Study on the Zoonotic Cycle of Tegumentary Leishmaniasis in Northeastern Brazil

Rev Inst Med Trop São Paulo 202264e60 Page 1 of 8 ORIGINAL ARTICLE httpdoiorg101590S16789946202264060 This is an openaccess article distributed under the terms of the Creative Commons Attribution License 1Centro de Vigilância em Saúde Ambiental Moreno Pernambuco Brazil 2Universidade Fernando Pessoa Faculdade de Ciência e Tecnologia Porto Portugal 3Fundação Oswaldo Cruz Instituto Aggeu Magalhães Recife Pernambuco Brazil 4Universidade Fernando Pessoa Unidade de Pesquisa em Energia Saúde e Ambiente Porto Portugal 5Universidade de Pernambuco Faculdade de Ciências Médicas Hospital Universitário Oswaldo Cruz Departmento de Dermatologia Recife Pernambuco Brazil Correspondence to Maria Edileuza Felinto de Brito Fundação Oswaldo Cruz Instituto Aggeu Magalhães Av Prof Moraes Rego sn Cidade Universitária CEP 50740465 Recife PE Brazil Email edileuzabritofiocruzbr Received 11 February 2022 Accepted 26 July 2022 Study on the zoonotic cycle of tegumentary leishmaniasis in an endemic area of a metropolitan region in the Northeastern region of Brazil Cláudio Júlio da Silva12 Juliana Figueirêdo da Costa Lima Suassuna Monteiro3 Karina Patrícia Baracho de Lima3 Cláudia Sofia de Assunção Gonçalves e Silva24 Éricka Lima de Almeida3 Samara Ferreira de Souza3 Ângela Cristina Rapela Medeiros5 Felipe Marinho Rocha de Macedo5 Sinval Pinto BrandãoFilho3 Stephane Naiara Carvalho dos Santos3 Maria Edileuza Felinto de Brito3 ABSTRACT This study was conducted to characterize the transmission cycle of the tegumentary leishmaniasis TL in an old colonization area at Pernambuco State Brazil The aims were to identify autochthonous cases sandflies fauna domestic animals as possible reservoir hosts and the Leishmania species involved in this endemic area A total of 168 suspected human cases of TL and 272 domestic animals canine feline equine goat and sheep were included The sandflies were captured and identified by species Patients were predominantly male and the average age was 37181 years old Of 85 patients who had skin lesions 256 of them had direct positive smears for TL and 34 isolates were identified as Leishmania Viannia braziliensis The confirmation for TL diagnosed by molecular detection PCR was almost three times more sensitive than the direct test p 0001 PR 272 associated with clinical examination The Kappa test on PCR between two different specimens biopsy and skin lesion swab was 608 p 0001 More than 200 specimens of sandflies 80 males and 159 females were captured and identified as Lutzomyia whitmani 996 and Lu evandroi 04 The detection of L V braziliensis by RealTime PCR in the blood of a captured fed female was positive in 593 of Lu whitmani Of the 272 domestic animals included 6176 were male n 168 Thirtysix animals 132 had lesions compatible with TL 34 dogs 1 cat and 1 sheep and 3 of them all dogs had lesions on the snout showing destruction of cartilage and mucosa The study suggests the participation of domestic animals as possible reservoirs However further studies are necessary to better understand the transmission cycle and take recommended measures in order to control the disease KEYWORDS Transmission cycle Ecoepidemiology Tegumentary leishmaniasis Leishmania Viannia braziliensis INTRODUCTION Tegumentary leishmaniasis TL is a zoonotic parasitic disease disseminated in the Old and New Worlds endemic in 92 countries with an estimate of one million new cases per year12 In Brazil there were 15484 new cases in 20193 In the Americas TL is transmitted through the bite of female phlebotomine sandflies of the Lutzomyia genus The clinical forms of the disease are cutaneous localized Silva et al Rev Inst Med Trop São Paulo 202264e60 Page 2 of 8 disseminated diffuse recidivans cutis and mucocutaneous and in some cases it may evolve to spontaneous healing except for the diffuse form14 Between 2007 and 2017 235301 cases were reported in all regions of Brazil and the North and Northeastern regions were the most affected with 101332 and 72395 cases respectively5 In the Northeast the predominance of cases occurs in locations with different agricultural plantations some remnants of the Atlantic rainforest and secondary vegetation whose aspects contribute to the spread of phlebotomines and the enzootic maintenance cycle with the involvement of wild and synanthropic reservoir hosts6 The modification of the natural environment caused by anthropic action leads