NS1 glycoprotein detection in serum and urine as an electrochemical screening immunosensor for dengue and Zika virus.

The incidence of infection by the dengue virus (DENV) has grown dramatically, reaching 128 countries in tropical and subtropical regions worldwide, with a pattern of hyper-endemicity. DENV is a mosquito-borne disease having four serotypes, one or two circulating in epidemic outbreaks. The diagnosis of DENV is challenging mainly due to the circulation of new viruses with remarkable similarities, such as Zika (ZIKV) that may cause fetal microcephaly. DENV affects 390 million people per year, but these numbers may be higher due to the underreported and misclassified cases. Recently, the NS1 nonstructural protein has been described in serum and urine of DENV and ZIKV patients, suggesting its use as a biomarker for screening since a negative NS1 sample confirms the absence of these infections. Herein, a label-free immunosensor comprising an assembled nanostructured thin film of carbon nanotube-ethylenediamine is described. The advantage of in situ electrosynthesis of polymer film is to allow major control of thickness and conductivity, in addition to designing the reactive groups for functionalization. A quartz crystal microbalance system was used to estimate the thickness of the polymeric film obtained. The anti-NS1 monoclonal antibodies were immobilized to carbon nanotubes by covalent linkage, permitting a high stability during measurements. Analytical responses to NS1 were obtained by differential pulse voltammetry (DPV), showing a linear range from 20 to 800 ng mL-1 and reproducibility of 3.0%, with a limit of detection (LOD) of 6.8 ng mL- 1. This immunosensor was capable of detecting ZIKV and DENV NS1 in spiked urine and real serum in a clinical range.Graphical abstract.

Can Zika Virus Infection in High Risk Pregnant Women Be Differentiated on the Basis of Symptoms?

Zika virus (ZIKV) infection in pregnancy is associated with congenital neurological abnormalities. Our understanding of the full clinical spectrum of ZIKV infection is incomplete. Using data from this prospective cohort study consisting of 650 women attending a high-risk pregnancy clinic during the Zika virus outbreak in Brazil, we investigated the extent to which specific symptoms can be utilized to differentiate ZIKV-infected pregnant women from those with other pregnancy-related problems. All were tested for ZIKV in urine by RT-qPCR. Demographic and clinical data including physical symptoms during follow-up were recorded and analyzed with respect to Zika virus exposure status. Forty-eight (7.4%) women were positive for ZIKV by RT-qPCR. The majority (70.8%) were asymptomatic, and only four ZIKV-positive women (8.3%) reported symptoms during pregnancy that met the WHO case definition. Zika-positive and -negative women reported similar frequencies of ZIKV-like symptoms (as per the WHO definition): fever (16.7% vs. 13.6%), arthralgia/arthritis (10.4% vs. 11.3%), rash (4.2% vs. 5.3%), and conjunctivitis (2.1% vs. 3.2%). Most pregnant women positive for ZIKV in urine are asymptomatic and do not deliver a baby with microcephaly. Physical symptoms alone did not differentiate between high-risk pregnant women positive or negative for ZIKV.

Zika Virus infection and Guillain-Barré syndrome in Northeastern Mexico: A case-control study.

BACKGROUND: Beginning August 2017, we conducted a prospective case-control investigation in Monterrey, Mexico to assess the association between Zika virus (ZIKV) and Guillain-Barré syndrome (GBS). METHODS: For each of 50 GBS case-patients, we enrolled 2-3 afebrile controls (141 controls in total) matched by sex, age group, and presentation to same hospital within 7 days. RESULTS: PCR results for ZIKV in blood and/or urine were available on all subjects; serum ZIKV IgM antibody for 52% of case-patients and 80% of controls. Subjects were asked about antecedent illness in the two months prior to neurological onset (for case-patients) or interview (for controls). Laboratory evidence of ZIKV infection alone (PCR+ or IgM+) was not significantly different between case-patients and controls (OR: 1.26, 95% CI: 0.45-3.54) but antecedent symptomatic ZIKV infection [a typical ZIKV symptom (rash, joint pain, or conjunctivitis) plus laboratory evidence of ZIKV infection] was higher among case-patients (OR: 12.45, 95% CI: 1.45-106.64). GBS case-patients with laboratory evidence of ZIKV infection were significantly more likely to have had typical ZIKV symptoms than controls with laboratory evidence of ZIKV infection (OR: 17.5, 95% CI: 3.2-96.6). This association remained significant even when only GBS case-patients who were afebrile for 5 days before onset were included in the analysis, (OR 9.57 (95% CI: 1.07 to 85.35). CONCLUSIONS: During ZIKV epidemics, this study indicates that increases in GBS will occur primarily among those with antecedent symptomatic ZIKV.

