**5. Clinical context**

*Biochemical Testing - Clinical correlation and Diagnosis*

ranges from 5 to 6 months [64, 65].

specificity to ZIKV epitopes [1].

risk for neuroinvasive disease [71].

**4.2 Plaque reduction neutralization test (PRNT)**

Zika viruses cocirculate besides several proposed ELISA tests, individuals previously exposed to other flaviviruses are not prevented to be misclassified. Another complication for patients experiencing a second flavivirus infection is a shorten IgM

This presents a diagnostic dilemma for patients living in regions in which flaviviruses are endemic, and reliable diagnostic tools that are able to discriminate between primary and secondary ZIKV or DENV infections are urgently needed [1, 62, 63]. Although the precise period of onset and duration IgM class antibodies in response to ZIKV is not well defined, data known by other flaviviruses suggest that IgM for ZIKV should start to be detectable 7 days following infection, in the majority of symptomatic patients (**Figure 2**). However, it has been recently reported in the literature that seroconversion may occur earlier for one-third of patients [1, 42]. While anti-ZIKV antibody titers decrease, IgM to ZIKV has a window of detection more than 12 weeks following infection for over 80% of individuals [42]. According to the modeling studies with other flaviviruses, including West Nile virus (WNV) and dengue (DENV), this serum persistence is consistent with the antibody responses following infection, suggesting that the mean time to IgM seronegativity

Apart from initial antibody response, IgG neutralizing antibodies (NA) normally develop after IgM response and should persist for years to decades following primary infection. It is believed that NA to ZIKV directed to a key epitope after infection should be highly specific in patients without prior exposure to flaviviruses; however, NA specificity, in the setting of past exposure to a closely related flavivirus, such as DENV, once shares diverse antigenic determinants, decrease

Another tool that is being proposed in the context of outbreaks is rapid point of care (POC) tests which are based on the IgM/IgG immunochromatography assay. They should be used carefully and are normally interpreted as qualitative screening tests, and further serological investigations are needed. When they present a positive result, the patient samples should be sent for plaque reduction neutralization test (PRNT), or in the case of pregnant women, RT-qPCR is recommended [66, 67]. Special attention should be given to the risk group composed of newborns, where the immunoenzymatic serological test is extremely important to define congenital infection. ZIKV IgG antibody detection in a newborn may reflect maternal infection due to the passive transfer of this class of immunoglobulins. Based on other congenital infections, uninfected newborns display IgG levels that decline gradually in the first months of life, and this corresponds to the time of elimination of the antibody transmitted by the mother [68]. For this reason, tests to detect IgM are commonly used to confirm infection in the child, and in the context of flavivirus infection, only ZIKV was demonstrated to cross the placental and fetal immunological barriers [69, 70]. ZIKV IgM positivity in CSF, after congenital infection, was described as a reliable tool to discriminate fetal infection, and it characterized high

Until now, the unique reference standard for NA detection related to flaviviruses

remains the plaque reduction neutralization test (PRNT). Although technically cumbersome, with several challenges: (1) long turnaround time of days to weeks; (2) requirement for live viral cultures; (3) technical-experience dependence; and (4) high degree of subjectivity, it offers the highest achievable level of specificity. This test initiates with a serial dilution of patient serum that is preincubated with live ZIKV or other closely related viruses (e.g., DENV), followed by deposition

antibody response that could misdiagnose the time for acute infection [1].

**90**

Zika virus (ZIKV) infection is usually an asymptomatic or a mild symptomatic disease in adults, with maculopapular and pruritic rash, fever, conjunctivitis, joint pain, headache, and muscle pain [74]. However, infection during pregnancy may be transmitted to the fetus and causes severe systemic fetuses' malformations, comprising the congenital Zika syndrome (CZS).

The clinical features of CZS have been described since 2015 Zika's outbreak in Brazil. They can be divided into structural and functional components [75]. The structural components are cranial morphology, brain, and ocular anomalies, as well as congenital contractures [75]. The functional component is related to neurologic sequelae [75]. Therefore, CZS may consist of (A) cranial morphology: severe microcephaly, overlapping cranial sutures, prominent occipital bone, redundant scalp skin, and neurologic impairment; (B) brain anomalies: thin cerebral cortices, abnormal gyral patterns, increased fluid spaces, subcortical calcifications, corpus callosum abnormalities, decreased white matter, and cerebellar hypoplasia; (C) ocular anomalies: macular scarring (**Figure 4A**), focal pigmentary retinal mottling (**Figure 4B**), and optic nerve hypoplasia or atrophy (**Figure 4A**); (D) congenital contractures: arthrogryposis and club feet; and (E) neurologic sequelae: marked early hypertonia, symptoms of extrapyramidal involvement, epilepsy, and irritability [75–77]. Early recognition and referral to multidisciplinary care may result in a better outcome for each one of the abnormalities described.

