Abstracts

DETECTION OF IgM ANTIBODIES TO Schistosoma mansoni GUT-ASSOCIATED ANTIGENS FOR THE STUDY OF THE DYNAMICS OF SCHISTOSOMIASIS TRANSMISSION IN AN ENDEMIC AREA WITH LOW WORM BURDEN

Herminia Y. KANAMURA(¹ (1 ) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, S. Paulo, SP, Brazil. (2 ) Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil. (3 ) Superintendência do Controle de Endemias - SUCEN, São Paulo, SP, Brazil. (4 ) Instituto Adolfo Lutz, Secretaria de Estado da Saúde de São Paulo, SP, Brazil. (5 ) Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Atlanta, GA, USA. ), Luiz Candido S. DIAS(² (1 ) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, S. Paulo, SP, Brazil. (2 ) Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil. (3 ) Superintendência do Controle de Endemias - SUCEN, São Paulo, SP, Brazil. (4 ) Instituto Adolfo Lutz, Secretaria de Estado da Saúde de São Paulo, SP, Brazil. (5 ) Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Atlanta, GA, USA. ), Carmem M. GLASSER(³ (1 ) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, S. Paulo, SP, Brazil. (2 ) Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil. (3 ) Superintendência do Controle de Endemias - SUCEN, São Paulo, SP, Brazil. (4 ) Instituto Adolfo Lutz, Secretaria de Estado da Saúde de São Paulo, SP, Brazil. (5 ) Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Atlanta, GA, USA. ),

Rita Maria da SILVA(⁴ (1 ) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, S. Paulo, SP, Brazil. (2 ) Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil. (3 ) Superintendência do Controle de Endemias - SUCEN, São Paulo, SP, Brazil. (4 ) Instituto Adolfo Lutz, Secretaria de Estado da Saúde de São Paulo, SP, Brazil. (5 ) Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Atlanta, GA, USA. ), Rosa Maria J. PATUCCI(³ (1 ) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, S. Paulo, SP, Brazil. (2 ) Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil. (3 ) Superintendência do Controle de Endemias - SUCEN, São Paulo, SP, Brazil. (4 ) Instituto Adolfo Lutz, Secretaria de Estado da Saúde de São Paulo, SP, Brazil. (5 ) Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Atlanta, GA, USA. ), Silvia G. CHIODELLI (⁴ (1 ) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, S. Paulo, SP, Brazil. (2 ) Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil. (3 ) Superintendência do Controle de Endemias - SUCEN, São Paulo, SP, Brazil. (4 ) Instituto Adolfo Lutz, Secretaria de Estado da Saúde de São Paulo, SP, Brazil. (5 ) Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Atlanta, GA, USA. ) & David G. ADDISS(⁵ (1 ) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, S. Paulo, SP, Brazil. (2 ) Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil. (3 ) Superintendência do Controle de Endemias - SUCEN, São Paulo, SP, Brazil. (4 ) Instituto Adolfo Lutz, Secretaria de Estado da Saúde de São Paulo, SP, Brazil. (5 ) Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Atlanta, GA, USA. ).

SUMMARY

For a period of 2 years, five follow-up measures of prevalence and incidence rates were estimated in a prospective study of S. mansoni infection in a group of schoolchildren who were living in a rural area of the Municipality of Itariri (São Paulo, Brazil), where schistosomiasis is transmitted by Biomphalaria tenagophila. Infection was determined by the examination of three Kato-Katz stool slides, and the parasitological findings were analyzed in comparison to serological data. In the five surveys, carried out at 6-month intervals (March-April and September-October), the prevalences were, respectively, 8.6, 6.8, 9.9, 5.8 and 17.2% by the Kato-Katz, and 56.5, 52.6, 60.8, 53.5 and 70.1% by the immunofluorescence test (IFT). Geometric mean egg counts were low: 57.8, 33.0, 35.6, 47.3 and 40.9 eggs per gram of feces, respectively. Of the total of 299 schoolchildren, who submitted five blood samples at 6-month intervals, one for each survey, 40% were IFT-positive throughout the study, and 22% were IFT-negative in all five surveys. Seroconversion from IFT negative to positive, indicating newly acquired S. mansoni infection, was observed more frequently in surveys carried out during March-April (after Summer holidays), than during September-October. Seasonal trends were not statistically significant for detection of S. mansoni eggs in stool. The results indicate that the use of IgM-IFT is superior to parasitological methods for detection of incidence of S. mansoni infection in areas with low worm burden.

