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Laboratory Diagnosis of Zika Virus Infection.

Zika virus (ZIKV) is an arthropod-borne virus discovered in 1947 in primates in the Zika Forest of Uganda during surveillance for yellow fever. (1) The virus was identified in association with relatively small clusters of human infections until 2007, when it caused an outbreak in Yap State, Micronesia, that affected three-quarters of the population. (2) In 2013, a larger ZIKV outbreak in French Polynesia occurred, (3,4) associated with cases of Guillain-Barre syndrome (GBS). (5,6) However, when a 20-fold increase in microcephaly was linked to a ZIKV epidemic in Brazil in 2015, the virus suddenly came to international attention. (7-9)

In February 2016, the World Health Organization declared the Zika epidemic a public health emergency of international concern and identified the development of reliable and accessible diagnostic tests a priority. In this brief review, the current status of laboratory testing, including recommended sample types, methods, result interpretation, and laboratory safety issues, will be discussed. This is a rapidly developing field, and the reader is advised to regularly consult the Centers for Disease Control and Prevention (CDC) Web site or his or her state health department for the most up-to-date recommendations.

LABORATORY DIAGNOSIS

Until recently, the laboratory diagnosis of ZIKV infection was limited to public health or research laboratories that prepared their own reagents. This limited diagnostic capacity created the need for prioritization of testing, with the highest priority given to pregnant women.

In addition to limited test availability, the laboratory diagnosis of ZIKV presents a number of other challenges. The virus cross-reacts serologically with other flaviviruses, such as dengue virus (DENV), West Nile virus, and yellow fever virus (Table 1). Thus, current or past infection, or even vaccination with another flavivirus, will often cause false-positive or uninterpretable antibody results. The 2007 outbreak in Micronesia was initially identified as DENV by a rapid immunoglobulin (Ig) M test. However, the Yap State Department of Health suspected a different pathogen and requested the assistance of the CDC, which subsequently identified ZIKV. (2)

Detection of viral RNA during acute infection by reverse transcription-polymerase chain reaction (RT-PCR) or other nucleic acid amplification tests (NAATs) provides more specific results, but ZIKV is detectable only briefly during acute illness. (10) Furthermore, the utility of molecular testing for the diagnosis of acute infection is limited, because 80% of patients have no symptoms to alert the patient or the clinician to infection, and therefore no frame of reference for onset date. With these limitations in mind, the approach to laboratory testing is discussed below and summarized in Tables 2 and 3.

Acute Infection

Zika virus RNA may be detectable in serum for several days prior to, as well as several days after, symptom onset.11 Very recently, the testing of whole blood has been reported to provide more prolonged detection of viremia, however, more studies are needed to confirm the advantages of testing this sample type. (36) In contrast, ZIKV IgM antibody begins to appear 4 to 7 days after onset of illness and persists for approximately 12 weeks. (10) Neutralizing antibodies, detected by plaque reduction neutralization tests (PRNTs), become detectable concurrently with IgM but persist for years. Although virus in serum declines as antibodies appear, ZIKV is shed in urine for longer, from 7 to 14 or more days after onset, and is usually at higher titer in urine than in serum. (12-14) Thus, molecular testing of urine in addition to serum is strongly recommended. Virus can also be detected in saliva, where it also tends to be shed for longer and at higher titer than in serum. (15) However, in studies where all 3 sample types were collected, testing saliva did not provide additional benefit above serum and urine. (14) Additionally, saliva has been found to be a less consistent sample type than urine, with a higher incidence of processing issues. The additional collection and testing of saliva is therefore not routinely recommended.

For patients with symptomatic disease, serum, whole blood, and urine should be submitted for molecular testing (usually by real-time RT-PCR) as soon as possible after onset of symptoms, preferably within the first few days. Different laboratories have varying time limits for the application of molecular ZIKV tests, ranging from within 7 to within 28 days following the onset of symptoms. For all negative tests, serum should be automatically reflexed for IgM testing. If serology is also negative, IgM testing should be repeated on a later serum sample because it may be too early to detect IgM. The rash illness of ZIKV can be indistinguishable from that of DENV and Chikungunya virus (CHIKV); thus, testing for all 3 viruses in acute symptomatic cases may be a consideration if infection occurred in an area where they were cocirculating.

Reports on prolonged ZIKV viremia in pregnant women16 have prompted the recommendation for laboratories to lift their usual time restrictions for molecular testing on pregnant patients. (17) Zika virus has also been detected in breast milk (18) and in semen. (19-21) Transmission via semen has been shown to occur as a consequence of oral, vaginal, and anal sex, before, during, and after onset of symptoms. (22) Male-to-female, female-to-male, and male-to-male cases of sexual transmission have been reported, and both the detection of virus in semen and delayed transmission (23) suggest that semen secretion can be ongoing for weeks to months after infection. Although ZIKV RNA can be detected in semen by RT-PCR, it is not a routinely performed test and is not recommended for assessing infectivity.