to the adaptation of vectors and reservoirs with the zoonotic disease cycle in the peridomicile areas affecting both humans and domestic animals78 In Brazil there are seven different predominant species of Leishmania and Leishmania Viannia braziliensis is the predominant species This species causes the most severe form of the disease which can compromise the patients mucosa and cartilage It is also the predominant etiologic agent of the disease in Pernambuco PE4689 The clinical diagnosis is based on anamnesis of the patient with clinical suspicion epidemiological data individual residing or transiting in an endemic area and laboratory tests46 The gold standard method for the laboratory diagnosis of TL is the direct parasitological examination by microscopy on a slide with impression of the biopsy or scraping of the lesion6 Molecular tests such as PCR and RealTime PCR qPCR are very important in diagnosis because of their high sensitivity and specificity10 The aim of this study was to characterize the zoonotic cycle through the description of several aspects of the eco epidemiology of TL in an endemic area of the Northeast of Brazil It also aimed to identify autochthonous cases characterize phlebotomine fauna and domestic animals as possible involved reservoir hosts MATERIALS AND METHODS A crosssectional study was conducted in the Moreno municipality Metropolitan Region of Recife PE Brazil in the Atlantic Forest area located at an altitude of 96 meters with a rainy tropical climate and an average annual rainfall of 13099 mm and approximately 63000 inhabitants where 17 are in urban households11 The majority of the studied region is covered by a monoculture of sugar cane or banana plantations The sample of autochthonous human cases included residents in endemic locations who sought the primary health care provided by Programa Saude da Familia PSF Family Health Program between 2015 and 2018 After the anamnesis data were collected for clinical and epidemiological diagnosis and clinical samples by biopsy or lesion scraping The samples were analysed for diagnostic confirmation at the Laboratory of Immunoparasitology of Aggeu Magalhaes InstituteFiocruzPE Case suspect individuals with compatible clinical signs such as the presence of a cutaneous ulcer with a granular background and infiltrated edges from an endemic area or someone who had visited it Case definition individuals with compatible clinical signs epidemiological data and positive parasitological or molecular tests The parasitological diagnosis was performed by visualizing the parasite in the smear through an optical microscope6 and the lesion fragments or scrapings obtained were inoculated in culture medium and in hamsters Mesocricetus auratus for the in vivo isolation of Leishmania spp8 The identification and characterization of the parasite was previously carried out through the analysis of the electrophoretic profile with isoenzymes MLEE812 Leishmania Collection of the Oswaldo Cruz Institute FIOCRUZ Rio de Janeiro CLIOCRJ The samples of domestic animals residing in Engenho Jardim and Engenho Cumaru Moreno included canines felines horses goats and sheep examined by a veterinarian from the Municipal Health Surveillance Center between 2017 and 2018 Blood and swab samples from ocular conjunctiva were collected and for those who had active lesion scarification puncture or swab of skin lesion was performed The sandflies were captured between January 2017 and December 2018 inside the houses where human or suspected cases of TL were notified For these captures the light traps model CDC Centers for Disease Control was installed in areas of banana plantations and in the remnants of the Atlantic Forest from the twilight period onwards13 The specimens were separated by gender and morphological identification according to Young and Duncan14 and stored in ethanol 70 at room temperature until the DNA extraction Each entire female body specimen was submitted for molecular testing by qPCR to detect natural infection For the molecular tests DNA extraction and purification from human biological samples were performed using the commercial kit QIamp DNA mini kit Qiagen Valencia USA according to the protocol of the manufacturer The extraction of DNA was performed individually according to Solano et al15 In order to confirm and verify the extraction efficiency some random samples had DNA quantified on the NanoDrop spectrophotometer Themo Scientific model 20002000c