Detecting vertical Zika transmission: emerging diagnostic approaches

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Detecting vertical Zika transmission: emerging diagnostic approaches for an emerged flavivirus Matt Collins, and Jesse Waggoner ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00003 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 6, 2019

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ACS Infectious Diseases

Detecting vertical Zika transmission: emerging diagnostic approaches for an emerged flavivirus Matthew Collins1, # and Jesse J. Waggoner1, 2

1

Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta,

GA, USA 2

Department of Global Health, Rollins School of Public Health, Atlanta, GA, USA

#

Corresponding Author, [email protected]

Matthew Collins, MD, PhD The Hope Clinic 500 Irvin Court, Suite 200 Decatur, GA 30030 Jesse Waggoner, MD Health Science Research Building, Room E-169 1760 Haygood Drive NE Decatur, GA 30322

ACS Paragon Plus Environment

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Zika virus was recently responsible for a massive epidemic that spread throughout Latin America and beyond. Though typically asymptomatic or self-limiting, the sheer numbers of Zika infections allowed identification of unexpected phenotypes including sexual transmission, Guillain-Barré syndrome, and teratogenicity. Thousands of infants in South, Central, and North America have now been born with microcephaly or one of a number of fetal anomalies constituting the congenital Zika syndrome (CZS). Diagnosing CZS is based on a combination of clinical risk assessment and laboratory testing (which includes determining whether the mother has experienced a possible Zika infection during her pregnancy). A newborn suspected of having congenital Zika infection (due to maternal Zika infection or a birth defect described in association with congenital Zika infection) is then specifically tested for presence of Zika virus in neonatal tissue or anti-ZIKV IgM in the blood or cerebrospinal fluid. Though the guidelines are clear, there is room for considerable practice variation to emerge from individualized patient-provider encounters, largely due to limitations in diagnostic testing for Zika. The natural history of Zika further obscures our ability to know who, when, and how to test. Molecular diagnostics are highly specific, but may not serve well those with asymptomatic infection. Serologic assays expand the diagnostic window, but are complicated by cross-reactivity among related flaviviruses and passive immunity transferred from mom to baby. Furthermore, existing and emerging diagnostic tools may not be widely available due to limitations in resources and infrastructure of health systems in affected areas. Improvements in assay parameters as well as advances in platforms and deployability hold promise for optimizing diagnostic approaches for congenital Zika infection. The diagnostic tools and technologies under development must be integrated with forthcoming clinical knowledge of congenital Zika infection to fully realize the value that laboratory testing holds for diagnosing in utero mother to child transmission but also for understanding, predicting, and managing the health outcomes due to congenital Zika infection. Keywords: Zika virus, vertical transmission, congenital infection, molecular diagnostics, serodiagnostics, flavivirus, maternal child health, TORCHZ


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Introduction Zika virus (ZIKV) infection during pregnancy can have devastating consequences. Data from Brazil in 2015 led to the recognition of ZIKV as a cause of microcephaly.1–3 Since that time, an array of congenital abnormalities – including some that only manifest in the postnatal period4 – due to ZIKV infection have been described and termed the congenital Zika syndrome (CZS).5–8 This was somewhat unexpected because related flaviviruses are not usually associated with vertical transmission. Transmission could proceed through a few different routes, which could differ by timing in pregnancy and route of maternal infection (sexual vs mosquito-borne), but ultimately culminate in the invasion of fetal tissue and replication inside fetal cells.9,10 The fetal Hofbauer cell has been identified as likely playing a critical role in establishing infection in the fetus.11–13 From there, both neurotropic and neurotrophic properties of ZIKV are central in mediating the ensuing neuropathogenesis in affected fetuses and infants.7,13–21 It has also been observed that pregnant females exhibit prolonged viremia, possibly attributable to ongoing ZIKV replication in the placenta or fetal compartment where the virus is ineffectively controlled.14,22–25

Epidemiologically, data from the United States Center for Disease Control (CDC) indicate that approximately 5% of pregnancies with possible maternal ZIKV infection result in pregnancy loss or birth defects;26,27 this includes returned travelers in the US and those residing in US territories such as Puerto Rico, where dengue has been endemic prior to the introduction of ZIKV. Infection in the first trimester carries aclear risk for CZS;28,29 however, infection in all three trimesters may still lead to adverse fetal outcomes (AFO).30 Moreover, symptoms reported by pregnant women are not reliable in predicting AFO since asymptomatic ZIKV infection in pregnancy can cause CZS.26,27,31 It is unknown why only a portion of ZIKV-exposed infants become infected, or why a portion of congenital ZIKV infections progress to CZS. For example, in French Guiana, a prospective study of pregnant women with confirmed ZIKV infections (by RT-PCR or IgM) documented maternal-fetal ZIKV transmission in 26% (76/291) of fetuses/infants with



