TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1995) 89,155-158 155

Visceral leishmaniasis in a new ecological niche near a major metropolitan area of Brazil

Sergio Cunhal, Miralba Freire 2, Conceicao Eulalio2, Jo& Critosvao 3, Eduardo Netto’, Warren D. Johnson, jr4, Steven G. Reed5 and Roberto Badaro 2,4 ‘Department of Preventive Medicine of the Federal University of Bahia, Salvador, Bahia, Brazil; 2Tropical Disease Laboratory of the University Hospital Professor Edgard Santos, University of Bahia, Salvador, Bahia, Brazil; 3Regional Directorate of Bahia, National Foundation of Health, Salvador, Bahia, Brazil; “Division of International Medicine, Cornell University Medical College, New York, NY 10021, USA; 51nfectious Disease Research Institute, Seattle, Washington, USA

Abstract In 1991, a community cross-sectional study was conducted in a village situated near the beach and close to Salvador, the capital city of Bahia, in Brazil, to determine the prevalence of visceral leishmaniasis since 1989. A serological survey was made of human and canine reservoirs and an intradermal skin test for leish- maniasis was used to assess cellular immune responses. Nearly 30% of the 243 individuals in the study area had positive skin tests and 14% had positive serology, the latter being compatible with recent infection; 29 of 460 dogs examined were seropositive. A possible association was observed between human infection and the presence of dogs in or near residences, but not between human infection and malnutrition. This report describes the evolution of a new focus of visceral leishmaniasis, its expansion toward a metropolitan area, and current measures taken to control the epidemic.

Keywords: leishmaniasis, visceral leishmaniasis, new focus, Brazil

Introduction At least 90% of the recorded cases of visceral leishma-

niasis (VL) in Brazil occur in the north-east section of the country. The state of Bahia reports the majority of cases, an average of 500-600 per year, mainly in the semi-arid interior regions. In 1989, an outbreak of VL was re- ported in the village of Monte Gordo, approximately 1 km from the ocean and 40 km north of Salvador, the capital city, with approximately 2.2 million inhabitants. This area is outside the general endemic area of VL in Bahia. Local health officials were concerned because this area had recently received waves of migrant workers and is frequently visited by tourists. Because there was con- cern that VL might spread to other areas around Salva- dor, a cross-sectional, community-based study was per- formed. This was restricted to the area of the village where most of the cases of VL had been reported, in order to determine the true prevalence of the disease and the autochthony of cases, and to gather data for the de- velopment of guidelines to halt the spread of the out- break. This report presents the results of the original study and the current status of infection and disease in the area. The significance of this study lies in the trend, observed in Brazil and elsewhere (COSTA et al., 1990), to- ward the urbanization of VL.

Methods Study area

The village of Monte Gordo, in Bahia, Brazil, has nearly 4000 inhabitants and is situated at sea level along the ‘Coca railway’, which links Salvador with coastal re- sorts frequented by tourists and vacationers (Fig. 1). The 17 cases of VL registered from 1989 to 1992 were mainly from a section of the village comprising 49 households (243 persons). There was a medical outpatient unit in the village with 2 doctors, but no laboratory facility. The study area was mapped, houses were identified by num- bers, and appropriate measures adopted for follow-up.

Study population The survey was carried out with the help of county

field workers who used a pre-coded questionnaire similar to one previously reported (BADARO et al., 1986a). Each house was numbered, and a census was conducted in which data were collected regarding sex, age, length of residence in the area, and migratory history. The presence of dogs was determined and information con-

Address for offprint requests: Laboratorio de Retrovirus, 6” Andar, Hospital Universitkio Professor Edgard Santos, Canela, Salvador, Bahia, Brazil.

MONTE GORDO 1999 y

.- CAMACARI 1990 lat. 12’45Y /c~@870

?

/

BARRA De JACUIPE 1992

Atlantic Ocean Fig. 1. Map showing Salvador and the coastal foci of visceral leishma- niasis in Bahia state, Brazil, with the year when first reported.

cerning socioeconomic status elicited. The questionnaire also asked about symptoms (fever, cough, weight loss) that had occurred within the prevrous 15 d and whether there was a history of VL. Children 12 years and younger were examined for hepato- or splenomegaly. Anthro- pometric measures (height and weight) were used for children 10 years and younger to evaluate nutritional status (WATERLOW et al., 1977; WHO, 1986).

