Cytologic Diagnosis of Leishmaniasis in HIV Infection DO NOT DUPLICATE

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1 Cytologic Diagnosis of Leishmaniasis in HIV Infection A Report of Eight Cases Blanca Vicandi, M.D., José A. Jiménez-Heffernan, M.D., Dip.R.C.Path., Pilar López-Ferrer, M.D., Luis Ortega, M.D., and José M. Viguer, M.D. BACKGROUND: Leishmania organisms are among the intracellular microorganisms with a tendency to develop in patients with the aquired immunodeficiency syndrome (AIDS). With increasing travel to endemic areas by patients with human immunodeficiency virus (HIV) infection, it is becoming a more-frequent diagnosis in nonendemic areas. CASES: Ten cytologic specimens from eight patients with leishmaniasis and AIDS were reviewed. Eight samples were obtained from lymph nodes through fine needle aspiration (FNA). Another sample was obtained after scraping a tongue ulcer. The last one was an ascitic fluid specimen. Smears showed numerous parasitized histiocytes with abundant intracellular Leishmania organisms (amastigotes). Extracellular microorganisms were also abundant. Diff-Quik stained smears allowed the Even in the presence of a bizarre clinical context, cytology may result in an easy and rapid identification of Leishmania amastigotes, allowing specific and prompt treatment. clear recognition of the characteristic morphologic appearence with a deep-staining area (nuclei) and paranuclear zone (kinetoplast). Intracellular organisms were round, while single, extracellular forms were a more elongated. CONCLUSION: The polymorphous clinical manifestations usually seen in patients suffering from leishmaniasis and AIDS constitute a diagnostic challenge that can be facilitated by cytopathologic examination. Cytology permits easy and rapid identification of Leishmania amastigotes, allowing a specific diagnosis and treatment. (Acta Cytol 2000;44: ) Keywords: leishmaniasis, HIV infection, aspiration biopsy. Leishmaniasis has been added to the list of infective From the Departments of Pathology, Hospital Universitario La Paz and Hospital La Zarzuela, Madrid, and Hospital de la Ribera, Alcira, Spain. Drs. Vicandi and López-Ferrer are Staff Cytopathologists, Department of Pathology, Hospital La Paz. Dr. Jiménez-Heffernan is Pathologist, Department of Pathology, Hospital La Zarzuela. Dr. Ortega is Pathologist, Department of Pathology, Hospital de la Ribera. Dr. Viguer is Chief, Cytology Section, Department of Pathology, Hospital La Paz. Address reprint requests to: José M. Viguer, M.D., Departamento de Anatomía Patológica, Hospital Universitario La Paz, Paseo de la Castellana 261, Madrid-28046, Spain. Financial Disclosure: The authors have no connection to any companies or products mentioned in this article. Received for publication November 15, Accepted for publication February 25, /00/ /$19.00/0 The International Academy of Cytology 835

2 836 Vicandi et al diseases associated with the acquired immunodeficiency syndrome (AIDS), and this is especially true for the Mediterranean countries. 1,12 Leishmania is a protozoan that produces a wide clinicopathologic pattern of disease, the most common of which is The high number of atypical clinical presentations and identification of new subspecies constitute a diagnostic challenge for the clinician and pathologist. visceral and cutaneous leishmaniasis. The infection is prevalent in Latin America, Africa, Asia and Mediterranean Europe. 4 The polymorphous clinical manifestations usually noted in adults suffering from AIDS constitute a diagnostic challenge that can be facilitated by cytopathologic examination. In this report we describe the finding of Leishmania in 10 cytologic specimens obtained from eight patients with AIDS. Materials and Methods Ten cytologic samples from eight patients with leishmaniasis and AIDS were reviewed. Eight were obtained from lymph nodes through fine needle aspiration (FNA) performed by cytopathologists using 23- to 25-gauge needles. Another sample was obtained after scraping a tongue ulcer. The last one was an ascitic fluid specimen. A portion of the smears were air dried with Diff-Quik, whereas the remainder were wet fixed with alcohol and stained with a modified Papanicolaou method. A total of 6 surgical biopsy specimens (from four patients), from bone marrow (n = 3), lymph node (n = 1), kidney (n = 1) and skin (n = 1), with leishmania were reviewed. Surgical specimens routinely were formalin fixed and paraffin embedded and processed for staining with hematoxylin and eosin. Methenamine silver, Giemsa stain and periodic acid Schiff stain were also used. Results of serologic techniques were available in four patients. Leishmanial antibody titers were measured by an enzyme-linked immunoabsorbent assay and indirect fluorescent antibody test. In vitro culture in the triple N medium of Novy, McNeal and Nicolle was performed on a bone marrow aspirate from patient 7. Results Patient Information Clinicopathologic features are summarized in Table I. All patients were young male adults suffering from AIDS. Seven patients presented with lymphadenopathies and one with a lingual ulcer. The peritoneal fluid specimen belonged to one of the former patients. In two cases FNA was performed as part of patient follow-up. In four patients different tissue biopsies permitted the histologic identification of Leishmania amastigotes. Leishmania antibody titers were positive in the four patients tested. The triple N culture obtained from patient 7 rendered a positive result with identification of promastigotes. Pathologic Findings Lymph node aspirates disclosed findings similar to those of hypercellular smears, with abundant histiocytes in a polymorphic lymphoid background. Histiocytes exhibited large amounts of cytoplasm that contained numerous Leishmania (amastigotes) organisms, which were also seen as isolated, extracellular forms (Figure 1). In all cases the parasitation was massive, with abundant microorganisms. Large, parasitized, multinucleated histiocytes were also seen. Smears from the lingual ulcer exhibited numerous squamous and inflammatory cells. Parasitized histiocytes were scarce and admixed with squamous cells with characteristic herpetic intranuclear inclusions. The ascitic fluid specimen showed a moderate number of parasitized histiocytes with few inflammatory cells. As opposed to those observed in lymph nodes, the number of amastigotes per cell was low, and no extracellular microorganisms were present. Both alcohol-fixed and air-dried smears showed amastigotes. Diff-Quik stained smears allowed clear recognition of the characteristic morphologic appearance with a deep-staining area (nuclei) and paranuclear zone (kinetoplast). Intracellular organisms were round, while single, extracellular forms showed a more-elongated shape. The recognition of elongated forms was possible in air-dried smears. Alcohol-fixed microorganisms showed a predominantly round form. Elongated forms reached 4 µm in diameter, while round forms measured 2 3 µm (in air-dried smears). Bone marrow, skin, renal and lymph node tissues showed numerous Leishmania amastigotes within

