Tropical Medicine and International Health

volume 2 no. 2 pp 140–152 february 1997

The epidemiology of cutaneous leishmaniasis insubtropical EcuadorRodrigo X. Armijos,1–4 M. Margaret Weigel,1–3 Ricardo Izurieta,1–3 Jose Racines,1,3,4 Camilo Zurita,1,3

Walter Herrera1 and Milton Vega1

1 Laboratorio de Inmunologı́a, Facultad de Ciencias Médicas, Universidad Central del Ecuador, Quito, Ecuador2 Direccion Nacional de Epidemiologı́a, Ministerio de Salud Publica, Quito, Ecuador3 Fundacion Internacional ‘‘Biociencias’’, Quito, Ecuador4 Departamento de Inmunologı́a, Instituto Nacional de Higiene, ‘‘Leopoldo Izquieta Perez’’, Quito, Ecuador

Summary An epidemiologic survey (n=466) was conducted in an area of subtropical rainforest innorth-west Ecuador with the following objectives: (1) to determine the prevalence ofcutaneous leishmaniasis (CL), (2) to identify the Leishmania species causing human disease,(3) to investigate the major clinical manifestations of leishmaniasis, (4) to study cellular andhumoral immune response indicators associated with disease status and (5) to identify riskfactors for CL. Fourteen per cent of subjects had parasitologically confirmed CL; 33% hadevidence of prior disease. However, 17.2% of subjects with a negative CL clinical historypresented with a positive Montenegro skin test (MST), indicating the possibility ofsubclinical infection. The species isolated from subject lesions were L. guyanensis (63%),L. panamensis (33%), and L. brazilensis (4%). Mean specific anti-Leishmania IgG and IgMOD serum levels were highest in subjects diagnosed with current CL, followed by those withprior CL, and were lowest in healthy subjects, respectively (0.56&0.27 vs 0.33&0.2 vs0.22&0.14; F-ratio=74; P<0.00001) and (665&270 vs 481&220 vs 301&128.5;F-ratio=37; P<0.00001). Likewise, subjects with present CL had measurably higher MSTreactions (13&6.7 mm) than those with prior CL (10.9&7.8 mm) or healthy individuals(2.4&2.5 mm; F-ratio=106; P<0.00001). Serum concentrations of IgG were predicted bylesion number (t=2.5; P=0.018), size (t=3.7; P=0.0006), and duration (t=3.5; P=0.0013).Furthermore, the MST induration size increased as a function of lesion number (t=3.0;P=0.005) and size (t=3.4; P=0.022). Subject age and sex did not predict serum IgG or IgMconcentrations or MST reactions in the 3 disease groups. Although no sex differences werefound with respect to clinical characteristics, children ¡12 years of age were almost 3 timesmore likely to have CL lesions or scars located on the face and head area compared toadults (OR=2.75; 95% CI=1.4–5.6, P=0.004). The risk factors associated with diseaseincluded age under 5 years (AOR=1.5; 95% CI=0.48–2.35), male gender in adults(AOR=2.8; 95% CI=1.1–7.8), and wood and/or cane exterior house walls (AOR=1.8; 95%CI=1.4–2.5). In contrast, electric home lighting was associated with decreased risk(AOR=0.7; 95% CI=0.4–2.3). The results suggest that it may be possible to modify aportion of the risk of CL by making changes in the housing environment which may help toreduce the amount of human–vector contact.

keywords cutaneous leishmaniasis; prevalence; immune status indicators; clinicalcharacteristics; risk factors; Ecuador

correspondence Dr R. X. Armijos, Associate Professor and Laboratory Director,Laboratorio de Inmunologı́a, Casilla 17-21-257, Facultad de Ciencias Médicas, UniversidadCentral del Ecuador, Quito. E-mail address: inmuno@ciuc.ecx.ec

