Leishmaniasis Workup

Historically, the diagnosis of leishmaniasis has been confirmed by isolating, visualizing, and culturing the parasite from infected tissue. For confirmation of cutaneous disease, procedures consist principally of performing biopsies and obtaining dermal scrapings and/or needle aspirates. The smears are stained in Leishman, Giemsa, or Wright stains and examined under oil immersion microscope.

For visceral disease, the parasite can be detected through direct evidence (amastigotes in tissue) from peripheral blood, bone marrow, liver, or splenic aspirates. The most sensitive method is splenic puncture, although iatrogenic complications can be serious, including potentially life-threatening hemorrhage. In current practice, the high sensitivity and specificity of the recombinant K39 assay has generally made such invasive procedures unnecessary.

In endemic areas, the diagnosis of leishmaniasis is often made based on the history and physical examination. Very few of the diagnostic tests discussed in the sections below are available in developing countries.

In both the localized cutaneous and mucocutaneous forms of leishmaniasis, cell-mediated immunity to the parasite is vigorous and organism density in the skin and/or mucosa is low, especially in long-standing disease (although very early in the disease large numbers of the parasites are frequently found). Therefore, growing organisms in culture can be difficult, as can finding them in pathologic specimens.

A Practical Guide for Laboratory Diagnosis of Leishmaniasis is available through the Centers for Disease Control and Prevention (CDC). Note that serologic testing is not discussed in the CDC’s guide.

Cutaneous leishmaniasis

In more than 70% of cutaneous leishmaniasis cases, microscopy of the parasite in Giemsa stains or histologic section can reveal the parasite and should be attempted first. Culture of the organisms is an option but is unreliable (approximately 40% sensitivity), because the organisms are difficult to isolate from the lesion, especially as the lesion becomes older. Consequently, the diagnosis often is epidemiologic (travel to endemic area, clinical picture, coupled with laboratory data).

The organism grows on liquid media with fetal calf serum (eg, Schneider Drosophila medium) (positive results in 1 wk) and Novy-MacNeal-Nicolle (NNN) medium (media available from the CDC), or a biphasic medium. Cultures can produce positive results in 1-3 weeks.

Systemic leishmaniasis

Considerable experience has been gained and success achieved with using bone marrow aspirates (and especially a small piece of the spiculated core tissue) for cultivating the parasite or for looking for macrophages filled with amastigotes in the stained bone marrow aspirate smears.

A variety of immunodiagnostic serologic tests have been developed to aid in the diagnosis of systemic leishmaniasis. However, the only successfully deployed serologic tests are limited to species of Leishmania that cause visceral disease. Limitations include false-negative serologic results due to inadequate titers of antibodies late in the course of the disease and false-positive results in the setting of other infectious or autoimmune diseases.

Serologic testing is useful with the indirect fluorescent antibody (IFA) test, which is 80-100% sensitive in patients with visceral leishmaniasis who are not infected with human immunodeficiency virus (HIV). However, IFA may cross-react in patients who have leprosy, tuberculosis, malaria, schistosomiasis, Chagas disease, and African trypanosomiasis. Serologic tests such as isoenzyme or monoclonal antibody analysis are not well established.

An enzyme-linked immunosorbent assay (ELISA) can be combined with IFA and/or Western blot to increase sensitivity and specificity. Polymerase chain reaction (PCR) is being used more frequently; it is more accurate in determining new-onset leishmaniasis than serum tests (92-99% sensitivity; 100% specificity).

Cutaneous and mucocutaneous leishmaniasis generally display normal laboratory values.

Hematologic tests

Complete blood cell (CBC) count

In patients with visceral leishmaniasis, the presence of (1) normocytic normochromic anemia, (2) leukopenia with decreased neutrophils and a relative monocytosis and lymphocytosis, and (3) thrombocytopenia may occur due to parasitic bone-marrow infiltration. The severity of pancytopenia may vary with only 1 or 2 cell lines decreased.

Coagulation studies

Prothrombin and partial thromboplastin times are generally normal in visceral leishmaniasis.

Peripheral blood smear

Amastigotes are revealed inside the circulating monocytes and neutrophils. However, these are often difficult to locate because of their small numbers.

L donovani is best detected by either (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. 

Leishmania donovani is one of the main Leishmania species that infects humans.

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Parasitologic diagnosis using peripheral blood smear and buffy coat smear is easier in patients with human immunodeficiency virus (HIV) coinfection, because parasites are more commonly found in the circulating monocytes of these patients.