Immunological studies Blood samples were obtained from each household and

levels of specific antibody determined by micro-enzyme- linked immunosorbent assay (ELISA), as previously de- scribed (BADARO et al., 1986b), to detect recent and/or active disease; titres al:100 were considered positive. Leishmania skin tests were performed on entire families, except for infants younger than 8 months. Induration of 5 mm or more by 48 h was considered positive (REED et al., 1986). Immunological testing of dogs was carried out by the National Foundation of Health (FNS), the federal agency charged with controlling outbreaks of infectious diseases in Brazil. The indirect immunofluorescent anti- body test was used with dog blood collected on filter paper, and samples with titres > 1:40 were considered positive.

156

Statistical analysis The Yates-corrected x2 test was used for proportions

and the t test for means. For nutritional assessment, the National Council for Health Sciences (NCHS) data (Jor- dan Anthropometric Software Package, Centers for Dis- ease Control, USA, 1987) was used, and children were sorted according to the Z cut-off score. Confidence inter- vals (95% CI) for association measures were estimated using the Taylor series method.

Results Data were collected for 45 d during June and July,

1991. Demographic data were obtained from 49 houses (243 persons), the entire population of the study area. Forty-two percent of the population (102 of 243) were children up to 12 years of age (Fig. 2). Twenty-two

65-90 -80.84 v) 75-79 E TO-74 U 65-69 w 60-64 * 55-59 -50-54 & 45-49 3 40-44 p 35-39

I 1 female 7 3

30 25 20 15 10 5 0 6 10 16 20 25

NUMBER Fig. 2. Age distribution of the entire population of the Monte Gordo vil- lage study population, Bahia state, Brazil, in 1991.

(44.9%) of the 49 households in the study area had mi- grated from other regions, but none from areas where VL was known to be endemic; 143 members (59%) of the study population had been born in the village; of those born elsewhere, nearly half had arrived before 1988. None of the houses had sewage disposal. Most of the adults worked in the area as fishermen, vendors, and tradesmen, generally without permanent employment. Twenty-seven (55%) of the households were illiterate. Only those with past or current VL and some of their relatives knew the hallmark signs of the disease (fever, anaemia, and enlargement of the abdomen).

Up to December of 1993, 30 of a total of 42 cases in the region had been confirmed parasitologically in Monte Gordo (both within and outside the study area). Most of the cases occurred in the summer, resembling the season- al pattern seen in endemic areas (Fig. 3).

7

x

5

III Jan Fob Mar Apr May Jun Jul Aug Sept Ott Nov Dee

MONTH Fig. 3. Seasonal distribution of parasitologically confirmed cases of vis- ceral leishmaniasis in Monte Gordo, Bahia state, Brazil, 1989-1993.

One hundred and fifty-two people were skin-tested. There was no significant age difference between those tested and those not tested (t test, P=O.7). Fifty percent of males and 28% of females were tested. Forty-eight persons (32%) were positive, corresponding to nearly 20% of the entire study population. There was no signifi-

Table 1. Age distribution of positive leishmanin skin test

Age group (years) No. No.

examined positive”

o-9 11 (21%) IO-19 z:: 18 (44%) 20-39

:i 6 (2S’Yo)

a40 13 (33%) Total 152 48 (32%)

“No significant difference between age groups (x2=5.71, 3 degrees of freedom, P=O*l).

cant difference between the percentages of positive and negative tests in the various age groups (Table 1). There were more positive persons among those who had lived in the area for 3 years or more than in those who had ar- rived since 1988. Many more people who were born in the village were positive (40 of 100) than those born in other places (8 of 52) (x*=8.49, PsO.01).

