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Development of diagnostic and point of care assays for a gammaherpesvirus infecting koalas.

ABSTRACT: The recent listing of koala populations as endangered across much of their range has highlighted the need for better management interventions. Disease is a key threat to koala populations but currently there is no information across the threatened populations on the distribution or impact of a gammaherpesvirus, phascolarctid gammaherpesvirus 1 (PhaHV-1). PhaHV-1 is known to infect koalas in southern populations which are, at present, not threatened. Current testing for PhaHV-1 involves lengthy laboratory techniques that do not permit quantification of viral load. In order to better understand distribution, prevalence and impacts of PhaHV-1 infections across koala populations, diagnostic and rapid point of care tests are required. We have developed two novel assays, a qPCR assay and an isothermal assay, that will enable researchers, clinicians and wildlife managers to reliably and rapidly test for PhaHV-1 in koalas. The ability to rapidly diagnose and quantify viral load will aid quarantine practices, inform translocation management and guide research into the clinical significance and impacts of PhaHV-1 infection in koalas.

SUBMITTER: Wright BR

PROVIDER: S-EPMC10234535 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Development of diagnostic and point of care assays for a gammaherpesvirus infecting koalas.

Wright Belinda R BR Jelocnik Martina M Casteriano Andrea A Muir Yasmine S S YSS Legione Alistair R AR Vaz Paola K PK Devlin Joanne M JM Higgins Damien P DP

PloS one 20230601 6

The recent listing of koala populations as endangered across much of their range has highlighted the need for better management interventions. Disease is a key threat to koala populations but currently there is no information across the threatened populations on the distribution or impact of a gammaherpesvirus, phascolarctid gammaherpesvirus 1 (PhaHV-1). PhaHV-1 is known to infect koalas in southern populations which are, at present, not threatened. Current testing for PhaHV-1 involves lengthy l ...[more]

PMID: 37262062

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Project description:The rapid detection and identification of infectious disease pathogens is a critical need for healthcare in both developed and developing countries. As we gain more insight into the genomic basis of pathogen infectivity and drug resistance, point-of-care nucleic acid testing will likely become an important tool for global health. In this paper, we present an inexpensive, handheld, battery-powered instrument designed to enable pathogen genotyping in the developing world. Our Microfluidic Biomolecular Amplification Reader (µBAR) represents the convergence of molecular biology, microfluidics, optics, and electronics technology. The µBAR is capable of carrying out isothermal nucleic acid amplification assays with real-time fluorescence readout at a fraction of the cost of conventional benchtop thermocyclers. Additionally, the µBAR features cell phone data connectivity and GPS sample geotagging which can enable epidemiological surveying and remote healthcare delivery. The µBAR controls assay temperature through an integrated resistive heater and monitors real-time fluorescence signals from 60 individual reaction chambers using LEDs and phototransistors. Assays are carried out on PDMS disposable microfluidic cartridges which require no external power for sample loading. We characterize the fluorescence detection limits, heater uniformity, and battery life of the instrument. As a proof-of-principle, we demonstrate the detection of the HIV-1 integrase gene with the µBAR using the Loop-Mediated Isothermal Amplification (LAMP) assay. Although we focus on the detection of purified DNA here, LAMP has previously been demonstrated with a range of clinical samples, and our eventual goal is to develop a microfluidic device which includes on-chip sample preparation from raw samples. The µBAR is based entirely around open source hardware and software, and in the accompanying online supplement we present a full set of schematics, bill of materials, PCB layouts, CAD drawings, and source code for the µBAR instrument with the goal of spurring further innovation toward low-cost genetic diagnostics.

Project description:IntroductionThe diagnosis and detection of pathogens such as Rickettsia and Orientia is a cause of major concern among the public health community. Unavailability of rapid, cost-effective diagnostic assays contributes to delayed diagnosis and timely treatment. Using the methodology of systematic reviewing and meta-analysis, the study aimed to synthesize and compare the diagnostic performances of all the available isothermal assays for the detection of classical rickettsial diseases.MethodsStudies were retrieved from PubMed and Scopus, and selection and screening were conducted using pre-determined inclusion and exclusion criteria. Analysis was performed using Meta-DiSc 2.0 for the assessment and comparison of diagnostic performance of the isothermal assays.ResultsOverall, six studies were selected as a part of this systematic review. All the selected studies (n = 6) optimized LAMP as their index test to detect scrub typhus. The quality assessment of the selected studies revealed only (n = 1) study to be of poor quality with a QUADAS-2 score of (<2). Meta-analysis revealed the pooled sensitivity of LAMP to be 66% [95% CI (0.40-0.85)] with a pooled specificity of 94% [95% CI (0.81-0.98)]. LAMP was estimated with a positive likelihood ratio of 8.3 [95% CI (3.8-18.1)] and a negative likelihood ratio of 0.3 [95% CI (0.2-0.7)] with a false positivity rate of 0.07 [95% CI (0.02-0.2)]. The diagnostic odds ratio was reported to be 21.96 [95% CI (10.2-47.3)]. Due to severe heterogeneity in the body of evidence (I 2 = 0.77), a meta-regression was performed with certain covariates to explore the potential causes. A case-control design was found to exaggerate the sensitivity {0.84 [95% CI (0.5-0.9)]} and specificity {0.73 [95% CI (0.6-0.8)]}.ConclusionThe findings reveal subpar performance of LAMP for the detection of scrub typhus. Active research and development focused on optimization of novel molecular diagnosis that are efficient, rapid, and cost-effective shall foster timely diagnosis and aid in reduction of the overall burden of scrub typhus.Protocol and registrationA detailed protocol of this review is registered and available in Prospero at: https://www.crd.york.ac.uk/prospero/. (registration number CRD42024511706).

Dataset Information

Development of diagnostic and point of care assays for a gammaherpesvirus infecting koalas.

Publications

Development of diagnostic and point of care assays for a gammaherpesvirus infecting koalas.

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