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

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

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

Sensitivity and specificity of Wb123 IgG and IgG4 LIPS.

*<p>Calculations (Sensitivity, Specificity, PPV, NPV) are shown when the positive was defined as sera from proven <i>Wuchereria bancrofti</i> infection and the negative was defined as either normal (Normals), <i>Loa loa</i>-infected (<i>L. loa</i>). <i>Onchocera volvulus</i>-infected (O. <i>volvulus)</i>, <i>Mansonella perstans</i>-infected (<i>M. perstans</i>) or infected with soil transmitted helminths (Other Helminths).</p><p>ND = not done.</p>**<p>Positive Predictive Value.</p>***<p>Negative Predictive Value.</p

Sequence alignment of Wb123.

<p>Sequence alignment of Wb123 with homologous proteins in <i>Brugia malayi</i> (Bm123), <i>Loa loa</i> (Ll123), and <i>Onchocerca volvulus</i> (Ov123). Boxed blue amino acids are those that show 100% conservation with those of Wb123.</p

Patient population.

*<p>Number of patients relates to the number for whom Wb123 serology was done; the number of patients for whom Mf and CAg were done is shown in their respective columns.</p>**<p>Not done.</p

Levels of Ab to Wb123 in Children from 1975 and 1992.

<p>Ab levels to Wb123 in children ≤11 years old by 2 year age divisions in 1975 (red dots) and 1992 (blue dots). Each dot represents a single individual and the black bars represent the geometric means for each group. The shaded grey area indicates values considered negative and p-values were calculated using the Mann Whitney U test.</p