Unequal burden of Zika-associated microcephaly among populations with public and private healthcare in Salvador, Brazil.

OBJECTIVES: To describe the differences in clinical presentation and relative disease burden of congenital Zika syndrome (CZS)-associated microcephaly at 2 large hospitals in Salvador, Brazil that serve patients of different socioeconomic status (SES). METHODS: Clinical and serologic data were collected prospectively from pregnant women and their infants, who delivered at 2 study centers during the 2015-2016 Zika virus (ZIKV) epidemic in Salvador, Brazil. RESULTS: Pregnant women from Salvador, Brazil delivering in a low SES hospital had 3 times higher ZIKV exposure rate than women at a high SES hospital. However, different SES hospitals had similar prevalence of infants with CZS-associated microcephaly (10% vs 6%, p = 0.16) after controlling for ZIKV exposure in their mothers. CONCLUSIONS: Our study supports the positive association between low SES, high maternal ZIKV exposure, and high rates of CZS-associated microcephaly.

Evaluation of immunoglobulin M-specific capture enzyme-linked immunosorbent assays and commercial tests for flaviviruses diagnosis by a National Reference Laboratory.

Zika and Dengue viruses present considerable immunological cross-reactivity, resulting in a troublesome serodiagnosis due to occurrence of false positive results. Due to Brazil's wide variety of circulating flaviviruses we aimed to access the use of in house serological tests adapted by National Reference Laboratory for Arboviruses in Brazil and evaluate commercial tests available. We evaluated in house IgM ELISAs for the individual detection of anti-ZIKV, -DENV, and -YFV IgM, against a panel of samples positive for dengue, zika, yellow fever, Rocio, Ilheus, Saint Louis encephalitis, West Nile and chikungunya. We also evaluated two commercial kits for dengue and zika IgM detection recommended by the Brazilian Ministry of Health in 2015. The sensitivity and specificity for the in house ZIKV IgM ELISA was 60.0 % and 88.6 % and for the in house DENV IgM ELISA was 100 % and 82.2 %, respectively. The in house YFV IgM ELISA presented 100 % for both sensitivity and specificity. The Novagnost Zika Virus IgM test presented a sensitivity of 47.3 % and specificity of 85.3 % and the Serion ELISA classic Dengue Virus IgM, 92.8 % and 58.9 %, respectively. Overall, both in house ELISAs for ZIKV and DENV adapted and evaluated here, presented better performances than the commercial kits tested.

Zika Virus Epidemic in Brazil. II. Post-Mortem Analyses of Neonates with Microcephaly, Stillbirths, and Miscarriage.

INTRODUCTION: The recent Zika virus(ZIKV) epidemic in Brazil was characterized by a range of different clinical presentations, particularly microcephaly, Guillain-Barré syndrome, and death. In this context, we determined the causal relationship between fatal microcephaly cases and ZIKV infection. METHODS: Twelve fatal cases of neonates, whose mothers were infected with ZIKV during pregnancy, were examined; cases included nine neonatal deaths due to microcephaly, one miscarriage, and two stillbirths. Tissue samples were obtained from all cases at necropsy and were submitted for virological investigation (RT-qPCR and virus isolation) and/or histopathology (hematoxylin and eosin staining) and immunohistochemical assay for the detection of ZIKV antigens. RESULTS: ZIKV antigens and/or ZIKV RNA were detected in tissue samples of all 12 cases examined. ZIKV was recovered in one case. Results of the virological and immunohistochemical analyses, as well as the anatomic abnormalities and histopathologic changes observed at necropsy on the 12 fatal cases, are presented. CONCLUSIONS: Data from these 12 cases provide strong evidence of the causal relationship between ZIKV and congenital disease in fetuses of women who were infected with the virus during pregnancy.

In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus.