Recent Exposure

Because 80% of ZIKV infections are asymptomatic and the virus is only transiently detectable in blood/serum and urine, diagnosis with RT-PCR is often not possible. However, IgM is reliably detected from approximately 2 to 12 weeks after exposure. Thus, the screening test of choice for recent subclinical infection is ZIKV IgM class capture enzyme-linked immunosorbent assay (MAC-ELISA). In the event of a positive or equivocal ZIKV MAC-ELISA test, more specific serologic testing by PRNT is required to compare neutralizing antibody titers to ZIKV and other flavivruses. (10)

Serologic cross-reactivity to other flaviviruses can be seen with the initial primary flavivirus infection, but titers to the infecting virus are usually higher. In contrast, when prior flavivirus infection or vaccination has occurred, the anamnestic response can yield high or higher titers to the previous virus, leading to uninterpretable or confusing results.

Congenital Infection of the Fetus

The primary means of detecting risk to the fetus is testing of the mother. Where testing indicates a woman has been infected during pregnancy, fetal ultrasound should be used to monitor for developing abnormalities, including microcephaly, disrupted brain development, intracranial calcifications and eye abnormalities, intrauterine grown restriction, and limb abnormalities. (24) Amniotic fluid has been reported as positive for ZIKV by RT-PCR in some pregnancies with affected fetuses. (25-27) However, the role of amniotic fluid testing in diagnosing ZIKV infection or disease in the fetus is not well defined because of the lack of data on sensitivity, optimal timing of testing, and predictive value of a positive result for clinical disease.

Congenital Infection of the Infant at Birth

When an infant is born to a mother with known or suspected ZIKV infection, infant serum, whole blood, placental tissue, umbilical cord, and cerebrospinal fluid (CSF) if obtained for another reason, should be sent for ZIKV IgM, RT-PCR, and histopathology, as appropriate. Importantly, several pieces of tissue should be sampled from various areas of both the fetal and maternal sides of the placenta for molecular testing.

Postmortem in the Fetus or Infant

For the diagnosis of ZIKV, both fresh and formalin-fixed tissues should be collected for molecular testing, histopathology, and immunohisto-chemical staining. Brain tissue should be carefully sampled, making every effort to maintain the brain architecture in order to evaluate neuropathology. Placenta should be sampled extensively, including any pathologic lesions. Samples of individual organs should be submitted, including heart, lungs, liver, kidneys, skeletal muscle, spinal cord, bone marrow, and eyes, as well as blood and body fluids if available. Photographs of grossly observed fetal malformations and organ abnormalities should be prepared, if feasible.

Neurologic Syndromes

In cases of suspected or diagnosed ZIKV infection exhibiting acute meningitis, encephalitis, or myelitis, samples of CSF, blood, and urine should be submitted for molecular testing and serology. For postinfectious syndromes, including GBS, virus may no longer be detectable. However, blood/serum, urine, and CSF should still be submitted for RT-PCR, and serum and CSF submitted for serology.

Past Infection

Currently, IgG testing is not routinely available at public health laboratories, where the focus is on the detection of acute illness or recent infection. Although IgM may persist for several months, detection is only reliable for 12 weeks following onset of symptoms. Thus, there is a potential role for IgG avidity testing to extend the detection of recent infection. An accurate IgG test would also be useful for population studies of seroprevalence and protection. Unfortunately, serologic cross-reactivity presents even more issues for IgG testing because of the longevity of potentially cross-reactive antibodies.

Blood Donor Testing

Retrospective testing of samples collected during the 2013 to 2014 outbreak in French Polynesia revealed 2.8% of blood donors were positive for ZIKV RNA by RT-PCR,11 and transmission of ZIKV by blood products from an asymptomatic, acutely infected donor has recently been documented in Brazil. (28) Thus, the US Food and Drug Administration (FDA) recommended in February 2016 that in areas with ongoing local transmission of ZIKV, blood collection cease unless screening by NAAT could be implemented. In addition, in areas where local transmission was not occurring, the following strategies were initiated: donor deferral for 4 weeks based on travel to or residence in areas with local ZIKV transmission, or sexual contact with at-risk persons. However, many blood collectors, including the Red Cross, began proactively using investigational NAATs. On August 26, 2016, the FDA issued revised guidance, recommending that all donations collected in the United States and its territories be tested for ZIKV, or be treated with an FDA-approved pathogen reduction device if available. Currently there are 2 investigational NAAT ZIKV assays available for testing donated blood. Additionally, licensed pathogen reduction technologies are available for plasma and platelets. For the most up-to-date information, consult the CDC Web site: http://www.cdc.gov/zika/ transmission/blood-transfusion.html.

