INTRODUCTION
Giardia intestinalis is the causative agent of diarrhea disease in humans and other mammals.1 This parasite has a worldwide distribution and its prevalence varies from 15% to 30%, mainly among nursery and primary school children in slum areas of developing countries.2 In Venezuela, a prevalence of up to 25% has been reported in populations at low socioeconomic levels. 3,4 The clearance of human G. intestinalis infection is dependent on antibodies directed against parasite proteins and T cell activity.5 In this regard, parasite-specific IgA antibodies in infected children has been evaluated by several investigators. 6,7 In previous studies, a 31-kD protein and 57-kD heat-shock protein (HSP) from a membrane fraction were recognized by specific IgA antibody to Giardia spp.6 Additionally, it was shown by immunoblotting that children with chronic infections with G. intestinalis did not show an IgA response to the 57-kD G. intestinalis HSP, which suggested an impairment of the switch from an IgM response to an IgG or IgA response.7 Few studies have evaluated the specific IgA antibody response in patients infected with G. intestinalis before and after treatment.8 The aim of this study was to explore the specific IgA and IgG antibody responses to G. intestinalis by enzyme-linked immunosorbent assay (ELISA) and Western blotting in serum of infected children with G. intestinalis before and after treatment with secnidazole.
MATERIALS AND METHODS
Study population
We studied 364 randomly selected schoolchildren (148 boys and 216 girls) ranging in age from 2 to 11 years (mean ± SD = 5.0 ± 3.0 years) from a disease-endemic sub-urban area of Caracas, Venezuela, as part of the Program of Primary Medical Attention of the Venezuelan Ministry of Health from November 1999 through July 2002. An initial report on this survey included detailed clinical data about response to treatment with secnidazole.3 Seven non-infected children from the same area (mean ± SD age = 5.0 ± 3.2 years) with no recent history of diarrheal illness and no parasites in stool samples at the time of evaluation were used as a control group.
Ethical approval
Parents or legal guardians provided written informed consent for participation of children. The study protocol (pre-evaluation, treatment and post-evaluation) was reviewed and approved by the Ethics Committee of the Institute of Biomedicine in Caracas, Venezuela.
Blood sampling and stool examination
Blood samples were obtained and serum samples were separated and stored at −20°C until use. Serum samples were taken on day 0 (i.e., before and 21 days after treatment). Examination of stool was performed with three consecutive stool samples from each child collected daily and placed in preservative solution (40% tincture of merthiolate, 5% formalin, 1% glycerol) for identification of eggs, cysts, or larvae of intestinal parasites.
Treatment
Children positive for G. intestinalis infection were treated with a single oral dose (30 mg/kg of body weight) of secnidazole suspension (Rhône-Poulenc, Rorer, France).
Culturing of G. intestinalis and preparation of soluble extract
Giardia intestinalis trophozoites of the Portland 1 strain (strain no. 30888; American Type Culture Collection, Manassas, VA) were grown axenically in TYI-S-33 medium at 37°C with 10% heat-inactivated bovine serum.9 Soluble extract was prepared from parasites and resuspended in 1 mL of phosphate-buffered saline (PBS) containing 0.2% Triton X-100. This suspension was sonicated (20 kilocycles/second with a 30-second burst) and centrifuged at 8,000 × g for 20 minutes at 4°C. Supernatants were used as soluble extract and stored at −80°C until use.