Zika virus (ZIKV) has recently caused a pandemic disease, and many cases of ZIKV infection in pregnant women resulted in abortion, stillbirth, deaths and congenital defects including microcephaly, which now has been proposed as ZIKV congenital syndrome. This study aimed to investigate the in situ immune response profile and mechanisms of neuronal cell damage in fatal Zika microcephaly cases. Brain tissue samples were collected from 15 cases, including 10 microcephalic ZIKV-positive neonates with fatal outcome and five neonatal control flavivirus-negative neonates that died due to other causes, but with preserved central nervous system (CNS) architecture. In microcephaly cases, the histopathological features of the tissue samples were characterized in three CNS areas (meninges, perivascular space, and parenchyma). The changes found were mainly calcification, necrosis, neuronophagy, gliosis, microglial nodules, and inflammatory infiltration of mononuclear cells. The in situ immune response against ZIKV in the CNS of newborns is complex. Despite the predominant expression of Th2 cytokines, other cytokines such as Th1, Th17, Treg, Th9, and Th22 are involved to a lesser extent, but are still likely to participate in the immunopathogenic mechanisms of neural disease in fatal cases of microcephaly caused by ZIKV.

Zika virus epidemic in Brazil. I. Fatal disease in adults: Clinical and laboratorial aspects.

BACKGROUND: Zika virus (ZIKV) was first detected in Brazil in May 2015 and the country experienced an explosive epidemic. However, recent studies indicate that the introduction of ZIKV occurred in late 2013. Cases of microcephaly and deaths associated with ZIKV infection were identified in Brazil in November, 2015. OBJECTIVES: To determine the etiology of three fatal adult cases. STUDY DESIGN: Here we report three fatal adult cases of ZIKV disease. ZIKV infection in these patients was confirmed by cells culture and/or real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and by antigen detection using immunohistochemical assay. Samples of brain and other selected organs taken at autopsy from three patients were also analyzed by histopathological and immunohistological examination. RESULTS: The first patient, a 36-year-old man with lupus and receiving prednisone therapy, developed a fulminant ZIKV infection. At autopsy, RT-qPCR of blood and tissues was positive for ZIKV RNA, and the virus was cultured from an organ homogenate. The second patient, a previously healthy female, 16 years of age, presented classic symptoms of Zika fever, but later developed severe thrombocytopenia, anemia and hemorrhagic manifestations and died. A blood sample taken on the seventh day of her illness was positive RT-PCR for ZIKV RNA and research in the serum was positive for antinuclear factor fine speckled (1/640), suggesting Evans syndrome (hemolytic anemia an autoimmune disorder with immune thrombocytopenic purpura) secondary to ZIKV infection. The third patient was a 20-year-old woman hospitalized with fever, pneumonia and hemorrhages, who died on 13days after admission. Histopathological changes were observed in all viscera examined. ZIKV antigens were detected by immunohistochemistry in viscera specimens of patients 1 and 3. These three cases demonstrate other potential complications of ZIKV infection, in addition to microcephaly and Guillain-Barre syndrome (GBS), and they suggest that individuals with immune suppression and/or autoimmune disorders may be at higher risk of developing severe disease, if infected with ZIKV.

West Nile Virus Encephalitis: The First Human Case Recorded in Brazil.

A Brazilian ranch worker with encephalitis and flaccid paralysis was evaluated in the regional Acute Encephalitis Syndromic Surveillance Program. This was the first Brazilian patient who met the Centers for Disease Control and Prevention (CDC) confirmation criteria for West Nile virus disease. Owing to the overlapping of neurological manifestations attributable to several viral infections of the central nervous system, this report exemplifies the importance of human acute encephalitis surveillance. The syndromic approach to human encephalitis cases may enable early detection of the introduction of unusual virus or endemic occurrence of potentially alarming diseases within a region.

Genetic analysis of members of the species Oropouche virus and identification of a novel M segment sequence.

