American tegumentary leishmaniasis is the generic name for a variety of cutaneous and mucocutaneous presentations of parasitosis caused by several species of the genus Leishmania. This is a widespread infection in the American continent, from the South of the United States to the North of Argentina. We herein describe the management of a patient with American tegumentary leishmaniasis in Mendoza, Argentina, a nonendemic area of South America, whose diagnosis and treatment were significantly delayed, because the patient did not report a recent history of travel to any known endemic areas. This case stresses the need for training health-care professionals in the diagnosis and treatment of not only endemic parasitosis within their work zones but also nonendemic parasitosis.Fil: Cargnelutti, Diego Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Borremans, Carlos Guillermo. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Tonelli, Rosa Lidia. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Carrizo, Liliana Carmen. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Salomón, María Cristina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentin

Borremans, Carlos Guillermo

Cargnelutti, Diego Esteban

Carrizo, Liliana Carmen

Salomón, María Cristina

Tonelli, Rosa Lidia

Journal of Microbiology Immunology and Infection

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

Journal of Microbiology, Immunology and Infection (2016) 49, 809e812Available online at www.sciencedirect.comScienceDirectjournal homepage: www.e- jmii .comCASE REPORTDiagnosis of Leishmania infection in anonendemic area of South AmericaDiego Esteban Cargnelutti a,b,*, Carlos Guillermo Borremans a,Rosa Lydia Tonelli a, Liliana Carmen Carrizo a,Marı́a Cristina Salomón aa Área de Parasitologı́a, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza,Argentinab Instituto de Medicina y Biologı́a Experimental de Cuyo (IMBECU), CCT-Mendoza, CONICET, Mendoza,ArgentinaReceived 4 August 2014; accepted 29 November 2014Available online 11 December 2014KEYWORDSArgentina;leishmaniasis;mucosal involvement;nonendemic area* Corresponding author. Área deCiencias Médicas, Universidad Nacionertador, Number 80, Mendoza, ArgentE-mail address: diegocargneCargnelutti).http://dx.doi.org/10.1016/j.jmii.20141684-1182/Copyright ª 2015, Taiwan SBY-NC-ND license (http://creativecomAmerican tegumentary leishmaniasis is the generic name for a variety of cutaneous and muco-cutaneous presentations of parasitosis caused by several species of the genus Leishmania. Thisis a widespread infection in the American continent, from the South of the United States to theNorth of Argentina. We herein describe the management of a patient with American tegumen-tary leishmaniasis in Mendoza, Argentina, a nonendemic area of South America, whose diag-nosis and treatment were significantly delayed, because the patient did not report a recenthistory of travel to any known endemic areas. This case stresses the need for traininghealth-care professionals in the diagnosis and treatment of not only endemic parasitosis withintheir work zones but also nonendemic parasitosis.Copyright ª 2015, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is anopen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Parasitologı́a, Facultad deal de Cuyo, Avenida del Lib-ina.lutti@hotmail.com (D.E..11.014ociety of Microbiology. Publishedmons.org/licenses/by-nc-nd/4.0/IntroductionLeishmaniasis is a parasitic disease caused by protozoa ofthe genus Leishmania, which affects a large population allover the world. Estimates suggest that more than 12 millionpeople are infected by the protozoan Leishmania. Thereare different species of Leishmania that can cause thedisease in different regions. American tegumentaryby Elsevier Taiwan LLC. This is an open access article under the CC).Figure 1. American tegumentary leishmaniasis. (A) Infection in the nose and lip regions that presented as edema and erythema.