Measurement of IgA and IgG antibodies to Giardia sp. by ELISA
Flat-bottomed, Poly Sorp polyvinyl microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 μL/well of soluble extract at concentration of 10 μg/mL in PBS, pH 7.4, and incubated overnight at 4°C. After washing with PBS containing 0.1% Tween 20 (PBS-T), 100 μL/well of individual serum samples diluted 1:50 for IgA and 1:200 for IgG in PBS-T were added and incubated overnight at 4°C. After washing, 100 μL/well of biotinylated-conjugated goat anti-Human IgA (α-chain; Sigma, St. Louis, MO) or biotinylated-conjugated goat anti-human IgG (heavy plus light chains) (GIBCO-BRL Life Technologies, Gaithersburg, MD) diluted 1:2,000 or 1:5,000 in PBS-T was added and incubated for 2 hours at 37°C. After washing, 100 μL/well of streptavidin-peroxidase (Zymed, Laboratories, South San Francisco, CA) diluted 1:2,000 was added and incubated for 30 minutes at 37°C. The color reaction was developed with 100 μL/well of tetramethylbenzidine (Sigma) and stopped after 20 minutes by addition of 100 μL/well of 2 N HCl. Optical density (OD) values were read in an ELISA reader (Multiskan MK 2; Titertek, Huntsville, AL) at 450 nm. The cut-off values, previously calculated on the basis of average OD values from seven negative control serum samples, were 0.12 for IgA and 0.10 for IgG and were equivalent to the mean ± 2 SD observed for G. intestinalis–free human sera.
Gel electrophoresis and immunoblotting
Soluble extract (350 μg/lane) was separated by preparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% polyacrylamide gel in sample buffer as described. 10 Proteins were transferred to a 0.45-μm nitrocellulose membrane (Sigma) by a method previously reported. 11 The membrane was preincubated in blocking buffer (50 mM Tris-Hl, 150 mM NaCl, 5% skimmed milk) at room temperature under constant agitation for 1 hour, cut into strips, and incubated with individual sera samples diluted 1:50 for IgA and 1:100 for IgG in blocking buffer, 0.05% Tween 20 overnight at 4°C. The strips were washed three times in blocking buffer, 0.05% Tween 20 and incubated for 2 hours with alkaline phosphatase–conjugated goat anti-human IgA (α-chain; Sigma) diluted 1:1,000 and goat-anti-human IgG (heavy plus light chains) (GIBCO-BRL) diluted 1:2,000 in blocking buffer. Strips were revealed with the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium kit (Vector Laboratories, Inc. Burlingame, CA), extensively washed in distilled water, and photographed.
Statistical analysis
The significance of difference of serologic values in the ELISA between groups before and after treatment was evaluated using the Student’s t-test. P values < 0.05 were considered significant. A level of protein reactivity (null, moderate, or high) determined by Western blotting was used as a variable with clinical symptoms (presence or absence of abdominal pain, fever, diarrhea, headache, weight loss, and flatulence). Relationships between these variables were analyzed using multiple correspondence analysis. These results were obtained using the statistical software S-Plus 2000 (AT&T Bell Laboratories, Murray Hill, NJ).
RESULTS
Parasite rate and effect of treatment
A total of 190 (52.2%) of 364 children were infected with intestinal parasites. Fifty-four (28.4%) of 190 children were infected with G. intestinalis and other intestinal parasites, and 34 (18.0%) of 190 children (19 boys, mean ± SD age = 4.3 ± 1.6 years and 15 girls, mean ± SD age = 5.2 ± 1.6 years) were infected only with G. intestinalis. Data from children infected only with G. intestinalis parasites were subjected to statistical analysis. All patients were cured after treatment, as confirmed by negative stool examination results and a significant reduction or elimination of clinical symptoms such as abdominal pain, diarrhea, flatulence, and fever.3
Measurement of antibody levels by ELISA
Specific IgG and IgA antibody levels against soluble extract of G. intestinalis before and 21 days after treatment with a single oral dose of secnidazole were measured by ELISA. Non-infected children show no detectable levels of specific IgG and IgA antibodies against Giardia spp. (Table 1). There was no significant decrease in OD values for specific IgG antibody levels after treatment. However, a significant decrease in OD values was found in boys (0.19 ± 0.1 before treatment versus 0.16 ± 0.06 after treatment; P = 0.0077). The reduction in OD values in girls was not significant (0.13 ± 0.04 before treatment versus 0.13 ± 0.034 after treatment; P = 0.17) (Table 1). However, a significant reduction in specific IgA antibody levels (1.16 ± 0.99 before treatment versus 0.94 ± 0.96 after treatment; P = 0.032) was observed in 26 of 34 children (Table 1). This reduction was significant in girls (1.24 ± 1.07 before treatment versus 0.9 ± 0.86 after treatment; P = 0.014; Figure 1A), but not in boys (1.1 ± 1.02 before treatment versus 0.98 ± 1.06 after treatment; P = 0.23; Figure 1B).
