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.

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.

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.