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Introduction: Acute febrile illnesses (AFI) are a frequent chief complaint in outpatients. Because the capacity to investigate the causative pathogen of AFIs is limited in low- and middle-income countries, patient management may be suboptimal. Understanding the distribution of causes of AFI can improve patient outcomes. This study aims to describe the most common etiologies diagnosed over a 16-years period in a national reference center for tropical diseases in a large urban center in Rio de Janeiro, Brazil., Methods: From August 2004-December 2019, 3591 patients > 12 years old, with AFI and/or rash were eligible. Complementary exams for etiological investigation were requested using syndromic classification as a decision guide. Results. Among the 3591 patients included, endemic arboviruses such as chikungunya (21%), dengue (15%) and zika (6%) were the most common laboratory-confirmed diagnosis, together with travel-related malaria (11%). Clinical presumptive diagnosis lacked sensitivity for emerging diseases such as zika (31%). Rickettsia disease and leptospirosis were rarely investigated and an infrequent finding when based purely on clinical features. Respiratory symptoms increased the odds for the diagnostic remaining inconclusive., Conclusions: Numerous patients did not have a conclusive etiologic diagnosis. Since syndromic classification used for standardization of etiological investigation and presumptive clinical diagnosis had moderate accuracy, it is necessary to incorporate new diagnostic technologies to improve diagnostic accuracy and surveillance capacity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bressan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Chikungunya-fever (CHIKF) remains a public health major issue. It is clinically divided into three phases: acute, post-acute and chronic. Chronic cases correspond to 25-40% individuals and, though most of them are characterized by long-lasting arthralgia alone, many of them exhibit persistent or recurrent inflammatory signs that define post-Chikungunya chronic inflammatory joint disease (pCHIKV-CIJD). We aimed to identify early clinical markers of evolution to pCHIKV-CIJD during acute and post-acute phases., Methodology/principal Findings: We studied a prospective cohort of CHIKF-confirmed volunteers with longitudinal clinical data collection from symptoms onset up to 90 days, including a 21-day visit (D21). Of 169 patients with CHIKF, 86 (50.9%) completed the follow-up, from whom 39 met clinical criteria for pCHIKV-CIJD (45.3%). The relative risk of chronification was higher in women compared to men (RR = 1.52; 95% CI = 1.15-1.99; FDR = 0.03). None of the symptoms or signs presented at D0 behaved as an early predictor of pCHIKV-CIJD, while being symptomatic at D21 was a risk factor for chronification (RR = 1.31; 95% CI = 1.09-1.55; FDR = 0.03). Significance was also observed for joint pain (RR = 1.35; 95% CI = 1.12-1.61; FDR = 0.02), reported edema (RR = 3.61; 95% CI = 1.44-9.06; FDR = 0.03), reported hand and/or feet small joints edema (RR = 4.22; 95% CI = 1.51-11.78; FDR = 0.02), and peri-articular edema observed during physical examination (RR = 2.89; 95% CI = 1.58-5.28; FDR = 0.002). Furthermore, patients with no findings in physical examination at D21 were at lower risk of chronic evolution (RR = 0.41, 95% CI = 0.24-0.70, FDR = 0.01). Twenty-nine pCHIKV-CIJD patients had abnormal articular ultrasonography (90.6% of the examined). The most common findings were synovitis (65.5%) and joint effusion (58.6%)., Conclusion: This cohort has provided important insights into the prognostic evaluation of CHIKF. Symptomatic sub-acute disease is a relevant predictor of evolution to chronic arthritis with synovitis, drawing attention to joint pain, edema, multiple articular involvement including small hand and feet joints as risk factors for chronification beyond three months, especially in women. Future studies are needed to accomplish the identification of accurate and early biomarkers of poor clinical prognosis, which would allow better understanding of the disease's evolution and improve patients' management, modifying CHIKF burden on global public health., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lázari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: A wide variety of viruses can cause rash diseases (RDs) or acute febrile illness (AFIs) in children, adolescents and adults; however, approximately 19% of RD cases and 40% of AFI cases remain without a defined etiology. Parvovirus B19 (B19V) and herpesvirus infection can also cause RD and/or AFI, and in some risk groups, these infections can become persistent (or latent) and may require hospital treatment. Since these infections do not have mandatory reporting, they can be hidden by other diseases, such as those caused by arboviruses (e.g., dengue virus). In this context, the aim of this study was to pursue the differential laboratory diagnoses of B19V and herpesvirus infections in patients with RD and AFI, without a defined etiology, seen in hospitals and/or reference centers for infectious diseases in Rio de Janeiro., Methods: A total of 114 participants were enrolled in the study, including 54 children and 60 adults. B19V infection was assessed by real-time PCR (qPCR) and ELISA (anti-B19V IgM and IgG). EBV was assessed through qPCR, and betaherpesviruses (HCMV, HHV-6 and HHV-7) were assessed through multiplex qPCR. Sociodemographic and clinical data were obtained from the medical record data of these participants., Results: The median age of children with RD was 2 years (interquartile range (IQR): 5), and 55.6% were male. Among adults with AFI, the median age was 38 years (IQR: 21), and 56.7% were female. Regarding RD patients, viral prevalence (and load) were 5.5%(104IU/mL), 3.4%(104IU/mL), 5.5%(104IU/mL) and 11.1%(105IU/mL) for B19V, EBV, HCMV and HHV-6 infection, respectively, and in AFI patients they were 6.6%(105IU/mL), 1.6%(103IU/mL), 3.3%(104IU/mL) for B19V, HCMV and HHV-6, respectively. HHV-7 was not detected in RD or AFI patients., Conclusion: These results suggest the importance of including B19V and herpesviruses in the differential laboratory diagnoses for patients with RD and AFI, not only for epidemiological purposes but also for the proper management of the patient., Competing Interests: The authors have declared that no competing interests exist