Detection of Zika virus in paired urine and amniotic fluid samples from symptomatic and asymptomatic women and their babies during a disease outbreak: association with neurological symptoms in newborns.

Paired maternal and newborn urine and amniotic fluid from 138 subjects collected during a Zika virus (ZIKV) outbreak was analyzed for ZIKV by gene amplification (RT-qPCR), and the findings were correlated with clinical symptoms and neurological anomalies in the babies. ZIKV was detected in 1 of 9 symptomatic women (11.1%) and in 19 of 129 asymptomatic women (14.7%). Neurological manifestations were present in 19 babies (13.7%), 10 of 20 (50%) positive and 9 of 119 (7.6%) negative (p < 0.001) for ZIKV. Twelve (8.6%) urines collected during gestation were ZIKV-positive; only 2 remained positive for ZIKV postpartum. Six (4.1%) newborn urines collected within 1 day of delivery were ZIKV-positive cases. In 3 of these cases, ZIKV was detected in mother's urine pre- and postpartum and in both mother's urine and babies' urine. Four of the amniotic fluid samples (2.9%) were ZIKV-positive. Among ZIKV-negative babies with neurological sequel, 87.5% were female; in contrast, 72.7% ZIKV-positive babies with neurological abnormalities were male (p = 0.019). We conclude that during a ZIKV outbreak, clinical symptoms and ZIKV detection in biological fluids are poor predictors of infection and adverse neurologic sequel in newborns.

Genomic Epidemiology Reconstructs the Introduction and Spread of Zika Virus in Central America and Mexico.

The Zika virus (ZIKV) epidemic in the Americas established ZIKV as a major public health threat and uncovered its association with severe diseases, including microcephaly. However, genetic epidemiology in some at-risk regions, particularly Central America and Mexico, remains limited. We report 61 ZIKV genomes from this region, generated using metagenomic sequencing with ZIKV-specific enrichment, and combine phylogenetic, epidemiological, and environmental data to reconstruct ZIKV transmission. These analyses revealed multiple independent ZIKV introductions to Central America and Mexico. One introduction, likely from Brazil via Honduras, led to most infections and the undetected spread of ZIKV through the region from late 2014. Multiple lines of evidence indicate biannual peaks of ZIKV transmission in the region, likely driven by varying local environmental conditions for mosquito vectors and herd immunity. The spatial and temporal heterogeneity of ZIKV transmission in Central America and Mexico challenges arbovirus surveillance and disease control measures.

Microcephaly caused by congenital Zika virus infection and viral detection in maternal urine during pregnancy.

Currently Latin America is undergoing a major epidemic of Zika virus, which is transmitted by Aedes mosquitoes. Concern for Zika virus infection has been increasing as it is suspected of causing brain defects in newborns such as microcephaly and, more recently, potential neurological and autoimmune complications including Guillian-Barré syndrome and acute disseminated encephalomyelitis. We describe a case of virus infection in a 25-year-old woman during the first trimester of her pregnancy, confirmed by laboratory tests only for the detection of viral particles in maternal urine, with imaging studies demonstrating the progression of cranial and encephalic changes in the fetus and later in the newborn, such as head circumference reduction, cerebral calcifications and ventriculomegaly.

Isolation of infectious Zika virus from a urine sample cultured in SIRC cells from a patient suspected of having rubella virus.