KEYWORDS: Schistosoma mansoni, Epidemiology, Immunodiagnosis, Seroconversion.

INTRODUCTION

In the State of São Paulo, Brazil, S. mansoni infection is endemic, in some known areas, but rates of transmission and worm burden are low. Therefore, detection of infection using only one fecal examination, as is usual in epidemiologic studies, has been shown to be insensitive^4,11.

In our experience with seroepidemiologic studies in different regions of São Paulo State, Brazil, we have found that S. mansoni seroprevalence rates are 2-4 times higher than prevalence determined by fecal parasitological methods. These differences seem to be mainly because of the poor sensitivity of the parasitological methods to detect patients with low egg counts^5,21,22. Seroprevalence values may also be higher because of the incapacity of the serological tests to discriminate between present and past infections^3,17,26,27. Our experience suggests that, in such areas of low endemicity, serologic results more closely approximate the true situation than do parasitologic results^{6, 14,16}. For schistosomiasis control programs, it is important to use the method that best estimates true schistosomiasis prevalence. This is important not only for a better epidemiological understanding, but also for more efficient coverage of chemotherapy in control programs.

The detection of IgM antibodies against gut-associated antigens by immunofluorescence test (IFT) on worm paraffin sections has been shown to be very sensitive and specific for the diagnosis of both acute and chronic S. mansoni infections²⁸. Also, the detection of IgA antibodies to the gut antigens has been demonstrated to be valuable for differential diagnosis of acute schistosomiasis¹⁸. The usefulness of the IgM-IFT on worm paraffin sections, by using blood samples collected on filter paper, has been tested for epidemiologic purposes and showed to be very efficient and practical. Slides with paraffin sections can be easily prepared and maintained at room temperature for many months without loss of antigenicity¹⁶.

In the present study we have examined the applicability of the IgM-IFT to understanding the dynamics of the schistosomiasis transmission in an area of low endemicity. The serological data were analyzed in comparison with the results of the fecal parasitological examination, following, for a period of 2 years, a group of about 600 schoolchildren living in a rural area of Municipality of Itariri (Sao Paulo, Brazil).

MATERIAL AND METHODS

Description of the study area and population

The climatic and geographical aspects of the study area have been described in a previous paper⁷. The landscape is hilly, with many small streams and plentiful vegetation. The climate is hot and wet, with a mean temperature at 18 °C in the coldest month (July), and temperatures varying from 25 to 35 °C during the day in the hottest months, with a mean temperature higher than 22 °C (December and January).

This project was carried out in a rural village of the Municipality of Itariri, with an area of 294 km² and population of about 11,400 inhabitants, in the Vale do Rio Ribeira de Iguape, near to the Southeast coast of the São Paulo State, in Brazil. According to the data on epidemiologic classification by site of infection²⁴, as collected by SUCEN (Superintendência do Controle de Endemias) of the São Paulo State Health Department, about 68% of the S. mansoni cases can be classified as autochthonous, with the infection being acquired in one of the municipalities around Itariri. The autochthonous schistosomiasis prevalence in the municipality of Itariri was 3.1%, ranging from 0 to 9.2% in the different villages, according to the parasitological survey carried out in 1990, when about 4,900 fecal samples were examined. The only intermediary host in the area is the snail Biomphalaria tenagophila. From April of 1991 to April of 1993, children from four primary schools were followed by parasitological and serological diagnostic methods. In São Paulo, Brazil, primary school education takes 8 years. The school year starts in February and ends in November, with a period of Summer Holiday from December to February, and Winter Holiday in July. Five surveys were carried out, one for each semester, with a 6-month interval between them. In each survey, one fecal sample was collected from all of the children who were registered in the four schools selected for this study; also capillary blood was collected from fingertip onto a filter paper. Informed consent was obtained from all individuals participating in the study and for children the consent of their parents was obtained.

