• Design, Production, and evaluation of recombinant fusion NIE/SsIR antigens for the diagnosis of human strongyloidiasis
  • Ghazal Ghaznavi,1,* Amir Savardashtaki,2
    1. Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
    2. Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran


  • Introduction: Strongyloidiasis is a neglected tropical disease caused by the intestinal nematode Strongyloides stercoralis. Unlike many other soil-transmitted helminths, this parasite has a unique autoinfective life cycle that allows it to persist in the human host for decades. In immunocompetent individuals, infection may remain asymptomatic or present with mild gastrointestinal disturbances; however, in immunocompromised patients—including transplant recipients, cancer patients undergoing chemotherapy, and individuals with autoimmune diseases requiring immunosuppressive therapy—the parasite can cause disseminated hyperinfection syndrome, a severe and often fatal condition. Accurate diagnosis of strongyloidiasis remains a major challenge, particularly in endemic regions. Conventional parasitological methods, such as direct stool microscopy or culture, have limited sensitivity due to intermittent and low-level larval excretion. Commercial serological assays, although more sensitive, often suffer from cross-reactivity with other helminth infections and may not be locally validated for endemic populations. The endemicity of strongyloidiasis in the northern and southern coastal regions of Iran, combined with the rising number of at-risk immunosuppressed individuals, underscores the urgent need for an improved, locally adapted, and highly specific diagnostic assay. To address this gap, we designed and evaluated a recombinant fusion antigen based on NIE and SsIR proteins for use in serological assays.
  • Methods: The coding sequences for NIE and SsIR antigens were retrieved from the NCBI database. A synthetic gene construct encoding a fusion protein of NIE and SsIR connected by a flexible linker was designed. Bioinformatic analyses included: Antigenicity prediction: using EMBOSS tools. B-cell epitope mapping: with BCPRED and ABCPRED servers. Physicochemical characterization: performed using PROTPARAM. Codon optimization for E. coli expression was carried out with OPTIMIZER and GENSCRIPT platforms, and the construct was cloned into the pET28 expression vector. Recombinant protein expression was induced in E. coli host cells, and the protein was purified using nickel-affinity chromatography. To evaluate diagnostic performance, serum samples were collected from three groups: (i) 30 patients with parasitologically confirmed S. stercoralis infection (via microscopy or stool culture), (ii) 50 healthy controls from non-endemic areas, and (iii) 50 patients infected with other intestinal parasites to assess cross-reactivity. Recombinant protein-based ELISA and Western blot assays were developed and optimized. Parallel testing with a commercial serological kit was performed to benchmark sensitivity and specificity.
  • Results: The recombinant NIE+SsIR fusion protein was successfully expressed in soluble form and purified to high yield and purity. Bioinformatic analysis confirmed favorable antigenic properties, high predicted stability, and multiple accessible B-cell epitopes. In ELISA assays, the recombinant antigen demonstrated strong reactivity with sera from S. stercoralis patients, while showing minimal reactivity with healthy controls or sera from individuals infected with other parasites. Western blot analysis corroborated these findings, with a distinct and specific band pattern detected exclusively in strongyloidiasis-positive sera. When compared to the commercial diagnostic kit, the recombinant NIE+SsIR fusion antigen achieved comparable, and in some cases superior, diagnostic accuracy. The assay displayed high sensitivity in detecting confirmed cases and high specificity, effectively minimizing cross-reactivity. Statistical analysis confirmed that the recombinant antigen-based assay is a reliable and robust tool for serodiagnosis of strongyloidiasis.
  • Conclusion: The present study demonstrates that the NIE+SsIR recombinant fusion protein is a promising candidate for the development of sensitive and specific serological assays for human strongyloidiasis. By combining two well-characterized antigens into a single construct, we achieved enhanced immunoreactivity while maintaining high specificity. This approach offers several advantages: improved diagnostic accuracy, reduced cross-reactivity, and adaptability for large-scale local use. The development of such a test has important public health implications, particularly in endemic areas of Iran where the burden of disease is underestimated and vulnerable populations are at risk of severe outcomes. Incorporating this antigen into standardized ELISA or rapid diagnostic platforms could facilitate early detection, guide timely treatment, and ultimately reduce morbidity and mortality associated with strongyloidiasis.
  • Keywords: Strongyloidiasis; Strongyloides stercoralis; Recombinant protein; NIE; SsIR