The ELISA was positive in 14 people, including 2 who already had VL; 10 of these individuals were older than 12 years. In 8, the skin tests were negative, denoting re- cent asymptomatic infection. Clinical examination of 80 (nearly 80%) of the children revealed the liver to be palp- able 2-3 cm below the costal margin, or l-2 cm from the xyphoid process. In one child, with a history of having had VL in 1990, splenomegaly of 7 cm was found (Table 2). None of those who had not had VL previously, but Table 2. CLinical and serological evaluation of 21 people in the study area who had positive serology or a history of recent visceral leishmaniasis

Age (years)

Skill Enlargement of History of test Liver Spleen

VL b-4 SerologP (cm) (4

15 29 26 14 11 63 74 43 52 18 78

8 6 4

<I 51 11 69

<l

No No No Yes Yes No No No Yes Yes No Yes No Yes Yes No No No Yes No No

10 20 20 5

20

ND ND

10 ND ND ND

ND

+ + +

+ + + + -

ND + -

I& - + + +

+ +

0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 7 2 0 0 0 0 0 0 0 2 0 0 0 0 0

‘+, positive; -, negative; ND, not done. bLiver and spleen size were measured below the costal margin.

who were serologically positive, regardless of the skin test result, had developed clinical disease by the time of writing.

Nutritional status, assessed in 59 of 72 children under 10 years of age, showed a relationship between height and age with a Z-score lower than -2 in 15 (25%), denot- ing stunting malnutrition. Other measures were normal. All of the 44 children not suffering from malnutrition were serologically negative; however, 8 had positive skin- tests, denoting past (but not recent) infection. None had a clinical history compatible with acute VL. Thirteen of the 15 children with malnutrition were skin-tested, but only 2 were positive and both had been treated recently for visceral leishmaniasis with pentavalent antimony; none was serologically positive.

Of 54 people who frequently used mosquito bed nets, 17 had positive skin-tests, compared to 31 of the 98 who

157

did not use nets, not a significant difference (x2=0.87, P=O.99; prevalence ratio=0.995 with 95% CI 0.61- 1.62). Seventeen of 40 people who used insecticides in their houses had positive skin-tests, versus 31 of the 112 who did not use them; again, there was no significant correlation or protective effect (x2=2.35, P=O.13; pre- valence ratio= 1.54 with 95% CI 0.96-2.45).

In the canine survey, 29 of 460 dogs found in the prin- cipal nucleus of the village were seropositive (6.3%). Of the 17 dogs which came from the study area, 7 were sero- positive (information on 2 animals was lost). Thus, within the study site, the prevalence of seropositive dogs (7115) was nearly 46%, corresponding to 24% of all the positive dogs (7/29) encountered in the village. However, despite the finding that a greater proportion of persons with positive skin tests had seropositive dogs in the house, there was no significant difference from those with negative skin-tests: 13 of 27 (47%) versus 35 of 125 (28%) (x2=3.29, P=O.O7; prevalence ratio of 1.72 with 95% CI 1.06-2.78).

Discussion Sandfly catches were made in the study area in 1992 by

entomologists of the National Foundation of Health and by World Health Organization representatives (Drs P. Desjeux and F. Modabber). The peridomestic species, Lutzomyia longipalpis, was found particularly in hen houses. Sandfly density was not measured. Although none of the flies tested was found to be infected? the ex- istence of human cases in which there was no history of having lived in endemic areas, associated with the presence of seropositive dogs having characteristic signs of VL, plus the presence of the vector, suggested the strong possibility of active transmission in the area. Fur- thermore, the majority of reported cases involved people who had been born in Monte Gordo and who had never lived in other areas.

Even assuming under-reporting of VL cases and the possibility that some cases in the past had not been rec- ognized, the number of VL cases appearing within a rela- tively short period and in a restricted area indicated the occurrence of an outbreak. The percentage of positive skin tests was far greater than expected, and was similar to that observed in areas with a longer history of VL transmission. There was no marked correlation between age and positive serology, denoting probable recent in- fection of adults as is common in a new focus. Further- more, earlier studies have demonstrated the importance of sub-clinical cases (BADARO et al., 1986c), and our sur- vey clearly documented this. In addition., the fact that hepatomegaly was found very commonly m this popula- tion may support the view that self-healing may occur in subclinical VL cases.