#### **Figure 4.**

*(A) Right eye retinography showing macular (black arrowhead) and peripheral (black arrow) scarring and optic nerve hypoplasia (white arrow); (B) left eye retinography showing focal pigmentary retinal mottling (black arrow).*

The diagnostic approach during prenatal care should be different in an endemic area and in a nonendemic area. Endemic area means residence in or travels to the affected area. In addition, in each of these two scenarios, the diagnosis is divided into symptomatic and asymptomatic pregnant women.

#### **5.1 Endemic area (residence or travel)**

#### *5.1.1 Symptomatic pregnant women*

The ZIKV infection is divided into acute and convalescent phases. The acute phase is within the first 7 days of the symptoms, and the convalescent phase is 2–12 weeks after [78]. As shown in **Figure 5**, during the acute phase, the possible primary tests are the ZIKV RT-qPCR and the Immunoglobulin (Ig) M. The first one can be obtained from serum, saliva, urine, or amniotic fluid. When it is detected in the serum or saliva, it is confined to 5–7 days after onset of symptoms, and when it is detected in urine, it lasts up to 3 weeks after onset of illness. The evidence is lacking on the diagnostic accuracy of RT-qPCR of amniotic fluid and on the optimal time to perform amniocentesis [74]. The IgM is detectable ≥4 days after onset of illness [78]. In the convalescent phase, the primary test is IgM and the second test is plaque reduction neutralization test (PRNT). However, the Zika virus RT-qPCR can also be performed during this phase.

If the primary results are **negative ZIKV RT-qPCR and negative ZIKV IgM** (red flowchart in **Figure 5**), there is no evidence of virus detection, but an intrauterine ultrasound scan (US) should be done. If there are any fetal development abnormalities, serological tests for others congenital infections could be done and the pregnant woman should be referred to a specialized care. The possible congenital infections are toxoplasmosis, rubella, cytomegalovirus (CMV), herpes virus, parvovirus B19, varicella, and syphilis, known by the acronym TORCH's syndrome (Sd).

#### **Figure 5.**

*Flowchart showing initially two clinical scenarios (symptomatic and asymptomatic pregnant women) and the different primary and secondary tests and results for Zika virus diagnosis during pregnancy. NAT: RT-qPCR nucleic acid test. IgM: Immunoglobulin M. US: Intrauterine ultrasound. PRNT: Plaques reduction neutralization test.*

**93**

*ZIKV Diagnostics: Current Scenario and Future Directions*

it should be considered to repeat the US at 28–30 weeks [74].

The possible fetal abnormalities are microcephaly, intracranial calcifications, ocular lesions and calcifications, ventriculomegaly, abnormal sulcation and gyration, abnormal cortical development (lissencephaly), cerebral atrophy, callosal dysgenesis, cerebellar atrophy, brainstem hypoplasia, microphthalmia and arthrogryposis, intrauterine growth restriction, and evidence of placental insufficiency [74]. If there is absence of fetal abnormalities, the pregnant can follow routine antenatal care and

If the primary results are **negative ZIKV RT-qPCR and non-negative ZIKV IgM**, the secondary test PRNT should be performed. It can be done for any other flavivirus that might be found in that geographic area [74]. If ZIKV PRNT ≤10 (white flowchart in **Figure 5**), there is no evidence of Zika virus infection. If ZIKV PRNT ≥10 and dengue virus PRNT ≥10 (blue flowchart in **Figure 5**), there is evidence of a flavivirus infection but no confirmation of a specific virus. Finally, if ZIKV PRNT ≥10 and dengue virus PRNT <10 (green flowchart in **Figure 5**), there is evidence of ZIKV infection. In any of the last two scenarios, the timing of infection cannot be determined. If the primary result is **positive ZIKV RT-qPCR** (green flowchart in **Figure 5**), acute ZIKV infection is confirmed and US should be done. If there is any fetal abnormality, the pregnant woman should be referred to a specialized care, and if there is no abnormality, routine antenatal care should be done, as well as a US follow-up

In endemic area of residence or traveling, the pregnant woman with possible ZIKV exposure should be first tested at initiation of prenatal care and three times during pregnancy [67]. The primary tests should be ZIKV RT-qPCR and ZIKV IgM serology. If available, US should be considered for fetal morphology assessment.