Waltham Massachusetts USA Rev Inst Med Trop São Paulo 202264e60 Study on the zoonotic cycle of tegumentary leishmaniasis in an endemic area of a metropolitan region in the Northeastern region of Brazil Page 3 of 8 The DNA from human samples and from the reference strain CLIOC IOCL0566 MHOMBR75M2903 of L V braziliensis was amplified by PCR using oligonucleotides B1B2 target of Leishmania kDNA of the Viannia subgenus performed according to de Bruijn and Barker16 Visualization of the amplification products was done by electrophoresis in 1 agarose gel stained with ethidium bromide17 Visualization of the DNA bands occurred in a photo documentation system for electrophoresis gel LPIX TouchLoccus Cotia SP Brazil For molecular diagnosis in domestic animals DNA from blood samples swab from lesion secretion and from ocular conjunctiva were amplified by qPCR as well as for the phlebotomine The kDNA fragments of the subgenus Viannia used as qPCR target had 138 base pairs and were amplified using a reference thermocycler QuantiStudio 5 Applied Biosystems Waltham Massachusetts USA using Software 7500 version 205 Applied Biosystems The qPCR reactions followed the conditions of Cavalcanti et al18 Weather data on temperature relative humidity and precipitation from the Pernambuco Water and Climate Agency were analyzed and compared with the monthly density of the collected sandflies Statistical analysis of frequencies Kappa tests Pearsons and Yates chisquare tests and Fishers Exact tests were performed using the IBM SPSS Statistical Software Version 200 IBM SPSS Armonk NY USA Comparison of proportions was calculated in MedCalc Statistical software version 20110 MedCalc Software Ostend Belgium considering α 5 The risk of positivity PR was calculated using the prevalence ratio and respective 95 confidence intervals CI The calculation to analyze the leukocyte and plasma samples submitted to qPCR was performed according to Medronho et al19 The study in humans was approved by the Ethics Committee of University Fernando Pessoa SSD18072016 with written informed consent The study on domestic animals was approved by the Ethics Committee on the Use of Animals of the Aggeu Magalhaes InstituteOswaldo Cruz Foundation in Pernambuco 1152017 with free and informed consent of the research by the tutors RESULTS The aspect of the endemic region studied can be viewed in Figure 1 where a house can be seen among remains of the Atlantic Forest and sugarcane and banana plantations 168 human cases with suspected TL were included aged between 5 and 74 years old mean 37181 with the majority 631 being male Of the suspected cases 112 667 were confirmed to have TL 48 286 did not have the disease and 8 48 had no laboratorial confirmation The clinical forms found were localized cutaneous 929 recidivans cutis 36 disseminated 27 and one that evolved to scar 08 Most patients had a single skin lesion 812 and the remaining had more than ten multiple lesions with diameters between 0505 cm and 9075 cm and evolution periods between 15 days and 10 months As for the location of the lesion the majority 759 occurred in the lower and upper limbs while the others were affected in different regions of the body Lesions of the type ulcerated 756 were presented as plaque crusted verrucous erythematous disseminated recidivans cutis and mucocutaneous while the others healed spontaneously Two patients had coinfection pyoderma gangrenosum and psoriasis Some patients had other diseases such as vascular disease stasis ulcer tuberculosis impetigo fungi chronic eczema and folliculitis After the diagnosis of TL all patients received specific treatment The results of the laboratorial tests are shown in Table 1 It was found that the molecular test by PCR identified 548 and the parasitological test by direct research was found in 256 of cases respectively The concordance of the PCR test between samples collected by tissue fragment biopsy and lesion swab was 608 Kappa value p 0001 The relation between gender age and skin color did not show any statistical differences in PR Having complete or incomplete primary education is a protective factor for infection by L V braziliensis compared to illiterates PR 075 CI 95 058099 The type of occupation seems to be associated with an increased risk of having the disease but it did not reach statistical significance Table 2 Of all the 239 specimens 80 males and 159 females of phlebotomine sandfly captured almost all were identified as Lu whitmani