Oropouche virus (OROV) is a public health threat in South America, and in particular in northern Brazil, causing frequent outbreaks of febrile illness. Using a combination of deep sequencing and Sanger sequencing approaches, we determined the complete genome sequences of eight clinical isolates that were obtained from patient sera during an Oropouche fever outbreak in Amapa state, northern Brazil, in 2009. We also report the complete genome sequences of two OROV reassortants isolatd from two marmosets in Minas Gerais state, south-east Brazil, in 2012 that contained a novel M genome segment. Interestingly, all 10 isolates possessed a 947 nt S segment that lacked 11 residues in the S-segment 3' UTR compared with the recently redetermined Brazilian prototype OROV strain BeAn19991. OROV maybe circulating more widely in Brazil and in the non-human primate population than previously appreciated, and the identification of yet another reassortant highlights the importance of bunyavirus surveillance in South America.

Emergence of new insect-restrictive viruses in the Amazon region.

The complete genome was determined for 12 viruses isolated from 8 different pools of mosquitoes (Culex sp. and Psorophora ferox) collected at Brejeira farm, Canaan dos Carajas, Para state in northern Brazil. Eight of the viruses were distantly related to Piura virus, hereafter designated as Brejeira virus; the other 4 were similar to Wallerfield virus.

Air travel is associated with intracontinental spread of dengue virus serotypes 1-3 in Brazil.

Dengue virus and its four serotypes (DENV-1 to DENV-4) infect 390 million people and are implicated in at least 25,000 deaths annually, with the largest disease burden in tropical and subtropical regions. We investigated the spatial dynamics of DENV-1, DENV-2 and DENV-3 in Brazil by applying a statistical framework to complete genome sequences. For all three serotypes, we estimated that the introduction of new lineages occurred within 7 to 10-year intervals. New lineages were most likely to be imported from the Caribbean region to the North and Northeast regions of Brazil, and then to disperse at a rate of approximately 0.5 km/day. Joint statistical analysis of evolutionary, epidemiological and ecological data indicates that aerial transportation of humans and/or vector mosquitoes, rather than Aedes aegypti infestation rates or geographical distances, determine dengue virus spread in Brazil.

Genomic and phylogenetic characterization of Brazilian yellow fever virus strains.

Globally, yellow fever virus infects nearly 200,000 people, leading to 30,000 deaths annually. Although the virus is endemic to Latin America, only a single genome from this region has been sequenced. Here, we report 12 Brazilian yellow fever virus complete genomes, their genetic traits, phylogenetic characterization, and phylogeographic dynamics. Variable 3' noncoding region (3'NCR) patterns and specific mutations throughout the open reading frame altered predicted secondary structures. Our findings suggest that whereas the introduction of yellow fever virus in Brazil led to genotype I-predominant dispersal throughout South and Central Americas, genotype II remained confined to Bolivia, Peru, and the western Brazilian Amazon.

Evaluation of an immunoglobulin M-specific capture enzyme-linked immunosorbent assay for rapid diagnosis of dengue infection.

Various assays have been developed to diagnose dengue virus infection, relying on techniques from the fields of serology and molecular biology. Many of these assays have been successful, but there is still an urgent need for accurate, simple and rapid diagnostic assays to diagnose dengue virus infection and to assist in patient management. Using a panel of well-characterized sera and a collection of retrospective samples obtained during the dengue epidemics that occurred in Belém, Brazil, between 2002 and 2009, a modified immunoglobulin M-specific capture enzyme-linked immunosorbent assay (Rapid-MAC-ELISA) was evaluated and compared with the "gold standard" MAC-ELISA, in order to assess the specificity, sensitivity, stability, reproducibility and cost-effectiveness of this new assay. These results demonstrated that the Rapid-MAC-ELISA is comparable to the MAC-ELISA in terms of sensitivity and specificity and is highly reproducible; additionally, it is easily performed, less expensive than other available formats and can be completed within three hours. Furthermore, the Rapid-MAC-ELISA can be used for the diagnosis of dengue virus infections in resource-limited areas where dengue is endemic.

Molecular epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon: genetic divergence and dispersal.