Background: Chikungunya (CHIKV) is a reemerging arboviral disease and represents a global health threat because of the unprecedented magnitude of its spread. Diagnostics strategies rely heavily on reverse transcriptase-polymerase chain reaction (RT-PCR) and antibody detection by enzyme-linked Immunosorbent assay (ELISA). Rapid diagnostic tests (RDTs) are available and promise to decentralize testing and increase availability at lower healthcare system levels., Objectives: We aim to identify the extent of research on CHIKV RDTs, map the global availability of CHIKV RDTs, and evaluate the accuracy of CHIKV RDTs for the diagnosis of CHIKV., Eligibility Criteria: We included studies reporting symptomatic individuals suspected of CHIKV, tested with CHIKV RDTs, against the comparator being a validated laboratory-based RT-PCR or ELISA assay. The primary outcome was the accuracy of the CHIKV RDT when compared with reference assays., Sources of Evidence: Medline, EMBASE, and Scopus were searched from inception to 13 October 2021. National regulatory agencies (European Medicines Agency, US Food and Drug Administration, and the Brazilian National Health Surveillance Agency) were also searched for registered CHIKV RDTs., Results: Seventeen studies were included and corresponded to 3,222 samples tested with RDTs between 2005 and 2018. The most development stage of CHIKV RDTs studies was Phase I (7/17 studies) and II (7/17 studies). No studies were in Phase IV. The countries that manufacturer the most CHIKV RDTs were Brazil (n = 17), followed by the United States of America (n = 7), and India (n = 6). Neither at EMA nor FDA-registered products were found. Conversely, the ANVISA has approved 23 CHIKV RDTs. Antibody RDTs (n = 43) predominated and demonstrated sensitivity between 20% and 100%. The sensitivity of the antigen RDTs ranged from 33.3% to 100%., Conclusions: The landscape of CHIKV RDTs is fragmented and needs coordinated efforts to ensure that patients in CHIKV-endemic areas have access to appropriate RDTs. Further research is crucial to determine the impact of such tests on integrated fever case management and prescription practices for acute febrile patients., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: SD is employed by FIND, the global alliance for diagnostics.

