Choice of Laboratory Tissue Homogenizers Matters When Recovering Nucleic Acid From Medically Important Ticks.

Ticks can vector and transmit many pathogens and pose a serious human health threat throughout the world. After collection, many diagnostic laboratories must mechanically disrupt tick specimens for diagnostic testing and research purposes, but few studies have evaluated how well-commercial tissue homogenizers perform this task. We evaluated four commercially available tissue homogenizers: The Bead Ruptor 24 Elite, the Bullet Blender Storm, the gentleMACS Dissociator, and the Precellys 24. We quantitatively compared maceration level, nucleic acid quality, quantity, amplification, and DNA shearing to determine which machines performed the best. The Bead Ruptor 24 Elite had the highest overall score when disrupting a single, uninfected adult Amblyomma americanum (Linnaeus) (Ixodida: Ixodidae) and performed well in follow-on tests including disrupting individual juvenile samples and detecting pathogens from infected samples.

Direct detection of Leishmania from clinical samples.

The ability to rapidly and accurately diagnose leishmaniasis is a military priority. Testing was conducted to evaluate diagnostic sensitivity and specificity of field-expedient Leishmania genus and visceral Leishmania specific dual-fluorogenic, hydrolysis probe (TaqMan), polymerase chain reaction assays previously established for use in vector surveillance. Blood samples of patients with confirmed visceral leishmaniasis and controls without the disease from Baringo District, Kenya, were tested. Leishmania genus assay sensitivity was 100% (14/14) and specificity was 84% (16/19). Visceral Leishmania assay sensitivity was 93% (13/14) and specificity 80% (4/5). Cutaneous leishmaniasis (CL) skin scrapes of patients from Honduras were also evaluated. Leishmania genus assay sensitivity was 100% (10/10). Visceral Leishmania assay specificity was 100% (10/10) from cutaneous leishmaniasis samples; no fluorescence above background was reported. These results show promise in a rapid, sensitive, and specific method for Leishmania direct detection from clinical samples.

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.