Assessment of Echinococcus granulosus Somatic Protoscolex Antigens for Serological Follow-Up of Young Patients Surgically Treated for Cystic Echinococcosis

Sera from CE patients.A total of 125 sera were collected from 25 young CE patients during a clinical follow-up period as defined in Table 1. All of the patients but one had been subjected to surgery, and some additionally underwent chemotherapy following the diagnosis of CE, which corresponded to the time point when the patient entered the present study. It has to be noted that some of the patients exhibited, at entrance, a relapse situation in that they had already been previously diagnosed with CE and then treated (Table 1). The CE cases used in the present study included 15 male and 10 female children or adolescents (age range, 4 to 15 years; average, 9 years). In each case, a first serum sample was obtained at the time of diagnosis of CE (or relapsing CE), prior to clinical intervention. The number of follow-up sera and time intervals were variable between the patients and are documented in Table 2. For most cases, the time points were 1 week after surgery; 1, 6, and 12 months after surgery; and then once a year. All serum specimens were stored at −20°C prior to laboratory testing. Clinical examinations were carried out in the pediatric surgery service of the E. P. S. Hospital of Monastir (Tunisia). Standard laboratory investigations were run in the Laboratory of Diagnostic Parasitology of Monastir University.

Patients' clinical files were retrospectively reviewed for the anatomical location and number of cysts, results of radiologic investigations according to World Health Organization guidelines (39), clinical complications, and putative benzimidazole treatment. Twenty of the 25 CE patients achieved a cure or a fully stabilized stage of infection (no disease) without any clinical or radiological evidence of recurrence or cyst activity during and at the endpoint of the study, with 2 patients (no. 5 and 6) showing total calcification of the initial lesions. Another 5 of the 25 CE patients were still sick at the end of the study, as demonstrated by persisting disease either upon the development of new cysts (patients 22 and 23) or enlargement of existing cysts (patients 21, 24, and 25). One case (patient 24) was considered inoperable and involved disseminated liver cysts. This patient was submitted to continuous albendazole treatment (Zentel; 10 mg/kg/day) for 5 years (Table 1). Thus, the grouping of the patients was retrospectively determined and based upon their clinical status at the endpoint of the study period (5 years).

Antigen preparation.Protoscoleces were collected from fresh fertile sheep liver cysts (G1 genotype) directly at the abattoir of Sousse, Tunisia. Viability was determined by the vital coloration approach with 0.2% eosin staining. Furthermore, viability was confirmed microscopically upon flame cell activity and peristaltic motility of the protoscoleces. Respectively validated batches of protoscoleces were submitted to several washing cycles with a sterile physiological sodium chloride solution, followed by three washings with phosphate-buffered saline (PBS; pH 7.2). Subsequently, the parasites were sedimented at 10,000 × g for 10 min and the pellet, supplemented with an equal volume of sterile PBS (pH 7.2), was stored at −80°C until further processing. A PSSA was obtained by performing three freezing-thawing cycles (37°C water bath, liquid nitrogen), followed by two sonications for 30 s at 57 W in a Sonifier B-12 cell disruptor (Bransonic Power Company, Danbury, CT). The treated fluid was sedimented at 10,000 × g at 4°C for 10 min, and the supernatant was supplemented with 1% of a 100 mM PMSF (phenylmethylsulfonyl fluoride) solution; subsequent storage was in liquid nitrogen until use. E. granulosus hydatid fluid (HF) antigen was obtained and processed as previously described (2), and the same batch of antigen as used in that previous study was used.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.PSSA was separated on a 5 to 20% linear gradient polyacrylamide gel as described by Gottstein et al. in 1986 (22), by using reducing and nonreducing conditions separately. Briefly, this was achieved by diluting PSSA in either (i) 10 mM Tris-HCl with 10% SDS and 9 M urea and heating it for 20 min at 65°C or (ii) a similar buffer but with 5% β-mercaptoethanol and heating it for 5 min at 100°C. Proteins were loaded at a concentration of 100 μg/gel (preparative 14-cm slot). Electrophoresis was run at 500 V for 2 h at 4°C.

