Florida Bureau of Public Health laboratories
Arbovirus trioplex PCR
BPHL has verified the FDA authorized (IVD EUA) qualitative real-time reverse transcription polymerase chain reaction (rRT-PCR) “CDC Trioplex Real-time RT-PCR Assay” to detect viral nucleic acid (RNA) from dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV).
Analyte(s)
Dengue (DEN), chikungunya (CHIK), and zika (ZIK)
Test Code
1550
Testing Location
Testing Department
Virology
Pre-approval Required?
Yes.
Requires prior approval from regional epidemiology and notification to the testing lab. Contact the local county health department (CHD) to start the process for approval.
Required Forms
BPHL Test Requisition, DH1847
Include all required fields, who authorized the testing, mosquito bite history, onset date, collection date, travel history, and symptoms.
Turn Around time
Three to 10 business days after receipt at the laboratory
Test note
Trioplex PCR assay tests for dengue, chikungunya, and zika in one test from acute serum, whole blood in EDTA, CSF, urine, and amniotic fluid. Serum collected less than 14 days post onset of symptoms or exposure. Whole blood in EDTA, CSF, urine, and amniotic fluid may only be tested alongside a patient-matched serum (paried sera) specimen. Amniotic fluid and urine are only approved for zika (with paired serum).
Minimum Volume
1-2 mL serum (preferred source), CSF, urine, and amniotic fluid
1 mL, (3-5 mL preferred volume) of whole blood in EDTA tube
Collection Media
Serum separator – Tiger top (red/gray) or red top
Whole blood in EDTA *Paired Sera Required with whole blood*
Sterile container with no media
Specimen Sources
Acute serum (preferred source), whole blood in EDTA, CSF, urine, and amniotic fluid
Amniotic fluid and urine are only approved for zika (with paired serum*)
Specimen Labeling
Specimen must be labeled with at least two unique patient identifiers, e.g., name and DOB.
Information on the specimen must match the requisition and/or test order.
Special SPecimen Preparation
*Paired Sera Collection:
1. First specimen (acute) collected in red top tube one to three days after onset of illness. Separate serum and store refrigerated at 28° C until second specimen is collected.
2. Second specimen (convalescent) collected in red top tube 10-14 days after first specimen.
3. Ship sera together overnight at 2-8° C
Storage Conditions
Store specimens at 2-8° C or frozen at -20° C or lower until time of shipment. Specimens should be sent to the lab within four days of collection.
Packaging and Shipping
Whole blood in EDTA with paired sera specimens should be shipped on frozen ice packs at 2-8° C. Samples should not arrive frozen.
CSF, tissue, serum, urine, and amniotic fluid specimens should be shipped on frozen ice packs at 2-8° C or frozen on dry ice at -20° C or lower. It the specimens were stored frozen, then they should be shipped frozen to avoid a freeze thaw cycle.
Separate specimens into different bags to help prevent cross contamination.
Include paperwork with specimens but separate them physically by placing the paperwork in the outer pocket of the biohazard bag.
unsatisfactory specimen
1. Requisition form was not fully completed with all required fields
2. No patient identification on specimen tube
3. Patient identifiers on the specimen do not match the requisition or test order
4. Specimen damaged or leaked in transit
5. Specimen was received outside the required temperature range
6. Specimen was received hemolyzed
Interferences and Limitations
Interpretation of rRT-PCR test results must account for the possibility of false-negative results. False-negative results can arise from: poor sample collection, degradation of viral RNA during shipping or storage, specimen collection conducted prior to symptom onset, specimen collection after nucleic acid can no longer be found in the patient (approximately 14 days post-onset of symptoms for serum, whole blood, and/or urine.)
Reference Range
Negative: Not detected
Result Indicator
No virus detected
Name of virus detected
Reference lab
CDC if needed
Reflex testing
None