**4. Clinical state**

In several regions of the country, such as in the South and Southeast, intense environmental changes occurred due to anthropic action and agricultural and pastoral activities, which led to the near disappearance of cutaneous Leishmaniasis in the late 40s. However, from the 70s and 80s Leishmaniasis has reappeared in these regions, with a significant increase in the number of new cases arising from endemic areas [1]. Transmission classically was due to the bite of an insect, the so-called insect vector. This insect, also called a sandfly, belongs in the Old World to the genus Phlebotomus, and in the New World, to the genus Lutzomyia [4].

The first cases of ATL in America date from 1885 and in Brazil, the first report was in 1909. In the period 1985-1999, there were 388,155 auctothon cases in Brazil of ALT; from 1999 to 2003, 33,872 cases of ATL a year were registered [1]. In the period from 2000 to 2009, an average of 24,684 confirmed cases of Leishmaniasis was registered in Brazil the Information System for Notifiable Diseases (SINAN) [14]. In 2003, the regions with the highest prevalence of LTA were

In Brazil, 23,399 confirmed cases of ATL were notified in 2009, of which 94.1% were new cases and 4.6% relapses. With respect to clinical manifestations, 93.7% of patients had the cutaneous clinical form and 6.2%, clinical mucosa. Of all patients, in 2009, only 73.5% [17, 23] were cured, 16 patients died due to ATL, and 122 died from other causes, noting that 21.2% there was no

It is estimated that every year there are new cases in Brazil and the growth of this number is due in part to the emergence and growth of AIDS and deforestation areas [2]. It mostly affects young and adult males [16]. The greater number of cases of American Tegumentary Leish‐ maniasis among men and adults suggests extra-domiciliary transmission in the economically active population, while this occurrence among women, children and people with non-

The transmission of ATL in the Amazon presents a clear seasonal variation, it being more intense in the rainy season, when the temperature, solar radiation and evaporation are lower and humidity higher, thus favoring an increase in the density of the phlebotominae, including the species involved in the cycle of the disease [1]. In endemic areas, there may be significant

In the Americas, 11 dermotropic species of *Leishmania* which cause the disease in humans are currently recognized and 8 species have been described as affecting only animals. However, in Brazil, 7 species, there being 6 of the subgenus *Viannia* and 1 of the subge‐ nus *Leishmania,* have been identified. The three main species are: *Leishmania ( Leishmania) amazonensis* - distributed throughout the primary and secondary forests of the Amazon (Amazonas, Pará, Rondônia, Tocantins and the southwest of Maranhão southwest), particularly in *igapó* and forest areas of the "swamp-forest" type. Its presence extends to the regions of the Northeast (Bahia), Southeast (Minas Gerais and São Paulo) and Midwest

agricultural occupations suggests intra- and or peridomiciliary transmission.

the North (14,200 cases) and the Northeast (8,005 cases) [5].

48 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

information on the evolution of 21.2% of the cases [14].

percentages of children with the disease [16].

**3. Etiological agents and vectors**

Tegumentary Leishmaniasis is more common than the visceral disease and is characterized in its classical form by the presence of a well-bounded ulcer with raised edges. [17] ATL is an initial infection of the skin (its site of preferential location) from which it can undergo propa‐ gation or a secondary process which goes on to manifest itself in the mucosae of the upper airways [5]. The incubation period of the disease in humans is, on average, 2 months, it being possible for there to be shorter (two-week) periods and longer periods (of up to 2 years) [16].

The disease breaks out, in general, during the first five years that follow the appearance of the skin lesion, but may do so even a few decades after the primary cutaneous lesion, the scar from which can still be seen. However, in some patients the disease appears primarily in the mucosa membrane, without leaving traces on the skin. [2]. The most common manifestation is leishmaniotic ulcer: a single skin ulcer or only in small numbers, with raised edges, framed and the absence of local pain. Other morphological features can also be identified, such as: infiltrated plaque, tubercule, nodule and verrucous vegetating lesion. When the mucosa is injured, it can present an infiltrated erythema, granular or ulcerated aspect. In order of frequency, mucosal lesions are manifested mainly in the nose, hard palate, pharynx and larynx, which they can present themselves with an erythematous-infiltrated, granular, ulcerated or polypoid aspect with a roughly rounded surface [5].

