by Anne Gaynor, manager of HIV, hepatitis, STD and TB programs, APHL

Public health laboratories around the US have been in the epicenter of Zika response efforts. Unlike many viruses, Zika virus requires multiple tests performed at various stages of exposure and/or infection depending on the case. Like most things related to Zika, testing guidance is complicated and evolving as experts learn more about this virus.

Below is an explanation of the three tests being used to test patients for Zika virus. Currently CDC guidance recommends testing for symptomatic pregnant women, symptomatic non-pregnant patients, asymptomatic pregnant women and pregnant women’s asymptomatic sexual partners with potential exposure. There is currently no testing guidance for asymptomatic patients who are not pregnant and whose partner is not pregnant. If you are concerned about Zika, talk with your healthcare provider.

Polymerase Chain Reaction (PCR)

Who is performing PCR testing for Zika virus infections?

• Almost all public health laboratories, several large commercial laboratories and some clinical laboratories are approved to perform PCR testing. Because PCR is a commonly used method for looking for many viruses and bacteria, there is more laboratory capacity for this test than the other Zika tests listed below. Developing new tests such as the one for Zika and initiating use of PCR tests is rather straightforward.
• PCR is very specific and fast relative to the other tests available (results can often be given within one day).

When would you begin with PCR testing?

• Symptomatic patients (pregnant or not) who are tested less than 14 days after symptom onset
• Asymptomatic pregnant women who were potentially exposed to Zika less than 14 days before testing (e.g., travelled to high-risk areas, were bitten by a mosquito in a high-risk area, sexual partner was potentially exposed, etc.)

What can PCR tell us?

• This test can tell us if a person currently has the virus in their system, which may indicate an acute infection or a very recently acquired infection.
• A positive result from PCR is reliable, so no follow up testing would be needed.

What can’t PCR tell us?

• This test cannot tell us if the person was infected more than 14 days ago (sometimes less). At that stage, the virus may no longer be detectable by PCR which could lead to a negative test result. For that reason, a negative result from PCR requires serology (see below).

Enzyme Linked Immunosorbent Assay (ELISA – serology)

Who is performing ELISA testing for Zika virus infections?

• Many public health laboratories and one large commercial laboratory are performing ELISA testing for Zika virus. While there is testing coverage for the entire country, there is less laboratory capacity for ELISA testing than with PCR. Over the past eight years – well before the arrival of Zika virus – funding cuts forced many public health laboratories to stop performing this type of test or limit the number of ELISA tests performed. As a result, fewer public health laboratories have the necessary capacity (space, staff, equipment) to bring this test on as quickly as the PCR, or, in some cases, at all. Additionally, developing a new ELISA test can take a substantial amount of time and resources. CDC has recently licensed their FDA-approved test for use by the four major commercial laboratories. One has started testing; three are expected to begin soon.
• ELISA is labor intensive and can take up to three days to perform a single test.

When would you perform ELISA testing?

• Symptomatic patients who had a negative PCR result
• Asymptomatic pregnant women who had a negative PCR result; ELISA would then be performed 2-12 weeks after their potential exposure
• Symptomatic patients who are tested 14 or more days after symptom onset
• Asymptomatic pregnant women who are tested 14 or more days after potential exposure (e.g., travelled to high-risk areas, were bitten by a mosquito in a high-risk area, sexual partner was potentially exposed, etc.)

What can ELISA tell us?

• This test tells us whether a person has antibodies to Zika virus. The type of antibody the Zika virus test looks for is generated by the body’s immune system within 7-10 days after infection and can last for several weeks.
• A negative ELISA result is reliable for ruling out recent Zika virus infection and would require no further testing.

What can’t ELISA tell us?

• ELISA can cross-react with antibodies to dengue virus and West Nile virus, causing false positives for Zika virus in some cases. This is because Zika virus, dengue virus and West Nile virus all belong to the flavivirus family. Because these family members are very similar, ELISA can be positive if the person has a current flavivirus infection. For this reason, a positive ELISA test result requires follow-up testing with PRNT (see below).

Plaque Reduction Neutralization Test (PRNT)

Who is performing PRNT testing for Zika virus infections?

• A small number of public health laboratories and CDC laboratories can perform this very specialized type of testing. PRNT is a highly complex test that requires laboratory scientists to grow the virus in their laboratory. While there is testing coverage for the entire country, laboratory capacity for PRNT is the lowest compared to PCR and ELISA.

When would you perform PRNT testing?

• Only after positive ELISA test results – the testing process would never begin with PRNT

What can PRNT tell us?

• PRNT is a different type of serology test that can detect antibodies from a recent or more distant Zika infection.
• PRNT can be used to help determine if the initial ELISA test was positive due to Zika infection or if there was a cross-reaction with dengue virus or another flavivirus. It can also help determine if the infection that led to the positive result on the ELISA was recent and potentially of concern or whether it was in the past and not involved in the patient’s current illness.

As stated previously, CDC continues to update testing guidance as we learn more about Zika virus and its effects. For more on information, please see the APHL and CDC Zika response webpages.

Photo via CDC: This image depicts Centers for Disease Control and Prevention (CDC), Microbiologist who was in the process of preparing reagents that would be used in the CDC-developed Zika IgM antibody-capture enzyme-linked immunosorbent assay (ELISA), referred to as the Zika MAC-ELISA test.