Evaluation of Trichinella spiralis Larva Group 1 Antigens for Serodiagnosis of Human Trichinellosis

Abstract

To identify Trichinella antigens suitable for high-specificity and high-sensitivity serodiagnosis of human trichinellosis, we evaluated assays using four antigens: (i) crude first-stage larval extract (CLE), (ii) O-deglycosylated CLE, (iii) tyvelose-bearing antigens (Trichinella spiralis larva group 1 [TSL-1] antigens) purified by US4 affinity chromatography and coupled directly to enzyme-linked immunosorbent assay (ELISA) plates (pTSL-1 antigens), and (iv) TSL-1 antigens immobilized on ELISA plates with the monoclonal antibody (MAb) US4 (cTSL-1 antigens). Assays using these antigens were compared by analysis of sera from healthy individuals (n = 224) (group 1), individuals with noninfectious intestinal pathologies (n = 114) (group 2), individuals with other parasitic infections (n = 107) (group 3), and individuals with confirmed trichinellosis (n = 42) (group 4). Our results indicate that capture ELISA using cTSL-1 antigens is the most effective method for serodiagnosis of human trichinellosis; this was the only method showing 100% specificity and 100% sensitivity at the patent stage of the infection, and it was also the most sensitive for sera obtained prior to patency in indirect immunofluorescence (IIF). Indirect ELISA with pTSL-1 antigens was also 100% specific but was slightly less sensitive, particularly with sera obtained before IIF patency. Inhibition ELISA with MAb US4 indicated (i) that in Trichinella-infected patients the immune response to TSL-1 antigens is directed mostly against tyvelose-containing epitopes (mean of 84.2% of total anti-TSL-1 immunoglobulin G1 [IgG1] antibody response [range, 51.3 to 97.6%]) and (ii) that in most individuals a large proportion of anti-CLE IgG1 antibodies (mean, 49.5%; range, 7.3 to 92.6%) are directed against tyvelose epitopes.

The genus Trichinella is currently considered to include five encapsulating species (T. spiralis, T. britovi, T. nelsoni, T. nativa, and T. murrelli) and three nonencapsulating species (T. pseudospiralis, T. papuae, and T. zimbabwensis), most of which have been implicated in human infections (20). The incubation period (2 to 45 days) and the frequency and intensity of symptoms depend on several factors, including (i) the Trichinella species causing the infection, (ii) the infective dose, and (iii) the individual response to the parasite (7, 20). Diagnosis of human trichinellosis may be difficult, particularly in nonepidemic cases, and requires detailed anamnesis, evaluation of nonspecific biological signs (eosinophilia, leukocytosis, alterations in electrolyte and protein levels, or increases in muscle enzymes such as creatine phosphokinase and lactate dehydrogenase), and immunological techniques for determination of circulating anti-Trichinella antibodies or, less frequently, circulating antigens (13, 19, 21). Over the last decades many techniques have been adapted for detecting antibodies against Trichinella antigens, such as bentonite flocculation, latex agglutination, indirect immunofluorescence (IIF), counterimmunoelectrophoresis, immunoblotting, and enzyme-linked immunosorbent assay (ELISA) (13). At present, however, ELISA techniques using either crude antigen preparations from muscle L1 larvae (10, 25, 28, 41), whole excretory-secretory antigens (ESA) (4, 9, 33, 42), or purified antigens (8, 18, 44) are the most widely used. IIF using cryosections of infected muscles or isolated larvae is also routinely used in some laboratories (17, 31, 37). In addition, antigens from adult worms and newborn larvae have been evaluated for immunodiagnosis (9, 27) but are not widely used.

The use of different antigenic fractions for the immunodiagnosis of human trichinellosis is a significant cause of confusion. In this regard, crude larval extracts (CLEs) may give rise to cross-reactivity if used for serodiagnosis of Trichinella infections in regions in which infections with other, antigenically related pathogens are present (3, 10). It is possible that the specificity of Trichinella CLE could be improved by O deglycosylation of the antigens, as occurs with Anisakis simplex CLE (23), but this possibility has not yet been investigated. ESA recovered by in vitro culture of muscle larvae for 18 h have proved to be more specific, but their recovery is laborious, and cross-reactivity problems may likewise arise. T. spiralis larva group 1 (TSL-1) antigens purified by affinity chromatography with monoclonal antibodies (MAbs) are sensitive and specific for serodiagnosis of Trichinella infections in pigs (2, 15, 43), but these antigens have not been tested in humans. To clarify this situation, in the present study we compared the diagnostic value of indirect and capture ELISAs using Trichinella CLE and tyvelose-containing TSL-1 antigens, with or without O deglycosylation, and sera from Trichinella-free individuals and from patients infected with T. britovi or T. spiralis. Finally, the role of tyvelose-containing epitopes in the recognition of Trichinella antigens by sera from patients with confirmed trichinellosis was also investigated.

