Introduction

Leishmaniasis is a neglected tropical disease caused by protozoa of the Leishmania genus and has two main clinical forms: tegumentary leishmaniasis (TL), which can be cutaneous and/or mucousal; and visceral leishmaniasis (VL).¹

Between 2007 and 2015, 6,801 cases of cutaneous leishmaniasis (CL) were registered in the state of Amapá.² CL can progress to severe although not fatal forms. Notwithstanding, between 2017 and 2018, the Amapá State Health Surveillance Superintendency recorded four deaths of patients with CL being treated with Glucantime^®3 (meglumine antimoniate) and 19 cases of canine VL.⁴ Also in 2018 the first autochthonous case of human VL in the state was confirmed: a riverside dweller aged 82 who was infected in the municipality of Mazagão and died from complications caused by cirrhosis of the liver.⁴

Surveillance of VL, which is caused by the etiological agent Leishmania infantum, favors early diagnosis and treatment, thus reducing risk of death. Nevertheless, it is uncommon for people with CL to die and death usually results from comorbidities exacerbated by the toxicity of Glucantime^®.⁵ Among the agents causing CL, Leishmania braziliensis and, principally, Leishmania guyanensis, show resistance to Glucantime^®, the prolonged use of which increases the risk of adverse events.⁶L. guyanensis occurs in the North of Brazil^1,7,8 but its frequency and distribution in Amapá are unknown.

The objective of this study was to investigate Leishmania species in a series of autochthonous cutaneous leishmaniasis cases in Amapá in 2018.

Methods

We conducted a descriptive study of a series of CL cases. The Brazilian state of Amapá has 670,000 inhabitants, distributed over 16 municipalities.9 One side of the state faces the Atlantic Ocean, while to the North it borders with Suriname and French Guiana, and to the south it borders with the Brazilian state of Pará. In 2018, the CL detection coefficient in Amapá was 68.7/100,000 inhabitants, with autochthonous cases in 14 of the state’s 16 municipalities.³ Phlebotomine insects, which are potential CL vectors, are abundant in this Amazon subregion,^8,10,11 where CL is endemic.^2,3

In order to collect data, we recruited individuals attending the Amapá Tropical Diseases Reference Center between January and May 2018. The inclusion criteria were: males and females over 12 years old, with primary lesions suggestive of CL for at least two weeks. Those with medical contraindication to anesthesia and/or biopsy or with some other laboratory diagnosis were excluded.

The Leishmania species identified in the study were assessed in relation to the following variables:

Leishmania variable measurement and diagnosis were based on leishmaniasis surveillance criteria defined in the Ministry of Health technical manual,1 as well as being based on molecular techniques.^12,13

Lesion exudates were used to confirm infection, obtained via swabs for smearing on glass slides, using Giemsa stain to examine for amastigotes under the microscope.¹Leishmania were characterized using DNA extracted from the skin (phenol-chloroform). Skin was collected via lesion biopsy using local anesthesia (2% lidocaine). The sample was kept in a flask containing 0.5mL of NET (NaCl 0.15mM; EDTA 50mM; Tris-HCl 0.1M/pH 7.5). In order to ensure the quality of the extracted skin/DNA samples, refrigerator temperature was controlled daily (2-8^oC). Polymerase chain reaction (PCR) was performed with subsequent sequencing of the 234 base pair target, i.e. Leishmania DNA heat shock-protein 70 gene coding region (hsp70-234).¹²

The following assays were performed:

We used BioStat 5.0 to produce frequency tables and to calculate means and standard deviations (available at: www.mamiraua.org.br).

The study project was approved by the Federal University of Amapá Research Ethics Committee (Certification of Submission for Ethical Appraisal [CAAE] No. 57036716.0.0000.0003, approved on 22/08/2016) and by the Evandro Chagas Institute Research Ethics Committee (CAAE No. 57036716.0.3001.0019, approved on 03/10/2016).

Results

Thirty-eight individuals with CL were included. They were 28 males and 10 females aged between 13 and 81 with a mean age of 34.4 years (standard deviation: 14.2), resident in ten municipalities in the state of Amapá. The largest number of cases (n=13) came from the municipality of Mazagão. The most frequent etiological agent among the participants was Leishmania guyanensis (32/38). Clinical/demographic aspects and etiology of the CL cases are shown in Table 1.

