Identification of Wb123 as an Early and Specific Marker of <em>Wuchereria bancrofti</em> Infection

<div><h3>Background</h3><p>The current antibody tests used for monitoring in lymphatic filariasis (LF) elimination programs suffer from poor specificity because of the considerable geographical overlap with other filarial infections such as <em>Loa loa (</em>Ll<em>)</em>, <em>Onchocerca volvulus (</em>Ov<em>)</em>, and <em>Mansonella</em> perstans (Mp).</p> <h3>Methods</h3><p>Using bioinformatics to assemble into contigs 2048 expressed sequence tags (ESTs) from the L3 infective larvae of <em>W. bancrofti</em> (Wb), these were next assessed for homology to known proteins and nucleotides and to similar assemblies of L3 larval ESTs of <em>B. malayi</em> (Bm – n = 5068), Ov (n<em> = </em>4166), and Ll (n<em> = </em>3315). Nineteen potential L3- and Wb- and/or Bm-specific antigens were identified. Sixteen of the 19 antigens could be expressed as fusion proteins with Renilla luciferase (Ruc); these were used in a rapid Luciferase Immunopreciptation System (LIPS) assay.</p> <h3>Results</h3><p>One of the 16 expressed antigens (Wb123) was both highly immunogenic and specific for Wb. Using Wb123-based IgG and IgG4 LIPS assays on well-defined sera from normal North Americans and those infected exclusively with intestinal helminths, we could detect all of the Wb-infected individuals (from diverse geographic regions) with 100% sensitivity and 100% specificity. Using sera from exclusively Ll-infected, Ov-infected Mp-infected or Bm-infected subjects as the negative comparator, the sensitivities were between 98–100% and the specificities ranged between 84–100% (for IgG anti-Wb123) and between 98–100% (for IgG4 anti-Wb123). Blinded assessments using panels of sera from various Wb-, Bm- or non-Wb helminth-infected subjects demonstrated equally high degrees of sensitivity and specificity.</p> <h3>Significance</h3><p>We have identified a Wb-encoded antigen that can be used both as a rapid, high throughput tool to diagnose individual <em>Wb</em> infections and as a sensitive method for early detection of recrudescent infections in areas of control and for mapping new areas of Wb transmission.</p> </div

Antibodies to Wb123 fail to normalize following definitive anti-filarial treatment.

<p>IgG (Top panel) and IgG4 (Middle panel) anti-Wb123 antibodies in two individual patients followed longitudinally over an 8 (red line) or 17 (blue line) year period following definitive anti-filarial chemotherapy. Circulating antigen levels in the same two subjects is shown (Bottom panel). Gray shaded boxes show the normal range for each assay.</p

Real-time PCR (qPCR) assays incorporating LLMF72 and LLMF269 targets.

<p>Shown are the results of Taqman qPCR assays using primer/probe sets for LLMF72 (circles) and LLMF269 (squares). Each point represents the mean and standard deviation of duplicate assays. Panel A: limiting dilutions of cDNA template created by reverse transcription of <i>L. loa</i> total RNA. Panel B: limiting dilutions of highly purified <i>L. loa</i> genomic DNA template. Panel C: total DNA extracted from whole blood spiked with limiting dilutions of intact <i>L. loa</i> microfilariae.</p

Primer and probe sequences for PCR assays.

<p>Primer and probe sequences for PCR assays.</p

Performance of Wb123-specific IgG LIPS assay in blinded analysis.

<p>Blinded analysis of IgG anti-Wb123 antibodies from samples obtained from <i>Wuchereria bancrofti</i>-infected patients from Haiti (Haiti MF pos), those from a Wb non-endemic region of Haiti (Haiti non-exposed), US non-travelers, those with other non-filarial parasitic infection, or those with <i>Brugia malayi</i> infection. Each dot represents an individual subject and the horizontal line is the GM. The dashed line represents the cutoff between negative and positive based on ROC analysis.</p

ROC Analysis Wb123 antibody responses.

<p>Receiver operating characteristic (ROC) analysis using serum samples from <i>Wuchereria bancrofti</i>-infected subjects (n = 43) and normal North American non-travelers (n = 50) for IgG (Left panel) and IgG4 (Right panel) anti-Wb123 antibodies.</p

Samples used to assess sensitivity and specificity of Wb123 LIPS.

*<p>Circulating antigen negative.</p

Wb123-specific IgG and IgG4 in <i>W. bancrofti</i> and related helminth infections.

<p>IgG and IgG4 antibodies to Wb123 distinguish <i>Wuchereria bancrofti</i>-infected subjects from those with related filarial and non-filarial helminth infection. IgG (Left panel) or IgG4 (Right panel) antibodies to Wb123 in individual serum samples from those with <i>Wuchereria bancrofti</i> (Wb), <i>Onchocerca volvulus</i> (Ov), <i>Loa loa</i> (Ll), <i>Mansonella perstans</i> (Mp), other helminth infections, or no infection (Normal). Each dot represents an individual sample and the horizontal line is the geometric mean (GM). The dashed line represents the cutoff between negative and positive based on ROC analysis.</p

Reverse transcriptase PCR (RT-PCR) of limiting dilutions of <i>L. loa</i> total RNA.

<p>Shown are concentrations of specific PCR product using primers for targets LLMF72 (circles, solid line) and LLMF269 (squares, dashed line). The lower X-axis indicates the number of <i>L. loa</i> microfilariae corresponding to the amount of RNA used as template (upper X-axis). Each point represents the result of a single assay.</p

Performance of LLMF72 qPCR assay with blood spots in comparison to microscopy.

<p>ND: not done.</p