? 1997 Blackwell Science Ltd140

Introduction

Leishmaniasis is considered a public health problemof global importance (WHO 1990; 1991; PAHO1994). The world-wide incidence of the cutaneousform of leishmaniasis is estimated at 1–1.5 millioncases per annum (PAHO 1994). The prevalence ofthe disease is reported to be increasing in manyLatin American countries (PAHO 1994), includingthe Republic of Ecuador where the disease isendemic in most of the nation’s 21 provinces(Armijos & Weigel 1995). Much of the increase inthe Pacific coastal and Amazonian regions ofEcuador during the past 20 years is related to popu-lation migration and economic development activi-ties including subsistence farming, cattle ranching,timber harvesting, and mineral and petroleumexploration (Armijos & Weigel 1995; Vasconez1992). One of the regions most affected is the largeexpanse of subtropical rainforest situated in thenorth-west of the country, which includes a largeportion of the Province of Pichincha. The Ministeriode Salud Publica (MSP) estimated the annual inci-dence of cutaneous leishmaniasis in rural areas ofthe province at 47.5/10000 between 1985 and 1989(MSP 1990). However, these data were collected bypassive detection methods. Moreover, they includedrural population groups in the High Andes wherethe disease is rare or non-existent but neglectedgeographically isolated groups in the subtropicallowlands where the prevalence of cutaneous leish-maniasis appeared to be high. In addition to the lackof reliable data regarding disease prevalence, therewas little known about which Leishmania specieswere responsible for causing human disease in thearea and the common clinical manifestations, cellularand humoral immune response and risk factorsassociated with cutaneous leishmaniasis. This infor-mation has practical value for disease control. Thus,the study was undertaken with the following majorobjectives: (1) to determine the prevalence of cutane-ous leishmaniasis, (2) to identify the Leishmaniaspecies associated with human disease, (3) to investi-gate the major clinical manifestations of the disease,(4) to study cellular and humoral immune responseassociated with cutaneous leishmanias, and (5) toidentify modifiable and non-modifiable risk factorsassociated with disease.

Materials and methods

Description of the study site

The study was conducted during an 18-monthperiod (1989–1991) in 26 agricultural hamletslocated in a remote primary subtropical rainforest.This area is situated in the north-west corner ofPichincha Province, Ecuador, approximately 125statute miles from the capital, Quito (Figure 1). Thesubtropical rainforest in which the study was con-ducted ranges in elevation from 500–1800 metresabove sea-level. The mean annual temperature rangeof the study area is 27–30)C, according to geo-graphic elevation. The major rainy season lasts fromDecember until April, during which time most of thesecondary roads and trails to the communities areimpassable except on foot or by pack animals.

Subject selection

The selection of the study site was based on preva-lence estimates of cutaneous leishmaniasis from theMinistry of Public Health which indicated that thedisease was endemic in the region (MSP 1985).Informal surveys by the study team in the precedingyear suggested that the actual prevalence was higherthan official figures indicated. Government censusdata estimated the total population of the area at3985 persons (INEC 1987). It was decided to sample

Figure 1 Map of Ecuador. Shaded area represents thelocation of the study site in north-west Pinchina Province.

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10% of the total population for the purpose ofobtaining the prevalence estimates and other data forthe survey. The study area was divided into equallyspaced sectors using highly detailed official militarygrid maps obtained from the Ecuadorian Institute ofMilitary Geography. Twenty-six map sectors wereselected using a random procedure (Kalton 1983). Arandom selection of prospective subjects from eachhousehold was made for each sector based on popu-lation size estimates obtained from the census data(INEC 1987). The prospective subjects were thencontacted and asked to participate in the survey.Individuals who agreed to participate then under-went clinical and laboratory examinations to deter-mine their leishmaniasis status. None of the adultsubjects nor the parents/guardians of minor subjectsselected for the study refused to participate. The sub-jects were placed into one of 3 clinical groups forthe purpose of the data analyses. The groupsincluded individuals without clinical or laboratoryevidence of current or past disease (Group 1), thosewith clinical evidence of prior disease (Group 2), andthose with parasitologically confirmed evidence ofcurrent disease (Group 3). Informed verbal consentrather than written consent was obtained from sub-jects due to the high illiteracy rate of the populationand their distrust of signing official documents thatthey could not read. Everyone in the area surveyedreceived the same level of free medical care duringthe visits, regardless of whether they participated inthe research portion of the study. The study wasapproved by the Ethics Committee of the Schoolof Medical Sciences in the Central University ofEcuador. Data were collected on a total of 466 indi-viduals or 11.5% of the total population. The datacollected included subject sociodemographic andother characteristics, leishmaniasis prevalence andclinical characteristics and risk factors. The datawere collected by questionnaire, clinical history,physical examination and laboratory analysis.

