Prevalence of Trypanosoma cruzi and Leishmania chagasi infection and risk factors in a Colombian indigenous population

Prevalência da infecção por Trypanosoma cruzi e Leishmania chagasi e fatores de risco numa população indígena da Colômbia

Trypanosoma cruzi and

Leishmania chagasi infection and risk factors in a Colombian indigenous population

Augusto CORREDOR ARJONA⁽¹⁾, Carlos Arturo ALVAREZ MORENO^(1,4), Carlos Alberto AGUDELO⁽¹⁾, Martha BUENO⁽³⁾, Myriam Consuelo LÓPEZ⁽¹⁾, Elvia CÁCERES⁽¹⁾, Patricia REYES⁽¹⁾, Sofia DUQUE BELTRAN⁽²⁾, Luis Eduardo GÜALDRON⁽²⁾ & Maria Mercedes SANTACRUZ⁽²⁾

SUMMARY

This study was carried out in order to obtain base-line data concerning the epidemiology of American Visceral Leishmaniasis and Chagas’ Disease in an indigenous population with whom the government is starting a dwelling improvement programme. Information was collected from 242 dwellings (1,440 people), by means of house to house interviews about socio-economic and environmental factors associated with Leishmania chagasi and Trypanosoma cruzi transmission risk. A leishmanin skin test was applied to 385 people and 454 blood samples were collected on filter paper in order to detect L. chagasi antibodies by ELISA and IFAT and T. cruzi antibodies by ELISA.

T. cruzi seroprevalence was 8.7% by ELISA, L. chagasi was 4.6% and 5.1% by IFAT and ELISA, respectively. ELISA sensitivity and specificity for L. chagasi antibodies were 57% and 97.5% respectively, as compared to the IFAT. Leishmanin skin test positivity was 19%. L. chagasi infection prevalence, being defined as a positive result in the three-immunodiagnostic tests, was 17.1%. Additionally, 2.7% of the population studied was positive to both L. chagasi and T. cruzi, showing a possible cross-reaction. L. chagasi and T. cruzi seropositivity increased with age, while no association with gender was observed. Age (p<0.007), number of inhabitants (p<0.05), floor material (p<0.03) and recognition of vector (p<0.01) were associated with T. cruzi infection, whilst age ( p<0.007) and dwelling improvement (p<0.02) were associated with L. chagasi infection. It is necessary to evaluate the long-term impact of the dwelling improvement programme on these parasitic infections in this community.

KEYWORDS:Visceral leishmaniasis; Chagas’ disease; Seroprevalence; Colombia.

INTRODUCTION

The Tripanosomatidae which affect man in America are from the Leishmania and Trypanosoma genera. The first combines different leishmaniasis species, within which Leishmania chagasi is found. The American Visceral Leishmaniasis’ (AVL) causative agent; within the second is found Trypanosoma cruzi, the causative agent of American trypanosomiasis. These parasitoses occupy shared ecological niches³¹ in many parts of America, from the south of the United States to Argentina, which allow simultaneous evaluation of the two entities.

In Colombia, AVL was described for the first time in 1944 by Gast Galvis in a patient from the State of Santander¹⁶. After notification of this AVL case, almost 20 years (1960) passed until two new cases were reported¹⁷. From 1981 onwards, more than 600 new cases have been recorded, which can be explained by amongst other aspects, the suspension of DDT spraying in L. chagasi synanthropic foci and an improvement in the diagnosis and reporting of the disease²³. The Tolima department is the region of the country where most visceral leishmaniasis cases have been registered since 1981; 37.3% corresponded to the municipality of Coyaima, which makes it one of the country’s most important foci⁵.

On the other hand, the estimated figures for human infection with T. cruzi in Colombia oscillate between 1,200,000 and 1,700,000 inhabitants and some 40,000 new cases occur every year, principally in regions situated below 2,000 metres above sea-level and which coincide with the distribution of the principal vector in Colombia, Rhodnius prolixus, in the departments of Guajira, Cesar, Santander, Boyacá, Arauca, Casanare, Cundinamarca, Tolima, Meta, Huila, Caquetá, Bolívar and Caldas^10,11. Some blood-bank seroprevalence studies from endemic zones in the departments of Santander, Norte de Santander, Tolima, Boyaca, Cundinamarca, Meta, Huila, Guajira, Cesar, Arauca, Casanare and Caqueta, show levels oscillating between 3% and 6%^7,18,19.

