A Novel Xenomonitoring Technique Using Mosquito Excreta/Feces for the Detection of Filarial Parasites and Malaria

<div><p>Background</p><p>Given the continued successes of the world’s lymphatic filariasis (LF) elimination programs and the growing successes of many malaria elimination efforts, the necessity of low cost tools and methodologies applicable to long-term disease surveillance is greater than ever before. As many countries reach the end of their LF mass drug administration programs and a growing number of countries realize unprecedented successes in their malaria intervention efforts, the need for practical molecular xenomonitoring (MX), capable of providing surveillance for disease recrudescence in settings of decreased parasite prevalence is increasingly clear. Current protocols, however, require testing of mosquitoes in pools of 25 or fewer, making high-throughput examination a challenge. The new method we present here screens the excreta/feces from hundreds of mosquitoes per pool and provides proof-of-concept for a practical alternative to traditional methodologies resulting in significant cost and labor savings.</p><p>Methodology/Principal Findings</p><p>Excreta/feces of laboratory reared <i>Aedes aegypti</i> or <i>Anopheles stephensi</i> mosquitoes provided with a <i>Brugia malayi</i> microfilaria-positive or <i>Plasmodium vivax</i>-positive blood meal respectively were tested for the presence of parasite DNA using real-time PCR. A titration of samples containing various volumes of <i>B</i>. <i>malayi</i>-negative mosquito feces mixed with positive excreta/feces was also tested to determine sensitivity of detection. Real-time PCR amplification of <i>B</i>. <i>malayi</i> and <i>P</i>. <i>vivax</i> DNA from the excreta/feces of infected mosquitoes was demonstrated, and <i>B</i>. <i>malayi</i> DNA in excreta/feces from one to two mf-positive blood meal-receiving mosquitoes was detected when pooled with volumes of feces from as many as 500 uninfected mosquitoes.</p><p>Conclusions/Significance</p><p>While the operationalizing of excreta/feces testing may require the development of new strategies for sample collection, the high-throughput nature of this new methodology has the potential to greatly reduce MX costs. This will prove particularly useful in post-transmission-interruption settings, where this inexpensive approach to long-term surveillance will help to stretch the budgets of LF and malaria elimination programs. Furthermore, as this methodology is adaptable to the detection of both single celled (<i>P</i>. <i>vivax</i>) and multicellular eukaryotic pathogens (<i>B</i>. <i>malayi</i>), exploration of its use for the detection of various other mosquito-borne diseases including viruses should be considered. Additionally, integration strategies utilizing excreta/feces testing for the simultaneous surveillance of multiple diseases should be explored.</p></div

Limits for the detection of <i>B</i>. <i>malayi</i> DNA in mosquito excreta/feces samples.

<p>Limits for the detection of <i>B</i>. <i>malayi</i> DNA in mosquito excreta/feces samples.</p

PCR positivity of excreta/feces from mosquitoes infected with <i>P</i>. <i>vivax</i>.

<p>PCR positivity of excreta/feces from mosquitoes infected with <i>P</i>. <i>vivax</i>.</p

Evaluation of extraction methods for the isolation of DNA from mosquito excreta/feces.

<p>Evaluation of extraction methods for the isolation of DNA from mosquito excreta/feces.</p

Illustrative output from RepeatExplorer analysis of <i>Necator americanus</i>.

<p>During “clustering” each nucleotide within a cluster is assigned a number. That number corresponds to how many individual next-generation sequencing reads that particular nucleotide appeared in. Using this output, a stretch of the most abundant nucleotides (depicted in green within the larger cluster’s sequence) is selected, and the corresponding nucleotides (highlighted in yellow) are selected as the candidate sequence from which the primers and probe are designed.</p

Workflow for repeat analysis.

<p>Output data from a next-generation sequencing run are uploaded to the RepeatExplorer Galaxy-based platform. During the QC and manipulation phase, the <i>FASTQ Groomer</i> tool is used to convert sequence reads into Sanger format. The <i>FASTQ</i>: <i>READ QC</i> tool is then used to verify the quality of the reads before removing unnecessary sequence (i.e. adapter sequences, etc.) from the ends of each read using the <i>FASTQ Trimmer</i> tool. The QC analysis is then repeated, and the <i>FASTQ to FASTA converter</i> tool is used to convert each read into FASTA format. Using these DNA sequence reads as input, sequences undergo clustering, during which an “all-to-all” sequence comparison is performed, and similar sequences are grouped together into clusters. Clusters containing the most highly repetitive sequences are then selected as putative diagnostic targets to be used for primer and probe-based real-time PCR assay design.</p

Comparative assay results for each species of parasite.

<p>Comparative assay results for each species of parasite.</p

Estimated Minimum Quantities of DNA in the Eggs of Each Species of STH.

<p>Estimated Minimum Quantities of DNA in the Eggs of Each Species of STH.</p

Selected primer and probe sequences for each multi-parallel assay.

<p>Selected primer and probe sequences for each multi-parallel assay.</p

Comparative probe testing.

<p>For each novel probe design, FAM-TAMRA and double quenched FAM-ZEN-IOWA BLACK probes were synthesized. Comparative testing revealed that double quenched probes outperformed traditional probes, as evidenced by lower Ct values and greater ΔRn values. The plot above demonstrates these findings with the amplification of three concentrations of <i>N</i>. <i>americanus</i> template DNA using both double quenched (yellow) and traditional (blue) probe designs.</p