Pediatric Leishmaniasis Workup

The parasite can be detected through direct evidence from peripheral blood, bone marrow, or splenic aspirates, as explained below. The smears are stained in Leishman, Giemsa, or Wright stains and examined under oil immersion microscope.

Peripheral blood smear

Amastigotes are revealed inside the circulating monocytes and neutrophils. However, they are often difficult to locate because of the small numbers. L donovani is best detected using the following methods: (1) creating thick film by producing a single straight leukocyte edge when making a peripheral smear or (2) centrifuging citrated blood and withdrawing the sediment, which is then smeared, dried, and stained.

Culture

Obtaining a culture is time consuming, and findings take approximately a month. Cultures are made using a Novy-McNeal-Nicolle medium. The medium is a rabbit-blood agar that has an overlay of Locke solution with added antibiotics. Two mL of blood is aseptically obtained from a vein and is diluted with 10 mL of citrated saline solution. This is centrifuged and the cellular deposit is inoculated into the water of condensation of nitrosonornicotine (NNN) medium and incubated at 22°C for 1-4 weeks. At the end of each week, a drop of condensation fluid is examined for promastigote forms.

Polymerase chain reaction

Parasite DNA can be identified using polymerase chain reaction (PCR).^[1]

Animal inoculation

This is a sensitive method but can take several weeks. The sample is inoculated intraperitoneally or intradermally into skin on the nose and feet. The parasites can be demonstrated from the ulcers and nodules that develop from the sites of inoculation.

Detection of hypergammaglobinemia

The aldehyde test and the antimony test were the initial tests used to diagnose kala azar.

Aldehyde test results reveal increased gamma globulin levels. Obtain approximately 1 mL of blood in a small glass tube and add 1-2 drops of 40% formalin. The formation of milky whitelike opacity and jellification indicates a positive result. Aldehyde test findings are not positive unless the disease has been present for at least 3 months.

Antimony test findings also depend on a rise in serum gamma globulin levels. Positive findings are indicated by a white flocculent precipitate observed when a urea stibamine solution comes in contact with serum.

Immunological tests

Before the use of specific leishmanial antigens, nonspecific antigens were used. These include the Witebsky, Klingenstein, Kuhn (WKK) antigen from the tubercle bacilli and an antigen from the Kedrowsky acid-fast bacillus. False-positive results occur in patients with tuberculosis, leprosy, and tropical Eosinophilia infection. The direct agglutination test, which detects the specific immunoglobulin M (IgM) antibody at an early stage, has been found to be useful in the detection of both clinical and subclinical infections. Because this test is easy to perform and the results are available in 24 hours, it can be used as a rapid test in primary care settings. Antibodies to the K39 antigen have been found to have high sensitivity and specificity in rapid diagnosis of visceral leishmaniasis, including a recent K39 immunochromatographic test and a K39 strip test.^[2]

Nonspecific tests

Specific leishmanial antigens prepared from cultures have been used in a number of tests. The direct agglutination test, immunofluorescent antibody test, complement fixation, and counterimmunoelectrophoresis are the various tests used in diagnosis of kala azar. Cross-reactions can occur with leprosy, Chagas disease, malaria, and schistosomiasis. Polymerase chain reaction (PCR) has also been used in the diagnosis of leishmaniasis using sequences from the variable region of kinetoplast DNA. A negative serological test result does not exclude the possibility of a leishmanial infection.

An immunochromatographic test for detection of anti–rK-39 antibodies has been reported with high sensitivity and specificity in diagnosis of visceral leishmaniasis and post–kala azar dermal leishmaniasis.

Leishmanin skin test (Montenegro test) is a delayed hypersensitivity reaction introduced in Montenegro, South America. It is performed by intradermally injecting 0.1 mL of killed promastigote antigen. The test results are available after 72 hours. The leishmanin skin test results are negative during active visceral leishmaniasis and usually become positive only after successful therapy. The test results are also positive in patients with dermal leishmaniasis. This test is useful only for epidemiological purposes, indicating prior exposure to infection.

Supportive tests

Hematological parameters include the following: blood examination findings reveal a normochromic normocytic anemia, leukopenia, neutropenia, thrombocytopenia, elevated gamma globulin levels, and a reversal of the albumin-globulin ratio.

Bone marrow aspiration is the most common sample obtained. Samples are obtained from the sternum or the iliac crest. Amastigote forms are revealed in plain film, and the promastigote forms are revealed in culture. Although safer than splenic puncture, the parasites are scant and may give a false-negative test result. Positivity rates ranging from 54-86% have been obtained using bone marrow.

When the spleen is enlarged considerably, splenic aspiration is one of the valuable methods for obtaining a positive result. As many as 98% of positive results have been obtained using splenic aspiration. Amastigote forms are revealed in stained specimens, and the promastigote forms are revealed in culture. Splenic puncture is associated with the risk of uncontrolled hemorrhage and, therefore, should be carried out only when bone marrow examination findings are inconclusive. Platelet counts and prothrombin times should be checked before the procedure.

Lymph node aspiration or biopsy may be helpful in the presence of enlarged lymph nodes. In cutaneous disease, tissue can be obtained with a 3-mm punch biopsy, lesion scrapings, or needle aspiration of the nonnecrotic edge of the lesion.

Leishmaniasis is a disease that involves the reticuloendothelial system. The parasitized macrophages disseminate infection to all parts of the body, especially to the spleen, liver, and bone marrow.

The spleen is enlarged, with a thickening of the capsule, it is soft and fragile, its vascular spaces are dilated and engorged with blood, and the reticular cells of Billroth are markedly increased and packed with amastigote forms of the parasite. However, no evidence of fibrosis is present.

In the liver, the Kupffer cells are increased in size and number and infected with amastigote forms of Leishmania. The bone marrow is hyperplastic, and parasitized macrophages replace the normal hemopoietic tissue.