Tropical medicine rounds
Oxford, UK International IJD Blackwell 1365-4632 45 Publishing, Publishing Journal Ltd, of Ltd. Dermatology 2004
Leishmaniasis recidiva cutis due to Leishmania (Viannia) panamensis in subtropical Ecuador: isoenzymatic characterization Leishmaniasis Calvopina Tropical medicine et al. recidiva rounds cutis in Ecuador
Manuel Calvopina, MD, MSc, DTM&H, Hiroshi Uezato, MD, PhD, Eduardo A. Gomez, MD, Masataka Korenaga, MSc, PhD, Shigeo Nonaka, MD, PhD, and Yoshihisa Hashiguchi, PhD
From the Department of Parasitology, School of Medicine, Kochi University, Kochi, Japan, Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan, and Departamento de Medicina Tropical, Facultad de Medicina, Universidad Catolica Santiago de Guayaquil, Ecuador Correspondence Manuel Calvopina, MD, MSc, DTM&H Department of Parasitology School of Medicine Kochi University Kochi 783-8505 Japan E-mail:
[email protected]
Abstract Background Information regarding leishmaniasis recidiva cutis (LRC), a clinical variant of cutaneous leishmaniasis, in the New World is scarce. LRC is characterized by slowly progressing lesion(s) that appear after a variable period of time, from months to years, in or around the scar of an apparently clinically healed sore. Patients and methods Six patients are reported who presented with crusted, papular lesions located on the edge of a healed scar, with a mean of 18.2 months of slowly progressive evolution. The isolated strains of Leishmania parasites were characterized by enzyme electrophoresis. Eleven enzyme systems were assayed. Skin biopsies from the active border of the lesions were taken for histopathology. Results Tissue sections showed a granulomatous, lymphohistiocytic, dermal infiltrate containing Langhans’ giant cells. The anamnestic data, together with the clinical and histopathologic findings, support the diagnosis of LRC. The isoenzyme profile of Leishmania parasites isolated from five of the six patients identified them as Leishmania (Viannia) panamensis. Conclusions These findings are the first reported evidence of LRC within the clinical spectrum of American tegumentary leishmaniasis in Ecuador, and of its causative agent. The existence of LRC has future implications for both disease treatment and vaccine development.
Introduction
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Leishmaniasis recidivans (LR) is also known as relapsing, lupoid, or chronic tuberculoid cutaneous leishmaniasis in the Old World,1 and as leishmaniasis recidiva cutis (LRC) in the New World.2 There are very few reports and very little knowledge about LRC in the New World; by contrast, LR is often observed in the Old World where it is usually caused by Leishmania (Leishmania) tropica.1 This variant of cutaneous leishmaniasis was first reported by Christopherson3 in 1923 and well described by Berlin4 in 1940. The lesion is characterized by a scar with peripheral activity that progresses slowly. Lesions typically recur at the site of an original ulcer, generally within 2 years and often within the edge of the scar, and last for several years.4,5 In the New World, there are only a few reports of LRC: 14 cases in Brazil and some observations from Colombia and Peru.2,6–10 L. braziliensis subspecies and L. (L.) amazonensis have been isolated from cases in Brazil and Colombia.6,7,9 Leishmaniasis is endemic and widely distributed in Ecuador. The disease is present in most of its recognized tegumentary forms: localized, diffuse, and disseminated cutaneous, and mucocutaneous or espundia.11,12 The localized cutaneous International Journal of Dermatology 2006, 45, 116–120
form, usually a single ulcerative lesion on an exposed area of the body, is the most common form in the three endemic regions of Ecuador. LRC, however, has not been reported from any of them. At least six species, all in the Viannia and Leishmania subgenera, cause cutaneous infections in Ecuador: L. (Viannia) braziliensis, L. (V.) guyanensis, L. (V.) panamensis, L. (L.) mexicana, L. (L.) amazonensis, and L. (L.) major-like. In tropical and subtropical areas, the predominant species are L. (V.) panamensis and L. (V.) guyanensis.12–14 The mucocutaneous form is present in the Amazon region and is caused by L. (V.) braziliensis.15 In this paper, we report for the first time the presence of LRC in Ecuador, and its causative agent. We describe the case histories, clinical characteristics, and histopathology of six patients in the subtropical rainforest of Ecuador, and identification of the Leishmania isolated. Patients and Methods Geographical area and patients The study was performed in 2002 in seven communities in the subtropical area north-west of the Pichincha Province, Ecuador. © 2004 The International Society of Dermatology
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This area with secondary subtropical rainforest forms part of the western branch of the Andes belt at an altitude of 500–1200 m above sea-level. Children from primary schools and their relatives with cutaneous lesions clinically suspected as leishmaniasis were eligible for inclusion in the study. Informed consent was obtained from patients and their parents to participate in the study. A comprehensive clinical history was taken and a physical examination was performed. Because skin test antigens were not available in the country, the Montenegro test could not be performed.
