Molecular Diagnosis of Dengue.

Several protocols for genomic amplification using reverse transcription followed by polymerase chain reaction (RT-PCR), important in the identification of the infecting serotype, have been used in the rapid diagnosis of Dengue Virus (DENV) infections. The qualitative protocol described by Lanciotti et al. (J Clin Microbiol 30: 545-551, 1992) suggested by WHO detects the four DENV serotypes simultaneously in one procedure "semi-nested," generating amplified products with specific sizes in base pairs for each serotype and it has been the most used in the past two decades. However, advances in molecular diagnosis have enabled the development of RT-PCR in real time (qRT-PCR) based on the use of dyes and probes (SYBR green and TaqMan), which is performed in a single step and is capable of providing quantitative data. In addition to quantification, the advantages of qRT-PCR over conventional RT-PCR include speed, greater sensitivity and specificity, and low rate of false positives. Several protocols for the diagnosis and/or quantification of DENV have already been described. Non-PCR-based methods such as reverse transcription loop-mediated isothermal amplification have shown high sensitivities and specificities. RT-PCR and qRT-PCR techniques can be performed using serum, plasma, infected cells, mosquitoes, fresh, and paraffin-embedded tissues. However, despite fast and accurate, they are limited to samples collected during the acute phase of infection (up to 7 days after the onset of symptoms) and require specialized equipment and trained staff.

Serological Diagnosis of Dengue.

A reliable and specific diagnosis is imperative in viral diagnosis, both for clinical management and surveillance, and to ensure that early treatment and control measures are carried out. The number of days of illness is important to choose the most appropriate method to be used and for the correct interpretation of the results obtained. Specific IgM is elicited after that period, indicating an active infection and usually lasts up to 3 months. However, in DENV secondary infections, IgM levels may be significantly lower or undetectable. After 10-12 days, a lifetime specific IgG is produced. Routinely, the laboratory diagnosis of DENV infections can be performed by viral isolation and/or detection of viral nucleic acid, serological assays for the detection of specific antibodies (IgM/IgG), antigen (NS1) and the detection of viral antigens in tissues, which are suitable during certain phases of the disease. For serological diagnosis, serum, plasma, or cerebrospinal fluid (CSF) samples may be investigated. If the test is carried out a few days after collection, the specimens can be stored at 4 °C, since the immunoglobulins are stable in serum or plasma. If the storage period is extended, the material must be kept at -20 °C or -70 °C. In serology, several methods can be used to detect specific viral antigens and/or antibodies, produced by the host in response to DENV infection. Routinely, serological tests include the hemagglutination inhibition (HI) assay, the plaque reduction neutralizing test (PRNT), the gold standard assay for dengue immune response characterization, and ELISAs to detect IgM (MAC-ELISA) and IgG (IgG-ELISA).

Increased circulating levels of High Mobility Group Box 1 (HMGB1) in acute-phase Chikungunya virus infection: Potential disease biomarker.

BACKGROUND: Chikungunya virus (CHIKV) causes a febrile syndrome with intense and debilitating arthralgia that can persist for several months or years after complete virus clearance. As there is no specific antiviral treatment or vaccine against CHIKV, identification of serological markers that help clinical management of CHIKV patients is urgent. The High Mobility Group Box 1 (HMGB1) protein is secreted to extracellular milieu and triggers an intense inflammatory process by inducing the overexpression of pro-inflammatory cytokines. HMGB1 plays an important role in several virus diseases as well as in rheumatoid arthritis. OBJECTIVES: This study focus on the investigation of HMGB1 serum levels in a sera panel from CHIKV-infected patients in an attempt to assess its potential as a biomarker for chikungunya clinical management. STUDY DESIGN: Eighty CHIKV-positive samples and 32 samples from healthy donors were subjected to a quantitative HMGB1 ELISA assay to assess the HMGB1 circulating levels. RESULTS: HMGB1 levels were significantly higher in CHIKV-positive samples (516.12 ng/mL, SEM ± 48.83 ng/mL) compared to negative control (31.20 ng/mL, SEM ± 3.24 ng/mL, p < 0.0001). Circulating levels of HMGB1 persisted elevated during the whole acute-phase of disease and correlated with virus titer (p < 0.05). CONCLUSIONS: The present study is the first to describe increased serum levels of HMGB1 in CHIKV infection and its positive correlation with virus titer, suggesting its potential use as a biomarker for diagnosis and treatment of chikungunya fever.

Viral and Prion Infections Associated with Central Nervous System Syndromes in Brazil.

Virus-induced infections of the central nervous system (CNS) are among the most serious problems in public health and can be associated with high rates of morbidity and mortality, mainly in low- and middle-income countries, where these manifestations have been neglected. Typically, herpes simplex virus 1 and 2, varicella-zoster, and enterovirus are responsible for a high number of cases in immunocompetent hosts, whereas other herpesviruses (for example, cytomegalovirus) are the most common in immunocompromised individuals. Arboviruses have also been associated with outbreaks with a high burden of neurological disorders, such as the Zika virus epidemic in Brazil. There is a current lack of understanding in Brazil about the most common viruses involved in CNS infections. In this review, we briefly summarize the most recent studies and findings associated with the CNS, in addition to epidemiological data that provide extensive information on the circulation and diversity of the most common neuro-invasive viruses in Brazil. We also highlight important aspects of the prion-associated diseases. This review provides readers with better knowledge of virus-associated CNS infections. A deeper understanding of these infections will support the improvement of the current surveillance strategies to allow the timely monitoring of the emergence/re-emergence of neurotropic viruses.

