Histopathological characteristics of cutaneous lesions caused by Leishmania Viannia panamensis in Panama.

Cutaneous leishmaniasis (CL) is an endemic disease in the Republic of Panama, caused by Leishmania (Viannia) parasites, whose most common clinical manifestation is the presence of ulcerated lesions on the skin. These lesions usually present a chronic inflammatory reaction, sometimes granulomatous, with the presence of lymphocytes, plasma cells and macrophages. This study describes the histopathological characteristics found in the skin lesions of patients with CL caused by Leishmania (V.) panamensis in Panama. We analyzed 49 skin biopsy samples from patients with clinical suspicion of CL, by molecular tests (PCR for subgenus Viannia and HSP-70) and by Hematoxylin-Eosin staining. Samples were characterized at the species level by PCR-HSP-70/RFLP. From the 49 samples studied, 46 (94%) were positive by PCR and were characterized as Leishmania (V.) panamensis. Of these, 48% were positive by Hematoxylin-Eosin staining with alterations being observed both, in the epidermis (85%) and in the dermis (100%) of skin biopsies. The inflammatory infiltrate was characterized according to histopathological patterns: lymphohistiocytic (50%), lymphoplasmacytic (61%) and granulomatous (46%) infiltration, being the combination of these patterns frequently found. The predominant histopathological characteristics observed in CL lesions caused by L. (V.) panamensis in Panama were: an intense inflammatory reaction in the dermis with a combination of lymphohistiocytic, lymphoplasmacytic and granulomatous presentation patterns and the presence of ulcers, acanthosis, exocytosis and spongiosis in the epidermis.

INTRODUCTION

Cutaneous leishmaniasis (CL) is a zoonotic disease caused by parasites of the genus Leishmania, which infects mammalian reservoirs through the bite of an infected vector of the genus Lutzomyia in the New World and Phlebotomus in the Old World , . Leishmaniasis is among the group of neglected tropical infections, leading to high levels of morbidity mainly among the most vulnerable social groups , . In Panama, it is considered an emerging health problem with an estimated 1,000-3,000 new cases per year . In 2011, the Pan American Health Organization (PAHO) reported Panama as the country with the highest incidence of CL per 100,000 habitants in the American continent .

CL presents a spectrum of clinical and histopathological manifestations that encompass different morphological states such as a nodule, plaque and ulcer; and which may persist as a chronic lesion or heal with a scar, depending on the infecting parasite species . The lesion may later recur . Commonly, the infection presents as cutaneous lesions in exposed areas of the patient, being the skin the main affected organ. Once the parasite invades the skin, after the innate immune response, the defense against infection is the cell-mediated immune response, which actively participates in the formation of granuloma eventually limiting the spread of the infectious agent and, in this way, controlling the infection .

Because the current CL treatment is complicated and is often associated with drugs that have adverse effects, diagnostic confirmation prior to treatment is important. The main diagnostic data are clinical and epidemiological; however, additional laboratory testing is required for definitive diagnosis. Several diagnostic tests are available to confirm the diagnosis: parasitological, immunological, histopathological, immunopathological and molecular . Histopathological techniques are important because they allow observing the parasite and studying the inflammatory infiltrate that is produced during infection by Leishmania parasites - . The cutaneous lesion of leishmaniasis is characterized by a chronic inflammatory reaction, sometimes granulomatous, with the presence of lymphocytes, plasma cells and macrophages. Leishmania amastigotes are found in the cytoplasm of macrophages as rounded organisms, with a round nucleus, a kinetoplast and surrounded by a clear halo , , . Two histological patterns have been reported in patients with active CL lesions. The first one is a non-specific chronic inflammatory reaction with a diffuse infiltrate of lymphocytes, plasma cells, macrophages, granulocytes and cell debris. The second one, a granulomatous reaction that contains histopathological elements found in a chronic inflammatory reaction, but associated with the presence of epithelioid cells and the formation of granulomas, frequently associated with giant cells. Vasculitis, neuritis and necrosis have also been observed. In scarred lesions, dermal fibrosis and perivascular infiltrates can be found . The first reports of CL in Panama were those described by Darling in 1911 , Herrick in 1911 , Darling and Connor in 1911 and Bates in 1913 , who considered the lesions clinically identical to the oriental sore. Fox in 1931 summarized the clinical and gross features of the disease as seen in Brazil, but did not stress the histological aspects, which were later summarized by Snow et al. in 1948 . However, there is only one detailed report from 1952 describing the histopathological changes in skin lesions of patients affected by CL in Panamá . The results showed a rather constant and distinctive histological pattern presented in 20 skin ulcer biopsies due to CL. The cardinal findings of epithelial hyperplasia, poor granulation, intense chronic inflammation of the dermal papillae, inflammatory cells located on sweat glands towards the periphery of the lesion, and lack of blood vessel changes, differentiate leishmaniasis from most cutaneous ulcers; however, positive diagnosis still requires the demonstration of the presence of parasites .

