Zika Virus Workup

Confirmation of Zika virus infection based on diagnostic testing is challenging due to test sensitivity, specificity, and the epidemiologic prevelance of Zika. 

Diagnostic testing of Zika virus (ZIKV) infection is based on molecular and serologic methods. ^[32] Nucleic acid amplification testing (NAAT) is standard diagnostic testing for confirmation of acute infection. A negative NAAT for Zika virus does not rule out infection due to transient viremia during active infection. FDA has issued emergency use authorization on Zika NAAT testing to be performed on serum, plasma, whole blood, cerebrospinal fluid, urine, or amniotic fluid. 

Serologic testing with immunoglobulin IgM can be performed as early as 7 days after symptom onset. A negative immunoglobulin IgM serologic test does not rule out infection due to lack of precise timing to detect presence of antibody response. Early or late antibody testing can result in a false negative result due to lack of antibody development or waning of antibody response post infection, respectively. IgM antibody can remain positive for up to 12 weeks or longer in patients with history of Zika virus infection making it difficult to interpret recent or acute infection. False-positive IgM test result can occur due to cross-reactivity with other flaviviruses (eg, yellow fever, dengue, Japenese encephalitis, West Nile). Zika virus IgM antibody assays can be used on serum, plasma, whole blood, or cerebrospinal fluid.

In presence of concern for cross-reactivity with other flaviviruses, plaque reduction neutralization tests (PRNTs) provide quantitative virus-specific antibody titers for dengue, Zika, and other flaviviruses. CDC uses a PRNT with a 90% cutoff value titer ≥10 in serum and ≥2 in cerebrospinal fluid (the typical starting dilutions) to define positive specimens. A neutralizing antibody titer ≥4- fold higher titers compared to other flavivirus titers is considered diagnostic. Differentiation from other flaviviruses and late diagnosis of Zika virus infection more than 3 months from illness is possible with PRNT. Diagnostic testing using PRNT >1 year past illness is challenging, as observed with 27% of persistent neutralizing antibody titers in patients in Florida with an inability to distinguish between dengue and Zika.

The last locally-acquired Zika nucleic acid amplification tests (NAAT)-confirmed case in the continental United States was in September 2017 and in the US territories was reported in May 2018. As the prevelance of Zika virus infection has declined, potential detection of false-positive test results is likely. 

The WHO recommends using the Brighton criteria to diagnose Guillain-Barré syndrome. ^[33]

Prompt diagnosis and laboratory confirmation of Zika virus infection is challenging.

Serologic Testing

Zika virus infection is diagnosed based on detection and isolation of Zika virus RNA from serum using nucleic acid amplification test (NAAT). The highest sensitivity of NAAT is during the initial week of illness, which is characterized by high viremia. After the initial week of illness, serologic testing for virus-specific immunoglobin M (IgM) and neutralizing antibodies against Zika virus infection can be performed using enzyme-linked immunosorbent assay (ELISA). ^[18]  Serum IgM antibody testing should be performed if NAAT results are negative, regardless of when the specimen was collected.

In patients within 7 days of symptom onset, a positive serum NAAT for Zika is suggestive of the presence of acute Zika virus infection. The NAAT should be repeated on newly extracted RNA from the same specimen to rule out false-positive test results.

In patients within 7 days of symptom onset, a negative serum NAAT and IgM antibody testing is suggestive of absence of Zika virus infection. 

In patients more than 7 days to 12 weeks from symptom onset, a negative IgM antibody testing is suggestive of absence of Zika virus infection.

In patients with indeterminate IgM antibody testing, repeat IgM testing or confirmatory PRNTs test should be performed. 

In patients with positive IgM antibody testing without positive NAAT, a confirmatory PRNTs test should be performed. 

A 4-fold higher titer based on PRNT results might not differentiate anti-Zika virus antibodies from cross-reacting antibodies in all persons with previous infection or vaccination against a related flavivirus.

If IgM testing is positive for Zika or dengue virus or returns equivocal results, the following PRNT interpretations apply:

• A PRNT titer ≥10 indicates evidence of infection with that specific flavivirus when the PRNT to the other flavivirus(es) tested is < 10.
• A PRNT titer < 10 to a specific flavivirus indicates an absence of infection with that virus.
• A positive PRNT result ( ≥10 to multiple flaviviruses) indicates evidence of recent flaviviral infection. 

The tables from CDC below provide interpretation of dengue and Zika virus diagnostic testing results in nonpregnant and pregnant patients with a clinically compatible illness and the risk for infection with both viruses. 

Testing for Zika Virus Infection in Pregnant Women

Pregnant women with symptoms and possible exposure to Zika virus should undergo molecular and serologic diagnostic testing for confirmation of Zika virus infection. A NAAT on serum and urine specimen and IgM antibody test on serum should be performed and collected within 12 weeks of symptom onset. The diagnostic testing algorithm below should be used to interpret diagnosis of Zika and distinguish from dengue virus infection.

Testing for Congential Zika Virus Infection

Molecular and serologic diagnostic testing for Zika virus is recommended for confirmation of diagnosis. Other than Zika virus RNA testing in infant's serum and urine and IgM antibodies in serum, cerebrospinal fluid testing for NAAT and IgM antibody testing is recommended to increase diagnostic yield. 

