Identification of Antigenic Epitopes of Strongyloides ...

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Antigen for Immunodiagnosis of Strongyloidiasis Using Western Blot ... Background: Microscopic diagnosis of strongyloidiasis depending on detection of larvae ...
Parasitologists United Journal (PUJ)

Vol. 3, No. 1 & 2 , 2010 65 - 74

Identification of Antigenic Epitopes of Strongyloides stercoralis Filariform Larval Antigen for Immunodiagnosis of Strongyloidiasis Using Western Blot Ola A. Ismail, Wafaa M. Zaki and Sherif M. Abaza Parasitology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt Received: June, 2010

Accepted: July, 2010

Abstract Background: Microscopic diagnosis of strongyloidiasis depending on detection of larvae in fecal samples is not sensitive, especially in asymptomatic patients, and development of reliable serological methods is imperative. Western blot (WB) technique showed promising results for the reactivity analysis in several parasitic infections. Objective: The objective of the present study is to identify relevant proteins of S. stercoralis filariform larvae (L3) using WB and a panel of serum samples for immunodiagnosis of strongyloidiasis. Material and Methods: S. stercoralis L3 were cultured from fecal samples of infected patients. The antigen was extracted and analyzed using SDS-PAGE. Sixty nine serum samples belonging to 3 groups of patients were analyzed and included in the study as: Group I (shedding S. stercoralis larvae in feces), group II (infected with other parasites), and group III (with negative parasitological results). Reactivity of the resulting bands of S. stercoralis L3 antigen was analyzed with the serum samples using WB technique. Results: Thirty four immunoreactive bands were detected in the WB analysis representing recognition of proteins with molecular weight (MW) varying from 19 to 214 kDa. Immunodominant proteins of 43, 41, 36, and 23/33 kDa were recognized respectively in 39%, 35%, 70% and 60% of sera from patients with confirmed strongyloidiasis; and in 29%, 21%, 21% and 30% of sera from those infected with other parasitic infections. One band (41 kDa) gave reaction with one serum sample from group III. Conclusion: It was concluded that the 43, 41, 36 and 32 kDa bands could be considered important tools for the development of diagnostic techniques for strongyloidiasis. Keywords: S. stercoralis, Strongyloidiasis, Immunodiagnosis, Filariform Larval Antigen, Western Blot.

Introduction Strongyloides stercoralis is an intestinal nematode with worldwide distribution, predominantly in tropical and subtropical countries and sporadically in temperate regions(1). The true prevalence of S. stercoralis is likely underestimated because infection is often subclinical. Currently, an estimated 100-200 million persons are infected worldwide in 70 countries(2). In Egypt, S. stercoralis prevalence was estimated in Dakahlia Governorate to be 1.5%(3) and a similar result was obtained in another study conducted on handicapped children in an Egyptian asylum for the blind(4). In both articles, diagnosis was performed using wet mount and routine concentration methods. Strongyloidiasis is a severe disease, but it can be clinically unapparent in the majority of patients with GIT-restricted infections(5). Nevertheless, systemic 65

invasion by larval stage of the parasite leads to a fatal hyperinfection syndrome and disseminated strongyloidiasis in immunocompromised patients(6). Dissemination of infection to other organs develops due to accelerated parasite reproduction via internal autoinfection(7). In the progressive stages of AIDS, as a result of immunosuppression or in the context of chemotherapy, S. stercoralis is capable of inducing overwhelming infection(8). A study conducted in 2009 highlighted the importance of considering strongyloidiasis in all patients from endemic regions on immunosuppressive drug therapy who presented with GIT symptoms and/or systemic Gram-negative bacterial infections without an obvious cause, and recommended that they should be subjected for appropriate investigations(9). Strongyloides stercoralis is one of the most difficult parasitic infections to diagnose(10). Definitive