Background: Chikungunya is a widely distributed, re-emerging tropical disease caused by the chikungunya virus (CHIKV). Little is known about the duration for which CHIK RNA are detectable in bodily fluids, especially genital secretions, and current evidence is based on small series or case reports. An understanding of viral dynamics across different body compartments can inform diagnostic testing algorithms and public health prevention interventions., Methodology: A prospective cohort study was conducted to assess the presence and duration of detectable levels of CHIKV RNA in blood, urine, saliva, semen, and vaginal secretions. Men and women (≥ 18 years) with a positive reverse transcriptase-polymerase chain reaction (RT-PCR) test for CHIKV in the acute phase (1-14 days) of the disease were included. After enrollment, clinical data and samples were collected every 15 days over the first 2 months, and a final collection was performed 3 months after recruitment. The Kaplan-Meier interval-censoring method and the parametric Weibull model were fitted to estimate the median time of viral persistence until the lack of CHIKV RNA detection among all body fluids. Punctual estimates of the median time of CHIKV RNA persistence for each fluid were estimated using a 95% confidence interval (CI)., Results: From April to December 2019, 170 participants were screened. Of these, 152 (100 women) were enrolled in the study. The median and interquartile range (IQR) ages for men and women were 39.3 (IQR: 26.9, 50.7) and 43.5 (IQR: 33.8, 53.6) years, respectively. CHIKV RNA was detected in 80.3% (122/152) of serum samples, 23.0% (35/152) of urine samples, 30.3% (46/152) of saliva samples, 14.3% (6/42) of semen samples, and 20.2% (20/99) of vaginal secretion samples. The median time until the loss of CHIKV RNA detection was 19.6 days (95% CI, 17.5-21.7) in serum, 25.3 days (95% CI, 17.8-32.8) in urine, 23.1 days (95% CI, 17.9-28.4) in saliva, and 25.8 days (95% CI, 20.6-31.1) in vaginal secretion. The number of semen samples available was too small to make statistical estimates, but a last positive sample was obtained from a participant 56 days after the onset of symptoms., Conclusions: CHIKV RNA could be detected in all bodily fluids studied, including genital secretions during the acute and convalescent phases and additional studies on viral infectivity in semen and vaginal secretions are warranted., Competing Interests: The authors have declared that no competing interests exist.

[This corrects the article DOI: 10.1371/journal.pone.0241426.].

Background: Zika virus (ZIKV) is a flavivirus associated with microcephaly and other fetal anormalities. However, evidence of asymptomatic ZIKV infection in pregnant women is still scarce. This study investigated the prevalence of Zika infection in asymptomatic pregnant women attending two public maternities in Maranhão state, Northeast Brazil., Methods: A total of 196 women were recruited at the time of delivery by convenience sampling from two maternity clinics in São Luís, Maranhão, Brazil, between April 2017 and June 2018. Venous blood, umbilical cord blood and placental fragments from maternal and fetal sides were collected from each subject. ZIKV infection was determined by reverse transcription polymerase chain reaction (RT-qPCR) for ZIKV and by serology (IgM and IgG). Nonspecific laboratory profiles (TORCH screen) were obtained from medical records., Results: The participants were mostly from São Luís and were of 19-35 years of age. They had 10-15 years of schooling and they were of mixed race, married, and Catholic. ZIKV was identified in three umbilical cord samples and in nine placental fragments. Mothers with positive ZIKV RT-qPCR were in the age group older than 19 years. Of the 196 women tested by ZIKV rapid test, 6 and 117 women were positive for anti-ZIKV IgM and anti-ZIKV IgG antibodies, respectively. Placental Immunohistochemistry study detected ZIKV in all samples positive by RT-PCR. The newborns did not show any morphological and/or psychomotor abnormalities at birth., Conclusions: Asymptomatic ZIKV infection is frequent, but it was not associated to morphological and/or psychomotor abnormalities in the newborns up to 6 months post-birth. Although pathological abnormalities were not observed at birth, we cannot rule out the long term impact of apparent asymptomatic congenital ZIKV infection., Competing Interests: The authors have declared that no competing interests exist.