complete data. Notably, 34/76 (45%) children had no signs of CZS or AFO. If children for whom only the placenta tested positive were removed from analysis, transmission occurred in 18%, but this eliminated a number of children from consideration who had severe manifestations at birth.32 The risk of infection did not differ by trimester of exposure.32 This work is consistent with high rates of congenital ZIKV infection that have been observed in studies of non-human primates as well.23 Additionally, there is concern that maternal ZIKV infection during pregnancy may still negatively impact the health of uninfected infants.33 The precise rates and risk factors for each of these events remains unclear. Hypotheses that viral genetics, host genetic/epigenetic factors,34,35 or maternal antibodies (Ab) elicited by prior flavivirus infection may be contributing factors require further investigation.

Reliable diagnostics are a cornerstone for most public health activities in response to the recent ZIKV epidemic as well as being central to the clinical care of pregnant women and their offspring at risk for ZIKV infection. The few years since ZIKV emerged in Latin America have been marked by rapid scientific discovery; however, substantial challenges remain. Diagnostic development has benefited from experience with other flaviviruses and unprecedented technologic advance. Several new diagnostic tests show promising performance in early studies, but issues such as specificity and standardization require further investigation before the field arrives at gold standards. Deployability is an additional consideration, particularly as large populations in resource limited settings are affected by Zika and related infections, and it is important that those populations fully benefit from advances in the field. In this article, we review general clinical virology of ZIKV to illustrate the need for diagnostic testing, approaches for arboviral testing along with their applications and shortcoming in the context of congenital ZIKV infection, and prospects for robust diagnostics that could be implemented by clinicians in diverse settings.


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Framework for diagnosing congenital ZIKV infection. Diagnosis of common congenital infections Diagnostic testing for congenital infectious diseases, particularly those associated with congenital anomalies and referred to as TORCH (Toxoplasmosis/Treponema, Other, Rubella, CMV, Herpes) infections, remains challenging.36 It is important to establish the diagnosis of potential congenital infections for clinical management of the pregnancy as well as public health reporting. Non-infectious causes of a fetal anomaly may need to be considered if TORCH testing is negative. Treatment of maternal HIV clearly prevents maternal-to-child transmission; however, maternal treatment of toxoplasmosis, rubella, and CMV has not consistently decreased transmission.37 Serologic testing of the newborn longitudinally can clarify the diagnosis of a congenital infection in some case, but this has the major disadvantage of prolonged diagnostic delays.36 The best approach relies on appropriate risk stratification based on maternal exposures, clinical findings, screening laboratory tests, vaccination history, and typically diagnostic testing of both mom and baby.38,39 A variety of molecular and serologic tests, which vary by infectious agent under consideration, are used to test the mother and the newborn. In utero testing of amniotic fluid or fetal blood may also be considered, for instance, in the setting of suspected congenital cytomegalovirus (CMV) infection. Prenatal imaging may be suggestive of congenital infection, but never obviates the need for laboratory testing and confirmation.

Previous experience with congenital infections may provide insight into the diagnostic strategies employed in CZS. CMV is the most frequently cited pathogen in the differential diagnosis for CZS, as it may cause some of the structural changes observed in severe CZS as well as intracranial calcifications.40,41 Prenatal ultrasound may provide important diagnostic information, but abnormalities are only detected in ~1/3 of fetuses infected by CMV during the first trimester.42,43 The diagnosis of fetal CMV infection is pursued when there is clinical suspicion for congenital infection and primary CMV infection is confirmed



in the mother. Recent maternal infection is indicated by the presence of CMV-specific IgM and low avidity IgG antibodies. IgM+ in isolation is not sufficient to establish primary infection, as there are frequent false positives with the assay and IgM may persist for many months following CMV infection. Persistence of low avidity IgG may also be prolonged. Conceptually, avidity increases over time in the presence of ongoing immune stimulation by CMV antigen (Ag), but the cut-offs defining high and low avidity are ambiguous. Finally, seroconversion for CMV during the pregnancy would provide sound evidence for primary CMV infection, but specimens from earlier in the pregnancy are often not available for testing as routine screening of maternal status for CMV is not recommended for many reasons.39 In the context of maternal primary CMV infection, PCR from a fetal specimen is diagnostic of congenital CMV infection with high specificity (97-100%) and good sensitivity after 21 weeks gestation and 6-7 weeks after maternal infection.39 A single IgM+ test from a fetal or neonatal specimen is more reliable in diagnosing congenital rubella and toxoplasma infection compared to other TORCH pathogens.38