Subject interviews

Subjects were questioned by trained interviewersusing a data collection instrument which elicitedspecific information regarding subject age, sex,marital status, primary occupation, family size,land and house ownership, level of technological

sophistication, residence and migration patterns,health care access, house construction materials,elevation of bedrooms, water and electricity sources,garbage and waste water elimination, use of metalbedroom window screens, placement of home gar-dens and orchards, and the presence of putativemammalian reservoirs in the area.

Clinical history and physical examination

A detailed clinical history, including disease andtreatment history, was taken for each subject. Thecomprehensive physical examination involved adetailed examination of the skin and mucous mem-branes and specifically concentrated on the detectionof present or past cutaneous leishmaniasis. The sizeof leishmaniasis lesions and scars was measuredusing the ‘ball-point pen technique’ (Sokal 1975).

Immune status indicators

The study collected data on humoral and cellularimmune status indicators. Montenegro or leishmaninskin test (WHO 1990) data results were available fora random subsample of 262 subjects or 56% of thesample. The Montenegro skin test used 100 ìl of6#106 heat-killed Leishmania mexicanapromastigotes/ml administered intracutaneously. Theresults were read and recorded 48 hours later usingthe ‘ball-point pen technique’ (Sokal 1975). Testswith an induration size ¢5 mm were classifiedpositive (WHO 1990).Specific anti-Leishmania IgG and IgM serum con-

centrations were measured by optic density in aMultiskan reader at 492 nm (El Safi & Evans 1989).It was planned to collect data on a random subset of85 and 55% of subjects for specific anti-LeishmaniaIgG and IgM concentrations, respectively. However,unanticipated technical errors reduced the final sam-ples to 341 (73.2%) for anti-Leishmania serum IgGand 147 (31.5%) for anti-Leishmania serum IgM.

Laboratory diagnosis of leishmaniasis

Several methods were employed to confirm the pres-ence of Leishmania amastigotes or promastigotes inall subjects with suspicious lesions. These tests,which took tissue samples from ulcers or nodules,

Tropical Medicine and International Health volume 2 no. 2 pp 140–152 february 1997

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142 ,C 1997 Blackwell Science Ltd

were performed under sterile conditions with caretaken to insure that the ulcer sites did not becomeinfected with bacterial or other organisms. Thedirect smear method used a sterile surgical blade toobtain dermal scrapings from the ulcer borders. Thedual tissue samples obtained were then placed on aglass slide and fixed with methanol. One was subse-quently stained with Giemsa to allow direct obser-vation of any parasite amastigotes present under anoil immersion microscope at #100 power (Weigleet al. 1987). The other sample was used to detectamastigotes using a direct immunofluorescent anti-body test (DIFMA) (Chico et al. 1995). The aspiratemethod utilized a 3-ml syringe and 22-gauge needlecontaining 100 ml sterilized PBS to obtain tissuefrom the ulcer border. It was then deposited into amodified Schneider’s NNN medium where parasiteamastigotes, if present, grew for one month.All Leishmania strains isolated by this method

were characterized by isoenzyme chemotaxonomicanalysis (Armijos et al. 1990; 1995). Celluloseacetate electrophoresis was used in the isoenzymepattern determinations. The sample parasite strainswere run in parallel with WHO reference strains.The isolates were characterized on at least 2 occa-sions for up to 20 enzymes. These included 8oxido-reductases, 7 transferases, 2 hydrolases, 2 iso-merases, and one lyase. The 3 enzymes recognized toidentify accurately parasites in the genus Leishmaniawere employed. These were glucose phosphate iso-merase (GPI-EC 5.3.1.9), mannose phosphate iso-merase (MPI-EC 5.3.1.8), and phosphogluconatedehydrogenase (6PGDH-EC 1.1.1.44). Ecuadorianand World Health Organization reference isolateenzyme data were compared and the results utilizedto identify individual Leishmania species.