Whether or not the area is a new focus, concern con- tinues regarding the origin of this outbreak. There are at least 2 hypotheses to explain this, and these are not mu- tually exclusive. First, there is the presence of the vector in Salvador and along the coast, as well as a nreviouslv described autochthon&ts case of canine VL in Salvador (SHERLOCK. 1964). Also. foxes. a naturallv infected reservoir of’leishmania sip. (LAINSON, 1983), occur in the region of Monte Gordo. Considering the relatively short flight range of Lutzomyia, it is possible that the parasite and the vector had existed in the area at low le- vels and in an intimate, confined cycle until there was an intrusion of human population. Second, it is possible that the parasite was brought to the area during the mi- grations which have become so common in the region re- cently. VL in Brazil is not considered to be an anthropo- notic disease; however, an epidemic could follow the introduction of infected domestic dogs (LAINSON, 1983). Further dramatic environmental changes, including modification of flora and fauna, brought about by the pressure for industrial and economic development and by continued migrations to the north coastal counties of Bahia, may create other ecological conditions favourable

to the perpetuation of the life cycle of the organism re- sponsible for VL.

The county of Camacari, which includes Monte Gordo, is attracting many new residents including immi- grants, primarily labourers at the nearby petrochemical plant and, more recently, tourists and people from Salva- dor who come to enjoy a new seashore resort. The popu- lation grew from 89 178 in 1980 to 140 060 in 1991. Par- ticipating in this population growth is the village of Monte Gordo which has been in existence since the 17th century. The socioeconomic profile of the people stu- died-low income, no formal job training, and wide- spread malnutrition among the children, which is a rec- ognized risk factor for VL (CERF et al., 1987breflects the effect of an increased immigrant population. Migra- tory waves have been identified before as a cause of tropi- cal disease outbreaks. PEREA et al. (1991) described a serious epidemic of VL in the Sudan brought about by civil war and economic collapse.

Malaria has been reported in our study area since 1980 (MARQUES et al., 1986). Insecticides were used to combat it, but were stopped as soon as the disease was brought under control. Cessation of insecticide application has been described earlier as a contributory factor in the de- velopment of outbreaks of leishmaniasis (SAF’JANOVA, 1971; BHATTACHARYA, 1981).

The recent appearance of VL in Monte Gordo seems to be related to a-process described as urbanization or do- mestication of natural zoonotic foci (MOTT et al.. 1991). In Brazil, one of the main examples of a VL outbreak in a large urban area was an epidemic in Terezina in the state of Piaui (COSTA et al., 1990), associated with migra- tory waves during the dry season and with economic downturns. After our survey, other cases occurred in Bahia near to Salvador-in Lauro de Freitas, Barra de Jacuipe, and Areia, with suspected cases in Itapoan-all within 10 km of the major coastal highway north of Sal- vador .

In order to prevent the expansion of VL, a control pro- gramme has been implemented which includes passive and active surveillance of cases, rapid diagnosis and treat- ment, regular measurement of vector density after spra- ying, and the elimination of infected dogs. In November 1992, a canine survey was conducted (during a rabies con- trol vaccination campaign) using the Falcon assay screen- ing test (FAST)-ELISA (ASHFORD et al., 1993), which per- mits rapid diagnosis of canine infections in the field.

Among 467 dogs screened, 54 were positive using a crude lysate antigen, and 33 were confirmed with a spe- cific cloned antigen (K39) which recognizes only L. dmo- vani-specific antibodies (BURNS et al., 1993). This survey was performed in a single day, and all positive dogs were killed within 15 d after being diagnosed. The impact of this intervention will be reported elsewhere. However, as mentioned earlier, the disease has moved along the rail- way toward Salvador. Unfortunately, it is possible that the dog-peridomiciliary and sylvatic links may have al- ready led to the urbanization of VL in Salvador.

Acknowledgements We thank Dr Eduardo Hage, public health officer of Bahia,

who first reported the cases in Monte Gordo and called our at- tention to the need for a study. Thanks also to Mr Natanael Dantas and Miss Eliete Santos, the 2 field workers, and to Miss Adair Russell for editorial assistance and for the preparation of this manuscript.

Supported by funding from the World Bank, UNDP, the World Health Organization and by research grants A116282, A130639, A126506, and TWO0018 from the National Institutes of Health, USA.

References Ashford, D. A., Badaro, R., Eulalio, C., Freire, M., Miranda,

C., Zalis, M. G. & David, J. R. (1993). Studies on the control of visceral leishmaniasis: validation of the Falcon assay screening test-enzyme-linked immunosorbent assay (FAST- ELBA) for field dia nosis of canine visceral leishmaniasis. AmericanJournal of 8 ropwal Medicine and Hygiene, 48, 1-8.