Serum reactivity assessed by immunoblotting
We examined potential changes in serum reactivity to G. intestinalis proteins after treatment with secnidazole for specific IgG and IgA antibody responses. The control group did not react with parasite proteins (Figure 1A). Serum of infected children recognized several protein bands between 14 kD and 170 kD, mainly 14-, 15-, 21-, 56-, 72-, 122-, 137-, and 170-kD proteins. Thirteen children did show any change in serum reactivity (non-responders) (Figure 2B). In contrast, 17 children show modifications in serum reactivity for parasite proteins (responders). These patients showed a reduction or disappearance of reactivity for the 14-, 122-, and 137-kD proteins (Figure 2C). Serum of three patients reacted with new protein bands after treatment (Figure 2D). The IgG antibody reactivity to the parasite proteins in infected children was also evaluated by Western blotting before and after treatment. No changes in serum reactivity for specific IgG antibodies to G. intestinalis were detected. An example of this pattern for two patients is shown in Figure 3.
Clinical status and serum reactivity
To determine the relationships between clinical status and antibody response to G. intestinalis antigens before and after treatment with secnidzole, multiple correspondence analysis was used. We found a significant reduction of abdominal pain, diarrhea, flatulencies, and fever after treatment (P < 0.05). However, no significant association between changes in the serum reactivity and reduction of clinical symptoms was detected.
DISCUSSION
Infection with G. intestinalis is one of the most common human infections, particularly in developing countries, which affects mainly preschool children and school children. 12 Poor sanitary conditions and the low socioeconomic status of most of the Venezuelan population favor the high prevalence of infections with intestinal parasites, among them G. intestinalis. Thus, infection with G. duodenalis is considered one of the major public health in our country. Therefore, application of masses treatment and serologic monitoring may be essential for control of these infections. It has been reported that secnidazole in a single dose is effective in eradication of giardiasis. 13–15 In previous studies, we showed significant parasitologic and clinical cure in children infected with G. intestinalis3 and increased mean hemoglobin levels after treatment with secnidazole. 16 In addition, detection of specific protein bands and serum antibody responses against Giardia species proteins by Western blotting and ELISA has been reported. 6,7,17–21 Nevertheless, little is known about effect of treatment on specific antibody responses.
Analysis by ELISA of specific IgA antibody before and after treatment with secnidazole showed a significant reduction in OD values of serum from infected children. This result was likely caused by a reduction in antigen load as a consequence of parasite elimination. This reduction was more significant in girls than in boys. This finding was probably caused by a hormonal-related effect on the antibody response. 22 No change in serum reactivity to G. intestinalis antigens was observed by immunoblotting in some patients. This finding might be caused by high antibody levels from repeated exposure to the parasite. It has been reported that the IgA antibody response to Giardia species can be detected and remain elevated even after infection has been cleared. 23 However, we observed changes in serum reactivity to 14-, 122-, and 137-kD proteins in some infected children. Specific IgG antibody levels against G. intestinalis antigens showed low OD values by ELISA and no significant changes in serum reactivity by Western blotting. These results suggest that evaluation of specific IgG antibody responses is not a suitable method for monitoring the humoral response to infection with G. intestinalis after treatment in a population in a parasite-endemic area. Previous studies have shown a decrease in specific antibody levels after treatment of patients infected with Giardia species.5,8
In Venezuela, although reinfection is frequent, treatment might induce an immunizing effect, as seen in other parasite models. An increase in the repertoire of antibody response against additional antigens may be an advantage in parasite-endemic populations. In addition, comparable results were observed in patients with other parasite infections. 24–28 For example, treatment with praziquantel of children infected with Schistosoma mansoni accelerated development of acquired immune response and modified the level of antibody. 28 Proteomic studies of G. intestinalis proteins by Palm and others 19 showed immunoreactivity in sera of infected patients for specific IgG to native proteins and reactivity to recombinant proteins in a population with acute giardiasis. However, in Venezuela exposure to parasites is more frequent than in countries where outbreaks are the current infection pattern. It has been reported that in populations in which exposure to the parasite occurs at an early age, the humoral immune response is rather uniform and differences between current and past infections cannot be established. 17 Consistently, we could not find a significant association between reduction of symptoms and changes in the serum reactivity after treatment. However, Western blotting could be used as an immunologic tool for monitoring the efficacy of treatment in the follow-up of patients with G. intestinalis infections. Therefore, assessment of community-based disease management strategies, such as mass chemotherapy, would be improved by identifying new protein markers whose levels decrease or become undetectable after elimination of the parasite.