Considering that the histopathological characteristics of these lesions, as well as their diagnostic and predictive value have been poorly documented in Panama, this study had the main aim to describe the histopathological findings of cutaneous lesions caused by Leishmania (V.) panamensis in patients from Panama.

MATERIALS AND METHODS

Study design

This study aimed to describe the histopathological features of skin biopsies from patients with CL in Panama. Patients with suspected CL were attended at the Tropical Medicine Clinic, Gorgas Memorial Institute of Health Studies, Panama City. This study was carried out from January 2012 to December 2012. All patients were adults, and accepted, freely and voluntarily, to participate in the study through the signing of informed consents. Patients were biopsied and the histopathological and molecular diagnosis of CL was performed. After CL diagnosis, all patients were treated with meglumine antimonate (Glucantime™) in a dose of 20 mg/kg/body weight, intramuscularly for 20 days, according to the Panamanian guidelines for Leishmaniasis control .

Biopsy collection

Forty-nine skin biopsy samples from patients with suspected CL attended at the Tropical Medicine Clinic, Gorgas Memorial Institute of Health Studies, were evaluated. Biopsy specimens were taken with a 5-mm Harris punch (Whatman International Ltd), preceded by the application of local anesthesia and asepsis , .

Molecular characterization

DNA extraction from the biopsy samples was performed using the Qiagen QIamp® DNA Blood Mini Kit (Qiagen, CA, USA) according to the manufacturer’s instructions. Two protocols based on the polymerase chain reaction (PCR) were used: a PCR to identify the subgenus Viannia (LV-B1 primers), which amplifies the kDNA region of the minicircle yielding a 750 pb fragment; and the Hsp-70 PCR (F25-R1310 primers) that amplifies a region from the Heat Shock Protein 70 gene yielding a product of 1,286 pb . In addition, samples were characterized by PCRHsp-70/RFLP analysis using the enzymes HaeIII and BccI to observe the distinct patterns of Leishmania species that have been previously described , .

Histopathological analysis

All samples were fixed in 10% buffered formalin, and processed within a period of not more than 48 h to dispose of the paraffin tissue block , , in the Laboratory of Pathology of Santo Tomas Hospital, Panama city. All tissue samples were dehydrated, cleared, embedded in paraffin, cut into 4–5 μm thick sections and stained with Hematoxylin-Eosin (HE) and Giemsa . Lesion sections were characterized microscopically based on the histological alterations found in the epidermis and in the dermis. At the same time, the intensity and distribution of inflammatory reactions in the dermis were evaluated, as well as the presence of different cell types present in the infiltrate, the formation of granuloma and the parasite load, among other morphological aspects. A comparative, semi-quantitative histopathological analysis of the HE-stained sections was performed, attributing crosses according to the intensity of the different characterized processes: (-) negative, (+) discrete, (++) moderate and (+++) intense .

RESULTS

Cutaneous lesions of 49 CL-suspected patients were analyzed using molecular and histopathological methods. Of the patients studied, 73% were male and 27% female. Most of the patients (96%) came from the province of Panama. The age range was 23 to 71 years with an average of 41 years old. Evolution time of lesions varied from 10 to 90 days with an average of 30 days. The number of lesions ranged from 1 to 8 with an average of 2 lesions per patient, mostly located in the upper limbs (60%). Out of the total samples, 94% (46/49) were positive by PCR (Viannia, Hsp-70). Positive PCR Hsp-70 samples (78%, 36/46) were characterized as L. (V.) panamensis by PCR-RFLP analysis.

