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Title Andean Leishmaniasis in Ecuador caused by Infection with LeishmaniaMexicana and L. Major-Like Parasites

Author(s)

Hashiguchi, Yoshihisa; Gomez, Eduardo A.; De Coronel, Vicenta V.;Mimori, Tatsuyuki; Kawabata, Masato; Furuya, Masato; Nonaka, Shigeo;Takaoka, Hiroyuki; Alexander, J. Bruce; Quizhpe, Aida M.; Grimaldi Jr.,Gabriel; Kreutzer, Richard D.; Tesh, Robert B.

Citation American Journal of Tropical Medicine and Hygiene, 44(2), pp.205-217;1991

Issue Date 1991-02

URL http://hdl.handle.net/10069/21856

Right Copyright © 1991 by The American Society of Tropical Medicine andHygiene

NAOSITE: Nagasaki University's Academic Output SITE

http://naosite.lb.nagasaki-u.ac.jp

Am. J.Trop.Med.Hyg..44(2),1991,pp.205—217(90-165)Copyright C 1991by The American Societyof Tropical Medicine and Hygiene

ANDEAN LEISHMANIASIS IN ECUADOR CAUSED BYINFECTION WITH LEISHMANIA MEXICANA AND

L. MAJOR-LIKE PARASITES

YOSHIHISAHASHIGUCHI,EDUARDO A. GOMEZ,VICENTAV. DE CORONEL,TATSUYUKI MIMORI, MASATO KAWABATA, MASATO F@JRUYA,SHIGEO NONAKA,

HIROYUKI TAKAOKA, J. BRUCE ALEXANDER, AIDA M. QUIZHPE, GABRIELGRIMALDI JR., RICHARD D. KREUTZER, @r@iROBERT B. TESH

Kochi Medical School. Nakoku, Kochi, /apan; Facultad de Medicina, Universidad CatolicaSantiagodeGuayaquil,Guayaquil,Ecuador;InstitutoNacionaldeHigieney MedicinaTropical,

Guayaquil, Ecuador; Kumamoto University School of Medicine, Kimamoto, /apan; NihonUniversitySchoolofMedicine,Tokyo,/apan;InstituteforLaboratoryAnimals.KochiMedicalSchool, Kochi, /apan; Nagasaki University School of Medicine, Nagasaki, /apan; Oita Medical

College, Hazama, Oita, /apan; Central Florida Research and Education Center, Sanford,Florida, USA; Hospital Cantonal Paute, Paute, Azuay, Ecuador; Institute Oswaldo Cruz,

Rio de /aneiro, Brazil; Youngstown State University, Youngstown, Ohio, USA;YaleUniversitySchoolofMedicine,New Haven,Connecticut,USA

Abstract.Between 1986 and 1988,epidemiologicstudieswere carriedout in a smallrural community in an Andean region of Ecuador, where cutaneous leishmaniasis is highlyendemic. A total of 25 human cases, positive for Leishmania parasites by culture and/orsmear, were examined. Fourteen of the cases were in infants less than one year of age,suggesting intradomiciliary transmission of the disease. Clinically, many of these cases weresimilar to descriptions of―uta,―a form of cutaneous leishmaniasis which occurs in Andeanregions of Peru and is reportedly caused by L. peruviana. Of the 11 positive cultures obtainedfrom human cases in the present study, eight were identified by molecular characterizationas L. mexicana and three were identified as L. major-like. Two additional isolates of L.mexicana were also made from an infected dog and from a sand fly, Lutzomyia ayacuchensis,livingin theregion,thusimplicatingthelatterspeciesas possiblereservoirandvector, respectively, of L. mexicana in this highland community. The significance andvalidity of recent isolates of L. major-like parasites from the New World are also discussed.

Cutaneous and mucocutaneous leishmaniasisare endemic in Ecuador; the disease occurs inboth lowland and Andean regions of the country.―2To date, five different species of the parasite (Leishmania braziliensis, L. panamensis, L.guyanensis, L. mexicana and L. amazonensis)have been isolated from Ecuadorian patients withthe disease.3'4 In a previous paper we reportedLeishmania isolates made from humans and wildmammals living in warm, humid lowland areason the Pacific Coast of Ecuador.3 Because of reports of leishmaniasis from other regions of thecountry, we decided to investigate the etiologyof the disease in a representative Andean focus.The present paper describes the results of thisstudy and reports human disease in highlandregions of Ecuador caused by L. mexicana andan unusual parasite closely related to L. major.

MATERIALS AND METHODS

Descrfption of the study area

The investigation was carried out in and aroundCanton Paute, a small rural community of about

2,000 people, which is situated at an altitude ofbetween 2, 300 and 2, 500 meters above sea level(2°46'S, 78°45'W) in Azuay Province, in theCentral Andean region of Ecuador (Fig. 1). Thestudy area is surrounded by rocky slopes, whichare covered with alpine vegetation consisting ofgrasses, low shrubs and Agave (Fig. 2A—D).Treecover is restricted to the lower mountain slopesand consists of sparse Eucalyptus groves. Theclimate during the dry season (May to October)is moderate, with temperatures during the dayreaching 15—25°Cbut dropping to as low as 6°Cat night because of the altitude. During the rainyseason (November to April), the humidity andtemperature are somewhat higher.

The outdoor activities of the local inhabitantsare confined mainly to daylight hours because ofthe low nocturnal temperatures; consequently,the people do not usually enter the surroundingcountryside for hunting, work or other purposesafter dark. Crops raised in the area include potatoes, corn, beans, lentils and lettuce; domestic

205

206 HASHIGUCHI AND OTHERS

Isolation of parasites

In order to identify the causative agent(s) ofcutaneous leishmaniasis in the area, an attemptwas made to isolate Leishmania parasites fromhuman cases, domestic dogs and potential sandfly vectors. The dogs and sand flies were obtainedfrom the Don Boscos housing project in Pauteand from three surrounding localities, Yumacay,Cenacuro and Tutucan, situated 1 to 3 km fromthe town (Fig. 2C). Material for culture was collected by syringe aspiration from cutaneous ulcers of human patients and from the livers ofdogs; this material was inoculated directly intotubes ofblood agar. Phlebotomine sand flies werecaptured, as described above, and then dissectedand examined for the presence of promastigotes;if positive, the gut contents were aspirated intoa syringe and inoculated into the nose of a Syrianhamster. If local swelling or an ulcerative lesionsubsequently developed at the inoculation site,the affected tissue was biopsied, triturated andthen cultured in tubes of blood agar.