MATERIALS AND METHODS

Parasites and antigens. (i) T. spiralis CLE.

Crude first-stage larval (L1) extract (CLE) from T. spiralis was obtained by suspension of worms (1:10, vol/vol) in phosphate-buffered saline (PBS) (phosphate buffer, 0.015 M; NaCl, 0.15 M [pH 7.2]) and then homogenization in a Braun S Potter homogenizer, followed by sonication in an ice water bath (100 W, five times for 30 s each) (Branson Ultrasonics Corp., Danbury, Conn.). The homogenate was centrifuged (3,000 × g for 30 min at 4°C), the resulting pellet was discarded, and the supernatant was centrifuged again (40,000 ×g for 60 min at 4°C). The soluble fraction obtained (CLE) was stored at −30°C until use. The protein concentration was estimated with the Bio-Rad Laboratories (Richmond, Calif.) protein assay with bovine serum albumin (BSA) (Sigma-Aldrich, Madrid, Spain) as protein concentration standard.

(ii) T. spiralis pTSL-1 antigens.

Affinity-purified TSL-1 antigens (pTSL-1 antigens) from T. spiralis were obtained by affinity chromatography as follows. MAb US4 (see below) was conjugated to cyanogen-bromide-activated Sepharose 4B beads (LKB-Pharmacia, Uppsala, Sweden) according to the supplier's instructions. CLE antigens (25 mg) in a total volume of 5 ml were passed into the affinity column, which was previously equilibrated with PBS. After passage of this fraction, the column was washed three times with PBS, and bound glycoproteins were then eluted with 0.1 M glycine-HCl (pH 2.5). The eluted fraction was neutralized with Tris, dialyzed against PBS, and stored at −30°C.

MAb.

The immunoglobulin G1 (IgG1)/κ MAb US4, specific for the tetrasaccharide [β-d-Tyv(1,3)β-d-GalNAc(1,4)α-l-Fuc(1,3)β-d-GlcNAc] present on TSL-1 antigens (14, 38), was obtained by fusion of spleen cells from T. spiralis-infected male NBF1 mice with P3-X63-Ag8.653 cells, as previously described (39). The MAb was partially purified by ammonium sulfate precipitation, and used to immobilize antigens in capture ELISA (see below).

Human sera. (i) Sera from healthy individuals (group 1).

A total of 224 sera from healthy individuals (18 to 35 years old) were provided by the Blood Transfusion Center of Galicia (Santiago de Compostela, Spain).

(ii) Sera from individuals with noninfectious diseases (group 2).

Sera from 114 patients with intestinal pathologies (including appendicitis, cancer, and liver and biliary diseases) were likewise used as controls to evaluate assay specificity. These sera were provided by Carmen Cuéllar of the Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.

(iii) Sera from individuals with infectious diseases other than trichinellosis (group 3).

A total of 107 sera from patients with one or more of the following parasitic diseases were used as controls to evaluate assay specificity: onchocerciasis (n = 9), hydatidosis (n = 1), other filariasis (n = 9), dracunculiasis (n = 10), schistosomiasis (n = 10), fascioliasis (n = 5), toxocariasis (n = 18), strongyloidiasis (n = 1), hymenolepiasis (n = 2), uncinariasis (n = 5), anisakiasis (n = 17), giardiasis (n = 3), and amoebiasis (n = 4). Other sera showing eosinophilia alone (n = 4) or increased total IgE (n = 6) were also included in this group. These sera were provided by Jose Mario Alonso of the Instituto de Medicina Regional, Universidad Nacional del Nordeste, Resistencia, Chaco, Argentina, and by the Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.

(iv) Sera from Trichinella-infected individuals (group 4).