The asymptomatic woman should follow the routine antenatal care. The symptomatic woman should do the primary tests ZIKV RT-qPCR and ZIKV IgM serology.

Additionally, the differential diagnosis is also crucial for the correct assessment and management of the disease. Signs of maternal disease, as well as intrauterine and neonatal findings, are similar in most of the TORCH's congenital infections [75, 80]. Moreover, some genetic syndromes (Sd), such as Aicardi-Goutières Sd, pseudo-TORCH Sd, and mutations in the JAM3, NDE1, and ANKLE2 genes can also

The main clinical findings in maternal TORCH infections include nonspecific signs, like fever, fatigue, malaise, headache, myalgia, arthralgia, lymphadenopathy, conjunctivitis, and making the ability to recognize the specific etiological agent difficult. Moreover, there are typical findings in some of these infections that can lead to the diagnosis. In herpes simplex virus infection, there is also painful genital ulcer, pruritus, and dysuria [80]. In primary syphilis infection, there is a firm, round, and painless chancre on external genitals and vagina, lasting 3–6 weeks [81]. In rubella, parvovirus B19, varicella, and ZIKV, cutaneous rash is an additional important sign [80, 82, 83]. The rash in rubella is maculopapular and usually progresses from the face to the body, fading within 2–4 days [80, 82, 83]. In parvovirus B19, the rash is also maculopapular, persisting until the 6th day and disappearing on the 7–9th day after the first appearance [80, 82, 83]. First, it occurs on the face, sparing nasal, and periorbital zones and second, on the trunk and extremities [83]. In varicella, it

The possible results of the laboratory tests and US are described above.

If available, US for fetal morphology assessment should be considered.

*DOI: http://dx.doi.org/10.5772/intechopen.82373*

every 4 weeks until birth [74, 79].

*5.1.2 Asymptomatic pregnant women*

be in the differential diagnosis [75].

**5.2 Nonendemic area**

*ZIKV Diagnostics: Current Scenario and Future Directions DOI: http://dx.doi.org/10.5772/intechopen.82373*

*Biochemical Testing - Clinical correlation and Diagnosis*

**5.1 Endemic area (residence or travel)**

*5.1.1 Symptomatic pregnant women*

also be performed during this phase.

into symptomatic and asymptomatic pregnant women.

The diagnostic approach during prenatal care should be different in an endemic area and in a nonendemic area. Endemic area means residence in or travels to the affected area. In addition, in each of these two scenarios, the diagnosis is divided

The ZIKV infection is divided into acute and convalescent phases. The acute phase is within the first 7 days of the symptoms, and the convalescent phase is 2–12 weeks after [78]. As shown in **Figure 5**, during the acute phase, the possible primary tests are the ZIKV RT-qPCR and the Immunoglobulin (Ig) M. The first one can be obtained from serum, saliva, urine, or amniotic fluid. When it is detected in the serum or saliva, it is confined to 5–7 days after onset of symptoms, and when it is detected in urine, it lasts up to 3 weeks after onset of illness. The evidence is lacking on the diagnostic accuracy of RT-qPCR of amniotic fluid and on the optimal time to perform amniocentesis [74]. The IgM is detectable ≥4 days after onset of illness [78]. In the convalescent phase, the primary test is IgM and the second test is plaque reduction neutralization test (PRNT). However, the Zika virus RT-qPCR can

If the primary results are **negative ZIKV RT-qPCR and negative ZIKV IgM** (red flowchart in **Figure 5**), there is no evidence of virus detection, but an intrauterine ultrasound scan (US) should be done. If there are any fetal development abnormalities, serological tests for others congenital infections could be done and the pregnant woman should be referred to a specialized care. The possible congenital infections are toxoplasmosis, rubella, cytomegalovirus (CMV), herpes virus, parvovirus B19, varicella, and syphilis, known by the acronym TORCH's syndrome (Sd).