and one female was Lu evandroi During the study period the presence of the sandfly species occurred predominantly in the months of August 2017 and July 2018 At first the room temperature varied between 20 and 28 ºC the relative humidity between 51 and 704 and the rainfall was 1232 mm with annual average Figure 1 Panoramic view of Cumaru Mill in Moreno municipality Silva et al Rev Inst Med Trop São Paulo 202264e60 Page 4 of 8 of 16198 mm Temperature average in the capture of July 2018 varied between 22 ºC and 26 ºC the relative humidity between 57 and 79 and the rainfall was 40 mm with an annual average of 13314 mm In the peridomicile phlebotomine infestation was more common in chicken coops 92 than in stables but in general shelters for domestic animals were close to homes and with poor hygiene conditions The specimens collected were submitted to qPCR for confirmation of the infection by L V braziliensis Among those classified as fed with visible blood in the abdomen n 27 593 n 16 were positive for the parasite Among unfed females no visible blood on the abdomen n 132 333 were positive n 44 for L V braziliensis The average concentration level of DNA of L V braziliensis among the fed female sandflies was 10072828 fg ranging from 0 to 119669 fg and in the nonfed 2791163 fg ranging from 0 to 119669 fg Among all captured females visibly fed and not fed the infection rate was 377 Of the 272 domestic animals included in the study 212 were dogs Canis familiaris 21 were cats Felis cattus 33 were horsesdonkeys Equus caballus Equus asinus 5 were goats Capra aegagrus hircus and 1 was a sheep Ovis aries They had different ages breeds and sizes More than 70 of all animals were medium or large and only 132 34 dogs 1 cat and 1 sheep had skin lesions Of the 272 animals in the study 255 blood samples and 192 ocular conjunctival swabs were collected and submitted to qPCR The positivity of the skin lesion smear in qPCR Table 1 Results of laboratory tests on human samples Laboratory tests n 168 Positive result n Negative result n Not carried out or contaminated n Parasitological Direct search smear slide In vivo culture In vitro culture Molecular Conventional PCR 43 256 20 119 14 83 92 548 42 250 23 137 5 30 55 327 83 494 125 744 149 887 21 125 Table 2 Characterization of the sample of cases with suspected TL according to the diagnosis and respective risk of positivity with a confidence interval of 95 Characteristics TL diagnosis Positive n Negative n Value of p PR CI 95 Total Gender n 160 Male Female 69 616 43 384 29 604 19 396 0971 102 082125 10 98 612 62 388 Age group n 158 5 to 18 19 to 29 30 to 49 50 to 74 19 170 24 214 41 366 28 250 10 218 7 152 18 391 11 239 0773 10 118 085164 106 078145 110 079152 29 184 31 196 59 373 39 247 Skin color n 160 White Mixed ethnicity Black 16 143 84 75 12 107 8 167 33 688 7 146 0694 10 127 05326 086 024302 24 150 117 731 19 119 Level of education n 160 Illiterate Incomplete or complete elementary school Completed high school or more 27 241 39 348 46 411 8 167 28 583 12 250 0021 10 075 058099 103 082129 35 219 67 419 58 362 Occupation n 160 Agricultural professions Other professions indoor or mixed environment No profession children and students 34 303 57 509 21 188 18 375 18 375 12 250 0293 108 043268 181 075439 10 52 325 75 469 33 206 There was no information for 2 individuals PR risk of positivity calculated as prevalence ratio Rev Inst Med Trop São Paulo 202264e60 Study on the zoonotic cycle of tegumentary leishmaniasis in an endemic area of a metropolitan region in the Northeastern region of Brazil Page 5 of 8 from domestic animals is described in Table 3 It was found that 174 animals 64 were positive for L V braziliensis of which 526 were asymptomatic Of the animals with a suspicious lesion 5 dogs were positive for sporotrichosis and 31 were positive for TL In the parallel analysis between the qPCR results and the presence or absence of a skin lesion there was 873 concordance of the negative results and 126 of the positive results Table 4 The comparison of positivity between the different biological samples collected from domestic animals showed greater positivity in the swab from the skin lesion 857 than in whole blood 733 Table 5 DISCUSSION According to the Health Surveillance Secretary of the Ministry of Health20 the Pernambuco State recorded an average of 38 cases of TL annually In 2015 there was a doubling of the number of cases in the Moreno municipality which on average