Validation of the easyscreen flavivirus dengue alphavirus detection kit based on 3base amplification technology and its application to the 2016/17 Vanuatu dengue outbreak

Abstract

Background: The family flaviviridae and alphaviridae contain a diverse group of pathogens that cause significant morbidity and mortality worldwide. Diagnosis of the virus responsible for disease is essential to ensure patients receive appropriate clinical management. Very few real-time RT-PCR based assays are able to detect the presence of all members of these families using a single primer and probe set. We have developed a novel chemistry, 3base, which simplifies the viral nucleic acids allowing the design of RT-PCR assays capable of pan-family identification.

Methodology/principal finding: Synthetic constructs, viral nucleic acids, intact viral particles and characterised reference materials were used to determine the specificity and sensitivity of the assays. Synthetic constructs demonstrated the sensitivities of the pan-flavivirus detection component were in the range of 13 copies per PCR. The pan-alphavirus assay had a sensitivity range of 10-25 copies per reaction depending on the viral strain. Lower limit of detection studies using whole virus particles demonstrated that sensitivity for assays was in the range of 1-2 copies per reaction. No cross reactivity was observed with a number of commonly encountered viral strains. Proficiency panels showed 100% concordance with the expected results and the assays performed as well as, if not better than, other assays used in laboratories worldwide. After initial assay validation the pan-viral assays were then tested during the 2016-2017 Vanuatu dengue-2 outbreak. Positive results were detected in 116 positives from a total of 187 suspected dengue samples.

Conclusions/significance: The pan-viral screening assays described here utilise a novel 3base technology and are shown to provide a sensitive and specific method to screen and thereafter speciate flavi- and/or alpha- viruses in clinical samples. The assays performed well in an outbreak situation and can be used to detect positive clinical samples containing any flavivirus or alphavirus in approximately 3 hours 30 minutes.

Fig 1. Example of the 3base mechanism.

The example sequences below show the increase in homology from 75% (“Before”) to 95% (“After”) via the 3base conversion where all C bases are detected as T bases.

Fig 2. A diagram showing the 3base conversion process at the nucleotide level.

The bisulphite reaction deaminates cytosine residues in nucleic acids to a uracil sulphonate. After treatment the base is then desulphonated to yield uracil. PCR amplification then converts this to a thymine, which results in essentially a 3base genome (A, G and T).

Fig 3. Results obtained using the pan-alphavirus assay (Cycle threshold (Ct) from a single PCR replicate).

The standard curve was plotted using the log10 concentration versus Ct value.

Fig 4. Results obtained using the pan-flavivirus assay (Cycle threshold (Ct) from a single PCR replicate).

The standard curve was plotted using the log10 concentration versus Ct value.

Fig 5. Weekly positive results from December to the 24^th March 2017.

Fig 6. Age distribution of positive samples.

Fig 7. Dengue positive samples per 1000.

The figure shows the population of each island in Vanuatu with the number of cases per 1000 island population included.