Data analysis

Subject clinical characteristics and other descriptivedata were analysed with standard techniques includ-ing frequency, percentage and mean&standarddeviation. Analysis of variance was used to examinethe relations of subject leishmaniasis status and theinduration size of the Montenegro skin test or anti-Leishmania IgG and IgM serum concentrations, con-trolling for age and sex. Multiple linear regressiontechniques were employed to explore the relations

between the Montenegro skin test reaction size andother continuous variables including leishmaniasislesion and/or scar number, size, and reported timeelapsed since disease onset. Likewise, the associa-tions of anti-Leishmania IgG and IgM with lesion orscar size and number and disease duration were ana-lysed with multiple linear regression techniques. Allthe regression analyses which utilized a backwardsstepwise procedure also took account of the influ-ence of leishmaniasis status, age and sex. Data onimmune status indicators and clinical characteristicswere explored for possible age and sex differencesusing various statistical techniques depending on thenature of the predictor and outcome variables. Theseincluded contingency table analysis with ÷2 andlogistic regression. The contribution of individualfactors to leishmaniasis risk were explored in theinitial analyses using contingency table analysis with÷2. The final models used multiple logistic regressionwith adjustment for potential confounders (Dawson-Saunders & Trapp 1990; Norusis 1992).

Results

Subject sociodemographic characteristics

The age of the 466 subjects ranged from 0.5 to 78years (x=21.5&17.7 years). The age distributionwas skewed toward younger individuals since 51%of the subjects were under the age of 16 years.Slightly more than half of the subjects were male(54.9%). Most adults (¢18 years) reported that theyhad lived in their present communities an average of10.4&8.2 years, the majority having migrated origi-nally from the rural Andes (48%), Pacific coastalplain (33%), or nearby subtropical areas (19%). Allof the children under the age of 5 years and 85%aged 5–17 years had been born in the study areas.The data indicated that most adult males wereinvolved in subsistence farming, including the culti-vation of bananas, citrus and other fruits, vegetables,coffee, cacao, cassava, and domestic livestock. Anumber also sold cattle and pigs or harvested timberto earn extra cash. The majority of women wereoccupied with domestic and child-care duties butmany also participated in family agriculturalendeavours. Mean family size was 6.1&2.7 persons.Most adult subjects reported owning their own

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home (72.2%) and the land they farmed (58.8%).The majority of subject homes had exterior wallsconstructed of locally available wooden planks, cane,and other plant materials. Only 22% of subjects hada house with brick or cement block exterior wallconstruction. The most common material utilized forroofing was zinc or asbestos sheeting (87%). Veryfew homes (12%) had metal bedroom windowscreens to keep out insects. Sixty per cent of homeslacked electrical power, instead using beeswax can-dles, kerosene, gasoline, or oil lamps for lighting.Less than 10% of subjects lived in homes that wereequipped with an inside toilet or latrine; most hadoutside latrines (63%) or lacked any type of facilities(30%). In half of the homes, domestic waste waterwas evacuated to open fields or nearby streams orrivers. Surface dumps were used by all but 24% ofhouseholds to dispose of waste. A little over one-third of these dumps were located less than 10 mfrom the homes of subjects; 60% were between 10and 20 m in distance. The fruit orchards and gar-dens of 58% of subjects were situated less than 10 maway from their residences. Subjects’ access to tradi-tional medical care was limited. Only 5% lived inclose proximity to a public or private hospital. Morethan one-third reported that the nearest medicalfacility, most typically a public rural health clinic,was more than one hour away. Seventy per cent ofsubjects or the parents/guardians of minor subjectsreported that leishmaniasis treatment was not avail-able at their nearest available medical facility.

Disease prevalence

Fourteen per cent of subjects (n=65) had evidence ofactive leishmaniasis and 33% of past disease. Sub-jects were confirmed as currently infected only if theLeishmania parasite was identified in tissue samplesby one or more of the 3 laboratory diagnostic meth-ods employed in the study. Specifically, 94.3, 52 and45% of the subject samples tested positive using theDIFMA, aspirate cultures and direct smear tech-niques, respectively. Figure 2 shows the relationshipbetween age and leishmaniasis status in the sample.The age group most likely to be affected with leish-maniasis was young children. Specifically, one-thirdof the infants and children under the age of 5 yearshad active lesions at the time of the study and

another 12% showed evidence of prior disease. Thefrequency of active and past disease was relativelystable in the other age groups, averaging around 10and 40%, respectively.