Electrophoretically resolved proteins were transferred onto nitrocellulose membranes as described by Gottstein et al. in 1986 (22). Nitrocellulose strips (3 mm wide) were then incubated with human sera diluted at 1:100 in PBS (pH 7.2) containing 0.3% Tween 20 and 5% skim milk powder overnight at 4°C. After three washing steps, the strips were incubated with goat anti-human immunoglobulin G (IgG)-horseradish peroxidase conjugate (diluted 1:400) for 2 h at room temperature (Sigma A6029). Immunoreactive bands were visualized with a chromogenic substrate including 3,3′-diaminobenzidine tetrahydrochloride (DAB) and 0.03% H₂O₂.

ELISA.Nunc Immulon ELISA microplate strips were coated by incubation at 100 μl per well with either PSSA or HF diluted in ELISA coating buffer (0.1 M NaHCO₃-Na₂CO₃ [pH 9.6] containing 0.02% NaN₃) at a concentration of 2.5 μg/ml (PSSA) or 10.0 μg/ml (HF). Incubation was overnight at 4°C. ELISA strips were washed three times with washing buffer (PBS, 3% Tween 20) and blocked with blocking buffer consisting of PBS (pH 7.2) with 0.05% bovine hemoglobin and 0.02% NaN₃. Sera were diluted 1:100 in blocking buffer, and alkaline phosphatase-labeled anti-human IgG conjugate (Sigma A5403) was used at a 1:2,500 dilution. All incubations were done at 37°C for 30 min. Plates were finally washed four times and subsequently incubated with the substrate for 15 min. The reaction was finally stopped with 10 μl of 3 N NaOH. Absorbances (A₄₀₅) were read with a microplate reader (Dynex Technologies Bioconcept). The results were expressed in arbitrarily defined antibody units (AU) as previously described (23). On each ELISA plate, we incorporated highly, intermediately, and weakly positive control sera. The intermediately and weakly positive controls quantitatively represented approximately 50 and 10% of the reactivity of the highly positive control serum, respectively. Considering these thresholds and based upon empirical experiences, we clustered all AU values into high, intermediate, and weak classes of reactivity.

Affinity purification of antibodies.Affinity purification of specific antibodies against the 27- and 28-kDa double bands was carried out as described by Müller et al. in 1992 (26). Briefly, the bands corresponding to the 27- and 28-kDa molecules were cut out from the nitrocellulose following SDS-PAGE and blotting of PSSA. This was achieved by the following steps. After blocking of the nitrocellulose with PBS containing 0.3% Tween 20 and 5% skim milk powder, cut strips were incubated with a pool of highly positive CE patient sera at a dilution of 1:10 in PBS with 0.3% Tween 20 overnight at 4°C. After washing with PBS-Tween three times for 20 min each, the bound antibodies were eluted with 900 μl of low-pH buffer (50 mM Tris, 50 mM glycine, 0.05% Tween 20, pH 2.6) for 5 min on ice with vortexing. The strips were then removed, and 100 μl of 1 M Tris base was added to the eluted antibody solution for neutralization. The solution was then sedimented (14,000 × g for 10 min) in order to remove potentially remaining nitrocellulose particles. Subsequently, bovine serum albumin was added to a final concentration of 0.1%. Affinity-purified antibodies were aliquoted and stored at −20°C. Affinity-purified antibodies were used for subsequent immunoblotting at a dilution of 1:50.

Antisera and conjugates.Biotin-labeled mouse anti-human IgG1, IgG2, IgG3, or IgG4 monoclonal antibody and streptavidin-alkaline phosphatase were used to determine IgG subclasses (Sigma catalog no. B6775, B3398, B3523, and B3648). Nitroblue tetrazolium chloride-5-bromo-4-chloro-3-indolylphosphate (BCIP) was used as the substrate for isotype-specific immunoblotting.

To demonstrate the whole antigenic profile of PSSA as a reference to the affinity-purified antibody profile, a hyperimmune rabbit anti-sheep HF polyclonal antibody was used. It was revealed with an anti-rabbit IgG antibody-alkaline phosphatase conjugate diluted 1:1,000 (Promega catalog no. S3731).

Protein concentrations.Protein concentrations were determined with the Bio-Rad Bradford protein assay with bovine plasma immunoglobulin as the standard.

Statistical analyses.Statistical analyses were done with the EpiInfo 6 program. A t test was used to compare the sensitivities of groups of patients during each follow-up period. P ≤ 0.05 was considered statistically significant. Differences were compared by chi-square tests or Fisher's exact probabilities with 95% confidence intervals. The agreement between the two tests was calculated with the kappa factor (κ). κ > 0.7 reflected concordance between ELISA and immunoblotting.

