VUELTA CICLISTA A ESPAÑA [Material gráfico]

Copia digital. Madrid : Ministerio de Educación, Cultura y Deporte, 201

Repurposed Automated Handheld Counter as a Point-of-Care Tool to Identify Individuals ‘At Risk’ of Serious Post-Ivermectin Encephalopathy

<div><p>Introduction</p><p>Administration of ivermectin (IVM) as part of mass drug administration (MDA) campaigns for onchocerciasis and/or lymphatic filariasis (LF) has been suspended in areas co-endemic for <i>Loa loa</i> due to severe post-treatment adverse events (SAEs) associated with high-burden of infection (>30,000 mf/ml). One simple approach for preventing SAEs is to identify and exclude individuals at risk from MDA. Here, we describe a repurposed hand-held automated cell counter (Scepter 2.0; HHAC) as a rapid, point-of-care method for quantifying microfilariae (mf) in the blood of infected individuals.</p><p>Methodology/Principal Findings</p><p>The quantification of microfilarial levels in blood of naturally infected humans, experimentally infected baboons, or mf-spiked human blood was tested using a microfluidic-based automated counter and compared to traditional calibrated thick-smears. We demonstrate that mf can be quantified in 20 µl of whole blood following lysis with 10% saponin within a minute of obtaining blood. There was a highly significant concordance between the counts obtained by the HHAC and those by microscopy for mf densities of >5,000 (p<0.0001, r_c = 0.97) or >30,000 per ml (p<0.0001, r_c = 0.90). Preliminary proof of concept field studies in Cameroon with 20 µl of blood from <i>L. loa</i> infected humans (n = 22) and baboons (n = 4) also demonstrated a significantly high concordance (p<0.0001, r_c = 0.89) with calibrated thick blood smears counts.</p><p>Conclusions/Significance</p><p>A repurposed HHAC is a portable, sensitive, rapid, point-of-care and quantitative tool to identify individuals with high levels of <i>L. loa</i> mf that put them at risk for SAEs following MDA. In addition, it provides ease of data storage and accessibility.</p></div

Signal to noise in HHAC.

<p>HHAC counts of uninfected normal human blood bank donors using 20 µl of blood samples (A). Increasing the blood volume (40 µl or 50 µl) does not change the noise (normal blood, solid dots) compared to <i>L. loa</i> microfilarial counts (open circles)(B).</p

Correlation of HHAC counts with microscopy counts of mf.

<p>Purified mf of <i>B. malayi</i> spiked in normal human blood was tested in triplicate with a 60 µm sensor at various concentrations (0 to 100,000 mf/ml): (A) serially diluted from 100,000 mf/ml or (B) blinded at various random concentrations. Microscopy counts* plotted on the x-axis and HHAC counts in triplicate on y-axis (x10³ mf/ml). ‘r_c’ denotes concordance correlation coefficient. * - Counts based on serial dilution.</p

Adaptation of HHAC for detecting mf.

<p>All samples were lysed and analyzed by the HHAC using the 60 µm sensor. Histograms shown are representative samples of normal human blood before (A) and after spiking with <i>B. malayi</i> mf (B); uninfected canine blood (C), <i>D. immitis</i> infected canine blood (D) and <i>B. malayi</i> infected feline blood (E); phosphate buffer saline (PBS) before (F) and after spiking with <i>B. malayi</i> mf (G).</p

Correlation of HHAC counts with microscopy counts of <i>L. loa</i> mf.

<p>Field-testing of <i>L. loa</i> infected human (open circles) and baboon (solid dots) blood counts by HHAC compared with calibrated thick-smear microscopic counts.</p

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></div

Sensitivity of the <i>L</i>. <i>loa</i> RF4-based LAMP assay.

<p>Dilutions of genomic <i>L</i>. <i>loa</i> DNA were amplified with the RF4 primer set and <i>Bst</i> 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of <i>L</i>. <i>loa</i> DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.</p

Species-specificity of the <i>L</i>. <i>loa</i> RF4-based LAMP assay.

<p>Turbidity curves generated with various DNAs (200 pg) amplified in the absence <b>(A)</b> or presence <b>(B)</b> of the V/DEF additive using the <i>L</i>. <i>loa</i> LAMP primer set with <i>Bst</i> 2.0. Each graph shows the results of two experiments.</p

Species-specificity of RF4 as determined by PCR.

<p>Agarose gels showing specific amplification of <i>L</i>. <i>loa</i> RF4 <b>(A)</b> or a conserved 244 bp actin gene fragment <b>(B)</b> from WGA <i>L</i>. <i>loa</i> (WGA Ll), genomic <i>L</i>. <i>loa</i> (g Ll), <i>W</i>. <i>bancrofti</i> (Wb), <i>O</i>. <i>volvulus</i> (Ov), <i>B</i>. <i>malayi</i> (Bm), <i>A</i>. <i>albopictus</i> (Aa), and <i>H</i>. <i>sapiens</i> (Hs) DNA. PCR <b>(A)</b> and low molecular weight <b>(B)</b> markers (MWM) were used (New England Biolabs).</p