Saint Louis encephalitis virus (SLEV), a member of the genus Flavivirus (family Flaviviridae), is an encephalitogenic arbovirus broadly distributed in the Americas. Phylogenetic analysis based on the full-length E gene sequences obtained for 30 Brazilian SLEV strains was performed using different methods including Bayesian and relaxed molecular clock approaches. A new genetic lineage was suggested, hereafter named genotype VIII, which co-circulates with the previously described genotype V in the Brazilian Amazon region. Genotypes II and III were restricted to São Paulo state (South-east Atlantic rainforest ecosystem). The analysis also suggested the emergence of an SLEV common ancestor between 1875 and 1973 (mean of 107 years ago), giving rise to two major genetic groups: genotype II, more prevalent in the North America, and a second group comprising the other genotypes (I and III-VIII), broadly dispersed throughout the Americas, suggesting that SLEV initially emerged in South America and spread to North America. In conclusion, the current study demonstrates the high genetic variability of SLEV and its geographical dispersion in Brazil and other New World countries.

Mayaro fever virus, Brazilian Amazon.

In February 2008, a Mayaro fever virus (MAYV) outbreak occurred in a settlement in Santa Barbara municipality, northern Brazil. Patients had rash, fever, and severe arthralgia lasting up to 7 days. Immunoglobulin M against MAYV was detected by ELISA in 36 persons; 3 MAYV isolates sequenced were characterized as genotype D.

Oropouche fever epidemic in Northern Brazil: epidemiology and molecular characterization of isolates.

BACKGROUND: Oropouche fever virus is an important arbovirus associated with febrile disease that re-emerged in 2006 in several municipalities of Pará State, Bragantina region, Amazon, Brazil, 26 years after the last epidemic. OBJECTIVE: To investigate an Oropouche fever outbreak in this region. STUDY DESIGN: A serologic survey and prospective study of acute febrile cases were performed in Magalhães Barata (urban and rural areas) and Maracanã (rural area) municipalities. Serology (IgM-ELISA and hemagglutination-inhibition [HI]), virus isolation, RT-PCR and real-time-PCR were used to confirm Oropouche virus (OROV) as responsible for the febrile outbreaks. RESULTS: Real-time-PCR showed high titers of OROV in acute-phase serum samples from febrile patients. From 113 of 119 acutely febrile patients with paired serum samples, OROV infections was confirmed by serologic conversion (n=76) or high titers (n=37) for both HI and IgM-ELISA. Patients had a febrile disease characterized by headache, chills, dizziness, photophobia, myalgia, nausea, and vomiting. Females and children under 15 years of age were most affected. Nucleotide sequencing of six OROV isolates identified that genotype II was associated with the human disease epidemic. CONCLUSIONS: Oropouche fever, which has re-emerged in the Bragantina region in eastern Amazon 26 years after the last epidemic, is caused by genotype II, a lineage previously found only in Peru and western Brazil.

Genetic characterization of orthobunyavirus Melao, strains BE AR633512 and BE AR8033, and experimental infection in golden hamsters (Mesocricetus auratus).

Melao virus (MELV) strains BE AR8033 and BE AR633512 were isolated from pools of Ochlerotatus scapularis mosquitoes in Belém, Pará State (1955), and Alta Floresta, Rondônia State (2000), Brazil, respectively. The aim of the present study was to molecularly characterize these strains and to describe the histopathological, biochemical and immunological changes in golden hamsters (Mesocricetus auratus) following intraperitoneal injection of MELV strains. Hamsters were susceptible to both of the MELV strains studied. Viraemia was observed 3-6 days post-infection (p.i.) for BE AR633512 and only on the second day p.i. for BE AR8033. Neutralizing antibodies against both strains were detected in blood samples obtained at 5 days p.i. and persisted up to 30 days p.i. Aspartate aminotransferase, alanine aminotransferase and blood urea nitrogen were significantly altered in animals infected with the two MELV strains, while creatinine was only altered in animals inoculated with BE AR633512. Histopathological changes were observed in the central nervous system, liver, kidney and spleen of hamsters, and infection was confirmed by detection of specific MELV antigens by immunohistochemistry. Strain BE AR633512 caused more severe tissue damage than strain BE AR8033, showing increased neurovirulence and pathogenicity. Genetic analysis based on the full-length sequences of the glycoprotein (Gn and Gc) and nucleocapsid protein (N) genes revealed high levels of homology between the MELV strains. Interestingly, the greatest genetic divergence was found for the Gn gene of strain BE AR633512, in which several synonymous and non-synonymous mutations causing changes in RNA secondary structure were observed. Further studies will be necessary to investigate the role of Gn and Gc mutations in the MELV pathogenicity.