In the municipality of Coyaima, R. prolixus was found in habitations and to be infected with T. cruzi and T. rangeli¹⁰. Some previous serological surveys revealed active trypanosome transmission²⁴. However, until the present, no studies have been initiated to allow the magnitude of american tripanosomiasis in this region to be established.

According to the characteristics of the region in question, it is necessary to establish the dynamics of the two entities’ transmission, by means of the evaluation of the different ecological, socio-economic and cultural factors, which influence its transmission chain. This study was intended to learn about T. cruzi and L. chagasi infection prevalence and their possible relationship with reservoirs, vectors and associated risk factors. Given that these parasitoses are determined, for the greater part, by housing conditions, the study allowed base-line information to be obtained concerning epidemiological aspects in order to evaluate the impact of a housing modification and improvement programme on the prevalence of these diseases. This programme was initiated by the government due to the 1991 epidemic in this municipality and consisted of peridomiciliary changes, latrine, farmyard and kitchen construction and domestic changes such as modifications to walls, roof and/or floor.

MATERIALS AND METHODS

The study was carried out in the municipality of Coyaima, located in the Tolima department, covering an area of 667 km². The town centre is located at 03º 48' 09'' latitude North and 75º 11' 54'' longitude West and 350 m altitude (Fig. 1)²¹. The average temperature is 28.2 ºC, with an annual rainfall of 1,502 mm. The surrounding land is distributed between thermic, hot and mild levels. Economic activities of greatest importance are agriculture, dairy-farming and mining. Principal crops are coffee, sesame seeds, maize and millo. Precious metal, lime and barita mines are being worked.

Fig. 1. Municipality of Coyaima, Tolima located in the centre of the country

According to the 1993 census, Coyaima has a population of approximately 23,303 inhabitants, 90% of whom belong to the Indian community and live in the rural zone. 84% of the inhabitants have unsatisfactory basic necessities and levels of illiteracy were 24.2% for men and 32.6% for women¹².

The study area was established in the following way:

During the 1977–1995 period, the Coyaima hospital attended 165 parasitologically confirmed cases of visceral leishmaniasis. From this information, the study area was established; the two localities from which the greatest number of cases came and in which the housing modification programme had been initiated at least two years before, due to the cholera epidemic, being selected. The localities of Doyare Centro and Doyare Esmeralda were thus chosen.

In the chosen localities, 242 homes (100% of the total) were surveyed during 1995-1996, concerning risk, socio-economic and environmental factors associated with L. chagasi and T. cruzi transmission such as: number of years as householders, overcrowding, type of housing, roof, floor and wall materials, public services to the housing, prevention measures taken for vector control such as spraying and use of mosquito-netting, vector presence and identification, reservoir presence, possession of animals and housing modification.

The survey revealed a population of 1,440 people of indigenous ethnology, with equal gender proportions (sexes) and showed that of the 242 homes, 124 had been included in the housing modification and improvement programme.

The procedure which was applied will be described next, previous written consent having been obtained from the 538 people who made up the sample. The sample presented the same age and gender distribution as the original population.

The Montenegro test was applied to the whole sample, reading of which was only possible for 385 people, 48 hours after inoculation, individuals with ³ 4 mm indurations being considered to be positive. The test was made with L. chagasi promastigote soluble extract, containing 25 m g/ml parasite protein, manufactured at the Instituto Nacional de Salud according to previously described methodology⁹.

Blood samples were taken from 454 people, from the sample’s total 538, on Whattman #3 filter paper on which tests to determine T. cruzi and L. chagasi antibodies were carried out utilizing ELISA and IFAT (Indirect Fluorescence Antibody Test) techniques, according to previously described methodology. IFAT was carried out following CAMARGO⁶; DE SOUZA & CAMARGO¹⁴ and CORREDOR & LÓPEZ’s procedures⁸. The ELISA was made according to BARTLETT et al.⁴, using T. cruzi and L. chagasi soluble antigen in 0.75 m g/ml and 500 m g/ml concentrations respectively and 1:1,000 in T. cruzi and 1:50 in L. chagasi. Optical densities greater than 0.4 and 0.5 were considered to be ELISA positive for T. cruzi and L. chagasi. Titres equal to or greater than 1:32 for the IFAT test were considered to be positive for L. chagasi. All individuals who proved to be positive, in any of the tests, were submitted to a clinical examination looking for possible signs of Chagas’ disease or AVL.