A great variety of viruses which cause exanthema share other clinical manifestations, making the etiologic identification a very difficult task, relying exclusively on the clinical examination. Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as congenital rubella syndrome (CRS). In the present report, we described the presence of Zika virus (ZIKV) particles in urine samples and also ZIKV isolation in SIRC cells from the urine of a patient in acute phase of suspected rubella disease. The 50-year-old unvaccinated woman living in Sao Paulo, Brazil, was admitted to the emergency room with fever, headache, rash, arthralgia and prostration. Urine samples were collected for virus isolation and RT-qPCR. SIRC and Vero cells were inoculated with urine samples during 7 days. RT-qPCR was performed using measles virus (MV) and RV primers and both were found to be negative. After this result, RT-qPCR was performed for parvovirus B19, herpes virus 6 and ZIKV. The urine sample and the isolate were positive by Real Time PCR for ZIKV and negative for all other viruses tested. The sequences isolated are from the Asiatic lineage.

Microcephaly caused by congenital Zika virus infection and viral detection in maternal urine during pregnancy / Microcefalia causada por infecção congênita pelo Zika virus e detecção viral na urina materna durante a gestação

Summary Currently Latin America is undergoing a major epidemic of Zika virus, which is transmitted by Aedes mosquitoes. Concern for Zika virus infection has been increasing as it is suspected of causing brain defects in newborns such as microcephaly and, more recently, potential neurological and autoimmune complications including Guillian-Barré syndrome and acute disseminated encephalomyelitis. We describe a case of virus infection in a 25-year-old woman during the first trimester of her pregnancy, confirmed by laboratory tests only for the detection of viral particles in maternal urine, with imaging studies demonstrating the progression of cranial and encephalic changes in the fetus and later in the newborn, such as head circumference reduction, cerebral calcifications and ventriculomegaly.

Resumo Atualmente, a América Latina está passando por uma grande epidemia de Zika vírus, transmitido por mosquitos Aedes. A preocupação pela infecção pelo Zika vírus vem aumentando, uma vez que é suspeita de causar defeitos cerebrais em recém-nascidos, como a microcefalia e, mais recentemente, potenciais complicações neurológicas e autoimunes, como síndrome de Guillian-Barré e encefalomielite disseminada aguda. Descrevemos um caso de infecção pelo vírus em uma mulher de 25 anos durante o primeiro trimestre de gestação, confirmado dentre os exames laboratoriais apenas pela detecção de partículas virais na urina materna, com estudos de imagens demonstrando a evolução das alterações cranianas e encefálicas no feto e no recém-nascido, como redução do perímetro cefálico, calcificações cerebrais e ventriculomegalia.

Diagnostic Validation of the RealStar® Zika Virus Reverse Transcription Polymerase Chain Reaction Kit for Detection of Zika Virus RNA in Urine and Serum Specimens.

With the Zika virus outbreak in South America starting in 2015 and its potential to cause malformation of the fetus in infected women, the need for diagnostic methods became obvious. Until now, only limited data are available on the diagnostic performance of commercial kits. Here, we present data comparing the RealStar® Zika Virus RT-PCR Kit 1.0 for detection of Zika virus from 208 serum and urine samples collected in French Guiana with a reference method. Of these, 114 samples tested positive with the RealStar® Kit and 111 with the reference method.

Immune Cell Dynamics in Rhesus Macaques Infected with a Brazilian Strain of Zika Virus.

Zika virus (ZIKV) is a mosquito-borne and sexually transmitted flavivirus that is associated with fetal CNS-damaging malformations during pregnancy in humans. This study documents the viral kinetics and immune responses in rhesus macaques infected with a clinical ZIKV Brazilian isolate. We evaluated the viral kinetics and immune responses induced after an i.v. infection with a Brazilian ZIKV clinical isolate (HS-2015-BA-01) in rhesus macaques for up to 142 d. ZIKV-specific Ab-secreting cells, germinal center reactions, and monocyte, dendritic cell, NK, and T cell frequencies were monitored. ZIKV loads were readily detected in plasma (until day 5 or 7), semen and urine (until days 7 and 14), and saliva (until day 42), but the viremia was rapidly controlled. No detectable clinical manifestations were observed. However, lymph node hyperplasia was clearly visible postviremia but was associated with low frequencies of ZIKV-specific Ab-secreting cells in lymph nodes and bone marrow, correlating with low Ab titers. CD14+/CD16- monocytes and myeloid CD11chi dendritic cells decreased in blood, whereas NK and T cell numbers were only marginally altered during the course of the study. ZIKV infection caused a significant lymphoid tissue activation but limited induction of ZIKV-specific B cells, suggesting that these parameters need to be considered for ZIKV vaccine design.