Background: Infection by SARS-CoV-2 in domestic animals has been related to close contact with humans diagnosed with COVID-19. Objectives: To assess the exposure, infection, and persistence by SARS-CoV-2 of dogs and cats living in the same households of humans that tested positive for SARS-CoV-2, and to investigate clinical and laboratory alterations associated with animal infection., Methods: Animals living with COVID-19 patients were longitudinally followed and had nasopharyngeal/oropharyngeal and rectal swabs collected and tested for SARS-CoV-2. Additionally, blood samples were collected for laboratory analysis, and plaque reduction neutralization test (PRNT90) to investigate specific SARS-CoV-2 antibodies., Results: Between May and October 2020, 39 pets (29 dogs and 10 cats) of 21 patients were investigated. Nine dogs (31%) and four cats (40%) from 10 (47.6%) households were infected with or seropositive for SARS-CoV-2. Animals tested positive from 11 to 51 days after the human index COVID-19 case onset of symptoms. Three dogs tested positive twice within 14, 30, and 31 days apart. SARS-CoV-2 neutralizing antibodies were detected in one dog (3.4%) and two cats (20%). In this study, six out of thirteen animals either infected with or seropositive for SARS-CoV-2 have developed mild but reversible signs of the disease. Using logistic regression analysis, neutering, and sharing bed with the ill owner were associated with pet infection., Conclusions: The presence and persistence of SARS-CoV-2 infection have been identified in dogs and cats from households with human COVID-19 cases in Rio de Janeiro, Brazil. People with COVID-19 should avoid close contact with their pets during the time of their illness., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To describe and estimate the frequency of pregnancy outcomes, clinical and laboratory characteristics of vertical transmission of CHIKV in the neonate., Study Design: We performed a systematic review evaluating the clinical presentation of perinatally-acquired CHIKV infection in neonates. The search was performed using Medline (via PubMed), LILACS, Web of Science, Scielo, Google Scholar and Open grey to identify studies assessing vertical transmission of CHIKV up to November 3, 2020. There were no search restrictions regarding the study type, the publication date or language. Studies with no documented evidence of CHIKV infection in neonates (negative RT-PCR or absence of IgM) were excluded., Results: From the 227 studies initially identified, 42 were selected as follows: 28 case reports, 7 case series, 2 cross-sectional studies and 5 cohort studies, for a total of 266 CHIKV infected neonates confirmed by serological and/or molecular tests. The vertical transmission rate was 50% in the Reunion Island outbreak, which was the subject of the majority of the studies; the premature delivery were reported in 19 (45.2%) studies; the rate of fetal distress was 19.6% of infected babies and fetal loss occurred in 2% of the cases. Approximately 68.7% of newborns were diagnosed with encephalopathy or encephalitis after perinatally acquired CHIKV. Most of the infected neonates were born healthy, developing CHIKV sepsis clinical syndrome within the first week of life., Conclusions: We alert neonatologists to the late manifestations of neonatal CHIKV infection, relevant to the management and reduction of morbidity. A limitation of our review was that it was not possible to carry out meta-analysis due to differences in study design and the small number of participants., Competing Interests: The authors have declared that no competing interests exist.

Background: Serological diagnosis of Zika virus (ZIKV) infection is challenging because of the antibody cross-reactivity among flaviviruses. At the same time, the role of Nucleic Acid Testing (NAT) is limited by the low proportion of symptomatic infections and the low average viral load. Here, we compared the diagnostic performance of commercially available IgM, IgAM, and IgG ELISAs in sequential samples during the ZIKV and chikungunya (CHIKV) epidemics and co-circulation of dengue virus (DENV) in Brazil and Venezuela., Methodology/principal Findings: Acute (day of illness 1-5) and follow-up (day of illness ≥ 6) blood samples were collected from nine hundred and seven symptomatic patients enrolled in a prospective multicenter study between June 2012 and August 2016. Acute samples were tested by RT-PCR for ZIKV, DENV, and CHIKV. Acute and follow-up samples were tested for IgM, IgAM, and IgG antibodies to ZIKV using commercially available ELISAs. Among follow-up samples with a RT-PCR confirmed ZIKV infection, anti-ZIKV IgAM sensitivity was 93.5% (43/46), while IgM and IgG exhibited sensitivities of 30.3% (10/33) and 72% (18/25), respectively. An additional 24% (26/109) of ZIKV infections were detected via IgAM seroconversion in ZIKV/DENV/CHIKV RT-PCR negative patients. The specificity of anti-ZIKV IgM was estimated at 93% and that of IgAM at 85%., Conclusions/significance: Our findings exemplify the challenges of the assessment of test performance for ZIKV serological tests in the real-world setting, during co-circulation of DENV, ZIKV, and CHIKV. However, we can also demonstrate that the IgAM immunoassay exhibits superior sensitivity to detect ZIKV RT-PCR confirmed infections compared to IgG and IgM immunoassays. The IgAM assay also proves to be promising for detection of anti-ZIKV seroconversions in sequential samples, both in ZIKV PCR-positive as well as PCR-negative patients, making this a candidate assay for serological monitoring of pregnant women in future ZIKV outbreaks., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: BW has received, outside the submitted work, fees for consulting and travel, in her role as a member of the Data Monitoring and Adjudication Committees for the Takeda dengue vaccine trials, and also personal fees from Roche for work on their Advisory Board on Severe Dengue.TJ has received, outside the submitted work, personal fees from Roche for work on their Advisory Board on Severe Dengue. All other authors have declared that no competing interests exist.