Cord blood testing is not recommended due to false-positive and false-negative test results. 

Following clinical and diagnostic algorithm should be followed as recommended by CDC:

CDC has provided guidance on interpretation of NAAT and IgM antibody testing for diagnosis of congenital Zika virus infection. 

Differentiation between dengue and Zika virus infection in infants can be performed by PRNT, if IgM test on infant's initial sample is nonnegative (nonnegative serology terminology varies by assay and might include “positive,” “equivocal,” “presumptive positive,” or “possible positive”) and NAAT is negative. PRNT does not distinguish between infant or maternal passive antibodies collected from infant during or immediately after birth. As maternal antibodies usually are not detected past 18 months, diagnostic testing past 18 months can help to diagnose congenital Zika virus infection.

In infants with nonnegative IgM and positive Zika neutralizing antibodies at birth, PRNT at age of ≥ 18 months can confirm or rule out congenital Zika virus infection: 

• A positive PRNT is suggestive of presumed congenital Zika virus infection. It is difficult to interpret timing of infection in infants traveling or residing in areas with Zika. 
• A negative PRNT is suggestive of absence of congenital Zika virus infection. 

For infants with clinical findings consistent with congenital Zika syndrome and maternal laboratory evidence of possible ZIKV infection during pregnancy, PRNT at age of ≥ 18 months can be considered if infant testing was negative (NAAT and IgM antibody) or not performed at birth.

Congenital Zika virus infection

A comprehensive workup is recommended in infants with clinical findings consistent with congenital Zika syndrome (CZS). CDC provides guidance on recommended evaluation for management of CZS. Standard evaluation includes comprehensive physical exam including growth parameters and developmental monitoring and screening; vision screening using tools as recommended by the American Academy of Pediatrics; and a newborn hearing screen at birth, preferably with automated auditory brainstem response (ABR). 

Further evaluation is recommended:

• Head ultrasound
• Comprehensive ophthalmologic exam performed by age 1 month by an ophthalmologist experienced in assessing and intervening in infants.
• Infants should be referred for automated ABR by age 1 month if the newborn hearing screen was passed using only otoacoustic emissions methodology
• Subspecialist consultation as seen in the following table to help with infant care

Close monitoring and prompt testing is recommended for any complications associated with congenital Zika syndrome:

• Diaphragmatic paralysis should be considered in an infant who develops respiratory distress or failure or who fails to wean from a ventilator.
• Swallowing dysfunction, such as difficulty breathing with feeding, coughing or choking during feeding, or extended feeding times, requires assessment for dysphagia.
• Signs of increasing intracranial pressure (eg, increasing head circumference, irritability, or vomiting) should prompt neuroimaging to assess for postnatal hydrocephalus.

A multidisciplinary team and regular well-child visits should be performed with routine preventative care and immunizations. A diagnostic ABR no longer is recommended at age 4–6 months for infants who passed the initial hearing screen with automated ABR due to lack of data suggesting delayed-onset hearing loss in infants with CZS.

Infants without clinical findings of CZS who were born to a mother with laboratory evidence of Zika virus infection or travel to area with Zika virus should be closely monitored during subsequent well-chils visit. Any clinical findings consistent with CZS should prompt appropriate subspecialist referrals for evaluation and management.

Diagnosis of congenital Zika virus infection during pregnancy can be performed using prenatal ultrasound and amniocentesis. However, interpretation of these tests is unknown. CDC recommends shared decision-making between the patient and provider before pursuing prenatal screening testing. 

Ultrasound examination specifically for detection of abnormalities related to congenital Zika virus infection are unknown due to variable sensitivity, specificity, and positive and negative predictive values. ^[34]  Per CDC, prenatal ultrasound findings associated with congenital Zika virus infection include intracranial calcifications at the gray-white matter junction, ventriculomegaly, abnormalities of the corpus callosum, microcephaly, and limb anomalies. Correlation of prenatal ultrasound findings suggestive of congenital Zika virus infection with maternal laboratory evidence of Zika virus or maternal exposure to Zika virus during pregnancy should be taken into consideration during interpretation and reporting. There are no recommended timings to conduct ultrasound. Abnormal prenatal ultrasound has been detected in the second and third trimester of pregnancy based on limited case reports. Frequency of prenatal ultrasound should be performed based on clinical judegement, patient preference, and the risk for Zika. 

Detection of a fetal anomaly should be followed by amniocentesis for evaluation of intrauterine Zika virus infection. Zika virus RNA can be found transiently in amniotic fluid, thus absence of RNA via NAAT does not rule out congenital Zika virus infection. The sensitivity and specificity of amniocentesis for determination of congenital infection and prediction of fetal abnormality is unknown. ^[35]

 

Detection of a fetal anomaly should be followed by amniocentesis for evaluation of intrauterine Zika virus infection. Zika virus RNA can be found transiently in amniotic fluid, thus absence of RNA via NAAT does not rule out congenital Zika virus infection. The sensitivity and specificity of amniocentesis for determination of congenital infection and prediction of fetal abnormality is unknown. ^[35]