In a report during the Zika outbreak that occurred in the state of Rio de Janeiro, Brazil, based on a large number of suspected and laboratory confirmed cases, the most common signs or symptoms were macular or maculopapular rash (97%), followed by pruritus (79%), prostration (73%), headache (66%), arthralgias (63%), myalgias (61%), nonpurulent conjunctivitis (56%) and lower back pain (51%). Fever, when present, was low grade and short-term. The authors suggest that pruritus, the second most common clinical sign presented by the confirmed cases, should be added to the PAHO case definition [26▪▪]. In another study, Jimenez Corona et al. analysed 93 autochthonous cases of Zika in Mexico. The main clinical features were fever (96.6%), rash (93.3%), nonpurulent conjunctivitis (88.8%), headache (85.4%) and myalgia (84.3%) .
Clinical characteristics and outcome data on ZIKV infection in young children are scarce, but based on limited information, ZIKV infection in children is mild and similar to that in adults [34,41].
The arboviral burden of diseases caused by cocirculation of DENV, CHIKV and ZIKV in the Americas [42,43] can lead to coinfections and has been reported previously [44,45]. The clinical spectrum of manifestations and the overall epidemiological relevance of those coinfections are still unknown and should be accessed.
Fatalities attributed to ZIKV are rare, excluding foetal losses among women infected during pregnancy and newborns with severe congenital ZIKV disease. However, because the current epidemic is rapidly evolving, some deaths related to ZIKV have been reported [17,37,38,46]. In October 2015, a 15-year-old girl previously diagnosed with sickle cell disease died with vaso-occlusion, triggered by inflammation and severe splenic sequestration . Another four deaths were reported in Colombia . Until May 2016, three people died from complications linked to the ZIKV, according to Brazilian health officials . In Puerto Rico, a 70-year-old man died of complications related to severe thrombocytopenia at the end of February 2016 .
The spectrum of the Zika clinical manifestations is increasing as the epidemic is spreading . Clinical manifestations have apparently changed since the large French Polynesian outbreak in 2013–2014 , when severe neurological complications were reported [47▪▪], followed by an increase in severe congenital malformations in the emergence in Brazil in 2015 .
Some pregnant cohorts were already established to study infant outcomes exposed to ZIKV [30▪,67]. Brasil et al.[30▪] published preliminary results of 88 pregnant women. Among the 72 women with confirmed ZIKV infection, 42 underwent prenatal ultrasonography, and foetal abnormalities were observed in 12 (29%); none of the 16 women with negative tests had foetal abnormalities. The abnormalities observed on ultrasonography varied widely [30▪].
Since 2015, several reports were published describing a wide range of congenital abnormalities probably associated with ZIKV infection in utero. The WHO  has started a process for defining the spectrum of this syndrome to map and analyse the clinical manifestations including visual abnormalities [69–71] and neuroimaging findings [72–75].
Apart from perinatal transmission, there is increased evidence of nonvector-borne forms of ZIKV transmission, including sexual transmission . Foy et al. described clinical and serologic evidence indicating that one scientist transmitted ZIKV to his wife probably by sexual contact after his return home. Venturi et al. retrospectively diagnosed a case of ZIKV infection leading to a secondary autochthonous case, probably by sexual transmission. D’Ortenzio et al. presented a case with more evidence supporting the hypothesis for ZIKV sexual transmission (either oral or vaginal). Deckard et al. described the first report of ZIKV transmission from an infected man to a sex partner through anal sex.
ZIKV is potentially transmissible via blood products and organ or tissue transplantation [84,85]. So far, there is no scientific evidence supporting that ZIKV could be transmitted through human saliva, breast milk and urine, although some studies have reported infectivity viral particles confirmed by the presence of a cytopathic effect onto Vero cells [86–88].
The diagnosis of Zika infections can be performed on clinical-epidemiological and laboratorial bases. Currently, ZIKV can be detected in distinct clinical specimens such as blood (plasma, serum), CSF, urine, saliva, breast milk, semen, vaginal secretion, amniotic fluid and tissues [28,39,59,78,89–96].