Principles for diagnosing arboviral infections Diagnosis of arboviral infections generally follows two non-mutually exclusive approaches: 1) virologic and 2) serologic.44,45 The former includes direct evidence of viral infection through isolation of live virus from clinical specimens, detection of viral protein Ag such as nonstructural protein-1 (NS1), or the detection of viral RNA using a nucleic acid amplification test (NAAT, of which reverse transcription-polymerase chain reaction (RT-PCR) is the most common). Serologic approaches depend on testing a subject’s blood for Abs that were elicited by recent or remote virus infection. Envelope protein (E) is a major target of antiflavivirus Abs, but responses are also directed to nonstructural proteins such as NS1.44,46,47 Two types of Abs may be detected. IgM is produced within the first 5-7 days of illness and is maintained at detectable levels for week to a few months in most cases.48 IgG appears days to even weeks later, depends on responding B cells undergoing class switch recombination (to change from IgM to IgG production), and is


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present for years after infection due to ongoing production by long-lived plasma cells.49 In addition to measuring precise Ab species, serologic assays may also measure functional properties related to a specific virus or viral Ag.50 The most notable of such assays for flaviviruses is plaque reduction neutralization testing (PRNT), which measures the capacity of Ab in serum to block infection of susceptible target cells in tissue culture.51

In general, NAAT testing is highly specific and preferred for acute infections. Sensitivity may be limited by presentation to medical care late in the course of acute illness. NAAT testing is rarely used to identify viremic individuals without symptoms in high transmission settings52 and is implemented under FDA guidance for screening blood products; however, this is generally not an effective approach for population surveillance or clinical screening of asymptomatic individuals.53 Serology testing provides a much broader diagnostic window, as IgG Ab responses are maintained for years; however, it can be days to more than a week for Ab to reach detectable levels following infection.44 Demonstration of seroconversion by testing an acute and convalescent sample can be diagnostic, but obtaining a second specimen 2-4 weeks after the first is often not practical and of no utility in clinical decision making at the time of presentation. Crossreactivity among Ab produced by related flaviviruses is also a notorious confounder of serologic testing.54

Challenges and current practice for diagnosing congenital Zika infection Several of the challenges to diagnosing congenial ZIKV infection mirror the longstanding limitations of testing for other TORCH pathogens. Confirmed ZIKV infection of a woman during pregnancy constitutes the state of ZIKV exposure from the standpoint of the fetus. Exposure may or may not result in congenital infection. For this discussion, it is assumed that maternal infection and fetal exposure are necessary for congenital infection to occur; however, even this assumption carries the caveat that ZIKV may theoretically be transmitted sexually to a pregnant woman and breech the placental barrier in an



ascending fashion without first causing systemic maternal infection and viremia.9 An estimated 50-80% of ZIKV infections are inapparent,55,56 which has a significant impact on screening algorithms and the interpretation of diagnostic test results, as subclinical infection carries the same risk for transmission as acute, symptomatic disease.26,57 For symptomatic ZIKV cases, a confirmed diagnosis requires laboratory testing to support clinical evaluation.58 Compared to DENV, symptoms such as rash (particularly an early, potentially pruritic rash) and conjunctivitis seem to be more prominent components of symptomatic ZIKV infections, which less commonly causes perturbations in hematologic lab tests.59–61 Nevertheless, ZIKV ultimately causes a non-specific acute illness that cannot be clinically distinguished from many alternate etiologies.62 The performance of case definitions may be positively skewed during an outbreak setting, but that should not distract from the critical role of reliable diagnostic tests in all aspects of clinical care and public health activities.

The concepts underlying both virologic and serologic diagnostic approaches to arboviral infections are applicable to diagnosing ZIKV infection in a fetus or newborn with the exception that maternal IgG is actively transported into fetal circulation – this has been specifically studied for the related flavivirus dengue63–65 – so assays that detect IgG levels or activity will portray the mother’s immune status as opposed to that of the baby. Of note, much of the epidemiologic data during the recent ZIKV epidemic include mothers with evidence of possible ZIKV infection. Diagnostic uncertainty surrounding the mother’s ZIKV infection status will be amplified in testing the fetuses or newborns of those mothers. Ambiguous test results typically occur when serologic testing fails to distinguish between recent ZIKV vs DENV infection due to cross-reactive Ab elicited by both infections.4,66 Even though neutralization testing is a more specific assay for defining flavivirus infections,51,67 titers may not always be discriminatory, as observed in the serologic assessment of the 2007 ZIKV outbreak in Micronesia.68 Careful analysis of relative neutralization titers to DENV and ZIKV within a sample could resolve many cases that arrive at the


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non-definitive label of “flavivirus infection,”69,70 but this is not easily translatable to large-scale clinical testing, and the severe consequences of misidentifying a ZIKV+ case favor conservative cut offs.

