A multiplex immunoassay using recombinant antigens provided a species-specific serodiagnosis for 80% of seropositive samples from travelers and immigrants evaluated for filarial infections or strongyloidiasis, according to a study published in The Journal of Infectious Diseases.
Investigators evaluated an all-in-one assay designed to measure IgG and IgG4 responses simultaneously to Brugia malayi adult antigen and seven recombinant antigens specific to Loa loa, Wuchereria bancrofti, Onchocerca volvulus and Strongyloides stercoralis.
For assay validation, the investigators used serum samples from patients with parasitologically confirmed single infections: 37 with loiasis, 27 with bancroftian filariasis, 195 with onchocerciasis and 40 with strongyloidiasis, along with 70 uninfected controls. Infection was confirmed through detection of microfilariae in blood or skin for the filarial infections and L3/L1 larvae in stool for strongyloidiasis. Cutoffs for each recombinant-antigen assay were set to achieve more than 98% specificity.
The multiplex panel included the recombinant antigens Ll-SXP1 for L. loa; Wb123 and Wb5 for W. bancrofti; Ov16 and OvOC3261 for O. volvulus; and Ss-NIE and Ss-IR for S. stercoralis. Serum or plasma was tested in duplicate on magnetic microspheres, with separate IgG and IgG4 measurements. Receiver operating characteristic analyses and the Youden J statistic were used to establish positivity cutoffs; sensitivity and specificity were then calculated for each assay.
For L. loa, Ll-SXP1 showed 98.7% specificity and 87.5% sensitivity with IgG and 100% specificity and 85.3% sensitivity with IgG4. For W. bancrofti, Wb123 and Wb5 had IgG sensitivities of 95% and 65%, respectively, and IgG4 sensitivities of 89% and 53.4%. IgG specificity was 99.7% for Wb123 and 99.9% for Wb5; IgG4 specificity was 100% for both.
The two O. volvulus antigens had IgG sensitivities of 78% for OvOC3261 and 84% for Ov16 and IgG4 sensitivities of 87.2% and 82%, respectively. IgG specificity was 99.5% for OvOC3261 and 99.7% for Ov16; IgG4 specificity was 100% for both.
For S. stercoralis, Ss-NIE and Ss-IR had IgG sensitivities of 74.7% and 84.6%, respectively, and IgG4 sensitivities of 77.9% and 60.5%. IgG specificity was 99.9% for Ss-NIE and 99.7% for Ss-IR; IgG4 specificity was 100% for both.
The assay was then prospectively used to test samples from 1,865 unique patients between 2016 and July 2025. Of those, 1,547 were seronegative for B. malayi adult antigen and all recombinant antigens, allowing the infections represented in the panel to be largely excluded. Among 318 seropositive samples, 253 were assigned a species-specific serodiagnosis: 99 for L. loa, 29 for W. bancrofti, 32 for O. volvulus and 93 for S. stercoralis. Thirty-one seropositive samples, or 10%, showed evidence of coinfection. In 65 samples, or 20% of the seropositive group, filarial antibody reactivity was detected but the infecting species could not be determined.
Additional analyses showed that 48 samples reactive to at least one recombinant antigen were negative for both IgG and IgG4 against B. malayi adult antigen. Species-specific serodiagnoses also were often based on positivity for only one antibody isotype, indicating additional value from using both IgG and IgG4 in the multiplex assay. Separately, 57 samples reactive to B. malayi adult antigen were positive for S. stercoralis recombinant antigens but nonreactive to all filarial recombinant antigens, which the investigators said suggested broader antigenic cross-reactivity between filarial parasites and S. stercoralis than previously appreciated.
The investigators also formulated a diagnostic algorithm that begins with recombinant-antigen reactivity. Reactivity to any recombinant antigen supports a species-specific serodiagnosis. Samples negative for recombinant antigens but positive for B. malayi adult antigen are classified as filarial-antibody positive, a finding that suggests exposure to a filarial parasite without identifying the species. Absence of reactivity to all antigens can largely exclude the infections represented in the panel, although the investigators cautioned that seronegativity can occur with marked immunosuppression or recent exposure or infection.
The authors noted several limitations. Antibody testing remains an imperfect proxy for active infection, cross-reactivity can produce false-positive results, antibodies may persist after definitive treatment and patients tested soon after exposure may not yet have seroconverted. Among immigrants, a positive antibody result may reflect prior rather than active infection and may warrant confirmation with antigen detection or molecular testing, when available.
The authors concluded that IgG- and IgG4-based serology provides a sensitive and complementary approach to diagnosing filarial infections in travelers and immigrants. “These data provide a new framework for clinically useful parasite-specific serology that helps to direct targeted therapeutic options,” they wrote.
The study was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. No conflicts of interest were reported.