The search for AVL infection in domestic reservoirs was carried out on 22 dogs belonging to houses where seropositive individuals or those reactive to leishmanina for L. chagasi were found. These dogs were evaluated by clinical examination and direct smear of the popliteal ganglion haemolymph.

Phlebotomine captures were undertaken for 10 days, inside the housing’s living space as well as in the peridomiciliary area where positive cases were registered. Forest samples were also taken from form-wooded areas close to the housing. These samples were taken during dawn and nightfall hours (approximately 8 hours/day/person) during the month of July, using automatic and manual aspirators on human and animal bait (chicken, pigs, cattle and dogs). Captured Phlebotomines were stored in 70% alcohol and information concerning day, hour and capture site were noted. The insects so collected were identified in the Universidad Nacional de Colombia Biology laboratories. Dissection was not used in the search for L. chagasi natural infection nor was housing inspected in the search for triatomine bugs.

For risk factor analysis, a house was considered positive when one or more individuals living in it were positive by any of the tests performed.

The proportions were compared by X² test and Fischer test when necessary. The EPIINFO programme version 6.04 ¹³ was used to make these tests.

RESULTS

Seroprevalence

The ELISA test was positive in 8.7% for T. cruzi antibodies. For L. chagasi, ELISA and IFAT positivity was 5.1% and 4.6% respectively. Montenegro Skin Test (MST) was 19.0 % (Table 1). If all individuals positive for any one of the three immunodiagnostic methods are considered to be L. chagasi positive cases, then positivity was 23.3%.

TABLE 1

a	L. chagasi INFECTION						T. cruzi INFECTION ELISA
	ELISA		IFAT		Montenegro Skin Test
a	#	%	#	%	#	%	#	%
POSITIVE	23	5.1	21	4.6	73	19.0	39	8.7
NEGATIVE	431	94.9	433	95.4	312	81.0	409	91.3
TOTAL	454	100	454	100	385	100	448	100

L. chagasi and T. cruzi infection positivity in an Indigenous Community. Coyaima. 1996.

The positivity of the MST (reactive individuals over population examined) showed a statistically significant progressive increase with age (p< 0.05) (Figure 2).

Fig. 2
- Montenegro skin test by age groups. Coyaima. 1996.

In the case of T. cruzi infection as evaluated by ELISA, positivity increased with age and then decreased in the 40-49 year old group (Figure 3). L. chagasi antibody detection was carried out by ELISA as well as IFAT, finding a progressive increase with age, however a diminution was observed in the 20-29 and 40-49 year old groups (Figure 3). The ELISA test showed a 57% sensitivity and 97.5% specificity as opposed to IFAT (routine test used for LVA diagnosis by National Institute of Health of Colombia) (Table 2). Serological positivity with both tests (IFAT and ELISA) had an 80.4% agreement with the Montenegro test (Table 3). 2.7% of the people were simultaneously positive for L. chagasi and T. cruzi, indicating a possible crossed reaction or a mixed infection since individuals were encountered who were serologically positive for T. cruzi and negative for L. chagasi as determined by MST.

TABLE 2

ELISA	IFAT
	POSITIVE	NEGATIVE	TOTAL
POSITIVE	12	11	23
NEGATIVE	9	422	431
TOTAL	21	433	454

Comparison between serological tests for L. chagasi antibody detection. Coyaima, 1996.

TABLE 3

SEROLOGY ELISA and/or IFAT	MONTENEGRO TEST	FREQUENCY	PERCENTAGE (%)
+	+	11	3.7
+	-	10	3.0
-	+	49	16.5
-	-	230	76.7
TOTAL		300	100

Comparison of Serology and Montenegro Skin Test in L. chagasi infection detection. Coyaima, 1996.

Fig. 3
- Seroprevalence by T. cruzi and L. chagasi by age groups.

No differences were observed between positivity with respect to gender for T. cruzi nor for L. chagasi. Serologically reactive individuals did not show important clinical alterations compatible with AVL or Chagas’ disease.

