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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Longitudinal assessments of hydatid cyst viability are commonly made using serial radiology with or without serial serology. However, there is no test for cure and follow-up surveillance is therefore required for many years. At the Hospital for Tropical Diseases (HTD) in London, an enzyme-linked immunosorbent assay (ELISA) measuring CE-specific total IgG is the main serologic test used for diagnosis and follow-up. However, our experience with this test is that although there may be a gradual decrease in optical density (OD) over time following treatment, the test result rarely becomes negative even after many years, even in those whose disease is apparently cured. Similarly, serial radiology is often unreliable in establishing parasitologic cure; cysts killed by chemotherapy may persist and post-operative defects cannot always be distinguished from viable cysts.
Previous studies have reported that IgG subclasses 1–4 are differentially expressed in patients with chronic helminthic infections.9–11 Similarly, cross-sectional studies of patients with CE using an ELISA incorporating partially purified hydatid cyst fluid antigen B have found a predominance of IgG1 and IgG4 expression in serum.12–15 Moreover, serum concentrations of IgG4 are greater in patients with symptomatic CE compared with those with asymptomatic disease.15 It has therefore been suggested that IgG subclass measurements may provide a more sensitive index of disease activity than CE-specific total IgG.
Patients at HTD with symptomatic CE are treated and followed-up for many years with detailed longitudinal monitoring of clinical, radiologic, and serologic parameters. In this study, we aimed to conduct a retrospective longitudinal analysis of IgG subclass expression in patients with treated CE to determine whether serum concentrations of these subclass antibodies provide a better correlate of CE activity compared with CE-specific total IgG.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment.
The combined medical and surgical approach to treatment of CE at this institution has been previously reported.16 Where possible, patients receive albendazole, 400 mg twice a day for 
3 months, combined with praziquantel, 40 mg/kg/day for two weeks prior to surgical intervention. A minority of patients in this series were treated by medical means alone, using repeated courses of albendazole in combination with a single course of praziquantel. The most common surgical procedure performed was cystectomy using a freezing cone to prevent intra-operative spillage of cyst contents. Two patients were treated surgically by the puncture, aspiration, injection, reaspiration (PAIR) procedure.17
Definitions. Cases. Cases of CE were defined as patients with 1) histologically confirmed CE or 2) radiologic imaging results consistent with CE and positive serology.
Cure.
A patient with CE was defined as having cured disease if the following three criteria were fulfilled: 1) complete surgical resection of all cyst material without spillage of cyst contents; 2) treatment with albendazole, 400 mg twice a day for 3 months plus praziquantel, 40 mg/kg/day for two weeks; and 3) no clinical or radiologic evidence of recurrence during 3 years of follow-up.
Inactive.
A patient with CE was defined as having inactive disease if the following three criteria were fulfilled: 1) surgical resection was not performed, was incomplete, or was associated with intra-operative spillage; 2) anthelminthic treatment with albendazole, 400 mg twice a day for 3 months plus praziquantel, 40 mg/kg/day for two weeks; and 3) there was no clinical or radiologic evidence of cyst activity or recurrence during 3 years of follow-up.
Relapse. Relapsed CE was defined as a recurrence of active CE following a period of quiescent disease as shown by the radiologic enlargement of existing cysts and/or the development of new daughter cysts.
Serology. At HTD, an ELISA incorporating crude E. granulosus horse hydatid cyst fluid antigen is the main serologic test routinely used to measure total CE-specific IgG antibody. Using cyst material from a horse rather than from a human overcomes the problem of reduced ELISA specificity due to contamination with human antigens. This assay has a sensitivity of approximately 90% and a specificity of approximately 96% in detecting hepatic CE among the selected patient population at HTD (Chiodini PL, unpublished data). Suspected cases with borderline positivity may be further tested with an immunoblot. Complement fixation tests were not performed in this study because our own experience is that this test lacks sensitivity and shows significant inter-test variability. The ELISAs were all optimized by checkerboard titration using defined positive and negative sera and standard laboratory methods. Samples from the same patient were analyzed concurrently. The OD cut-off for each of the five assays was determined as the mean + 3 SD of the negative control sera (n = 20).