The 1^st, 3^rd and 5^th surveys were carried out respectively in 1991, 1992 and 1993, between March and April of each year, i.e., soon after the Summer Holiday. The 2^nd and 4^th surveys were carried out in the second semester, between September and October, respectively of 1991 and 1992, i.e., after the Winter Holiday.

All S. mansoni egg positive children, after each positive stool, were treated with Oxamniquine, in a single dose of 20-25mg/kg-body weight.

Immunofluorescence test (IFT)

IgM antibodies to S. mansoni gut antigens on Rossman's solution fixed adult worm paraffin sections were detected by IFT, according to the technique previously described ²⁸. Several drops of capillary blood from a fingertip were collected from each individual onto a Whatman #3 filter paper, dried at room temperature and stored in a plastic bag at -20°C. For the IFT, the dried blood spot discs were cut from the filter by a punch with 8 mm diameter. The paper discs were eluted in 120 µl of 0.01 M pH7.2 PBS (Phosphate buffered saline), and incubated overnight at 4 °C. The serum dilution in each eluate after removal of the paper disc was estimated to be 1:16 ¹².

Fecal examination

In each survey, the Kato-Katz technique¹⁹ was performed on one fecal specimen from each schoolchild. S. mansoni egg counts were expressed in eggs per gram (epg), using the average of the egg counts obtained from three slides for each individual. Geometric mean of the egg counts was calculated for each survey for the S. mansoni egg positive samples only. Eggs of other helminth species were also recorded, and the results published elsewhere¹⁶.

Statistical analyses

Data from the five surveys were entered into a microcomputer using the Epi-Info database package² and the same program was used to analyze the results from each survey. Incidence and prevalence rates were compared by the Chi square test using Yate^'s correction.

RESULTS

A total of 987 schoolchildren, 5 to 18 years old, were enrolled in the study. The number of stool and blood samples examined for each survey and the percentage positive for both methods are shown in Table 1. The number of blood specimens collected exceeds the number of stool specimens for all surveys. Geometric mean egg counts were low: 57.8, 33.0, 35.6, 47.3 and 40.9 epg, respectively for the 1^st to 5^th surveys. The percentages of children positive for S. mansoni infection by stool examination were 8.6% and 9.9% for the 1^st and 3^rd surveys (after the summer), compared with 6.8% and 5.8% for the 2^nd and 4^th surveys, respectively (after the winter). The difference between these two groups was statistically significant (p = 0.004). In addition, the prevalence of infection in the 5^th survey, 17.2%, was significantly greater than that of the previous four surveys (p<0.000001). Similarly, seroprevalence was higher for the 5^th survey than for the previous four surveys (p < 0.000001).

Of the 987 children who participated in the study, 299 were followed from the 1^st to the 5^th survey, submitting five blood samples to the serological test; from them 296 submitted both fecal and blood specimens to all five surveys (Table 2). Parasitologic and serologic prevalence data obtained for these 296 children (Table 2) were similar to the prevalence data obtained for all the children who were registered in the four schools and examined each semester (Table 1).

Of the 299 children, who submitted five blood samples for serologic testing, 121 (40.5%) remained IFT-positive throughout the study, and 65 (21.8%) had negative IFT results in all five surveys (Table 3). A total of 63 children seroconverted from IgM-IFT-negative to positive (with no subsequent IFT-negative specimens), indicating new acquisition of S. mansoni infection. Of the total of 87 (65+22) children, with negative results for IgM-IFT in the first four surveys, 22 (25.3%) seroconverted between the 4^th and 5^th surveys. Of the 122 (65+22+3+32) children, who were IgM-IFT negative in first two surveys, 32 (26.2%) seroconverted between the 2^nd and 3^rd surveys. These rates of seroconversion were observed during the summer months, and they were higher (p < 0.000001) than the rates for the other two intervals observed during the winter months: 4.7% and 3.3 %. The former value correspond to 6 of 128 (65+22+3+32+6) children, who seroconverted between the 1^st and 2^nd surveys, and the latter to 3 of 90 (65+22+3) children, who seroconverted between the 3^rd and 4^th surveys. Fifty children (3+39+8) had one or more positive specimens followed by one or more negative specimens in a variety of patterns; 39 (78%) of these children were positive on both, the initial and final specimens (Table 3).

