Leishmaniasis Diagnostics

Leishmaniasis is diagnosed by demonstrating the parasite in tissue (amastigotes in Giemsa stained smears or aspirates, promastigotes in culture), by molecular methods, and by serology and antigen tests in visceral disease.

Visceral leishmaniasis (kala azar, Leishmania donovani and L. infantum) is diagnosed by splenic aspirate (sensitivity 93 to 99%), bone marrow aspirate (53 to 86%) or lymph node aspirate (53 to 65%), and by the rK39 rapid immunochromatographic test (sensitivity 85 to 100% in the Indian subcontinent and 80 to 90% in East Africa, specificity 90 to 99%).

Cutaneous and mucosal leishmaniasis are diagnosed by smear, culture, histology and PCR from the edge of the lesion (PCR sensitivity over 95%, with species identification by sequencing or restriction analysis, which is needed for treatment choice).

About 700 000 to 1 million new cases of cutaneous and 50 000 to 90 000 of visceral leishmaniasis occur each year, in over 90 countries, with most visceral cases in India, Bangladesh, Nepal, Sudan, South Sudan, Ethiopia, Kenya and Brazil. Untreated visceral leishmaniasis is fatal in over 95% of cases within 2 years.

It causes prolonged fever, weight loss, massive splenomegaly, pancytopenia, hypergammaglobulinaemia and hypoalbuminaemia. Post-kala-azar dermal leishmaniasis appears in 5 to 15% of cases in India and 50% in Sudan after treatment. Coinfection with HIV greatly raises the risk of visceral disease and relapse.

Indications. Fever for over 2 weeks with splenomegaly and cytopenias in a person from or returning from an endemic area. Chronic non-healing skin ulcers, nodules or plaques after sandfly exposure. Destructive nasal or oropharyngeal lesions (mucosal leishmaniasis). Unexplained pancytopenia, hypergammaglobulinaemia and hepatosplenomegaly.

Immunocompromised patients with unexplained fever and cytopenias. Relapse after treatment. Test of cure is clinical, with parasitological confirmation in doubtful relapse.

Specimen. Visceral: splenic aspirate (most sensitive, with a risk of haemorrhage, so done only with platelet count over 40 times 10 to the power 9/L, a prothrombin time within 5 seconds of control, ultrasound control and experienced staff), bone marrow aspirate (safer), lymph node aspirate, or buffy coat. Blood in EDTA for PCR (sensitivity over 90%).

Serum for rK39 and direct agglutination. Urine for antigen (KAtex). Cutaneous: clean the lesion, scrape or aspirate the raised edge (not the necrotic centre), prepare smears and a biopsy for histology, culture (Novy-MacNeal-Nicolle or Schneider medium) and PCR.

Reference values. No Leishmania amastigotes on smear, culture or PCR. Serology and rK39 negative. Direct agglutination titre under 1 in 800 (cut offs vary by region).

Interpretation.

• Amastigotes (Leishman-Donovan bodies, 2 to 4 micrometres, with nucleus and kinetoplast) in smears or aspirates: confirms infection, graded from 0 (no parasites in 1000 fields) to 6 (over 100 in each field).
• Positive rK39 with compatible illness: visceral leishmaniasis is probable, but the test is positive in 10 to 30% of healthy people in endemic areas and stays positive for years, so it is unreliable for relapse. Negative in 10 to 20% of East African and HIV-coinfected cases.
• Positive direct agglutination titre (1 in 6400 or more with a compatible picture): visceral leishmaniasis.
• Positive urinary antigen (latex agglutination): supports disease, with moderate sensitivity (64 to 100%).
• PCR positive: confirms the diagnosis, identifies species (L. donovani, L. infantum, L. major, L. tropica, L. braziliensis, L. mexicana), and is quantitative for monitoring.
• Pancytopenia, raised total protein with polyclonal increase in gammaglobulins, low albumin, raised ferritin, and haemophagocytic features on marrow: supportive of visceral leishmaniasis.
• Species in cutaneous disease predicts outcome: L. braziliensis (New World) risks mucosal disease and needs systemic treatment, L. major and L. mexicana often heal on their own, and L. tropica may be persistent.
• Negative smear with positive serology in a patient with HIV: serology can be falsely negative, so PCR or splenic aspirate is needed.

Critical values and findings needing immediate action.

• Confirmed visceral leishmaniasis: start liposomal amphotericin B (or other regimens as locally available) without delay, especially with severe anaemia, bleeding, bacterial sepsis or malnutrition.
• Haemophagocytic lymphohistiocytosis syndrome with visceral leishmaniasis: urgent treatment of leishmaniasis, and supportive care.
• Mucosal leishmaniasis threatening the airway: urgent systemic therapy and ENT assessment.
• Coinfection with HIV: start antiretroviral therapy, give secondary prophylaxis, and expect relapses.
• Severe anaemia (haemoglobin under 7 g/dL), platelet count under 20 times 10 to the power 9/L with bleeding, or neutropenia with infection: transfusion and antibiotics.
• Treatment failure or relapse: repeat parasitological testing and consider drug resistance.

Interferences and pitfalls.

• Splenic aspiration is hazardous, with risk of fatal bleeding in under 0.1% when done correctly, so is reserved for experienced staff.
• Smear sensitivity depends on the number of parasites, stain quality and the reader. A negative smear does not exclude disease.
• rK39 and other antibody tests cannot separate active disease from past infection or asymptomatic infection, and they stay positive after cure.
• Antibody tests cross-react with Trypanosoma cruzi (Chagas disease) and occasionally with malaria and tuberculosis. Antibody levels are low in HIV and other immunosuppression.
• Cutaneous lesions superinfected with bacteria may hide parasites, and secondary infection is treated before sampling.
• Culture needs special media and takes 1 to 4 weeks, and contamination is common.
• PCR needs a validated assay, and species typing must be matched to local species.
• Differential diagnosis of fever and splenomegaly includes malaria, typhoid, brucellosis, tuberculosis, schistosomiasis, lymphoma, leukaemia, histoplasmosis, hepatosplenic candidiasis and tropical splenomegaly.
• Treatment toxicity (nephrotoxicity and hypokalaemia with amphotericin, pancreatitis and cardiotoxicity with antimonials) needs monitoring.
• Post-kala-azar dermal leishmaniasis can be a reservoir for transmission, and diagnosis by skin smear or PCR is needed.

Special groups.

• Children: severe disease, malnutrition, and rapid deterioration, with cutaneous disease on the face.
• Pregnancy: liposomal amphotericin B is preferred, and antimonials and miltefosine are contraindicated.
• HIV-positive patients: atypical presentations, parasites in unusual sites (gut, lung), lower antibody sensitivity, frequent relapse and need for secondary prophylaxis.
• Travellers and military personnel: cutaneous and mucosal disease after exposure in endemic areas.
• Transplant recipients: reactivation of latent infection.
• Regions: visceral leishmaniasis in the Indian subcontinent (L. donovani, anthroponotic), East Africa (L. donovani), the Mediterranean, Middle East and South America (L. infantum, with dogs as reservoirs). Cutaneous leishmaniasis in the Middle East, North Africa, Central Asia, and the Americas.

Follow up tests. Full blood count with differential, liver function, albumin and total protein with globulin, creatinine and electrolytes (before and during amphotericin), ECG in antimonial and miltefosine therapy, HIV test, ferritin and triglycerides in suspected haemophagocytic syndrome, blood cultures and tuberculosis tests in prolonged fever, malaria film,

abdominal ultrasound for spleen size, repeat bone marrow or spleen examination for relapse, species typing by PCR, ophthalmology, and test of cure at 6 months and 12 months.