Levels of Giardia intestinalis–specific IgG and IgA antibodies (optical density values) in children by enzyme-linked immunosorbent assay before and after treatment with secnidazole
Levels of specific IgA antibodies to Giardia intestinalis measured by enzyme-linked immunosorbent assay in 34 infected children after (A) and before (B) treatment with Secnidazole. Upper panel: girls; bottom panel: boys. Optical density was measured at 490 nm.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 1; 10.4269/ajtmh.2009.80.11
Levels of specific IgA antibodies to Giardia intestinalis measured by enzyme-linked immunosorbent assay in 34 infected children after (A) and before (B) treatment with Secnidazole. Upper panel: girls; bottom panel: boys. Optical density was measured at 490 nm.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 1; 10.4269/ajtmh.2009.80.11
Levels of specific IgA antibodies to Giardia intestinalis measured by enzyme-linked immunosorbent assay in 34 infected children after (A) and before (B) treatment with Secnidazole. Upper panel: girls; bottom panel: boys. Optical density was measured at 490 nm.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 1; 10.4269/ajtmh.2009.80.11
Analysis by Western blot of specific IgA antibodies to Giardia intestinalis antigens after (A) and before (B) treatment with secnidazole. A, Serum samples from seven non-infected control children. B, Thirteen infected children whom did not show a change in reactivity to G. intestinalis antigens. C, Serum of 17 children with a change in reactivity to proteins of 14, 122, and 137 kD. D, Serum of three children that recognized other proteins after treatment.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 1; 10.4269/ajtmh.2009.80.11
Analysis by Western blot of specific IgA antibodies to Giardia intestinalis antigens after (A) and before (B) treatment with secnidazole. A, Serum samples from seven non-infected control children. B, Thirteen infected children whom did not show a change in reactivity to G. intestinalis antigens. C, Serum of 17 children with a change in reactivity to proteins of 14, 122, and 137 kD. D, Serum of three children that recognized other proteins after treatment.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 1; 10.4269/ajtmh.2009.80.11
Analysis by Western blot of specific IgA antibodies to Giardia intestinalis antigens after (A) and before (B) treatment with secnidazole. A, Serum samples from seven non-infected control children. B, Thirteen infected children whom did not show a change in reactivity to G. intestinalis antigens. C, Serum of 17 children with a change in reactivity to proteins of 14, 122, and 137 kD. D, Serum of three children that recognized other proteins after treatment.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 1; 10.4269/ajtmh.2009.80.11
Analysis by Western blot of proteins immunoreactive with specific IgG antibodies against Giardia intestinalis antigens in two infected and cured children (patients 102 and 109) before treatment (a), after treatment (b), and in a control (c).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 1; 10.4269/ajtmh.2009.80.11
Analysis by Western blot of proteins immunoreactive with specific IgG antibodies against Giardia intestinalis antigens in two infected and cured children (patients 102 and 109) before treatment (a), after treatment (b), and in a control (c).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 1; 10.4269/ajtmh.2009.80.11