As shown in Table 4, of the 160 schoolchildren who submitted fecal samples to all five surveys, only 57 (35.6%) had at least one fecal sample positive for S. mansoni eggs, and 103 children (64.4%) remained parasitologically negative throughout the follow-up study. No differences in the incidence rates by the parasitologic Kato-Katz method was observed in the different surveys, except for the 5^th survey.

Among the 103 children who consistently tested negative on Kato-Katz examination of stool, 45 (43.7%) had IgM antibodies present at the time of the 1^st survey, indicating previous contact with S. mansoni or possible infection not detected by the Kato-Katz technique. All of the nine Kato-Katz-positive children in the 1^st survey and five positive in the 2^nd survey were IgM-IFT-positive in the 1^st survey. Among the children who were Kato-Katz-positive in the 3^rd, 4^th and 5^th surveys, respectively, three, two and 9 children had no IgM antibodies at the time of the 1^st survey, but all seroconverted during the follow-up. All of the children with two or more fecal samples positive for S. mansoni eggs, except one (who was Kato-Katz positive in the 4^th and 5^th surveys), were IgM-IFT-positive in the 1^st survey.

In nine non-treated children, selected because of their positive result for the IgM-IFT but negative for the Kato-Katz technique in the first survey, new fecal samples were collected to confirm the S. mansoni infection. In one case, the infection was confirmed by the finding of one egg on one of three Kato-Katz slides of a second fecal sample. In six children, negative for all three Kato-Katz slides of the second fecal sample, the confirmation was possible only by the spontaneous sedimentation technique, when one to 60 slides had to be examined to find at least one S. mansoni egg. In the last two children, S. mansoni eggs were detected only by the spontaneous sedimentation techniquein a third fecal sample.

DISCUSSION

In the present study, immunofluorescence test (IFT) was performed on Rossman's fixed adult S. mansoni worm paraffin sections for detection of IgM antibodies to gut-associated polysaccharide antigens²⁵, in a group of schoolchildren living in a village of the municipality of Itariri (São Paulo State, Brazil). This study was planned in order to evaluate the potential of the IgM-IFT, compared to the parasitologic method, as a tool for studying the dynamics of schistosomiasis transmission in an area of low endemicity for schistosomiasis. In previous papers^{18, 28}, the IgM-IFT was shown to be very sensitive for detecting both acute and chronic S. mansoni infections, and also very specific when tested on clinically healthy individuals. When compared to an ELISA for detection of IgG antibodies to total worm protein antigens, although detecting different classes of antibodies against different antigenic structures of the S. mansoni worms, the IgG-ELISA and the IgM-IFT showed similar diagnostic potential for both acute and chronic forms of schistosomiasis²⁹. When applied in field studies, the IgM-IFT on worm paraffin sections displayed a good specificity in an area with high prevalence of other helminth infections, and proved useful for mapping and differentiating areas with and without active transmission of S. mansoni¹⁶. The IgG-IFT, as used in previous epidemiological studies, on frozen sections of S. mansoni worms⁶ or egg granulomata²¹, has some limitations related to the difficulties for the storage and transportation of the antigen sections, which must be conserved frozen to keep their antigenic activity. The high stability of the Rossman's fixed adult S. mansoni worm paraffin sections, at room temperature, and the possibility of detecting IgM antibodies in blood samples collected on filter paper, with good sensitivity and specificity, support the use of the IgM-IFT for epidemiologic purposes.