Zika virus (ZIKV) has been detected in blood, urine, semen, cerebral spinal fluid, saliva, amniotic fluid, and breast milk. In most ZIKV infected individuals, the virus is detected in the blood to one week after the onset of symptoms and has been found to persist longer in urine and semen. To better understand virus dynamics, a prospective cohort study was conducted in Brazil to assess the presence and duration of ZIKV and related markers (viral RNA, antibodies, T cell response, and innate immunity) in blood, semen, saliva, urine, vaginal secretions/menstrual blood, rectal swab and sweat. The objective of the current manuscript is to describe the cohort, including an overview of the collected data and a description of the baseline characteristics of the participants. Men and women ≥ 18 years with acute illness and their symptomatic and asymptomatic household contacts with positive reverse transcriptase-polymerase chain reaction test for ZIKV in blood and/or urine were included. All participants were followed up for 12 months. From July 2017 to June 2019, a total of 786 participants (284 men, 502 women) were screened. Of these, 260 (33.1%) were enrolled in the study; index cases: 64 men (24.6%), 162 (62.3%) women; household contacts: 12 men (4.6%), 22 (8.5%) women. There was a statistically significant difference in age and sex between enrolled and not enrolled participants (p<0.005). Baseline sociodemographic and medical data were collected at enrollment from all participants. The median and interquartile range (IQR) age was 35 (IQR; 25.3, 43) for men and 36.5 years (IQR; 28, 47) for women. Following rash, which was one of the inclusion criteria for index cases, the most reported symptoms in the enrollment visit since the onset of the disease were fever, itching, arthralgia with or without edema, non-purulent conjunctivitis, headache, and myalgia. Ten hospitalizations were reported by eight patients (two patients were hospitalized twice) during follow up, after a median of 108 days following symptom onset (range 7 to 266 days) and with a median of 1.5 days (range 1 to 20 days) of hospital stay. A total of 4,137 visits were performed, 223 (85.8%) participants have attended all visits and 37 (14.2%) patients were discontinued., Competing Interests: The authors have declared that no competing interests exist.

Background: The signs and symptoms of Zika virus infection are usually mild and self-limited. However, the disease has been linked to neurological complications such as Guillain-Barré syndrome and peripheral nerve involvement, and also to abortion and fetal deaths due to vertical transmission, resulting in various congenital malformations in newborns, including microcephaly. This review aimed to describe the o signs and symptoms that characterize the congenital Zika syndrome., Methods and Findings: A systematic review was performed with a protocol and described according to the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. The search strategy yielded 2,048 studies. After the exclusion of duplicates and application of inclusion criteria, 46 studies were included. The main signs and symptoms associated with the congenital Zika syndrome were microcephaly, parenchymal or cerebellar calcifications, ventriculomegaly, central nervous system hypoplasia or atrophy, arthrogryposis, ocular findings in the posterior and anterior segments, abnormal visual function and low birthweight for gestational age., Conclusions: Zika virus infection during pregnancy can cause a series of changes in the growth and development of children, while impacting the healthcare system due to the severity of cases. Our findings outline the disease profile in newborns and infants and may contribute to the development and updating of more specific clinical protocols., Competing Interests: The authors have declared that no competing interests exist.

Circumsporozoite protein (CSP) is the primary pre-erythrocytic vaccine target in Plasmodium species. Knowledge about their genetic diversity can help predict vaccine efficacy and the spread of novel parasite variants. Thus, we investigated pvcsp gene polymorphisms in 219 isolates (136 from Brazilian Amazon [BA], 71 from Rio de Janeiro Atlantic Forest [AF], and 12 from non-Brazilian countries [NB]). Forty-eight polymorphic sites were detected, 46 in the central repeat region (CR), and two in the C-terminal region. Also, the CR presents InDels and a variable number of repeats. All samples correspond to the VK210 variant, and 24 VK210 subtypes based on CR. Nucleotide diversity (π = 0.0135) generated a significant number of haplotypes (168) with low genetic differentiation between the Brazilian regions (Fst = 0.208). The haplotype network revealed similar distances among the BA and AF regions. The linkage disequilibrium indicates that recombination does not seem to be acting in diversity, reinforcing natural selection's role in accelerating adaptive evolution. The high diversity (low Fst) and polymorphism frequencies could be indicators of balancing selection. Although malaria in BA and AF have distinct vector species and different host immune pressures, consistent genetic signature was found in two regions. The immunodominant B-cell epitope mapped in the CR varies from seven to 19 repeats. The CR T-cell epitope is conserved only in 39 samples. Concerning to C-terminal region, the Th2R epitope presented nonsynonymous SNP only in 6% of Brazilian samples, and the Th3R epitope remained conserved in all studied regions. We conclude that, although the uneven distribution of alleles may jeopardize the deployment of vaccines directed to a specific variable locus, a unique vaccine formulation could protect populations in all Brazilian regions., Competing Interests: The authors have declared that no competing interests exist.