TEST METHODS

Zika virus testing for clinical purposes in the United States must be performed using assays and reagents that have been validated and approved by the CDC, or those that have emergency use authorization (EUA) by the FDA. Because of the potential consequences of positive test results on decisions regarding termination of pregnancy, laboratory-developed tests cannot be offered without the submission of assay validation data to the FDA and the subsequent EUA for their use. To aid in the development of test kits by commercial companies, the FDA has ZIKV reference material available for validation studies. More information is available at the FDA Web site at http://www.fda.gov/ downloads/EmergencyPreparedness/Counterterrorism/ MedicalCountermeasures/MCMIssues/UCM507010.pdf.

Additionally, the CDC is developing panels of sera from patients who have had recent ZIKV infection, to assist with the development of commercial serology assays.

Clinical test methods currently rely most prominently on the detection of IgM, neutralizing antibody, or the detection of viral RNA by NAAT (Table 4). Virus isolation for ZIKV is performed by very few laboratories and has been difficult to achieve from most primary clinical specimens. Diagnosis by histopathology, immunohistochemistry, and electron microscopy of tissue is covered in a separate article in this issue.

Antibody Response

Although serology is the most commonly applied method for diagnosing both recent and past infections, current antibody tests suffer from extensive cross-reactivity due to the substantial sequence and structural homology between the surface proteins of ZIKV and other flaviviruses, particularly DENV. A study of 9 Thai patients with documented recent DENV infections found all sera were cross-reactive with ZIKV, including when tested using neutralization tests, in the absence of current or prior ZIKV infection. (29) Because ZIKV is circulating in countries with transmission of dengue viruses and also where yellow fever or dengue vaccines are commonly administered, the inability to reliably differentiate flavivirus antibody responses is a serious impediment to accurate diagnosis and surveillance. Thus, a high priority in research and test development is identifying unique epitopes for each virus in order to develop more specific diagnostic tests.

MAC-ELISA Testing.--A MAC-ELISA test developed at the CDC Arbovirus Diagnostic Laboratory at Fort Collins, Colorado, has been given EUA by the FDA and is provided to US public health laboratories for diagnostic testing. The MAC-ELISA test is used for the qualitative detection of ZIKV IgM antibodies in serum or CSF. Cerebrospinal fluid can only be tested alongside a patient-matched serum specimen. Zika virus IgM is usually detectable by day 4 following onset of illness and persists for 12 or more weeks following initial infection. If a positive or equivocal result is obtained, additional testing using PRNT must be performed by the CDC or a CDC-designated laboratory for confirmation. Negative MAC-ELISA results from samples collected in the appropriate window have a strong negative predictive value for recent ZIKV infection. However samples collected very early in the course of infection should be also tested by RT-PCR, and if negative, a convalescent serum should be submitted.

In its current format the MAC-ELISA is a very labor-intensive test, requiring the on-site preparation of 96-well reaction plates coated with anti-human IgM. Following incubation with diluted specimen, they are then reacted with CDC-provided ZIKV culture lysate, recombinant antigen, or uninfected cell culture lysate for background determination. Subsequent incubation with a flavivirus chimeric monoclonal antibody is then followed by a color reaction. Each component of the MAC-ELISA must be standardized and validated prior to use in the laboratory, and restandardized when new lot numbers of reagents are introduced. For test specimens, a screening dilution of 1:400 is used, and testing of the sample against the uninfected antigen must fall within an acceptable range. Results are interpreted based on the ratio of color intensity in the ZIKV-positive antigen well (P) and that in the control well (N). Specimens with P/N values of greater than or equal to 3.0 are reported as "presumptive Zika IgM positive" and are forwarded to the CDC or an approved laboratory for confirmatory testing with PRNT. P/N values between 2.0 and 3.0 are considered equivocal, and further tests (RT-PCR and/or PRNT, or collection of a convalescent serum sample) should be performed. The most common cause of false-positive results is cross-reactivity with antibodies to other flaviviruses, especially DENV. However, IgM assays can be false positive for other reasons as well. (30)

Although Zika IgG ELISA and avidity testing would be useful additions to the diagnostic tools, reliable assays are not yet available.