In these assays, 200-µL volumes were used throughout unless otherwise stated. Immunolon-II high-binding microtiter plates (Dynatech, Chantilly, VA) were coated overnight at room temperature with centrifuged crude hydatid cyst fluid (9 µg/mL). Plates were washed four times with phosphate-buffered saline, pH 7.2, 0.05% Tween (PBS-Tween) and the wells were then blocked with 3% non-fat milk in PBS-Tween for 60 minutes at room temperature. For the assays for total IgG, IgG1, IgG2, IgG3, and IgG4, patient sera were diluted in PBS-Tween 1:500, 1:200, 1:100, 1:50, and 1:50, respectively. Diluted sera were incubated in duplicate wells of the micro-titer plate for 90 minutes at 30°C and the plates were then washed four times with PBS-Tween. The different assays were then continued as follows.
Total IgG assay. Rabbit anti-human horseradish peroxidase-conjugated IgG (Dako, Ely, Cambridgeshire, United Kingdom) diluted 1:4,000 in PBS-Tween was added to each well and the plate was incubated for 90 minutes at 30°C. The plate was then washed four times with PBS-Tween. A substrate solution of 4 mg/ml of o-phenylenediamine dihydro-chloride (Sigma, St. Louis, MO) plus 0.005% hydrogen peroxide was added and incubated at room temperature for 15–20 minutes. Color development was stopped with 2.5M sulfuric acid (30 µL) and absorbance values (ODs) were measured at 490 nm with an ELISA plate reader.
IgG subclass assays. For the IgG subclass assays, monoclonal mouse anti-human IgG1, IgG2, IgG3, and IgG4 (Sigma) diluted 1:500, 1:2,500, 1:1,000 and 1:2,500, respectively, was added to each well. The plates were then incubated for 90 minutes at 30°C and washed four times with PBS-Tween. Peroxidase-conjugated goat anti-mouse Fc-specific IgG (Sigma) was used as the secondary antibody and was diluted 1:24,000, 1:10,000, 1:4,000, and 1:24,000 in PBS-Tween for the IgG1, IgG2, IgG3, and IgG4 assays, respectively. Plates were incubated for 60 minutes at 30°C and the microtiter plate was then washed four times with PBS-Tween. Substrate solution was then added and the plate was developed and read as described for the total IgG assay.
Western blots. Crude E. granulosus antigen from horse hydatid cyst fluid was separated alongside molecular weight markers using polyacrylamide gel electrophoresis according to standard procedures.18 The 12% acrylamide resolving gel was overlaid with a 5% stacking gel. The separated antigens were transferred onto a nitrocellulose (NC) membrane (Hybond-P; Amersham Pharmacia Biotech, Little Chalfont, Bucks, United Kingdom) using a Semi-Phor semi-dry transfer unit (Hoefer Scientific Instruments, San Francisco, CA). Before immunodetection, the NC membrane was blocked with 3% non-fat milk in 50 mM PBS (Blotto). The NC membrane was washed three times with 50 mM PBS, 0.05% Tween, cut into strips approximately 4 mm wide, and placed in a multi-channel plastic tray. One milliliter of a diluted serum (1:50 in blotto) was applied to each NC strip and incubated for one hour at room temperature. The blots were then washed three times with PBS, 0.05% Tween, and goat anti-human IgG conjugated with horseradish peroxidase diluted 1:500 in blotto was added to each NC strip and incubated for one hour at room temperature. The strips were washed again. Color development of the bands was achieved by incubating each NC strip for 30 minutes with chloronaphthol (3% 4-chloro-1-naphthol in absolute alcohol) diluted 1:100 in 50 mM Tris-HCl, pH 7.6, 0.03% hydrogen peroxide. The reaction was terminated by dilution of the substrate in a large volume of distilled water.