**92**

**Figure 5.**

*neutralization test.*

*Flowchart showing initially two clinical scenarios (symptomatic and asymptomatic pregnant women) and the different primary and secondary tests and results for Zika virus diagnosis during pregnancy. NAT: RT-qPCR nucleic acid test. IgM: Immunoglobulin M. US: Intrauterine ultrasound. PRNT: Plaques reduction*  The possible fetal abnormalities are microcephaly, intracranial calcifications, ocular lesions and calcifications, ventriculomegaly, abnormal sulcation and gyration, abnormal cortical development (lissencephaly), cerebral atrophy, callosal dysgenesis, cerebellar atrophy, brainstem hypoplasia, microphthalmia and arthrogryposis, intrauterine growth restriction, and evidence of placental insufficiency [74]. If there is absence of fetal abnormalities, the pregnant can follow routine antenatal care and it should be considered to repeat the US at 28–30 weeks [74].

If the primary results are **negative ZIKV RT-qPCR and non-negative ZIKV IgM**, the secondary test PRNT should be performed. It can be done for any other flavivirus that might be found in that geographic area [74]. If ZIKV PRNT ≤10 (white flowchart in **Figure 5**), there is no evidence of Zika virus infection. If ZIKV PRNT ≥10 and dengue virus PRNT ≥10 (blue flowchart in **Figure 5**), there is evidence of a flavivirus infection but no confirmation of a specific virus. Finally, if ZIKV PRNT ≥10 and dengue virus PRNT <10 (green flowchart in **Figure 5**), there is evidence of ZIKV infection. In any of the last two scenarios, the timing of infection cannot be determined.

If the primary result is **positive ZIKV RT-qPCR** (green flowchart in **Figure 5**), acute ZIKV infection is confirmed and US should be done. If there is any fetal abnormality, the pregnant woman should be referred to a specialized care, and if there is no abnormality, routine antenatal care should be done, as well as a US follow-up every 4 weeks until birth [74, 79].

#### *5.1.2 Asymptomatic pregnant women*

In endemic area of residence or traveling, the pregnant woman with possible ZIKV exposure should be first tested at initiation of prenatal care and three times during pregnancy [67]. The primary tests should be ZIKV RT-qPCR and ZIKV IgM serology. If available, US should be considered for fetal morphology assessment. The possible results of the laboratory tests and US are described above.

#### **5.2 Nonendemic area**

The asymptomatic woman should follow the routine antenatal care. The symptomatic woman should do the primary tests ZIKV RT-qPCR and ZIKV IgM serology. If available, US for fetal morphology assessment should be considered.

Additionally, the differential diagnosis is also crucial for the correct assessment and management of the disease. Signs of maternal disease, as well as intrauterine and neonatal findings, are similar in most of the TORCH's congenital infections [75, 80]. Moreover, some genetic syndromes (Sd), such as Aicardi-Goutières Sd, pseudo-TORCH Sd, and mutations in the JAM3, NDE1, and ANKLE2 genes can also be in the differential diagnosis [75].

The main clinical findings in maternal TORCH infections include nonspecific signs, like fever, fatigue, malaise, headache, myalgia, arthralgia, lymphadenopathy, conjunctivitis, and making the ability to recognize the specific etiological agent difficult. Moreover, there are typical findings in some of these infections that can lead to the diagnosis. In herpes simplex virus infection, there is also painful genital ulcer, pruritus, and dysuria [80]. In primary syphilis infection, there is a firm, round, and painless chancre on external genitals and vagina, lasting 3–6 weeks [81]. In rubella, parvovirus B19, varicella, and ZIKV, cutaneous rash is an additional important sign [80, 82, 83]. The rash in rubella is maculopapular and usually progresses from the face to the body, fading within 2–4 days [80, 82, 83]. In parvovirus B19, the rash is also maculopapular, persisting until the 6th day and disappearing on the 7–9th day after the first appearance [80, 82, 83]. First, it occurs on the face, sparing nasal, and periorbital zones and second, on the trunk and extremities [83]. In varicella, it

is initially pruritic and maculopapular, following a vesicular phase until crushing occurs, usually about 5 days later [84]. Finally, the rash in ZIKV infection is pruritic, descending, and maculopapular, beginning proximally and spreading to the extremities with resolution occurring within 1–4 days of onset [14, 74].

Nonspecific intrauterine US findings in TORCH infections are intrauterine growth restriction, abnormal fluid imbalance (ascites, hydrops, pericardial effusion, pleural effusion, and oligo-polyhydramnios), hepatosplenomegaly and hepatic calcifications, echogenic bowel, echogenic kidneys, and limb deformities [80, 85]. Cerebral brain calcification, although relatively common in most of these infections, can be an important distinguishing factor due to its location [80, 85]. In congenital toxoplasmosis, the calcifications are diffuse and widely distributed, and in rubella, they are at basal ganglia [80, 85]. In CMV congenital infection, the calcifications are usually punctate and periventricular or cortical [80, 85]. Conversely, in ZIKV, the calcifications are larger and denser, usually appearing at the gray and white matter junction [80, 86].

