Context.—Localized cutaneous leishmaniasis (CL) typically presents as papules, crusted nodules, plaques, or noduloulcerative lesions. Atypical CL does not show these features or mimic malignant lesion. In atypical forms, CL may be overlooked because of its similarity to other dermal diseases.
Objective.—To compare conventional, molecular, and immunohistochemical methods in the diagnosis of typical and atypical CL.
Design.—The kinetoplast DNA, nested, polymerase chain reaction assay and immunohistochemical methods were compared and validated against conventional methods, including cytology and pathology, using 100 specimens of typical and atypical lesions of suspected CL.
Results.—Compared with other methods, polymerase chain reaction of the kinetoplast DNA showed the highest sensitivity (typical positive, 100%, 67 of 67; atypical positive, 94%, 31 of 33) and specificity (100%), followed by immunohistochemistry (typical positive, 97%, 65 of 67, with 100% specificity; atypical positives, 94%, 31 of 33, with 100% specificity), and cytology (typical positive, 79%, 53 of 67, with 100% specificity; atypical positive, 58%, 19 of 33, with 100% specificity), followed by pathology (typical positive, 70%, 47 of 67, with 100% specificity; atypical positive, 42%, 14 of 33, with 100% specificity). In addition, polymerase chain reaction enabled identification of 98% (98 of 100) of the positive samples that included strains of Leishmania major (99% [99 of 100] cases) and Leishmania tropica (1% [1 of 100] cases).
Conclusions.—Because cytology is cheap and easy to perform with high sensitivity, it is the preferred, primary approach for typical CL, but cytology and pathology do not have sufficient sensitivity for diagnosis of atypical CL cases. Nested polymerase chain reaction and immunohistochemistry are sensitive tests for diagnosis of both typical and atypical CL and are recommended as complementary tests in suspected CL with negative conventional microscopy results.
Cutaneous leishmaniasis (CL) has recently grown into a major public health problem in 88 countries, including Iran.1,2 The clinical spectrum of the disease manifestation in Iran includes CL, localized Leishmania lymphadenitis, diffuse multiorgan involvement (kala azar), and, rarely, mucosal leishmaniasis.3,4 In the endemic areas, diagnosis of CL is often made by epidemiologic and lesion characteristics, but the diagnosis has to be confirmed by microscopic demonstration of the parasite, which is mostly done for the initial diagnosis (scraping, fine-needle aspiration, and touch smears), in the tissue and/or by sample culture to avoid potential misdiagnosis.5 Culture techniques require sophisticated laboratory setups, are time-consuming, and harbor the risk of contamination.6 Serology tests cannot distinguish between past and current infections, and diagnosis can be complicated by the cross-reaction of antibodies with trypanosomiasis, tuberculosis, and toxoplasmosis.7 Another limitation of these methods is due to the variability of host sensitivities because antibody titers may vary with the infecting species, tissue tropism, and the immunocompetence of the host.8 In the present study, cytology, pathology, immunohistochemistry (IHC), and nested polymerase chain reaction (PCR) were applied for diagnosis of typical and atypical forms of CL.
MATERIALS AND METHODS
One hundred patients who had clinical manifestations of the typical (papules, crusted nodules, plaques, or noduloulcerative lesions) and atypical (eczematoid, lupoid, nodular, warty, or verrucous, multiinfection, erythematous, diffuse, and volcanic ulcer or mimicking of a malignant lesion) variants of cutaneous lesions formed the basis of this study. The patients presented to the Parasitology Department between April 2010 and March 2011 from different rural and urban areas of Fars Province, Iran. Cytology, pathology, IHC, and nested PCR were performed for diagnoses of CL.
The Ethics Committee of the Faculty of the Medical School, University of Shiraz Medical Sciences (Shiraz, Iran) approved the study, and the author group collected written, informed consent from all patients.
Histopathology and Cytology Examination
One skin biopsy specimen from the borders of the lesions or scars of 100 patients was collected, using a disposable punch (Kai Industries, Tokyo, Japan) of 3 or 4 millimeters in diameter for each patient. The patients had not received any type of previous treatment. After careful sterilization of the biopsy site, local anaesthesia with 2% lidocaine was applied. A tissue imprint was provided from each sample before the samples were fixed in buffered formalin and stained by Giemsa for touch smear examination. The tissue samples were fixed in 10% buffered formalin, dehydrated with graded ethanol, cleared with xylene, impregnated with paraffin wax, sectioned at 5 μm thick, stained with hematoxylin-eosin, and studied by a light microscope (Olympus, Tokyo, Japan).
Cytology smears from all patients were collected from different parts, including the center and border of the ulcer and beneath the scab, using a disposable sterile lancet. Multiple smears were made on slides and were both air dried and alcohol-fixed and were stained by Wright-Giemsa methods.