ELISA.Posttreatment evaluation of antibody levels in CE patients was carried out from the time of diagnosis (day 0 [D0]) until the end of the follow-up period (Table 1). For the PSSA ELISA, antibody levels were arbitrarily clustered into highly positive (70 to 180 AU), intermediately positive (25 to 65 AU), weakly positive (1 to 20 AU), and negative (<1 AU) groups; for the HF ELISA, the arbitrary clusters were as follows: 85 to 185 AU, highly positive; 25 to 80 AU, intermediately positive; 1 to 20 AU, weakly positive; <1 AU, negative (Table 3).

At the time of diagnosis, D0 (prior to surgery), PSSA ELISA results exhibited the whole range, from high to intermediate to negative, of antibody levels for both CCE and NCCE patients, and the same held true for the HF ELISA results. The posttreatment evolution of IgG antibody levels in the PSSA ELISA showed that in many cases immunoglobulin levels increased first during the 6 months following surgery, reaching their highest level at 1 month postoperation. Subsequently, levels started to progressively decrease, reaching seronegativity in 4 (20%) of 20 CCE patients (no. 1, 7, 12, and 20) within 1 and 5 years of serological follow-up, compared to only 3 (15%) of 20 CCE patients (no. 1, 12, and 14), as determined with the HF ELISA. These latter sera were also negative in the PSSA ELISA, and the patients were followed up for another few years, although seronegativity had already been reached at 1, 2, and 3 years after treatment (Table 2). Besides, the PSSA ELISA yielded two patients (no. 4 and 14) who were negative at the time of initial diagnosis and who remained seronegative for the whole follow-up period, i.e., 3 and 2 years postoperation, respectively.

Moreover, the majority of patients who remained PSSA ELISA seropositive at the end of the follow-up period showed a faster decrease in IgG levels compared to the kinetics obtained in the HF ELISA, and the remaining antibodies at the end of the study were mostly at low levels (1 to 20 AU). Two CCE patients (no. 5 and 6) whose antibody levels were still high (106 and 95 AU) in the HF ELISA at the end of the follow-up period showed a more pronounced decrease in the IgG level in the PSSA ELISA, reaching relatively lower levels of 47 and 57 AU at the end of the study, respectively. The final clinical examination of these cases revealed the presence of fully calcified cysts; therefore, these patients were correctly considered clinically cured of hydatidosis.

With regard to the overall outcome of the PSSA and HF ELISAs in reference to the study endpoint situation, 75% of the patients with cured cysts (CCE) were still seropositive by both ELISAs at 4 to 5 years postsurgery. Therefore, the endpoint determination of the antibody level alone did not provide a sufficient prognostic value to decide for individual patients if further treatment needed to be added or treatment needed to be continued (Table 3).

In NCCE patients with still active CE, the postoperative serologic response exhibited only a slight increase or decrease in antibody levels in the PPSE ELISA (Table 2). In all five cases, antibody levels in both ELISAs subsequently remained high until the end of the follow-up period. Thus, the endpoint seropositivity was still 100%.

In general, the HF ELISA exhibited higher antibody levels, as well as higher rates of seropositivity, than the PSSA ELISA at diagnosis and during the whole follow-up period (Tables 2 and 3).

Immunoblotting.The PSSA immunoblot profiles of sera from 25 CE patients (20 CCE and 5 NCCE) were, with respect to the banding pattern, qualitatively and quantitatively very heterogeneous within each patient group. The overall pattern, including bands ranging between 9 and 150 kDa, also fluctuated considerably during the follow-up time course for many individual patients. Interestingly, however, we noticed a double band at 27 and 28 kDa that appeared, under both reducing and nonreducing conditions, to be linked to clinical findings. In CCE patients, this double-band activity progressively disappeared; however, the effect did not start before 6 months postsurgery (Fig. 1A and 2A). In some of the cases, the 27-kDa band disappeared before the 28-kDa band (data not shown). Conversely to CCE patients, the respective bands remained strongly recognizable in all of the five NCCE patients with a still active cyst(s) and stayed strongly reactive until the end of the follow-up period (Fig. 1B and 2B).

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FIG. 1.

Temporal evolution of 27- and 28-kDa band recognition by immunoblotting in CCE (A) and NCCE (B) patients after treatment. D0, serum samples collected before surgery; D7, serum samples collected 1 week after surgery; 1 M, serum samples collected 1 month after surgery; 6 M, serum samples collected 6 months after surgery; 12 M, serum samples collected 12 months after surgery, and so on. *, P = 0.012; ¥, P = 0.022.