Current CDC guidance recommends testing for congenital ZIKV infection only for women with a symptomatic illness consistent with ZIKV. Testing of asymptomatic pregnant women in the absence of ongoing potential exposure to ZIKV is discouraged, but can be offered on a case-by-case basis. Women with ongoing risk for ZIKV infection should be offered testing 3 times during pregnancy with a NAAT. If maternal testing is not negative or fetal imaging is consistent with ZIKV-associated anomalies, additional diagnostic testing is recommended.71

Infants born to mothers with positive ZIKV testing during their pregnancy and for those with congenital abnormalities consistent with CZS regardless of maternal ZIKV testing should undergo intensive evaluation.4 The diagnosis of congenital ZIKV infection is confirmed by the detection of ZIKV RNA in the neonate. Multiple specimen types are often tested by NAAT including blood and potentially CSF, though CSF results have frequently been negative,72 and infants may not have detectable RNA in serum/whole blood, urine, or saliva.32,73–75 ZIKV RNA detection in amniotic fluid is transient and molecular results may be discrepant between amniotic fluid and placental tissue.76 A non-negative IgM test in the infant defines probably congenital ZIKV infection. PRNT is not obligatory, but if performed, a result of ZIKV PRNT90 < 10 would identify a false positive IgM (again, in cases where NAAT is negative), and the case is unlikely to represent a congenital Zika infection.4. Placental tissue can also be submitted for immunohistochemistry and NAAT testing, with positive results confirming maternal infection but not congenital ZIKV infection.75 Placental ZIKV combined with detection of ZIKV-associated birth defects is sufficient to diagnose CZS.71 Cord blood testing is not routinely recommended for testing of congenital ZIKV infections because of potential for false positive and false negative results.77 Limitations to cord blood testing have also been



described for West Nile virus.78 Nevertheless, cord blood testing is an active area of investigation due to several advantages for clinical and surveillance testing. In contradistinction to typical diagnostic development, for congenital infection, specificity and timing of infection are less of a concern in congenital infections as no prior infection or exposure is relevant. This caveat fades if testing is delayed and postnatal infections with ZIKV or pathogens such as DENV become a possibility, which may confuse the interpretation of ZIKV test results.

Additional recommended follow-up for infants with potential congenital ZIKV infection includes developmental screening, follow-up imaging by ultrasound and/or MRI, and hearing and ophthalmological evaluation.74 Accurate laboratory tests that more confidently confirm and exclude ZIKV infection in those fetuses/neonates at risk for CZS are needed to improve both clinical care and lower cost and healthcare utilization. Currently, low rates of ZIKV testing and follow-up for infants with ZIKV-associated birth defects or born to mothers with confirmed ZIKV infection26,27 represents a concerning logistical barrier to fully evaluating and implementing effective diagnostics for congenital ZIKV infection. Preconception counseling represents an additional clinical conundrum in the setting of imperfect diagnostics. In fact, one remarkable feature of the Zika epidemic is that several health organizations and countries recommended women and couples to postpone pregnancy for up to two year.79 There are not data available that allow systematic delineation of risk for congenital or sexual transmission, thus, no approved test can completely rule out the possibility that ZIKV will complicate conception after a potential ZIKV exposure. Rather, providers should engage patients in shared decision-making discussions concerning the risks and benefits to conception and not offer diagnostic testing for this purpose.71,80,81

Progress in ZIKV diagnostic development


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A number of diagnostic methods for ZIKV have been developed, with a rapid expansion of available tests following the introduction of ZIKV into the Western Hemisphere.45,82 Current testing modalities and specimen types are summarized in Figure 1. All current methods have been developed and evaluated for the diagnosis of ZIKV infection, and in particular, for the diagnosis of symptomatic cases. Assays have not been specifically developed or evaluated for the detection or prognosis of congenital ZIKV infection, though certain results have been studied for these outcomes. Early on in the epidemic, a complicated regulatory framework developed in the United States surrounding the use of ZIKV diagnostic tests. This was likely motivated initially by the need to limit inappropriate testing in the setting of limited availability of validated diagnostics. However, this may have also resulted in underestimates of ZIKV incidence among populations at risk for infection.83

Virologic methods Viral isolation: Culture-based methods for ZIKV detection and other methods for virus propagation (mosquito or mouse inoculation) are generally not used for clinical diagnosis.45 ZIKV can be cultured in several cell lines, such as Vero cells or the C6/36 Aedes albopictus mosquito cell line. However, these methods may not be available in many clinical laboratories, require weeks to produce a result, and are less sensitive than other techniques. Although the isolation of ZIKV may be important for research purposes and strain characterization, this does not provide additional diagnostic information in the setting of potential congenital infection.