had 50 cases per year unpublished data The municipality is considered a lowrisk area for TL in terms of infection from 2018 to 2020 35 cases were confirmed in the region It had an average incidence of 1167year between 2018 and 2020 with a ranging from 8 in 2020 cases to 16 in 2018 Despite the lowrisk area for TL Moreno is among the seven municipalities with the highest incidence of annual cases in the Pernambuco State20 In the present study the individuals were mostly male of an economically active age with a pattern of populations vulnerable to infection by Leishmania spp in rural areas21 The disease affects different ages including children92223 A previous study in the same population found a positivity Table 3 qPCR results for detection of Leishmania spp in blood samples conjunctival swab or lesion swab from domestic animals residing in the study regions Domestic animal Biological sample Positive Result n Not detected n Not collected n Total Dog Blood 141 665 63 297 8 38 212 Swab 38 179 115 543 59 278 Cat Blood 11 524 1 48 9 428 21 Swab 5 238 8 381 8 381 Horse Donkey Blood 19 576 14 424 0 00 33 Swab 5 151 16 485 12 364 Goat Blood 2 40 3 60 0 00 5 Swab 1 20 3 60 1 20 Sheep Blood 1 100 0 00 0 00 1 Swab 0 00 1 100 0 00 Total Blood 174 64 81 298 17 62 272 Swab 49 18 143 526 80 294 Table 4 qPCR result as a function of the existence or not of a skin lesion compatible with TL qPCR parallel analysis Skin lesion Yes n No n Total n Positive 24 126 167 874 191 100 Negative 9 127 62 873 70 100 Total 33 126 229 874 262 100 Parallel analysis means that every type of biological sample was analyzed in parallel when all qPCR results were negative the overall qPCR result was negative when at least one qPCR result in any biological sample was positive the overall qPCR result was positive it was not possible to obtain qPCR results of 10 animals Table 5 Comparison of positivity between different biological samples of domestic animals qPCR Type of biological sample Value of p Chisquared Difference Confidence interval 95 Positivity in qPCR Whole blood leukocytes plasma 733 Whole blood vs conjunctival swab 0001 9840 475 38715512 Conjunctival Swab 258 Whole blood vs skin lesion swab 0464 054 124 24972546 skin lesion swab 857 Parallel analysis means that every type of biological sample was analyzed in parallel when all qPCR results were negative the overall qPCR result was negative when at least one qPCR result in any biological sample was positive the overall qPCR result was positive Silva et al Rev Inst Med Trop São Paulo 202264e60 Page 6 of 8 of 224 with a positive Montenegro skin test for previous infection24 Twenty years later this study found a 667 positivity for TL infection unpublished data Regarding the characteristics of the disease several clinical forms of TL were identified with a predominance of the simple ulcerated form which is corroborated by other studies421 In Brazil 90 of reported cases present the cutaneous form whereas the mucocutaneous form varies from 3 to 6 of the cases depending on the region2325 Clinical manifestations are variable and dependent on the associated Leishmania species and the hosts immune response16 Patients from the locations of this study presented coinfection with some bacteria species pyoderma gangrenosum psoriasis and other diseases such as vascular diseases stasis ulcer impetigo chronic eczema folliculitis STDs tuberculosis some with dermatological manifestations like TL This makes the diagnosis of TL difficult6 hence the need for specific and sensitive diagnostic tests for TL25 Direct examination results were higher than those from the Leishmania isolates obtained in culture26 The isolates obtained from hamsters Mesocricetus auratus had a higher positivity than the isolates from culture medium but without statistical significance The Leishmania species circulating in the region was identified in previous studies as L V braziliensis4827 and confirmed by specific qPCR tests L V braziliensis is the predominant species throughout the country and is present in several municipalities in Pernambuco State48928 The molecular tests used have high sensitivity and specificity in addition to being fast and effective161729 PCR has detection sensitivity up to 1 fentogram 1015g of Leishmania DNA which corresponds to 110 of the parasite16 This method has been standardized in tissue samples invasive methods to identify Leishmania species in subclinical cases of the disease or with low parasite