Parasite species

The Leishmania species encountered in the culturesand aspirates obtained from patients with activelesions were L. guayanesis (62.7%), L. panamensis(33.3%) and L. brazilensis (4%). Two subjects hadlesions that harboured more than one Leishmaniaspecies while 3 others were infected with differentparasite species located in separate lesions.

Clinical characteristics

The majority of the 65 active disease cases diagnosed(14%) presented with cutaneous ulcers, although thenodular form was observed in 9 subjects. Multiplelesions were common. Subjects with current diseasehad an average of 2.6&s.d. 1.9 lesions (range 1–14)with a mean surface area of 8.4&s.d. 19.4 cm2

(range 0.2–117 cm2). The reported mean time oflesion duration at the time of the study was6.5&s.d. 8.9 months. The mean number of healedscars recorded for subjects diagnosed with prior dis-ease was 1.8&s.d. 1.2. These ranged in size from0.55 to 41.9 cm2 (x=5.7&s.d. 5.8 cm2). The sub-jects with prior disease reported that their leishman-iasis scars had healed an average of 8.6&s.d. 8.5months prior to the time of the study. The locationsmost frequently affected by leishmaniasis lesions orscars were the bodily extremities (53.8%); scars werealmost equally distributed on legs and feet and/or

Figure 2 Age-specific prevalences of cutaneousleishmaniasis (n=466). /, Active disease; ,, past disease.

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arms and hands. Twenty-three per cent of the lesionsand scars observed were situated in the face andhead area. Sex or age differences were not found tobe associated with the size or number of lesions orscars. However, compared to adult subjects, children¡12 years of age were almost 3 times more likely tohave lesions or scars in either the face or head area(OR=2.75; 95% CI 1.4–5.6; P=0.004). In contrast,adults were more likely to have leishmaniasis ulcersor scars below the waist, especially on the legs orlower arms (OR=2.42; 95% CI 1.14–5.2; P=0.01).

Cellular and humoral immunity indicators

Subject leishmaniasis status was found to predictseveral indicators of cellular and humoral immunestatus. For example, as is shown in Figures 3–5,mean specific anti-Leishmania IgG and IgM serumlevels as well as mean Montenegro skin test reaction

sizes were highest in subjects diagnosed with currentdisease, followed by those with prior disease, andwere lowest in healthy subjects. The analyses con-trolled for subject age and sex. There was some evi-dence of possible subclinical infection since 17.2% ofsubjects who had no obvious prior clinical history forleishmaniasis or physical evidence of scarring causedby the same presented with a positive Montenegroskin test. The number and mean size of leishmaniasislesions were also found to be predictive of serum IgGconcentrations, controlling for age and sex (Figures 6and 7). Likewise, a positive association was identifiedbetween the reported time since the onset of leish-maniasis lesions and mean specific anti-LeishmaniaIgG serum concentrations (Figure 8). Furthermore,the size in the Montenegro skin test was found toincrease both as a function of lesion number (Figure9) and mean lesion size (Figure 10). These analysescontrolled for the influence of subject age and sex.The data were explored for possible age or sex dif-ferences regarding cellular and humoral immunityindications. However, no measurable age differenceswere found in anti-Leishmania IgG or IgM serumlevels or in the induration size of the Montenegroskin test controlling for leishmaniasis status. Like-wise, these 3 indicators of immune status in adultswere not differentiated by sex when subject leish-maniasis status was controlled for in the analyses.

CL risk factors

As Table 1 indicates, children under the age of 5years had almost 2.5 times the risk of developing

Figure 3 Association between subjects’ disease status andanti-Leishmania IgG serum OD concentrations. F-ratio=73;P<0.0001.

Figure 4 Association between subjects’ disease status andanti-Leishmania IgM serum OD concentrations.F-ratio=41.0; P<0.0001.

Figure 5 Association between subjects’ disease status andMontenegro skin test size of induration. F–ratio=106;P<0.00001.

