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)
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Panos Vandris
