ATLANTA – A novel pyrosequencing (PSQ)–based reverse-transcription polymerase chain reaction (RT-PCR) assay improves and expands diagnostic capabilities for Zika virus infection, according to findings in 60 patients diagnosed with the virus in 2016 and 2017.
Dr. Julu Bhatnagar
The PSQ assay provides rapid, specific, and cost-effective detection of the virus in tissues of congenital and pregnancy-associated infections, and, compared with serum-based assays, extends the time frame for Zika virus detection, Julu Bhatnagar, PhD, reported in a presentation at the International Conference on Emerging Infectious Diseases.
Dr. Bhatnagar and her colleagues from the Centers for Disease Control and Prevention in Atlanta developed the assay and evaluated it using RNA extracted from formalin-fixed, paraffin-embedded placental/fetal tissues from 53 women with varying pregnancy outcomes, and brain tissues from seven infants with microcephaly who died. In all of the tissue samples, which were received between January 2016 and August 2017, Zika virus was previously identified by conventional RT-PCR and Sanger sequencing.
The PSQ assay detected and sequence confirmed Zika virus in tissues from all 60 patients, whereas 40 negative control samples, including tissues from dengue- and chikungunya virus–confirmed cases, all tested negative.
In addition, the PSQ assay detected Zika virus in placental tissues from three other cases that were previously negative by the conventional tissue-based RT-PCR, thereby demonstrating better sensitivity of the PSQ assay in comparison to conventional tissue RT-PCR, said Dr. Bhatnagar, who is molecular pathology team leader in the Infectious Diseases Pathology Branch, Division of High-Consequence Pathogens and Pathology at the CDC’s National Center for Emerging and Zoonotic Infectious Diseases.
“Importantly, PSQ results can be obtained in 1 day and at half the cost of Sanger sequencing,” she said.
The findings are important because Zika virus infection during pregnancy can cause microcephaly and is associated with pregnancy loss. Laboratory diagnosis of the virus is challenging for pregnancy-associated infections because of the short duration of viremia, she explained.
However, prolonged detection of Zika virus RNA in placental, fetal, and neonatal brain tissue has been reported.
Dr. Bhatnagar was the first author on a 2016 study published in Emerging Infectious Diseases that provided confirmation of the linkage of Zika virus with microcephaly and that suggested its association with adverse pregnancy outcomes and provided evidence of Zika virus replication and persistence in fetal brain and placenta.
“This article highlights the value of tissue analysis to expand opportunities to diagnose Zika virus congenital and pregnancy-associated infections and to enhance the understanding of mechanism of Zika virus intrauterine transmission and pathogenesis,” she and her colleagues wrote in that article. “In addition, the tissue-based RT-PCRs extend the time frame for Zika virus detection and particularly help to establish a diagnosis retrospectively, enabling pregnant women and their health care providers to identify the cause of severe microcephaly or fetal loss.”
Those findings led to the hypothesis that the PSQ assay evaluated in the current study would provide better opportunities for detection, particularly in cases where serum RT-PCR or serologic testing is negative because of testing performed outside the optimal testing window, she said.
Indeed, the novel assay not only allows for an extended time frame for Zika virus detection, it also provides insights into viral tissue tropism and persistence, she noted.
According to the CDC, no local mosquito-borne Zika virus transmissions have been reported in the continental United States in 2018, but transmission is still a threat internationally, and those traveling outside of the continental United States should find out if they are traveling to an area with risk of Zika.