The medium used for culture was slightlymodified from that described by Walton and others.7 It contained 40 g of Difco blood agar base(Difco Laboratories, Detroit, MI), 1,000 ml ofdistilled water and 20% defibrinated rabbit blood.Two ml of melted medium were poured intoglass culture tubes, and the tubes were sealedwith rubber caps. These blood agar slants wereleft at room temperature for several hours toallow formation of condensation fluid, and thenwere stored at 4°Cuntil used. Prior to use, anoverlay of sterile saline (0.9%) was added to eachtube. Two drops of a 20% gentamycin solutionwere sometimes added to combat bacterial contamination.

Parasitecharacterization

Most of the Leishmania isolates made duringthis study were characterized by three differentmolecular techniques: isozyme electrophore515,8.10 indirect radioimmune assay using specific

monoclonal antibodies,' ‘‘@and restriction endonucleasefragmentpatternsofkDNA.'6―8―9These procedures are done routinely in our laboratories (G.G., Jr. and R.D.K.) for the identification of Leishmania parasites; the techniqueshave been described in detail in previous publications.3' 8-19

PACIFICOCEAN

Fiouar 1. Mapof the Republicof Ecuador,showing locationof the studyarea(Paute),principalcities,and other localities whereL. mexicana or L. majorlike parasites were isolated. Shaded area on map indicatestheAndeanregionofthecountryandelevations

@ 1,000 metersabove sea level.

animals present in the community are dogs, guinea pigs, rabbits, cattle, horses and chickens. Thewild mammal fauna of the area is somewhat depauperate and consists of mainly rats, mice, rabbits, opossums, mustelids and wild canines.

Sand fly collections

Collections of phlebotomine sand ifies weremade between the hours of 18:00 and 21:00, byaspirating the insects as they came to bite humanvolunteers sitting around the rocks and animalburrows where resting sand ffies had been encountered during the day. The collection techniques were described in detail in two earlierpublications.5'6

Examination of patients with dermal lesions

Laboratoryand clinicalexaminationsof thesubjects were performed in the town at the Hospital Cantonal Paute. All necessary informationon subjects was recorded on registration cards prepared by the authors. Specimens for smear andculture were taken from persons with suspectedleishmanial ulcerous lesions visiting the hospital.House visits were also made in the communityto search for unreported leishmaniasis cases; subjects with suspicious dermal lesions were taken tothe hospital for further detailed examinations.

PERU

207ETIOLOGY OF ANDEAN LEISHMANIASIS IN ECUADOR

Fioui@2. Generallandscapeofthestudyarea(CantonPaute).A.Entrancetothetown:Yumacay mountainwhere the sand ffies were collected is indicated by arrow. B. Center and outskirts of Paute, showing humandwellings.C. Viewof Paute from Yumacay mountain, showingthe high rocky hills surrounding the town. Arrowsindicate the sites where positive leishmaniasis cases lived; letters identify various sites in the community. a =Yumacay, b = Don Boscos,c = Paute proper, d = Tutucan. D. Ecologicfeaturesof Yumacay mountain illustratinglow shrubs and rocky terrain above Paute.

of age. None of the adults seen with dermallesions were diagnosed as having Leishmaniainfection.

Most of the positive lesions (88%) occurred onthe face, with the remainder on the ear, arm andfoot (Table 1). The localization of leishmaniallesions to these exposed sites is probably a reflection of the cool climate and the fact that mostof the rest of the body is covered at night, particularly in small children. The number of lesionsper person ranged from 1 to 9; only 24% of thecases had more than two lesions. Almost all ofthe subjects had small, localized lesions, measuring less than 5 mm in diameter (Figure 3B).

RESULTS

Clinical and epidemiologic studies

As shown in Table 1, a total of 25 personswith active cutaneous leishmaniasis were identified; these subjects all had ulcerous lesions withabundant amastigotes present in smear specimens. No sex difference in the incidence of thedisease was observed among this group of subjects: 13 were males and 15 were females. Interestingly,allofthesubjectswerechildren;theirages ranged from 3 months to 9 years. Fourteenof these children (56%) were less than one year

Dura

Age°SexNumber of lesionsSize of lesions° (number)Site of lesionstionm(months)Smear#Culture result#(identification)4YM15x2face5+06YF115

x 10face14++ (L.major-like)12MF14x4face14+03MF25x3,2x2face3+05YF13

x 3face7++ (L.mexicana)7MF15x4face3+025MF12x2face24+039MF23

x 3(2)face3+0l2MM23x2,2x2face2+020MF44x3,3x2

2 x 2(2)face6+09YM17x5foot4+0SMM15

x 5arm3++ (L.major-like)5YM25x4,3x2ear,arm2+0SMM25

x 4, 4 x 3face4++ (L.mexicana)lOMM23x2,2x2face4+09YM12x2face3+01

lMM11 x 2face7++ (L.mexicana)1lMF13 x 2face9++ (L.mexicana)1OMF33

x 3 (3)face8++ (L.mexicana)lOMM33x2,2x2(2)face4+01OMM45

x 5, 2 x 21 x 1 (2)face4++

(L.mexicana)11MM22

x 2(2)face9++(L.major-like)SMM13x 2face4++ (L.mexicana)7MF95x 4, 3 X 3 (2)

1 x1(6)face4+09MF54x 4, 2 x 2(20)

1 x 1 (2)face2++(L. mexicana)

208 HASHIGUCHIAND OTHERS

TAmE 1

Clinicalandparasitologicalfindingsin25 human casesofcutaneousleishmaniasisseeninPaute,Departmentof Azuay, Ecuador between July 1986 and August 1988

* Y — years; M months.