Data analysis.
An OD at diagnosis 0.25 was taken as the cut-off above which an assay might potentially provide useful information for post-treatment follow-up. The ELISA ODs increased in a proportion of the patients in the three months following surgical treatment of CE cysts, presumably because of antigen release. Depending on which was the greater, either the OD at diagnosis or the post-operative (< 3 months) OD was selected as the baseline value for post-treatment serologic follow-up. The proportion of patients with a change in OD (
OD) 0.2 and 0.5 during follow up were determined for each assay. Among patients with cured or inactive disease, group comparisons were made between peak ODs and ODs of the latest follow-up serum samples. Since the ODs were normally distributed, parametric statistical tests were used for group comparisons. Prism version 2.0 software (GraphPad Software Inc., San Diego, CA) was used for statistical analysis.
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RESULTS |
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3 months, plus praziquantel, 40 mg/kg/day for two weeks). In addition, 25 of the patients underwent surgical procedures. The mean ± SD duration of follow-up after diagnosis was 5.5 ± 2.6 years (range = 3–12 years). Fifteen patients fulfilled the case definition for cure’ and 11 for inactive disease. Relapse occurred in two patients. The mean number of serial serum samples analyzed per patient was 8.1 (range = 3–34).
Serology at diagnosis.
Cut-off ODs (mean + 3 SD) derived from analysis of negative control sera were low for each of the assays: total IgG (0.217), IgG1 (0.028), IgG2 (0.168), IgG3 (0.163), and IgG4 (0.009) (Figure 1
). Among the cases of CE studied, the number with positive serology (cut-off level) at diagnosis was 28 of 28 (total IgG), 28 of 28 (IgG1), 26 of 28 (IgG2), 13 of 28 (IgG3), and 23 of 28 (IgG4). Thus, the total IgG, IgG1, and IgG2 assays had greater diagnostic sensitivity than the IgG3 and IgG4 assays. Similarly, the mean ± SD ODs obtained with the total IgG, IgG1, and IgG2 ELISAs (0.956 ± 0.204, 0.805 ± 0.326, and 1.216 ± 0.539, respectively) were significantly greater than those of the IgG3 and IgG4 assays (0.389 ± 0.608 and 0.524 ± 0.395, respectively) (Figure 1).
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0.25 obtained with the total IgG, IgG1, and IgG2 ELISAs were 28 of 28, 25 of 28 and 26 of 28, respectively, compared with 12 of 28 and 17 of 28 with the IgG3 and IgG4 ELISAs, respectively. These results indicated that measurement of IgG3 and IgG4 subclasses would not be useful for post-treatment follow-up of a significant proportion of patients.
Serology in patients with cured CE.
Patients with cured disease (n = 15) were followed-up for a mean ± SD of 4.9 ± 1.8 years. The mean OD obtained with each of the five ELI-SAs decreased substantially between the peak values during active disease and values when cured (Table 1). The greatest mean decrease in OD (OD) was observed with the IgG2 assay and this was significantly greater than the mean OD with the total IgG assay.
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OD 0.2 was seen in the great majority of patients with the total IgG, IgG1, and IgG2 assays, but in comparatively few with the IgG3 and IgG4 assays (Table 1
). When the total IgG, IgG1, and IgG2 assays were compared, the proportion of patients with a OD 0.5 was much smaller with the total IgG assay. In view of this observation, serial longitudinal changes in IgG1, IgG2, and total IgG were plotted for the patients (n = 8) in whom the convalescent total IgG OD remained > 0.5 despite cure (Figure 2). In the majority of these cases, the dynamic range of the IgG2 ODs was greater than that of the total IgG and IgG1 ELISAs. Furthermore, in some of the patients (e.g., H1, H4, and H12), ODs for the total IgG and IgG1 ELISAs remained persistently elevated post-treatment, whereas a much greater reduction was seen in ODs obtained with the IgG2 assay.