(B) Granulomatous lesion on the soft palate.810 D.E. Cargnelutti et al.leishmaniasis is the generic name for a variety of cutaneousand mucocutaneous presentations of parasitosis caused bydifferent species of Leishmania.1 This infectious disease isa serious health problem. In endemic areas, high morbidityrates are reported, whereas in nonendemic areas it isusually misdiagnosed as some other similar granulomatousdermal diseases. Diagnosis of the infection is complicatedbecause of the high costs of laboratory supplies and su-perinfection of the lesions. The main problem, however, isthe insufficient training of health-care professionals innonendemic areas.2American tegumentary leishmaniasis is a widespreadinfection in the American continent, from the South of theUnited States to the North of Argentina.3 The provinces ofSalta, Jujuy, Tucumán, Catamarca, Santiago del Estero,Chaco, Formosa, Misiones, and Corrientes constitute theendemic area in Argentina. Although species of Lutzomyia(a vector of this disease) have been detected in the prov-inces of Santa Fe and Entre Rı́os, so far there are no reportsof infection in animals or humans.4A common question asked by doctors whenever a patientpresents with atypical clinical symptoms for the region is“Where have you been?” When investigating canine leish-maniasis cases, the veterinary surgeons usually inquireabout the origin or movement of animals. A large propor-tion of leishmaniasis had been diagnosed in patients whoeither have been to or were born in an endemic area. Suchquestions should also be asked at the clinics of developingcountries where nonautochthonous cases may appear.In this report, we will describe the management of anAmerican tegumentary leishmaniasis case that was detec-ted in a nonendemic area. The diagnosis was significantlydelayed because the patient did not report any recenthistory of travel to any known endemic areas.Case reportOur case is a 19-year-old female patient who had nevertraveled outside the province of Mendoza, Argentina. As anantecedent to the present episode, at the age of 14, thepatient suffered upper lip trauma while practicing sports,which caused edema and erosion in the affected area (i.e.,the lips). At that time, she was diagnosed with supportgranulomatous cheilitis. She was treated with minocyclinefor a brief period in another institution, but the treatmentwas discontinued due to pharmacodermy. The patientmentioned that the lesions significantly increased in sizeduring her two pregnancies, and that she did not receive anyfurther treatment. In March 2013, she came to us forconsultation because of severe granulomatous infiltration inthe hard palate and soft upper and lower lips, which wasassociated with edema and mucosal erosions for 4 years(Fig. 1A and B). A nasal endoscopy showed granulomas in thenostrils, mouth, palate, and anterior and posterior pillars.In April 2013, we performed a biopsy of the mucosa ofthe upper lip and the left posterior palate. Biopsy resultsshowed intense inflammatory infiltrate with abundantlymphocytes, plasma cells with Russell bodies, and histio-cytes with vacuolated cytoplasm. Rhinoscleroma wassuspected.Therefore, we requested additional tests to be per-formed on the patient: The patient’s complete blood countwas normal. The skin test for tuberculosis was negative.Her chest X-ray was normal. The venereal disease researchlaboratory result was negative (i.e., nonreactive). For thebacteriologic study, we collected mucus samples from thepalate and lip. Neither alcohol-resistant acid bacillus norfungal elements were observed in the sample. In addition,Giemsa and ZiehleNeelsen staining were negative. Gramstaining showed a few Gram-positive cocci. Staphylococcusaureus and Streptococcus viridans were isolated from thesample. She also underwent serology testing for deep my-coses. However, the mucus sample was negative for Coc-cidioides, Paracoccidioides, Histoplasma, and Aspergillus.The sample was negative for tumor markers as well.In April 2013, despite failing to isolate Klebsiella rhino-scleromatis in culture, there was a clinical suspicion ofrhinoscleroma based on biopsy results. The patientreceived ciprofloxacin (500 mg every 12 hours) for 4 monthsand cefixime (400 mg every 24 hours) for 1 month. A slightimprovement in edema and erythema was observed inupper lip on the 1st month control visit. The patientremained stable on successive control visits.Leishmania infection in a nonendemic area 811In July 2013, the patient presented with hoarseness andpersistent granulomatous lesions on the palate, infiltrationof the lower third of the face, and protrusion of both lipswithout significant improvement despite treatment withciprofloxacin for the past 4 months.From a public health institution in the