In this paper, the significantly higher IgM-IFT prevalence rates obtained in comparison to the parasitological data, in all five surveys (Table 1), might actually indicate an area with low worm burden, and not necessarily low endemicity. The geometric mean egg counts, varying from 33.0 to 57.8 epg, obtained by the examination of three Kato-Katz slides for each stool sample, also suggest a low worm burden in the infected individuals. If greater number of slides for each stool sample were examined¹¹, the mean egg count in the studied area would probably be lower than 20 epg. The limit of sensitivity of the method, as shown by Katz et al.²⁰, is at that level. The comparative analysis of the results of the serologic test and the parasitologic follow-up (Table 4) suggests the superiority of the serological test and this lack of sensitivity of the Kato-Katz method for detecting individuals with low worm burden, as already reported by different authors^1,4,6,11.

The tendency to significantly higher parasitologic prevalence rates observed for the post-Summer surveys, when compared to the post-Winter surveys (Table 1) indicates some seasonal influence on S. mansoni transmission in the area. The occurrence of seasonal transmission was clearly detected by the serological follow-up study, since the results of the IgM-IFT showed significant differences of the seroconversion taxes in the post-Summer and post-Winter surveys, statistically higher in the former (Table 3). The positive IgM-IFT result in a schoolchild who showed negative result in the previous survey is undoubtedly indicative of recently acquired infection. In experimentally infected baboons, IgM antibodies to S. mansoni gut antigens were detected by IFT around the second week after infection, thus, many weeks before the appearance of S. mansoni eggs in the feces (data not published). The possibility of detecting S. mansoni infection in such a very early phase after contact with the cercariae can provide a sensitive method to determine incidence of the disease, certainly earlier than the parasitologic method. For this method, we must wait for seven weeks or more for the eggs to be found in the feces. Also, the number of eggs in the feces in this early phase is very small, and the parasitologic methods can fail to detect them. In our study, the parasitologic follow-up data (Table 4) did not show different incidence rates in the post-Summer and post-Winter surveys as did the serological data (Table 3), except for the 5^th survey. The increase in incidence during this last period of the study was unexplained. Some exceptional climatic condition must have favored the transmission of schistosomiasis in the area. Therefore, the parasitologic techniques are not as sensitive as the serologic methods for determining schistosomiasis incidence rates, and the IgM-IFT must be certainly more efficient than Kato-Katz technique in S. mansoni surveillance programs, especially in endemic areas with low worm burdens.

In different epidemiologic studies carried out in schistosomiasis endemic areas of the São Paulo State^5,6,21, or outside of Brazil^8,10,13,15, the authors have obtained higher prevalence indices by serologic than parasitologic methods. The lack of sensitivity of the parasitologic Kato-Katz method for detecting individuals with low egg output cannot fully explain the exceptionally higher serological prevalence rates, in comparison to the parasitologic ones, observed in the present investigation. Rather, the high sensitivity of the IgM-IFT to detect very low infections, which would not be detected by fecal examination, might be an important explanation for such a great difference observed between the serologic and parasitologic methods in this study. Also, the potential of the IgM-IFT to detect recent infection, even before the appearance of eggs in the feces, probably contributes to this difference. The repeated negative results of fecal examinations in some serologically positive children showed how difficult the parasitological confirmation of the S mansoni infection can be in residents of endemic areas with low worm burdens. Also, the possible presence of gut-specific IgM antibodies in some previously infected and self or drug-cured individuals must be considered. S. mansoni infection has been reported since 1953 in the municipality of Itariri, and selected treatment with Hycanthone or Oxamniquine has been performed. In fact, about 20 % of the children enrolled in this project had received previous treatment with one of the above-mentioned drugs, and the serologic prevalence was higher in the previously treated children than in those who had never received treatment¹⁶. Serologic tests based on the detection of circulating antigens would not be subject to these limitations, but such tests have not so far proved to be sufficiently sensitive for diagnosis of individuals with low worm burden⁹.Other factors like contact with cercariae of other parasite species must also be considered and better investigated in the area. The possibility of cross reactivity with other helminth antigens seems unlikely, given the good specificity demonstrated by the IgM-IFT when applied on a rural population of a different village of Itariri. In this village autochthonous schistosomiasis cases had never been detected, but the prevalence rates for other helminth species were as high as the village where the present study was carried out¹⁶.

