Dermatologic Manifestations of Leishmaniasis Workup

Cutaneous lesions

Skin scrapings can be obtained from the base of an active ulcer, or a 4-mm biopsy specimen can be obtained from the edge of a suggestive lesion or ulcer. A diagnosis to the genus Leishmania is made if amastigotes are identified in a touch preparation or histopathology section from cutaneous lesions or in biopsy specimens of infected tissue. 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). 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.

Direct visualization of the organism is diagnostic but 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 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 diagnostic sensitivity of microscopic identification of leishmanial amastigotes is typically 75-85% but 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.

See the images below.

Polymerase chain reaction (PCR) is now routinely used in experienced laboratories as a rapid diagnostic technique.^{[8, 9]} Even in remote locations and under harsh conditions, this technique has proven its worth, as evidenced by the US military's recent experience with Leishmania infection in Iraq. Validated genus-specific PCR primers exist, and approval of this assay by the US Food and Drug Administration (FDA) is being sought so it can be used in worldwide facilities certified by the College of American Pathologists. Species-specific PCR probes allow for rapid speciation in confirmed cases of leishmaniasis, and some are currently undergoing final validation. A word of caution is warranted, however, because many laboratories throughout the world offer species-level PCR diagnostics, but 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.

In the United States, FDA approval of these assays should be sought or they 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 vitro cultures of tissue are regularly obtained to aid in 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 currently available in only 2 reference laboratories in the United States (ie, 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.

In vivo diagnosis of Leishmania organisms can also be achieved by inoculating clinical specimens into golden hamsters or certain highly susceptible mouse strains. Although results are not available for weeks to months, they are useful in diagnosing the disease, especially in difficult cases.

Mucocutaneous leishmaniasis or visceral leishmaniasis

Aspirates or touch preparations of mucosal or visceral tissue dermal scrapings may be processed similar to cutaneous samples, as described above.

Systemic leishmaniasis

Visceral leishmaniasis has been diagnosed for a very long time using the same methodology described above for cutaneous lesions but applied to either splenic, hepatic, or bone marrow aspirates. 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. 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.

The most promising serologic test to date is a Leishmania chagasi recombinant amastigotes K39 (rK39)–based antigen test system that has been used with an enzyme-linked immunosorbent assay, a direct agglutination test, and even a nitrocellulose dipstick test. The degree of conservation of the K39 gene is high among isolates of the Leishmania donovani family, including L chagasi and Leishmania infantum. Therefore, this test is useful for most recognized cases of visceral leishmaniasis. Assays based on rK39 antigen are highly sensitive and specific and have been validated in several large studies worldwide. This currently 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.^{[10, 11]}

Ancillary tests important in the diagnosis of visceral leishmaniasis include determinations of the CBC count with differential; liver function tests; and lipase, amylase, gamma globulin, and albumin tests.

See the image below.

Imaging studies have little to no value in the diagnosis of any of the forms of leishmaniasis.

Skin testing

Similar to the purified protein derivative testing (PPD for Mycobacterium tuberculosis), Montenegro leishmanin skin testing (LST) has been used for decades to determine previous or current exposure to Leishmania parasites. LST 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. LST is not applicable to immunologically anergic patients with widely disseminated cutaneous disease. Because LST is not standardized, the FDA has not approved it; therefore, LST is not available as a diagnostic tool in the United States. Efforts to standardize this test and make it available through the FDA clearance process have been ongoing for more than 2 decades.

Identification of parasite species

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.

Procedures associated with leishmaniasis consist principally with performing biopsies, obtaining dermal scrapings, and/or needle aspirates for cutaneous disease to confirm the diagnosis. 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 rK39 assay (requiring only 20 µL of serum) has made this generally unnecessary.

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 up-regulated, 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.

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.

Recidivans cutaneous leishmaniasis is usually difficult to confirm because of the rarity of organisms and because of its histologic similarity to lupus vulgaris.

Aspirates from bone marrow, lymph nodes, or the spleen are typically obtained to make a histologic diagnosis of kala azar. As with diagnosis with skin samples, diagnosis based on these aspirates depends on identification of Leishmania parasites, which are usually plentiful in macrophages.