Differential Frequencies of HLA-DRB1, DQA1, and DQB1 Alleles and Haplotypes Are Observed in the Arbovirus-Related Neurological Syndromes.

BACKGROUND: We took advantage of the 2015-2016 Brazilian arbovirus outbreak (Zika [ZIKV]/dengue/chikungunya viruses) associated with neurological complications to type HLA-DRB1/DQA1/DQB1 variants in patients exhibiting neurological complications and in bone marrow donors from the same endemic geographical region. METHODS: DRB1/DQA1/DQB1 loci were typed using sequence-specific oligonucleotides. In silico studies were performed using X-ray resolved dimer constructions. RESULTS: The DQA1*01, DQA1*05, DQB1*02, or DQB1*06 genotypes/haplotypes and DQA1/DQB1 haplotypes that encode the putative DQA1/DQB1 dimers were overrepresented in the whole group of patients and in patients exhibiting peripheral neurological spectrum disorders (PSD) or encephalitis spectrum disorders (ESD). The DRB1*04, DRB1*13, and DQA1*03 allele groups protected against arbovirus neurological manifestation, being underrepresented in whole group of patients and ESD and PSD groups. Genetic and in silico studies revealed that DQA1/DQB1 dimers (1) were primarily associated with susceptibility to arbovirus infections; (2) can bind to a broad range of ZIKV peptides (235 of 1878 peptides, primarily prM and NS2A); and (3) exhibited hydrophilic and highly positively charged grooves when compared to the DRA1/DRB1 cleft. The protective dimer (DRA1/DRB1*04) bound a limited number of ZIKV peptides (40 of 1878 peptides, primarily prM). CONCLUSION: Protective haplotypes may recognize arbovirus peptides more specifically than susceptible haplotypes.

Lessons Learned at the Epicenter of Brazil's Congenital Zika Epidemic: Evidence From 87 Confirmed Cases.

Congenital Zika virus infection has stimulated great international concern. A prospective case series of 87 infants with laboratory-confirmed congenital Zika syndrome (CZS) at the epicenter of the Brazilian Zika epidemic in Pernambuco state is presented. Mothers were interviewed for symptoms of possible Zika virus (ZIKV) infection during pregnancy, and fetal ultrasounds were obtained. Infant cerebrospinal fluid (CSF) samples were tested for ZIKV-specific antibodies, and sera were screened for other congenital infections. Neuroimaging and ophthalmologic evaluations were also performed. Sixty-six mothers (76%) reported symptoms of ZIKV infection during gestation. Fetal ultrasounds were available from 90% of the mothers, and all demonstrated brain structural abnormalities. All of the CSF samples tested positive for ZIKV immunoglobulin M. The majority of infants (89%) were term; the mean birth weight was 2577 ± 260 g, and the mean head circumference was 28.1 ± 1.8 cm. Severe microcephaly, defined as head circumference 3 SD below the mean for sex and gestational age, was found in 72 (82%) infants. All infants had an abnormal neurological exam, and 18 (20.7%) had arthrogryposis. The main abnormalities detected in computed tomography scans were calcifications (99%), followed by ventricular enlargement (94%), cortical hypogyration (81%), and less commonly, cerebellar hypoplasia (52%). Unilateral diaphragm paralysis was identified in 3 infants. Maternal young age, term infant, small for gestational age, and the presence of ophthalmologic abnormalities were significantly associated with a smaller head circumference Z score. Our findings, based on laboratory-confirmed ZIKV infection, add valuable evidence for the understanding of CZS.

1H Nuclear Magnetic Resonance Metabolomics of Plasma Unveils Liver Dysfunction in Dengue Patients.