Neutralizing Antibody.--For more definitive results, MAC-ELISA-positive or equivocal samples are tested for neutralizing antibody by PRNT. In this method, serial dilutions of patient serum are incubated for an hour at room temperature with a standard challenge dose of infectious virus to allow virus-specific antibody, if present, to bind the virus. The serum-virus mixtures are then inoculated onto cell culture monolayers, allowed to adsorb, then overlaid with a semisolid medium. After a defined incubation period, monolayers are fixed and stained, and virus-induced plaques are enumerated (Figure). If neutralizing antibody is present, infectious virus will be neutralized, and the number of plaques will be reduced. The end point is defined as the dilution of serum that results in a 90% reduction in viral plaques. In addition to ZIKV, tests for antibodies to other cocirculating flaviviruses, or vaccine viruses if the patient has been vaccinated, should be performed, and results compared. The entire process can take 3 to 4 weeks.

Result interpretation for ZIKV serology tests is complex. Guidelines have been published in the Morbidity and Mortality Weekly Report. (31)

Nucleic Acid Detection

Zika virus is an RNA virus, and a number of conventional and real-time RT-PCR tests have been published in the literature and reviewed elsewhere. (32,33) Possibly one of the most extensively used tests was a real-time assay developed by the CDC in response to the Yap State outbreak.10 As originally described, it incorporates reactions targeting the premembrane and envelope genes of the virus. In a Lab Diagnosis of Zika Virus Infection--Landry & St. George protocol subsequently distributed by the CDC in 2016 to public health laboratories, the premembrane assay was replaced with one for the NS2B gene. The envelope gene assay is currently incorporated in the Trioplex assay, a multiplex assay developed at the CDC for the detection of ZIKV, DENV, and CHIKV that has been given EUA by the FDA.

Although there have been very few comparative clinical studies of the performance of different ZIKV conventional and real-time RT-PCR assays, a multiplex RT-PCR for ZIKV, CHIKV, and DENV evaluated in clinical trials in Nicaragua was recently found to be more sensitive for ZIKV detection than the single-plex CDC ZIKV envelope gene assay. (34)

A number of commercial NAAT tests have now been given EUA for clinical use by the FDA (Table 5). Many more are in various stages of development and approval, including some that can be performed on highly simplified platforms. Allowance has been made for the use of investigational tests to screen blood donations for ZIKV, and the FDA has created reference materials to assist with the development and validation of NAATs.

Virus Isolation

Although established cultures of ZIKV grow well in LLCMK2 cells, Vero E6 cells, and others, producing plaques in a week or less as well as in suckling mice, (35) the virus has been very difficult to isolate from primary specimens. This is believed to be due at least in part to the almost universally low to very low viral loads in clinical specimens. In practice, culture methods are reserved for specialized reference laboratories, because RT-PCR and other molecular methods are more sensitive, rapid, and avoid the biohazards of working with infectious virus.

LABORATORY SAFETY

Samples collected from patients with suspected arbovirus infections may contain high titers of potentially hazardous viruses, especially CHIKV, and should be handled with appropriate precautions. Serum and CSF samples should be heat inactivated for 30 minutes at 56[degrees]C prior to serologic testing. Samples that may contain CHIKV should be inactivated for 2 hours, because of the possibility of very high CHIKV titers in blood.

The biosafety level classification of ZIKV varies in different countries. In the United States, molecular and serologic procedures can be performed in BSL-2 facilities with BSL-3 practices. All manipulations should be performed within a Class II or higher biologic safety cabinet, and pregnant women should be excluded from working with potentially ZIKV-positive samples.

Samples tested by molecular methods are routinely added to lysis buffer for nucleic acid extraction, and molecular lysis buffers are variable in their viral inactivation properties. Therefore, care should be taken to ensure that either the method being used is one that completely inactivates virus, or that lysed samples are being handled with an assumption of the ongoing presence of live virus. Virus isolation should only be attempted in experienced arbovirus reference laboratories, and ZIKV cultures should not be handled by pregnant women.

CONCLUSIONS

The rapid and accurate diagnosis of ZIKV infection is an international priority. Recently, a number of commercial NAAT tests have received EUA for clinical diagnostic use from the FDA. Although this has expanded the availability of ZIKV diagnostic services and alleviated some of the testing burden on public health laboratories, NAAT has limited utility in ZIKV diagnosis because most patients are either asymptomatic or present for testing after the brief period of likely viral shedding.

The greatest need and most difficult challenge is the development of commercially available antibody tests for the specific and sensitive diagnosis of recent ZIKV infection. Research is urgently needed to identify ZIKV epitopes that do not cross-react with other flavivirus antigens. New information is emerging at a rapid pace, and with ongoing public-private and international collaborations as well as government support, it is hoped that rapid progress will be made in developing robust and widely applicable diagnostic tools.

The authors thank Alan Dupuis and Laura Kramer, PhD, for providing photographs of ZIKV PRNT assays, and Susan Stramer, PhD, for helpful comments on testing policies for blood donors.