The Usefulness of a Duplex RT-qPCR during the Recent Yellow Fever Brazilian Epidemic: Surveillance of Vaccine Adverse Events, Epizootics and Vectors.

From 2016 to 2018, Brazil faced the biggest yellow fever (YF) outbreak in the last 80 years, representing a risk of YF reurbanization, especially in megacities. Along with this challenge, the mass administration of the fractionated YF vaccine dose in a naïve population brought another concern: the possibility to increase YF adverse events associated with viscerotropic (YEL-AVD) or neurological disease (YEL-AND). For this reason, we developed a quantitative real time RT-PCR (RT-qPCR) assay based on a duplex TaqMan protocol to distinguish broad-spectrum infections caused by wild-type yellow fever virus (YFV) strain from adverse events following immunization (AEFI) by 17DD strain during the vaccination campaign used to contain this outbreak. A rapid and more accurate RT-qPCR assay to diagnose YFV was established, being able to detect even different YFV genotypes and geographic strains that circulate in Central and South America. Moreover, after testing around 1400 samples from human cases, non-human primates and mosquitoes, we detected just two YEL-AVD cases, confirmed by sequencing, during the massive vaccination in Brazilian Southeast region, showing lower incidence than AEFI as expected.

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.

BALB/c mice infected with DENV-2 strain 66985 by the intravenous route display injury in the central nervous system.

Dengue is a mild flu-like arboviral illness caused by dengue virus (DENV) that occurs in tropical and subtropical countries. An increasing number of reports have been indicating that dengue is also associated to neurological manifestations, however, little is known regarding the neuropathogenesis of the disease. Here, using BALB/c mice intravenously infected with DENV-2 strain 66985, we demonstrated that the virus is capable of invading and damaging the host's central nervous system (CNS). Brain and cerebellum of infected animals revealed histological alterations such as the presence of inflammatory infiltrates, thickening of pia matter and disorganization of white matter. Additionally, it was also seen that infection lead to altered morphology of neuroglial cells and apoptotic cell death. Such observations highlighted possible alterations that DENV may promote in the host's CNS during a natural infection, hence, helping us to better understand the neuropathological component of the disease.

Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study.

BACKGROUND: The incidence of microcephaly in Brazil in 2015 was 20 times higher than in previous years. Congenital microcephaly is associated with genetic factors and several causative agents. Epidemiological data suggest that microcephaly cases in Brazil might be associated with the introduction of Zika virus. We aimed to detect and sequence the Zika virus genome in amniotic fluid samples of two pregnant women in Brazil whose fetuses were diagnosed with microcephaly. METHODS: In this case study, amniotic fluid samples from two pregnant women from the state of Paraíba in Brazil whose fetuses had been diagnosed with microcephaly were obtained, on the recommendation of the Brazilian health authorities, by ultrasound-guided transabdominal amniocentesis at 28 weeks' gestation. The women had presented at 18 weeks' and 10 weeks' gestation, respectively, with clinical manifestations that could have been symptoms of Zika virus infection, including fever, myalgia, and rash. After the amniotic fluid samples were centrifuged, DNA and RNA were extracted from the purified virus particles before the viral genome was identified by quantitative reverse transcription PCR and viral metagenomic next-generation sequencing. Phylogenetic reconstruction and investigation of recombination events were done by comparing the Brazilian Zika virus genome with sequences from other Zika strains and from flaviviruses that occur in similar regions in Brazil. FINDINGS: We detected the Zika virus genome in the amniotic fluid of both pregnant women. The virus was not detected in their urine or serum. Tests for dengue virus, chikungunya virus, Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus, HIV, Treponema pallidum, and parvovirus B19 were all negative. After sequencing of the complete genome of the Brazilian Zika virus isolated from patient 1, phylogenetic analyses showed that the virus shares 97-100% of its genomic identity with lineages isolated during an outbreak in French Polynesia in 2013, and that in both envelope and NS5 genomic regions, it clustered with sequences from North and South America, southeast Asia, and the Pacific. After assessing the possibility of recombination events between the Zika virus and other flaviviruses, we ruled out the hypothesis that the Brazilian Zika virus genome is a recombinant strain with other mosquito-borne flaviviruses. INTERPRETATION: These findings strengthen the putative association between Zika virus and cases of microcephaly in neonates in Brazil. Moreover, our results suggest that the virus can cross the placental barrier. As a result, Zika virus should be considered as a potential infectious agent for human fetuses. Pathogenesis studies that confirm the tropism of Zika virus for neuronal cells are warranted. FUNDING: Consellho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Impact of the emergence and re-emergence of different dengue viruses' serotypes in Rio de Janeiro, Brazil, 2010 to 2012.