Histopathology, parasite isolation, and cultivation Samples were taken from the active part of the lesion(s). Tissue obtained by needle aspiration was cultured in USMARU medium. Skin biopsies (diameter, 3 mm) were taken with a sterile punch, under xylocaine anesthesia, and fixed in 10% formalin for histopathologic examination. All isolates and reference strains were grown in USMARU biphasic medium prepared following the method of Shamsuzzaman et al.,16 and were passaged no more than four times after isolation. Bulk culture was in RPMI medium with 20% inactivated fetal calf serum, as described previously.16 Briefly, Leishmania parasites were inoculated in RPMI 1640 medium containing 2 mM L-glutamine, 0.18–0.3% sodium bicarbonate, penicillin (100 U/mL), and streptomycin (50 µg/mL). Fixed biopsy samples were embedded in paraffin, sectioned, stained with Giemsa and hematoxylin and eosin, and examined for histologic characteristics and the presence of amastigotes. Isoenzyme electrophoresis and identification of Leishmania isolates Cellulose acetate electrophoresis analysis of the isoenzyme profiles of the Leishmania isolates was performed according to previously described methods.17 Extracts were analyzed for 11 enzyme systems: four oxido-reductases, two isomerases, four transferases, and one hydrolase. To better ascertain the species attribution of the stocks surveyed, six selected World Health Organization (WHO) reference strains pertaining to different species were included in the study: L. (V.) braziliensis MHOM / BR / 75/M2904; L. (V.) panamensis MHOM/PA/ 71/LS94; L. (V.) guyanensis MHOM/BR/75/M4147; L. (L.) mexicana MNYC/ BZ /
62/M379; L. (L.) amazonensis MHOM/BR/ 73 / M2269; and L. (L.) chagasi MHOM / BR/74 / M2682.
Results Morphologic diagnosis
The characteristics of the patients and their lesions are presented in Table 1. The six patients studied are from 29 confirmed cutaneous leishmaniasis cases. All six patients had lesions that had clinically healed completely 1–3 years previously leaving atrophic scars, but presenting later with two or more reddish papules expanding at the periphery. Physical examination revealed an atrophic scar with a mean diameter of 3.3 cm, with confluent, small, red–brown, scaly papules surrounding it. Lesions were localized on various body sites (Table 1 and Fig. 1). No patient had received prior chemotherapy. Histopathology
Histologically, the lesions showed epidermal hyperplasia and a granulomatous dermal infiltrate composed of lymphohistiocytic cells, a few plasma cells, and Langhans’ giant cells. Some tubercle formation with epithelioid cells was seen, but there were no areas of necrosis (Fig. 2). Biopsies from Patients 2 and 4 also showed fibrosis. Amastigotes were not visible in any specimen. Parasite identification
Leishmania parasites were cultured from the lesions of five patients, speciated by their zymodeme patterns, and compared with those of WHO New World Leishmania reference strains. All five stocks were identified as L. (V.) panamensis, based on the similarity with the enzyme profile of the WHO reference strains (Fig. 3). Discussion In Ecuador, the most common cutaneous lesions of leishmaniasis are ulcerative, but papules, plaques, nodules, and erysipeloid forms have also been described.11 Here, we report, for the first time, six cases of LRC, a cutaneous variant
Table 1 Characteristics of patients and leishmanial lesions, and parasite identification
Case
Sex
Age (years)
Location of lesion
Clinical features
Size of lesion (mm)
Disease evolution and reactivation time (months)
Isoenzyme identification
1 2 3 4 5 6
Male Female Female Female Male Male
10 42 14 10 4 10
Abdomen Right elbow Left leg Left leg Left forearm Face
3 crusted papules around a scar 2 reddish papules around a scar 1 papule around a large scar 1 papule around a scar 6 reddish papules around a scar 5 crusted papules around 2 scars
35 × 30 20 × 15 40 × 25 35 × 30 50 × 40 45 × 43/33 × 30
30/14 36/24 24/6 60/36 18/5 36/24
L. (V.) panamensis L. (V.) panamensis L. (V.) panamensis L. (V.) panamensis L. (V.) panamensis Not isolated
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Figure 3 Isoenzyme pattern for phosphogluconate
Figure 1 Photograph of Patient 6 showing two extensive
atrophic scars on the face, with five small red–brown papules surrounding them
Figure 2 Histopathology of a papular recidivans lesion showing
a typical granulomatous infiltrate composed of histiocytes and multinucleated giant cells (arrow), surrounded by lymphocytes with scattered neutrophils and eosinophils. No caseation necrosis nor amastigotes were seen (hematoxylin and eosin, ×200)