Basically, it is possible to do the staging of the lesions that have occurred in the ATL by taking into consideration the time of onset, extent and spread of the lesion, and grouping them into: 1). Primary infections: which characterizes the primary accident (initial injury) or leishmaniotic cancer, found at the site of the bite, and after the incubation period (two weeks to one year), erythematous papules appear that progress to forming ulcers with serosanguinous crusts. 2) The onset of secondary Leishmaniasis ranges from one to three months after the primary infection, involving the skin, lymph nodes, lymphatic organs and mucosa and by contiguity, the mucous membranes of the nose, lips, eyelids and genitals are affected when the primary or secondary lesions settle near these regions. 3) Tertiary Leishmaniasis requires a longer period to appear, generally, five to ten years after the initial lesion and is characterized by the presence mainly of naso-oro-pharyngeal, laryngeal and ocular lesions, and it is in this period tertiary that the primary infection has already disappeared and the secondary one, in general, is still present [5]. The clinical presentation exhibits polymorphism and the spectrum of severity of the signs and symptoms is also variable, although there is a certain correspondence between the different clinical presentations and the different species of the parasite [7].

Mucosal Leishmaniasis appears under the following clinical forms: 1) Late –this is the most common form. Classically, it is associated with multiple or long-lasting skin lesions, sponta‐ neous cures or insufficient CL treatments; 2) Of undetermined origin - when the ML is clinically isolated, it not being possible to detect any other evidence of prior CL; 3) Concomitant - when the mucosal lesion occurs at a distance, but at the same time as the active skin lesion (not contiguous to the natural orifices); 4) Contiguous – this occurs by direct propagation of the skin lesion, located next to natural orifices, to the mucosa of the aerodigestive tracts. The skin lesion may be in activity or healed at the time of diagnosis; 5) Primary – this occurs, possibly, by the bite of the vector in the mucosa or semimucosa of lips and genitals [7.8].

It is believed that untreated mucosal lesions are progressive, there being few reports of spontaneous cicatrizations of these lesions which, even if treated, may leave permanent sequelae [22]. There are several hypotheses that attempt to explain the predilection of the nasal mucosa: direct contact of the hand with the skin lesions and scratching one´s nose afterwards, the epithelium of the anterior part of the nasal cavities offer conditions to the location of Leishmanias and the lower temperature in the anterior area of the nasal septum, due to the presence of a current of inspiratory air. The transition zone between the squamous epithelia and the pseudostratified vibrating, in the anterior part of the nasal septum and the lower turbinate head, is the "*locus minoris resistentia*" to the Leishmaniasis process. However, the most consistent hypothesis says that *Leishmania* requires lower temperatures for its growth. Thus, since the anterior area of the nasal septum is cooler due to the inspiratory airflow, there would be a predilection for proliferation of the parasites [13]. The association of low temperature with Leishmaniasis may, in part, be explained by the documentation *in vitro* that macrophages grown at 29°C are less able to destroy *Leishmania* than macrophages cultured at 33°C [3].

which can still be seen. However, in some patients the disease appears primarily in the mucosa membrane, without leaving traces on the skin. [2]. The most common manifestation is leishmaniotic ulcer: a single skin ulcer or only in small numbers, with raised edges, framed and the absence of local pain. Other morphological features can also be identified, such as: infiltrated plaque, tubercule, nodule and verrucous vegetating lesion. When the mucosa is injured, it can present an infiltrated erythema, granular or ulcerated aspect. In order of frequency, mucosal lesions are manifested mainly in the nose, hard palate, pharynx and larynx, which they can present themselves with an erythematous-infiltrated, granular, ulcerated or

Basically, it is possible to do the staging of the lesions that have occurred in the ATL by taking into consideration the time of onset, extent and spread of the lesion, and grouping them into: 1). Primary infections: which characterizes the primary accident (initial injury) or leishmaniotic cancer, found at the site of the bite, and after the incubation period (two weeks to one year), erythematous papules appear that progress to forming ulcers with serosanguinous crusts. 2) The onset of secondary Leishmaniasis ranges from one to three months after the primary infection, involving the skin, lymph nodes, lymphatic organs and mucosa and by contiguity, the mucous membranes of the nose, lips, eyelids and genitals are affected when the primary or secondary lesions settle near these regions. 3) Tertiary Leishmaniasis requires a longer period to appear, generally, five to ten years after the initial lesion and is characterized by the presence mainly of naso-oro-pharyngeal, laryngeal and ocular lesions, and it is in this period tertiary that the primary infection has already disappeared and the secondary one, in general, is still present [5]. The clinical presentation exhibits polymorphism and the spectrum of severity of the signs and symptoms is also variable, although there is a certain correspondence between the different clinical presentations and the different species of the parasite [7].