Mendoza prov-ince, the patient was referred to the Área de Parasitologı́aat the School of Medicine of the National University of Cuyo(Mendoza, Argentina) to exclude Leishmania infection indifferential diagnosis, because the patient did not show anysigns or symptoms suggestive of this infection thus far.Lesions were cultured by an aspirate technique inaccordance with the procedure reported by Romero et al,5but with some minor modifications. For the culture, 10-mLcapacity tubes with rubber caps filled with a biphasic cul-ture medium were used. The solid phase consisted of 4 mLof a blood agar-based mediumdNovyeMacNealeNicollemedium with blood agar base, to which 5% defibrinatedrabbit blood was added at 40C after the agar hardened.The liquid phase was 500 mL isotonic saline (0.9%) con-taining 100 U/mL penicillin and 100 mg/mL streptomycin.Before sample collection, the puncture site was vigor-ously cleaned with 70% ethanol. A local anesthetic (100 mLof 2% lidocaine) was injected with a 1-mL syringe equippedwith a 13-G needle. The aspiration puncture was madeusing a commercially available needle holder (Vacutainer).A 21-G needle at a 45-degree angle was then inserted intothe dermis at the ulcer border by rotary movement. Thesample was collected in the tube containing the biphasicculture medium by suction.The culture tubes were incubated at 26C. For parasiteexamination, the contents of each tube were gently ho-mogenized, and approximately 10 mL of the suspension wasobserved under an optical microscope (40 magnification).The cultures were examined every 3 days to detect promas-tigotes under the optical microscope. After 6 days, Leish-mania spp. promastigotes were observed (Fig. 2A and B).DiscussionThe diagnosis of American tegumentary leishmaniasis basedon clinical aspects is usually inaccurate because many otherFigure 2. Leishmania spp. promastigotes: (A) under opticconditions such as fungal and mycobacterial infections,sarcoidosis, and neoplasms imitate the behavior of this typeof leishmaniasis. In addition, serological and histopatho-logical methodologies have a limited diagnostic certainty,because of their low sensitivity levels. Cell-mediated im-mune response measured by delayed-type hypersensitivityoften identifies positive cases more accurately. Neverthe-less, the results depend on the immunological status of thepatient, the period since the patient acquired the infec-tion, the kind of Leishmania species, and the quality of thereagents used. Despite the high specificity of the skin test,it is not possible to differentiate current from past in-fections using this method.6Although polymerase chain reaction has been proved tobe highly effective in the diagnosis of leishmaniasis,7 thedisadvantages to this approach are the high cost, theavailability of reagents and equipment, and the pooradaptability to field conditions.Currently used parasitological diagnostic methodologiesinvolve the direct observation of amastigotes in stainedsmears or cultures of samples from skin lesions. The tradi-tional culture methods consist of a biphasic culture systemof blood agar with liquid overlay.8 Therefore, there is aneed to establish simpler procedures for obtaining thesamples from cutaneous leishmaniasis patients, becausethe complexity of the collection significantly restricts itsmassive use in public health institutions. Culturing of as-pirates from the American tegumentary leishmaniasis lesionis an alternative methodology because it is easily per-formed, economical, and had a high positivity rate, rangingfrom 28.6% to 89.0% in studies that evaluated patientsinfected with different Leishmania species.5,9In the Argentine Republic, the endemic area correspondswith the tropical and subtropical region north of thecountry in a broad geographical region crossing Argentinafrom east to west. This territory covers nine provinces ofthe Argentine Republic (Salta, Jujuy, Tucumán, Catamarca,Santiago del Estero, Chaco, Formosa, Misiones y Corrientes)with an approximate extension of 497,000 km2.The endemic area extends from a longitude of 66 W to54 E; and from a latitude of 22 N to 29 S. A recent studyreported the presence of Lutzomyia (a vector of thismicroscopy (40 magnification). (B) Zoom of image A.812 D.E. Cargnelutti et al.disease) in the provinces of Santa Fe and Entre Rı́os, but sofar there are no reports of infection in