Supportive Data

The following validation data supports the use of this assay for clinical testing.

Accuracy/Diagnostic Sensitivity and Specificity:

Clinical specimens, commercial samples, and a blinded panel of positive and negative samples provided by the Centers for Disease Control and Prevention (CDC) were used for the accuracy experiments. Testing was completed per the manufacturer's instructions, using the easy Mag (bioMerieux) for RNA extraction.

-Ninety-five clinical serum specimens received from the Mayo Infectious Disease Serology Laboratory (IDS) were tested using the Altona RealStar (ARS) Chikungunya (CHIK) RT-PCR Kit 2.0 assay. These specimens had been submitted through Mayo Clinic Laboratories and sent to an external laboratory for serology (IgM IFA) and reverse transcription polymerase chain reaction (RT-PCR) testing in 2014.

-Sixteen vials of human plasma from donor units collected in Puerto Rico extracted using the QIAGEN QIAamp Viral RNA Mini Kit.

-Spiking studies: To supplement the results, negative cerebrospinal fluid (CSF) specimens were spiked with viral RNA of CHIK and tested in a blinded fashion. The spiking material was heat inactivated (HI) CHIK culture fluid (CF). The ARS CHIK results were compared to the consensus results of the ARS CHIK, a published assay by Lanciotti(1) and a commercial assay, the Bio-Rad ZDC Multiplex RT-PCR Assay (ZDC). The gold standard was considered the consensus between 2 of the 3 PCR assays.

Results:

The ARS CHIK RT-PCR Kit detected 9 more chikungunya virus-positive specimens from the patient samples received from IDS than the Lanciotti and ZDC RT-PCR assays.

-Eight of the nine ARS CHIK+/Lanciotti-/ZDC- results were positive by chikungunya IgM EIA and/or chikungunya RT-PCR at another commercial reference lab, and therefore considered likely true positives. Final consensus sensitivity and specificity were 100% and 98% respectively.

-There was 100% agreement with among the ARS CHIK, Lanciotti, and CDC Trioplex RT-PCR assays using a CDC validation panel.

-The CSF specimens spiked with HI chikungunya virus CF at low concentrations gave expected results in 44/44 specimens.

Analytical Specificity:

No cross-reactivity was observed with the ARS CHIK RT-PCR Kit when tested against a comprehensive specificity panel, which included 32 bacterial, fungal, and viral organisms from culture collections along with well characterized laboratory strains causing similar disease states, closely related organisms, or from organisms commonly found in the specimens tested. This included West Nile virus (lineage 1 and 2), dengue virus (types 1, 2, 3, and 4), tick-borne encephalitis virus, yellow fever virus, Japanese encephalitis virus, Zika virus, and poliovirus. The manufacturer tested additional organisms as listed in the package insert.