load following up on treatment and distinction between active and past infections29 It was demonstrated that PCR is better to define the patient diagnosis of TL in all types of the analyzed biological samples Therefore the combination of minimally invasive sample collection procedures such as the swab of lesion salivary fluid and blood with molecular PCR test for diagnostic confirmation of TL is very important252931 Especially when compared with classic parasitological tests with lower positivity102632 and need for invasive collection lesion puncture and biopsy The concordance in PCR between the tissue fragments and swab samples from the lesion had a very significant index according to Cohens Kappa31 which demonstrates the effectiveness of the swab collection technique in relation to biopsy31 The divergence of results can be attributed to the low number of parasites in the lesion or in the exudate as the parasites are not evenly distributed in the lesion which can lead to false negatives32 Amplification of L V braziliensis DNA by PCR in blood samples from patients was found to be low compared to the tissue biopsy samples due to the scarcity of circulating parasites in this biological sample33 The sandflies species most involved in the transmission in Brazil are Lu whitmani Lu intermedia Lu umbratilis Lu wellcomei Lu flaviscutellata and Lu migonei34 In the present results the predominance of Lu whitmani was observed as the main species and probable vector in this region Captured species were done in chicken coops and stables located in the peridomicile The positivity to L V braziliensis in these sandflies measured by qPCR in the studied endemic region was very high These results strongly indicate the natural infection by L V braziliensis in Lu migonei Among fed phlebotomine positivity was 593 and in those without visible blood not fed 333 The low number of captured phlebotomine may be due to the use of herbicides and fire as mentioned by Nasser and Will35 These factors cause changes in the feeding habits when adapting to the environment modified by anthropic action The climate changes also interfere with the biological behaviour of the fauna and consequently with their feeding role modifying the frequency of blood meals36 According to Brito et al24 the expansion of agriculture and construction of houses close to the forests favors the risk of infection due the presence of domestic and synanthropic animals naturally infected close to the houses On the other hand the adaptation of phlebotomine to the modified environment increases transmissibility as well as the expansion of the zoonotic cycle of TL through the contact between these hosts and the human population more frequently Lu whitmani is the most important species in the transmission of TL in Brazil Our results showed positivity for L V braziliensis in specimens collected from Lu whitmani An entomological survey in 2001 collected 444 specimens of Lu whitmani in this municipality also showing the predominance of this species in eleven other locations in the municipality unpublished data Between 2011 and 2012 another study confirmed the presence of the species in the intradomicile and peridomicile with 3071 specimens collected of which 2919 Lu whitmani 122 Lu evandroi and 30 Lu choti Of the 37 sandflies species identified in Pernambuco Lu whitmani was the most predominant37 The domestic animals examined in this study showed a high positivity rate for TL through qPCR especially in blood samples which confirms the high prevalence of infection in the region even with some asymptomatic Rev Inst Med Trop São Paulo 202264e60 Study on the zoonotic cycle of tegumentary leishmaniasis in an endemic area of a metropolitan region in the Northeastern region of Brazil Page 7 of 8 animals Dogs were the species with highest percentage of positivity in the blood As they transit through more regions and different areas of the peridomicile they may have a greater chance of being bitten Equidae are also parasitized by Leishmania spp Several studies have already confirmed the presence of L V braziliensis in these domestic animals residing in endemic areas93839 A sheep with a lesion like TL uncommon symptomatic condition was also identified confirmed by direct examination for TL A similar case occurred in South Africa where a sheep presented a lesion that evolved to spontaneous healing7 According to DantasTorres40 the diagnosis of asymptomatic domestic animals is important