With regard to the number of lesions, 20 individuals had multiple lesions while 18 had a single lesion. In 37/38 cases, lesions were of the ulcer type, mostly with a 5-6cm diameter, predominantly located on the upper limbs (n=9) and lower limbs (n=11). Predominant clinical manifestations were crust, secondary infection and satellite lesions. The characteristics of the lesions of the individuals with CL and respective etiological agents identified following sequencing and analysis of the Leishmania hsp70-234 gene region are described in Table 2.

This study contains the first report of L. infantum associated with CL in Amapá: a 35year-old woman infected in the municipality of Porto Grande, with multiple varied size verrucous cutaneous lesions on her body, including her thighs and legs, without visceral disease

Discussion

The most frequent etiological agent among the five species identified in individuals with CL in Amapá was L. guyanensis: 32 of the 38 cases analyzed. The hsp70-234 Leishmania marker discriminates six pathogens in the Amazon.12,14 The specificity of hsp70-234 has not been previously evaluated for L. lindenbergi, the seventh pathogen that causes human CL in the Amazon.8 Some species identified in this study are worthy of note: (i) Leishmania amazonensis, in addition to localized CL it can cause diffuse (anergic) leishmaniasis which is rarely curable; and (ii) Leishmania guyanensis and L. braziliensis, which induce cutaneous/mucous lesions that are hard to deal with,1,15 having a high degree of hypersensitivity and which may appear months or years after clinical cure of the primary lesion.1 Only long-term medical follow-up will ensure effective diagnosis and treatment of relapses.15 Even in the absence of mucous involvement, knowing what the pathogen is enables a prognosis to be defined.

Those who had CL caused by L. braziliensis and L. naiffi had a single ulcer. Leishmania braziliensis is widely distributed in Brazil.¹L. naiffi infection in humans is limited to the states of Amazonas, Acre, Pará, Rondônia and Mato Grosso,¹⁶ with no complications having been reported until our study revealed the first case of CL caused by L. naiffi in Amapá.

L. infantum is phylogenetically close to Leishmania donovani, the agent that causes VL and post-kala-azar dermal leishmaniasis in the Old World (Europe);¹⁷ occurrence of cutaneous lesions caused by L. infantum in immunocompetent individuals is uncommon in the Americas, especially in Brazil.^14,18

L. infantum has been isolated in the skin of Africans with CL, producing a lesion histologically distinct from that caused by L. major.¹⁹ Since 2015, cases of CL caused by L. infantum have been recorded in different Brazilian states.^14,18 In Africa and the Americas, the clinical characteristics of CL vary according to the etiological agent.^1,19 Our study highlights the pleomorphism of CL caused by L. infantum, alerting as to the need for early diagnosis²⁰ and, in this sense, the importance of surveillance for preventing also human VL in Amapá.

CL hotspots have been identified in Amapá, principally in areas of agricultural, plant extractivism and prospecting/mining settlements,² where males of productive age are most affected. The majority of the individuals we analyzed had multiple primary lesions, suggesting diverse vector bites. This is a recognized behavior of Nyssomyia umbratilis, the agent that transmits L. guyanensis.^1,8 In these cases, prolonged treatment with Glucantime^® would be necessary; however, there is considerable morbidity and even deaths associated with the toxicity of this chemotherapy.^3,5,21,22

Although Glucantime^® is the first drug option for treating CL in Brazil, Pentamidine Isethionate^® is recommended where L. guyanensis is predominant.¹ Study participants infected with L. guyanensis came from nine of the ten municipalities considered in the sample, suggesting widespread distribution of this species in Amapá, where vector fauna are abundant.^8,10,11

L. braziliensis and L. guyanensis infection caused by Leishmania RNA virus subtype 1, found in Peru, Bolivia, French Guiana, Suriname and Brazil, would explain the high virulence and resistance of these species to the drug.²³ Biological factors of CL need to be monitored, especially in frontier regions.

With regard to treatment, with effect from 2017 Pentamidine Isethionate^® has been considered preferential to Glucantime® for systemic treatment of CL in areas where L. guyanensis is predominant in Brazil. In cases of (i) kidney, heart and liver failure, (ii) people who have had kidney, heart and liver transplants, (iii) pregnant women and people more than 50 years old, the use of liposomal Amphotericin B® is recommended. Glucantime® would be the first option only for intralesional use in these areas.¹ The results found by our study support the new guidelines for treatment of CL in Amapá adopted with effect from 2018.²⁴

Our study confirmed the predominance of L. guyanensis infection in Amapá, frequently associated with multiple lesions, as was as providing the first reports of the presence of Leishmania naiffi and L. infantum in clinical samples of CL in the state of Amapá.