NS1 Ag: The detection of flavivirus NS1 Ag, which is a viral encoded protein that is secreted in the mammalian host bloodstream by virus-infected cells, is a virologic method for diagnosing acute ZIKV infection. The earliest evidence that NS1-based assays may perform with acceptable specificity came in a retrospective analysis of acute serum specimens from RT-PCR-confirmed ZIKV cases in French Guyana,



which were tested by two DENV NS1 Ag tests. One or both assays were negative in 65 of 65 ZIKV PCR+ samples tested.84 A set of rapid NS1 assays has been developed for the detection ZIKV and the four DENV serotypes. In the initial evaluation, the ZIKV NS1 assay had a sensitivity and specificity of 81% and 86%, respectively. ZIKV NS1 concentrations are ~10-fold lower than concentrations in acute dengue, which may improve the apparent specificity of DENV NS1 assays but also increases volume requirements for optimal ZIKV NS1 detection.85 Similar to those used for DENV,86 multi-modal kit-based assays that detect a combination of Ag and Ab (ex, ZIKV NS1 Ag, and IgM and IgG Ab to ZIKV) will likely soon be available, but will require field validation.

NAAT: A large number of NAATs have been developed for ZIKV detection, with a rapid expansion in the number of available tests since 2015.45,87–100 No assays have been cleared by the US Food and Drug Administration (FDA) for the detection of ZIKV. Rather, 14 assays have been granted Emergency Use Authorization (EUA) following the decision on 26 February 2016 that there was “significant potential for a public health emergency” from ZIKV in the Americas.101,102 Notably, if the EUA declaration is terminated, any EUAs issued under that declaration will no longer remain in effect. A full accounting and description of available NAATs is beyond the scope of this review, but there are important characteristics, advantages and disadvantages to note.

RT-PCR results are generally thought of as highly accurate and reliable. However, results from an external quality assessment of ZIKV molecular detection in 2017 highlight the complexity of this diagnostic modality. This evaluation found that only 20/50 (40%) participating labs (and 28/85 (32.9%) results from individual assays) correctly identified all samples in the panel.103 Results indicated that all protocols were variable, without a clear advantage to commercial over laboratory-developed tests. With few notable exceptions, published figures for NAAT analytical sensitivity are similar.90,91,94,95,104 The Aptima Zika Virus


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Assay, which is a clear outlier and based on transcription mediated amplification, has a reported limit of detection of 11.5-17.9 copies/mL of specimen.105 It remains unclear if such sensitive analytical detection will translate into a significant increase in clinical sensitivity for acute infection. NAATs with EUA have been evaluated using a panel of FDA Reference Material, which includes two Asian-lineage strains of ZIKV and a dilution series to measure of assay sensitivity. Overall, assay performance has not been well studied for contemporary African-lineage strains,100 though in the aforementioned quality assessment, no lineagespecific differences were observed.103

Instrumentation is a key consideration for NAAT testing. RT-PCR and real-time RT-PCR (rRT-PCR) account for the majority of published assays to date. Conventional methods, which require the use of gel electrophoresis for amplicon detection, have largely been replaced by real-time methods that utilize either probe-based detection chemistry90,91,94 or a DNA intercalating agent.106,107 Real-time assays, which include a number of laboratory-developed methods, can often be optimized for use on a variety of instruments, allowing for the adaptation of published methods to existing laboratory infrastructure. Of methods that have received EUA, the Altona RealStar Zika Virus RT-PCR Kit has been evaluated on the largest number of instruments (7).94 However, other methods, such as the Aptima Zika Virus Assay105 or Abbot RealTime Zika, require the use of a specific, proprietary instrument or may only be performed at specific laboratories (Quest, ARUP).

A number of NAATs have been specifically designed for

implementation in resource limited settings or small clinics that may be removed from sophisticated laboratory facilities. These include isothermal methods, such as reverse transcription-recombinase polymerase amplification (RT-RPA)89,96 and RT-loop mediated amplification (RT-LAMP),87,88,93,99 and novel designs that utilize toehold switches and/or CRISPR for product detection.98 Assay performance times are generally shorter for isothermal methods, which can produce a result in as little as 15 minutes. 87–89,93,99 However, turn-around-time may be limited by the need for nucleic acid extraction. Additional touted



benefits to these methods have yet to be fully realized, as they require specific kits for performance, which may be more expensive and difficult to obtain than those for rRT-PCR, or they have only demonstrated proof-of-concept performance and require further clinical evaluation.