References

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(11.) Musso D, Nhan T, Robin E, et al. Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to February 2014. Euro Surveill. 2014;19(14): pii: 20761.

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(13.) Shinohara K, Kutsuna S, Takasaki T, et al. Zika fever imported from Thailand to Japan, and diagnosed by PCR in the urines. J Travel Med. 2016;23(1): pii: tav011.

(14.) Bingham AM, Cone M, Mock V, et al. Comparison of test results for Zika virus RNA in urine, serum, and saliva specimens from persons with travel-associated Zika virus disease-Florida, 2016. MMWR Morb Mortal Wkly Rep. 2016;65(18):475-478.

(15.) Musso D, Roche C, Nhan TX, Robin E, Teissier A, Cao-Lormeau VM. Detection of Zika virus in saliva. J Clin Virol. 2015;68:53-55.

(16.) Driggers RW, Ho CY, Korhonen EM, et al. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. N Engl J Med. 2016; 374(22):2142-2151.

(17.) Oduyebo T, Igbinosa I, Petersen EE, et al. Update: interim guidance for health care providers caring for pregnant women with possible Zika virus exposure-United States, July 2016. MMWR Morb Mortal Wkly Rep. 2016;65(29): 739-744.

(18.) Dupont-Rouzeyrol M, Biron A, O'Connor O, Huguon E, Descloux E. Infectious Zika viral particles in breastmilk. Lancet. 2016;387(10023):1051.

(19.) Atkinson B, Hearn P, Afrough B, et al. Detection of Zika virus in semen. Emerg Infect Dis. 2016;22(5):940.

(20.) Mansuy JM, Dutertre M, Mengelle C, et al. Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen? Lancet Infect Dis. 2016; 16(4):405.

(21.) Matheron S, D'Ortenzio E, Leparc-Goffart I, Hubert B, de Lamballerie X, Yazdanpanah Y. Long lasting persistence of Zika virus in semen [published online ahead of print July 28, 2016]. Clin Infect Dis. doi: 10.1093/cid/ciw509.

(22.) Brooks JT, Friedman A, Kachur RE, LaFlam M, Peters PJ, Jamieson DJ. Update: interim guidance for prevention of sexual transmission of Zika virus--United States, July 2016. MMWR Morb Mortal Wkly Rep. 2016;65(29):745-747.

(23.) Turmel JM, Abgueguen P, Hubert B, et al. Late sexual transmission of Zika virus related to persistence in the semen. Lancet. 2016;387(10037):2501.

(24.) Society for Maternal-Fetal Medicine Publications Committee. Ultrasound screening for fetal microcephaly following Zika virus exposure. Am J Obstet Gynecol. 2016;214(6):B2-B4.

(25.) Besnard M, Eyrolle-Guignot D, Guillemette-Artur P, et al. Congenital cerebral malformations and dysfunction in fetuses and newborns following the 2013 to 2014 Zika virus epidemic in French Polynesia. Euro Surveill. 2016; 21(13). doi: 10.2807/1560-7917.ES.2016.21.13.30181.

(26.) Calvet G, Aguiar RS, Melo AS, et al. Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study. Lancet Infect Dis. 2016;16(6):653-660.

(27.) Perez S, Tato R, Cabrera JJ, et al. Confirmed case of Zika virus congenital infection, Spain, March 2016. Euro Surveill. 2016;21(24). doi: 10.2807/15607917.ES.2016.21.24.30261.

(28.) Cunha MS, Esposito DL, Rocco IM, et al. First complete genome sequence of Zika virus (flaviviridae, flavivirus) from an autochthonous transmission in Brazil. Genome Announc. 2016;4(2): pii: e00032-16.

(29.) Priyamvada L, Quicke KM, Hudson WH, et al. Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus. Proc Natl Acad Sci U S A. 2016;113028:7852-7857.

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(32.) Waggoner JJ, Pinsky BA. Zika virus: diagnostics for an emerging pandemic threat. J Clin Microbiol. 2016;54(4):860-867.

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(34.) Waggoner JJ, Gresh L, Mohamed-Hadley A, et al. Single-reaction multiplex reverse transcription PCR for detection of Zika, chikungunya, and dengue viruses. Emerg Infect Dis 2016;22(7):1295-1297.

(35.) Way JH, Bowen ET, Platt GS. Comparative studies of some African arboviruses in cell culture and in mice. J Gen Virol. 1976;30(1):123-130.

(36.) Lustig Y, Mendelson E, Paran N, et al. Detection of Zika virus RNA in whole blood of imported Zika virus disease cases up to 2 months after symptom onset, Israel, December 2015 to April 2016. Euro Surveill. 2016;21(26). doi:10.2807/ 1560-7917.