Size of lesion in mm.m@).,@r@uonof disease at time patient was first seen.# Positive smear or culture (+); Negative culture (0). Identification based on molecular characterization of isolates, a of isolates, as noted in text.

The incidence of new leishmaniasis cases inthe community varied according to the time ofyear; the majority ofsubjects were infected during the rainy season (November to April). Theduration of the disease was recorded as the timeperiod between the earliest awareness of the infection and the date offirst hospital examination(Table 1). This period varied in length from 2 to24 months, although most of the lesions (88%)were ofless than 12 months duration. This timewas undoubtedly influenced by the age of subjectpopulation, since many ofthe cases were in children less than 1 year ofage. Parents of 3 infants(ages 3, 8 and 12 months) reported that the infections were noted shortly after birth at 3, 30and 15 days, respectively. The homes of thesesubjects were situated close to the rocky habitat(rock crevices) where sand flies were observedresting during the day and where the insects were

collected from human bait at night. The proximity of sand flies to houses and the young ageofmany ofthe subjects suggest that these insectsprobably enter human dwellings at night to feedand that intradomiciliarytransmission ofthe disease is occurring in Paute.

A total of 11 Leishmania isolates were madein Paute from the 25 human cases with amastigote-positive smears (Table 1). Three of theseisolates were identified as L. major-like and eightwere identified as L. mexicana (Tables 1 and 2).An effort was also made to isolate parasites fromdomestic dogs in the area. Two of3O dogs (6.7%)examined from Don Boscos and Yumacay werepositive on smear for leishmanial infection; parasites cultured from one ofthese positive animalswere identified as L. mexicana. All ofthe isolateswere characterized by two or more of the molecular techniques described above.

209ETIOLOGY OF ANDEAN LEISHMANIASIS IN ECUADOR

FIGuRE3. Representative lesions of cutaneous leishmaniasis patients in Paute. A 9-month old girl with 5lesions (4 x 4, 2 x 2, 2 x 2, 1 x 1 and 1 x 1 mm) of2 monthsdurationon herface. B. Two small lesions (3x 2 and2 x 2 mm)onthefaceofa 10-montholdboy.C.A somewhatlargerlesion(15 x 10mm),ontheface of an 11-monthold male infant.

During other studies on the epidemiology ofleishmaniasis in Ecuador, two additional isolatesof L. mexicana and the L. major-like parasitewere made from subjects living in Alausi, Department of Chimborazo, and Quininde, Department of Esmeraldas, respectively (Figure 1).Alausi is a highland community (elevation 2,500meters), located about 80 km north of Paute;Quininde is situated in the Pacific lowlands. Thelatter two parasite strains were included with theeight L. mexicana and three other L. major-likehuman isolates from Paute in the comparativemolecular studies described below. Both of thesubjects from whom these strains were isolatedhad single, localized cutaneous lesions.

During1988,apreliminarysurveyofthesandfly fauna of the Paute study area was made. Diurnal collections revealed the presence of twoclosely related species, Lutzomyia ayacuchensis,Caceres and Bianchi Galati and Lu. osornoi Ristorcelli and Van Ty. The former was more abundant in human bait collections. Two of 97(2.1%)Lu. ayacuchensis collected at that time from Yumacay and Cenacuro had promastigotes in theirguts.6 Parasites were present in the anterior andposterior portions of the midgut but were notfound in the hindgut, indicating that they weresuprapylarian in their development. One of thesesamples was successfully cultured and subsequently identified as L. mexicana. These prelim

Stock Designation4 Clinical form' Geographic origin Parasite species

210 HASHIGUCHI AND OTHERS

TAm.a 2

Origin and identification of Leishmania reference strains and 15 isolates from Ecuador which were characterizedby monoclonai antibodies', isoenzyme2 and/or schizodeme (kDNA)3 analyses in this study

Reference strainsLl85 MHOM/BR/77/LTBOO16Ll86 MHOM/VE/001L20L562 MHOM/PA/7l/LS94*L564 MORYIPA/68/GML3L565 MHOM/BR/75/M4147L566 MHOM/BR/75/M2903L568 MHOM/VEJ74/PM@H3*L569 MHOM/BR173/M2269L571 MHOM/SU/S8/str.OD@L577 MNYG'B1162/M379L580 MHOM/ET/72/Ll00@L581 MHOM/SU/73/5-ASKHL867 MHOM/VEJ6O/LtRodL880 MHOM/BR/87/IM3 147L88 1 IHAR/CO/85/CL500L886 MHOM/IL'79/LRC-L25 1L888 MCHO/EC/82/LsplL892 MPOT/EC/87/G-03L894 MHOM/EC/87/G-07Ll023 MHOM/BR/8 l/M6426°

EcuadorianisolatesL890 MHOM/EC/86/PauteL895 MHOM/EC/87/G-09Ll222 MHOM/E@Y88/Paute1Ll223 MHOM/EC/88/Paute6L1224 MHOM/EC/88/Paute7Ll225 MHOM/EC/88/Paute23L1226 MHOM/EC/88fPaute24Ll 227 MHOM/EC/88/Paute2SLl228 MHOM/EC/88/Paute27Ll229 MHOM/EC/88/Paute29Ll230 MHOM/EC/88/PautelO3L123l MHOM/EC/88IPautel15Ll233 MCAN/EC/88/Pautelnu2Ll363 IAYA/EC/89/PAI1L1364 MHOM/EC/89/AL1

