Humoral Immune Responses in Patients with Acute Schistosoma mansoni Infection Who Were Followed Up for Two Years After Treatment

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1 304 CLINICAL ARTICLES Humoral Immune Responses in Patients with Acute Schistosoma mansoni Infection Who Were Followed Up for Two Years After Treatment Ana L. T. Rabello, Maria Monica A. Garcia, From the LaboratOrio de Esquistossomose, Centro de Pesquisas Rene Rogerio A. Pinto da Silva, Roberto S. Rocha, Rachou-Fundacäo Oswaldo Cruz-FIOCRUZ, and the Centro and Naftale Katz Especializado em Ultrasonografia, Belo Horizonte, Brazil Eighteen patients with acute Schistosoma mansoni infection were followed up for 2 years after with praziquantel or oxamniquine. Cure rates, clinical features, abdominal ultrasonographic findings, and specific humoral responses were determined at 2-, 6-, and 24-month follow-ups. Fourteen patients (77.8%) were considered parasitologically cured. Levels of IgA antibody to soluble egg antigen (SEA) and IgM antibody to keyhole limpet hemocyanin (KLH) became negative or decreased to the cutoff level for chronic infection 2 months after, while levels of IgG antibody to KLH declined between 12 and 24 months after. Levels of IgM and IgG antibodies to saline worm adult protein as well as IgM and IgG antibodies to SEA remained positive during the follow-up period. Discrete lymph node enlargement and hepatomegaly were still present in six of the eight cured children 2 years after, while complete regression was observed in adults. In our group of patients, in addition to presenting with more intense clinical manifestations, children were cured less often and had slower abatement of symptomatology after than adults. Acute schistosomiasis is a toxemic disease that follows the active penetration of Schistosoma cercariae in an immunologically naive vertebrate host. Six to 8 weeks after the first contact with contaminated water, infected patients present with a syndrome frequently including fever, diarrhea, toxemia, and hepatosplenomegaly. The serological definition of acute disease can be reliably made on the basis of the presence of high levels of IgG or IgM antibody to keyhole limpet hemocyanin (KLH) [1-5] or high levels of IgA antibody to soluble egg antigen (SEA) [1]. Supported by epidemiological and clinical aspects, serology plays an important role in the differentiation between acute and chronic schistosomiasis and many other diseases that manifest as fever, hepatosplenomegaly, and intestinal symptoms [1]. Hospitalization may be necessary for patients presenting with more severe clinical manifestations. Intense toxemia, fever, vomiting, and diarrhea frequently provoke dehydration. Clinical support and strict attention should be provided to the acutely infected patient. Only a modest number of scientific publications have addressed acute schistosomiasis in humans, probably because it is frequently misdiagnosed and because of the rarity of the Received 5 September 1995; revised 5 August Grant support: Conselho Nacional de Pesquisas (CNPq-Brasil), PAPES/ FIOCRUZ. Reprints or correspondence: Dr. Ana Rabello, Centro de Pesquisas Rene Rachou, Av. Augusto de Lima, 1715, Belo Horizonte, MG, Brasil. Clinical Infectious Diseases 1997; 24: by The University of Chicago. All rights reserved /97/ $02.00 disease. Studies on post follow-up are rare in the literature; only a few case reports [6-8] have been published. In a previous study [1], the relation between the morbidity (clinical findings) of acute Schistosoma mansoni infection and humoral immune responses was characterized. It has been demonstrated that morbidity (measured by the clinical/sonographic index) is more severe in patients with high-level egg output, irrespective of age or intensity of water contact. It has also been shown that IgA antibodies to SEA are predominantly observed during the acute phase of the infection and that increased levels of IgM and IgG antibodies to KLH and IgA and IgM antibodies to SEA correlate positively with morbidity after adjustment for age and intensity of water contact [1]. In the present study, cure rate, clinical features, abdominal ultrasonographic findings, and specific humoral responses were determined 2, 6, and 24 months after specific chemotherapy for 18 patients with acute schistosomiasis who were described in a previous report [1]. Patients and Methods Patients. A population of 25 previously healthy individuals from the same extended family (mean age ± SD, 21.6 ± 15.5 years; 12 females [48.0%]; 13 males [52.0%]) came into contact with water infested with S. mansoni cercariae. Eighteen patients, six adults and 12 children (age, less than 15 years) had the acute clinical syndrome and were followed up 2, 6, and 24 months after. Acute schistosomiasis was diagnosed on the basis of epidemiological and clinical findings [1] and the documentation of S. mansoni eggs in stools by the Kato-Katz method [9] or by qualitative sedimentation [10].