Mucosal Leishmaniasis appears under the following clinical forms: 1) Late –this is the most common form. Classically, it is associated with multiple or long-lasting skin lesions, sponta‐ neous cures or insufficient CL treatments; 2) Of undetermined origin - when the ML is clinically isolated, it not being possible to detect any other evidence of prior CL; 3) Concomitant - when the mucosal lesion occurs at a distance, but at the same time as the active skin lesion (not contiguous to the natural orifices); 4) Contiguous – this occurs by direct propagation of the skin lesion, located next to natural orifices, to the mucosa of the aerodigestive tracts. The skin lesion may be in activity or healed at the time of diagnosis; 5) Primary – this occurs, possibly,

It is believed that untreated mucosal lesions are progressive, there being few reports of spontaneous cicatrizations of these lesions which, even if treated, may leave permanent sequelae [22]. There are several hypotheses that attempt to explain the predilection of the nasal mucosa: direct contact of the hand with the skin lesions and scratching one´s nose afterwards, the epithelium of the anterior part of the nasal cavities offer conditions to the location of Leishmanias and the lower temperature in the anterior area of the nasal septum, due to the presence of a current of inspiratory air. The transition zone between the squamous epithelia and the pseudostratified vibrating, in the anterior part of the nasal septum and the lower turbinate head, is the "*locus minoris resistentia*" to the Leishmaniasis process. However, the most

by the bite of the vector in the mucosa or semimucosa of lips and genitals [7.8].

polypoid aspect with a roughly rounded surface [5].

50 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

It occurs more often in males and at age bands usually higher than CL, which is probably due to its character of secondary complication [8]. The involvement of the nasal and/or mucosa is usually more severe and thus may cause sequelae and death [20].

The initial lesion is characterized by a whitish nodulation without ulceration which is usually observed in the cartaliginous septum (Kiesselbach's area), the floor and side wall, specifically on the head of the inferior turbinate. This impairment classified as stage I of the disease, represents a very early stage of inflammation and does not resemble, from the clinical standpoint, any other nasal disease. Subsequently, a fine granular lesion appears, character‐ ized by superficial ulceration, documented at the anterior septum, inferior turbinates and floor of the nasal cavity (Stage II) [17]. At first, there is hyperemia and edema of the mucosa of the anterior septum, with the establishment of nodulations [3].

In Stage III, or the stage of deep ulceration, tissue reaction is more intense, with aberrant granulation tissue and infiltration of the mucosa, thus widening the nasal septum. There is sometimes edematous infiltration of the nasal pyramid itself. In this phase, hematic crusts can be observed on the septum, the inferior turbinates and the floor of the nasal cavities. These lesions are characterized by excessive fragility, as they bleed very easily when the mucosa is touched.

Clinically, the patient may complain of soreness at the level of the nasal pyramid, sanguinolent rhinorrhea and emission of hematic crusts. Nasal obstruction is a frequent symptom in this phase of the disease. From the external point of view, due to the inflammatory process that involves the cartilages and subcutaneous cell tissue and the very skin of the base of the nasal pyramid, the nose takes on the aspect known as tapir-nose.

Stage IV of the disease is characterized by the cartilaginous involvement of the anterior septum with necrosis and, sometimes, impairment of the columella. It is at this stage that perforation of the cartilaginous septum is established, also with marked infiltration of the posterior septum. In more advanced forms (Stage V), total destruction of the columella may occur and may drop, thus transforming the nasal cavity into a similar cloaca and some‐ times there is perforation of the dorsum of the nasal pyramid [17]. For some researchers, the specific destruction of the nasal cartilage could also indicate an autoimmune reaction that would explain why some patients undergo severe tissue destruction and others only present mucosal involvement years later [21]. In some cases there may be total destruc‐ tion of the anterior septum, only the entire columella remaining, with the nose being sealed. Extensive crusts of a hemorrhagic aspect or even resulting from the drying of mucous secretion caused by enlargement of the internal nasal structure can be observed as a consequence of the tissue injury, represented by the destruction of the cartilaginous septum and inferior turbinates. On this occasion, there is elimination of sanguinolent discharge and the presence of crusts is accentuated [17]. The patient's death usually occurs because of aspiration or respiratory failure [5].

