera from leprosy, schistosomiasis, malaria, cutaneous leishmaniasis, and Chagas disease

Direct Agglutination test (DAT)

Direct Agglutination test (DAT) uses trypsin-treated Coomasie blue-stained promastigotes as antigen

Strip test

Immunochromatographic strip test employs Leishmania rK39 antigen in the diagnosis of visceral leishmaniasis

Aldehyde test of Napier

• Aldehyde test of Napier is done by adding a drop of 40% formalin to 1ml to 2 ml of serum in a test tube

• within 2 to 20 minutes, if jellification occurs, changing the serum to a milky white opacity like of a boiled egg, the aldehyde test of Napier is positive

• negative in cases of cutaneous leishmaniasis

• disadvantages include cross-reactivity with cases of African trypanosomiasis, liver cirrhosis, schistosomiasis japonica, and multiple myeloma

Antimony test

Upon addition of urea stibamine solution into the patient's serum, antimony test results in the formation of a white flocculent precipitate in positive cases of leishmaniasis caused by Leishmania donovani.

Molecular test

The molecular tests used for diagnosis of Leishmania donovani are:

• DNA probes

• PCR

Leishmania skin test (Montenegro test)

• Leishmania skin test (Montenegro test) delayed hypersensitivity test

• gives negative results in active Leishmania donovani infections

• indicates prior exposure in positive cases

• positive skin test results are seen only 6 to 8 weeks after the cure

• 0.2 ml of Leishmania antigen is injected intradermally and read after 48-74 hours

• induration and erythema of 5mm diameter or larger are seen in positive cases