All laboratory-developed NAATs require RNA extraction for optimal performance, although direct sample testing has been reported for certain isothermal methods.89,93,108 The need for extraction is significant as it increases test complexity, places additional demands on laboratory infrastructure, and accounts for 6075% of the per-test cost for rRT-PCR. This “hidden” cost is rarely discussed during assay evaluations. Lowcomplexity, sample-to-answer platforms are available for ZIKV testing; however, these require specialized instruments and reagents, which raises cost and limits accessibility even for high-resource laboratories.105 To truly expand capacity to perform NAAT, the need for simple, rapid, and inexpensive RNA extraction must be addressed.96,99

Serologic methods The serologic diagnosis of flaviviruses such as ZIKV can be a complicated matter, particularly in areas where multiple related viruses co-circulate.66,109 While extensive work in serodiagnostic development for other flaviviruses, particularly DENV, provides crucial precedent for the serologic diagnosis of ZIKV, an exhaustive review of that field is not possible here.44,50,67,110,111 As discussed above, IgM and neutralization testing have been the only assays applied to diagnosis of congenital Zika by the CDC; however, other types of assays are considered here as these may be incorporated into the diagnostic approaches for maternal and/or congenital ZIKV in the future.

IgM: IgM testing by MAC-ELISA48,112,113 has featured prominently in clinical diagnostic algorithms for pregnant women, newborns, and suspected cases in general – however, detection of ZIKV-specific IgM


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was already plagued by cross-reactivity with DENV, and has since fallen out of favor due to emerging data on the persistence of this response beyond the first few months of convalescence, making it more difficult to attribute the test result to a recent infectious event.71 However, in the context of diagnosing congenital infection, the IgM assay retains value in identifying probable in utero ZIKV infections.77 Because IgM does not cross the placenta, IgM detected in a neonate (blood or CSF) is of fetal origin and consistent with congenital infection. Moreover, cross-reactivity is much less relevant in this setting since it is extremely unlikely that another flavivirus is the cause of the positive IgM result.

Independent researchers have compared the performance of the CDC-MAC ELISA and commercial assays. One analysis in a well-defined specimen panel found that this assay had a sensitivity of 70.1% and a specificity of 82.8% (both lower than these same parameters of in in-house MAC-ELISA).114 Another found the InBios IgM ELISA to display sensitivity up to 100%, but the specificity in DENV-immune specimens was poor.115 The specificity of IgM may be improved by using NS1 Ag rather than inactivated virus or virus-like particles.116 One issue with IgM-based assays is that IgM responses can be blunted or absent in secondary flavivirus infection, and this phenomenon may account for the low sensitivity of ZIKV IgM tests reported in some studies that included a highly DENV-immune population.117 Additionally, some evaluations have been performed using specimens from European or Canadian residents with flavivirus vaccination or travel-related infections.118,119 While these studies are sound, their generalizability to populations in flavivirus-endemic populations is likely limited.

NS1 serology: Studies of serologic responses to NS1 showed that DENV-immune sera exhibited low frequency of cross-reactivity to ZIKV NS1 and that ZIKV infection elicited a ZIKV NS1-specific Ab response (minimal cross-reactive binding to DENV NS1), though with delayed kinetics compared to anti-E Ab.120 Two notable studies – one a direct Ag binding ELISA121 and one a competition assay called NS1 BOB122 – have



applied this principle and tested their assays in relatively large panels of sera to define sensitivity (among known ZIKV cases) and specificity (using a combination of DENV- or other flavivirus-immune sera and healthy controls). This is an important advance in the field as these assays can be performed in lab without sophisticated analytic equipment or biosafety capacity for live virus culture. The turnaround time is also favorable at several hours to 2 days.

Neutralization testing: Because of the time, resources, and safety requirements for neutralization testing, perhaps the best improvement would be to remove this assay from diagnostic algorithms. Optimizations of Ab-binding assays could obviate the need for neutralization assays and make reliable ZIKV diagnostic tools more widely available.116,123,124 However, neutralization assays will likely continue to be used as a confirmatory test.