The earliest signs and symptoms of mucosal Leishmaniasis are nasal obstruction, epistaxis and the establishment of granulomas in the anterior nasal septum. As the disease evolves, patients begin to present a leishmaniotic facies known as "tapir nose" due to edematous infiltration of the lining and supporting structures of the nose [3]. Lesions reach the cartilaginous nasal septum and may extend to the lateral wall and floor, the region of the palate, uvula, and less frequently, involvement of the pharynx, larynx, vocal cords and upper lip occur with varying degrees of infiltration, granulation and ulceration [17]. The infiltration of the soft palate reaches the proportions of a real tumor. The whole palate is changed: the uvula is reduced to a shapeless mass, with an irregular, vegetating surface. In the palatal vault, lobed prominences are formed, separating themselves by sinuous furrows and ulcerated erosions.

More rarely it can involve the gum and dental interstices, where voluminous and prominent granulations develop, and reach the upper lip. The tongue is usually spared [5]. The manifes‐ tations of the mucosal diseases include involvement of pillars and uvula with an increase in volume, hyperemia, roughness and superficial ulcers [12]. The pharynx is the second site of involvement when mucosal lesions caused by *L. braziliensis* set in. As in the nose, the lesion initially observed at the level of the mucosa of the pharynx takes on a lumpy aspect; however, here, there is a much more intense edematous infiltration, especially of the uvula and secon‐ darily of the tonsillar pillars, extending also to the mucosa of the posterior pharyngeal wall.

The appearance can be observed at this stage of granulation tissue that is a little redundant intermingled with the lumpy aspect of the mucosa. The next stage is characterized by the appearance of abundant granulation tissue, which causes important tissue destruction, also involving the lymphoid tissue of Waldeyer's lymphatic ring at the level of tonsils. Areas with a tenuous fibrin layer mix in with the granulation tissue of a vegetating aspect.

Because of the intense tissue aggression, in the specific post-treatment healing process, the presence of abundant fibrous tissue, with the formation of true whitish cords can be docu‐ mented. These completely deform the configuration of the anatomical structures of the velum of the palate and the posterior wall of the pharynx, leading to full stenosis in the communica‐ tion of the oropharynx with the nasopharynx [3]. In the mouth, the hard palate is often involved, with dissemination of the process to the soft palate, uvula and pharynx. The proliferating infiltrative process can cause fusion of the uvula, pillars, lateral cords and posterior wall, causing obliteration of the nasopharynx. Deformity and narrowing of the lumen of the oropharynx may occur due to fibrosis of the tonsils [5].

Laryngeal mucosa is the 3rd site of election when mucosal Leishmaniasis sets in. As in the pharynx, the mucosal lesions present the same characteristics of finely granular tissue. There may be in situations of greater inflammation, the presence of granulation tissue with a vegetating aspect, covered at times with a tenuous fibrin layer, involving the mucosa that covers the cartilage of the epiglottis, extending to the mucosa of the structures of the laryngeal vestibule and vocal folds. At this stage, dysphonia characterized by a muffled voice is always present, which draws attention to the impairment of the organ [3]. Pharyngolaryngeal involvement can be intense, to the point of causing dysphagia, dyspnea, dysphonia, odyno‐ phagia, and coughing [15]. The hypopharynx, larynx, epiglottis, arytenoid cartilages and the posterior commissure of the vocal cords are covered by a lesion with a vegetating aspect, which sometimes come to join up. These granulations often regress and eventually disappear, making the surface affected by a smooth and slightly whitish coloration.

the presence of crusts is accentuated [17]. The patient's death usually occurs because of

The earliest signs and symptoms of mucosal Leishmaniasis are nasal obstruction, epistaxis and the establishment of granulomas in the anterior nasal septum. As the disease evolves, patients begin to present a leishmaniotic facies known as "tapir nose" due to edematous infiltration of the lining and supporting structures of the nose [3]. Lesions reach the cartilaginous nasal septum and may extend to the lateral wall and floor, the region of the palate, uvula, and less frequently, involvement of the pharynx, larynx, vocal cords and upper lip occur with varying degrees of infiltration, granulation and ulceration [17]. The infiltration of the soft palate reaches the proportions of a real tumor. The whole palate is changed: the uvula is reduced to a shapeless mass, with an irregular, vegetating surface. In the palatal vault, lobed prominences are formed,

