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Zika virus: old friend, new ways to diagnose. How researchers are facing this new challenge

In 10 seconds? Zika virus is not a new disease, in fact the first case was described in 1947 in Uganda. After 2007, the virus appeared in Africa, Asia and Pacific Islands. More over, as the symptons are very similar to other diseases, the CDC estimates that several cases were not documented. The recent epidemic in South America in the last years putted Zika virus in the spotlight leading to several questions, how to diagnose is one of them.

How to diagnose zika? The diagnostic is made through an urine/blood test associated with a questionnaire about the person's travel habits. The problem is that even before collecting samples to make a diagnostic, doctors face a variety of challenges. First, most of people will not develop any symptoms or present mild symptoms, most of them common to several diseases. Second, as the transmission of zika virus can be through different ways, try to map the places someone has travelled is not enough. For example you can get zika virus by having unprotected sex. Third, tests are not specific for zika.

What are researchers current doing to face those challenges? After the recent epidemic in South America, it was clear the need to develop new ways to diagnose. The techniques availabe were either cross reacting with virus from the same family or taking too long to have the result. In this sense several studies were made to improve those methods. We current have tests that don’t show cross reaction with dengue virus but the work is non stop and probably we will have new discoveries soon.

What is the importance of Zika virus? Zika virus became "famous" after its association with microcephaly and more recently, with Guillain-Barré syndrome. Several studies showed that the increase of children born with microcepahly was associated with mothers infected with the virus in the first trimester. And, recently, in some adults the disease progressed to a more serious condition called Guillain-Barré syndrome. The last one has yet to be confirm.


Results of a Zika Virus (ZIKV) Immunoglobulin M-Specific Diagnostic Assay Are Highly Correlated With Detection of Neutralizing Anti-ZIKV Antibodies in Neonates With Congenital Disease.

Abstract:  Usually, immunoglobulin M (IgM) serologic analysis is not sufficiently specific to confirm Zika virus (ZIKV) infection. However, since IgM does not cross the placenta, it may be a good marker of infection in neonates. We tested blood from 42 mothers and neonates with microcephaly and collected cerebrospinal fluid (CSF) specimens from 30 neonates. Molecular assays were performed for detection of ZIKV, dengue virus, and chikungunya virus; IgM enzyme-linked immunosorbent assays and plaque-reduction neutralization tests (PRNTs) were performed to detect ZIKV and dengue virus. No control neonates without microcephaly were evaluated. Among neonates, all 42 tested positive for ZIKV IgM: 38 of 42 serum specimens (90.5%) were positive, whereas 30 of 30 CSF specimens (100%) were positive. ZIKV IgM-specific ELISA ratios, calculated as the mean optical density (OD) of the test sample when reacted on viral antigen divided by the mean OD of the negative control when reacted with viral antigen, were higher in CSF specimens (median, 14.9 [range, 9.3-16.4]) than in serum (median, 8.9 [range, 2.1-20.6]; P = .0003). All ZIKV IgM-positive results among the neonates were confirmed by the detection of neutralizing antibodies. Mother/neonate pairs with primary ZIKV infection had neutralizing antibodies to ZIKV only, and mother/neonate pairs with ZIKV virus infection secondary to infection with another flavivirus had high titers of neutralizing antibodies to ZIKV. Among secondary infections, median titers in serum were 2072 (range, 232-12 980) for mothers and 2730 (range, 398-12 980) for neonates (P < .0001), and the median titer in CSF was 93 (range, 40-578) among neonates (P < .0001). Among neonates, detection of ZIKV IgM in serum is confirmatory of congenital ZIKV infection, and detection of ZIKV IgM in CSF is confirmatory of neurologic infection. Therefore, we recommend testing for ZIKV IgM in neonates suspected of having congenital ZIKV infection and performance of PRNTs in equivocal cases.

Pub.: 08 Dec '16, Pinned: 16 Apr '17

A Multiplex Microsphere Immunoassay for Zika Virus Diagnosis.