CLDCLCLVLCLCLCLCLCLCLDCLCLDCLYL

CLVLVLCLCL

Bahia,BrazilVenezuelaCanal Zone, PanamaDarien, PanamaPara, BrazilPara, BrazilPara, BrazilPara, BrazilAzerbaijan, USSRCayo, BelizeWollo, EthiopiaTurkmen,USSRVenezuelaAmazonas,BrazilSantander,ColombiaJericho,IsraelGuayas,EcuadorBolivar,EcuadorPichincha,EcuadorPara, Brazil

CL Azuay,PauteCL Esmeraldas,QuinindeCL Azuay, PauteCL Azuay, PauteCL Azuay,PauteCL Azuay, PauteCL Azuay,PauteCL Azuay, PauteCL Azuay, PauteCL Azuay, PauteCL Azuay, PauteCL Azuay,PauteCL Azuay,Paute— Azuay, Paute

CL Chimborazo,Alausi

L. amazonensisL. major-likeL. panamensisL. aristidesiL guyanensisL. braziliensisL. venezuelensisL. amazonensisL. tropicaL. mexicanaL.aethiopicaL. majorL major-likeL. amazonensisL. colombiensisL majorL. equatorensisL.amazonensisL. panamensisL. lainsoni

L. major-likeL major-likeL. mexicanaL. mexicanaL. mexicanaL. mexicanaL. major-likeL. mexicanaL. mexicanaL. mexicansL. mexicansL. major-likeL. mexicansL.mexicansL. mexicana

Leishmania species classified by monoclonal antibodies according to their serodeme type.‘Leishmaniaspecies classified by enzyme electrophoresis according to their enzyme patterns.‘Leishmaniaspecies classified according to their restriction-endonuclease fragment patterns of kDNA.‘Designationcode: Host (M for Mammalia CAN - cams familians; CHO - Choloepus hoffmannr, DAS - Dasypus novemcinctus; HOM -

Homo sapiens;NYC - Nyctomyssumichrasw,ORY - Or,vzomyscapfto@,andPOT - Potusfiavus-,IforInsecta:AYA - Lutzomyiaayacuchensis',and LIAR - Lutzomyia hartmanl) /country of origin /year of isolation /original code.

‘CL—cutaneous leishm*niaais DCL —diffuse cutaneous leishmsniaa@içand VL —visceral leishmsnissis.* WHO Leishniania reference strain.

inary data suggested that Lu. ayacuchensis mightbe a vector of this parasite in the Paute area.Accordingly, in September, 1988, a 13-monthlongitudinal study was initiated to determine theseasonal variation in Leishmania infection ratesand the biting activity of Lu. ayacuchensis inCanton Paute. The results of this latter studywere published elsewhere.3'6 Of 2,600 Lu. ayacuchensis collected from human bait over the13-month period, 95 (3.7%) were found to benaturally infected with promastigotes. Although

these parasite-positive insects were not cultured,the promastigotes were always observed in thesand fly midgut, indicating that they were probably members of the mexicana complex.

Laboratory studies

Isoenzyme electrophoresis. Ten of the Pauteisolates, (eight from humans and one each froman infected dog and a sand fly) and the singlehuman isolate from Alausi had identical allo

211ETIOLOGYOF ANDEANLEISHMANIASISIN ECUADOR

123 45612 3 6

0-

V

LL

+

0@0

MPICM

1 231— -1------

4 5 6

v-iV V

•PCDHFIGURE 4. Diagrammatic representations of the electrophoretic patterns (allomorphs) of glucose phosphate

isomerase (GPI), mannose phosphate isomerase (MPI) and 6-phosphogluconatedehydrogenase(6 PGDH),distinguishingthe2 groupsofEcuadorianisolatesand showingthesimilaritiesintheenzymeprofilesofeachgroupwith L. mexicans and L. majorreferencestrains.Lane 1 representsthe allomorphsof 72 L. mexicansstrains;lane 2 shows allomorphsof the Pauteand Alausi L. mexicans strainslisted in Table 2; lane 3 of 165L. amazonensis strains; lane 4 of 69 L. major strains; lane 5 of L. major-like strains Ll23l from Paute, L895fromQuininde,andthe referencestrainL867 fromVenezuelalane6 of 54 L. tropicaisolates.0 is origin.Anodeis at the top of each diagram.The arrowidentifiesthe most frequentallomorph.

morphs (bands of enzyme activity observed byelectrophoresis); these enzyme electrophoreticprofiles were similar to those possessed by theWHO L. mexicana reference strain (stock codeL577 in Table 2) for the enzymes GPI, MPI, and6 PGDH (Figure 4). The latter enzymes are useful in distinguishing most Leishmani&°and indicate that the aforementioned isolates were L.mexicana.

One of the remainingthree human isolates fromPaute (L123 1), plus the single strain from a subjectin Quininde (L895)were examined by isozyme electrophoresis. These two isolates wereextremely homogeneous and had identical elec

trophoretic profiles for each of the enzymes tested (up to 20 enzymes). Their profiles were distinctfrom thoseof any of the WHO referencestrains or from other stocks of the L. mexicana,L. braziliensis, and L. donovani complexes reportedpreviously@―°forthesame enzymes. Incontrast, the enzyme electrophoretic profiles ofthese two isolates were similar to those possessedby 68 L. major reference strains, including theWHO reference strains L58l and L886 (Figure4). A similar profile was also produced by parasite strain L867 which was originally isolatedfrom a case of diffuse cutaneous leishmaniasis(DCL) in Venezuela and was used in another