More rarely it can involve the gum and dental interstices, where voluminous and prominent granulations develop, and reach the upper lip. The tongue is usually spared [5]. The manifes‐ tations of the mucosal diseases include involvement of pillars and uvula with an increase in volume, hyperemia, roughness and superficial ulcers [12]. The pharynx is the second site of involvement when mucosal lesions caused by *L. braziliensis* set in. As in the nose, the lesion initially observed at the level of the mucosa of the pharynx takes on a lumpy aspect; however, here, there is a much more intense edematous infiltration, especially of the uvula and secon‐ darily of the tonsillar pillars, extending also to the mucosa of the posterior pharyngeal wall.

The appearance can be observed at this stage of granulation tissue that is a little redundant intermingled with the lumpy aspect of the mucosa. The next stage is characterized by the appearance of abundant granulation tissue, which causes important tissue destruction, also involving the lymphoid tissue of Waldeyer's lymphatic ring at the level of tonsils. Areas with

Because of the intense tissue aggression, in the specific post-treatment healing process, the presence of abundant fibrous tissue, with the formation of true whitish cords can be docu‐ mented. These completely deform the configuration of the anatomical structures of the velum of the palate and the posterior wall of the pharynx, leading to full stenosis in the communica‐ tion of the oropharynx with the nasopharynx [3]. In the mouth, the hard palate is often involved, with dissemination of the process to the soft palate, uvula and pharynx. The proliferating infiltrative process can cause fusion of the uvula, pillars, lateral cords and posterior wall, causing obliteration of the nasopharynx. Deformity and narrowing of the lumen

Laryngeal mucosa is the 3rd site of election when mucosal Leishmaniasis sets in. As in the pharynx, the mucosal lesions present the same characteristics of finely granular tissue. There may be in situations of greater inflammation, the presence of granulation tissue with a vegetating aspect, covered at times with a tenuous fibrin layer, involving the mucosa that covers the cartilage of the epiglottis, extending to the mucosa of the structures of the laryngeal vestibule and vocal folds. At this stage, dysphonia characterized by a muffled voice is always present, which draws attention to the impairment of the organ [3]. Pharyngolaryngeal

a tenuous fibrin layer mix in with the granulation tissue of a vegetating aspect.

of the oropharynx may occur due to fibrosis of the tonsils [5].

separating themselves by sinuous furrows and ulcerated erosions.

aspiration or respiratory failure [5].

52 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

There is generalized laryngeal inflammation particularly in the piriform sinuses. The vocal folds appear to be moving well, but phonation is weak and the muscular control of tension can be harmed by granulomatous formation and subsequent fibrosis. Even after successful treatment, the voice rarely returns to normal and the lumen of the larynx may be reduced [5]. Painful dysphagia in degrees of greater or lesser intensity prevents the normal feeding of the individual, with consequent impairment of general condition and, in very advanced cases, becoming cachexia. In post-treatment healing, the deformities that these cartilages present are very evident, such as fibrous tissue which is also whitish, thereby completely modifying the anatomy of the organ, except for a residual permanent dysphonia [3].

Complications include pneumonia due to aspiration, bacterial infections, secondary myiasis, cachexia due to difficulties in swallowing, laryngeal edema and asphyxiation, which may be lead on to the patient's death mainly due to respiratory failure and sepsis [5]. The presentation of the clinical form with lesions exclusive of mucosa of the larynx and trachea is relatively uncommon [2]. The ear is not usually affected in mucosal Leishmaniasis. However, the involvement of the mucosa of the nasopharynx leads to impairment of the pharyngeal orifice of the Eustachian tube, situated on its sidewall. A process of otitis media with effusion (secretory otitis media chronic) can be established in these cases.

The sensation of blocked ear, tinnitus and hearing loss are complaints in these cases [3]. Morbidity of the skin and ear cartilage occurs because it is a place of lower temperature, apt for the growth of *Leishmania*, besides being an area exposed to the inoculation of the vectors. The external ear commonly presents an increase in volume, ulcers with raised edges, some‐ times covered with crusts, and may appear as an infiltrated plate, tubercule, vegetating warty nodule and lesion, on course in the end to mutilating the ear [5].

Mucosal Leishmaniasis can compromise the labial mucosa and gingival margin. This is a rarer manifestation of the disease [3]. The lesion in some individuals heals early, sometimes without seeking medical attention. Others remain for months with the lesion in activity and the healing process is slow. This phenomenon can be explained by the rapid or late establishment of a specific immune response which is effective in eliminating the parasite.