Forty-eight percent (22/46) of the samples were positive to amastigotes by microscopic evaluation of histological sections. In patients with CL lesions of 30 days or less (early lesions), 11 were positive and 13 were negative; and in patients with lesions of more than 30 days of infection (late lesions), 11 were positive and 11 were negative. Moreover, the amount of parasites varied between discreet (+) and intense (+++) in the two groups. Amastigote forms were observed in typical histiocyte phagocytic vacuoles, visible in HE staining (Figure 1). The parasite load presented a variable distribution in both, superficial and middle dermis and even deeper in the subcutaneous fat, with occasional signs of endarteritis.

Figure 1

Amastigotes forms of Leishmania inside macrophages vacuoles in the dermis of patients affected by localized cutaneous leishmaniasis caused by L. (V.) panamensis (red arrows). (Hematoxilin-Eosin staining)

The histopathological analysis from the 46 skin biopsies of patients with clinical and laboratory diagnosis of localized CL caused by L. (V.) panamensis showed morphological alterations in both, epidermis and dermis. Alterations of the epidermis were observed in 85% (39/46) of skin biopsies; and were mainly characterized by acanthosis in 41% (19/46), spongiosis in 39% (18/46), exocytosis in 35% (16/46), parakeratosis in 17% (8/46) and pseudoepitheliomatous hyperplasia in 7% (3/46) of the cases (Figure 2). The presence of ulcers in the fundus, border and surface of epidermis was observed in 43% (20/46) of skin biopsies (Table 1). However, the histopathological alterations in the superficial and deep dermis occurred in 100% of the cases and were characterized mainly by a lymphoplasmocytic inflammatory infiltrate of variable intensity, with diffuse or focal distribution. The inflammatory infiltrate was intense in 39% (18/46), moderate in 57% (26/46) and mild in 4% (2/46) of the cases with diffuse distribution in 37% (17/46) and focal in 63% (29/46) of the cases. Milder infiltrates were preferentially located in superficial dermis with tendency of perivascular distribution. The inflammatory infiltrate was characterized by predominance of lymphocytes (61%), followed by histiocytes (22%) and plasma cells (20%). Granulomatous outline or well-formed epithelioid granulomas were observed in 46% (21/46) of the cases, with the presence of multinucleated giant cells in 24% (5/21) and focal necrosis area in 14% (3/21) of these cases. The presence of Leishmania amastigote forms in macrophage cytoplasm was observed in 48% (22/46) of HE-stained histological sections and the parasite load varied from mild, moderate to intense (Table 2). According to different inflammatory cells predominance in the dermal tissue response, it was possible to classify them into three histopathological patterns: lymphohistiocytic infiltration in 50% (23/46), lymphoplasmocytic in 61% (28/46) and granulomatous in 46% (21/46), being the combination of these patterns frequently found (Figure 3).

Figure 2

Paraffin histological section of the skin lesion of a patient affected by localized cutaneous leishmaniasis caused by L. (V.) panamensis showing in A) acanthosis (black arrow), and in B) spongiosis (yellow arrow) and lymphohistiocytic exocytosis (blue arrow) (Hematoxilin-Eosin staining)

Table 1

Histopathological findings observed in the epidermis of patients with cutaneous leishmaniasis caused by Leishmania (V.) panamensis in Panama

	EPIDERMIS
	Ulcer	Acanthosis	Spongiosis	Exocytosis	Paraqueratosis	Hyperplasia
Number	20/46	19/46	18/46	16/46	8/46	3/46
Percentage (%)	43	41	39	35	17	7

Table 2

Histopathological findings observed in the dermis of patients with cutaneous leishmaniasis caused by Leishmania (V.) panamensis in Panama