animals or humans.4Mendoza city (32530 LS, 68490 LW), capital of theMendoza province, is located in the foothills that extend tothe east of the final slopes of the Andes and is bordered byChile. The city is 750 m above sea level. It is a semiaridtemperate zone, although the mountain ranges provide acertain degree of aridity with scarce rainfall (averageannual rainfall, 250 mm; average annual temperature,18.8C). The city of Mendoza is more than 800 km awayfrom the area of transmission of the American tegumentaryleishmaniasis and lies in a completely different ecologicalregion to those where sand fly insect vectors are found.During discussions, the patient denied several timeshaving traveled outside the province of Mendoza. Once thediagnosis was established, the patient had to be hospital-ized for treatment. The patient’s mother mentioned thatthe girl had been born and remained in the province of Saltauntil the age of 3 years. The infection in our case was thusasymptomatic until 9 years of age, after which she devel-oped upper lip trauma as a manifestation of this disease.This parasitic disease is an exotic infection in Mendoza,and in some cases, the provincial public health system hasnot been able to establish the diagnosis of the infectionaccurately.10 However, the present case of Americantegumentary leishmaniasis that had an unconventionalmucocutaneous presentation for the Argentine Republicwas diagnosed and treated in an efficient and accurate wayby the academicescientific staff at the School of Medicineof the National University of Cuyo, Mendoza province.This case demonstrates the importance of teamworkbetween the health and academic institutions that allowedfor the correct diagnosis of a nonendemic parasitosis inMendoza. In addition, this case stresses the need fortraining health-care professionals in the diagnosis andtreatment of not only endemic parasitosis within their workzones but also nonendemic parasitosis. Globalization ofparasitosis due to the migration of populations increasesthe need for continuous training in the approach to andmanagement of nonendemic parasitosis.Conflicts of interestAll contributing authors declare no conflicts of interest.AcknowledgmentsThis work was carried out with the financial support of theSecretarı́a de Ciencia, Técnica y Posgrado of the Uni-versidad Nacional de Cuyo (Project No. 06/J417).References1. WHO Expert Committee on the Control of Leishmaniases.Control of the leishmaniases. Geneva, Switzerland: WHOPress; 2010. p. 5e12.2. Escobar MA, Martinez F, Scott Smith D, Palma GI. Americancutaneous and mucocutaneous leishmaniasis (tegumentary): adiagnostic challenge. Trop Doct 1992;22:69e78. 63e4.3. Salomón OD, Sosa-Estani S, Ramos K, Orellano PW, Sanguesa G,Fernández G, et al. Tegumentary leishmaniasis outbreak inBella Vista City, Corrientes, Argentina during 2003. Mem InstOswaldo Cruz 2006;101:767e74.4. Salomón OD, Orellano PW, Quintana MG, Perez S, Sosa Estani S,Acardi S, et al. Transmission of tegumentary leishmaniasis inArgentina. Medicina (B Aires) 2006;66:211e9.5. Romero GA, Sampaio RN, Macedo Vde O, Marsden PD. Sensi-tivity of a vacuum aspiratory culture technique for diagnosis oflocalized cutaneous leishmaniasis in an endemic area ofLeishmania (Viannia) braziliensis transmission. Mem InstOswaldo Cruz 1999;94:505e8.6. Berman JD. Human leishmaniasis: clinical, diagnostic, andchemotherapeutic developments in the last 10 years. ClinInfect Dis 1997;24:684e703.7. Toz SO, Culha G, Zeyrek FY, Ertabaklar H, Alkan MZ,Vardarlı AT, et al. A real-time ITS1-PCR based method in thediagnosis and species identification of Leishmania parasitefrom human and dog clinical samples in Turkey. PLoS Negl TropDis 2013;7:e2205.8. Marzochi MC, Teixeira PC, Marzochi KB, da Conceição NF,Coutinho W, de Brito DB. Vacuum aspiratory puncture systemfor Leishmania culturing, isolation and transport. Preliminaryreport. Rev Inst Med Trop Sao Paulo 1993;35:301e3.9. Weigle KA, Labrada LA, Lozano C, Santrich C, Barker DC. PCR-based diagnosis of acute and chronic cutaneous leishmaniasiscaused by Leishmania (Viannia). J Clin Microbiol 2002;40:601e6.10. Martı́n-Sánchez J, Navarro-Mari JM, Pasquau-Liaño J,Salomón OD, Morillas-Márquez F. Visceral leishmaniasis causedby Leishmania infantum in a Spanish patient in Argentina:what is the origin of the infection? Case report. BMC Infect Dis2004;4:20.