2 CID 1997;24 (March) Follow-up of Acute Schistosomiasis 305 All patients were treated days after water contact with a single oral dose of praziquantel (50 mg/kg of body weight for adults; 60 mg/kg of body weight for children) or oxamniquine (15 mg/kg of body weight for adults; 20 mg/kg of body weight for children). Follow-up evaluation included clinical and sonographic examination, determination of blood cell counts, at least three stool examinations, and serology. Antibody levels in serum samples from two groups of controls were compared with those in serum samples from patients with acute disease. Controls were 14 patients with chronic schistosomal infection, which was defined by positive results of stool examination and presentation with the clinical form of intestinal disease (age range, years; fecal egg count range, ), and 12 individuals without infection, which was defined as no history of previous and negative results of stool examination (age range, 9-53 years). Ultrasonography. Abdominal ultrasonography was performed by the same author in all cases; the technique has been described elsewhere [11]. Antibody detection. Antibody responses were evaluated by previously described ELISAs for IgG and IgM antibodies to SEA, saline worm adult protein (SWAP), and KLH and IgA antibody to SEA [1, 12]. Statistical analysis. Statistical analysis was performed with SAS software [13]. All variables were individually assessed with the W test of normality. Three-way comparisons of mean antibody levels were made by the F test, and two-way comparisons were made by Scheffe's test. An a error of <5% was considered significant [14]. Results Cure rate and symptoms after. Fourteen (77.8%; six adults [100%] and eight children [66.7%]) of 18 patients were considered cured after ; cure was defined as negative results of stool examination during the follow-up period. Of the noncured patients, one 7-year-old boy presented with abdominal pain and sporadic diarrhea, and one 10-yearold boy complained of mild weakness 2 months after. Two girls (1 and 7 years old, respectively) who had eggs in their stools were asymptomatic at 6 months. Noncured patients were retreated with oxamniquine and considered parasitologically cured after 2 months of re. Abdominal ultrasonography. A gradual reduction of the number and size of enlarged abdominal lymph nodes was observed in all 14 cured patients. Two years after, none of the adult patients had visible lymph nodes. However, six of the eight children who were treated and cured had discrete lymph node enlargement and hepatomegaly. WBC and eosinophil counts in peripheral blood The absolute eosinophil count in peripheral blood significantly decreased 2 months after for all cured and for three noncured patients. The WBC count decreased at 6 months (figure 1). Serum antibody levels. A significant decrease was observed in levels of IgM antibody to SWAP at 2 months and in levels of IgG antibody to SWAP at 24 months (figures 2A and 2B). However, the mean did not reach the negative level for controls. In the individual analysis, after 6 months, one patient had levels of IgM antibody to SWAP that were lower than the cutoff level for the negative control group, and two patients had negative levels of IgM and IgG antibodies to SWAP after 2 years of follow-up. Levels of IgM antibody to KLH significantly decreased at 2 months (figure 2C). The same was observed only for levels of IgG antibody to KLH at 24 months, when levels of both Igs decreased to the cutoff level for chronic infection (figure 2D). Of the 14 cured patients, all 14 had negative levels of IgM antibody to KLH and 11 had negative levels of IgG antibody Figure 1. Eosinophil and WBC counts before and 2, 6, and 24 months after of 14 patients with acute Schistosoma mansoni infection who were treated and cured (0) and four patients with acute S. mansoni infection who were treated, not cured, and retreated at month 2 ( 0 ). A: eosinophil counts (F = 2.13 and P =.11); B: WBC counts (F = 21.7 and P <.0001). = mean of individual values; * = significant differences between time point and month 0 (Scheffe's test included data for the 14 patients who were treated and cured).