UNLABELLED: Dengue, due to its global burden, is the most important arthropod-borne flavivirus disease, and early detection lowers fatality rates to below 1%. Since the metabolic resources crucial for viral replication are provided by host cells, detection of changes in the metabolic profile associated with disease pathogenesis could help with the identification of markers of prognostic and diagnostic importance. We applied (1)H nuclear magnetic resonance exploratory metabolomics to study longitudinal changes in plasma metabolites in a cohort in Recife, Brazil. To gain statistical power, we used innovative paired multivariate analyses to discriminate individuals with primary and secondary infection presenting as dengue fever (DF; mild) and dengue hemorrhagic fever (DHF; severe) and subjects with a nonspecific nondengue (ND) illness (ND subjects). Our results showed that a decrease in plasma low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) discriminated dengue virus (DENV)-infected subjects from ND subjects, and also, subjects with severe infection even presented a decrease in lipoprotein concentrations compared to the concentrations in subjects with mild infection. These results add to the ongoing discussion that the manipulation of lipid metabolism is crucial for DENV replication and infection. In addition, a decrease in plasma glutamine content was characteristic of DENV infection and disease severity, and an increase in plasma acetate levels discriminated subjects with DF and DHF from ND subjects. Several other metabolites shown to be altered in DENV infection and the implications of these alterations are discussed. We hypothesize that these changes in the plasma metabolome are suggestive of liver dysfunction, could provide insights into the underlying molecular mechanisms of dengue virus pathogenesis, and could help to discriminate individuals at risk of the development of severe infection and predict disease outcome. IMPORTANCE: Dengue, due to its global burden, is the most important mosquito-borne viral disease. There is no specific treatment for dengue disease, and early detection lowers fatality rates to below 1%. In this study, we observed the effects of dengue virus infection on the profile of small molecules in the blood of patients with mild and severe infection. Variations in the profiles of these small molecules reflected the replication of dengue virus in different tissues and the extent of tissue damage during infection. The results of this study showed that the molecules that changed the most were VLDL, LDL, and amino acids. We propose that these changes reflect liver dysfunction and also that they can be used to discriminate subjects with mild dengue from those with severe dengue.

Description of 13 Infants Born During October 2015-January 2016 With Congenital Zika Virus Infection Without Microcephaly at Birth - Brazil.

Congenital Zika virus infection can cause microcephaly and severe brain abnormalities (1). Congenital Zika syndrome comprises a spectrum of clinical features (2); however, as is the case with most newly recognized teratogens, the earliest documented clinical presentation is expected to be the most severe. Initial descriptions of the effects of in utero Zika virus infection centered prominently on the finding of congenital microcephaly (3). To assess the possibility of clinical presentations that do not include congenital microcephaly, a retrospective assessment of 13 infants from the Brazilian states of Pernambuco and Ceará with normal head size at birth and laboratory evidence of congenital Zika virus infection was conducted. All infants had brain abnormalities on neuroimaging consistent with congenital Zika syndrome, including decreased brain volume, ventriculomegaly, subcortical calcifications, and cortical malformations. The earliest evaluation occurred on the second day of life. Among all infants, head growth was documented to have decelerated as early as 5 months of age, and 11 infants had microcephaly. These findings provide evidence that among infants with prenatal exposure to Zika virus, the absence of microcephaly at birth does not exclude congenital Zika virus infection or the presence of Zika-related brain and other abnormalities. These findings support the recommendation for comprehensive medical and developmental follow-up of infants exposed to Zika virus prenatally. Early neuroimaging might identify brain abnormalities related to congenital Zika infection even among infants with a normal head circumference (4).

High frequency of pre-existing neutralizing antibody responses in patients with dengue during an outbreak in Central Brazil.

BACKGROUND: This study aims to identify dengue neutralizing antibody response in patients with dengue from a well-characterized cohort during an outbreak in central Brazil, 2012-2013. METHODS: We analyzed paired samples from 40 patients with severe dengue and 20 patients with dengue. Eligibility criteria were: IgM, NS1Ag and/or RT-PCR positivity and positive IgG result. Plaque reduction neutralization test (PRNT50) from DENV-1 to DENV-4 was performed to identify serotype-specific NAbs response. An infecting serotype was defined as ≥4-fold increase in DENV NAbs in paired samples. Monotypic response was classified as PRNT50 ≥ 1/20 to only one DENV serotype, and multitypic response was considered to be PRNT50 ≥ 1/20 to two or more serotypes simultaneously. RESULTS: Patients were mainly adults. Virological dengue infection was confirmed by RT-PCR: DENV-4(n = 14) and DENV-1(n = 10). Forty-four out of 60(73.3 %) patients had NAbs to DENV-4, DENV-1(68.3 %), DENV-2(68.3 %) and DENV-3(61.6 %) respectively. Fifteen percent of the cases presented monotypic response, whereas 85 % had multitypic response. DENV-4 infected-patients presented the greatest difference in PRNT50 titers compared with other serotypes. Pre-existing DENV NAbs was not correlated with disease severity. This was the first time that DENV-4 was implicated in an epidemic in the region. CONCLUSION: Our data indicates high exposure of multiple DENV serotypes in all age groups in the pre-dengue vaccine era and also previous to Zika virus introduction in Brazil.