The main goal of this manuscript was to investigate the neurodevelopment of children exposed by Zika virus in the intrauterine period who are asymptomatic at birth. Newborns with documented Zika virus exposure during the intrauterine period who were asymptomatic at birth were followed in the first two years of life for neurodevelopment using Bayley III test. Children were classified as having normal or delayed neurodevelopment for age based on most recent Bayley III evaluation results. Eighty-four infants were included in the study. The first Bayley III evaluation was performed at a mean chronological age of 9.7±3.1 month; 13 children (15%) had a delay in one of the three domains, distributed as follow: 10 (12%) in the language domain and 3 (3.5%) in the motor domain. The most recent Bayley III evaluation was performed at a mean age 15.3±3.1 months; 42 children (50%) had a delay in one of the three domains: 4 (5%) in cognition, 31 (37%) in language, and 20 (24%) in motor performance. There were no statistical differences in Gender, Gestational Age, Birth Weight and Head Circurference at birth between children with normal and delayed neurodevelopment for age. A very high proportion of children exposed ZIKV during pregnancy who were asymptomatic at birth demonstrated a delay in neurodevelopment, mainly in the language domain, the first two years of life., Competing Interests: The authors have declared that no competing interests exist.

Background: Although malaria cases have substantially decreased in Southeast Brazil, a significant increase in the number of Plasmodium vivax-like autochthonous human cases has been reported in remote areas of the Atlantic Forest in the past few decades in Rio de Janeiro (RJ) state, including an outbreak during 2015-2016. The singular clinical and epidemiological aspects in several human cases, and collectively with molecular and genetic data, revealed that they were due to the non-human primate (NHP) parasite Plasmodium simium; however, the understanding of the autochthonous malarial epidemiology in Southeast Brazil can only be acquired by assessing the circulation of NHP Plasmodium in the foci and determining its hosts., Methodology: A large sampling effort was carried out in the Atlantic forest of RJ and its bordering states (Minas Gerais, São Paulo, Espírito Santo) for collecting and examining free-living NHPs. Blood and/or viscera were analyzed for Plasmodium infections via molecular and microscopic techniques., Principal Findings: In total, 146 NHPs of six species, from 30 counties in four states, were tested, of which majority were collected from RJ. Howler monkeys (Alouatta clamitans) were the only species found infected. In RJ, 26% of these monkeys tested positive, of which 17% were found to be infected with P. simium. Importantly, specific single nucleotide polymorphisms-the only available genetic markers that differentiate P. simium from P. vivax-were detected in all P. simium infected A. clamitans despite their geographical origin of malarial foci. Interestingly, 71% of P. simium infected NHPs were from the coastal slope of a mountain chain (Serra do Mar), where majority of the human cases were found. Plasmodium brasilianum/malariae was initially detected in 14% and 25% free-living howler monkeys in RJ and in the Espírito Santo (ES) state, respectively. Moreover, the malarial pigment was detected in the spleen fragments of 50% of a subsample comprising dead howler monkeys in both RJ and ES. All NHPs were negative for Plasmodium falciparum., Conclusions/significance: Our data indicate that howler monkeys act as the main reservoir for the Atlantic forest human malarial parasites in RJ and other sites in Southeast Brazil and reinforce its zoonotic characteristics., Competing Interests: The authors have declared that no competing interests exist.