Sera from 42 patients showing epidemiological and clinical data compatible with trichinellosis were provided by Teresa Gárate. These patients were from central and southern Spain and had been infected in three independent trichinellosis outbreaks between 2000 and 2003. The Trichinella species responsible for the outbreaks (Table 1) were T. britovi (patients 1 to 26, first outbreak), T. britovi (patients 27 to 39, second outbreak), and T. spiralis (patients 40 to 42, third outbreak). Trichinella species identification was done at the Laboratorio de Parasitología (Instituto de Salud Carlos III, Madrid, Spain) by using species-specific US5 and US9 MAbs (39) and/or randomly amplified polymorphic DNA (36), using isolated L1 larvae. All of these subjects had shown indications of trichinellosis during confirmed trichinellosis outbreaks; indications were (i) positivity in IIF assay and/or (ii) presence of two or more clinical symptoms or signs (myalgia, fever, orbital edema, eosinophilia, and/or increased muscle enzymes). Note that when several sequentially obtained samples were available for a single patient, only the first sample that tested positive in ELISA with one or more of the antigens analyzed, or in IIF, was used for the experiments included in Fig. 1. However, all sequentially obtained serum samples were included in the experiments presented in Fig. 2.

TABLE 1.

Clinical characteristics of the group of patients with trichinellosis (n = 42) and results of IIF assay for these patients

Patient	Outbreaka	IIF resultb	Clinical symptomsc
1	I	1/1280	PFE, F, M, E
2	I	1/160	PFE, F, M, E
3	I	>1/160	PFE, F, E
4	I	>1/1280	NA
5	I	>1/1280	PFE, F, M
6	I	B	PFE, M, E
7	I	>1/160	PFE, M, E
8	I	1/160	PFE, F, E, CPK
9	I	1/160	PFE, M, E
10	I	>1/160	PFE, M, F, E, CPK, LDH
11	I	>1/160	PFE, M, CPK, LDH
12	I	>1/160	F, M, CPK, LDH
13	I	>1/160	PFE, F, M, CPK, LDH
14	I	<1/20	PFE, F, M
15	I	>1/160	F, M, CPK, LDH
16	I	>1/160	PFE, F, M, CPK, LDH
17	I	>1/160	NA
18	I	1/40	NA
19	I	B	F, M
20	I	>1/160	NA
21	I	>1/160	NA
22	I	>1/160	PFE, E
23	I	>1/100	PFE, E
24	I	B	F
25	I	>1/160	F
26	I	>1/160	F
27	I	>1/160	ND
28	II	1/160	M
29	II	>1/160	ND
30	II	>1/160	ND
31	II	1/160	ND
32	II	>1/160	ND
33	II	1/160	ND
34	II	>1/160	ND
35	II	1/160	ND
36	II	>1/160	ND
37	II	1/160	ND
38	II	1/20	ND
39	II	1/20	ND
40	III	1/40	ND
41	III	1/80	ND
42	III	B	ND

^aOutbreaks I and II were caused by T. britovi, whereas outbreak III was caused by T. spiralis.

^bIn IIF assay, a serum was considered positive if clear fluorescence was observed with a serum dilution of 1:20 or higher, negative if fluorescence was not detected with a serum dilution of 1:20, and borderline (B) if only a very faint or irregular fluorescence was observed with a serum dilution of 1:20.

^cPFE, palpebral and facial edema; F, fever; M, myalgia; E, eosinophilia; CPK, raised serum creatine phosphokinase levels, LDH, raised serum lactate dehydrogenase levels, ND, no clinical data available.

FIG. 1.

Serum IgG1 responses to CLE antigens (A), O-deglycosylated CLE antigens (B), pTSL-1 antigens (C), and cTSL-1 antigens (D). Each antigen was assayed with a panel of serum samples as indicated on the x axis: healthy subjects (group 1 [G1]), patients with noninfectious pathologies (group 2 [G2]), patients with infectious diseases other than trichinellosis (group 3 [G3]), and trichinellosis patients (group 4 [G4]). Each circle is the OD obtained for a single subject. Dotted horizontal lines are cutoffs defined as the mean of the results for negative subjects (groups 1, 2, and 3) plus 4 standard deviations (see text). Solid horizontal lines are cutoffs defined as the maximum ODs obtained in analysis of negative sera (upper-limit value) (see text).

FIG. 2.

