Abstract
.Current diagnostic protocols of acute Zika virus (ZIKV) infection focus on detection of viral RNA in serum or urine using reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, detecting infection can be a challenge, given that 80% of people with acute ZIKV infection are asymptomatic, and the window to detect viremia in serum is short. The ability to extend that window is needed to detect ZIKV at later time points after infection, particularly in high-risk individuals such as pregnant women. We evaluated RNA extraction methods to optimize detection of ZIKV in various body fluids using RT-qPCR as a means of improving the analytical sensitivity of detection. We optimized methods for ZIKV RNA recovery from a number of body fluids by spiking with three varying concentrations of virus, then comparing recovery with that of spiked buffer control. RNA extraction protocols were adjusted as necessary for maximum RNA recovery. Adjustment of the elution step was essential for improved ZIKV RNA recovery from whole blood, saliva, vaginal secretions, and breast milk. Optimal recovery from urine samples required the addition of Urine Conditioning Buffer, and the use of RLT Plus buffer and RNeasy Mini Spin Columns was necessary for RNA extractions from semen samples. Optimized QIAamp MinElute Virus Spin Kit (QIAGEN, Valencia, CA) protocol followed by the singleplex ZIKV RT-qPCR assay provided a reliable method for detection of ZIKV RNA in a variety of biological samples. Improved diagnostics are crucial for timely detection and diagnosis, particularly during pregnancy when the consequences of ZIKV infection can greatly impact the developing fetus.
Highlights
The extraction protocol optimized as before did not improve the recovery of Zika virus (ZIKV) RNA in urine and semen specimens (Supplemental Tables 1 and 2)
A series of alternative extraction protocols were tested for semen specimens and eventually one was selected that yielded 89% of the replicates within two cycle threshold (Ct) units of the expected Ct values and an average of 29% ZIKV RNA recovery (Figure 2; Supplemental Table 1)
Using QIAamp MinElute Virus Spin Kit as basic RNA extraction method, we evaluated protocol variations to optimize detection of ZIKV in various body fluids using reverse transcription quantitative polymerase chain reaction (RT-qPCR), as means of improving the analytical sensitivity of detection
Summary
Zika virus (ZIKV), a member of Flavivirus genus, emerged in the Americas in 2015 and is a significant public health concern.[1,2,3] The virus has been known to circulate with limited reported activity in the Old World since before the middle of the twentieth century.[2,3] It first appeared in the continental New World in Brazil in March 2015, initiating an explosive outbreak that resulted in millions of infections throughout South, Central, and North America, including the U.S territory of Puerto Rico, within 1 year.[1,2,3] Most of the clinical cases reported in the U.S states have been associated with travel to the affected areas.[4]. The number of locally acquired ZIKV cases in the United States is about 20 times lower than travel-associated cases, with limited autochthonous transmission confined to Florida and south Texas.[4]
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