66 diagnosis of strongyloidiasis usually depends on the demonstration of S. stercoralis larvae in the feces or duodenal fluid(2,11). Yet, it was reported that a single stool examination failed to detect larvae in up to 70% of cases(12). Moreover, in a majority of uncomplicated cases where the intestinal worm load is low and the larval output is minimal and irregular, standard parasitological methods (wet mount, and concentration techniques) had low sensitivity even when repeated several times(11). Baermann technique and agar-plate fecal cultures were reported to be more sensitive, but time consuming(13). ELISA, is available nowadays for detection of serum IgG against S. stercoralis antigen, but it shows cross-reactivity with hookworms, filarial worms, Schistsoma spp., Paragonimus and Echinococcus spp.(14). Moreover, an indirect immunofluorescence antibody test (IFAT) was developed, with a wide range of reported sensitivities and specificities(15). This led the investigators to claim that assessing the accuracies of serological assays is limited primarily by the absence of a gold standard method, rendering the utility of immunoassays for diagnosis and for follow-up controversial. Antigenic preparation became a crucial part of the process for the establishment of reliable immunological tests. Antigens prepared from a saline extract of S. stercoralis larvae or from heterologous parasites, e.g. S. ratti and venezuelensis have been used for diagnosis of strongyloidiasis(16). The antigenic identity of 8 strains of S. venezuelensis was evaluated by IFAT, ELISA and immunoblot (IB). The results revealed average sensitivity (93%) and specificity (100%), for both IFAT and ELISA, while in IB, anti-S. stercoralis IgG recognized a single antigenic fraction of 45 kDa. These results registered the importance of recombinant antigen synthesis that could be useful in immunodiagnosis and vaccine development against strongyloidiasis(17). A new coproantigen detection system for S. venezuelensis diagnosis in immunosuppressed rats was reported using ELISA and anti-L3 polyclonal antibody produced in rabbits(18). The authors claimed that their results may represent a first step in the development of a rapid coproantigen detection kit. Despite the great advances in using these antigens for the diagnosis of S. stercoralis, advanced research is necessary to compare the

Ismail et al., immunodominant antigenic components of those species with S. stercoralis(19). Therefore, the identification and characterization of specific S. stercoralis antigenic epitopes is vital for the development of recombinant proteins that could be used in immunodiagnosis of strongyloidiasis. In the present work, the goal was to identify relevant proteins of S. stercoralis L3 using WB in a panel of serum samples, and to evaluate their sensitivity, specificity, and accuracy. Material and Methods Study type: A case control study. Sample size: To determine the number of samples in each group, the following equation was applied(20): N = 2 (Zα√2Pq + Zβ√2(P1q1 + P2q2) (P1-P2)2 Where N is number of samples in each group, P1 is the estimated event in group I (1.5 %)(3), P2 is the estimated event in group II (3.1%)(21), P is the median of P1 and P2, q=1-P, q1=1-P1 and q2=1-q2. Zα is the standard normal deviate corresponding to the probability (1.96) with confidence = 0.05 (95%). Zβ is the standard normal deviate corresponding to the probability (1.28) with confidence = 0.20 (80%). The sample size was composed of 23 subjects in each group. Patients: Patients of the present study were from residents of the inpatient wards of Suez Canal University Hospital, Ismailia, Egypt. They were subjected to routine parasitological investigations (stool and urine analysis and blood film examination). Stool samples were examined using direct examination, formalinether concentration, modified acid-fast stain, modified trichrome stain and the agar plate culture technique(22). Accordingly, a total of 69 individuals ranging in age from 10 to 60 years of both sexes were enrolled over the period from April 2008 to June 2009. Patients were classified into 3 groups (23 patients in each). Group (I) included patients shedding S. stercoralis larvae (9 of them were immunocompromised from Oncology Department inpatients). Group (II) included patients harboring other parasites than Strongyloides: E. vermicularis (5), A. lumbricoides (4), A. duodenale (4), G. lamblia (3), E. histolytica/dispar (3), S. mansoni (2) and Taenia spp. (2). Group (III) included inpatients with negative results in three consecutive fecal samples collected every other day

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Western Blot in Diagnosis of Strongyloidiasis and with no history of strongyloidiasis. A blood sample (3-5 ml) was withdrawn from each enrolled subject, and sera were stored at -20oC till used in WB. Isolation and collection of L3(23): S. stercoralis L3 were selectively obtained by culture of stool specimens collected from infected individuals. The stool samples were mixed with finely ground wood charcoal, moistened with water, spread in several Petri dishes and incubated at 25°C for 3-5 days. Petri dishes were examined after 48 hours for recovery of L3 under dissecting microscope. Larvae were then harvested using Baermann technique, concentrated by centrifugation for 5 min at 1000 g and stored at -20°C until use for antigen preparation. Antigen preparation(24): Larvae were homogenized using Potter homogenizer (Pyrex Laboratory Glassware, Corning, USA). The parasite extract was disaggregated by ultrasonic process, incubated overnight at 4°C, and then ultra-centrifuged at 10,000×g for 30 min at 4°C. The supernatant fluid was collected and dialyzed with cut off of 2 kDa at 4°C. Polyethylene glycol (Ubichem, Germany) was then added on the dialysis membrane for fluid absorption and protein concentration. Protein concentration of the antigen extract was determined(25) and the preparation was kept at -20°C until use. Electrophoresis and electrophoretic transfer: SDSPAGE analysis of antigenic extract was performed(26) using Mini-protean 3 vertical gel electrophoresis (BioRad System; USA); and then, the gels were transferred to nitrocellulose membranes using a transfer apparatus(27). The membranes were cut into strips and incubated with the collected sera diluted at 1:200 for 2 hours at room temperature with gentle shaking. After washing (3 times, 5 min each), they were incubated with peroxidaseconjugated goat anti-human IgG (Sigma, USA), and then exposed to the substrate (amino-9-Ethyl Carbazole; AEC) for 30 minutes. The strips were rinsed thoroughly with distilled water to stop the reaction, and examined for color development at the site of positive reacting fractions using software Gel pro-analyzer. Statistical analysis: Statistical analysis was performed using the Statistics for Windows software (State soft Inc, 1993). The frequencies of components recognized by IgG in WB among the different groups were compared using Fisher exact test (analysis between two proportions by Z statistics when frequency is less than 5). Differences were statistically significant at level of 5% (P < 0.05). Sensitivity, specificity, accuracy, positive