The emergence of chikungunya virus (CHIKV) has raised serious concerns due to the virus' rapid dissemination into new geographic areas and the clinical features associated with infection. To better understand CHIKV dynamics in Rio de Janeiro, we generated 11 near-complete genomes by means of real-time portable nanopore sequencing of virus isolates obtained directly from clinical samples. To better understand CHIKV dynamics in Rio de Janeiro, we generated 11 near-complete genomes by means of real-time portable nanopore sequencing of virus isolates obtained directly from clinical samples. Our phylogenetic reconstructions indicated the circulation of the East-Central-South-African (ECSA) lineage in Rio de Janeiro. Time-measured phylogenetic analysis combined with CHIKV notified case numbers revealed the ECSA lineage was introduced in Rio de Janeiro around June 2015 (95% Bayesian credible interval: May to July 2015) indicating the virus was circulating unnoticed for 5 months before the first reports of CHIKV autochthonous transmissions in Rio de Janeiro, in November 2015. These findings reinforce that continued genomic surveillance strategies are needed to assist in the monitoring and understanding of arbovirus epidemics, which might help to attenuate public health impact of infectious diseases., Competing Interests: The authors have declared that no competing interests exist.

Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas., Competing Interests: The authors have declared that no competing interests exist.

Aedes albopictus, originally considered as a secondary vector for arbovirus transmission, especially in areas where this species co-exist with Aedes aegypti, has been described in most regions of the world. Dispersion and domiciliation of Ae. albopictus in a complex of densely urbanized slums in Rio de Janeiro, Southeastern Brazil, was evidenced. In this study, we tested the hypotheses that 1) Ae. albopictus distribution in urban slums is negatively related to distance from vegetation, and 2) these vectors have taken on a domestic life style with a portion of the population feeding, ovipositing, and resting indoors. To do this, we developed an integrated surveillance proposal, aiming to detect the presence and abundance of Aedes mosquitoes. The study, based on a febrile syndrome surveillance system in a cohort of infants living in the slum complex, was performed on a weekly basis between February 2014 and April 2017. A total of 8,418 adult mosquitoes (3,052 Ae. aegypti, 44 Ae. albopictus, 16 Ae. scapularis, 4 Ae. fluviatilis and 5,302 Culex quinquefasciatus) were collected by direct aspiration and 46,047 Aedes spp. eggs were collected by oviposition traps. The Asian tiger mosquito, Ae. albopictus, was aspirated in its adult form (n = 44), and immature forms of this species (n = 12) were identified from the eggs collected by the ovitraps. In most collection sites, co-occurrence of Ae. aegypti and Ae. albopictus was observed. Key-sites, such as junkyards, thrift stores, factories, tire repair shops and garages, had the higher abundance of Ae. albopictus, followed by schools and households. We collected Ae. albopictus at up to 400 meters to the nearest vegetation cover. The log transformed (n+1) number of females Ae. albopictus captured at each collection point was inversely related to the distance to the nearest vegetation border. These results show that Ae. albopictus, a competent vector for important arboviruses and more commonly found in areas with higher vegetation coverage, is present and spread in neglected and densely urbanized areas, being collected at a long distance from the typical encounter areas for this species. Besides, as Ae. albopictus can easily move between sylvatic and urban environment, the entomological monitoring of Ae. albopictus should be an integral part of mosquito surveillance and control. Finally, key-sites, characterized by high human influx and presence of potential Aedes breeding sites, should be included in entomological monitoring.

Background: During 2015-16 Brazil experienced the largest epidemic of Zika virus ever reported. This arthropod-borne virus (arbovirus) has been linked to Guillain-Barré syndrome (GBS) in adults but other neurological associations are uncertain. Chikungunya virus has caused outbreaks in Brazil since 2014 but associated neurological disease has rarely been reported here. We investigated adults with acute neurological disorders for Zika, chikungunya and dengue, another arbovirus circulating in Brazil., Methods: We studied adults who had developed a new neurological condition following suspected Zika virus infection between 1st November 2015 and 1st June 2016. Cerebrospinal fluid (CSF), serum, and urine were tested for evidence of Zika, chikungunya, and dengue viruses., Results: Of 35 patients studied, 22 had evidence of recent arboviral infection. Twelve had positive PCR or IgM for Zika, five of whom also had evidence for chikungunya, three for dengue, and one for all three viruses. Five of them presented with GBS; seven had presentations other than GBS, including meningoencephalitis, myelitis, radiculitis or combinations of these syndromes. Additionally, ten patients positive for chikungunya virus, two of whom also had evidence for dengue virus, presented with a similar range of neurological conditions., Conclusions: Zika virus is associated with a wide range of neurological manifestations, including central nervous system disease. Chikungunya virus appears to have an equally important association with neurological disease in Brazil, and many patients had dual infection. To understand fully the burden of Zika we must look beyond GBS, and also investigate for other co-circulating arboviruses, particularly chikungunya.