as they may be reservoirs of Leishmania spp The knowledge of ecoepidemiology of the studied region transmission control through early diagnosis and treatment are measures which collaborate with prevalence and incidences decrease of leishmaniasis according to goals established by PAHOWHO for 20303 Also the main measures of control for this disease according to the Brazilian Ministry of Health are avoiding living close to remnants of forest netting on doors and windows and applying repellent on the body6 CONCLUSION In conclusion our findings demonstrate that the epidemiology of TL associated with L V braziliensis in this municipality of the Zona da Mata region has different characteristics when compared with previous studies in the same region over time425 to those observed in other regions of Brazil In this sense more studies are necessary especially on the role of domestic animals to support their real contribution to the disease transmission cycle and maintenance and also to contribute to the adoption of TL control recommendations in endemic areas It is also recommended that within the scope of the Primary Health Care program and epidemiological surveillance community health agents promote campaigns aimed to improve the spread of information on the transmission cycle signs and symptoms of disease and its care to the human populations living in the endemic areas of the country CONFLICT OF INTERESTS None to declare REFERENCES 1 Burza S Croft SL Boelaert M Leishmaniasis Lancet 201839295170 2 World Health Organization Leishmaniasis status of endemicity of cutaneous leishmaniasis 2020 cited 2022 Aug 16 Available from httpsappswhointneglecteddiseases ntddataleishmaniasisleishmaniasishtml 3 Pan American Health Organization Leishmaniases epidemiological report in the Americas cited 2022 Aug 16 Available from httpsirispahoorghandle10665251742 4 Brito ME Andrade MS DantasTorres F Rodrigues EH Cavalcanti MP Almeida AM et al Cutaneous leishmaniasis in northeastern Brazil a critical appraisal of studies conducted in State of Pernambuco Rev Soc Bras Med Trop 201245 4259 5 Melo MG Morais RC Goes TC Silva RP Morais RF Guerra JA et al Clinical and epidemiological profiles of patients with American cutaneous leishmaniasis from the states of Pernambuco and Amazonas Brazil Rev Soc Bras Med Trop 202053e20200083 6 Brasil Ministério da Saúde Departamento de Vigilância das Doenças Transmissíveis Manual de vigilância da leishmaniose tegumentar Brasília Ministério da Saúde 2017 cited 2022 Aug 16 Available from httpbvsmssaudegovbrbvs publicacoesmanualvigilancialeishmaniosetegumentarpdf 7 van der Lugt JJ Carlyon JF de Waal DT Cutaneous leishmaniasis in a sheep J S Afr Vet Assoc 199263747 8 Brito ME Andrade MS Mendonça MG Silva CJ Almeida EL Lima BS et al Species diversity of Leishmania Viannia parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil Trop Med Int Heal 200914127886 9 BrandãoFilho SP Brito ME Carvalho FG Ishikaw EA Cupolillo E FloeterWinter L et al Wild and synanthropic hosts of Leishmania Viannia braziliensis in the endemic cutaneous leishmaniasis locality of Amaraji Pernambuco State Brazil Trans R Soc Trop Med Hyg 2003972916 10 Pena HP Belo VS XavierJunior JC TeixeiraNeto RG Melo SN Pereira DA et al Accuracy of diagnostic tests for American tegumentary leishmaniasis a systematic literature review with metaanalyses Trop Med Int Health 202025116881 11 Instituto Brasileiro de Geografia e Estatística BrasilPernambuco Moreno cited 2022 Aug 16 Available from httpscidades ibgegovbrbrasilpemorenopanorama 12 Cupolillo E Grimaldi G Jr Momen H A general classification of New World leishmania using numerical zymotaxonomy Am J Trop Med Hyg 199450296311 13 McNelly J The CDC trap as a special monitoring tool In Proceedings of the SeventySixth Annual Meeting of the New Jersey Mosquito Control Association 19892633 cited 2022 Aug 16 Available from httpsvectorbiorutgersedu outreachcdctraphtm 14 Young DG Duncan MA Guide to the identification and geographic distribution of Lutzomyia sand flies in Mexico Silva et al Rev Inst Med Trop São Paulo 202264e60 Page 8 of 8 the West Indies Central and South America Diptera Psychodidae Gainesville Associated Publishers 1994 15 Solano P Duvallet G Dumas V Cuisance D Cuny G Microsatellite markers for genetic population studies in Glossina palpalis Diptera Glossinidae Acta Trop 19976517580 16 de Bruijn MH Barker DC Diagnosis of New World leishmaniasis specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA Acta Trop 1992524558 17 Green MR Sambrook J Molecular cloning a laboratory manual 4th ed New York Cold Spring Harbor 2012 18 Cavalcanti MP DantasTorres F Albuquerque SC Morais R Brito MC Otranto D et al Quantitative real time PCR assays for the detection of Leishmania Viannia braziliensis in animals and humans Mol Cell Probes 2013271228 19 Medronho RA Carvalho DM Bloch KV Luiz RR Werneck GL editores Epidemiologia São Paulo Atheneu 2002 20 Brasil Ministério da Saúde DATASUS Leishmaniose tegumentar americana casos confirmados notificados no Sistema de Informação de Agravos de Notificação Pernambuco cited 2022 Aug 16 Available from httptabnetdatasusgovbrcgi tabcgiexesinannetcnvltapedef 21 Pezente L Benedetti M Perfil epidemiológico da leishmaniose Tegumentar Americana no Estado de Roraima Amazônia Brasil entre 2007 e 2016 Braz J Health Rev 20192173442 22 Vasconcelos JM Gomes CG Sousa A Teixeira AB Lima JM Leishmaniose tegumentar americana perfil epidemiológico diagnóstico e tratamento Rev Bras Anal Clin 2018502217 23 World Health Organization Working to overcome the global impact of neglected tropical diseases first WHO report on neglected tropical diseases cited 2022 Aug 16 Available from httpsappswhointirishandle1066544440 24 Brito ME Silva CJ Silva CM Salazar PR Coutinho JS Reis LC et al Clinical epidemiological profile of American tegumentary leishmaniasis at the Pinto Sugar Mill in Moreno Municipality Greater Metropolitan Recife Pernambuco State Brazil Cad Saude Publica 20082424458 25 Thakur S Joshi J Kaur S Leishmaniasis diagnosis an update on the use of parasitological immunological and molecular methods J Parasit Dis 20204425372 26 Corvalan FH Sampaio RN Brustoloni YM Andreotti R Lima Junior MS DNA identification of Leishmania Viannia braziliensis in human saliva from a patient with American cutaneous leishmaniasis J Venom Anim Toxins Incl Trop Dis 20111798102 27 Moreira MA Luvizotto MC Garcia JF Corbett CE Laurenti MD Comparison of parasitological immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs Vet Parasitol 200714524552 28 Rodrigues EH Brito ME Mendonça MG Werkhäuser RP Coutinho EM Souza WV et al Evaluation of PCR for diagnosis of American cutaneous leishmaniasis in an area of endemicity in northeastern Brazil Brazil J Clin Microbiol 20024035726 29 Martins L Alexandrino A Guimarães G Detection of Leishmania braziliensis DNA in American tegumentary leishmaniasis patients Rev Saude Publica 2010445714 30 Brito ME Almeida EL Medeiros AC Werkhäuser RP Alexandre JL Sá BS et al Leishmania Viannia braziliensis isolated from the saliva of patients in a cutaneous leishmaniasis endemic area of northeastern Brazil Mem Inst Oswaldo Cruz 2018113e170250 31 Adams ER Gomez MA Scheske L Rios R Marquez R Cossio A et al Sensitive diagnosis of cutaneous leishmaniasis by lesion swab sampling coupled to qPCR Parasitology 201414118917 32 Landis JR Koch GG The measurement of observer agreement for categorical data Biometrics 19773315974 33 Shahbazi F Shahabi S Kazemi B Mohebali M Abadi AR Zare Z Evaluation of PCR assay in diagnosis and identification of cutaneous leishmaniasis a comparison with the parasitological methods Parasitol Res 2008103115962 34 Abbate JM Maia C Pereira A Arfuso F Gaglio G Rizzo M et al Identification of trypanosomatids and blood feeding preferences of phlebotomine sand fly species common in Sicily Southern Italy PLoS One 202015e0229536 35 Nasser N Will E Perfil epidemiológico da Leishmaniose Tegumentar Americana no município de BlumenauSC Arq Catarin Med 2017462838 36 Silva CJ Pereira SV Apolinário EJ Santos GL MeloSantos MA Silva AF et al Culicidae fauna Diptera Culicidae survey in urban ecotonal and forested areas from the Moreno municipality Pernambuco State Brazil Rev Soc Bras Med Trop 2018515237 37 Sales KG Costa PL Morais RC Otranto D BrandãoFilho SP Cavalcanti MP et al Identification of phlebotomine sand fly blood meals by realtime PCR Parasit Vectors 20158230 38 Guimarães VC Costa PL Silva FJ Silva KT Silva KG Araújo AI et al Phebotomine sandflies Diptera Psychodidae in São Vicente Férrer a sympatric area curtaneous and visceral leishmaniasis in the state of Pernambuco Brazil Rev Soc Bras Med Trop 2012456670 39 Vieira AF Alvarenga TF Oliveira SF Coelho EC Teixeira MC Moscon LA et al Leishmaniose em equídeos revisão Pubvet 20201415 40 DantasTorres F SimõesMattos L Brito FL Figueredo LA Faustino MA Leishmaniose felina revisão de literatura Clin Vet 2006113240