Abstract: Rapid and accurate diagnosis of infectious agents is essential for patient care, disease control, and countermeasure development. The present serologic diagnosis of Zika virus (ZIKV) infection relies mainly on IgM-capture ELISA which is confounded with the flaw of cross-reactivity among different flaviviruses. In this communication, we report a multiplex microsphere immunoassay (MIA) that captures the diagnostic power of viral envelope protein (that elicits robust, yet cross-reactive antibodies to other flaviviruses) and the differential power of viral nonstructural proteins NS1 and NS5 (that induce more virus-type specific antibodies). Using 153 patient specimens with known ZIKV and/or dengue virus (DENV; a closely related flavivirus) infections, we showed that (i) ZIKV envelope-based MIA is equivalent or more sensitive than IgM-capture ELISA in diagnosing ZIKV infection, (ii) antibody responses to NS1 and NS5 proteins are more ZIKV-specific than antibody response to envelope protein, (iii) inclusion of NS1 and NS5 in the MIA improves the diagnostic accuracy when compared with the MIA that uses envelope protein alone. The multiplex MIA achieves a rapid diagnosis (turnaround time<4h) and requires small specimen volume (10μl) in a single reaction. This serologic assay could be developed for use in clinical diagnosis of ZIKV infection and for monitoring immune responses in vaccine trials.

Pub.: 18 Jan '17, Pinned: 16 Apr '17

A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients.

Abstract: The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV) infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT) that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV) diagnosis that can attain the "gold standard" of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

Pub.: 12 Mar '17, Pinned: 16 Apr '17


Abstract: Zika virus (ZKV) infection is a huge public health problem in Brazil because of the increased incidence of microcephaly in neonates from infected mothers. Detection of specific IgG antibodies in maternal serum samples constitutes an important approach for diagnosing ZKV infection and evaluating its relationship with neonatal microcephaly. However, as there is no serological test produced in Brazil to detect IgM and IgG antibodies against ZKV, we sought to examine specific IgG in serum samples from patients or suspected mothers to detect previous infection and to test for specificity with regard to flaviviral infections occurring in the same area. Brazilian Zika virus native antigens were obtained from infected Vero cell layers or free virions in the culture medium and then used in ELISA. We tested sera from eight ZKV RNA-diagnosed infected patients (ZKVR), seven neonates with microcephaly and their mothers after delivery (MM), 140 dengue virus IgM-positive (DM) and IgG (DG)-positive patients, and 100 yellow fever (YF)-vaccinated patients. According to the ELISA, ZKVR samples were mostly positive (7/8), and all the MM serum samples were positive for ZKV IgG (7/7). In contrast, cross-reactions for dengue or yellow fever-vaccinated patients were observed, including DM (48/95), DG (10/45) or YF (3/100) serum samples; however, these cross-reactions exhibited low antigen avidity so that 6 M urea largely removed this cross-reactivity, with only a few cross-reacting samples remaining (8/140). ELISA based on extracted virions was much more specific, with all ZKVR (8/8) and MM sera being positive for ZKV IgG (7/7) and only borderline cross-reactivity found for DM (6/95), DG (3/45) or YF (4/100)-vaccinated serum samples. This technique (ELISA) can identify specific IgG in ZKV-infected patients and may be helpful in diagnosing congenital infetions after maternal RNA virus clearance or in epidemiological studies.

Pub.: 17 Dec '16, Pinned: 16 Apr '17

Testing for Zika Virus (ZIKV) Infection in Pregnancy: Key Concepts to Deal with an Emerging Epidemic.