Work at CDC has improved specificity in neutralization tests assessing secondary flavivirus cases (ZIKV infection occurs in a DENV-immune host) by depleting IgG (and only considering the IgM component of neutralization activity). This appears to be most effective in the 3-12 week time period, but may have utility at even later time periods as IgM has been observed to persist for many months in some individuals.125 The other principle improvements in neutralization testing involve using VLPs or reporter systems that do not depend on working with infectious, wild type ZIKV and automating the analysis workflow to make high-throughput assays even more efficient.126–129

Multiplex testing Given the overlap in clinical presentation and co-circulation of ZIKV, DENV and CHIKV, a number of multiplex molecular tests have been developed for the detection and differentiation of these pathogens in a single reaction.90,91,130–132 The clinical utility of these methods has been demonstrated in endemic


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settings,62,133 and the potential benefit in the setting of congenital infection was shown in a recent report, in which a high proportion of children born to mother in the ZIKV-negative group had CHIKV.30 In a large evaluation, two multiplex molecular assays demonstrated similar performance for ZIKV detection,134 and both have demonstrated similar sensitivity and specificity compared to monoplex assays.90,91 For similar reasons, flavivirus Ag has been included in multiplex serologic assays seeking to decipher the etiologic agent responsible for syndromic presentations such as acute febrile illness in the tropics135 or tick-borne infection.136 A new IgM assay points to the value of multiplexing in addition to automation to maximize testing efficiency and minimize turnaround time.137 Another approach focused completely on detection of IgG binding to Ag from a diverse set of pathogens with the goal of assessing control intervention effectiveness via iterative IgG-based serologic surveillance.138 The New York State Health Department developed a microsphere immunoassay for ZIKV in response to the high testing burden and diagnostic inaccuracy of existing assays. This work revealed that ZIKV E Ag were particularly useful for achieving high sensitivity, while Ag from nonstructural proteins were required to improve assay specificity and performance, all with an turnaround time of 99% decrease in confirmed cases in the US and US Territories). While this is a relief in the short term, ZIKV has become endemic in many locations throughout the world and cases of CZS may continue to occur sporadically,163 or additional epidemics may occur in areas where


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there is a susceptible population and competent mosquito vectors. A refined understanding of congenital ZIKV infection and the pathophysiology of CZS is needed to accurately assess risks to the fetus and target prevention efforts.

Gauging the risk of exposure to the fetus begins with improved methods for detecting and timing maternal infections that may be asymptomatic or even precede the pregnancy. For mothers who present to care with a positive anti-ZIKV IgM, NAATs performed on whole blood – where ZIKV RNA remains detectable for weeks to months164 – and avidity testing of specific anti-ZIKV IgG may provide measures to determine the timing of infection in relation to the pregnancy and fetal development. Avidity testing of antibodies to CMV and Toxoplasma has been utilized in pregnancy, but this technique has not been widely studied in arboviral infections or other acute viral illnesses.140165,166 Although improvements to available diagnostic methods may expand access to arbovirus testing, incremental changes to existing technologies are not expected to have a dramatic impact on the detection or management of CZS cases. The risk of CZS does not correlate with maternal disease severity, serum viral load, the duration of viremia, or pre-existing flavivirus antibodies.57,167 Therefore, new approaches are needed to identify biomarkers, genes or epigenetic changes, and/or transcriptomic signatures among pregnant women at high risk for ZIKV transmission and in non-invasive/minimally-invasive specimens from fetuses/infants with CZS.

In order to transform the diagnostic landscape, determinants of transmission to the fetus and the protective role of the placenta need to be better characterized. This may prove particularly vexing, however, as there may not be overt placental pathology in all ZIKV cases,168 even in the setting of CZS/AFO. In a study of twins born during the ZIKV epidemic in Brazil, 6/7 dizygotic pairs were discordant for CZS (both monozygotic pairs were concordant)es- Neural progenitor cells from the twins demonstrated significant difference in gene expression. When infected with ZIKV in vitro, neural progenitor cells from



the twin with CZS permitted higher ZIKV viral loads and had decreased cell growth compared to cells of the unaffected sibling.35 This data is similar to findings from a study of ZIKV transmission in Rag1 deficient mice, which demonstrated that not all pups from an infected dam were infected with ZIKV despite ZIKV RNA detection in all placentas.34 However, animal models have provided conflicting data in this regard. In a macaque model, ZIKV was transmitted to 4/4 fetuses despite maternal infection occurring at different time-points during pregnancy.23 Consistent with findings from human autopsy cases, no specimen type was positive for ZIKV RNA in all fetuses.73,75,169 It is conceivable, then, that human fetal infection is very common following maternal placental infection, but expression of the CZS phenotype may result from a stochastic process or genetic/epigenetic determinants in the fetus.23,34,35