Genetic ancestry and income are associated with dengue hemorrhagic fever in a highly admixed population.

To test whether African ancestry is protective for severe dengue, we genotyped 49 hospitalized cases of dengue hemorrhagic fever (DHF) as well as 293 neighborhood cases of dengue fever and 294 asymptomatic controls in Salvador, Bahia, Brazil. Ancestry-informative markers and 282 unlinked SNPs not associated with the clinical presentation of dengue were used to estimate ancestry. After controlling for income, both self-defined Afro-Brazilian ethnicity and African ancestry were protective for DHF (P=0.02, OR=0.28 and P=0.02, OR=0.13, respectively). Income or an index of income indicators, however, was also independently associated with the diagnosis of DHF.

Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum.

We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Genetic divergence and dispersal of yellow fever virus, Brazil.

An analysis of 79 yellow fever virus (YFV) isolates collected from 1935 to 2001 in Brazil showed a single genotype (South America I) circulating in the country, with the exception of a single strain from Rondonia, which represented South America genotype II. Brazilian YFV strains have diverged into two clades; an older clade appears to have become extinct and another has become the dominant lineage in recent years. Pairwise nucleotide diversity between strains ranged from 0% to 7.4%, while amino acid divergence ranged from 0% to 4.6%. Phylogenetic analysis indicated traffic of virus variants through large geographic areas and suggested that migration of infected people may be an important mechanism of virus dispersal. Isolation of vaccine virus from a patient with a fatal case suggests that vaccine-related illness may have been misdiagnosed in the past.

Isolations of yellow fever virus from Haemagogus leucocelaenus in Rio Grande do Sul State, Brazil.

Following howling monkey (Alouatta caraya) deaths and yellow fever (YF) antigen detection by immunohistochemistry in the liver sample of a dead monkey in April and May 2001 in the municipalities of Garruchos and Santo Antônio das Missões, Rio Grande do Sul State, Brazil, epidemiological field investigations were initiated. Two strains of YF virus were isolated in suckling mice from 23 Haemagogus (Conopostegus) leucocelaenus Dyar & Shannon mosquitoes collected from the study sites. The YF virus was isolated from this species in the 1930s in Brazil and in the 1940s in Colombia. No human cases were reported during the current epizootic outbreak. The YF virus isolation and the absence of Hg. (Haemagogus) janthinomys Dyar from the area suggest that Hg. leucocelaenus may be a secondary YF vector and play an important role in the epidemiology of this disease in the Southern Cone.

Isolation of dengue 2 virus from a patient with central nervous system involvement (transverse myelitis).

A dengue fever case is described in a 58-year-old male patient with febrile illness and thrombocytopenia complicated by neurological involvement characterized by transverse myelitis followed by weakness of both legs and flaccid paralysis. Muscle strength was much diminished and bilateral areflexia was observed. Dengue 2 (DEN-2) virus was isolated and the patient sero-converted by hemagglutination-inhibition and IgM-ELISA tests. The RT-PCR test was positive to DEN-2 in acute phase serum and culture supernatant, but negative in the cerebrospinal fluid. After three weeks of hospitalization the patient was discharged. No other infectious agent was detected in the blood and cerebrospinal fluid samples. The patient had full recovery from paralysis six months after the onset of DEN-2 infection.