The cytology smears, touch prints, fine-needle aspiration, and histologic sections were examined under different magnifications. The smear was considered negative if amastigotes were not found on ×100 magnification (oil immersion field [OIF]) of the microscope. The density of amastigotes was quantified using a semiquantitative scale: grade I, 1 amastigote/whole slide to 1 amastigote/OIF in a total of at least 100 OIFs; grade II, 2 to 10 amastigotes/OIF in a total of at least 50 OIFs; grade III, 11 to 20 amastigotes/OIF in a total of at least 50 OIFs; grade VI, more than 21 amastigotes/OIF in a total of at least 10 OIFs.9
Review of the 40 Leishmania-positive cytologic smears and histologic sections of 100 patients was conducted in details blindly by 3 pathologists and was scored according to Table 1 by differentiation of the various types of inflammatory cells (plasma cells, eosinophils, lymphocytes, segmented neutrophils, monocytes, mast cells, multinucleated giant cells, and binucleated histiocytes), free Leishman-Donovan bodies (L-Ds), and granulomatous reaction. Two hundred cells were counted in each smear and the results expressed as a percentage. The interrelationship between various inflammatory cells, L-Bs, and the cells containing Leishman bodies was evaluated.
The following monoclonal antibodies were kindly provided for this experiment by Special Programme for Research and Training in Tropical Disease, World Health Organization (Geneva, Switzerland): IS2-2B4 (A11), specific for Leishmania tropica, and XLVI-5B8- B3 (T1) (Hamburg, Germany), specific for Leishmania major.10,11
Sections 3 μm thick were used for the IHC analysis. The slides were deparaffinized in xylol, rehydrated, and treated with 3% hydrogen peroxide solution for 10 minutes at room temperature to quench the endogenous peroxides. The antigen retrieval was conducted by pretreatment with microwaving (power 100 for 10 minutes; then, power 20 for 20 minutes) using a 10-mmol/L concentration of citrate buffer (pH 6.0). The primary antibody was applied for 1 hour (diluted 1:200). The detection system used was Envision+ (DakoCytomation, Glostrup, Denmark) and developed with diaminobenzidine (DakoCytomation). 3,3′-Diaminobenzidine–hydrogen peroxide was applied as the chromogen, and hematoxylin was used as the counterstain.
DNA Extract From Paraffin Blocks and Smears
One-fourth of each 3- and 4-mm-diameter punch biopsy was placed into Eppendorf tubes. Deparaffinization was conducted by adding 1 mL of xylene to microtubes containing tissue sections, shaken for 20 minutes, and centrifuged for 5 minutes at 5000g, and then, the flow through was discarded. For rehydration, that step was repeated by adding 0.5 mL xylene and 0.5 mL ethanol, followed by 100%, 75%, and 50% ethanol for 2 minutes. The kinetoplast DNA (kDNA) from the smears and tissue samples of the cutaneous lesions was extracted using phenol-chloroform–isoamyl alcohol, as previously described by Noyes et al.12
Nested PCR Assay
Two rounds of nested PCR were performed to amplify the variable segments present in the smear scrapings on the minicircle kDNA from the Leishmania species, and histologic sections were applied, with a slight modification. Special primers related to the variable regions of kDNA were used in the PCR analysis according to the method of Noyes et al.12 The external primers CSB2XF (CGA GTA GCA GAA ACT CCC GTT CA) and CSB1XR (ATT TTT CGC GAT TTT CGC AGA ACG) were used in the first round of PCR testing, and the internal primers 13Z (ACT GGG GGT TGG TGT AAA ATAG) and LiR (TCG CAG AAC GCC CCT) were used in the second round of PCR tests. Each 25-μL, first-round reaction mixture contained 200 μM of each deoxynucleoside triphosphate (Roche, Penzberg, Germany), 1.0 U Taq polymerase (CinnaGen, Tehran, Iran), a 5-μL template DNA, 1.5 mM MgCl2, 50 mM Tris-HCl (pH 7.6), 10 mM CSB1XR, and 10 mM CSB2XF. The thermocycler used (Eppendorf AG, Hamburg, Germany) was set to provide 5 minutes at 94°C, followed by 35 cycles each of 30 seconds at 94°C, 1 minute at 55°C, and 1 minute at 72°C, with a final extension for 5 minutes at 72°C. The products of the first round of the PCR test were diluted 1:9 with ultrapure water, and then, 1 μL of those dilutions were used as the templates for the second round of PCR, which used the same conditions and reaction mixture as the first round, except that LiR and 13Z were used as the primers. The PCR products were analyzed by 1.5% agarose gel electrophoresis. As the positive controls, the DNA extracted from the promastigote cultures of the reference strains of L major (MCAN/IR/97/LON490) and L tropica (MHOM/IR/89/ARA2) was also run on each gel. The size of each detected amplicon was estimated by comparison with a 100 base pairs (bp)–1500-bp molecular-weight “ladder” (Roche, Penzberg, Germany) run on the same gel.