Analysis by Western blot of proteins immunoreactive with specific IgG antibodies against Giardia intestinalis antigens in two infected and cured children (patients 102 and 109) before treatment (a), after treatment (b), and in a control (c).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 1; 10.4269/ajtmh.2009.80.11
Address correspondence to Juan C. Jiménez, Laboratory of Immunopathology, Institute of Biomedicine, Faculty of Medicine, Central University of Venezuela, Caracas, Venezuela. E-mail: jcjimenez488@hotmail.com
Authors’ addresses: Juan C. Jiménez, Laboratory of Immunopathology, Institute of Biomedicine, Faculty of Medicine, Central University of Venezuela, Caracas, Venezuela and EA3609 Parasitology-Mycology Service, Faculty of Medicine, Lille 2 University, University Hospital Centre and Federative Research Institute 142, Lille Pasteur Institute, Lille, France. Anthony Pinon, Predictive Microbiology Service, Centre Européen de Recherches sur l’Eau, l’Environnement, l’Aliment et la Toxicologie, Lille Pasteur Institute, Lille, France. Daniel Dive and Monique Capron, Institute National de la Santé et de la Recherche Médicale Unite 547–Schistosomiasis, Malaria and Inflammation, Lille Pasteur Institute, 1 Rue du Professor-Calmette BP245, 59019 Lille, France. Eduardo Dei-Cas, EA3609 Parasitology-Mycology Service, Faculty of Medicine, Lille 2 University, University Hospital Centre and Institute for Research Institute 142, Lille Pasteur Institute, Lille, France. Jacinto Convit, Laboratory of Immunopathology, Institute of Biomedicine, Faculty of Medicine, Central University of Venezuela, Caracas, Venezuela.
Financial support: This study was supported by the Ecology of Pathogenic Eukaryotic Microorganisms Project (EA-3609, Lille, 2 University, and Institue for Food Research 142), and The Programa de Promoción al Investigador del Ministerio de Ciencia y Tecnología, Venezuela.
REFERENCES
- 2↑
Ahmed MM, El-Hady M, Morsy TA, 1990. Parasitic infections and hemoglobin levels among children of different socioeconomic classes in Abha, Saudi-Arabia. J Egypt Soc Parasitol 20 :61–67.
- 3↑
Di Prisco MC, Jimenez JC, Rodriguez N, Costa V, Villamizar J, Silvera A, Carrillo M, Lira C, Zerpa E, Lopez Y, 2000. Clinical trial with secnidazole in a single dose in Venezuelan children infected by Giardia intestinalis. Invest Clin 41 :179–188.
- 4↑
Miller SA, Rosario CL, Rojas E, Scorza JV, 2003. Intestinal parasitic infection and associated symptoms in children attending day care centers in Trujillo, Venezuela. Trop Med Int Health 8 :342–347.
- 5↑
Heyworth MF, Vergara JA, 1994. Giardia muris trophozoite antigenic targets for mouse intestinal IgA antibody. J Infect Dis 69 :395–398.
- 6↑
Char S, Shetty N, Narasimha M, 1991. Serum antobidy response in children with Giardia lamblia and infection of an immunodominant 57-kilodalton antigen. Parasite Immunol 13 :329–337.
- 7↑
Char S, Cevallo AM, Yamson P, 1993. Impaired IgA response to Giardia heat-shock antigen in children with persistent diarrhoea and giardiasis. Gut 34 :38–40.
- 8↑
Cervetto JL, Ramonet M, Nahmod LH, 1987. Giardiasis. Functional, immunological and histological study of the small bowel. Therapeutic trial with a single dose of tinidazole. Arq Gastroenterol 24 :102–112.
- 9↑
Keister DB, 1983. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77 :487–488.
- 10↑
Laemmli UK, 1970. Cleavege of structural proteins during the assembly of the head of bactriophage T4. Nature 227 :680–685.
- 11↑
Towbin H, Staehlin T, Gordon J, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76 :4350–4354.