Stockcode Species'Monoclonal

antibodies'M2M3MiM8P9VlTlT2T3T4T8TlOReference

strainsL569L.amazonensis32.012.713.72.42.01.81.22.10.80.91.01.6L564L.aristidesi26.91.40.81.61.41.01.41.81.41.61.30.9L577L.mexicans1.61.826.232.91.61.21.57.37.71.24.41.0L568L.venezuelensis1.21.00.81.22.07.43.65.210.41.62.31.1L867L.major-like1.21.31.61.0830.837.633.740.216.08.32.4L568L.major1.21.52.12.41.51.738.434.841.017.712.32.2L571L.tropica1.00.72.01.01.41.68.418.922.65.92.720.8L580L.aethiopica1.01.21.61.21.61.33.67.616.91.11.31.0Ecuador

isolates4Ll222L.mexicans1.61.86.64.41.91.612.019.326.46.36.81.9L1233L.mexicans2.12.01.83.92.11.814.22333139.76.81.8Ll224L.mexicana1.71.71.84.31.81.78.718,324.45,36.11.4L1363L.mexicans1.01.63.45.21.41.122.319.624.810.35.61.4Ll364L.mexicans1.52.02.44.21.51.617.723.626.75.64.61.2L890L.major-like2.62.31.81.26.41.826.138.042.310.0731.6L895L.major-like1.71.21.01.25.81.336.913.69.87.25.00.8L1226L.major-like1.00.81.01.6431.08.218.225.03.64.81.0L123lL. major-like0.81.00.71.36.21.622.722.123.89.25.61.2

212 HASHIGUCHI AND OTHERS

T@aii 3A comparisonofthebinding'ofLeishmarnaspecies-specificmonoclonslantibodiestoreferencestrainsandselected

Leishmania isolatesfrom Ecuador

Results shown express the ratio ofcpm boundantibody/cpm bound control; values >3 were considered positive and appear in boldface type.2 Stock identification (classification) also established by isoenzyme and/or schizodeme (kDNA) analyses.

3 From the hybridoma clones: M2, IX-2H7-ElO; M3, IX-5H9.ClO; Mi, LXVIII.lD7-B8; M8, LXVIII-4D8-E3; M9, XLV-2B5-H7; P9, CXIII

2E9-D12; Vi, CLXXVI-3Cl-F4; Tl, XLVI-5B8-B3; T2, XLV1-4H12-C2; T3, XLVI-5A5-D4; T4, LXVIII-1A4-Gl; T8, LXVII-3E12-F8; and Tb,XCIV-bH2-A8.

4 No significant cross-reactivity was obtained to any ofthese strains with a bai@e panel of monocbonal antibodies species-specific for I. braziliensis

or L donovani complex parasites.

study'7 as a reference strain of “L.pifanoi.―Characterization of strain L867 by monoclonalantibodies and kDNA analyses is described below.

Reactivity with monoclonal antibodies. The 13Ecuadorian Leishmarna isolates were also characterized by serodeme analysis, using alarge panel ofspecies-specific monoclonal antibodies. Thespecificity ofthese monoclonal antibodies, whichwere derived for species complexes of Leishmania, have been described before.' â€â€˜@As shownin Table 3, some ofthe monoclonals which boundto reference strains of the L. mexicans and L.tropica complexes also bound to representativeparasite strains from Ecuador. In general, thestocks characterized biochemically as L. mexicana reacted wealdy with one or both of themonoclonal antibodies designated M7 (LXVIIIlD7-B8) and M8 (LXVIII-4D8-E3), which arespecific for this parasite peci3 The remainingfour Ecuadorian isolates (L890, L895, Ll226 andL123l), two ofwhich could not be distinguishedfrom the WHO L. major reference strains byzymodeme analysis, reacted with monoclonalsTi, T2, T3, T4, T8, and TlO that were originally

produced against parasites in the L. major—L.tropica ‘@ However, these latter four Ecuadorian isolates also reacted with monoclonalP9 (Table 3), which was derived from and isspecific for strain L867, described earlier as “L.pifanoi.―7 The Old World L. major strains didnot react with the P9 monoclonal antibody. Similarly, the L. mexicans isolates also reacted withthe L. major-derived monoclonals, but they didnot react with the P9 monoclonal. Based on theseresults, it appears that the P9 monoclonal antibody is specific for a group of New World parasites which are L. major-like.

Schizodeme analysis ofkDNA. A comparisonof kDNA fragment patterns from strains representing selected Leishmania complexes and Ecuadorian parasite isolates was done by schizodeme analysis of endonuclease Msp I, Hinf I(Figure 5), Alu I, Mbo I, Rsa I, and Taq I (Figure6) digests of kDNAs, fractioned by gradient acrylamide gel electrophoresis. With these digests,sequence microheterogeneity in mini-circle DNAis revealed and can be compared between different parasites. As the degree of heterogeneitywithin mini-circles varies amongspecies or strains

213ETIOLOGY OF ANDEAN LEISHMANIASIS IN ECUADOR

ofLeishmania, the fingerprint obtained with eachof the restriction enzymes is unique for each ofthese parasites.'6' 15-m9As shown in Figures 5 and6, the Ecuadorian isolates which were analyzedin this study can be clustered into two distinctgroups of parasites, according to the major sequence classes (minicircle fragments or digests)released by the six restriction endonucleases tested. One ofthesegroups (L890, L895, Ll226 andLl23l) had kDNA fragment patterns very similar to those possessed by parasite strain L867.On the basis of their kDNA “fingerprints,―theabove L. major-like isolates are distinct from theOld World L. major strains L58 1 and L886. Theyare more closely related to Leishmania strainLl86, a parasite which was isolated from a DCLcase in Venezuela, and to a group of strains'6previously described from Brazil as “L.majorlike―(unpublished data).

The second group of Ecuadorian Leishmaniaisolates(L1225,L1227, L1228, Ll233, L1363and L1364) have very similar kDNA fragmentpatterns, and are distinct from the L. major-likegroup (Figures 5 and 6). The “fingerprints―ofthis second group are more like those of the L.mexicana reference strain (L577). For this reasonwe feel that parasites in the latter group representstrains of L. mexicana.

