Genome Filtering for New DNA Biomarkers of <i>Loa loa</i> Infection Suitable for Loop-Mediated Isothermal Amplification

<div><p><i>Loa loa</i> infections have emerged as a serious public health problem in patients co-infected with <i>Onchocerca volvulus</i> or <i>Wuchereria bancrofti</i> because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for <i>L</i>. <i>loa</i>. The pipeline identified ~140 new <i>L</i>. <i>loa</i> specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the <i>L</i>. <i>loa</i> genome and maps to a <i>L</i>. <i>loa-</i>specific region of the long terminal repeats found at the boundaries of <i>Bel/Pao</i> retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified <i>L</i>. <i>loa</i> but not <i>W</i>. <i>bancrofti</i>, <i>O</i>. <i>volvulus</i>, <i>Brugia malayi</i>, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25–30 minutes and as little as 0.060 pg of <i>L</i>. <i>loa</i> DNA (~1/1600^th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the <i>L</i>. <i>loa</i> genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal.</p>