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FIG. 2.

Follow-up by immunoblotting, exemplified with sera from 4 out of 20 CCE (A) and 5 NCCE (B) patients under reducing conditions (nonreducing conditions not shown). D0, serum samples were collected before surgery; D7, serum samples were collected 1 week after surgery; 1 M, serum samples were collected 1 month after surgery; 6 M, serum samples were collected 6 months after surgery; 1Y, serum samples were collected 1 year postsurgery; 2Y, serum samples were collected 2 years postsurgery, and so on. ¥, sera taken 1 month after a second surgery.

Taking the presence of at least one band of the 27- and 28-kDa doublet marks as an indicator of still-active cysts, most of the CCE patients were in accordance with this criterion by exhibiting a significant decrease from 62% at 6 months postsurgery (detectability rate among 20 CCE patients) to 18% at 3 years postsurgery (Fig. 1A). Only one patient (no. 5) showed persistent anti-28 kDa antibody activity even at 5 years postsurgery. The clinical examination of this case yielded the presence of a fully calcified cyst, and the patient was thus correctly classified as clinically cured of hydatidosis. Moreover, patients 4 and 14, who both were negative in the PSSA ELISA at the time of diagnosis and during the whole follow-up period, were both also negative by immunoblotting with regard to 27- and 28-kDa band recognition.

Among the 14 CCE patients who remained positive in the PSSA ELISA, 11 became negative by 27- and 28-kDa immunoblotting, and none of the PSSA ELISA-negative samples was positive by 27- and 28-kDa immunoblotting. Thus, at the end of the follow-up period, an overall immunoblot negativity was found in 15 (75%) of 20 patients.

Conversely, all NCCE patients still reacted with at least one of the doublet bands even at 5 years postsurgery (Fig. 2). A statistically significant difference between the two clinical groups was observed at 12 and 24 months postsurgery (P = 0.012 and 0.022, respectively).

No significant correlation was revealed between the PSSA ELISA and immunoblotting results of CCE patients (κ = 0.33). We could not calculate κ for the NCCE patients because the number of samples was too low.

Immunoaffinity analyses.In order to check if the 27- and 28-kDa molecules can also be found as metabolic products excreted or secreted into the cyst HF, affinity-purified antibodies specific for each of the doublet 27- and 28-kDa bands were used for immunoblotting with cyst HF antigen. A corresponding antibody-binding activity was completely lacking in HF. Moreover, we could demonstrate that affinity-purified antibodies specific to the 28-kDa band cross-reacted with the 27-kDa band and vice versa (Fig. 3A) when applied to PSSA.

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FIG. 3.

(A) Immunoblotting of HF (lanes a and b) and PSSA (lanes c to p) probed with affinity-purified antibodies specific to 28-kDa (lanes a and c) and 27-kDa (lanes b and d) bands, as revealed with anti-human IgG-peroxidase conjugate. Lanes e to g: immunoblotting of PSSA probed with polyclonal rabbit anti-sheep HF (lane e), negative control rabbit serum (lane f), and conjugate control (lane g), as revealed with anti-rabbit IgG-alkaline phosphatase conjugate. (B) Immunoblotting of PSSA probed with affinity-purified antibodies specific for either the 27-kDa or the 28-kDa band (lanes h to k) and revealed with anti-human immunoglobulin specific to IgG1 (lane h), IgG2 (lane i), IgG3 (lane j), or IgG4 (lane k). Lanes l to p: sera from five NCCE patients (lane l), serum from patient 21 taken 1 year postsurgery (lane m), serum from patient 22 taken 1 year postsurgery (lane n), serum from patient 23 taken 2 years postsurgery (lane o), serum from patient 24 taken 5 years postsurgery (lane p), serum from patient 25 taken 2 years postsurgery. These strips were revealed with an anti-human IgG4 conjugate.

Finally, affinity-purified antibodies obtained from a pool of highly seropositive CE patients were also used for the identification of the isotype(s) involved in antigen-binding activity. Immunoblotting revealed that the anti-27- and -28-kDa reactivity of CE patients was restricted to the IgG4 subclass. Moreover, five serum samples collected from the NCCE patients at the end of the respective follow-up periods were individually tested for their IgG4 subclass reactivity. All five sera exhibited strong recognition of the 27- and 28-kDa doublet band (Fig. 3B).