DISCUSSION

Leishmania pifanoi was originally reportedfrom Venezuela202' where it was associated witha form of diffuse cutaneous leishmaniasis (DCL).Based upon the unusual clinical and epidemiologic features of this disease, the parasite associatedwith itwas initiallydescribedas a newspecies(L.pifano:).However, more recentmolecular studies suggest that DCL in Venezuela iscaused by a heterologous group of parasites.Studies comparing isoenzymes,'°' 22 buoyantdensities of both nuclear and kinetoplastDNA,22'23and restriction enzyme fragment patterns,23 have shown that several Venezuelan parasite strains tentatively identified as “L.pzfanoi,―including a designated WHO reference strain(MHOM/VE/57/LL1), couldnotbereliablydifferentiatedfrom L mexicana.On theotherhand,the MHOM/VE/00/L54 strain of “L.pifanoi,―which was also isolated from a case of DCL inVenezuela, has been shown to be similar to L.amazonensis by monoclonal antibodies,'3 isoenzyme and kDNA analyses.24Four othermore

recent isolates from DCL cases in Venezuela werealso found to be indistinguishable from L. amazonensis by biological criteria and enzyme electrophoresis.25 In contrast, the MHOM/VE/00/L20 strain of “L.pifanoi―26was distinct fromother species of the L. mexicans complex byzymodeme analysis (Table 2). This latter strainwas also found to be phenotypically similar toreference strains of L. major, but it could bedifferentiated by kDNA restriction enzyme profil'6 The isolate L867, which was used in ourstudy, has also previously been defined as “L.pifanoi,― based on its characteristic reactivitypatterns with specific monoclonal bodi'7However, results of the present study (Table 3and Figures 4 & 5) indicate that the L867 strainfrom Venezuela as well as the four Ecuadorianstrains(L867,L895, L1226 and L1231) areverysimilar to the L. major reference strains. It alsoappears that monoclonal P9, which was originally made to L867, is specific for this group ofNew World L. major-like parasites.

The possibility of this latter group of parasitesbeing contaminated with L. major seems highlyunlikely, given the distinct patterns obtained withmonoclonal antibody P9 (Table 3) and kDNAanalyses (Figures 4 and 5). Instead, our data confirm a previously reported study,'6 indicating thatL major-likeparasitesoccurnaturallyintheNewWorld. Work is now in progress to better definethe phylogenetic relationship between these parasites and Old World L. major strains.

The Venezuelan L. major-like parasite, L867,was originally isolated from a case of DCL. It isnoteworthy that only one of the four Ecuadoriansubjects yieldingL major-like parasites had morethan 1 ulcerative lesion (Table 1);all were simplecutaneous lesions.

Ten isolates of L. mexicana were made fromPaute and one from Alausi. Corredor and others27recently reported the isolation of L. mexicanafrom three subjects living in Pueblo Rico, Department of Risaralda, and Samaniego, Department of Narino, in the neighboring country ofColombia. The latter two towns are situated inthe western Andean cordillera of Colombia atapproximately 1,550 meters elevation;27 thismountain range is contiguous with the monatains encompassing Paute and Alausi. The distance between these four towns (approximately730 km in a straight line from Pueblo Rico toPaute) suggests that L. mexicans may have afairly wide geographic distribution in the Andean

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FIGURES. Acrylamide gradient (5-12%) gel electrophoresis comparison of kDNA fragment patterns, generatedwiththerestrictionenzymesMspI (a,b)and HinfI (c,d),amongselectedspeciescomplexesof Leishmaniaand representativeparasitestrainsfrom Ecuador.The stock codes of the strainsstudiedby kDNA analysisareindicatedabove the lanes;informationon theiroriginis given in Table2. Theapparentsizesof kDNA fragmentsareestimatedrelativeto fragmentsfroma Hae IIIdigestof 0 x 174 RF DNA and theirmolecularweightsareindicatedinbasepairs(bp)besidethefigures.UndigestedkDNA networkisvisibleintheslotsatthetopofthe gels, whereaspresumptivemaxicircleDNA fragmentsand minicircleoligomersmigrateat 6 kb and arevisiblejust below. The linearizedminicirclesmigrateat about0.8 kb and, in some lanes, the minicircledigestsappearasbands(ladders)between0.05and0.7kb.

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regionofnorthernSouthAmerica.Becauseofitsaltitude and temperate climate, this Andean region is ecologically quite distinct from the warmlowland areas where L. mexicans generally occurs in Central America and Mexico.2

There are several other unusual features aboutthe Paute focus of cutaneous leishmaniasis. First,active ulcers were observed only in children (Table 1), indicating that the disease is highly endemic in the community. Second, many of the

215ETIOLOGY OF ANDEAN LEISHMANIASIS IN ECUADOR

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from selected species complexes of Leishmania and representative parasite strains from Ecuador. DNA wasfractionedasdescribedinFig.1.The stockcodesoftheLeishmaniastrainsanalyzedareindicatedabovethelanes, and their origins are shown in Table 2. Molecular weights are indicated in bp beside the figures.

active infections were in infants less than oneyear of age, implying intradomiciliary transmission. The latter mode of transmission is furthersuggested by the finding of infected anthropophiuic sand flies in rock crevices near humandwellings and by the low night-time temperatures which tend to keep children indoors duringthe period of maximum sand fly biting activity.

Another interesting aspect of leishmaniasis in

Paute is its clinical similarity to “uta,―a cutaneous form of the disease described in Peru.29“Uta―is prevalent among inhabitants of thewestern slopes and valleys of the Peruvian Andesat elevations between 600 and 3,000 meters abovesea level;29this disease is generally thought to becaused by L. peruvians.28 However, recent molecular studies' @.@ @‘suggest that L. peruvians isnot a distinct species, but probably a variant of

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216 HASHIGUCHI AND OTHERS

L. braziiensis. The clinical manifestations of theL. mexicans and L major-like infections in Paute(Table 1and Figures 3A—C)are indistinguishablefrom case descriptions of “uta.―28Furthermore,the ecology of the Paute area is quite similar tothat of the “uta―-endemicregion of Peru.28' 29These latter findings suggest that “uta―as a chinical entity is not restricted to Peru, and that thedisease is probably caused by more than oneLeishmania species.