A number of strategies have demonstrated protective efficacy against the development of vertical transmission of ZIKV in animal models, including passive immunization with neutralizing antibodies,170 treatment with hydroxychloroquine (an inhibitor of autophagic activity in human trophoblasts),171 and vaccination.172,173 Placental and fetal infection is markedly decreased in vaccine trials, though not completely prevented. Currently, it is unknown whether immunity following natural infection (or vaccination) will prevent future ZIKV infections and the potential for vertical transmission in the pregnant human host.174 Additional layers of complexity include possibilities for sexual transmission and the emergence of a different lineage into a previously-exposed population (such as the African lineage in the Americas).175 Phase III efficacy trials of ZIKV vaccines have not been feasible as a result of the dramatic decrease in ZIKV incidence after 2016. As such, vaccine approval may not occur in the near future and the development of alternative prevention strategies is warranted though may be prohibitively expensive to garner interest from the biotechnology sector. Questions remain regarding the optimal incorporation of available diagnostic methods into testing algorithms during pregnancy and the early identification of symptomatic infections among exposed fetuses. In addition to diagnostic results, could prognostic


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information be gleaned from assays that are already available and those under development? This could significantly improve management of ZIKV-exposed infants. Resolution of these questions remains critical, as the prevention of CZS will likely require a combination of strategies, including vaccination and the continued surveillance of pregnant women for ZIKV exposures that pose a risk to the developing child.

Author Contributions MHC and JJW searched the literature, drafted and edited the manuscript. Conflict of Interest The authors report no conflicts of interest. Acknowledgements None Abbreviations CZS

congenital Zika syndrome

ZIKV

Zika virus

CDC

Center for Disease Control

AFO

adverse fetal outcome

Ab

antibody

CMV

cytomegalovirus

Ag

antigen

NS1

nonstructural protein-1

NAAT

nucleic acid amplification test

RT-PCR

reverse transcription-polymerase chain reaction



E

envelope protein

PRNT

plaque reduction neutralization testing

FDA

Food and Drug Administration

EUA

emergency use authorization

rRT-PCR

real-time reverse transcription-polymerase chain reaction

RT-RPA

reverse transcription-recombinase polymerase amplification

RT-LAMP

reverse transcription-loop mediated amplification

CSF

cerebrospinal fluid


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Key Terms Deployability: Here, we use this term to describe an assay that has essential qualities and characteristics to move readily from validation to implementation. Thus, deployability takes into account factors such as cost and production that would determine if an assay could be used on large scale, but it also considers whether an assay could be used in the context in which it is needed. For example, a scalable assay with high performance characteristics and low cost would still not be optimal if it required sophisticated analytic equipment or greater than basic biosafety containment laboratory space, neither of which would be widely available in resource limited settings. Antigen (Ag): A molecule, substance, or organism that is specifically recognized by the immune system. Here, Ag usually refers to viral proteins or unique three-dimensional structural configurations that specifically interact with antibodies (Ab) produced by B cells, but Ag also can refer to pathogen-derived peptides that are displayed on the surface of host cells in the context of major histocompatibility complex (MHC) molecules and specifically recognized by T cells via interaction between their T cell receptors and the MHC-Ag complex. Virologic diagnostic: A laboratory assay that diagnosis a virus infection by detecting components of the viral pathogen during acute infection. The components could be infectious virus (viral culture), viral protein (nonstructural protein-1 (NS1) antigen) or specific fragments of the genome (RNA for Zika). Serologic diagnostic: A laboratory assay that confirms or supports recent or remote infection by detection of antibodies that react with Ag from the pathogen of interest. Usually serologic assays are performed on a serum specimen, but we use the term broadly to encompass any such assay that detect antibody responses in other body fluids including plasma, saliva, eluate from dried whole blood spots, cerebrospinal fluid, and genital and mucosal secretions.


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Nucleic acid amplification test (NAAT): A collection of assays that specifically amplify and detect unique sequences of genetic material of pathogens (RNA or DNA), reverse transcriptase – polymerase chain reaction (RT-PCR) being the most common NAAT used for Zika detection. Congenital Zika syndrome (CZS): A distinct patter of birth defects observed in fetuses and infants with congenital Zika infection, which is characterized by one or more of the following: microcephaly, decreased brain tissue, subcortical calcifications, retinal injury, contractures including clubfoot and arthrogryposis, and hypertonia.



Figure 1. Diagnostic modalities for maternal-fetal ZIKV transmission including non-invasive methods (blue boxes) and those requiring invasive sampling (red boxes). Specimen-method combinations for which assays have EUA are indicated (*). Only a single assay has obtained EUA for amniotic fluid (the CDC Trioplex assat, †). No anti-ZIKV IgG assays have received EUA. Figure adapted (authors superimposed diagnostic information in boxes) with permission of March of Dimes, © 2019 March of Dimes, a not-forprofit, section 501c(3).


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Detecting vertical Zika transmission: emerging diagnostic approaches

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