Abstract: Zika virus (ZIKV) is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Following epidemics in Micronesia and French Polynesia during the past decade, more recent ZIKV infection outbreaks were first reported in South America as early as May of 2013, and spread to now 50 countries throughout the Americas. Although no other flavivirus has previously been known to cause major fetal malformations following perinatal infection, reports of a causal link between ZIKV and microcephaly, brain and ocular malformations, and fetal loss emerged from hard hit regions of Brazil by October 2015. Among the minority of infected women with symptoms, clinical manifestations of ZIKV infection may include fever, headache, arthralgia, myalgia and maculopapular rash; however, only one out of every four to five people who are infected have any symptoms. Thus, clinical symptom reporting is an ineffective screening tool for the relative risk assessment of ZIKV infection in the majority of patients. As previously occurred with other largely asymptomatic viral infections posing perinatal transmission risk (such as HIV or CMV), we must develop and implement rapid, sensitive, and specific screening and diagnostic testing for both viral detection and estimation of timing of exposure. Unfortunately, despite an unprecedented surge in attempts to rapidly advance perinatal clinical testing for a previously obscure arbovirus, there are several ongoing hindrances to molecular and sonographic based screening and diagnosis of congenital ZIKV infection. These include: (1) difficulty in estimating the timing of exposure for women living in endemic areas, and thus limited interpretability of IgM serologies; (2) cross-reaction of IgM serologies with other endemic flaviruses, such as dengue (DENV); (3) persistent viremia and viruria in pregnancy weeks to months after primary exposure; and (4) fetal brain malformations and anomalies preceding the sonographic detection of microcephaly. In this commentary, we discuss screening and diagnostic considerations which are grounded not only in the realities of current obstetrical practice in a largely global population, but in basic immunology and virology. We review recent epidemiologic data pertaining to risk of congenital ZIKV malformations based on trimester of exposure, and consider side by side with emerging data demonstrating replication of ZIKV in placental and fetal tissue throughout gestation. We discuss limitations to ultrasound based strategies which rely largely or solely on the detection of microcephaly, and provide alternative neurosonographic approaches for the detection of malformations which may precede or occur independent of a small head circumference. This expert review provides information that is of value for the: 1) obstetrician, maternal-fetal medicine specialist, midwife, patient and family in cases of suspected ZIKV infection; 2) reviews the methodology for laboratory testing to explore the presence of the virus and the immune response; 3) ultrasound based assessment of the fetus suspected to be exposed to ZIKV with particular emphasis on the central nervous system; and 4) identifies areas ready for development.

Pub.: 28 Jan '17, Pinned: 16 Apr '17

Zika virus infection during pregnancy and microcephaly occurrence: a review of literature and Brazilian data

Abstract: In November of 2015, the Ministry of Health of Brazil published an announcement confirming the relationship between Zika virus and the microcephaly outbreak in the Northeast, suggesting that infected pregnant women might have transmitted the virus to their fetuses. The objectives of this study were to conduct a literature review about Zika virus infection and microcephaly, evaluate national and international epidemiological data, as well as the current recommendations for the health teams. Zika virus is an arbovirus, whose main vector is the Aedes sp. The main symptoms of the infection are maculopapular rash, fever, non-purulent conjunctivitis, and arthralgia. Transmission of this pathogen occurs mainly by mosquito bite, but there are also reports via the placenta. Microcephaly is defined as a measure of occipto-frontal circumference being more than two standard deviations below the mean for age and gender. The presence of microcephaly demands evaluation of the patient, in order to diagnose the etiology. Health authorities issued protocols, reports and notes concerning the management of microcephaly caused by Zika virus, but there is still controversy about managing the cases. The Ministry of Health advises notifying any suspected or confirmed cases of children with microcephaly related to the pathogen, which is confirmed by a positive specific laboratory test for the virus. The first choice for imaging exam in children with this malformation is transfontanellar ultrasound. The most effective way to control this outbreak of microcephaly probably caused by this virus is to combat the vector. Since there is still uncertainty about the period of vulnerability of transmission via placenta, the use of repellents is crucial throughout pregnancy. More investigations studying the consequences of this viral infection on the body of newborns and in their development are required.

Pub.: 18 Apr '16, Pinned: 27 Apr '17