The isolations ofL. mexicans from a dog andfrom Lu. ayacuchensis are noteworthy, since bothare new hosts records for this parasite. In Mexicoand Central America, forest rodents are thoughtto be the important reservoir ofL. meic32The isolation ofthis parasite from a local canineraises the possibility that dogs could be a domestic reservoir ofL. mexicana in Paute. Interestingly, dogs have long been thought to be themajor reservoir of L. peruvians (L. braziliensis)and of―uta―in Andean regions of Peru.28'32Sandfly isolations of L. mexicans have been madebefore from Lu. olmeca olmeca and Lu.ylephiletor,2theformerspeciesisthoughttobethe principal vector. Our isolation of this parasitefrom Lu. ayacuchensis, a man-biting speciesfound around human dwellings in Paute,6 addsanother potential vector to the list.

Acknowledgments:We wish to thank Dr. Diane McMahon-Pratt,Yale UniversitySchoolof Medicine,forproviding the monoclonal antibodies used in this study.We also express appreciationto our collaboratorsattheInstitutoNacionalde HygieneyMedicinaTropical,Guayaquil,Ecuador.

Financialsupport:OverseasScientificResearchProgramof the Ministryof Education,Scienceand Culture, Japan (grants 61041059, 62043055 and63041096); the UNDP/World Bank/WHO SpecialProgrammeforResearchandTraininginTropicalDiseases; the Brazilian Nacional Council of Scientific Development and Technology (CNPq), reference no.204011/88.4; the National Institutesof Healthof theUnited States (grants AI-2 1049 and AI-23004); andthe MacArthurFoundation (MolecularParasitologyProgram).

Authors' addresses: Yoshihisa Hashiguchi, Department of Parasitology,Kochi MedicalSchool, Nankoku, Kochi, 781—52,Japan;EduardoA. Gomez, Departmento de Parasitologia,Facultad de Medicina,Universidad Catolica,Santiagode Ouayaquil,Apartado 4671, Guayaquil,Ecuador,VicentaV. de Corond, Departmento de Parasitologia, Instituto Nacionalde Higiene y Medicina Tropical, Apartado 3961, Gunyaquil, Ecuador;Tatsuyuki Mimori, DepartmentofParasiticDiseases, Kumamoto University School ofMedicine,Kumamoto860, Japan;MasatoKawabata,

Department of Clinical Pathology, Nihon UniversitySchool of Medicine, Tokyo 173, Japan; Masato Furuya, Institute for Laboratory Animals, Kochi MedicalSchool, Kochi 781—51,Japan;Shigeo Nonaka, Department ofDermatology, Nagasaki University SchoolofMedicine, Nagasaki 852, Japan; Hiroyuki Takaoka,Division of Medical Zoology, Oita Medical College,Hazama, Oita 879—56,Japan; J. Bruce Alexander,Central Florida Research and Education Center, Sanford, Florida 32771-9608, USA; Aide M. Quizhpe,Hospital Cantonal Paute, Paute, Azuay, Ecuador; Gabriel Grimaldi Jr., Department oflmmunology, Instituto Oswaldo Cruz, CEP 21045 Rio de Janeiro, Brazil;Richard D. Kreutzer, Department ofBiology, YoungstownStateUniversity,Youngstown,Ohio44555, USA;Robert B.Tesh, Department ofEpidemiology & PublicHealth, Yale University School ofMedicine, New Hayen, Connecticut06510, USA.

REFERENCES

1. Hashiguchi Y, Gomez EA, 1987. A brief reviewofleishmarnasis in Ecuador. Hashiguchi Y, ed.Studies on New World leishmaniasis and itstransmission, with particular reference to Ecuador: Res Rep Ser No 1. Kochi, Japan: KyowaPrintingCo., 5—32.UI: 8916555

2. Grimaldi 0 Jr, Tesh RB, McMahon-Pratt D, 1989.A reviewofthe geographicdistributionand epidemiology ofleishmaniasis in the New World.AmJTropMedHyg4l:687—725. UI: 90265130

3. Mimori T, GrimaldiG Jr, KreutzerRD, GomezEA, McMahon-PrattD, Tesh RB, HashiguchiY, 1989. Identification,using isoenzyme electrophoresis and monoclonal antibodies, ofLeishmania isolated from humans and wild animals of Ecuador. Am J Trop Med Hyg 40:154—8.

4. ArmijosRX, ChicoME,CruzME,GuderianRH,Kreutzer RD. Berman JD, Rogers MD, Grog!M, 1990. Human cutaneous leishmaniasis inEcuador identification of parasites by enzymeelectrophoresis.Am J TropMedHyg 42: 424—8. UI 90252908

5. Gomez EA, CoronelVV de, HashiguchiY, 1990.SeasonalvariationintheinfectionrateswithLeishmania and in the biting activity of the sandflyLuayacuchensisin anAndeanleishmaniasisendemic area of Ecuador. HashiguchiY, ed.Studies on New World Leishmaniasis and itstransmission,withparticularreferencetoEcuador: Res Rep Ser No 2. Kochi, Japan: KyowaPrintingCo., 49—60.UI 8916555

6.TakaokaH,GomezEA,AlexanderJB,HashiguchiY, 1990. Natural infections with Leishmaniapromastigotes in Lutzomyia ayacuchensis (Diptera: Psychodidae) in an Andean focus of Ecuador.JMedEntomol27:701—2.UI 90355155

7. Walton BC, Shaw JJ, Lainson R, 1977. Observationsonthein vitrocultivationof Leishmaniabrazihiensis. J Parasitol 63: 1118—9.UI78068440

8. KreutzerRD, ChristensenHA, 1980. CharacterizationofLeishmaniaspp.by isozymeelectro

217ETIOLOGY OF ANDEAN LEISHMANIASIS IN ECUADOR

phoresis. Am I Trop Med Hyg 29: 199—208.UI 80172968

9. Kreutzer RD. Semko ME, Hendricks LD, WrightN, 1983. Identification of Leisb.mania spp. bymultiple isozymeanalysis. Am J TropMed Hyg32:703—15.UI 83280475