Liver function tests (LFTs)

Patients with visceral leishmaniasis may exhibit mild elevations in alkaline phosphatase, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels.

Other tests

Hypogammaglobinemia, circulating immune complexes, and rheumatoid factors are present in sera of most patients with visceral leishmaniasis. Rarely, immunocomplex deposition in the kidneys may lead to mild glomerulonephritis. However, renal failure is not a feature of visceral leishmaniasis.

Ancillary tests important in the diagnosis of visceral disease include measurements of lipase, amylase, gamma globulin, and albumin.

The aldehyde test and the antimony test were the initial tests used to detect hypogammaglobinemia and diagnose visceral leishmaniasis. Findings include elevated gamma globulin levels and a reversal of the albumin-globulin ratio.

For the aldehyde test, 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 that is observed when a urea stibamine solution comes in contact with serum.

Cutaneous leishmaniasis

For cutaneous leishmaniasis, take a 3-mm punch or wedge biopsy sample from a cutaneous sore from the raised edge of an active lesion where parasites are present. Avoid samples from the necrotic center. Additional tissue can be obtained through saline aspiration, tissue scrapings, or slit incisions.

Once tissue is obtained, send touch preparations, tissue impression slides, and formalin-fixed paraffin sections for hematoxylin and eosin staining. Send touch preparations and aspirations for Giemsa staining, as well. Direct visualization of amastigotes with their red rodlike cytoplasmic kinetoplast is diagnostic and helps distinguish them from other parasites (see the image below). Brown-Hopps staining has a higher sensitivity than other staining techniques.

Finding an organism in a tissue sample depends on the parasitic burden, the efficacy of the host's immune response, any coexisting bacterial contamination of the ulcer, and the age of the lesion (findings in older lesions are frequently nondiagnostic).

Mucocutaneous leishmaniasis

For mucocutaneous leishmaniasis, tissue can be obtained through dental scrapings or mucosal granuloma biopsy, although parasites may be difficult to isolate. A nonspecific granulomatous reaction often is observed. Giemsa stain may show the organisms.

Visceral leishmaniasis

Historically, bone marrow, liver, or splenic aspirates were the key to the laboratory diagnosis of visceral disease, but in current practice the high sensitivity and specificity of the recombinant K39 assay has generally made such invasive unnecessary.

The safest and most common way to obtain tissue is through bone-marrow aspiration obtained from the sternum or the iliac crest, although splenic aspiration may be used in cases that are difficult to diagnose. 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 of 54-86% have been obtained using bone marrow.

Splenic aspiration has a higher sensitivity than bone-marrow aspiration—as many as 98% of positive results have been obtained using splenic aspiration—but this procedure should be attempted only by experienced physicians. Splenic puncture is associated with the risk of uncontrolled hemorrhage and, therefore, should be carried out only when bone marrow examination findings are inconclusive. Contraindications include low platelet count, abnormal prothrombin time, and a spleen that is palpable 4 cm or less below the costophrenic angle.

Additional tissue can be obtained through liver biopsy and lymph node dissection.

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.

Organism identification

Direct visualization of the Leishmania organism is diagnostic, but this can be difficult in tissue sections because of its small size (2-4 mm) and because of subtle distinguishing characteristics on routine hematoxylin and eosin (H&E) stains. Diagnosis is usually much easier using Giemsa-stained touch preparations.

Regardless of the method of preparation, identification often requires an experienced pathologist and lengthy searches using high magnification, particularly when organisms are sparse. Giemsa, Brown-Hopps, Gram, or Leishman stains are all used to enhance Leishmania organisms on touch preparations, tissue aspiration, or biopsy samples.

The parasite consists of a nucleus and a kinetoplast surrounded by a cell wall. Visualization of all 3 features (ie, nucleus, cell membrane, and kinetoplast) is required to make a diagnosis based on microscopy findings.

Amastigotes in a macrophage at 1000× magnification. Inset shows the cell membrane and points out the nucleus and kinetoplast, which are required to confirm that the inclusion seen in a macrophage is indeed an amastigote.

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Free amastigotes near a disrupted macrophage. On touch preparations like this (Giemsa stain, original magnification × 1000), the amastigotes are easier to identify than on other preparations. These stains clearly demonstrate the cell membrane, nucleus, and kinetoplast; all 3 are required for definitive diagnosis.

