VITN Blog #3: ELISA and RT-qPCR as diagnostic tools for Zika

The Zika epidemic that spread like wildfire in Central and South America produced extraordinary levels of panic among local populations and anxiety in nearby countries such as the United States. Some athletes even withdrew from the 2016 Olympic Games held in Rio de Janeiro for fear of contracting the virus. Zika infection does not generally leave long-lasting effects in either men or women not trying to conceive, but the most devastating images to emerge from the epidemic were of infants born to Zika-infected mothers. Many suffer from microcephaly, i.e. small head size due to abnormal brain development. These children often experience severe developmental delays, but one symptom not as acknowledged in the popular media is epilepsy, a seizure disorder.

This study examined the prevalence and severity of epilepsy in a cohort of infants with congenital Zika infection. About two thirds of infants did have some type of epilepsy, and the authors went on to characterize the seizure type, use of antiepileptic drugs, and electroencephalographic features. One notable characteristic was that the infants’ epilepsy was usually early-onset and drug-resistant, indicating a systemic and persistent disruption of normal brain function due to viral action on the growing fetus. However, I was most interested in the two techniques the authors used to diagnose Zika infection in the serum and cerebrospinal fluid samples obtained from the infants and their mothers: enzyme-linked immunosorbent assay (ELISA) for IgM antibody capture and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assay for detecting Zika’s genome.

In class, we discussed the distinction between IgM, IgG, and IgA antibodies. IgM are found as a result of recent infection, IgG as a result of previous exposure to disease or vaccination, and IgA in bodily secretions (live, attenuated vaccine produces IgA while inactivated vaccine does not). Dr. Ben Pinsky had briefly summarized the mechanism of ELISA: place antigen on a solid support, add the patient serum, add an antibody conjugated to an enzyme, add the substrate of that enzyme, and measure the resulting color change. ELISA is a common protocol in serology, which seeks to identify antibodies in the blood serum. Dr. Pinsky mentioned several problems with this approach, including cross-reactivity between antibodies to related viruses (e.g. Zika and another member of the Flaviviridae family) and the persistence of IgM antibodies past the acute period of infection. Therefore, IgM antibody capture cannot be used on its own for diagnosis of Zika.

We are all familiar with the basic biochemical principles underlying PCR, which uses DNA as its starting material. RT-qPCR exploits these same rules, but with RNA instead. Under the Baltimore classification, Zika virus is in Group IV: it has positive-sense single-stranded RNA. As a result, PCR cannot be used for amplification of Zika’s genetic material, creating a need for RT-qPCR. Since RT-qPCR is dependent on nucleic acids, it is more specific than serology (according to Dr. Pinsky, such molecular diagnostic tools are steadily replacing older, more conventional ones like ELISA). RT-qPCR first uses reverse transcriptase to generate complementary DNA (cDNA) from the viral RNA template. That cDNA can then undergo PCR for amplification and further analysis such as sequencing for identification of Zika. Interestingly, RT-qPCR can be performed in just one step (in which reverse transcription and PCR occur in a single tube) or two, in which the reactions are separate. The number of hours that RT-qPCR takes varies depending on the chosen assay, but its analytical sensitivity makes it extraordinarily valuable for diagnosis.

The use of these two diagnostic techniques in this study demonstrates how important technological advances are to scientific investigation, especially in the field of infectious disease.

Study: “Epilepsy Profile in Infants with Congenital Zika Virus Infection” (https://www.nejm.org/doi/full/10.1056/NEJMc1716070)

- Panos Vandris