Development and validation of an arthropod maceration protocol for zoonotic pathogen detection in mosquitoes and fleas.

Arthropod-borne diseases remain a pressing international public health concern. While progress has been made in the rapid detection of arthropod-borne pathogens via quantitative real-time (qPCR), or even hand-held detection devices, a simple and robust maceration and nucleic acid extraction method is necessary to implement biosurveillance capabilities. In this study, a comparison of maceration techniques using five types of beads followed by nucleic acid extraction and detection were tested using two morphologically disparate arthropods, the Aedes aegypti mosquito and Xenopsylla spp. flea, to detect the zoonotic diseases dengue virus serotype-1 and Yersinia pestis. Post-maceration nucleic acid extraction was carried out using the 1-2-3 Platinum-Path-Sample-Purification (PPSP) kit followed by qPCR detection using the Joint Biological Agent Identification and Diagnostic System (JBAIDS). We found that the 5mm stainless steel beads added to the beads provided in the PPSP kit were successful in macerating the exoskeleton for both Ae. aegypti and Xenopsylla spp. Replicates in the maceration/extraction/detection protocol were increased in a stepwise fashion until a final 128 replicates were obtained. For dengue virus detection there was a 99% positivity rate and for Y. pestis detection there was a 95% positive detection rate. In the examination of both pathogens, there were no significant differences between qPCR instruments, days ran, time of day ran, or operators.

Real-time Leishmania genus master mix: a platform compatibility and stability study.

Performing diagnostics and vector-pathogen surveillance in austere environments is challenging. On-site diagnostic/detection mitigates vector-borne disease complications during military or humanitarian deployments to disease endemic locals. The mobile molecular diagnostic platform, Joint Biological Agent Identification and Diagnostic System (JBAIDS; BioFire Diagnostics Inc., Salt Lake City, UT, USA), rapidly identifies biothreat pathogens. Although ideal for remote diagnostics, the platform was validated for specific pathogens of insignificant epidemiological consequence. Recognizing the JBAIDS's remote diagnostic/detection versatility, we tested a Leishmania genus real-time PCR master mix validated for use on the SmartCycler® (Cepheid, Sunnyvale, CA, USA) for concomitant use on the JBAIDS. We evaluated assay sensitivity, precision, and specificity of one or more Leishmania spp. on the JBAIDS and found that the JBAIDS produces superior detection sensitivity and specificity compared to the SmartCycler®. We also examined the storage stability of a bulk lot preparation of the Leishmania genus real-time PCR master mix on the SmartCycler® to ensure that long periods of frozen storage that would translate to a field environment with the JBAIDS were not detrimental to the reagent. We found that the bulk master mix maintains its stability over a 13-month time period. Overall, these studies confirm JBAIDS's versatility and demonstrate a streamlined assay development approach where reagents are compatible with both platforms.

Leveraging arthropod-borne disease surveillance assays for clinical diagnostic use.

Researchers at the Walter Reed Army Institute of Research have taken a joint service approach to filling an identified diagnostic capability gap by leveraging a vector surveillance assay. Specifically, the Army took a field-stable real-time polymerase chain reaction assay, developed by the Air Force, for dengue virus surveillance in arthropod vectors and collaborated with Navy researchers for utility in human diagnostics. As current Department of Defense diagnostic PCR assays employ the Joint Biological Agent Identification and Diagnostic System, the dengue assay was tested for use on this platform. The low rates of false negative and false positive dengue samples in clinical matrices demonstrate excellent utility as a human diagnostic assay. Overall, converting an arboviral vector surveillance assay to human diagnostic assay and potentially vice versa is both cost effective and labor reducing. Codevelopment with harmonization of vector surveillance and diagnostics offers monetary and resource advantages to the Department of Defense and should be considered as a path forward in times when downsizing threatens assay development and pathogen discovery.