In this study, we used 2 positive characteristics as the gold standard for “typical Leishman body” detection in cytology or histology and for a “positive PCR test result.” There was no difference between patients that would influence the test results. Thirty negative controls were chosen as noninfectious, inflammatory skin disorders from nonendemic areas with proven immunofluorescence studies.
Data analyses were done using the SPSS 16.0 software package (formerly SPSS Inc [now IBM Corporation], Chicago, Illinois). All data are expressed as mean (SD). Differences among cell numbers at different sites, clinical features, and grades were tested by 2-way analysis of variance using the general linear model procedure. Finally, multiple linear regression analyses, with cell type as the dependent variable, were performed to control for effects of confounding independent variables (lesion sites, clinical features, and grades). A P value less than .05 was considered to indicate statistical significance.
Of the 100 patients clinically suspected to be infected by Leishmania parasites, 60 (60%) were male. The patients belonged to a heterogeneous population from Fars Province, southern Iran, and their ages ranged from 0.7 to 84 years. The duration of the disease varied from 15 days to 4 years. Most patients had lesions over exposed parts of the body, most commonly, the arms, followed by legs, face, and trunk. Most of the patients had multiple lesions involving more than one anatomic region, whereas less than 10% of them had only a single lesion. The clinical presentation was of 2 types: (1) typical (66%; 66 of 100), and (2) atypical (34%; 34 of 100) including erythematous leishmaniasis, volcanic ulcers (Figure 1), and multi-infections (Figure 2), with lupoid, diffuse, eczematous, verrucous, and nodular patterns.
Cytologic examination, including touch smears and fine-needle aspirations, detected Leishmania amastigotes in 72 of the 100 patients (72%) who were suspected of having either typical or atypical CL. The cytologic findings of 40 positive smears (40%) for Leishmania consisted of plasma cells, neutrophils, eosinophils, mast cells, lymphocytes, vacuolated macrophages, macrophages, macrophages loaded with L-Bs, multinucleated giant cells, binucleated histiocyte cells, granuloma, and free L-Bs.
Quantified amastigote density in 40 positive touch smears for Leishmania were classified by 3 independent observers. Differentiation of different types of inflammatory cells of the frothy positive smears with different grades and 5 negative smears are summarized in Table 1.
Of the 100 patients who underwent tissue biopsies, the biopsies of 61 (61%) were reported as leishmaniasis. The other 39 biopsies (39%) were inconclusive and reported as granulomatous reaction, inflammation, and ulceration with secondary bacterial infection, with 1 (1%) reported as squamous cell carcinoma. Histology of the lesions indicated a spectrum ranging from almost normal cutaneous cells to individual cell necrosis associated with infiltration of diffuse parasitized macrophages in grade IV to activated macrophages giving rise to well-formed epithelioid, tuberculoid granulomatous reactions with diffuse necrosis in grade I. The middle of the spectrum showed parasitized macrophages with ill-formed granulomatous reactions in groups II and III, with or without focal necrosis.
The histologic findings were ulceration and granulation tissue formation, granulomatous inflammation, acute inflammation, multinucleated giant cells with many intracytoplasmic L-Bs (Figure 3), numerous multinucleated giant cells, pseudoepitheliomatous hyperplasia with keratin pearls, and massive infiltration of lymphoplasma cells, neutrophils, macrophages, and macrophages loaded with L-Bs in submucosal areas of epithelium.
Immunohistochemistry and Molecular Findings
As the antibody used in the IHC technique was reactive to amastigote antigens, the parasite was highlighted by this technique (Figure 4). All specimens, including typical and atypical cases of CL, with a confirmed microscopic diagnosis of CL were confirmed by the anti-L major and L tropica monoclonal antibodies. Conventional, molecular, and IHC results of typical and atypical CL are summarized in Table 2.
The fragments specific to mini-circle kDNA of L major (560–590 bp long), and L tropica (750 bp long), were amplified from the 97 (97%) and 1 (1%) of 100 samples by second-round PCR assay, respectively (Figure 5). In 2 patients (2%) and in the negative controls, no amplicons were detected. All 100 people (100%) examined were identified as patients with CL with a positive result in at least 1 of the 4 (25%) techniques performed. All 30 non-Leishmaniases inflammatory skin lesions (100%) were negative for Leishmania by all methods. Clinical data, microscopic diagnosis, and results of PCR and immunostaining studies in patients with CL are presented in Table 3.