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The diagnostic sensitivity of microscopic identification of leishmanial amastigotes is typically 75-85%, but it may vary widely, depending on the size of the inoculum, the clinician's experience in sampling a proper area, the experience of the laboratory personnel in making smears or making tissue sections, and the observer's experience in finding the parasite.

Most Leishmania experts believe the ideal way to microscopically identify the parasite is with direct touch preparations from the lesion or biopsy tissue stained with Giemsa rather than with routine tissue sections.

Hepatosplenic features in visceral disease

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.

Cutaneous disease

Localized cutaneous leishmaniasis is characterized by irregular acanthosis, with or without epidermal ulceration, and dense dermal infiltrate of mixed inflammatory cells, particularly plasma cells, lymphocytes, and histiocytes. Early in the course of localized disease, organisms may be numerous and found readily in the cytoplasm of macrophages. As the lesion ages and as delayed-type immunity is upregulated, the infiltrate is replaced by noncaseating granulomata in which few or no organisms can be seen.

Ulcerated lesions are often secondarily infected by bacteria, in which case histologic changes may be nonspecific. Results with biopsy specimens obtained from old (>6 mo), partially treated, or low-burden infections are frequently nondiagnostic.

Diffuse cutaneous leishmaniasis occurs in individuals with poor cellular immunity to Leishmania parasites. Histologic diagnosis is straightforward in these cases. The dermis contains sheets of macrophages containing great numbers of amastigotes, with few lymphocytes or plasma cells.

Leishmaniasis recidivans is usually difficult to confirm because of the rarity of organisms and because of its histologic similarity to lupus vulgaris.

Post–kala-azar dermal leishmaniasis has a variable histology that is determined by the degree of host immunity and the parasite load. Granulomatous histology is seen with low numbers of organisms, whereas diffuse histiocytic or xanthomatous infiltrates may be seen with numerous organisms.

Visceral leishmaniasis

Definitive diagnosis of visceral disease is made by observing the parasite (more specifically, amastigotes in tissue) on stained Giemsa smears or by observing the culture of bone marrow, splenic, hepatic, or lymph node aspirates.

Light-microscopic examination of a stained bone marrow specimen from a patient with visceral leishmaniasis—showing a macrophage (a special type of white blood cell) containing multiple Leishmania amastigotes (the tissue stage of the parasite). Note that each amastigote has a nucleus (red arrow) and a rod-shaped kinetoplast (black arrow). Visualization of the kinetoplast is important for diagnostic purposes, to be confident the patient has leishmaniasis. (Credit: CDC/DPDx) Source: Centers for Disease Control and Prevention. Parasites home: leishmaniasis. Resources for health professionals: http://www.cdc.gov/parasites/leishmaniasis/health_professionals/

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In vitro cultures of tissue are regularly obtained to aid in leishmaniasis diagnosis and to help identify difficult Leishmania species. This technique has approximately the same diagnostic sensitivity as that of pathologic evaluation, but special laboratory capabilities and technical skills are required. These are available in only 2 reference laboratories in the United States (ie, the Centers for Disease Control and Prevention [CDC], Walter Reed Army Institute of Research).

The value of this method is that the species of the parasite can be identified on the basis of long-standardized isoenzyme patterns on cellulose acetate electrophoresis after the parasite is grown in vitro using both the New World methods of Kreutzer and the Old World methods of Dedet.

Specimens may be cultured on Novy-MacNeal-Nicolle (NNN) medium (rabbit-blood agar that has an overlay of Locke solution with added antibiotics), rabbit blood agar, Schneider Drosophila medium, or a multitude of specialized media to induce promastigote growth. Cultures usually take a few days to 2 weeks to demonstrate growth. Positive culture results occur approximately 75% of the time.

Additional cultures can be performed by inoculating tissue into the footpad and nose of hamsters or certain highly susceptible mouse strains (ie, in vivo cultures via animal inoculation). This is a sensitive method, especially in difficult cases, but results can take several weeks to months.

With successful culture, the parasite can be sent to specialized facilities or the Centers for Disease Control and Prevention (CDC) for polymerase chain reaction (PCR), isoenzyme electrophoresis, or monoclonal antibody speciation.

Specific leishmanial antigens prepared from cultures have been used in a number of tests. Serologic detection of antibodies to recombinant K39 antigen (eg, K39 immunochromatographic test, K39 strip test) ^[16] (see the following image) using a direct agglutination test (DAT), immunofluorescence assay (IFA), or enzyme-linked immunosorbent assay (ELISA) has been shown to be highly sensitive and specific in diagnosing visceral leishmaniasis and post–kala-azar dermal leishmaniasis. ^[17] A nitrocellulose dipstick test has also been used with K39 testing.