characterized by periods of reactivation in a healed primary lesion, and identify L. (V.) panamensis as the causative agent. This species has been reported to be the most prevalent in subtropical Ecuador, the region from which our patients originated, but normally causes simple cutaneous ulcers.12–14 In the neighboring country, Colombia, L. (V.) panamensis has also been implicated in the mucocutaneous form.18 In International Journal of Dermatology 2006, 45, 116–120
dehydrogenase (6-PGDH) enzyme. Lane 1, L. (L.) mexicana (MNYC/ BZ/ 62 / M379). Lane 2, L. (L.) amazonensis (MHOM / BR /73/ M2269). Lane 3, L. (L.) chagasi (MHOM / BR / 74 / M2682). Lane 4, Case 1. Lane 5, Case 2. Lane 6, Case 3. Lane 7, Case 4. Lane 8, Case 5. Lane 9, another patient with a cutaneous ulcer from the same area. Lane 10, L. (V.) braziliensis (MHOM / BR /73/ M2904). Lane 11, L. (V.) panamensis (MHOM / PA /1971/LS94). Lane 12, L. (V.) guyanensis (MHOM / BR /75/ M4147)
previous studies on leishmaniasis in Ecuador, we have also observed several cases clinically resembling LRC from the Pacific coastal lowlands and in the Andean foci, where L. (L.) mexicana and L. (L.) major-like are the etiologic agents.19 Variation in the clinical forms of cutaneous leishmaniasis is thought to be associated with the parasite species, host immune response, and the saliva of the sandflies, yet the mechanisms are poorly understood. In the Old World, recidivans disease is mostly linked with L. (L.) tropica and rarely with L. (L.) major. Previous reports on the characterization of strains in the New World have provided evidence that LRC disease is produced by L. braziliensis complex and L. (L.) amazonensis.6,7,9 Our findings show that L. (V.) panamensis can also be the etiologic agent of LRC. Patients with LR type display strong skin hypersensitivity to Leishmania antigen and, histopathologically, a well-organized granulomatous reaction, as demonstrated in our patients. Hence, we suggest that this clinical form is mostly associated with the individual immune response rather than the species, supporting the concept that LR is a sequela of an incomplete immune response to an earlier episode of leishmaniasis. There is some debate as to whether the recurrent lesions are due to relapse or reinfection.9 In our patients, reinfection is unlikely because the lesions recurred at the border of the old scar of a previous ulcer suspected to be due to cutaneous leishmaniasis. Furthermore, in this population, the incidence of leishmaniasis is low. Hence, the proportion of recurrent cases due to relapse, rather than reinfection, is likely to be © 2004 The International Society of Dermatology
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relatively high.20 The persistence of living parasites around or in “cured” leishmaniasis has been demonstrated in several studies,9,21,22 and was found in five of our six patients, suggesting that reactivation is the most likely mechanism. Clinical data also support reactivation. Intracellular Leishmania are probably seldom, if ever, entirely eradicated even from healthy hosts by antileishmanial chemotherapy,23 and may be more likely to persist if therapy is insufficient or incomplete,24 or if the infection has healed without treatment,25 as occurred in our patients. It must be presumed that, in LRC, dormant parasites persist in the healed lesion and are reactivated by a certain stimulus. The nature of this stimulus is unknown. Saravia et al.9 have demonstrated a relatively weak cutaneous delayedtype hypersensitivity response to leishmanin skin test antigens in patients with recurrent leishmaniasis, and have suggested that it may be a risk factor for relapse in Colombia. Others reports have suggested that local trauma and topical corticoids might reactivate lesions after many years.21 Nutritional status, low dietary iron intake, and reduced mean hemoglobin values have been shown to be risk factors for acquiring cutaneous leishmaniasis in the population studied,26 but we do not know if they are also risk factors for reactivation. Finally, our findings suggest that LRC may not be uncommon in New World leishmaniasis, but may be underreported. Furthermore, the presence of LRC as an important cause of morbidity in this subtropical region has future implications for treatment regimens and immunoprophylaxis. Acknowledgments This study was supported by a Grant-in-aid for overseas scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant no. 10037385). We thank Professor Anthony Bryceson for reviewing the manuscript and Lorena Quinteros for field assistance.