3 306 Rabello et al. CID 1997;24 (March) Figure 2. Levels of IgM and IgG antibodies to saline worm adult protein (SWAP) and levels of IgM and IgG antibodies to keyhole limpet hemocyanin (KLH) before and 2, 6, and 24 months after of 14 patients with acute Schistosoma mansoni infection who were treated and cured (0) and four patients with acute S. mansoni infection who were treated, not cured, and retreated at month 2 ( 0 ). A: levels of IgM antibody to SWAP (F = 7.77 and P =.0002); B: levels of IgG antibody to SWAP (F = and P <.0001); C.. levels of IgM antibodies to KLH (F = and P <.0001); D: levels of IgG antibody to KLH (F = 6.84 and P =.0006). * = significant differences between time point and month 0 (Scheffe's test included data for the 14 patients who were treated and cured); = mean of individual values;... = cutoff level for chronic infection; = cutoff levels for acute infection. to KLH 2 years after ; one patient had acute-phase levels of IgG antibody to KLH, and one patient had chronicphase levels of IgG antibody to KLH. Levels of IgM and IgG antibodies to SEA significantly decreased after 2 months of (figures 3A and 3B). Levels of IgG antibody to SEA were above the cutoff levels for chronic infection up to 24 months after. One and two patients had negative levels for IgM antibody to SEA at 6 and 24 months after, respectively. Levels of IgA antibody to SEA decreased significantly 2 months after (figure 3C), when five of the 14 patients who were treated and cured had antibody levels below the cutoff level for chronic infection. After 6 months, eight patients had negative levels of IgA antibody, and three had chronic-phase levels of IgA antibody. After 24 months, 10 patients had negative levels, 2 had chronicphase levels, and 2 had acute-phase levels of IgA antibody. Discussion The clinical and immune aspects of acute toxemic schistosomiasis before have been discussed elsewhere [1]. The therapeutic efficacy of schistosomicidal drugs for acute disease has been suggested to be reduced because of their relative inactivity against immature worms [17]. However, although most of these drugs are relatively inactive in mice 3 to 4 weeks after infection, at the earliest stage of patency (5 to 6 weeks after infection) a high cure rate can be achieved [15, 16]. Different cure rates, varying from 45% among children [17] to 90% among adults [18], have been observed with the use of oxamniquine as for acute schistosomiasis. Praziquantel was associated with a 90% cure rate in a study of adult patients treated 3 months after infection [18]. One patient in our study was treated with praziquantel (60 mg/kg of body weight) and prednisone (1 mg/d for 5 days beginning 2 days before the start of oxamniquine therapy) 37 days after water contact on the basis of clinical and epidemiological features. He had symptoms and S. mansoni eggs in his stools 60 days after. Re with oxamniquine alone was efficient therapy for this patient. We have suggested elsewhere [1] that the morbidity rates among children with acute schistosomiasis are higher than those among adults. In this follow-up study, children were cured less often and had slower abatement of clinical symptoms than adults. Nevertheless, no difference was observed in the specific humoral response between the age groups. It has been demonstrated that persons who have acute clinical symptomatology during their first exposure to S. mansoni infection also present with a distinguishing humoral immune response [1-3]. During this phase, there is intense necrosis with an inflammatory reaction around the eggs, resulting in granuloma formation in the liver, lymph nodes, and intestines. The size of the granulomas reduce as the disease proceeds toward chronicity [19]. Abdominal ultrasonography reveals lymph node enlargement in patients with acute disease but not in patients with chronic infection [11]. It has also been shown that children with acute clinical infection have more intense symptomatology [1]. In this study, we observed that resolution of lymph node enlargement was slower in children than in adult patients who were treated and cured (table 1).