English

Diagnosis of Leishmania infection in a nonendemic area of South America

American tegumentary leishmaniasis is the generic name for a variety of cutaneous and mucocutaneous presentations of parasitosis caused by several species of the genus Leishmania. This is a widespread infection in the American continent, from the South of the United States to the North of Argentina. We herein describe the management of a patient with American tegumentary leishmaniasis in Mendoza, Argentina, a nonendemic area of South America, whose diagnosis and treatment were significantly delayed, because the patient did not report a recent history of travel to any known endemic areas. This case stresses the need for training health-care professionals in the diagnosis and treatment of not only endemic parasitosis within their work zones but also nonendemic parasitosis

Tonelli, Rosa Lydia

Elsevier - Publisher Connector 

Diagnosis of Leishmania infection in a nonendemic area of South America 

CONICET Digital

Journal of Microbiology, Immunology and Infection (2016) 49, 809e812Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.e- jmii .comCASE REPORTDiagnosis of Leishmania infection in a
nonendemic area of South America
Diego Esteban Cargnelutti a,b,*, Carlos Guillermo Borremans a,
Rosa Lydia Tonelli a, Liliana Carmen Carrizo a,
Marı´a Cristina Salomo´n aa A´rea de Parasitologı´a, Facultad de Ciencias Me´dicas, Universidad Nacional de Cuyo, Mendoza,
Argentina
b Instituto de Medicina y Biologı´a Experimental de Cuyo (IMBECU), CCT-Mendoza, CONICET, Mendoza,
ArgentinaReceived 4 August 2014; accepted 29 November 2014
Available online 11 December 2014KEYWORDS
Argentina;
leishmaniasis;
mucosal involvement;
nonendemic area* Corresponding author. A´rea de
Ciencias Me´dicas, Universidad Nacion
ertador, Number 80, Mendoza, Argent
E-mail address: diegocargne
Cargnelutti).
http://dx.doi.org/10.1016/j.jmii.2014
1684-1182/Copyright ª 2015, Taiwan S
BY-NC-ND license (http://creativecomAmerican tegumentary leishmaniasis is the generic name for a variety of cutaneous and muco-
cutaneous presentations of parasitosis caused by several species of the genus Leishmania. This
is a widespread infection in the American continent, from the South of the United States to the
North of Argentina. We herein describe the management of a patient with American tegumen-
tary leishmaniasis in Mendoza, Argentina, a nonendemic area of South America, whose diag-
nosis and treatment were significantly delayed, because the patient did not report a recent
history of travel to any known endemic areas. This case stresses the need for training
health-care professionals in the diagnosis and treatment of not only endemic parasitosis within
their work zones but also nonendemic parasitosis.
Copyright ª 2015, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).Parasitologı´a, Facultad de
al de Cuyo, Avenida del Lib-
ina.
lutti@hotmail.com (D.E.
.11.014
ociety of Microbiology. Published
mons.org/licenses/by-nc-nd/4.0/Introduction
Leishmaniasis is a parasitic disease caused by protozoa of
the genus Leishmania, which affects a large population all
over the world. Estimates suggest that more than 12 million
people are infected by the protozoan Leishmania. There
are different species of Leishmania that can cause the
disease in different regions. American tegumentaryby Elsevier Taiwan LLC. This is an open access article under the CC
).
Figure 1. American tegumentary leishmaniasis. (A) Infection in the nose and lip regions that presented as edema and erythema.
(B) Granulomatous lesion on the soft palate.
810 D.E. Cargnelutti et al.leishmaniasis is the generic name for a variety of cutaneous
and mucocutaneous presentations of parasitosis caused by
different species of Leishmania.1 This infectious disease is
a serious health problem. In endemic areas, high morbidity
rates are reported, whereas in nonendemic areas it is
usually misdiagnosed as some other similar granulomatous
dermal diseases. Diagnosis of the infection is complicated
because of the high costs of laboratory supplies and su-
perinfection of the lesions. The main problem, however, is
the insufficient training of health-care professionals in
nonendemic areas.2
American tegumentary leishmaniasis is a widespread
infection in the American continent, from the South of the
United States to the North of Argentina.3 The provinces of
Salta, Jujuy, Tucuma´n, Catamarca, Santiago del Estero,
Chaco, Formosa, Misiones, and Corrientes constitute the
endemic area in Argentina. Although species of Lutzomyia
(a vector of this disease) have been detected in the prov-
inces of Santa Fe and Entre Rı´os, so far there are no reports
of infection in animals or humans.4
A common question asked by doctors whenever a patient
presents with atypical clinical symptoms for the region is
“Where have you been?” When investigating canine leish-
maniasis cases, the veterinary surgeons usually inquire
about the origin or movement of animals. A large propor-
tion of leishmaniasis had been diagnosed in patients who
either have been to or were born in an endemic area. Such
questions should also be asked at the clinics of developing
countries where nonautochthonous cases may appear.