Marie Louise Landry, MD; Kirsten St. George, PhD

Accepted for publication September 1, 2016.

Published as an Early Online Release October 20, 2016.

From the Departments of Laboratory Medicine and Medicine, Yale University School of Medicine, New Haven, Connecticut (Dr Landry); and the Laboratory of Viral Diseases, Wadsworth Center, New York State Department of Health, Albany (Dr St. George).

Dr St. George receives research support from ThermoFisher (Holtsville, New York) for the evaluation of new Zika virus diagnostic assays and for research on Zika and other viruses. She also has a royalty-generating collaborative agreement with Zeptometrix (Buffalo, New York) as well as research support and a collaborative research agreement with Akonni Biosystems (Frederick, Maryland). Dr Landry has no relevant financial interest in the products or companies described in this article.

Reprints: Marie Louise Landry, MD, Departments of Laboratory Medicine and Medicine, Yale University School of Medicine, PO Box 208035, New Haven, CT 06520-8035 (email: marie.landry@yale.edu).

Please Note: Illustration(s) are not available due to copyright restrictions.

Caption: Plaque reduction neutralization test of acute (A) and convalescent (B) patient sera against Zika virus (ZIKV). Serial dilutions of patient serum were each incubated with 100 plaque-forming units of ZIKV for 1 hour at room temperature, then the serum-virus mixtures were inoculated onto Vero monolayers. Following an incubation of several days, cultures were fixed and stained, and virus-induced plaques were enumerated. Plaque counts lower than 10 were considered negative. In the examples shown here, the titers for both acute and convalescent sera are 1:160, indicating exposure to ZIKV, but the timing of exposure is inconclusive.
Table 1. Arthropod-Borne Flaviviruses With
Potential Serologic Cross-Reactivitya

Flavivirus

Zika virus
Dengue virus types 1-4
Yellow fever virus
West Nile virus
St Louis encephalitis virus
Japanese encephalitis virus
Powassan virus

 (a) Chikungunya, Eastern equine encephalitis, Western equine
encephalitis, and Venezuelan equine encephalitis viruses are
members of the Togavirus family and usually do not cross-react
serologically with Zika.

Table 2. Clinical Scenarios in Which Diagnostic Zika
Virus (ZIKV) Testing May Be Indicated

Scenarioa            Samples           Method

Acute            Serum             NAAT, IgM (b)
illness

                 Whole blood       NAAT

                 Urine             NAAT

Recent           Serum             IgM (b)
exposure
(past
2-12 wk)

                 Whole blood       NAAT

Congenital       Maternal serum,   IgM, (b)
(prenatal)       whole blood       IgG, NAAT

                 Amniotic fluid    NAAT

Congenital       Both fresh and    NAAT,
(postmortem)     formalin-fixed    histopathology,
                 tissue:           IHC
                 placenta,
                 fetal brain,
                 eyes, and
                 samples of
                 each major
                 organ

Congenital       Maternal          IgM, (b) NAAT
(infant)         serum, blood

                 Infant serum,     IgM, NAAT
                 CSF if
                 obtained

                 Amniotic fluid    NAAT

                 Placenta,         NAAT,
                 umbilical cord    histopathology,
                                   IHC

Past             Serum             IgG
infection

Blood donor      Blood             NAAT
testing

Neurologic       Serum, CSF,       NAAT, IgM,
complications    whole blood       (b) IgG
(eg, GBS,
myelitis,
meningo-
encephalitis)

                 Urine             NAAT

Scenarioa            Samples              Comments

Acute            Serum             NAAT is more
illness                            definitive than
                                   serology if
                                   positive. Virus is
                                   usually detected in
                                   serum for 4-7 d after
                                   onset of symptoms

                 Whole blood       Because of the
                                   similarity of
                                   illnesses caused by
                                   ZIKV, DENV, and CHIKV,
                                   and their
                                   cocirculation in
                                   many regions, NAAT for
                                   all 3 viruses may be
                                   used for serum

                 Urine             Urine has higher
                                   viral load than
                                   serum and is
                                   often positive for
                                   14 d or longer

Recent           Serum             IgM should be reliably
exposure                           present from 2 to 12
(past                              wk after acute
2-12 wk)                           infection

                 Whole blood       Cross-reactivity
                                   between related
                                   flaviviruses is
                                   problematic, and all
                                   positive IgM results
                                   must be confirmed by
                                   PRNT A negative ZIKV
                                   IgM result should
                                   exclude recent
                                   infection unless
                                   the specimen was
                                   collected less than
                                   3 wk after exposure