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leishmaniasis compared to other age groups. How-ever, subjects aged 11–20 years were more likely toshow evidence of previous disease (OR=1.9, 95%CI=1.15–3.16, P=0.01). Although gender had noapparent influence on leishmaniasis risk in childrenand adolescents under the age of 18 years, adultmales had a disease risk almost triple that of adultfemales. No significant relations were identifiedbetween infection and length of residence, familysize, marital status, land and house ownership,reported primary occupation, and other sociodemo-graphic indicators.Subjects whose homes had exterior walls con-

structed of natural plant materials were at signifi-cantly higher risk of leishmaniasis compared to thosewith walls made of cement blocks or bricks (Table1). Those who had electric lighting had reduced riskfor the disease. Although individuals whose resi-dences were equipped with piped-in water and insidetoilets were found to have a decreased CL risk in theinitial analyses, these variables were not retained inthe final logistic regression model when adjustmentswere made for the type of exterior wall constructionand home illumination. No significant associationswere found between the risk for disease and thetype of material used for home roofing (i.e., zinc or

asbestos versus plant fibres), the presence of metalbedroom window screens, distance of the family

Figure 6 Association between lesionnumber and anti-Leishmania IgGserum OD concentrations. t=2.5;P=0.18.

Figure 7 Association between mean lesion size andanti-Leishmania IgG serum OD concentration. t=3.7;P=0.0006.

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fruit orchard or waste dump from the house, or themethod of waste disposal.Subjects were also queried about the presence of

putative domestic and wild mammalian reservoirs in

and around their homesteads and local hamletsincluding dogs (Canis familaris), anteaters (Taman-dua tetradactla), opossums (Didelphis marsupialis),kinkajous (Potos flavus), armadillos (Dasypus

Figure 8 Association betweenreported time since first appearanceof the disease and anti-LeishmaniaIgG serum OD concentrations. t=3.5;P=0.0013.

Figure 9 Association between lesionnumber and Montenegro skin testsize of induration. t=3; P=0.005.

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novemcinctus), agoutis (Dasyprocta punctata), pacas(Agouti paca), and ground squirrels (Scirus vulgaris),among others. However, since 85% of subjectsreported that these animals were present and 15%were uncertain of their living in the area, theplanned statistical analyses could not be performed.

Discussion

The data confirmed that cutaneous leishmaniasis isendemic in the subtropical lowland populationstudied. Almost half of the 466 subjects surveyedhad evidence of either current or previous disease.

The prevalence of leishmaniasis was 14%. Thisfigure is higher than that reported previously usingpassive case detection methods in rural PichinchaProvince by the Ministry of Public Health. Theobserved prevalence was also higher than that notedrecently for nearby populations living in tropicalrainforest areas in the adjacent provinces ofEsmeraldas (Kroeger et al. 1992; Weigel et al. 1996)and Manabi (Alava et al. 1992). The data also con-firmed findings from previous reports indicating thatthe distribution of Leishmania species causinghuman disease in the region is complex (Armijoset al. 1990; 1995).

Figure 10 Association between meanlesion size and Montenegro skin testsize of induration. t=3.4; P=0.022.

Risk factors AOR 95% CI P-value

Age* (<5 years) 1.47 0.48–2.35 0.03Gender1: Adult males 2.83 1.05–7.81 0.02Home exterior wall construction:Wood/cane vs cement/brick 1.77 1.38–2.53 0.02Home lighting source2

Electric vs non-electric source 0.67 0.43–2.25 0.03

AOR, Adjusted odds ratio; CI, confidence interval.1 Adjusted for home exterior wall construction type and home lighting source.2 Adjusted for home exterior wall construction type.

Table 1 Multiple logistic regressionanalysis of the association of subjectsociodemographic and habitatcharacteristics with the risk forcutaneous leishmaniasis