Primary dengue haemorrhagic fever in patients from northeast of Brazil is associated with high levels of interferon-ß during acute phase.

Dengue is an acute febrile disease caused by the mosquito-borne dengue virus (DENV) that according to clinical manifestations can be classified as asymptomatic, mild or severe dengue. Severe dengue cases have been associated with an unbalanced immune response characterised by an over secretion of inflammatory cytokines. In the present study we measured type I interferon (IFN-I) transcript and circulating levels in primary and secondary DENV infected patients. We observed that dengue fever (DF) and dengue haemorrhagic fever (DHF) patients express IFN-I differently. While DF and DHF patients express interferon-α similarly (52,71 ± 7,40 and 49,05 ± 7,70, respectively), IFN- ß were associated with primary DHF patients. On the other hand, secondary DHF patients were not able to secrete large amounts of IFN- ß which in turn may have influenced the high-level of viraemia. Our results suggest that, in patients from our cohort, infection by DENV serotype 3 elicits an innate response characterised by higher levels of IFN- ß in the DHF patients with primary infection, which could contribute to control infection evidenced by the low-level of viraemia in these patients. The present findings may contribute to shed light in the role of innate immune response in dengue pathogenesis.

Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil.

Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons.

Development of optical and electrochemical immunodevices for dengue virus detection.

Dengue virus (DENV) is the most prevalent global arbovirus, exhibiting a high worldwide incidence with intensified severity of symptoms and alarming mortality rates. Faced with the limitations of diagnostic methods, an optical and electrochemical biosystem was developed for the detection of DENV genotypes 1 and 2, using cysteine (Cys), cadmium telluride (CdTe) quantum dots, and anti-DENV antibodies. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), surface plasmon resonance (SPR), atomic force microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR) were employed to characterize the immunosensor. The AFM and SPR results demonstrated discernible topographic and angular changes confirming the biomolecular recognition. Different concentrations of DENV-1 and DENV-2 were evaluated (0.05 × 106 to 2.0 × 106 PFU mL-1), resulting in a maximum anodic shift (ΔI%) of 263.67% ± 12.54 for DENV-1 and 63.36% ± 3.68 for DENV-2. The detection strategies exhibited a linear response to the increase in viral concentration. Excellent linear correlations, with R2 values of 0.95391 for DENV-1 and 0.97773 for DENV-2, were obtained across a broad concentration range. Data analysis demonstrated high reproducibility, displaying relative standard deviation values of 3.42% and 3.62% for Cys-CdTe-antibodyDENV-1-BSA and Cys-CdTe-antibodyDENV-2-BSA systems. The detection limits were 0.34 × 106 PFU mL-1 and 0.02 × 106 PFU mL-1, while the quantification limits were set at 1.49 × 106 PFU mL-1 and 0.06 × 106 PFU mL-1 for DENV-1 and DENV-2, respectively. Therefore, the biosensing apparatus demonstrates analytical effectiveness in viral screening and can be considered an innovative solution for early dengue diagnosis, contributing to global public health.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3.

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast-E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli. RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Mannose-binding lectin levels and MBL2 gene polymorphisms are associated with dengue infection in Brazilian children at the early ages.