In this report, we will describe the management of an
American tegumentary leishmaniasis case that was detec-
ted in a nonendemic area. The diagnosis was significantly
delayed because the patient did not report any recent
history of travel to any known endemic areas.Case report
Our case is a 19-year-old female patient who had never
traveled outside the province of Mendoza, Argentina. As an
antecedent to the present episode, at the age of 14, the
patient suffered upper lip trauma while practicing sports,which caused edema and erosion in the affected area (i.e.,
the lips). At that time, she was diagnosed with support
granulomatous cheilitis. She was treated with minocycline
for a brief period in another institution, but the treatment
was discontinued due to pharmacodermy. The patient
mentioned that the lesions significantly increased in size
during her two pregnancies, and that she did not receive any
further treatment. In March 2013, she came to us for
consultation because of severe granulomatous infiltration in
the hard palate and soft upper and lower lips, which was
associated with edema and mucosal erosions for 4 years
(Fig. 1A and B). A nasal endoscopy showed granulomas in the
nostrils, mouth, palate, and anterior and posterior pillars.
In April 2013, we performed a biopsy of the mucosa of
the upper lip and the left posterior palate. Biopsy results
showed intense inflammatory infiltrate with abundant
lymphocytes, plasma cells with Russell bodies, and histio-
cytes with vacuolated cytoplasm. Rhinoscleroma was
suspected.
Therefore, we requested additional tests to be per-
formed on the patient: The patient’s complete blood count
was normal. The skin test for tuberculosis was negative.
Her chest X-ray was normal. The venereal disease research
laboratory result was negative (i.e., nonreactive). For the
bacteriologic study, we collected mucus samples from the
palate and lip. Neither alcohol-resistant acid bacillus nor
fungal elements were observed in the sample. In addition,
Giemsa and ZiehleNeelsen staining were negative. Gram
staining showed a few Gram-positive cocci. Staphylococcus
aureus and Streptococcus viridans were isolated from the
sample. She also underwent serology testing for deep my-
coses. However, the mucus sample was negative for Coc-
cidioides, Paracoccidioides, Histoplasma, and Aspergillus.
The sample was negative for tumor markers as well.
In April 2013, despite failing to isolate Klebsiella rhino-
scleromatis in culture, there was a clinical suspicion of
rhinoscleroma based on biopsy results. The patient
received ciprofloxacin (500 mg every 12 hours) for 4 months
and cefixime (400 mg every 24 hours) for 1 month. A slight
improvement in edema and erythema was observed in
upper lip on the 1st month control visit. The patient
remained stable on successive control visits.
Leishmania infection in a nonendemic area 811In July 2013, the patient presented with hoarseness and
persistent granulomatous lesions on the palate, infiltration
of the lower third of the face, and protrusion of both lips
without significant improvement despite treatment with
ciprofloxacin for the past 4 months.
From a public health institution in the Mendoza prov-
ince, the patient was referred to the A´rea de Parasitologı´a
at the School of Medicine of the National University of Cuyo
(Mendoza, Argentina) to exclude Leishmania infection in
differential diagnosis, because the patient did not show any
signs or symptoms suggestive of this infection thus far.
Lesions were cultured by an aspirate technique in
accordance with the procedure reported by Romero et al,5
but with some minor modifications. For the culture, 10-mL
capacity tubes with rubber caps filled with a biphasic cul-
ture medium were used. The solid phase consisted of 4 mL
of a blood agar-based mediumdNovyeMacNealeNicolle
medium with blood agar base, to which 5% defibrinated
rabbit blood was added at 40C after the agar hardened.
The liquid phase was 500 mL isotonic saline (0.9%) con-
taining 100 U/mL penicillin and 100 mg/mL streptomycin.
Before sample collection, the puncture site was vigor-
ously cleaned with 70% ethanol. A local anesthetic (100 mL
of 2% lidocaine) was injected with a 1-mL syringe equipped
with a 13-G needle. The aspiration puncture was made
using a commercially available needle holder (Vacutainer).
A 21-G needle at a 45-degree angle was then inserted into
the dermis at the ulcer border by rotary movement. The
sample was collected in the tube containing the biphasic
culture medium by suction.
The culture tubes were incubated at 26C. For parasite
examination, the contents of each tube were gently ho-
mogenized, and approximately 10 mL of the suspension was
observed under an optical microscope (40 magnification).
The cultures were examined every 3 days to detect promas-
tigotes under the optical microscope. After 6 days, Leish-
mania spp. promastigotes were observed (Fig. 2A and B).
Discussion
The diagnosis of American tegumentary leishmaniasis based
on clinical aspects is usually inaccurate because many otherFigure 2. Leishmania spp. promastigotes: (A) under opticconditions such as fungal and mycobacterial infections,