A review of Zika virus infections in pregnancy and implications for antenatal care in Singapore.

Given the consensus that there is a causal relationship between Zika virus (ZIKV) infection in pregnancy and congenital Zika syndrome (CZS), clinicians must be prepared to manage affected patients despite the numerous gaps in current knowledge. The clinical course in pregnancy appears similar to that in non-pregnant women, although viraemia may be prolonged. ZIKV infection can be diagnosed by serum and urine reverse transcription-polymerase chain reaction, but commercially available serological tests are currently unreliable in dengue-endemic regions. Although vertical transmission can occur at any time during gestation, first- and second-trimester infections have the highest risk of developing central nervous system anomalies. Aberrant fetal growth and pregnancy loss may also occur. Serial ultrasonography should be conducted for infected cases. Without a vaccine, pregnant women should be advised to minimise mosquito bites and reduce sexual transmission risk. Overall, the absolute risk of CZS arising amid a ZIKV outbreak appears relatively low.

Prolonged Detection of Zika Virus in Vaginal Secretions and Whole Blood.

Infection with Zika virus is an emerging public health crisis. We observed prolonged detection of virus RNA in vaginal mucosal swab specimens and whole blood for a US traveler with acute Zika virus infection who had visited Honduras. These findings advance understanding of Zika virus infection and provide data for additional testing strategies.

Persistent Zika Virus Detection in Semen in a Traveler Returning to the United Kingdom from Brazil, 2016.

Zika virus is normally transmitted by mosquitos, but cases of sexual transmission have been reported. We describe a patient with symptomatic Zika virus infection in whom the virus was detected in semen for 92 days. Our findings support recommendations for 6 months of barrier contraceptive use after symptomatic Zika virus infection.

Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil.

BACKGROUND: Zika virus (ZIKV) is an emergent threat provoking a worldwide explosive outbreak. Since January 2015, 41 countries reported autochthonous cases. In Brazil, an increase in Guillain-Barré syndrome and microcephaly cases was linked to ZIKV infections. A recent report describing low experimental transmission efficiency of its main putative vector, Ae. aegypti, in conjunction with apparent sexual transmission notifications, prompted the investigation of other potential sources of viral dissemination. Urine and saliva have been previously established as useful tools in ZIKV diagnosis. Here, we described the presence and isolation of infectious ZIKV particles from saliva and urine of acute phase patients in the Rio de Janeiro state, Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Nine urine and five saliva samples from nine patients from Rio de Janeiro presenting rash and other typical Zika acute phase symptoms were inoculated in Vero cell culture and submitted to specific ZIKV RNA detection and quantification through, respectively, NAT-Zika, RT-PCR and RT-qPCR. Two ZIKV isolates were achieved, one from urine and one from saliva specimens. ZIKV nucleic acid was identified by all methods in four patients. Whenever both urine and saliva samples were available from the same patient, urine viral loads were higher, corroborating the general sense that it is a better source for ZIKV molecular diagnostic. In spite of this, from the two isolated strains, each from one patient, only one derived from urine, suggesting that other factors, like the acidic nature of this fluid, might interfere with virion infectivity. The complete genome of both ZIKV isolates was obtained. Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak. CONCLUSIONS/SIGNIFICANCE: The detection of infectious ZIKV particles in urine and saliva of patients during the acute phase may represent a critical factor in the spread of virus. The epidemiological relevance of this finding, regarding the contribution of alternative non-vectorial ZIKV transmission routes, needs further investigation.