10. Kreutzer RD. Souraty N, Semko ME, 1987. Biological identities and differences among Leishmania species and sub species. Am J Trop MedHyg36: 22—32.UI 87125502

11. Jaffe CL, Bennett E, Grimaldi G Jr. McMahonPratt D, 1984. Production and characterizationofspecies-specific monoclonal antibodies againstLeishmania donovani for immunodiagnosis. JImmunol 133:440—7.UI 84214111

12. Pratt DM, Bennett E, Grimaldi G, Jaffe CL, 1985.Subspecies- and species-specific antigens ofleishmania mexicanacharacterized by monoclonal antibodies. J Immunol 134: 1935—40.UI85106290

13. Grimaldi GJr, DavidJR, McMahon-Pratt D, 1987.Identification and distribution of New WorldLeishmania species characterized by serodemeanalysis using monoclonal antibodies. Am JTropMedHyg36: 270—87.UI 87154108

14. McMahon-Pratt D, Bennett E, David JR. 1982.Monoclonal antibodies that distinguish subspeciesofLeishmaniabraziliensis. Jlmmunol 129:926—7.UI 82267067

15. Jaffe CL, McMahon-Pratt D, 1983. Monoclonalantibodies specific for Leishmarna tropica. I.Characterization of antigens associated withstage- and species-specific determinants. JImmum@!131: 1987—93.UI 84009127

16. Momen H, Grimaldi G Jr, Pacheco RS, Jaffe CL,McMahon-PrattD,MarzochiMC, 1985.Brazilian Leishmania stocks phenotypically similarto Leishmania major. Am J Trop Med Hyg 34:1076—84.UI 86212794

17.Pan AA, McMahon-PrattD, 1988.Monoclonalantibodies specific for the amastigote stage ofLeishmaniapifanoi.I.Characterizationofantigens associated with stage- and species-specificdeterminants. J Immunol 140: 2406—14.UI88170826

18. Lopes UG, Momen H, Grimaldi G Jr, MarzochiMC, Pacheco RS, Morel CM, 1984. Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral andcutaneous leishmaniasis. JParasitol 70:89—98.UI 84242239

19. Pacheco RS, Lopes UG, Morel CM, Grimaldi 0Jr, Momen H, 1986. Schizodeme analysis ofLeishmania isolates and comparison with somephenotypic techniques. Rioux JA, ed. Leishmania taxonomy and phylogeny. MontpelliecIMEEE, 57—65.

20.Medina R, Romero J,1959. Estudioclinicoyparasitologico de una nueva cepa de Leishmania.ArchVenezPaso!ParasitolMed3: 298—326.

21.MedinaR,Romero J,1962.Leishmaniapifanoi

a. sp. El agent causal de la leishmaniasis tegumentaria difl'usa. Arch VenezMed Trop 4; 349-353.

22. Chance ML, Gardener PJ, Peters W, 1977. Biochemical taxonomy of Leishmania as an ecological tool. Coioques internationaux du centredela recherchescientifique, No 239, EcologiedesLeishmanioses. Paris: Editions du CNRS, 53—62.

23. Barker DC, Butcher J, 1983. The use of DNAprobes in the identification ofleishmaniasis: discrimination between isolates ofthe Leishmaniamexicans and L. braziliensis complexes. TransR Soc Trop Med Hyg 77: 285—97.UI 8401866

24. Momen H, Grimaldi 0 Jr. 1984. On the identityof Leishmania mexicana pifanoi and L mcxicana garnhami [letter] Trans R Soc Trop MedHyg 78:701—2.UI 85066787

25. Scorza Th', Delgado 0, 1982. Amastigote morphometry and development of 4 isolates ofLeishmania mexicans pifanoi from Venezuelain Lutzomyia townsendi. Mem Inst Oswa!doCruz77:217—27.UI 83191567

26. Miles MA, Povoa MM, de Souza AA, Lainson R,Shaw JJ, 1980. Some methods for the enzymatic characterization of Latin-AmericanLeishmania with particular reference to Leishmania mexicana amazonensis and subspecies ofLeishmania hertigi. TransR Soc Trop Med Hyg74:243—52.UI 80214865

27. Corredor A, Kreutzer RD, Tesh RB, Boshell J,Palau MT. Caceres E, Duque S, Pelaez D, Rodriguez 0, Nichols 5, and others, 1990. Distribution and etiology of leishmaniasis in Columbia. Am J Trop Med Hyg 42: 206—14.UI90196458

28.LumbrerasH, GuerraH, 1985. LeishmaniainPeru. Chang K-P, BrayRS, eds. LeishmaniasisAmsterdam: Elsevier, 297—311.

29.HerrerA,l957.VerrugayutaenelvalledeHuaillacayan (Depto. de Ancash). I. Determinacionde los limites altitudinales de la zona endemicay de la incidencia de ambas enfermedades. RevMed Exp 11:40—49.

30.Romero GO, AranaM, LopezM, MontoyaI,BohiR, Campos M, Arevalo J, Uanos A, 1987.Characterization of Leishmania species fromPeru. Trans R Soc Trop Med Hyg 81: 14-24.UI 88178899

31. Lopez M, Montoya Y, Arena M, Cruzalegui F,Braga J, Llanos-Cuentas A, Romero 0, ArevaloJ, 1988. The use of nonradioactive DNA Probesfor the characterization of Leishmania isolatesfrom Peru. Am J Trop Med Hyg 38: 308-314.UI 88181354

32. Lainson R, Shaw JJ, 1987. Evolution, classification and geographical distribution. Peters W,Killick-Kendrick R, eds. The Leishmaniases inBiologyand Medicine,BiologyandEpidemiology. London: Academic Press, Vol 1, 1-120.