sarcoidosis, and neoplasms imitate the behavior of this type
of leishmaniasis. In addition, serological and histopatho-
logical methodologies have a limited diagnostic certainty,
because of their low sensitivity levels. Cell-mediated im-
mune response measured by delayed-type hypersensitivity
often identifies positive cases more accurately. Neverthe-
less, the results depend on the immunological status of the
patient, the period since the patient acquired the infec-
tion, the kind of Leishmania species, and the quality of the
reagents used. Despite the high specificity of the skin test,
it is not possible to differentiate current from past in-
fections using this method.6
Although polymerase chain reaction has been proved to
be highly effective in the diagnosis of leishmaniasis,7 the
disadvantages to this approach are the high cost, the
availability of reagents and equipment, and the poor
adaptability to field conditions.
Currently used parasitological diagnostic methodologies
involve the direct observation of amastigotes in stained
smears or cultures of samples from skin lesions. The tradi-
tional culture methods consist of a biphasic culture system
of blood agar with liquid overlay.8 Therefore, there is a
need to establish simpler procedures for obtaining the
samples from cutaneous leishmaniasis patients, because
the complexity of the collection significantly restricts its
massive use in public health institutions. Culturing of as-
pirates from the American tegumentary leishmaniasis lesion
is an alternative methodology because it is easily per-
formed, economical, and had a high positivity rate, ranging
from 28.6% to 89.0% in studies that evaluated patients
infected with different Leishmania species.5,9
In the Argentine Republic, the endemic area corresponds
with the tropical and subtropical region north of the
country in a broad geographical region crossing Argentina
from east to west. This territory covers nine provinces of
the Argentine Republic (Salta, Jujuy, Tucuma´n, Catamarca,
Santiago del Estero, Chaco, Formosa, Misiones y Corrientes)
with an approximate extension of 497,000 km2.
The endemic area extends from a longitude of 66 W to
54 E; and from a latitude of 22 N to 29 S. A recent study
reported the presence of Lutzomyia (a vector of thismicroscopy (40 magnification). (B) Zoom of image A.
812 D.E. Cargnelutti et al.disease) in the provinces of Santa Fe and Entre Rı´os, but so
far there are no reports of infection in animals or humans.4
Mendoza city (32530 LS, 68490 LW), capital of the
Mendoza province, is located in the foothills that extend to
the east of the final slopes of the Andes and is bordered by
Chile. The city is 750 m above sea level. It is a semiarid
temperate zone, although the mountain ranges provide a
certain degree of aridity with scarce rainfall (average
annual rainfall, 250 mm; average annual temperature,
18.8C). The city of Mendoza is more than 800 km away
from the area of transmission of the American tegumentary
leishmaniasis and lies in a completely different ecological
region to those where sand fly insect vectors are found.
During discussions, the patient denied several times
having traveled outside the province of Mendoza. Once the
diagnosis was established, the patient had to be hospital-
ized for treatment. The patient’s mother mentioned that
the girl had been born and remained in the province of Salta
until the age of 3 years. The infection in our case was thus
asymptomatic until 9 years of age, after which she devel-
oped upper lip trauma as a manifestation of this disease.
This parasitic disease is an exotic infection in Mendoza,
and in some cases, the provincial public health system has
not been able to establish the diagnosis of the infection
accurately.10 However, the present case of American
tegumentary leishmaniasis that had an unconventional
mucocutaneous presentation for the Argentine Republic
was diagnosed and treated in an efficient and accurate way
by the academicescientific staff at the School of Medicine
of the National University of Cuyo, Mendoza province.
This case demonstrates the importance of teamwork
between the health and academic institutions that allowed
for the correct diagnosis of a nonendemic parasitosis in
Mendoza. In addition, this case stresses the need for
training health-care professionals in the diagnosis and
treatment of not only endemic parasitosis within their work
zones but also nonendemic parasitosis. Globalization of
parasitosis due to the migration of populations increases
the need for continuous training in the approach to and
management of nonendemic parasitosis.
Conflicts of interest
All contributing authors declare no conflicts of interest.Acknowledgments
This work was carried out with the financial support of the
Secretarı´a de Ciencia, Te´cnica y Posgrado of the Uni-
versidad Nacional de Cuyo (Project No. 06/J417).
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Diego Esteban Cargnelutti

Carlos Guillermo Borremans

Rosa Lydia Tonelli

Liliana Carmen Carrizo

María Cristina Salomón

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Diagnosis of Leishmania infection in a nonendemic area of South America

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