	DERMIS – INFLAMMATORY INFILTRATE									Amastigote
	INTENSITY			DISTRIBUTION		CELLULAR TYPE
	Intense	Moderate	Discrete	Diffuse	Focal	*PMN	Lymphocyte	Plasma cell	Macrophage
Number	18/46	26/46	2/46	17/46	29/46	38/46 (-) 8/46 (+) 0/46 (++) 0/46 (+++)	0/46 (-) 2/46 (+) 15/46 (++) 29/46 (+++)	2/46 (-) 14/46 (+) 20/46 (++) 10/46 (+++)	1/46 (-) 19/46 (+) 15/46 (++) 11/46 (+++)	24/46 (-) 15/46 (+) 2/46 (++) 5/46 (+++)
Percentage (%)	39	57	4	37	63	83 (-) 17 (+) 0 (++) 0 (+++)	0 (-) 4 (+) 33 (++) 63 (+++)	4 (-) 30 (+) 44 (++) 22 (+++)	2 (-) 41 (+) 33 (++) 24 (+++)	52 (-) 33 (+) 4 (++) 11 (+++)

*PMN: Polimorphonuclear

Figure 3

Inflammatory patterns observed in cutaneous lesions caused by Leishmania (V.) panamensis. A) Intense and diffuse lymphohistiocytic inflammatory pattern; B) Intense lymphoplasmocytic inflammatory pattern; C) Granulomatous inflammatory pattern; D) Presence of multinucleated giant cells observed in the granulomatous reaction (Hematoxilin-Eosin staining)

DISCUSSION

At least 95% of leishmaniasis cases reported in Panama correspond to cutaneous lesions of chronic evolution . The clinical diagnosis of these lesions is often complicated, even for physicians experienced in the recognition and treatment of this parasitic disease. On the other hand, it is common to send histological samples of patients with suspected CL lesions to pathology units of reference Panamanian health institutions in which pathologists must have previous experience and academic background. However, studies aimed to characterize CL histopathological findings in Panama are scarce and consequently necessary for the diagnostic and prognostic support of these parasitic infections that are so frequent in this country .

In this work, evaluation by histopathology of 46 patient’s lesions with CL previously characterized by molecular tests was achieved. However, amastigotes were only detected in 48% (22/46) of biopsies. Amastigotes identified within histiocytes presented a variable distribution along the superficial, middle and deeper layers of dermis. This relatively low diagnostic sensitivity has also been reported in studies with other Leishmania species which are present in South America , - . Nevertheless, in these previous reports, it was considered that histopathological tests were important to describe the inflammatory infiltrate associated to leishmaniasis . According to the literature, histopathology is usually the method with the lowest diagnostic sensitivity (30% -60%) for CL , , . There are several factors that can explain the difficulties of CL diagnosis by histopathological analysis. One of them occurs in the later stages of the cutaneous infection when granulomas predominate and parasite-filled histiocytes gradually disappear , . In this regard, it has been reported that parasites are very difficult to find in lesion biopsies with 5-7 months of evolution , . Thus, we evaluate 23 patients with early lesions (up to 30 days) and 22 with late lesions (31-90 days). However, we did not find statistical differences between the two groups regarding amastigote detection and parasite load. Another situation occurs when there are several opportunistic infections in the same lesion or the same cell type in necrotic areas, making it difficult to observe amastigote forms. The same happens when parasites are free in connective tissues . Occasionally, the histopathological analysis fails to detect amastigotes even in early lesions which are rarely reported in the epidermis .

Chronic nonspecific and/or granulomatous inflammation are the main histopathological manifestations of cutaneous and mucocutaneous leishmaniasis in the New World , . The associated infiltration of plasma cells and vascular alterations are suggestive but not diagnostic features of the disease , , . The specific diagnosis is only possible through the demonstration of parasites in examined tissues . As already mentioned, parasites are generally scarce and difficult to demonstrate in more chronic lesions, thus, immunostaining techniques could be employed to increase the sensitivity of the histopathological diagnosis , . However, there are studies, using immunostaining techniques, that demonstrated that it is possible to obtain higher sensitivities in CL histopathological diagnosis , , . In this sense, Sotto et al. analyzed 40 biopsies of human patients with cutaneous or mucocutaneous leishmaniasis in South America, evidencing the presence of parasites in 20% of biopsies in histological sections stained by HE, while by indirect immunofluorescence of paraffin-embedded tissues, the positivity increased to 89.28% and by the immunoperoxidase reaction, it was possible to visualize the parasite in 64.51% of biopsies.