BACKGROUND: The mannose-binding lectin (MBL) plays an important role in innate immunity. Genetically determined variations in serum levels of MBL may influence the susceptibility and clinical outcome of dengue infection in early life. METHODS: We investigated the MBL2 gene polymorphisms and serum levels of MBL (total and functional) in children with asymptomatic (n=17) and symptomatic (n=29) primary dengue infections and age-matched uninfected children (n=84) enrolled in a birth cohort with dengue in Brazil. Polymorphisms of the MBL2 gene were assessed by reverse transcription-polymerase chain reaction (RT-PCR), whereas the enzyme-linked immunosorbent assay (ELISA) was used to quantify serum levels of MBL. RESULTS: We found that the X allele and YX genotype in the MBL2 were more frequent in the dengue cases than in the control group. Likewise, the LXPA haplotype was exclusively found in dengue cases, thus probably related to dengue infection in our setting. Moreover, we found a higher frequency of the O allele and AO genotype in the control group. Serum levels of total and functional MBL were higher in dengue naïve infants than in dengue cases. CONCLUSIONS: MBL2 variants related to lower production of serum MBL were associated with dengue infection in infants, whereas intermediate to high levels of total and functional serum MBL were associated with protection against dengue infection. These findings highlight the role of MBL2 variants and serum levels of MBL in the susceptibility of children to dengue disease at early ages.

Peripheral spectrum neurological disorder after arbovirus infection is associated with HLA-F variants among Northeastern Brazilians.

INTRODUCTION: Non-classical class I human leukocyte antigens (HLA) molecules are known to modulate the function of cytotoxic cells (NK and T CD8+) during viral infection by interacting with inhibitory/activating receptors. However, little is known about the HLA-E/-F genetic variability on arbovirus infections. METHODS: We evaluated by massive parallel sequencing the full HLA-E/-F genetic diversity among patients infected during the arbovirus (ZIKV, DENV, and CHIKV) outbreak leading to a broad range of neurological complications in the Brazilian State of Pernambuco. In parallel, healthy blood donors from the same area were also studied. Plink and R software were used for genetic association study. To limit the false-positive results and enhance the reliability of the results, we adopted P-values <0.01 as significant levels. RESULTS: Compared to controls, the HLA-F alleles: -1610 C (rs17875375), +1383 G (rs17178385), and +3537 A (rs17875384), all in complete linkage disequilibrium with each other (r2 = 1), were overrepresented in patients presenting peripheral spectrum disorders (PSD). The HLA-F*Distal-D haplotype that harbored the -1610 C allele exhibited a trend increase in PSD group. No associations were found for HLA-E. CONCLUSIONS: Our findings showed that the HLA-F genetic background seems to be more important than HLA-E on the susceptibility to PSD complications.

Cytokines and Soluble HLA-G Levels in the Acute and Recovery Phases of Arbovirus-Infected Brazilian Patients Exhibiting Neurological Complications.

Severe neurological complications following arbovirus infections have been a major concern in seasonal outbreaks, as reported in the Northeast region of Brazil, where the same mosquito transmitted Zika (ZIKV), Dengue (DENV), and Chikungunya (CHIKV) viruses. In this study, we evaluated the levels of 36 soluble markers, including cytokines, chemokines, growth factors, and soluble HLA-G (Luminex and ELISA) in: i) serum and cerebrospinal fluid (CSF), during the acute phase and two years after the infection (recovery phase, only serum), ii) the relationship among all soluble molecules in serum and CSF, and iii) serum of infected patients without neurological complications, during the acute infection. Ten markers (sHLA-G, IL-10, IL-22, IL-8, MIP-1α, MIP-1ß, MCP-1, HGF, VEGF, and IL-1RA) exhibited differential levels between the acute and recovery phases, with pronounced increases in MIP-1α (P<0.0001), MCP-1 (P<0.0001), HGF (P= 0.0001), and VEGF (P<0.0001) in the acute phase. Fourteen molecules (IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13, IL-15, IL-17A, IFN-α, TNF, and G-CSF) exhibited distinct levels between arbovirus patients presenting or not neurological complications. IL-8, EGF, IL-6, and MCP-1 levels were increased in CSF, while RANTES and Eotaxin levels were higher in serum. Soluble serum (IL-22, RANTES, Eotaxin) and CSF (IL-8, EGF, IL-3) mediators may discriminate putative risks for neurological complications following arbovirus infections. Neurological complications were associated with the presence of a predominant inflammatory profile, whereas in non-complicated patients an anti-inflammatory profile may predominate. Mediators associated with neuroregeneration (EGF and IL-3) may be induced in response to neurological damage. Broad spectrum immune checkpoint molecules (sHLA-G) interact with cytokines, chemokines, and growth factors. The identification of soluble markers may be useful to monitor neurological complications and may aid in the development of novel therapies against neuroinflammation.