Using our gold standard model, compared with other methods, PCR of the kDNA showed the highest sensitivity (typical, 100%, 67 of 67; atypical, 94%, 31 of 33) and specificity (100%) in the confirmed positive samples, followed by the IHC (typical, 97%, 65 of 67, with 100% specificity; atypical, 94%, 31 of 33, with 100% specificity), cytology (typical, 79%, 53 of 67, with 100% specificity; atypical, 58%, 19 of 33, with 100% specificity), and pathology (typical, 70%, 47 of 67, with 100% specificity; atypical, 42%, 14 of 33, with 100% specificity).
The results of the present study revealed remarkable variation in the clinical manifestations of CL caused by L major. The clinical presentation in 33% of the patients (33 of 100) was atypical. Localized CL typically presents as papules, crusted nodules, plaques, or noduloulcerative lesions. However, it may occasionally present with atypical presentations, such as with a zosteriform pattern, as hyperkeratotic psoriasiform, as eczematoid, or as erysipeloid, lupoid, sporotrichoid, nodular, warty, impetiginized, and acneiform lesions.13,14
There was an inverse correlation between the number of L-Bs with the number of neutrophils, lymphocytes, granulomatous reactions, and multinucleated giant cells. An inverse relationship between the number of L-Bs and the percentage of lymphocytes and neutrophils has been reported previously15 in the lesions of the patients with CL. Therefore, the presence of a granulomatous reaction, with a high percentage of macrophages and with multinucleated giant cells, is an important suggestive clue for the diagnosis of this disease in those cases that have negative results from histologic and cytologic tests and in patients who live in the endemic areas and are clinically suspected of having CL. Presence of granulomatous reactions and multinucleated giant cells in most of the patients with grades I, II, and microscopically negative lesions showed an inverse relation between the number of L-Bs and these chronic inflammatory cells. Such findings in the negative smears of patients who showed clinical manifestations of mucosal leishmaniasis are in accordance with the previous observation by Shirian et al.16
Demonstration of the parasite is important in the diagnosis of leishmaniasis.17 Detection of the parasites in the tissue sections in those patients who have been infected with only a few organisms or who are in the chronic and/or necrotic stages of the disease is histologically difficult because the cytopathologic results in such cases may be mistaken for nonspecific, chronic inflammation and/or granulomatous reactions.18,19 Complete forms of an L-B, nucleus, and kinetoplast can be seen at the cytologic level. Although there were no L-Bs in the cytologic sections of some patients, the smears from those patients showed several granulomas, and the diagnosis was confirmed by nested PCR. In those patients with intense inflammatory response, particularly in those treated and who were parasite free, cytology may be suggestive or inconclusive for leishmaniasis.3,17 Failure to detect L-Bs by histology and cytology methods may result from presence of high percentage of neutrophils due to contamination of lesions.
Leishmania major and L tropica were detected in 97 cases (97%) and 1 case (1%), respectively, of typical and atypical CL by nested PCR assay. Therefore, this is a sensitive test for the detection of Leishmanial DNA in tissues and smears of both typical and atypical CL cases. Nonetheless, based on the findings of this study, L major is the predominant species responsible for CL in this region. Fars province, southern Iran, is a hyperendemic region of leishmanisis.1,20 Negative PCR results in 2 patients (2%) could be due to either degradation of the protozoa during cell necrosis and lyses21 or technical errors during DNA extraction.
The IHC preparations presented a better visualization of the amastigotes in relation to the hematoxylin-eosin staining for CL diagnosis. The chromogenic substrate produces a precipitate layer at the site of the antibody-antigen reaction and results in a thick, diffuse, brown layer overlaying that complex, giving the amastigotes a slightly larger appearance than is appreciated on hematoxylin-eosin staining, so the Leishmania organisms are readily detectable at lower-power views. Possibly the main reason for the negative results in 4 IHC samples (4%) could be the location of the lesion that was selected for tissue sampling. These tissue sections had either minimum infection or were free of infection.
In conclusion, because cytology is cheap and easy to perform, it is the preferred primary approach for typical CL, but microscopic examination, including cytology and pathology, does not always have sufficient sensitivity for diagnosis of atypical CL cases. Nested PCR and IHC are sensitive tests for the diagnosis of leishmaniasis in tissues and smears of both typical and atypical CL cases; however, PCR is indicated as a second line in negative cytology or for species identification.
We thank the authorities of the Veterinary School, Shiraz University and Medical School, Shiraz University of Medical Sciences, and the Institute of Experimental Pathology, University of Muenster, for their support. We also thank M. R. Tabandeh, PhD, from Shahid Chamran University; A. Saeedzadeh, PhD, and K. Daneshbod, MD, from the Dr Daneshbod Pathology Laboratory; and J. Brosius, PhD; T. Rozhdestvensky, PhD; and G. Randau, MD, Institute of Experimental Pathology, University of Muenster, for their help and advice.
The authors have no relevant financial interest in the products or companies described in this article.