4 CID 1997;24 (March) Follow-up of Acute Schistosomiasis 307 Figure 3. Levels of IgM, IgG, and IgA antibodies to soluble egg antigen (SEA) before and 2, 6, and 24 months after of 14 patients with acute Schistosoma mansoni infection who were treated and cured (0) and four patient with acute S. mansoni infection who were treated, not cured, and retreated at month 2 ( 0 ). A: levels of IgM antibody to SEA (F = 6.05 and P =.0013); B: levels of IgG antibody to SEA (F = and P <.0001); C: levels of IgA antibody to SEA (F = and P <.0001). * = significant differences between time point and month 0 (Scheffe's test included data for the 14 patients who were treated and cured); = mean of individual values;... = cutoff level for chronic infection; = cutoff levels for acute infection. In acute infection, the eosinophil count is remarkably high (figure 1). In vitro, these cells have the capacity to kill schistosomula [20] and to participate as effector cells against parasite eggs [21]. An increase in the circulating eosinophil count is observed between 3 and 8 weeks after infection, concomitant with schistosomulum migration, worm maturation, egg laying, and the appearance of these cells in the granuloma [22]. In this study, a significant decrease in the eosinophil count in patients with acute schistosomiasis was observed 2 months after, which coincided with the decrease in levels of IgM antibodies to SWAP, KLH, and SEA and in levels of IgG and IgA antibodies to SEA. Yuesheng et al. [4] demonstrated that levels of IgM and IgG antibodies to KLH in patients with acute Schistosoma japonicum infection decreased 6 months after. In the present study, levels of IgM antibody to KLH decreased significantly 2 months after, but decreased levels of IgG antibody to KLH were observed only after 2 years of. Levels of all three specific serological markers of acute infection (IgA antibody to SEA and IgM and IgG antibodies to KLH) became negative or decreased to the cutoff level for chronic infection, while levels of antibodies not specifically related to the acute phase remained high (positive). Most circulating IgA antibody is produced in the bone marrow in the form of IgA 1 monomers, whereas secretory IgA antibody is produced mainly at mucosal sites. It has been suggested that the IgA system in humans is composed of two relatively independent synthetic centers but that IgA 1 B cells originate in the mucosa and secondarily colonize the marrow to form a subordinate locus of IgA-producing B cells [23]. The secretory component of secretory IgA antibody renders the IgA molecule to be less susceptible to proteolytic digestion and more mucophilic. The increase in the level of circulating IgA antibody in response to mucosal stimulus can also be explained by the hepatic transport of secretory IgA antibody and by the mucosal homing that permits traffic in the mucosal system, thus redirecting circulating IgA antibody to the mucosal tissues. Although it is possible that the egg deposition in mucosa constitutes the main stimulus causing the increase in the level of IgA antibody, the clear relation between IgA response and disease needs clarification. Different immune functions are certainly involved with IgG and IgM antibodies to KLH and IgA antibody to SEA in acute schistosomiasis. The antibody response to KLH seems to be related to the common carbohydrate epitopes shared between this nonspecific antigen and the schistosomulum surface. Schistosomulum migration and worm maturation do not occur in host mucosa. Nevertheless, egg deposition and elimination through intestinal mucosa are intense. It is worth mentioning that there may be no detectable IgA response to KLH in acute schistosomiasis (data not shown). The differential rates of decreased levels of serum IgG antibody to KLH and IgA antibody to SEA corroborate this hypothesis. The stimulus for IgG antibody to KLH lasts until between 12 and 24 months, as demonstrated in this report. On the contrary, after appropriate, the stimulus for IgA antibody to egg antigen ceases at about 60 days. It is not known whether an increase in the level of IgA antibody to SEA occurs when patients from areas of endemicity are first infected with S. mansoni. In chronic infection, although the intestinal mucosa is also affected, the IgA response to egg antigens is not strong.

5 308 Rabello et al. CID 1997; 24 (March) Table 1. Descriptive analyses of enlargement of abdominal lymph nodes and resolution of enlargement after chemotherapy for 18 patients with acute schistosomiasis. Adults Children Variable Before (n = 6) 6-9 mo after (n = 5) 24 mo after (n = 6) Before (n = 12) 6-9 mo after (n = 8)* 24 mo after (n = 8) No. (%) of patients with lymph node enlargement 5 (83.3) 1 (20.0) 0 12 (100.0) 8 (100.0) 6 (75.0) Mean no.t of enlarged lymph nodes Largest sizes NOTE.... = data not evaluated. * At the time of the 24-month follow-up, the abdominal lymph nodes of three of the four children who had not been cured and who had been retreated 6 to 12 months later were still enlarged. t Four categories were defined: absent (0), few (1), several (2), and numerous (3). $ Measure of the largest diameter of the largest lymph node. In summary, we suggest that in addition to presenting with more intense clinical manifestations, children are cured less often and have slower abatement of symptomatology after for acute schistosomiasis than adults. It has also been shown that the specific humoral immune responses related to acute schistosomiasis, IgG and IgM antibodies to KLH and IgA antibody to SEA, decrease with different kinetics up to 24 months after. Acknowledgments The authors are grateful to Dr. Andrew Simpson, Dr. Kenneth Gollob, and Dr. Philip Loverde for their critical review of this manuscript and to Aureo Almeida Oliveira and Adelu Chaves for their technical assistance. References 1. Rabello ALT, Garcia MM, Pinto da Silva RA, Rocha RS, Chaves A, Katz N. Humoral immune responses in acute schistosomiasis mansoni: relation to morbidity. Clin Infect Dis 1995;21: Mansour MM, Omer Ali P, Farid Z, Simpson AJG, Woody JW. 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