Zika-related adverse outcomes in a cohort of pregnant women with rash in Pernambuco, Brazil.

BACKGROUND: While Zika virus (ZIKV) is now widely recognized as a teratogen, the frequency and full spectrum of adverse outcomes of congenital ZIKV infection remains incompletely understood. METHODS: Participants in the MERG cohort of pregnant women with rash, recruited from the surveillance system from December/2015-June/2017. Exposure definition was based on a combination of longitudinal data from molecular, serologic (IgM and IgG3) and plaque reduction neutralization tests for ZIKV. Children were evaluated by a team of clinical specialists and by transfontanelle ultrasound and were classified as having microcephaly and/or other signs/symptoms consistent with congenital Zika syndrome (CZS). Risks of adverse outcomes were quantified according to the relative evidence of a ZIKV infection in pregnancy. FINDINGS: 376 women had confirmed and suspected exposure to ZIKV. Among evaluable children born to these mothers, 20% presented with an adverse outcome compatible with exposure to ZIKV during pregnancy. The absolute risk of microcephaly was 2.9% (11/376), of calcifications and/or ventriculomegaly was 7.2% (13/180), of additional neurologic alterations was 5.3% (13/245), of ophthalmologic abnormalities was 7% (15/214), and of dysphagia was 1.8% (4/226). Less than 1% of the children experienced abnormalities across all of the domains simultaneously. Interpretation: Although approximately one-fifth of children with confirmed and suspected exposure to ZIKV in pregnancy presented with at least one abnormality compatible with CZS, the manifestations presented more frequently in isolation than in combination. Due to the rare nature of some outcomes and the possibility of later manifestations, large scale individual participant data meta-analysis and the long-term evaluation of children are imperative to identify the full spectrum of this syndrome and to plan actions to reduce damages.

Endocrine Dysfunction in Children with Zika-Related Microcephaly Who Were Born during the 2015 Epidemic in the State of Pernambuco, Brazil.

Congenital viral infections and the occurrence of septo-optic dysplasia, which is a combination of optic nerve hypoplasia, abnormal formation of structures along the midline of the brain, and pituitary hypofunction, support the biological plausibility of endocrine dysfunction in Zika-related microcephaly. In this case series we ascertained the presence and describe endocrine dysfunction in 30 children with severe Zika-related microcephaly from the MERG Pediatric Cohort, referred for endocrinological evaluation between February and August 2019. Of the 30 children, 97% had severe microcephaly. The average age at the endocrinological consultation was 41 months and 53% were female. The most frequently observed endocrine dysfunctions comprised short stature, hypothyroidism, obesity and variants early puberty. These dysfunctions occurred alone 57% or in combination 43%. We found optic nerve hypoplasia (6/21) and corpus callosum hypoplasia (20/21). Seizure crises were reported in 86% of the children. The most common-and clinically important-endocrine dysfunctions were pubertal dysfunctions, thyroid disease, growth impairment, and obesity. These dysfunctions require careful monitoring and signal the need for endocrinological evaluation in children with Zika-related microcephaly, in order to make early diagnoses and implement appropriate treatment when necessary.

Neurological disease in adults with Zika and chikungunya virus infection in Northeast Brazil: a prospective observational study.