Overall, the laboratorial diagnosis of ZIKV infection relies on the same usual strategies used for other arboviruses, with viral genome detection by RT-PCR tests on acute-phase samples and serology (ELISA and immunofluorescence) for detection of specific antibody against the virus. The virus isolation may also be used in acute samples, but it is a more laborious and time-consuming approach, also requiring a more robust infrastructure, not available in most laboratories. However, as many other flaviviruses, results based on more routinely used serological tests may be compromised by a cross-reactivity in convalescent samples due to previous flaviviruses infections. For a more reliable result, flaviviruses diagnosis should test paired acute and convalescent samples, collected 2–3 weeks after the onset of symptoms. Usually, the choice of the laboratorial approach used will depend on the goal of the analysis, laboratory infrastructure, technical expertise and sampling availability.
During the acute phase of the disease, virus or viral nucleic acid detection can be performed. The virus isolation, despite not being performed on a routine basis, can be accomplished using mosquitoes cells (such as AP-61, Aedes pseudoscutellaris; C6/36, Aedes albopictus) or mammalian cell lines (such as BHK, VERO), directly from infected mosquitoes or by innoculation into newborn mice [97–100]. Even though the virus characterization is important, the isolation may be difficult due to the low viremia during the acute phase of ZIKV infection .
The most commonly and widely used diagnostic technique for ZIKV diagnosis is based on the virus molecular detection by conventional or real-time RT-PCR, and some protocols have been described for detecting ZIKV within the group [101–104] or specific to the virus [94,105–107]. The viral genome detection by molecular techniques provides a definitive diagnostic result, but as viremia is transient, this approach is most reliable if performed within the first week of the disease . By using quantitive RT-PCR, the RNA viral load in blood (7.28 × 106 to 9.3 × 108 copies RNA/ml), urine (2.5 × 103 to 8 × 106 copies RNA/ml), semen (1.1 × 107 to 2.9 × 107 copies RNA/ml) and breast milk (2.9 × 104 to 2 × 106 copies RNA/ml) could be determined [5,20,89,90,94,108]. Although the ZIKV genome has been detected on amniotic fluid, cord blood, CSF and placenta by RT-PCR, the method's sensitivity on those specimens is unknown .
Anti-ZIKV IgM and IgG antibodies can be detected by serological assays. However, the results should be carefully analysed due to false-positive results from possible cross-reactivity with other flaviviruses. The virus-specific IgM may be detectable more than 5 days after onset of symptoms. In some case, these biomarkers, indicative of an active infection, may rise as early as day 3 of illness and last over 2 months [91,94,110]. Seroconversion represented by at least a four-fold increase in paired sera, acute and convalescent sera, is desirable for a more reliable result. In a flavivirus-naive patient, a minimal cross-reactivity is obtained, but a significant cross-reactivity in patients exposed to a previous flavivirus infection is observed, even on yellow fever vaccinated patients.
Despite the gold standard for the specific detection of antiflavivirus anitbodies, the plaque-reduction test (PRNT) , useful for cross-reactive results on ZIKV infection where other flavivirus circulate, the assay is arduous and time-consuming, and specialized infrastructure and lab personnel are needed due to the live virus manipulation.
For the diagnosis of Zika congenital infection, CDC recommends the performance of both molecular and serological tests, as it is not known which one would be more reliable for the condition investigation . With tissue availability, immunohistochemistry assays may also be performed for viral antigen detection . Furthermore, aiming to diagnose congenital ZIKV infections, CSF, placenta, blood cord and/or umbilical cord tissues from neonates or unborn foetuses shall be analysed.
Our current understanding of the pathophysiology of ZIKV infection needs to be strengthened and epidemiological studies and animal models of the disease need to be developed. Public policies need to be implemented, effective preventive measures such as vaccines created and improving efforts to reduce transmission through vectors and more studies on sexual transmission are necessary. Accurate, portable and inexpensive point-of-care tests are in need to better identify cases, especially where other arboviruses cocirculate. Due to the difficulties in propagating the virus, antigen availability in many laboratories poses a burden for the diagnosis, and the development and use of recombinant antigens are desirable.
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