Leishmania episode 1a: attack of the clones
By Kiran Motaparthi, MD
February 10, 2021
Vol. 3, No. 6
Cutaneous leishmaniasis (CL), although not life-threatening, can progress to a disfiguring disease with prominent scarring. Humans serve as reservoirs for > 20 species of Leishmania, which are transmitted as flagellate promastigotes by sandflies. Within humans, the organisms convert to nonflagellate amastigotes. In the United States, “New World” disease may be imported following travel to an endemic region such as Mexico, Central and South America, and Texas. “Old World” disease occurs in Asia, Africa, and the Middle East. First-line systemic therapy for CL includes pentavalent antimonials, miltefosine, and liposomal amphotericin B; these are effective but potentially toxic treatments. (1) In leishmaniasis, small 2-4 micrometer rounded amastigote parasites are identified within enlarged histiocytes. In New World leishmaniasis, a “Marquee sign” in which organisms line up around histiocyte borders may be observed. Organisms have a nucleus and a kinetoplast, which is often difficult to identify in tissue sections. Evaluation of routine hematoxylin & eosin (H&E)-stained sections is only up to 70% sensitive for the detection of amastigotes. (2) The Giemsa stain is historically described to improve identification of amastigotes, but in practice this special stain adds little to routine H&E-based interpretation. (3)
Recently, CD1a immunostain has been used to identify amastigotes (Figure 1), demonstrating a peripheral “membrane-like” pattern with accentuation in one of the poles, leaving the unstained nucleus in the center (Figure 2). (4, 5) As antigen-presenting cells, Langerhans cells (CD1a-positive dendritic cells) phagocytize amastigotes before triggering T-cell mediated immunity. Most likely, antigen transfer of CD1a occurs during exocytosis. Alternatively, there may exist cross-reactivity between surface antigens of Leishmania and the CD1a epitope. However, this is less likely given that promastigotes cultured outside of hosts are CD1a-negative. (3, 5)
However, there are several caveats with the practical or routine use of this antibody stain:
There is variability in sensitivity based on the clone of anti-CD1a antibody used: MTB1 (Novocastra/Leica Biosystems) is variably positive depending on the species of Leishmania, but the clone 010 (Dako) is consistently negative. (5)
The species and hence geographic region influence detection by this method: CD1a (MTB1) is highly sensitive for the detection of L. infantum, (5) L. tropica, and L. major. (4) CD1a (MTB1) can also enable diagnostic confirmation in cases of Old World CL without identifiable amastigotes on H&E-stained sections. (6) However, CD1a (MTB1) is variably sensitive for New World species such as L. panamensis (3) and insensitive for L. braziliensis and L. peruviana. (7)
If amastigotes encounter Langerhans cells in the epidermis or papillary dermis, antigen transfer can occur. However, amastigotes that are injected into the deep dermis or new populations of amastigotes are less likely to be processed by CD1a-positive dendritic cells and will therefore be CD1a-negative. (3,5)
As with any stain, technical and interpretive difficulties may arise. Rare amastigotes present in H&E-stained sections may be lost in sections prepared for CD1a staining. (7) Dendritic cell cytoplasmic prolongations may mimic CD1a-positive organisms, appearing as oval fragments the size of amastigotes. (8)
Superior methods for detection of Leishmania are readily available in the United States. Culture and polymerase chain reaction (PCR) are up to 80% and 97% sensitive, respectively, and permit definitive diagnosis in challenging cases without identifiable organisms. Of importance for treatment selection, PCR permits speciation. (2) If a dermatologist suspects CL, at least 2 punch biopsies should be obtained: 1 for histopathology and 1 for PCR and culture. While the biopsy for histopathology should be placed in formalin, the biopsy for PCR/culture should be placed in a non-fixative preservative such as Roswell Park Institute Medium or buffered saline. The CDC provides media, histopathology, PCR, and culture free of charge. (9) See resources and instructions from the CDC (PDF).
Point to Remember: When a trained eye and routine H&E-stained sections are insufficient, PCR increases the diagnostic yield and may influence therapy. In settings where access to PCR and culture are limited, the use of a specific CD1a clone (MTB1) is sensitive for the detection of Old World species and provides variable support for the diagnosis of New World disease.
Our editor's viewpoint
Warren R. Heymann, MD
As I am writing this commentary travel bans are in effect related to the coronavirus situation. I hope that by the time this is published, that epidemic is under control, and that people can again travel worldwide without restriction. Under normal circumstances any dermatologist can see tropical disorders, leishmaniasis being among the most frequent. I have diagnosed cases — both New World and Old World — in my career. I cannot say enough about how obliging the CDC is in assisting with the diagnosis and treatment of patients with cutaneous leishmaniasis. By all means, request a CD1a stain if the H&E is equivocal and your suspicion is high for cutaneous leishmaniasis. Your next step, post haste, is to contact the CDC.
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Centers for disease control and prevention: practical guide for specimen collection and reference diagnosis of leishmaniasis (Online). 2016. Available at: https://www.cdc.gov/parasites/leishmaniasis/resources/pdf/cdc_diagnosis_guide_leishmaniasis_2016.pdf Accessed 24 February 2020.
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