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); decreases from the peak value were statistically significant for all assays except for the IgG3 ELISA. The greatest decrease in mean OD was seen with the IgG2 ELISA and a decrease 0.2 or 0.5 occurred in a greater proportion of patients tested with this assay compared with the others (Table 2). The mean ODs in the inactive group were far smaller than those in the cured group with the IgG1 (P < 0.001) and the total IgG (P < 0.001) assays. In contrast, the decrease in OD with the IgG2 assay was of similar magnitude when the cured and inactive groups were compared (P = 0.130).
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). Relapse of disease in patient H2 was accompanied by a substantial increase in CE-specific total IgG, IgG1, and IgG2. However, relapse of disease in patient H31 was accompanied by a marked increase in OD only with the IgG2 ELISA; the ODs of the total IgG and IgG1 remained < 0.4.
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). Western blots to detect CE-specific total IgG in the serum at the time of diagnosis of CE showed multiple bands of approximately 110–120, 38, 24, 16, and 8 kD. In contrast, the blots to detect IgG2 showed a dominant 8-kD band and weak bands of 38 and 100–120 kD. Following treatment, these bands in the IgG2 blots were barely visible but reappeared with a dominant 8-kD band during the patient’s disease relapse. Thus, we demonstrated for patient H2 that CE-specific IgG2 antibodies predominantly bound to the 8-kD subunit of E. granulosus antigen B at diagnosis and during disease relapse. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, an ELISA was used that incorporated crude E. granulosus hydatid cyst fluid rather than purified antigen (antigen B or antigen 5) as used in previous studies.12,13,15,19 Our rationale was that the broad diversity of antigens present in crude hydatid cyst fluid might enhance detection of subclass antibodies with different antigen specificities. Despite using crude hydatid cyst fluid in the ELISAs, background OD values among the negative controls were low. Furthermore, a wide range of ODs was observed in all four assays used and were in general higher than those generated in assays incorporating purified antigen B. Although some previous studies12–15 found a predominance of IgG1 and/or IgG4 antibody responses in patients with symptomatic CE at diagnosis, our data agree with those of Ramzy and others, who using crude hydatid cyst fluid as antigen, found that ELISAs for total IgG, IgG1, and IgG2 antibodies were the most sensitive.20 In the present study, low IgG3- and IgG4-specific antibody levels in a significant proportion of patients with CE at diagnosis effectively excluded these antibody subclasses as useful serologic markers for post-treatment follow-up.
Although there is no definitive test of cure among patients treated for symptomatic advanced CE, we believe that our case definition provided a very high likelihood of cure. These data confirm our previous clinical experience that detection of CE-specific total IgG antibody levels, while providing the basis for a sensitive diagnostic test, are unhelpful in serologic follow-up in a significant proportion of patients with CE. In contrast, in the present study, the broad dynamic range of serum IgG2 antibody concentrations and the correlation with treatment response and with relapse suggest that serum CE-specific IgG2 antibody concentrations may provide a useful index of post-treatment disease activity.
Eleven of the patients with CE were classified as having inactive disease because they were treated by medical means alone or because it was not possible to remove all cysts in some patients with extensive disease. However, all received chemotherapy and showed a clinical and radiologic response to treatment without relapse over many years of follow-up. It is likely that many of these patients were actually cured of their disease. The presence of residual dead cyst material may have provided an ongoing antigenic stimulus to antibody production and, in keeping with this, the post-treatment changes in antibody levels (ODs) for patients classified as having inactive disease were in general smaller than those with cured disease (Tables 1 and 2). The notable exception to this was the IgG2 assay, which showed similar post-treatment ODs in both cured and inactive disease groups. It is possible that IgG2 concentrations may correlate more closely with cyst viability and may be independent of the presence of persistent dead cyst material. More prolonged follow-up of this cohort of patients will establish whether indeed this is the case.