Congenital       Maternal serum,   Negative results in
(prenatal)       whole blood       the mother exclude
                                   infection in the
                                   fetus, unless
                                   specimen is collected
                                   too soon after
                                   exposure to detect
                                   IgM and too late
                                   to detect RNA

                 Amniotic fluid    Positive NAAT on
                                   amniotic fluid
                                   suggests
                                   infection in the
                                   fetus, although
                                   correlation with
                                   disease is not
                                   well defined

Congenital       Both fresh and    Tissue architecture
(postmortem)     formalin-fixed    should be
                 tissue:           maintained. Samples
                 placenta,         should be routed
                 fetal brain,      for routine
                 eyes, and         evaluation or
                 samples of        cytogenetics as
                 each major        indicated
                 organ

Congenital       Maternal
(infant)         serum, blood

                 Infant serum,     Infant serum should
                 CSF if            be obtained within
                 obtained          2 d of birth

                 Amniotic fluid

                 Placenta,         IHC can be performed
                 umbilical cord    on formalin-fixed
                                   placenta and
                                   umbilical cord

Past             Serum             Cross-reactivity with
infection                          other flaviviruses
                                   is a concern, and
                                   test interpretation
                                   can be extremely
                                   difficult

Blood donor      Blood             Used to detect
testing                            acute viremia and
                                   infectivity
                                   of blood or blood
                                   products

Neurologic       Serum, CSF,       Optimal test depends
complications    whole blood       on timing of sample
(eg, GBS,                          collection and
myelitis,                          whether neurologic
meningo-                           symptoms are due to
encephalitis)                      acute ZIKV infection,
                                   or are postinfectious

                 Urine

Abbreviations: CDC, Centers for Disease Control and Prevention;
CHIKV, Chikungunya virus; CSF, cerebrospinal fluid; DENV, dengue
virus; GBS, Guillain-Barre syndrome; Ig, immunoglobulin; IHC,
immunohistochemistry; NAAT, nucleic acid amplification test; PRNT,
plaque reduction neutralization test.

(a) For the latest guidance, consult the CDC Web site at
http://www.cdc.gov/zika.

(b) Positive ZIKV IgM result may be due to cross-reactive
antibodies from infection with DENV or a pathogen other than ZIKV.
Confirmatory PRNT is required for all ZIKV IgM-positive samples.
However, PRNT results can be difficult to interpret and may not
always distinguish recent from past ZIKV infection, or distinguish
ZIKV from other flavivirus or multiflavivirus infections.

Table 3. Specimens and Tests for Zika Virus
Laboratory Testing (a)

                                       NAAT (RT-PCR) (b)

Patient             MAC-ELISA With     Serum/Whole     Urine
Criteria          PRNT Confirmation:      Blood
                      Serum (b)

Symptomatic              Yes               Yes          Yes

Asymptomatic             Yes               Yes          Yes
pregnant woman

                  NAAT (RT-PCR) (b)

Patient           CSF (c)   Tissue/
Criteria                    Other (d)

Symptomatic       If CNS      Yes
                  disease

Asymptomatic      N/A         N/A
pregnant woman

Abbreviations: CNS, central nervous system; CSF, cerebrospinal
fluid; MAC-ELISA, immunoglobulin (Ig) M antibody capture
enzyme-linked immunosorbent assay;N/A, not applicable; NAAT,
nucleic acid amplification test; PRNT, plaque reduction
neutralization test; RT-PCR, reverse transcription-polymerase chain
reaction.

(a) For serum and other fluids, submit 1.0 mL minimum. For tissue,
submit 0.5 to 1 cm minimum.

(b) Transport fluids at 4[degrees]C on cold packs or frozen
at -70[degrees]C on dry ice; transport tissues frozen at -70[degrees]C
on dry ice.

(c) Antibody studies may also be performed on CSF.

(d) Samples of amniotic fluid, placenta, umbilical cord, brain, and
other tissues or fluids may be tested from potentially affected
fetus or infant.

Table 4. Zika Virus (ZIKV) Diagnostic Methods

Method                Uses                 Limitations

ZIKV
  NAAT (eg,   Detect virus in body   Lack of controls and
  RT-PCR)     fluids, tissues, and   samples for validation
              blood products         studies

  Antigen     Used in IHC of fixed   Sensitivity and
              tissues                specificity not well
                                     defined; subjective

  Virus       Isolate virus from     Specialized methods,
  isolation   body fluids and        including cell cultures,
              tissues                mosquito cultures, and
                                     suckling mice. Not
                                     sensitive

Zika
antibody
  IgM MAC-    IgM for recent         Cross-reactivity with
  ELISA       exposure (past         flaviviruses and higher
              2-12 wk)               false-positive rate
                                     with IgM assays in
                                     general. Infections
                                     occurring more than
                                     12 wk prior to
                                     collection may not
                                     be detected