3 Volume 44, Number 5/September October Leishmaniasis in HIV Infection Table I Clinicopathologic Data on Patients with Leishmaniasis and AIDS Case no. Age/sex Cytologic sample Clinical diagnosis Other tissue locations Other 1 25/M Lingual ulcer (exfoliative) Herpetic disease Bone marrow biopsy 2 30/M Cervical lymph node (FNA) Mycobacteriosis, Bone marrow, renal biopsy + Serologic test leishmaniasis Ascitic fluid (exfoliative) Nonspecific ascitis 3 27/M Cervical lymph node (FNA) Mycobacteriosis 4 32/M Cervical lymph node (FNA) Mycobacteriosis 5 31/M Axillary lymph node (FNA) Leishmaniasis Skin biopsy + Serologic test 6 42/M Cervical lymph node (FNA) Mycobacteriosis Lymph node, bone marrow + Serologic test biopsy Cervical lymph node (FNA) Leishmaniasis; treatment, follow-up 7 34/M Axillary lymph node (FNA) Leishmaniasis; treatment, Cultured from bone follow-up marrow aspirate 8 24/M Cervical lymph node (FNA) Nonspecific + Serologic test histiocytes. They were easily visible with hematoxylin and eosin and Giemsa stain. Discussion The infective stage of leishmaniasis involves a slender, flagellated protozoan that is transmitted through the bite of infected sandflies. They are phagocytosed by macrophages and transform into round amastigotes that lack flagella but contain a large, mitochondrionlike structure, the kinetoplast. 4,17 The extent and severity of the disease depend on the Leishmania species and the host immune response. Cell-mediated immunity controls the infection with a granulomatous response and few parasites. However, anergic hosts, such as those with AIDS, have diffuse lesions composed of macrophages stuffed with parasites. Leishmania is among the intracellular microorganisms (mycobacteria, Histoplasma, Toxoplasma and trypanosomes) with a tendency to develop in patients with AIDS. Those patients develop visceral leishmaniasis with systemic disease that mainly affects the mononuclear phagocytic system with hepatosplenomegaly, lymphadenopathy and bone marrow involvement. More rarely, the digestive tract and skin are parasitized. 17 Many of the first descriptions of leishmaniasis and AIDS came from the Mediterranean countries. 1,12 This is not surprising since this is an endemic area for the cutaneous and visceral forms of the disease. Despite many reports describing the presence of Leishmania infection on tissue biopsies, few cytologic descriptions of leishmaniasis in AIDS are available, and most are isolated case reports in non HIV-infected patients. 5,8,9,18,20,22,24 FNA and exfoliative cytology are powerful tools for the evaluation of infectious disorders in patients with AIDS. Cytology permits the exclusion of malignancy, the procurement of material for microbio- Figure 1 Many Leishmania organisms (amastigotes) within a histiocyte. Extracellular oval to elongated forms are also visible (Diff-Quik, 200).

