Zika, chikungunya and dengue viruses are co-circulating in Brazil. They share the same mosquito vector and their infections cause diseases with similar signs and symptoms, which can lead to misdiagnosis based on clinical grounds. In addition, if available, a reliable differential diagnosis is important for anticipating, preventing and managing complications. For example, patients with dengue should be monitored for hemorrhagic fever, patients with chikungunya should be monitored and treated for acute arthralgias and post-infectious chronic arthritis and, Zika virus infection during pregnancy can result in birth defects. Concisely, these viruses cause acute illness with similar clinical pictures, but patient management and disease complications are specific.

Moreover, because Zika is closely related to dengue, serologic samples may cross-react in tests for either virus. However, RNA detection methods such as RT-qPCR can reliably and specifically distinguish the three viruses because the assay is designed to target virus-specific genomic sequences, which virtually elimina tes the problem of cross-reaction. The main limita tion of the RT-qPCR is that virus RNA can be detected in the blood for 5 to 7 days after the onset of signs and symptoms. This narrow detection opportunity limits the period available for a new specimen collection to test for additional viruses. These observations suggest that the one-bug-one-RT-qPCR approach can be less effective than a broad-spectrum assay, which provides a fast, specific and definitive diagnostic answer for patients.

Recently, we validated an assay where Zika, chikungunya and dengue viruses are assessed simultaneously by RT-qPCR, but in independent reaction wells. The study was presented at the 65th AACC annual meeting under the title “Simultaneous  detection  of  Zika,  chikungunya  and  dengue  viruses  in  EDTA - plasma by RT-qPCR: if their vector is versatile their detection assays also should be”. For the three viruses, the proposed assay showed limits of detection below 100 copies/mL, presented acceptable precision for positive (> 72 copies/mL) and negative (<72 copies/mL) results and the agreement with the comparative methods ranged from 90-100%. In summary, the assay showed high sensitivity and very good precision and accuracy.

Two months after the test was released for Brazil’s Federal District population,  1135  patients  requested  the  assay.  The  breaking  down  of  this number revealed that 35.9% tested positive: 22.6% for dengue, 10.7% for Zika and 2.6% for chikungunya. Co-infection was rare; only one case of dengue and Zika virus co-infection was observed. Additionally, many people come to the laboratory  with  a  suspected  Z ika  virus  infection  and  leave  with  dengue  or chikungunya positive report (and vice versa), which exemplifies the above-cited possibility of misdiagnosis based on clinical grounds. Indeed, the distribution of positive results by patient’s neighborhood revealed that the three viruses co- circulate in all district’s  areas. This observation adds  further support for  the requirement  of  a  combined  assay for  these  viruses  and  justifies statement: “if their vector is versatile their detection assays also should be.”

Furthermore, the laboratory’s daily routine  and experience with these assays allowed us to make the following observations: a) Zika virus never showed as high viral loads as dengue and chikungunya; b) For the three viruses, low-viral loads are more frequent than high-viral loads. Thus, the assays must be highly sensitive; otherwise they will be prone to false-negative results; c) EDTA-plasma has less genomic DNA background than serum, due to many reasons it facilitates virus RNA detection.

In conclusion, when Zika, chikungunya and dengue (multiple arboviruses) circulate in the same geographical region, the simultaneous detection for these viruses by RT-qPCR is advantageous because a sensitive and rapid diagnosis of the appropriate virus will benefit patients.