Results obtained by using the different assays to analyze sera from 10 patients with confirmed trichinellosis. In all cases sample B shows patency in IIF assay; sample A was obtained 5 to 46 days previously. Note that these are clinical data, so we do not know when infection occurred. Black circles indicate a positive results, white circles indicate a negative result, and dotted circles indicate a borderline result. ELISA 1, ELISA with CLE antigens; ELISA 2, ELISA with O-deglycosylated CLE antigens; ELISA 3, ELISA with pTSL-1 antigens; ELISA 4, ELISA with cTSL-1 antigens.

Mild alkali antigen hydrolysis.

O-glycans from Trichinella CLE antigens were released by mild alkali treatment as described by MacDonald et al. (24). Briefly, 2 ml of CLE (5.8 mg/ml) was incubated with 2 ml of 0.02 M NaOH at 50°C for 24 h. The sample was neutralized by PBS dialysis at 4°C for 12 h. The O-deglycosylated antigens were stored at −20°C until use.

Immunoassays. (i) Indirect ELISA and inhibition by US4.

The ELISA plates were incubated overnight at 4°C with 1 μg of antigen (CLE, O-deglycosylated CLE, or pTSL-1 antigens) per well in 100 μl of PBS. After incubation, the plates were washed with distilled water (dH₂O), and the remaining active groups were blocked with (per well) 200 μl of 1% dry skim milk in Tris-buffered saline (TBS) (pH 7.4) containing 0.02% Tween-20 (TBS-T) for 2 h at 37°C. After a washing step with dH₂O, the plates were incubated for 1 h at 37°C with 100 μl of a 1:100 dilution of human serum per well. After washing again, each well was incubated with 100 μl of a 1:1,000 dilution of fluorescein isothiocyanate (FITC)-conjugated mouse anti-human IgG1 (Sigma-Aldrich) for 1 h at 37°C, and bound Ig-FITC was detected by treatment 100 μl of a 1:1,000 dilution of peroxidase-conjugated rabbit anti-FITC (Dako Diagnosticos SA, Barcelona, Spain) per well for 1 h at 37°C. Finally, the plates were washed with dH₂O, and 100 μl of a substrate solution containing o-phenylenediamine and urea hydrogen peroxide in phosphate-citrate buffer (Sigma Fast o-phenylenediamine dihydrochloride tablet sets) was added to each well and incubated for 30 min at room temperature. The reaction was stopped with 3 N H₂SO₄ (25 μl/well), and optical densities (ODs) were measured at 492 nm.

Inhibition by US4 in indirect ELISA was assayed by blocking tyvelose epitopes contained in CLE antigens with an excess of MAb US4 diluted 1:500 in TBS-T containing 1% dry skim milk (100 μl/well). Controls for maximal reactivity were obtained by adding 1% dry skim milk-TBS-T (100 μl/well) to ELISA wells instead of US4. Following a washing step with dH₂O, 100 μl of a 1:100 human serum dilution was added to each well, and the plates were incubated at 37°C for 1 h. The plates were then washed again, and bound human IgG1 antibodies were detected as described above.

(ii) Capture ELISA and US4 inhibition.

The ELISA plates were coated with a 1:1,000 dilution of partially purified MAb US4 to capture the specific antigens (cTSL-1 antigens), incubated overnight at 4°C, and then blocked as for indirect ELISA. After a washing step with dH₂O, 100 μl of a 1:500 dilution (1 μg) of CLE was added to each well and incubated for 1 h at 37°C. After a second wash with dH₂O, a 1:100 dilution of each serum was added to the wells, and the plates were incubated again at 37°C for 1 h. The plates were then washed again, and bound human IgG1 antibodies were detected as described for indirect ELISA.

Inhibition by US4 in capture ELISA was analyzed by blocking free tyvelose epitopes on immobilized TSL-1 antigens by adding an appropriate amount of MAb US4 (1: 500 dilution) and incubating at 37°C for 1 h. Controls for maximal reactivity were done with 1% dry skim milk in TBS-T (100 μl/well) instead of MAb US4. Following a washing step with dH₂O, 100 μl of a 1:100 dilution of human serum were added to each well, and the plates were incubated at 37°C for 1 h. The plates were then washed again, and bound human IgG1 antibodies were detected as described above.

(iii) IIF.

IIF was performed as described by Sulzer (45). A serum was considered to be positive when it gave a clear fluorescence pattern at a dilution of 1:20 or higher.