  IgG ELISA   Past infection         Cross-reactivity with
                                     flaviviruses and other
                                     viruses

  PRNT        Differentiate          Uses live virus to
              antibody reactivity    assess neutralizing
              for various            antibodies in patient
              flaviviruses           serum and spinal fluid

Method              Clinical              Availability
                 Applicability
ZIKV
  NAAT (eg,   Most accurate and      Available in public
  RT-PCR)     rapid test             health laboratories,
                                     but submission
                                     requires approval.
                                     Several commercial
                                     kits have received
                                     EUA;NAAT is
                                     increasingly available
                                     at commercial
                                     laboratories

  Antigen     Identify               Available only at CDC
              distribution of
              viral antigen in
              tissues

  Virus       For research.          Performed only in
  isolation   Detects and            highly specialized
              facilitates study      public health or
              of variants as         research labs. Not
              well as unexpected     routinely done
              pathogens

Zika
antibody
  IgM MAC-    Main screening test    Initially available
  ELISA       for at-risk            only in public health
              pregnancies            laboratories that
                                     prepare plates and
                                     standardize assay on
                                     site. Now offered at
                                     commercial
                                     laboratories; to date,
                                     one MAC-ELISA kit
                                     has received EUA

  IgG ELISA   Useful to determine    Not available
              immunity if accurate

  PRNT        Essential to assess    Performed only in
              specificity of IgM     highly specialized
              MAC-ELISA              public health or
                                     research labs

Abbreviations: CDC, Centers for Disease Control and Prevention;
ELISA, enzyme-linked immunosorbent assay; EUA, emergency use
authorization from US Food and Drug Administration; Ig,
immunoglobulin; IHC, immunohistochemistry; MAC-ELISA, IgM antibody
capture ELISA;NAAT, nucleic acid amplification test; PRNT, plaque
reduction neutralization test; RT-PCR, reverse
transcription-polymerase chain reaction.

Table 5. Zika Virus (ZIKV) Diagnostic Assays With Emergency
Use Authorization From the Food and Drug Administration (FDA)

Test                    Assay                       Approval
Category

Molecular    CDC Trioplex Real-time        Approved for Zika,
(a)          RT-PCR Assay                  chikungunya, and dengue
                                           testing on serum and CSF

                                           Additionally approved for
                                           Zika testing on whole
                                           blood and amniotic fluid

             Zika Virus RNA Qualitative    Approved for Zika testing
             Real-time RT-PCR (Focus       on serum
             Diagnostics, Cypress,
             California)

             RealStar Zika virus RT-PCR    Approved for Zika testing
             kit US (Altona Diagnostics    on serum and urine
             GmbH, Hamburg, Germany)

             Aptima Zika Virus Assay       Approved for Zika testing
             (Hologic Inc, Marlborough,    on serum and plasma
             Massachusetts)

             Zika Virus Real-time          Approved for Zika testing
             RT-PCR test (Viracor-IBT,     on serum, plasma, and
             Lee's Summit, Missouri)       urine

             VERSANT Zika RNA 1.0 Assay    Approved for Zika testing
             (kPCR) Kit (Siemens           on serum, plasma, and
             Healthcare Diagnostics        urine
             Inc, Newark, Delaware)

             xMAP MultiFLEX Zika RNA       Approved for Zika testing
             Assay (Luminex Corp,          on serum, plasma, and
             Austin, Texas)                urine

             LightMix Zika rRT-PCR         Approved for Zika testing
             Test (Roche Molecular         on serum and plasma
             Systems, Pleasanton,
             California)

Serology     CDC Zika MAC-ELISA for        Approved for Zika testing
             the detection of IgM          on serum and CSF

             ZIKV Detect IgM Capture       Approved for Zika testing
             ELISA (InBiOS, Seattle,       on serum
             Washington)

Abbreviations: CDC, Centers for Disease Control and Prevention;
CSF, cerebrospinal fluid; Ig, immunoglobulin; MAC-ELISA, IgM
antibody capture enzyme-linked immunosorbent assay; rRT-PCR,
real-time reverse transcription-polymerase chain reaction; RT-PCR,
reverse transcription- polymerase chain reaction.

(a) US FDA also allows the use of an investigational test from
Roche Molecular Systems or the Aptima Assay from Hologic, under IND
protocols, to screen blood donations in areas with active
mosquito-borne transmission of ZIKV.
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Author:Landry, Marie Louise; St. George, Kirsten
Publication:Archives of Pathology & Laboratory Medicine
Date:Jan 1, 2017
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