BACKGROUND: Since 2015, the arthropod-borne viruses (arboviruses) Zika and chikungunya have spread across the Americas causing outbreaks, accompanied by increases in immune-mediated and infectious neurological disease. The spectrum of neurological manifestations linked to these viruses, and the importance of dual infection, are not known fully. We aimed to investigate whether neurological presentations differed according to the infecting arbovirus, and whether patients with dual infection had a different disease spectrum or severity. METHODS: We report a prospective observational study done during epidemics of Zika and chikungunya viruses in Recife, Pernambuco, a dengue-endemic area of Brazil. We recruited adults aged 18 years or older referred to Hospital da Restauração, a secondary-level and tertiary-level hospital, with suspected acute neurological disease and a history of suspected arboviral infection. We looked for evidence of Zika, chikungunya, or dengue infection by viral RNA or specific IgM antibodies in serum or CSF. We grouped patients according to their arbovirus laboratory diagnosis and then compared demographic and clinical characteristics. FINDINGS: Between Dec 4, 2014, and Dec 4, 2016, 1410 patients were admitted to the hospital neurology service; 201 (14%) had symptoms consistent with arbovirus infection and sufficient samples for diagnostic testing and were included in the study. The median age was 48 years (IQR 34-60), and 106 (53%) were women. 148 (74%) of 201 patients had laboratory evidence of arboviral infection. 98 (49%) of them had a single viral infection (41 [20%] had Zika, 55 [27%] had chikungunya, and two [1%] had dengue infection), whereas 50 (25%) had evidence of dual infection, mostly with Zika and chikungunya viruses (46 [23%] patients). Patients positive for arbovirus infection presented with a broad range of CNS and peripheral nervous system (PNS) disease. Chikungunya infection was more often associated with CNS disease (26 [47%] of 55 patients with chikungunya infection vs six [15%] of 41 with Zika infection; p=0·0008), especially myelitis (12 [22%] patients). Zika infection was more often associated with PNS disease (26 [63%] of 41 patients with Zika infection vs nine [16%] of 55 with chikungunya infection; p≤0·0001), particularly Guillain-Barré syndrome (25 [61%] patients). Patients with Guillain-Barré syndrome who had Zika and chikungunya dual infection had more aggressive disease, requiring intensive care support and longer hospital stays, than those with mono-infection (median 24 days [IQR 20-30] vs 17 days [10-20]; p=0·0028). Eight (17%) of 46 patients with Zika and chikungunya dual infection had a stroke or transient ischaemic attack, compared with five (6%) of 96 patients with Zika or chikungunya mono-infection (p=0·047). INTERPRETATION: There is a wide and overlapping spectrum of neurological manifestations caused by Zika or chikungunya mono-infection and by dual infections. The possible increased risk of acute cerebrovascular disease in patients with dual infection merits further investigation. FUNDING: Fundação do Amparo a Ciência e Tecnologia de Pernambuco (FACEPE), EU's Horizon 2020 research and innovation programme, National Institute for Health Research. TRANSLATIONS: For the Portuguese and Spanish translations of the abstract see Supplementary Materials section.

Laboratory diagnosis of Zika virus.

The recommendations for laboratory diagnosis of ZIKV infection are the detection of viral-RNA by molecular methods, detection of ZIKV-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies by serologic tests and the plaque reduction neutralization test (PRNT) for confirmation of positive IgM results, in pregnant women. In the acute phase of disease ZIKV may be detected in blood (whole blood, serum, plasma), urine, saliva, cerebrospinal fluid (CSF), and other fluids; in urine, the virus may be detected over a longer period, viz., 15-20 days from the onset of symptoms. An accurate laboratory diagnosis requires combining serologic data to molecular testing, as well as clinical and epidemiological criteria, especially for pregnant women and children born with Zika congenital syndromes.

Association of Severe Hydrocephalus With Congenital Zika Syndrome.

Importance: Hydrocephalus is a treatable but potentially fatal complication that has not been previously described in congenital Zika syndrome (CZS). Objective: To describe the clinical features and imaging findings in 24 patients with congenital Zika syndrome (CZS) who developed hydrocephalus. Design, Setting, and Participants: This case series included patients with hydrocephalus who were born in October and November 2015 and followed up until mid-2017 in the 2 largest national referral centers for CZS in Brazil. The participants included consecutively enrolled children with a clinical and laboratorial diagnosis of CZS who developed clinical and/or image findings suggestive of hydrocephalus and who were confirmed to experience increased intracranial hypertension during ventriculoperitoneal shunt procedures. Main Outcomes and Measures: To retrospectively describe clinical and image findings in these 24 patients. Results: This multicenter cohort included 308 patients with CZS; 24 consecutive children were enrolled in this study. These children were aged between 3 to 18 months, and 13 of 24 (54%) were female. All patients presented with at least 1 positive test result for anti-Zika antibodies in cerebrospinal fluid or serum and had classic signs of CZS. At the time of hydrocephalus diagnosis, only 14 of 24 patients (58%) had symptoms and signs suggestive of hydrocephalus (mainly worsening seizures, vomiting, irritability, and/or sudden increase of head circumference percentile). Two of 24 patients (8%) had no symptoms suggestive of hydrocephalus but were found to have reduced brain volume on repeated imaging. Cerebellar or brainstem hypoplasia on baseline imaging were found in 18 of 23 patients (78%). At the second computed tomographic scan, all patients showed a marked increase of ventricular volume, compatible with communicating hydrocephalus, and reduction of brain tissue that was visibly worse than on baseline imaging for the 23 patients with repeated scans. Conclusions and Relevance: We present evidence that hydrocephalus is a complication of CZS in at least a proportion of patients. The clinical spectrum of this condition continues to evolve, but given that presenting signs and symptoms of hydrocephalus can be challenging to recognize in CZS, we provisionally recommend that high suspicion and appropriate monitoring for hydrocephalus should be part of the standard care of patients with CZS.