BACKGROUND: Rio de Janeiro (RJ) has been of major importance for the epidemiology of dengue viruses (DENVs) in Brazil. After the DENV 1-4 introductions in 1986, 1990, 2000 and 2011, respectively, the state has suffered explosive epidemics. We aimed to describe laboratorial, epidemiological and clinical aspects due to the emergence and re-emergence of distinct DENV in a 2-year period. METHODS: Suspected dengue cases (n=2833), including 190 fatal cases, were submitted to virus isolation, RT-PCR and non-structural 1 (NS1) antigen capture ELISA, IgM antibody-capture (MAC)-ELISA and IgG-ELISA. RESULTS: Case confirmation was 47.5%. MAC-ELISA confirmed 32.6% of the cases, RT-PCR confirmed 56.3%; DENV was recovered in 33.1% of samples inoculated and NS1 ELISA confirmed 27.5% of the cases. DENV-2 was prevalent in 2010, DENV-1 in 2011 and DENV-4 in 2012. Individuals infected by DENV-3 and over 65 years-old, and children 15 years-old and under infected by DENV-2 had a significantly higher risk of developing a severe disease. Fatal cases confirmed (n=67) were due to DENV-1 (26.8%), DENV-2 (14.9%), DENV-3 (2.9%) and DENV-4 (7.4%). CONCLUSIONS: It has been shown here that viral emergences or re-emergences may play different roles in the disease epidemiology, especially when many serotypes co-circulate.

First report of multiple lineages of dengue viruses type 1 in Rio de Janeiro, Brazil.

BACKGROUND: In Brazil dengue has been a major public health problem since DENV-1 introduction and spread in 1986. After a low or silent co-circulation, DENV-1 re-emerged in 2009 causing a major epidemic in the country in 2010 and 2011. In this study, the phylogeny of DENV-1 strains isolated in RJ after its first introduction in 1986 and after its emergence in 2009 and 2010 was performed in order to document possible evolutionary patterns or introductions in a re-emergent virus. FINDINGS: The analysis of the E gene sequences demonstrated that DENV-1 isolated during 2009/2010 still belong to genotype V (Americas/Africa) but grouping in a distinct clade (lineage II) of that represented by earlier DENV-1 (lineage I). However, strains isolated in 2011 grouped together forming another distinct clade (lineage III). CONCLUSIONS: The monitoring of DENV is important to observe the spread of potentially virulent strains as well to evaluate its impact over the population during an outbreak. Whether explosive epidemics reported in Brazil caused mainly by DENV-1 was due to lineage replacement, or due the population susceptibility to this serotype which has not circulated for almost a decade or even due to the occurrence of secondary infections in a hyperendemic country, is not clear. This is the first report of multiple lineages of DENV-1 detected in Brazil.

Recombinant polypeptide antigen-based immunoglobulin G enzyme-linked immunosorbent assay for serodiagnosis of dengue.

We have developed an indirect enzyme-linked immunosorbent assay for detection of anti-dengue virus (DENV) immunoglobulin G antibodies using four recombinant DENV envelope polypeptides as antigens, which demonstrated a sensitivity of 89.4% and a specificity of 93.3%. These easily produced antigens are a feasible, cost-effective alternative for generating reagents for dengue serological tests.

Adenoviruses associated with acute gastroenteritis in hospitalized and community children up to 5 years old in Rio de Janeiro and Salvador, Brazil.

Acute gastroenteritis is a major source of morbidity and mortality among young children in developed and developing countries. Human adenoviruses (HAdVs), and in particular species F, are related to childhood diarrhoea worldwide. This study presents the results obtained during an investigation of HAdVs causing acute gastroenteritis in children hospitalized in Rio de Janeiro, RJ, Brazil, from April 1996 to September 2003, as well as in children with diarrhoea living in the slums of Salvador, BA, Brazil, from October 2001 to September 2003. A total of 3060 stool samples was analysed by an enzyme immunoassay for rotavirus and adenovirus (EIARA) and 61 (2%) were found to be positive. HAdV presented with low prevalence throughout the year, with a slight but not significant increase in incidence in late summer and early autumn. Children up to 2 years of age were the most frequently affected (79% of all positive samples). All positive samples were analysed further by generic and species-specific HAdV PCR protocols, confirming 100% specificity of this rapid and inexpensive EIARA. Species F was the most prevalent (65%), despite the occurrence of species A (12%), C, D and co-infection F/D (5% each) and species B and co-infections F/A, F/C and B/D (2% each). In order to type the species F strains as HAdV-40 or -41, generic PCR and a HinfI restriction digest were performed. HAdV-40 and -41 were found to represent 62% (23/37) and 38% (14/37), respectively. These results demonstrated that a combination of generic and species-specific PCRs is useful and reliable for HAdV species and type identification directly from faecal specimens. The results confirmed the endemism of human adenoviruses, mainly species F, in children as aetiological agents of diarrhoea, although the limited sensitivity of EIARA as a screening method may have underestimated their prevalence.

Molecular typing of dengue virus type 2 in Brazil.

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).