References 1 World Health Organization. The Leishmaniases. WHO Tech Rep Series 1984; 701: 9–140. 2 Paes-Oliveira M. Leishmaniasis recidiva cutis. Anais Bras Dermatol 1977; 52: 353–359. 3 Christopherson JB. Case of leishmaniasis of the skin. Proc R Soc Med 1923; 16: 48–54. 4 Berlin C. Leishmania recidiva cutis. Leishmanid Arch Dermatol Syphilol 1940; 41: 874–886. 5 World Health Organization. Control of the Leishmaniases. WHO Tech Rep Series 1990; 793: 9–158. 6 Bittencourt AL, Costa JM, Carvalho EM, et al. Leishmaniasis recidiva cutis in American cutaneous leishmaniasis. Int J Dermatol 1993; 32: 802–805.
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7 Costa JML, Lago EL, Magalhaes AV, et al. Leishmaniose recidiva cutis causada por Leishmania Viannia braziliensis. Anais Bras Dermatol 1996; 71: 329 –333. 8 Oliveira-Neto MP, Mattos M, da Silva Freitas, et al. Leishmaniasis recidiva cutis in New World cutaneous leishmaniasis. Int J Dermatol 1998; 37: 846–849. 9 Saravia NG, Weigle K, Segura I, et al. Recurrent lesions in human Leishmania braziliensis infection – reactivation or reinfection? Lancet 1990; 336: 398–402. 10 Beaver PC, Jung RC, Cupp EW. Clinical Parasitology, 9th edn. Philadelphia: Lea & Febiger, 1984: 67–68. 11 Nonaka S, Hosokawa A, Maruno M, Hashiguchi Y. Clinical survey of cutaneous leishmaniasis in Ecuador. In: Studies on the New World Leishmaniasis and its Transmission with Particular Reference to Ecuador, Res Rep Series 6. Kyowa, Japan: Kyowa Printing Co. Japan, 2001: 69 –81. 12 Armijos RX, Weigel MM, Izurieta R, et al. The epidemiology of cutaneous leishmaniasis in subtropical Ecuador. Trop Med Intern Health 1997; 2: 140 –152. 13 Bañuls AL, Jonquieres R, Guerrini F, et al. Genetic analysis of Leishmania parasites in Ecuador: Are Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis distinct taxa? Am J Trop Med Hyg 1999; 61: 838–845. 14 Mimori T, Matsumoto T, Calvopina M, et al. Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World. Acta Tropica 2002; 81: 197–202. 15 Calvopina M, Guevara A, Armijos R, Hashiguchi Y. Clinical features of mucocutaneous leishmaniasis in the Amazonian region of Ecuador. In: Studies on the New World Leishmaniasis and its Transmission with Particular Reference to Ecuador, Res Rep Series 6. Kyowa, Japan: Kyowa Printing Co. Japan, 2001: 82–89. 16 Shamsuzzaman SM, Furuya M, Korenaga M, et al. Use of urine samples from healthy humans, nephritis patients or other animals as an alternative to foetal calf serum in the culture of L. (L.) donovani in vitro. Ann Trop Med Parasitol 1999; 93: 613–620. 17 Kreutzer RD, Christensen HA. Characterization of Leishmania spp. by isozyme electrophoresis. Am J Trop Med Hyg 1980; 29: 199–208. 18 Osorio LE, Castillo CM, Ochoa MT. Mucosal leishmaniasis due to Leishmania (Viannia) panamensis in Colombia: clinical characteristics. Am J Trop Med Hyg 1998; 59: 49–52. 19 Hashiguchi Y, Gomez EA, de Coronel VV, et al. Andean leishmaniasis in Ecuador caused by infection with Leishmania mexicana and L. major-like. Am J Trop Med Hyg 1991; 44: 205–217. 20 Davies CR, Reithinger R, Campbell-Lendrum D, et al. The epidemiology and control of leishmaniasis in Andean countries. Cad Saude Publica 2000; 16: 925–950. 21 Marovich MA, Lira R, Shepard M, et al. Leishmaniasis recidivans. Recurrence after 43 years: a clinical and immunologic report after successful treatment. Clin Infect Dis 2001; 33: 1076–1079.
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22 Spithill TW, Grumont RJ. Identification of species, strains and clones of Leishmania by characterization of kinetoplast DNA minicircles. Mol Biochem Parasitol 1984; 12: 217– 236. 23 Murray HW. Treatment of visceral leishmaniasis (kala azar): a decade of progress and future approaches. Int J Infect Dis 2000; 4: 158–177.
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24 Cannavo SP, Vaccaro M, Guarneri F. Leishmaniasis recidiva cutis. Int J Dermatol 2000; 39: 205–207. 25 Marsden PD. Mucosal leishmaniasis (“espundia” Escomel, 1911). Trans R Soc Trop Med Hyg 1986; 80: 859–876. 26 Weigel MM, Armijos RX, Zurita C, et al. Nutritional status and cutaneous leishmaniasis in rural Ecuadorian children. J Trop Pediatr 1995; 41: 22–28.
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