Association between microcephaly, Zika virus infection, and other risk factors in Brazil: final report of a case-control study.

BACKGROUND: A Zika virus epidemic emerged in northeast Brazil in 2015 and was followed by a striking increase in congenital microcephaly cases, triggering a declaration of an international public health emergency. This is the final report of the first case-control study evaluating the potential causes of microcephaly: congenital Zika virus infection, vaccines, and larvicides. The published preliminary report suggested a strong association between microcephaly and congenital Zika virus infection. METHODS: We did a case-control study in eight public maternity hospitals in Recife, Brazil. Cases were neonates born with microcephaly, defined as a head circumference of 2 SD below the mean. Two controls without microcephaly were matched to each case by expected date of delivery and area of residence. We tested the serum of cases and controls and the CSF of cases for detection of Zika virus genomes with quantitative RT-PCR and for detection of IgM antibodies with capture-IgM ELISA. We also tested maternal serum with plaque reduction neutralisation assays for Zika and dengue viruses. We estimated matched crude and adjusted odds ratios with exact conditional logistic regression to determine the association between microcephaly and Zika virus infection. FINDINGS: We screened neonates born between Jan 15 and Nov 30, 2016, and prospectively recruited 91 cases and 173 controls. In 32 (35%) cases, congenital Zika virus infection was confirmed by laboratory tests and no controls had confirmed Zika virus infections. 69 (83%) of 83 cases with known birthweight were small for gestational age, compared with eight (5%) of 173 controls. The overall matched odds ratio was 73·1 (95% CI 13·0-∞) for microcephaly and Zika virus infection after adjustments. Neither vaccination during pregnancy or use of the larvicide pyriproxyfen was associated with microcephaly. Results of laboratory tests for Zika virus and brain imaging results were available for 79 (87%) cases; within these cases, ten were positive for Zika virus and had cerebral abnormalities, 13 were positive for Zika infection but had no cerebral abnormalities, and 11 were negative for Zika virus but had cerebral abnormalities. INTERPRETATION: The association between microcephaly and congenital Zika virus infection was confirmed. We provide evidence of the absence of an effect of other potential factors, such as exposure to pyriproxyfen or vaccines (tetanus, diphtheria, and acellular pertussis, measles and rubella, or measles, mumps, and rubella) during pregnancy, confirming the findings of an ecological study of pyriproxyfen in Pernambuco and previous studies on the safety of Tdap vaccine administration during pregnancy. FUNDING: Brazilian Ministry of Health, Pan American Health Organization, and Enhancing Research Activity in Epidemic Situations.

Characterization of a dengue patient cohort in Recife, Brazil.

From 2004 to 2006, 658 patients with suspected dengue virus infections were enrolled in a clinical dengue cohort established in Recife, Pernambuco, located at the northeastern region of Brazil. A total of 2,364 blood samples were collected, and serum, plasma, and cells were cryopreserved. Among the suspected cases, 354 (54%) were confirmed as acute DENV-3 infection based on reverse transcription-polymerase chain reaction, virus isolation, and ELISA-IgM. According to WHO criteria, 29.4% of the positive acute cases were classified as dengue fever (DF) and 8.2% of the cases as dengue hemorrhagic fever (DHF), grade 1 or 2. The DHF cases represent 100% of those confirmed in Recife during the period of the study. The dengue cases that did not fulfill the definition of either DHF or DF were classified as DF complicated and accounted for 44.0% of the cases. All the acute cases were classified as either primary or secondary acute dengue virus infections. Secondary infection was predominant in patients with DF; however, there was no predominance of either primary or secondary infections in patients with DHF.