Date Published: February 17, 2019
Publisher: The American Society of Tropical Medicine and Hygiene
Author(s): Rodion Gorchakov, Rebecca M. Berry, Shital M. Patel, Hana M. El Sahly, Shannon E. Ronca, Kristy O. Murray.
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.
Zika virus (ZIKV), a member of Flavivirus genus, emerged in the Americas in 2015 and is a significant public health concern.1–3 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.1–3 Most of the clinical cases reported in the U.S. states have been associated with travel to the affected areas.4 Since 2015, more than 5,000 such travel-associated cases have been documented, mostly in 2016. 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
Initial testing of the spiked matrices with RNA extracted by the QIAamp MinElute Virus Spin Kit manufacturer’s protocol yielded poor recovery values for all sample types, except for serum and saliva (Figures 1–3; Supplemental Table 4). Because most of the tested matrices contained high amounts of cellular DNA/RNA, the extraction protocol was optimized by addition of 5 minutes incubation at 56°C at the elution step. Slow loading on the extraction column (3,000 rpm) was also added to the protocol to potentially improve recovery of sample nucleic acids. These modifications restored recovery of ZIKV RNA in whole blood, vaginal secretions, and breast milk samples and maintained full recovery in saliva samples (Figure 1; Supplemental Table 4).
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 RT-qPCR, as means of improving the analytical sensitivity of detection. We have shown that several adaptations of the extraction protocol, in combination with the previously published TaqMan ZIKV 1107 RT-qPCR assay, provide a reliable method for detection of ZIKV RNA in a variety of specimens. Serum and saliva swab specimens had optimal recovery of ZIKV RNA with the original manufacturer’s protocol and did not require additional extraction adaptations.