ELISA calculations.

To evaluate the performance of the diagnostic assays, we calculated sensitivity as TP/(TP + FN) and specificity as TN/(TN + FP), where TP, TN, FP, and FN are the numbers of true positives, true negatives, false positives, and false negatives, respectively. From the sensitivity and specificity values determined for each antigen, we calculated the Youden's J index (48) as an overall measure of the reliability of the diagnostic test. Interassay variability was assessed by testing a positive control serum on 4 to 5 consecutive days; on each day the control serum was tested in duplicate, and mean absorbance was determined and used to calculate the interassay coefficient of variation (CV). Statistical significances were assessed by t tests.

RESULTS

Cutoff calculations for Trichinella antigens.

To assess the suitability of the selected antigens for serodiagnosis of human trichinellosis, we first calculated the cutoff values (mean plus 4 standard deviations) for each antigen tested (CLE, O-deglycosylated CLE, pTSL-1 antigens, and cTSL-1 antigens). For these calculations we combined the ODs obtained from the three groups of negative subjects: healthy individuals (group 1), patients with noninfectious diseases (group 2), and patients with other infectious diseases (group 3) (see Materials and Methods). These cutoff values were 1.084 for CLE (n = 445), 0.332 for O-deglycosylated CLE (n = 445), 0.141 for pTSL-1 antigens (n = 252), and 0.098 for cTSL-1 antigens (n = 395). The maximal serum OD registered for sera from these groups was considered to be the upper limit for negativity. The upper-limit values for the antigens were 1.69 (CLE), 0.66 (O-deglycosylated CLE), 0.170 (pTSL-1 antigens) and 0.159 (cTSL-1 antigens). Serum ODs located between the cutoff and the corresponding upper-limit value were considered to be borderline values, while those higher than the upper limit were considered to be positive.

Recognition of Trichinella CLE antigens.

The results presented in Fig. 1A showed, as expected, maximal cross-reactivity when using Trichinella CLE in indirect ELISA. With this antigen, the sera from healthy subjects (n = 224 individuals) gave ODs ranging from 0.0 to 1.19 (mean = 0.124). For sera from patients with noninfectious diseases (n = 114 patients), we observed a significant increase in ODs with respect to healthy subjects, with ODs ranging from 0.0 to 1.31 (mean = 0.170; P < 0.01). For sera from patients with infectious diseases other than Trichinella (n = 107 patients), the mean OD was 0.348 (range, 0.0 to 1.692). Maximal cross-reactivities were obtained with sera from patients with onchocerciasis or dracunculiasis (n = 19 patients), with mean ODs of 0.751(P < 0.0001 with respect to the other patients [n = 88] in group 3). Assuming a cutoff of 1.084, previously obtained with negative sera, the specificity of the assay was 98.9%, but the sensitivity was unacceptably low; specifically, the data presented in Fig. 1A show that only 24 sera from the 42 subjects from group 4 (infected) gave ODs higher than the cutoff (57.1% sensitivity; J index, 0.56), and only 16 of 42 exceeded the upper-limit OD of 1.69 (38.1% sensitivity; J index, 0.381). The interassay CV was 2.7%.

Recognition of O-deglycosylated Trichinella antigens.

Figure 1B shows the results obtained with the four group of sera in indirect ELISA with O-deglycosylated CLE antigens. As can be seen, treatment of the antigen reduced the cross-reactivity observed with untreated CLE. The ODs obtained with sera from healthy individuals were relatively low (maximal OD, 0.183; mean, 0.037), and similar results were obtained with sera from patients with noninfectious diseases (maximum OD, 0.213; mean, 0.072). Sera from patients with infectious diseases other than those caused by Trichinella showed significantly higher ODs (maximum OD, 0.66; mean, 0.114; P < 0.0001 with respect to healthy individuals). As with CLE antigens, maximal cross-reactivities were obtained with sera from patients with onchocerciasis or dracunculiasis (n = 19 patients), with a mean OD of 0.246 (P < 0.0001 with respect to the other patients in group 3). Assuming the same cutoff value obtained with negative sera (OD = 0.332), the specificity of the assay was the same as for CLE antigens (98.9%). However, in spite of the reduced cross-reactivity, the OD positivities with O-deglycosylated antigens were still unacceptably low; only 20 of 42 gave ODs values higher than the cutoff value (sensitivity, 47.6%; J index, 0.46), and only 13 of 42 gave ODs higher than the upper-limit value (sensitivity, 30.9%; J index, 0.39). The interassay CV was 2.6%.