Housing and risk factors.

1. 100% of the housing studied did not have a drinking-water supply, nor electric light and only housing included in the modification programme had excretion disposal facilities (latrines or fixed sanitary ware).

2. 50.6% of the houses presented earth floors and 90.1% bahareque (wooden-slat) walls.

3. 84.7% and 84.5% of the population did not use repellent nor mosquito netting respectively.

4. 86.3% recognized the vectors of the entities studied and 91% had seen wild reservoirs near their housing.

Table 4 describes the housing’s principal characteristics and different risk factors for risks associated with T. cruzi and L. chagasi positivity. Statistically significant associations were encountered with vector presence and identification, number of inhabitants per house and flooring material in T. cruzi infection, whilst with L. chagasi only association with housing modification was found (Table 4).

TABLE 4

ASSOCIATED RISK FACTORS	CASE INFECTION T. cruzi		CASE INFECTION L. chagasi
	X2	P	X2	P
Housing Modification	2	0.15	5.32	0.02*
Wall Material	0	0.99	2.52	0.11
Flooring Material Cement Earth	4.32	0.03*	1.69	0.19
Housing Fumigation	0.51	0.47	0.53	0.96
Use of mosquito netting	0.01	0.93	0.56	0.45
Possesion of animals	0.01	0.93	0.48	0.49
Possesion of dogs	1.11	0.29	0.03	0.85
Vector Identification	5.65	0.01*	3.27	0.07
# inhabitants per house < 2 inhabitants > 2 inhabitants	3.86	0.049*	0.43	0.5

Relationship between housing characteristics and risk factors associated with T. cruzi and L. chagasi infection. Coyaima, 1996.

Entomology

In the entomological study 42 Lutzomyia samples were found, which were identified as being L. cayannensis and L. longipalpis. L. longipalpis was found with greater frequency, a specie demonstrated to be the principal vector of L. chagasi in America. It is interesting to note that in spite of the low number of samples collected, various of these were found in the housing’s walls, demonstrating intradomiciliary habits. In the majority of the homes farmyards with domestic animals (chicken, pigs) were found near the housing, a situation which increased the lutzomyias population, facilitating transmission. However, it is necessary to carry out vectorial studies in different seasons of the year to determine the months of greatest density.

Regarding the domestic reservoir study, the 22 dogs studied proved to be negative.

DISCUSSION

The 19% positivity found in the Montenegro Skin Test, is low when compared to results obtained in other zones of the country. In Córdoba 40%²⁹ was found and 51.2% in Cundinamarca⁹. However, this positivity is similar to that found in endemic regions of Africa^26,28. As this test has high specificity and cutaneous leishmaniasis was not found in the zone studied, the allergic index, as well as the increase in positivity level with age, confirms active L. chagasi infection transmission.

L. chagasi serological prevalence was found to be similar to that observed in other seroepidemiological studies^3,15,25. Serological values diminished over time whilst cellular immunity measured by Montenegro test remained positive²⁸. 3.0% of the T. cruzi and L. chagasi serologically positive but MST negative people could be explained by the possible crossed reactions between Leishmania and Trypanosoma as has been previously reported³⁰. Additionally, when comparing these serological tests, ELISA and IFAT, with the Montenegro test high concordance is observed, 80.4%, if compared with that published by BADARÓ et al., showed a 47% concordance in Brazil³.

L. chagasi infection in the majority of cases is not clinically manifested, which explains the high positivity in immunodiagnostic tests and the low presence of clinical cases of AVL. Thus, in this study, the positive people were not found to be ill and showed that they had not previously presented a picture compatible with AVL. However, it must not be forgotten that infected patients do not present the disease, thanks to the immune system’s efficient action; but, once this system is altered Leishmania behaves as an opportunistic parasite as is shown in AIDS patients, in the world’s different endemic zones^1,2.

If the IFAT is considered to be the gold-standard, it is seen that ELISA has low sensitivity and high specificity for detecting L. chagasi antibodies as has been reported by other authors^{20, 22}.

When comparing positivity by gender, no difference was shown in any age group, which suggests that the exposure risk is equal amongst women and men.