4 838 Vicandi et al logic cultures and, in many cases, the identification of specific pathogens. Pneumocystis, Aspergillus, Cryptococcus, Candida, Histoplasma, Coccidioides, mycobacteria, Toxoplasma, Cryptosporidium, herpesvirus, cytomegalovirus and JC virus are among the most common infectious diseases that can be diagnosed cytologically. 19,21 The identification of Leishmania amastigotes on cytologic specimens is usually easy, with few differential diagnostic considerations focused on other intracellular parasites (Table II). Histoplasmosis may present with lymphadenopathies and show organisms of the same diameter (2 4 µm) and shape. 2 The deeply stained chromatin of the fungal cells appears as an eccentric, oval or crescentic mass within the unstained cytoplasm. Sometimes a clear halo surrounds the organisms. As opposed to Leishmania, no paranuclear body (kinetoplast) is seen. Yeasts can be easily recognized with specific fungal stains performed on alcohol-fixed material. 13,23 Toxoplasmosis in AIDS patients shows a predilection for the brain, heart and lungs. It usually parasitizes somatic cells rather than phagocytic cells. Cytologic specimens may reveal free tachyzoites and cysts. 7,25 The former measure 5 7 µm and show a crescent, ovoid shape with a single central nucleus. No kinetoplast is seen. The crescentic morphology as well as the presence of central nuclei is visible in cytologic smears, allowing a specific diagnosis. Trypanosoma cruzi is a kinetoplastid, intracellular protozoan that causes American trypanosomiasis (Chagas disease). The infection, which has a great tendency to involve the heart, is Table II Differential Diagnosis of Intracellular Microorganisms in HIV Infection exacerbated by HIV infection. 15 Rarely does it present with generalized lymphadenopathy or splenomegaly. Morphologically, amastigotes from Trypanosoma may be very similar to those of Leishmania. However, the former have no predilection for phagocytic cells and instead parasitize myocardial and ganglion cells. Two bacterial diseases are also characterized by the presence of large, parasitized macrophages. Rhinoscleroma typically affects the upper respiratory tract, and lesions disclose macrophages containing rod-shaped bacteria ( Mikulicz cells ), better visualized with Giemsa or Wartin-Starry stain. When abundant, they may be seen with conventional hematoxylin and eosin staining. Although rare, the infection has been described in association with HIV infection. 16 Cytologic features have been described 26 and differ from Leishmania amastigotes in the bacilar shape and staining properties. Granulomatosis inguinale (Donovaniosis) is a chronic ulceronecrotizing infection of the anogenital area caused by Calymmatobacterium granulomatis, an encapsulated coccobacillus. Biopsy and cytologic smears permit the demonstration of Donovan bodies, which are seen as small, round, encapsulated bodies inside the cytoplasm of the histiocytes. Some of the microorganisms are paired, and others have a distinct, clear capsule. They are best demonstrated with Giemsa or Wartin-Starry stain. 3,6,10 In our series, most patients presented with lymph node involvement. Even in endemic areas, Leishmania organisms are a rare causative agent of lymphadenopathies when compared to other HIV- Organism Size (µm), shape Structure Parasitation Common organs involved Stains Leishmania 2 4, Round to elongated Protozoan with Phagocytic Bone marrow, lymph nodes, Giemsa, HE, (amastigotes) nucleus, kinetoplast cells liver, spleen, skin reticulum stain Histoplasma 2 4, Yeastlike, round to Fungal cell with cap- Phagocytic Lung, lymph nodes GMS, PAS oval sule, halo effect cells Toxoplasma 5 6, Crescentic Protozoan with cen- Somatic Brain, lung Giemsa, HE (tachyzoites) tral nucleus cells Trypanosoma 2 5, Round to oval Protozoan with Somatic Heart, brain Giemsa, HE (amastigotes) kinetoplast cells Mycobacteria 1 10, Curved bacilli Bacteria Phagocytic Lymph nodes, lung, diges- Ziehl-Neelsen cells tive tract, liver Calymmatobacterium 0.6 2, Coccobacilar Encapsulated bacteria Phagocytic Anogenital region Giemsa, WS granulomatis cells Klebsiella 2 3, Rod-shaped Encapsulated bacteria Phagocytic Upper respiratory tract Giemsa, WS rhinoscleromatis bacillus cells HE = hematoxilin-eosin, GMS = Gomori methenamine silver, PAS = periodic acid Schiff stain, WS = Warthin-Starry stain.

5 Volume 44, Number 5/September October Leishmaniasis in HIV Infection related disorders. Therefore, except for those cases in which a previous diagnosis of leishmaniasis is present (usually in a bone marrow biopsy), it is an unexpected finding. In those cases, such as 1 and 2, which presented with atypical clinical forms, the value of the cytologic findings is even greater. Peritoneal involvement by Leishmania is very rare, and to our knowledge there is only one reference to it in the medical literature. 14 Leishmaniasis should not be viewed as an exotic disorder exclusive of endemic countries. With the increase in travel to endemic areas and of patients with HIV infection, it is becoming a more-frequent diagnosis in nonendemic areas. 11,17 The high number of atypical clinical presentations and identification of new subspecies constitute a diagnostic challenge for the clinician and pathologist. 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