Development of a rapid serological assay for the diagnosis of strongyloidiasis using a novel diffraction-based biosensor technology.

BACKGROUND: Strongyloidiasis is a persistent human parasitic infection caused by the intestinal nematode, Strongyloides stercoralis. The parasite has a world-wide distribution, particularly in tropical and subtropical regions with poor sanitary conditions. Since individuals with strongyloidiasis are typically asymptomatic, the infection can persist for decades without detection. Problems arise when individuals with unrecognized S. stercoralis infection are immunosuppressed, which can lead to hyper-infection syndrome and disseminated disease with an associated high mortality if untreated. Therefore a rapid, sensitive and easy to use method of diagnosing Strongyloides infection may improve the clinical management of this disease. METHODOLOGY/PRINCIPAL FINDINGS: An immunological assay for diagnosing strongyloidiasis was developed on a novel diffraction-based optical bionsensor technology. The test employs a 31-kDa recombinant antigen called NIE derived from Strongyloides stercoralis L3-stage larvae. Assay performance was tested using retrospectively collected sera from patients with parasitologically confirmed strongyloidiasis and control sera from healthy individuals or those with other parasitoses including schistosomiasis, trichinosis, echinococcosis or amebiasis who were seronegative using the NIE ELISA assay. If we consider the control group as the true negative group, the assay readily differentiated S. stercoralis-infected patients from controls detecting 96.3% of the positive cases, and with no cross reactivity observed in the control group These results were in excellent agreement (κ = 0.98) with results obtained by an NIE-based enzyme-linked immunosorbent assay (ELISA). A further 44 sera from patients with suspected S. stercoralis infection were analyzed and showed 91% agreement with the NIE ELISA. CONCLUSIONS/SIGNIFICANCE: In summary, this test provides high sensitivity detection of serum IgG against the NIE Strongyloides antigen. The assay is easy to perform and provides results in less than 30 minutes, making this platform amenable to rapid near-patient screening with minimal technical expertise

Box and whiskers plot of seven groups of sera tested for anti-NIE IgG antibodies.

<p>The plots summarize the results of gold standard <i>Strongyloides</i> samples (n = 54) and six control groups comprised of healthy individuals (n = 7) as well as those with trichinosis (n = 8), filariasis (n = 9), schistosomiasis (n = 9), echinococcosis (n = 6) and amebiasis (n = 8). The lower and upper boxes represent the samples in the second and third quartile respectively while the error bars above and below the box correspond to the 95^th and 5^th percentiles. The horizontal lines separating the boxes represent the median and the diamond denotes the mean. X represents the minimum and maximum values. A significant difference was observed between the gold standard <i>Strongyloides</i> and all control groups (<i>p</i><0.001).</p

Serum dilution optimization.

<p>A series of different dilutions of <i>Strongyloides</i> positive serum were analyzed to determine the optimal serum concentration for use in dot-serology assays. Dilutions of 1∶10 and 1∶20 generated the highest antibody signal with no differences between the two dilutions (<i>p</i> = 0.17), while a significant decrease in signal intensity was observed between 1∶20 and 1∶50 dilutions (<i>p</i><0.001). A serum dilution of 1∶20 was determined to be optimal for the dot-based <i>Strongyloides</i> assay. Data represent mean ± SD.</p

Schematic representation of a dotLab biosensor.

<p>(A) Each sensor consists of a contiguous array of 8 assay locations spotted on the bottom of a 10 µL flow channel where reagents and samples are introduced. Each assay location is comprised of a repeating pattern of capture molecules arranged in a defined series of parallel lines creating a diffraction grating. (B) Illumination of an assay spot with a laser generates a predictable diffraction image. The intensity of the diffraction image is monitored in real time by a photodiode detector. (C) Increases in the height (<i>h</i>) of the diffraction grating due to molecular binding events results in a proportionate increase in the diffraction image intensity (Δ<i>DI</i>).</p

Representative trace of a dot-based serological analysis of a <i>Strongyloides</i> positive serum sample.

<p>The dotLab mX System outputs a real time trace displaying each reagent incubation and wash step in the assay. Note the binding curves representing the immobilization of NIE@D conjugate and serum anti-NIE antibodies. Significant signal amplification is achieved using anti-human IgG antibody conjugated gold nanoparticles (GNP). The three superimposed traces represent the results of a single assay performed with three spot monitoring.</p