Recognition of TSL-1 antigens in indirect ELISA.

We next evaluated the suitability of TSL-1 antigens for trichinellosis diagnosis. We first tested the sera from the three groups of negative samples against affinity-purified TSL1 antigens coupled to ELISA plates. The results obtained are presented in Fig. 1C. These antigens show minimal reactivity with sera from healthy subjects (n = 88 individuals) and from patients with noninfectious diseases (n = 71 patients), and only 1 of 93 sera from infected patients gave an OD higher than the cutoff calculated for this assay (0.141). With this cutoff, the specificity of the assay was 99.6%. Sensitivity was evaluated by testing the sera from 36 trichinellosis patients (six sera could not be tested because of low sample volume) and was found to be 100% (J index, 0.996). Using the upper-limit value for negativity (i.e., 100% specificity), one of these sera was classified as borderline, slightly reducing the sensitivity of the assay to 97.2% (J index, 0.972). The interassay CV was 4.0%.

Recognition of TSL-1 antigens in capture ELISA.

The results obtained in immunoassays using cTSL-1 antigens (Fig. 1D) compare favorably with those observed in ELISA assays using pTSL-1 antigens. The ODs obtained for healthy subjects (n = 181 individuals) were in all cases lower than the cutoff value (0.098), and only 2 of 112 sera from patients with noninfectious diseases and 2 of 102 sera from patients with infectious diseases slightly exceeded this cutoff, resulting in a specificity of 99%. However, using the upper limit for negativity (OD = 0.159; 100% specificity), all serum samples from trichinellosis patients (n = 42 patients) tested positive, thus reaching the maximal J index value of 1. The interassay CV was 5.6%.

Comparison of the different antigens for early detection of trichinellosis.

Serum samples from 10 of the infected patients were taken at various times postinfection, including a period before IIF patency (serum samples A in Fig. 2). We used these sera to assess which of the different serodiagnostic methods can detect Trichinella infection earliest. As shown in Fig. 2, ELISA with cTSL-1 antigens identified some A sera (patients 3, 4, and 6 to 10) that were not identified or identified as borderline by IIF. These results suggest that ELISA with cTSL-1 antigens is more sensitive than IIF or ELISA with CLE, allowing infections to be detected somewhat earlier. Note also that ELISA with cTSL-1 antigens appears to be more sensitive than ELISA with pTSL-1 antigens, since this assay identified all of the four A sera that were borderline by IIF, while only one of those sera was positive by ELISA with pTSL-1 antigens. Moreover, of the six A sera classified as negative by IIF, ELISA with cTSL-1 antigens classified three as positive and three as borderline, whereas none of these sera were classified as positive in ELISA with pTSL-1 antigens. ELISA with CLE or O-deglycosylated CLE gave unacceptable results.

Role of tyvelose-containing epitopes as targets for the anti-Trichinella antibody response in humans.

In view of the results detailed in the previous section, we tentatively concluded that, as in experimental infections in rodents, Trichinella antigens induce relatively few specific antibodies during early stages of the infection. By contrast, during the muscle phase of the parasite, there is abundant antibody induction, dominated by tyvelose-containing antigens (11, 46). Since the antigenicity of tyvelose epitopes in humans has not been characterized in detail, we calculated the percentages of antityvelose antibodies in serum samples from each patient by means of an ELISA in which tyvelose epitopes were selectively blocked with MAb US4. The inhibition study was done with CLE (in indirect ELISA) and cTSL-1 antigens (in capture ELISA) and with sera from 36 patients from outbreaks I and II (T. britovi; n = 33 patients) and outbreak III (T. spiralis; 3 patients). The results showed that all patients produced detectable IgG1 antibodies against TSL-1 antigens and that most of these antibodies recognize tyvelose-containing epitopes (inhibition range, 51.3 to 97.6%) (Fig. 3). However, considering all anti-Trichinella antibodies present in serum samples of each patient, the percentages of IgG1 antibodies reactive with tyvelose were more variable (US4 inhibition values ranging from 7.3 to 92.6%), although only 3 of 36 (8.3%) showed percentages of antityvelose antibodies below 15% (Fig. 3).