The T. cruzi positivity found indicates its active transmission in the region. Even though it is true that the crossed reaction with L chagasi can explain some seropositives, 6% were only positive for T. cruzi. This figure is low when compared to highly endemic Colombian regions where a 26% positivity has been found¹¹. However, to evaluate the problem’s real magnitude, it is necessary to do further serological studies and to carry out electrocardiographic evaluation on positive individuals.

No correlation between housing modification and T. cruzi infection was found, whilst in L. chagasi infection greater infection was observed in the modified houses. This could be attributed to the short time elapsed between the modification of the housing and this survey, which is not useful for measuring the impact of housing modification although it can be employed as baseline data for the long-term assessment of the housing modification program. Additionally a positive relationship between triatomine recognition and T. cruzi seropositivity was found.

In general, the studied area’s socio-economic conditions are precarious constituting the principal risk factor in the transmission of AVL as well as of Chagas’ disease.

RESUMO

Prevalência da infecção por Trypanosoma cruzi e Leishmania chagasi e fatores de risco numa população indígena da Colômbia

Este estudo foi realizado para obter a linha de base da epidemiologia da Leishmaniose Visceral Americana e da Doença de Chagas numa comunidade indígena, onde o governo está desenvolvendo um programa de melhoramento da habitação. A coleta de dados referentes aos fatores sócio-econômicos e do meio ambiente associados ao risco de transmissão de Leishmania chagasi e Trypanosoma cruzi foi feita por meio de respostas a questionário endereçado aos componentes acima mencionados. O inquérito foi realizado em 242 unidades domiciliárias (1440 indivíduos). Foi realizada a prova de Montenegro em 385 indivíduos e colhidas 454 amostras de sangue em papel de filtro, para pesquisar o teor de anticorpos contra L. chagasi por meio das técnicas de ELISA e IFI e o teor de anticorpos contra T. cruzi por meio de ELISA.

A prevalência sorológica foi de 8,7% para T. cruzi, 4,6% e 5,1% para L. chagasi por meio de IFI e ELISA, respectivamente. Ao se comparar estas duas provas foi encontrado que por meio de ELISA a sensibilidade e especificidade para detecção de anticorpos contra L. chagasi foi de 57% e 97% respectivamente. Os resultados da intradermo-reação de Montenegro revelaram uma positividade de 19%. Os resultados dos três testes de imunodiagnóstico mostraram uma prevalência da infecção por L. chagasi de 17,1%. Além disso, 2,7% da população estudada apresentou reações sorológicas positivas para os dois parasitos, evidenciando uma possível reação cruzada. A soropositividade para L. chagasi e T. cruzi aumentou com a idade, e não houve associação com o gênero. Idade (p<0,007), número de moradores (p<0,05), tipo de piso (p<0,03) e o reconhecimento do vetor (p<0,01) foram associados com a infecção por T. cruzi. Entretanto, na infecção por L. chagasi foi encontrada associação com a idade (p<0,007) e o melhoramento da habitação (p<0,02). Recomenda-se avaliar o impacto do programa de melhoramento da habitação sobre estas infecções parasitárias nesta comunidade num prazo longo.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Santiago Nicholls, the Instituto Nacional de Salud’s subdirector of Epidemiology, Dra. Gloria Palma from the School of Medicine, Universidad del Valle for their technical assistance and Coyaima and the Tolima department’s local health authorities for the logistical help provided when carrying out this work. This project was financed through an agreement between the Ministerio de Salud-Universidad Nacional de Colombia No. 000352/95.

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Correspondence to: Dr. Carlos Arturo Alvarez, Instituto de Salud en el Trópico, Of. 318, Ciudad Universitaria, Bogotá, Colombia.

Email: [e-mail]; [e-mail]

Received: 14 October 1998

Accepted: 21 June 1999

(1)

Public and Tropical Health Department, Instituto de Salud en el Trópico, Universidad Nacional, Bogotá, Colombia.

(2)

Parasitology Laboratory, Instituto Nacional de Salud, Bogotá, Colombia.

(3)

Biology Department, Facultad de Ciencias, Universidad Nacional, Bogotá, Colombia.

(4)

Infectious Diseases Unit, Internal Medicine Department, Universidad Nacional, Bogotá, Colombia.

Publication Dates

• Publication in this collection
  09 Nov 1999
• Date of issue
  July 1999

History

• Accepted
  21 June 1999
• Received
  14 Oct 1998