Morphological analysis of lesions showed a variable parasite load (discrete to intense) and inflammatory infiltrates of variable intensity with diffuse or focal distribution. Inflammatory infiltrates were characterized by lymphocytes predominance, followed by histiocytes and plasma cells. The epidermis showed acanthosis, spongiosis, exocytosis, parakeratosis and pseudoepitheliomatous hyperplasia. In addition, lesions demonstrated the presence of ulcers, focal necrosis area and well-organized granulomas. The presence of well-organized granulomas with multinucleated giant cells is a factor related to the immune system attempt to eliminate parasites . Similar histopathological features have been described in previous studies which analyzed biopsies obtained from patients infected with species from the Viannia subgenus , , - .

In the present study, 63% (29/46) of the tissue inflammatory response was found to be focal in the tissue, in contrast to data reported by Isaza et al. , showing that inflammatory response was diffuse (62.5%) in biopsies of patients coming from endemic areas of the State of Antioquia in Colombia. These patients had CL caused by L. (V.) panamensis. In addition, Isaza et al. described a predominance of macrophages and lymphocytes in the inflammatory infiltrate in accordance with our study. At the epidermis level, we found the presence of exocytosis in 35% of the samples. This cellular process was also observed (69.5%) in the study by Isaza et al. The presence of the exocytosis phenomenon emphasizes the importance of the epidermis in the immunoregulation of the disease .

The histological spectrum of CL has had variations in its classification in different types. For example, Kurban et al. and Mansour et al. , suggested two histological patterns in biopsies of patients with CL: the first one corresponding to lesions with less than one year of duration associated with a diffuse infiltrate, and the second one corresponding to lesions of more than one year of duration with granulomatous infiltrate. On the other hand, Venkataram et al. reported four histological patterns suggestive of CL. Moreover, Magalhães et al. and Ridley et al. described up to five histopathological patterns. In the present study, three main histopathological patterns were identified: the first one with a lymphohistiocytic inflammatory response found in 50% of samples, the second one with a lymphoplasmacytic response, present in 61% of samples and the third one with a granulomatous response, found in 46% of samples. It is important to mention that sometimes these patterns appear combined. For example, Azogue describes a histiolymphoplasmocytic response in 66% of CL cases associated with granulomatous reactions and Magalhães et al. described that 40.9% of CL cases had characteristic histiolymphoplasmocytic responses with cellular exudative reaction patterns and disorganized granulomatous reactions in 26.1% of biopsies. In contrast, Isaza et al. did not find granulomatous infiltrates in his study.

The histopathological and immunological host response to infection caused by parasites of the genus Leishmania, depends largely on the infecting parasite species , . In this sense, Silveira et al. described the clinical and immunopathological spectrum of the American CL, caused by different dermotropic species of the parasite responsible for the disease in the New World. While L. (V.) braziliensis presents a clear tendency to lead to localized cutaneous leishmaniasis (moderate T-cell hypersensitivity response), progressing to mucocutaneous leishmaniasis (prominent Th1 type immune response), L. (Leishmania) amazonensis shows an opposite tendency, leading infection to the anergic T-cell hyposensitivity pole, in association with a Th2 type of cellular immune response. The cellular immune response of the host also influences the histopathological aspect of skin lesions. Localized cutaneous leishmaniasis caused by L. (L.) amazonensis shows a dense inflammatory infiltrate of vacuolated macrophages in the dermis with a large number of amastigote forms, giving to the infiltrate the appearance of a macrophagic granuloma. This morphological aspect of the skin lesion differs from the localized cutaneous leishmaniasis cases caused by L. (V.) braziliensis and other species of the subgenus Viannia, where there is a predominance of a more modest infiltrate of macrophages with a generally scarce parasite load, however, lymphocytes and plasma cells are more frequent in the presence of well-formed epithelioid granulomas , , .

CONCLUSIONS

In conclusion, the histopathological changes observed in localized CL caused by L. (V.) panamensis in Panama were characterized by an intense inflammatory reaction in the dermis with diffuse predominance and lymphohistiocytic, lymphoplasmocytic and granulomatous presentation patterns, as well as the presence of ulcers, acanthosis, exocytosis and spongiosis in the epidermis. However, none of them are specific to establish the diagnosis by histopathology. Under the conditions described above, the sensitivity of the histopathological technique in the diagnosis of CL caused by L. (V.) panamensis was 48%.