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Subjects with present disease were found to havethe highest anti-Leishmania IgG and IgM serumlevels as well as larger Montenegro skin test indura-tion sizes compared to healthy and previously dis-eased individuals. Consistent with Minori et al.(1987), a strong positive correlation was observedbetween lesion size and IgG levels. IgG concen-trations were positively associated with both lesionnumber and duration. Lesion number and size pre-dicted the induration size in the Montenegro skintest. These data indicate that both cellular andhumoral immune responses are present in subjectswith cutaneous leishmaniasis. Although the preciserole of antibodies in conferring protection is not wellunderstood, the results suggested that antibody levelshave a positive relationship with the amount of tis-sue destruction in ulcers caused by leishmaniasis.Two non-modifiable risk factors, male gender (in

adults) and young age, were found to be associatedwith leishmaniasis. Specifically, adult men ¢18 yearsof age were found to have a markedly increased riskfor disease, almost 3 times that of their female coun-terparts. A higher leishmaniasis incidence for menwas also reported in the nearby tropical lowlands ofnorth-west Ecuador (Alava et al. 1992; Nonaka etal. 1990). In addition, a number of authors havereported the existence of male–female differences inleishmaniasis risk for other diverse Latin Americanpopulations (Castes et al. 1993; Dourado et al.1989; Le Pont et al. 1989; Torres et al. 1989; Dedetet al. 1989). It is possible that sex differences may beresponsible for the observed male–female distinctionin risk since women are reported to have generallyenhanced immune responses compared to men,including a more robust cellular immune statuswhich is related to hormonal influences (Brabin &Brabin 1992; Beckage 1991; Lahita 1984). Cutane-ous leishmaniasis is mainly controlled by cellularimmune mechanisms (WHO 1990). On the otherhand, in our study, no distinctions in disease riskwere found between male and female adolescentswho would be expected to exhibit hormonal profilessimilar to adults. Furthermore, if inherent biologicaldifferences were causal, then one might also expectto see sex differences in the clinical characteristicsand in the outcome of individuals with the disease.However, no significant differences were detectedbetween adult men and women with respect to

leishmaniasis lesion number, size, type or duration.Likewise, females and males with current disease didnot differ as to the parasite species identified fromtheir lesions. Neither cellular nor humoral indicatorsof immune status measured in the study were differ-entiated by sex in either adults or adolescents.On the other hand, it has been speculated that

much of the observed excess risk in men may be theresult of gender-specific occupational roles whichincrease male exposure to the vector in rainforestsand other ‘hot-spots’. There are new data which sup-port the gender role hypothesis. A recent study con-ducted in the same endemic area of Ecuador whichcollected 24-hour activity data indicated that menwere more likely than women to be outdoors duringthe times when the vector was likely to be active,that is, between sunset and sunrise (M. M. Weigel,unpublished data). They also were more likely towork in places where the vector was likely to bepresent and disturbed, e.g. in the rainforest or fields.In contrast, adult women were more likely to beengaged in indoor activities including cooking overwood fires in smoky kitchens during these hours.Smoke has been reported to have a protective effectagainst the transmission of other tropical diseasescaused by mosquito and sandfly vectors (Vlassoff &Bonilla 1992).The age group found to be at greatest risk for

contracting leishmaniasis were children under theage of 5 years. It is clear that a portion of theelevated risk of young children is due to their rela-tive lack of acquired immunity to leishmaniasis com-pared to other groups. However, it is also evidentthat exposure to the vector had to occur, since theydeveloped the disease. Thus, although the study dataindicated that extra-domiciliary activities wereassociated with leishmaniasis risk, there was alsoevidence that intradomiciliary and peridomiciliaryexposure to the vector was responsible since infants,toddlers and preschoolers typically spend most oftheir time with their mothers and other female care-takers in and around the home. Other authors havealso reported a high prevalence of cutaneous leish-maniasis in Ecuadorian children. Alava and associ-ates (1992) found a high proportion of cases inchildren under the age of 10 years in an endemicarea of tropical rainforest in Manabi Province. Simi-larly, Hashiguichi and Gomez (1990) reported that

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leishmaniasis was more common in children under10, especially those under 2 years, and they alsoidentified several cases which occurred in neonatesless than one month of age. In another study, it wasnoted that 56% of new leishmaniasis cases occurredin babies under the age of one (Hashiguchi &Gomez 1992). It appears that clothing customs, play,sleep or other habits of young children affect theprobability of their being bitten by the sandfly vec-tor. Reductions in cellular immune status associatedwith chronic malnutrition may be associated withincreased disease risk in children (Weigel et al.1995).The data identified two modifiable risk factors