- 12↑
Miotti PG, Gilman RH, Pickering LK, 1985. Prevalence of serum and milk antibodies to Giardia lamblia in different populations of lacting women. J Infect Dis 152 :1025–1031.
- 13↑
Rastegar-Lari A, Salek-Moghaddam A, 1996. Single-dose secnidazole versus 10-day metronidazole therapy of giardiasis in Iranian children. J Trop Pediatr 42 :184–185.
- 14
Cimerman BL, Camilo Coura C, Salle JM, Gurvitz R, Rocha RS, Bandeira S, Cimerman S, Katz N, 1997. Evaluation of secnidazole gel and tinidazole suspension in the treatment of giardiasis in children. Braz J Infect Dis 1 :241–247.
- 15↑
Escobedo AA, Canete R, Gonzalez ME, Pareja A, Cimerman S, Almirall P, 2003. A randomized trial comparing mebendazole and secnidazole for the treatment of giardiasis. Ann Trop Med Parasitol 97 :499–504.
- 16↑
Jimenez JC, Rodriguez N, Di Prisco MC, Lynch NR, Costa V, 1999. Haemoglobin concentrations and infection by Giardia intestinalis in children: effect of treatment with secnidazole. Ann Trop Med Parasitol 93 :823–827.
- 17↑
Granot E, Spira T, Fraser D, Deckelbaum RJ, 1998. Immunologic response to infection with Giardia lamblia in children: effect of different clinical settings. J Trop Pediatr 44 :241–246.
- 18
Hasan SMT, Maachee M, Córdova OM, Diaz de la Guardia R, Martins M, Osuna A, 2002. Human secretory immune response to fatty acid-binding protein fraction from Giardia lamblia. Infect Immun 70 :2226–2229.
- 19↑
Palm JE, Weiland ME, Griffiths WJ, Ljungstrom I, Svard SG, 2003. Identification of immunoreactive proteins during acute human giardiasis. J Infect Dis 187 :1849–1859.
- 20
Reiner DS, Gillin F, 1992. Human secretory and serum antibodies recognize environmentally induced antigens of Giardia lamblia. Infect Immun 60 :637–643.
- 21↑
Soliman MM, Ruhi Taghi-Kalani F, Ahmed A, Abou-Shady A, Salama A, El-Mageid A, Hondousa A, Hegazi MM, Belosevic M, 1998. Comparison of serum antibody responses to Giardia lamblia of symptomatic and asymptomatic patients. Am J Trop Med Hyg 58 :232–239.
- 22↑
Craig WR, Walker W, Alexander J, 2001. Sex-associated hormones and immunity to protozoan parasite. Clin Microbiol Rev 14 :476–488.
- 24↑
Anam K, Afrin D, Banerjee N, Pramanik SK, Guha RP, Goswami SK, Saha N, Ali N, 1999. Differential decline in Leishmania membrane antigen-specific immunoglobulin G (IgG), IgM, IgE and IgG subclass antibodies in Indian kalaazar patients after chemotherapy. Infect Immun 67 :6663–6669.
- 25
Isaza DM, Restrepo M, Mosca W, 1997. Western blot analysis of Leishmania panamensis antigens in sera of patients with American cutaneous leishmaniasis. J Clin Microbiol 35 :3043–3047.
- 26
Kumar Pai PK, Tripathi K, Pandey HP, Sundar S, 2002. Western blot analysis of the humoral immune response to Leishmania donovani polypeptides in cases of human visceral leishmaniasis: its usefulness in prognosis. Clin Diag Lab Immunol 9 :1119–1123.
- 27
Mutapi FP, Ndhlovu D, Hagan P, 1998. Chemotherapy accelerates the development of acquired immune response to Schistosoma haematobium infection. J Infect Dis 178 :289–293.
- 28↑
Noya O, Losada S, Alarcón de Noya B, 1995. Effect of chemotherapy on immune response to egg antigens of Schistosoma mansoni in chronically infected children from areas of low transmission. Parasite Immunol 17 :111–117.