In clinical practice, early detection of relapse of CE may be difficult. In patient H2, a marked increase in concentrations of total IgG, IgG1, and IgG2 antibodies all correlated with disease relapse (Figure 3). In contrast, it was striking that specific IgG2 was the only serologic correlate of disease relapse in patient H31 (Figure 3). Had IgG subclass measurements been made in this patient during follow-up, the increasing concentration of IgG2 over several years might have alerted the clinician to the disease activity before it became clinically and radiologically apparent. This information may have led to earlier treatment and thereby limited the morbidity associated with this relapse.
In contrast to our results, previous studies of the correlation of serum concentrations of IgG subclass antibody responses in patients with symptomatic CE have indicated that IgG4 antibodies might correlate well with the activity of CE.15,19 However, in light of the present findings, it is interesting to note that the IgG2 antibody response has previously been observed in patients with cysts undergoing natural involution, appearing on ultrasound as a solid mass with or without calcification12 (classified as type IV or V cysts, respectively).4 Several factors may underlie the differences between our data and those of previous studies. First, previous studies have been limited by a cross-sectional design, whereas in the present study the detailed clinical and radiologic follow-up of our cohort of patients with CE over many years permitted longitudinal analysis of changes in IgG subclass antibody responses over time. Second, we used crude horse hydatid cyst antigen, whereas recent studies have most commonly used partially purified hydatid cyst fluid antigen B,14,15,19 which appears to preferentially bind IgG4 antibodies.14,21 Indeed, the lack of standardized assays in serologic studies of CE make it difficult to make comparisons between different laboratories. Standardization would greatly facilitate progress of research in this field. Furthermore, the development of the World Health Organization standardized radiologic classification of cysts4 will further help comparability of studies.
Since our assays used crude hydatid cyst fluid as antigen, we proceeded to determine which was the immunodominant antigen recognized by the IgG2 subclass. The lipoprotein antigen B and antigen 5 are the two major immunodominant antigens that have been identified in E. granulosus cysts and are the most widely used antigens in current assays for immunodiagnosis of CE.22 Antigen B is a polymeric lipoprotein with a molecular mass of 120 kD that dissociates into 8-, 16-, and 24-kD subunits under reducing conditions on immunoblots, whereas antigen 5 is a very high molecular mass lipoprotein complex that dissociates into 38 and 22–24-kD subunits. We found that IgG2 predominantly bound to the 8-kD subunit of antigen B. Thus, the difference between our results and previous data is not simply explained by use of crude cyst fluid as antigen instead of partially purified antigen B.
In conclusion, using ELISAs that incorporated crude E. granulosus horse hydatid cyst fluid antigen and CE-specific total IgG, IgG1, and IgG2 were found to be sensitive correlates of disease at the time of clinical presentation. However, the IgG2 antibody response was the best correlate of disease activity during post-treatment follow-up. Further prospective evaluation of the IgG2 antibody response in patients during treatment is warranted using standardized serologic and radiologic reporting methods.
Received October 24, 2003. Accepted for publication December 1, 2003.
Acknowledgment: We are grateful for the administrative assistance provided by Margaret Armstrong.
Authors’ addresses: Stephen D. Lawn, Department of Infectious Diseases, St. George’s Hospital Medical School, London SW17 ORE, United Kingdom, Telephone: 44-208-725-5828, Fax: 44-208-725-3487, E-mail: stevelawn{at}yahoo.co.uk. John Bligh, Hospital for Tropical Diseases, London WC1E 6AU, United Kingdom. Philip S. Craig, Cestode Zoonoses Research Group, School of Environment and Life Sciences, University of Salford, Salford M5 4WT, United Kingdom. Peter L. Chiodini, Hospital for Tropical Diseases, London, United Kingdom and Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), IgG1 (•), and IgG2 (
) antibodies among patients (n = 8) in whom cystic echinococcosis (CE) was cured by surgery and anthelminthic chemotherapy but in whom the CE-specific total IgG enzyme-linked immunosorbent assay (ELISA) optical density (OD) values remained 