and negative predictive values (PPV and NPV) were determined(20). Sensitivity = A/(A+C)x100, specificity = D/(B+D)x100, PPV = A/(A+B) x 100, NPV = D/(C+D) x 100, and accuracy = (A+D)/(A+B+C+D) x 100, where A=True positive, B=False positive, C=False negative and D=True negative. Ethical consideration: The study was reviewed and approved by the ethical committee of scientific research, Faculty of Medicine, Suez Canal University. A written consent was obtained from each participant enrolled in the study. Relevant anti-parasitic drugs were prescribed to infected patients, followed by stool re-examination to ensure parasite elimination.

Results Detection of S. stercoralis L3 in patients of group (I) was achieved using 3 different methods: wet mount, concentration methods and agar plate culture. It was found that wet mount detected 5 cases, and the concentration technique detected 15 cases, with sensitivity of 21.6% and 65.2%, respectively. The agar plate culture was found to be the most sensitive one (100%). Using SDS-PAGE, analysis of S. stercoralis L3 antigen revealed eight major bands with MW ranging from ≤ 104 to 13.8 kDa. The bands observed were at 104, 83, 75, 50.6, 40.5, 31, 25 and 13.8 kDa (Figure 1). Using WB, S. stercoralis L3 antigen was allowed to react with sera of the enrolled groups. Different reactivity profiles were observed, representing recognition of proteins with MW that varied from 19 to 214 kDa. Based on intensity of immunoreactivity, this appeared to be the most highly immunodominant antigen. Infection-specific immunodominant antigenic components were defined as the ones recognized by at least 25% of cases(19). Serum samples obtained from S. stercoralis patients (group I) (Figures 2 and 3), presented a high frequency of reactivity with four protein bands corresponding to 43 kDa (9/23, 39%), 41 kDa (8/23, 34.8%), 36 kDa (16/23, 69.6%) and 32/33kDa (14/23, 60.9%). Applying Fisher-exact test to the frequencies of reactivity with these bands among the different groups, reactivity with 36 and 32/33 kDa bands occurred significantly (P < 0.001) more frequently in group I than in groups II and III. On the other hand, significance levels at 0.004 and 0.03

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were obtained on statistical analysis of reactivity frequencies of bands 43 and 41, respectively (Table 1). Reactivity of the selected bands (43, 41, 36 and 32/33 kDa) with sera of group II showed that 43kDa reacted with 6 samples (infected with A. duodenale, and E. vermicularis, 3 samples each), band 41 kDa reacted with 5 samples (4 A. duodenale and one E. vermicularis) and band 36 kDa reacted with 5 samples (one A. duodenale, 2 from each of A. lumbricoides and S. mansoni) (Figure 4). The duplicate band of 32 gave reaction with only one

sample from a patient infected with A. duodenale. On the other hand, only one serum sample in group III gave reaction with the 41 kDa protein band of (Figure 5). Sensitivity, specificity, accuracy, PPV and NPV of the most frequent selected bands, were calculated (Table 2). The most sensitive band was 36 kDa, while the most specific one was a duplicate band of 32⁄33 kDa. The latter band showed the highest percentage of both PPV and accuracy, and NPV was highest in band 36 kDa.

Table (1): Frequency of reactivity of the three groups with the most frequent bands . $ *#+ *"! ! %  "+   *" % /   /   /   &   0    0 4&    5     6    1 2 3 !) 1 2 3 !) 1 2 3!) . 6:7/ 9:7/ >:7/ 5>6 ,) 78 A:7/