FIG. 3.

Percentages of anti-cTSL-1 IgG1 antibodies (black bars) and anti-CLE antibodies (hatched bars) reacting with tyvelose epitopes. Results are for 36 patients with confirmed trichinellosis. The percentage of the response due to tyvelose epitopes was estimated as the percent inhibition of the total response by the tyvelose-specific MAb US4.

DISCUSSION

In recent decades, considerable efforts have been dedicated to developing reliable methods for the serodiagnosis of human trichinellosis (13, 19, 21, 30). ELISA techniques are the most frequently used (13), but a wide variety of antigens are used, and there is some confusion about which Trichinella antigens are most suitable for serodiagnosis in terms of sensitivity, specificity, and ease of use. In the present study we attempted to resolve this question by comparing serum antibody responses of four groups of individuals (healthy, affected by noninfectious pathologies, infected with parasites other than Trichinella, and infected with Trichinella) against selected Trichinella antigens. TSL-1 antigens (tested in indirect ELISA and capture ELISA) were chosen since they bear immunodominant tyvelose epitopes, which are the only epitopes so far identified as being specific for Trichinella (1, 32), and although they are the most abundant components of ESA preparations, to our knowledge they have never been used for serodiagnosis of human infections. CLE antigens (both native and O deglycosylated) were also included for comparative purposes. We considered only IgG1 antibodies, since these are the most frequent and become detectable early during Trichinella infections (22).

The results presented here show that CLE antigens are not useful for serodiagnosis of human trichinellosis in terms of specificity and sensitivity, since (i) many sera from healthy individuals (group 1) and from patients with other pathologies (groups 2 and 3) showed reactivities that overlapped with those obtained with sera from infected patients (group 4) and (ii) the number of sera testing positive with CLE was clearly lower than the number testing positive with TSL-1 antigens (Fig. 1). The low specificity of Trichinella CLE preparations for diagnosis of human trichinellosis has been reported previously (28, 44); however, it is believed that CLE preparations may be useful for early detection of Trichinella infections (28, 41, 44). Although it is expected that some of the antigens contained in CLE preparations may be recognized earlier than TSL-1 antigens, the data presented here suggest that such CLE antigens either are not specific (e.g., phosphorylcholine) (18, 40) or have immunogenicity below the threshold of the response induced by immunodominant cross-reactive antigens.

The specificity of Trichinella CLE antigens can be improved by mild alkali O deglycosylation, but antibodies to antigens thus treated still retain unacceptable cross-reactivity, mainly with sera from patients infected with filariae and Dracunculus, which are closely related antigenically (5, 16). These data suggest that only some of the cross-reacting epitopes contained in CLE glycoproteins are O-glycans in nature. Other immunodominant epitopes (e.g., phosphorylcholine), present on N-glycan structures (29), are also readily released by alkaline treatment (unpublished results), thus ruling out the possibility that this epitope is involved in the residual cross-reactions observed with filariae and Dracunculus after alkaline treatment. Furthermore, in addition to the specificity problem, the sensitivity of ELISA with O-deglycosylated CLE dropped drastically after alkaline treatment, probably because of the destruction of relevant Trichinella epitopes contained in CLE preparations. Several previous studies have reported differing sensitivities of peptides and glycoproteins to mild alkali treatment (23), depending on primary sequence and amino acid substitutions.

Unlike CLE antigens, the TSL-1 antigens used in indirect ELISA (previously isolated by affinity for MAb US4) and capture ELISA (captured with MAb US4 from the CLE fraction) both showed good sensitivity and specificity in testing of sera from muscle-phase Trichinella-infected patients. The best results were obtained with cTSL-1 antigens, which had 100% sensitivity and 100% specificity (J index = 1), versus 97.2% sensitivity (one serum out of 36 tested as borderline) and 100% specificity for pTSL-1 antigens (J index = 0.972). These results are also clearly better than those obtained with classic IIF (Table 1), in which 5 of 42 sera (11.9%) tested as borderline (very faint or irregular fluorescence with serum diluted 1:20) and 1 of 42 (2.4%) tested as negative (no fluorescence with serum diluted 1:20). In view of these results, it seems that TSL-1 antigens captured by immobilized antityvelose MAbs (cTSL-1 antigens) are the most useful antigens for serodiagnosis of human trichinellosis, combining high specificity, high sensitivity, and ease of use. Since Trichinella TSL-1 antigens contain many tyvelose epitopes borne on tri- and tetra-antennary structures (35, 47), the sensitivity of the method is not reduced by the blockage of some tyvelose epitopes during the capture procedure. Indeed, it seems likely that the capturing antibodies may act as spacing arms that facilitate the access of specific antibodies present in the serum. This hypothesis is supported by the fact that O-deglycosylated CLE antigens from Trichinella are poorly recognized by MAb US4 in indirect ELISA, while these antigens block the reactivity of US4 against native CLE antigens in capture ELISA with an efficacy similar to that of native CLE antigens (results not shown).