The cure of Leishmaniasis is not sterile. It has been possible to isolate viable parasites of ALT scars in individuals who have been cured for several years, a fact confirmed in experimental studies using an animal model. This phenomenon could thus explain the appearance of late relapses as well as the onset of the disease in immunocompromised patients, such as AIDS (Acquired Immunodeficiency Syndrome) [7].

### **5. Diagnosis**

It is very hard to detect Leishmaniasis in the initial stage [15]. The long interval between the onset of symptoms and etiological diagnosis of the mucosal form of ATL may reflect the limitation of the training of most physicians in the proper approach to mucosal Leishmaniasis [2]. A laryngoscopy exam usually demonstrates an extensive inflammatory component, with erythema and edemas evident. The granulomatous aspect associated with the presence of ulcers is common, and may present purulent exudate. In the advanced disease, tissue destruc‐ tion can be striking. As a protocol of etiological investigation on suspicion of granulomatous bodies that are difficult to access such as the larynx, laboratory tests and imaging should be requested, and should a diagnostic uncertainty be maintained, a biopsy of the lesions is recommended for histological study. If the appearance of the lesion suggests malignant neoplasm, research using noninvasive and invasive tests should occur simultaneously so as not to delay diagnosis [15].

The ENT examination associated with the Montenegro test remains the most important element for diagnosis, although it is usually of a presumptive character [20]. The encounter of *Leishmania* is the gold standard for the diagnosis of ATL [21]. The diagnosis can be confirmed by various tests: 1) Direct investigation of the parasite, which can be done by scraping the ulcerated surface or by compression of the slide on the wounded area of the lesion. The material is stained with Giemsa or Wright [2]. The direct parasitological examination is the procedure of first choice because it is faster, less expensive and easy to perform [16]. It gives good results in initial lesions, without associated bacterial infection [2], 2) Montenegro intradermoreaction: This translates the response of cell delayed hypersensitivity [16]. It consists of intradermal injection of 0.1 ml of antigen prepared from *Leishmania* promastigotes, with a reading after 48 hours. The test is considered positive that produces an induration of 5 mm or more. However, the positivity of the test indicates that the person has already been sensitized but is not necessarily a carrier of the disease [2]; 3) Histopathological examination of the tegumentary lesion [2]. The Biopsy can be performed with a "punch" of 4 mm in diameter, or a wedge, with the use of a scalpel. In ulcerated lesions, the whole edge of the whole lesion should be preferred, This, in general, shows a tumified and hyperemic aspect [16]; 4) Serology (indirect immuno‐ fluorescence or ELISA); they have good sensitivity but can give a reaction crossed with Chagas disease and visceral Leishmaniasis, this being the cause of false-positive results, thus reducing its specificity [2].

The most commonly used techniques for antibodies are: indirect immunofluorescence (IIF), counterimmunoelectrophoresis (CIE ), ELISA and Western blot. The Western blot technique has a superior sensitivity to the other serologic techniques [70.6%), a sensibility of 70.3% and a precision of 72.7%. In the immunocompetents, the specificity and sensitivity are 100% [4]; 5) Immunohistochemical techniques (immunostaining with anti *Leishmania* antibodies); they permit evidence of the parasite in histological sections; 6) Method of culture: culture takes place in Novy-MacNeal-Nicolle medium from the biopsy or aspirate [4]. They are not practical for diagnosis, especially of *Leishmania brasiliensis*, since it does not grow easily in culture media; in addition, bacterial or fungal contamination often complicate this procedure [2]. Research can be done into *Leishmania* in other affected organs such as the spleen, liver and lymph node, and whenever there is a suspected diagnosis, into the pleural fluid, bronchoalveolar lavage, intestinal biopsy, skin, etc. Hepatic and spleen biopsies are used as a last resort due to the increased risk of potentially serious complications such as hemorrhaging [4].

Cases are confirmed according to the following criteria: 1) Residence, arrival in or moving away from the area with confirmation of transmission and presence of the parasite in direct and/or indirect parasitological exams; 2) Residence, arrival in or moving away from the area with confirmation of transmission and intradermoreaction of Montenegro (MRI) positive, 3) Residence, arrival in or moving away from the area with confirmation of transmission with other methods of positive diagnosis [19].