158

Badar6, R., Jones, T. C., Lorenco, I+, Cerf, B. J., Sampaio, D., Carvalho, E. M., Rocha, H., Teureira, R. & Johnson, W. D., jr (1986a). A prospective study of visceral leishmaniasis in an endemic area of Brazil. 3ouwtal of Infectious Diseases, 154, 639-649.

Badar6, R., Reed, S. G., Barral, A., Orge, G. & Jones, T. C. (1986b). Evaluation of the micro enzyme-linked immunosorb- ent assay (ELISA) for antibodies in American visceral leishma- n&is: anti en responses. X

selecrion for detection of infection-specific mencanJourna1 of Tropical Medicine and Hygiene,

35.72-78. --7.- .-.

Badar6, R., Jones, T. C., Carvalho, E. M., Sampaio, D., Reed, S. G., Barral, A., Teixeira, R. &Johnson, W. D., jr (1986~). New oersoectives on a subclinical form of visceral leishma- niasis. 3ournal of Infectious Diseases, 154,1003-1011.

Bhattacharya, P. K. (1981). Distribution of kala-azar in India. Transactions of the Royal Societ), of Tropical Medicine and Hy- &ne. 75.333-334.

B&&‘J. -a., jr, Shreffler, W. G., Benson, D. R., Ghalib, H. W., Badar6, R. & Reed, S. G. (1993). Molecular charac- terization of a kinesin-related antigen of Leishmania chagasi that detects soecific antibodv in African and American vis- ceral leishma&asis. Proceed&s of the National Academy of Sciences of the USA, 90,775-779.

Cerf, B. J., Jones, T. C., Badar6, R., Sampaio, D., Carvalho, E. M.. Rocha. H., Teixeira. R. & lohnson, W. D.. ir (1987). Malnutrition as a &sk facto; for severe visceral leish&nia&. 3ownal of Infectious Diseases, 156,1030-1032.

Costa, C. H. N., Pereira, H. F. & Araujo, M. V. (1990). Epide- mia de leishmaniose visceral no estado do Piaui, Brasil 198& 1986. Rev&a de Saude Publica, Srio Paulo, 24,361-372.

Lainson, R. (1983). The American leishmaniases: some observa- tions on their ecology and epidemiology. Transactions of the

Royal Society of Tropical Medicine and Hygiene, 77,56%596. Marques, A. C., Pinheiro, E. A. & Souza, A. G. (1986). Urn es-

tudo sobre a disperslo de cases de malaria no Brasil. Revisra Brasileira de Malariologia e Doencas Tropicais, 38,5 1-75.

Mott, K. E., Desjeux, I’:, Moncayo, A., Ranque, P. & De Raadr, P. (1991). Parasitoses et urbamsation. Bulletin of the World Health Organization, 69, %16.

Perea, W. A., Ancelle, T., Moren, A., Nagelkerke, M. & Son- dorp, E. (1991). Visceral leishmaniasis in southern Sudan. Transactions of the Royal Society of Tropical Medicine and Hy- giene, 85,48-53.

Reed, S. G.. Badarci, R., Masur, H., Carvalho, E. M., Lorenco, R., Lisboa, A., Teixeira, R., Johnson, W. D., jr & Jones, T. C. (1986). Selection of a skin test antigen for American vis- ceral leishmaniasis. AmericanJournal of-Tropical Medicine and Hygiene, 35,79-85.

Safjanova, V. M. (1971). Leishmaniasis control. Bulletin of the World Health Organization, 44,561-566.

Sherlock. I. A. (1964). Notas sobre a transmissio da leishma- niose iisceral ‘no Biasi]. Re-vista Brasileira de Malariologia e Doeqas Tropicais, 16, 19-26.

Waterlow, J. C., Buzina, R., Keller, W., Lane, J. M., Nicha- man. M. Z. & Tanner. I. M. (19771. The oresentation and use of height and weight data ior comparing the nutritional status of groups of children under the age of 10 years. Bulletin of the World Health Organization, 55,489-498.

WHO (1986). Use and inte retation of anthro ometric indica- tors of nutritional status. bulletin of the Workz!Health Organiz- ation, 64,929-941.

Received 23 June 1994; accepted for publication 30 June 1994

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