Illustration of one form of the rK39 test for the serologic diagnosis of visceral leishmaniasis. It is an easy, very sensitive, and specific test for visceral disease. In this case, the dipstick second from the left shows a positive result and all the rest show reaction only at the control line.

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Recombinant K39 reactivity appears to correlate with active visceral disease caused by L donovani, L chagasi, and L infantum and is absent in cutaneous and mucocutaneous infections. Studies confirmed its diagnostic utility in India and Brazil but showed limited utility in Sudan because of regional species variance. ^{[18, 19, 20]} It is important to note that cross-reactions can occur with leprosy, Chagas disease, malaria, and schistosomiasis.

K39-based antigen testing is the only FDA-cleared rapid serologic test available that has undergone the necessary rigors of scientific testing to reliably trust for diagnosing visceral leishmaniasis. ^{[21, 22]}

DAT detects the specific immunoglobulin M (IgM) antibody at an early stage and has been found to be useful in the detection of both clinical and subclinical leishmaniasis 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.

Before the use of specific leishmanial antigens, nonspecific antigens were used. These include the Witebsky, Kingenstein, 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.

Over the past several years, significant advances in polymerase chain reaction (PCR) techniques have allowed for the highly sensitive and rapid diagnosis of specific Leishmania species. PCR can identify parasite DNA using sequences from the variable region of kinetoplast DNA. ^[23] However, a negative serologic test result does not exclude the possibility of a leishmanial infection.

Although limited to military and reference laboratories, leishmanial PCR diagnosis is becoming more widely available in developing-world laboratories and field sites. ^{[24, 25]} Even in remote locations and under harsh conditions, this technique has proven its worth, as evidenced by the US military's experience with Leishmania infection in Iraq. Molecular genetic studies can be carried out in various specimens including blood, bone marrow, or tissue. ^[26]

Validated genus-specific PCR primers exist, and approval of this assay by the US Food and Drug Administration (FDA) has been sought so that it can be used in worldwide facilities certified by the College of American Pathologists (CAP). Species-specific PCR probes allow for rapid speciation in confirmed cases of leishmaniasis, and some are undergoing final validation.

Note: Although many laboratories worldwide offer species-level PCR diagnostics, few have undergone the scientific rigor of complete validation necessary to assure accuracy of these species diagnostics. Many of these assays are suspect and may be misleading.

Consequently, in the United States, FDA approval of these assays should be sought, or the assays should be performed in certified laboratories in order to be confident of the results. Difficult cases should be referred to reference laboratories in the United States for rapid diagnosis and speciation, such as the Walter Reed Army Institute of Research Leishmania Diagnostic Laboratory or the Leishmania Diagnostic Laboratory at the US Centers for Disease Control and Prevention (CDC).

In visceral leishmaniasis, an elevated serum immunoglobulin level with polyclonal spike may be present on serum plasma electrophoresis (SPEP). Visceral disease was traditionally diagnosed based on the addition of formaldehyde to a serum sample (aldehyde test), which would increase the viscosity secondary to excessive immunoglobulins.

Cellulose acetate electrophoresis is a well-standardized method for determining the species of parasites grown from clinical samples. Although this test is standardized, it requires experience and special facilities; therefore, it is available only in highly specialized diagnostic facilities.

No skin tests for leishmaniasis are approved for use in the United States, primarily due to the lack of standardization of these tests.

The Leishmanin skin test (LST), also known as the Montenegro skin test (for its introduction in Montenegro, South America) is similar to the purified protein derivative (PPD) used for Mycobacterium tuberculosis. This test has been used in the developing world to determine delayed-type hypersensitivity reactions.

Killed promastigotes are injected intradermally; a 5-mm area of induration over 48-72 hours suggests past infection (ie, results are negative during active visceral leishmaniasis; positive results occur 2-3 months after infection, usually after successful therapy. The test results are also positive in patients with post–kala-azar dermal leishmaniasis).

The 2 main drawbacks of this test are that acute infections cannot be identified (in endemic regions, >70% of the population will test positive), because it remains positive for life, and those who are immunosuppressed (immunologically anergic patients) may not mount a response. Thus, this test is not used to distinguish between active and resolved disease, but it can be useful in evaluating known naive populations that become immunologically responsive to leishmanial antigens (ie, epidemiologic purposes).