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Molecular detection of Zika virus in blood and RNA load determination during the French Polynesian outbreak

Zika virus (ZIKV) viremia is reported as low and transient; however, these estimates rely on limited data. We report RNA loads in sera collected from symptomatic patients during the 2013‐2014 French Polynesian ZIKV outbreak. We performed molecular detection of ZIKV RNA in sera from 747 patients presenting with suspected acute phase ZIKV infection. Among patients with confirmed infection, we analyzed the duration of viremia, assessed viral RNA loads and recorded the main clinical symptoms. A total of 210/747 (28.1%) sera tested positive using a ZIKV‐specific RT‐PCR. Viral RNA loads in symptomatic patients that ranged from 5 to 3.7 × 106 copies/mL (mean 9.9 × 104 copies/mL) were not related to a particular clinical presentation, and were significantly lower than those previously obtained from asymptomatic ZIKV infected blood donors. The rate of detection of ZIKV RNA in sera from suspected cases of acute phase ZIKV infection was low. ZIKV RNA loads were lower in symptomatic patients compared to asymptomatic blood donors and were lower than RNA loads usually reported in dengue infections. As there is no abrupt onset of symptoms in ZIKV infections, we suggest that infected patients sought for medical attention when viremia was already decreasing or had resolved.

At the time of the study, the recommendation for acute phase ZIKV infection was based on molecular detection of ZIKV RNA during the acute phase of infection using reverse transcription (RT)-PCR in blood samples collected within the first week post-symptom onset (PSO). 7,24 Since then, this recommendation has been enlarged to the second week PSO, and serodiagnosis is also performed if RT-PCR is negative. 25 Serology-based diagnosis is limited by the cross-reactivity of antibodies developed in response to infections with other flaviviruses, especially dengue virus (DENV) 7,25 and requires confirmation by sero-neutralization tests which can only be performed by highly trained and specialized laboratories. 4,7 However, crossreactivity has been observed even using sero-neutralization assays, especially in secondary arbovirus infections. 24 In French Polynesia, as most of the patients were previously infected by DENV, ZIKV serodiagnosis was not implemented. 26 ZIKV viremia is usually reported as low and transient, making diagnosis by RT-PCR a challenge. 4,27 We report the data obtained using molecular based detection of ZIKV RNA from suspected cases of ZIKV infections and levels of ZIKV RNA loads observed for confirmed ZIKV infection cases.
These viral loads were compared with those previously published for asymptomatic ZIKV RNA positive blood donors in French Polynesia. 28 5 all sera were also tested using a DENV-specific RT-PCR, as previously reported. 30 ZIKV RNA loads (number of RNA copies/mL) were determined from sera that tested positive by RT-PCR by comparison to a standard curve obtained from serial dilutions of a known concentration of ZIKV RNA synthetic transcript, as previously reported. 14,28,29 Viral loads assessed in sera from symptomatic patients were compared using the same quantification method to those previously obtained in sera from    ZIKV RNA loads were assessed in 57 sera found positive by RT-PCR for which remaining sera was available (Supplemental Table S1). Detailed results for number of days PSO, and clinical data (fever, rash, asthenia, conjunctivitis, arthralgia/myalgia) were available for 42 patients.

| METHODS
ZIKV RNA loads that ranged from 5 to 3.7 × 10 6 copies/mL (mean 9.9 × 10 4 copies/mL) were not significantly different between males and females (P = 0.21) and between patients aged under and over 18 years (P = 0.12) ( Table 2) and were not significantly different from patients presenting with and without fever, rash, asthenia, conjunctivitis, or arthralgia/myalgia (Table 3). In contrast, ZIKV RNA loads were significantly lower compared to ZIKV RNA loads from 26 asymptomatic French Polynesian blood donors that were previously published (RNA loads ranging from 2.5 × 10 3 to 8.1 × 10 6 copies/mL, mean 7.3 × 10 5 copies/mL) (P < 0.0001) (Supplemental Table S2). 28 The analysis of ZIKV RNA loads from five blood donors that became symptomatic post-donation (ranging from 3 × 10 3 to 5.2 × 10 6 , mean 1.1 × 10 6 copies/mL) showed no significant difference with those obtained from the 21 blood donors who remained asymptomatic (P = 0.95), but they were significantly higher than those found in symptomatic patients (P = 0.004). The sensitivity of our method was the same as previously reported on sera tested in Yap, 4 estimated at 25 and 100 copies per assay for partial M/E and E encoding genes, respectively. 29 As previously reported, using the same primers and probes, ZIKV RNA was more frequently detected in saliva than in blood collected at the same time during acute phase ZIKV infection, but the use of saliva did not increase the window of detection of ZIKV RNA. 31 A study conducted from persons with travel-associated ZIKV disease concluded that urine also increased the rate of ZIKV RNA detection during the acute phase of infection and extended the window of ZIKV RNA detection. 32 Our results and those previously published 32 suggest that, during the first days PSO, the higher detection rates by RT-PCR in urine and saliva compared to blood are probably related to higher ZIKV RNA loads in these fluids, suggesting that a higher sensitivity could be reached during the acute phase of ZIKV infection by testing urine or saliva instead of blood. Though the most important challenge to ZIKV diagnosis is the short window during which RNA can be detected in serum.

| DISCUSSION
In contrast to other mosquito-borne diseases such as dengue, there is no abrupt onset of clinical symptoms in ZIKV infections. 7 In dengue, flushing is common on day 1 or 2 post-onset of fever and rash usually appears between day 2 and 6 after onset of fever. 33    Despite a trend for symptomatic WNV-positive donors to maintain higher levels of WNV RNA in whole blood compared to asymptomatic WNV-positive donors, the difference was not statistically significant. 43 For ZIKV, RNA loads were found higher in asymptomatic blood donors 28 than in symptomatic ZIKV-infected patients, but there was no difference in the levels of viral loads at the time of blood donation between blood donors who remained asymptomatic compared to those who developed symptoms.
In dengue, viral loads were found significantly higher in dengue hemorrhagic fever compared to dengue fever in some studies studies, [44][45][46] and not different in others. 47 According to the protocol implemented in Yap, 4

CONFLICTS OF INTEREST
Authors disclose any financial and other conflict of interests.