According to the data in Table 3, a very few individuals (1%) showed definitive seroconversion from IgM-IFT positive to negative status; on the other hand, 39 of them (13%) who showed seroconversion from positive to negative IgM-IFT, returned to positive status in following surveys. These results might suggest that, in our studied area, the children are repeatedly stimulated by a continuous contact with the infective cercariae; but, because of their resistance to new infections, there is no development of mature worms, and consequently no egg production. The higher number of seroconversions from negative to positive IgM-IFT results in post-Summer surveys, when compared to post-Winter surveys, can be explained by the higher chance of children to being exposed to the risk of contact with cercariae during the hot days of Summer holidays. Marçal Jr. et al.²³, studying the risk factors for S. mansoni infection, concluded that recreational activities like swimming, playing and fishing in the river were the main reasons for schistosomiasis transmission in the area, and these activities are more related to the Summer season.

The high sensitivity of the IgM-IFT to detecting schistosomiasis in a very early phase after infection can be used as a powerful tool for studying the dynamics of schistosomiasis transmission in endemic areas with low worm burden.

RESUMO

Detecção de anticorpos IgM contra antígeno de tubo digestivo do Schistosoma mansoni para estudo da dinâmica de transmissão da esquistossomose em área de baixa endemicidade.

Dados de prevalência e incidência da esquistossomose foram estimados, acompanhando-se um grupo de escolares residentes em área rural do município de Itariri (São Paulo, Brasil), por um período de 2 anos, com cinco inquéritos, um a cada semestre, realizados no primeiro semestre de cada ano entre março e abril e no segundo, entre setembro e outubro. O hospedeiro intermediário do Schistosoma mansoni na área é a Biomphalaria tenagophila. A infecção pelo S. mansoni foi determinada pelo método parasitológico de Kato-Katz, através do exame de três lâminas, e os resultados analisados comparativamente aos da reação de imunofluorescência para detecção de anticorpos IgM (RIF-IgM). Foram encontrados nos cinco inquéritos índices de prevalência de 8,6%, 6,8%, 9,9%, 5,8% e 17,2% pelo método parasitológico, e de 56,5%, 52,6%, 60,8%, 53,5% e 70,1%, pelo sorológico. As médias geométricas do número de ovos por grama de fezes (opg) foram baixas: 57,8, 33,0, 35,6, 47,3 e 40,9 opg, respectivamente do 1º ao 5º inquérito. Dos 299 escolares que se submeteram à RIF-IgM nos cinco inquéritos, 40% apresentaram-se positivos e 22% negativos no decorrer do acompanhamento. Soroconversão de negativo para positivo pela RIF-IgM, indicando provável infecção recente, foi observado com maior frequência nos inquéritos realizados no primeiro semestre (pós-verão), que no segundo semestre. A pesquisa de ovos nas fezes não mostrou sensibilidade para detectar essa sasonalidade. Os resultados indicam a superioridade da RIF-IgM para determinação de incidência de S. mansoni em áreas de baixa endemicidade.

ACKNOWLEDGEMENTS

We gratefully appreciate the excellent help provided by the Scientific Researcher Maria Ivani P. G. Silva and the technician Eliane Timóteo Garcia. This work received financial support from FAPESP (Proc. Saúde Coletiva 92/2142-9), and partial support from CNPq.

Correspondence to: Dr. Herminia Y. Kanamura, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Prof. Lineu Prestes 580 (Bloco 17), 05508-900 São Paulo, SP, Brasil. Phone: 55 11 8l8.3633, Fax: 55 11 813.2197. E-mail: kanamura@usp.br

Received: 30 January 1998

Accepted: 13 July 1998

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