associated with leishmaniasis. Specifically, subjectswhose homes had exterior walls constructed ofcement blocks or bricks rather than plant materialshad a disease risk only 40% of that of persons withwooden or cane walls. The latter type of construc-tion which typically contains open gaps in the wallsmost likely facilitates the entry of the sandfly vector.In this region of Ecuador, wood and wood/canehomes are normally elevated on wooden poles 1–2 mabove the ground. This type of construction helps tominimize the risk of flooding during the rainy winterseason and deters the entry of snakes and otherunwanted animals. However, like the exterior walls,the floors of the elevated wooden and cane homeshave cracks which may allow the vector into thedwelling. Cracks in building construction have beenreported as a positive risk factor for leishmaniasis inother geographical areas (WHO 1990). Contrary toour a priori hypothesis, the use of impervious roof-ing materials such as zinc or asbestos and metal bed-room window screens apparently did not confer anysignificant independent measure of protection againstcontracting leishmaniasis, probably because the vec-tor is readily able to enter through the gaps in wallsand floors.It is unclear why subjects who resided in dwellings

with electric lighting, independent of the type ofexterior wall construction, were at reduced risk forleishmaniasis. One possible explanation may lie inthe placement of the lighting source inside the home.Although both electric and non-electric sources mayattract the sandflies, electric light bulbs typicallyhang from the ceiling while lanterns and candles aretypically placed on tables or shelves and, thus, are in

close proximity to the human inhabitants. On theother hand, anectodal reports from area residentsindicate that many leave their electric lights on allnight in the belief that sandflies and mosquitoes willbe attracted to the light rather than the humans.The practice of clearing vegetation from around

the perimeters of homes and other buildings and thecreation of forest-free zones has been suggested as auseful measure to diminish the probability of vector–human contact as well as to reduce the number ofsylvanic disease reservoirs (WHO 1990). Most resi-dents cleared native trees and bushes outside theirhouses. However, they then replanted their nearbyhome orchards and gardens. In fact, 60% of the sub-jects in the current study had fruit trees and gardenssituated less than 10 m from their dwellings. It hasbeen hypothesized that orchards and gardens placedin close proximity to homes increase the risk forinfection. However, we found no differences in infec-tion risk between subjects with orchards and gardensplaced less than 10 m from the home and those withtrees and plants located further away. Although it ispossible that maintaining a small cleared space 20 mor less around homes may decrease the risk of vectorcontact, this practice may confer less protection thanmight be expected since many Lutzomyia speciesreadily enter and bite the occupants of dwellings¡200 m from the jungle’s edge (Young & Arias1992). In this area, it is common to maintain dogs,cats, horses, mules, donkeys, guinea-pigs, and otherdomestic animals in and around the home. Many ofthese species have been implicated as possible peri-domestic disease reservoirs (WHO 1990; Grimaldiet al. 1987; 1993) or nutritional attractants (i.e.provide blood meals).In conclusion, cutaneous leishmaniasis is a signifi-

cant public health threat in this region of north-westEcuador. The data verify an overlapping distributionof the Leishmania species causing human disease.Control of leishmaniasis is difficult since the smallcommunities in the area are located within a densesubtropical rainforest with high Lutzomyia density(Biol R. Sandoya, personal communication) where aminimum of 3 of the Leishmania species causinghuman disease are present. Elimination of the rain-forest or putative mammalian reservoirs and large-scale spraying of insecticides are neither practicalnor desirable. Although gender and age are not

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modifiable variables, it may be possible to reduce therisk for disease in these groups by making behav-ioural changes which minimize exposure to the vec-tor, such as avoiding the times and places where thevector is known to be present in large numbers, useof protective clothing, insect repellents, andpermethin-impregnanted bednets (Kroeger et al.1993). The findings of this study suggest that altera-tions in the housing environment may reduce intraor peri-domiciliary exposure to the sandfly vector.The use of relatively impermeable building materialssuch as bricks, cement blocks, or adobe for theexterior walls and floors of homes and other struc-tures may help reduce the risk of acquiring leishman-iasis infection. Where this is not practical or eco-nomically feasible, the placement of mud or clay inthe gaps and holes of cane or wood exterior wallsand flooring may help reduce entry of the vector intodwellings.

Acknowledgements

This work was supported in part by AID ESR 518-0058 and the Ecuadorian Ministries of Public Healthand Finance and AID DAN 5058G55-6054.

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