As an alternative to cTSL-1 antigens, pTSL-1 antigens may also be a good choice, as previously reported for swine trichinellosis (2, 15, 43), although with these antigens the OD responses are probably lower than those with cTSL-1 antigens if anti-Trichinella antibodies are present at low concentrations in the test. Therefore, discrimination between negative serum samples and positive serum samples with low reactivity is more difficult. In line with this, testing of sera obtained at different times postinfection (Fig. 2) indicated than ELISA with cTSL-1 antigens is markedly more sensitive that ELISA with pTSL-1 antigens or IIF. Although the clinical records for these samples do not specify the week postinfection at which they were obtained, by analogy with experimental infections in rodents and swine, it seems probable that most of them were in fact obtained at the beginning of the muscle phase, when antityvelose antibody production starts (11, 12). We thus suggest that ELISA with cTSL-1 antigens will be useful for confirming Trichinella infections and for monitoring established cases of trichinellosis (e.g., for evaluating the efficiency of antihelminth therapy) (26) but not for early diagnosis proper (i.e., during the intestinal and migratory phases of the infection). This limitation is common to all currently available methods for serodiagnosis of trichinellosis.

Interestingly, ELISA inhibitions with MAb US4 (3) showed (i) that human IgG1 antibody responses against TSL-1 antigens are directed almost exclusively against tyvelose-containing epitopes and (ii) that antibodies specific for tyvelose-containing epitopes account for a large proportion of anti-T. spiralis reactivity in most patients (more than 30% in 27 of 36 patients), although there was marked variation among individuals, with values ranging from 7.3 to 92.6% (similar to the results obtained in a preliminary study with only five patients by Denkers et al. [12]). These results explain the good sensitivity and specificity of capture ELISA using cTSL-1 antigens, but more importantly, they provide a method for determining whether a serum reacting with TSL-1 antigens or whole Trichinella antigens actually contains antibodies reacting with specific tyvelose epitopes. In our view, the good specificity and sensitivity obtained with TSL-1 antigens, together with the possibility of confirming serodiagnosis by means of selective inhibition with US4 or equivalent MAbs, make this assay more useful than the recently proposed assay of Bruschi et al. (6), which is based on the use of synthetic tyvelose-BSA conjugate. The assay of Bruschi et al. gives low OD responses and has been reported to give false-positive results in horses (34). Although cTSL-1 antigens and synthetic tyvelose-BSA conjugate have not yet been compared directly, it is possible that the poor results obtained with this latter may be due to one or more of the following causes: (i) a suboptimal hapten-protein relationship in tyvelose-BSA conjugates, (ii) an inappropriate spatial conformation of tyvelose epitopes compared with the antennary arrangement of tyvelose epitopes on TSL-1 antigens, (iii) nonspecific reactions due to the artificial covalent bond between the synthetic disaccharide [β-d-Tyv(1,3)β-d-GalNAc] and the carrier protein, or (iv) poor affinity of human or animal antibodies for the disaccharide compared with the complete natural tetrasaccharide [β-d-Tyv(1,3)β-d-GalNAc(1,4)α-l-Fuc(1,3)β-d-GlcNAc] present on the TSL-1 antennae. The last possibility is supported by the fact that some of the currently available antityvelose MAbs (including US4) recognize the tetrasaccharide [β-d-Tyv(1,3)β-d-GalNAc(1,4)α-l-Fuc(1,3)β-d-GlcNAc]-BSA but not the disaccharide [β-d-Tyv(1,3)β-d-GalNAc]-BSA used in the above-mentioned study by Bruschi et al. (6, 14, 38). Future studies comparing cTSL-1 antigens and tyvelose-BSA conjugates by using the same serum samples may clarify this question.