As well as seen with maternal clinical signs and intrauterine US findings, TORCH congenital infection can also produce a similar neonatal clinical presentation, although the features rarely occur simultaneously [87, 88]. The common clinical features include growth retardation, prematurity, jaundice, anemia, hepatosplenomegaly, microcephaly, cerebral calcifications, chorioretinitis, cataracts, microphthalmia, and others [87, 88]. However, clinical findings are more associated, although not exclusively, with a determined etiological agent. Cardiac lesions and deafness have been described in CMV and rubella congenital infections. Cicatricial skin lesions and limb hypoplasia are seen in varicella virus congenital infection [87]. Petechial or purpuric form exanthema (blueberry muffin spot) is typical of CMV infection [80, 82]. Chorioretinitis, hydrocephalus, and cerebral calcifications comprise the characteristic triad of congenital toxoplasmosis [89].

Finally, clinical signs of CZS can also be found in other TORCH infections. Cranial ZIKV morphology and brain anomalies can occur in congenital CMV infection [75]. Congenital contractures can appear in congenital rubella, varicella, and Coxsackie B infections [75]. Neurologic sequelae are seen in most TORCH infections [75]. Ocular anomalies, such as pigmentary mottling and chorioretinal scars, can manifest differently in each of these congenital infections. In rubella, the pigment mottling is usually diffused compared with the focal pigment mottling seen in Zika virus infection (**Figure 4B**) [77]. Chorioretinal lesions in toxoplasmosis can present with active exudative retinitis or regressed macular or peripapillary retinal scar [77]. In ZIKV congenital infection, chorioretinal lesions are atrophic and colobomatouslike and are found in the macula or retinal periphery (**Figure 4A**) [77]. Optic nerve hypoplasia, commonly seen in ZIKV congenital infection, is seldom seen in rubella, toxoplasmosis, herpes, and CMV congenital infections [77].

#### **6. Conclusions**

Human clinical diagnosis of ZIKV infection in regions where other arboviruses circulate, mainly DENV and CHIKV, has become a daunting task; therefore, laboratory confirmation is crucial for conclusive diagnosis. Detection of virus genome by RT-qPCR is helpful and demonstrated to be a reliable tool; however, the limited window for virus detection, low viral load, and stability restricted the use of these methodologies. Antibody screening is also hampered by cross-reactivity among other flaviviruses.

Due to impact of ZIKV infection, especially for pregnant women, a confident ZIKV serology test is urgently needed, and this will promote a better prenatal

**95**

provided the original work is properly cited.

*ZIKV Diagnostics: Current Scenario and Future Directions*

The authors declare no conflict of interest.

follow-up, especially in endemic areas. Nonetheless, as mentioned above, there are typical findings to aid the correct diagnosis and, thus, the appropriate management

We thank Fernandes Figueira Institute Pediatric Infectious Disease Team for helpful discussions during this work and field expertise from ZIKVIRUSIFF Cohort

This work was partially supported by Fernandes Figueira Institute Research Program (Programa de Incentivo à Pesquisa II), Rio de Janeiro State Research Grant (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro—grant E-26/201.840/2017), National Research Grant (Conselho Nacional de Desenvolvimento Científico e Tecnológico—grant 441098/2016-9), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—

study registered on clinicaltrials.gov with identifier NCT03255369.

*DOI: http://dx.doi.org/10.5772/intechopen.82373*

and counseling of the disease.

**Acknowledgements**

Finance Code 001.

**Author details**

Zilton Vasconcelos1

Daniela Prado Cunha1

Rio de Janeiro, Brazil

**Conflict of interest**

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Institute of Microbiology Paulo de Góes, Federal University of Rio de Janeiro,

, Andrea Zin1

, Luiza Neves1

and

\*, Renata Campos Azevedo2

1 Fernandes Figueira Institute, Fiocruz, Rio de Janeiro, Brazil

\*Address all correspondence to: zvasconcelos@iff.fiocruz.br

follow-up, especially in endemic areas. Nonetheless, as mentioned above, there are typical findings to aid the correct diagnosis and, thus, the appropriate management and counseling of the disease.