Noroviruses are the leading cause of acute gastroenteritis (AGE) in all age groups worldwide. Despite the high genetic diversity of noroviruses, most AGE outbreaks are caused by a single norovirus genotype: GII.4. Since 1995, several different variants of norovirus GII.4 have been associated with pandemics, with each variant circulating for 3 to 8 years. The Sydney&#95;2012 variant was first reported in Australia and then in other countries. A new variant, GII.P16-GII.4, was recently described in Japan and South Korea and then in the USA, France, Germany and England. In our study, 190 faecal specimens were collected from children admitted to a paediatric hospital and a public health facility during a surveillance study of sporadic cases of AGE conducted between January 2015 and July 2016. The norovirus was detected by RT-qPCR in 51 samples (26.8%), and in 37 of them (72.5%), the ORF1-2 junction was successfully sequenced. The new recombinant GII.P16-GII.4 Sydney was revealed for the first time in Brazil in 2016 and predominated among other strains (9 GII.Pe-GII.4, 3 GII.P17-GII.17, 1 GII.Pg-GII.1, 1 GII.P16-GII.3 and 1 GII.PNA-GII.4). The epidemiological significance of this new recombinant is still unknown, but continuous surveillance studies may evaluate its impact on the population, its potential to replace the first recombinant GII.Pe-GII.4 Sydney 2012 variant, and the emergence of new recombinant forms of GII.P16.

The burden of arboviruses in the Americas is high and may result in long-term sequelae with infants disabled by Zika virus infection (ZIKV) and arthritis caused by infection with Chikungunya virus (CHIKV). We aimed to identify environmental drivers of arbovirus epidemics to predict where the next epidemics will occur and prioritize municipalities for vector control and eventual vaccination. We screened sera and urine samples (n = 10,459) from residents of 48 municipalities in the state of Rio de Janeiro for CHIKV, dengue virus (DENV), and ZIKV by molecular PCR diagnostics. Further, we assessed the spatial pattern of arbovirus incidence at the municipal and neighborhood scales and the timing of epidemics and major rainfall events. Lab-confirmed cases included 1,717 infections with ZIKV (43.8%) and 2,170 with CHIKV (55.4%) and only 29 (<1%) with DENV. ZIKV incidence was greater in neighborhoods with little access to municipal water infrastructure (r = -0.47, p = 1.2x10-8). CHIKV incidence was weakly correlated with urbanization (r = 0.2, p = 0.02). Rains began in October 2015 and were followed one month later by the largest wave of ZIKV epidemic. ZIKV cases markedly declined in February 2016, which coincided with the start of a CHIKV outbreak. Rainfall predicted ZIKV and CHIKV with a lead time of 3 weeks each time. The association between rainfall and epidemics reflects vector ecology as the larval stages of Aedes aegypti require pools of water to develop. The temporal dynamics of ZIKV and CHIKV may be explained by the shorter incubation period of the viruses in the mosquito vector; 2 days for CHIKV versus 10 days for ZIKV.

Background: Zika is a new disease in the American continent and its surveillance is of utmost importance, especially because of its ability to cause neurological manifestations as Guillain-Barré syndrome and serious congenital malformations through vertical transmission. The detection of suspected cases by the surveillance system depends on the case definition adopted. As the laboratory diagnosis of Zika infection still relies on the use of expensive and complex molecular techniques with low sensitivity due to a narrow window of detection, most suspected cases are not confirmed by laboratory tests, mainly reserved for pregnant women and newborns. In this context, an accurate definition of a suspected Zika case is crucial in order for the surveillance system to gauge the magnitude of an epidemic., Methodology: We evaluated the accuracy of various Zika case definitions in a scenario where Dengue and Chikungunya viruses co-circulate. Signs and symptoms that best discriminated PCR confirmed Zika from other laboratory confirmed febrile or exanthematic diseases were identified to propose and test predictive models for Zika infection based on these clinical features., Results and Discussion: Our derived score prediction model had the best performance because it demonstrated the highest sensitivity and specificity, 86·6% and 78·3%, respectively. This Zika case definition also had the highest values for auROC (0·903) and R2 (0·417), and the lowest Brier score 0·096., Conclusions: In areas where multiple arboviruses circulate, the presence of rash with pruritus or conjunctival hyperemia, without any other general clinical manifestations such as fever, petechia or anorexia is the best Zika case definition.

Objective: The aim of this study was to describe the use of dengue warning signs by pediatric healthcare staff in the Brazilian public health care system., Methods: Cross-sectional study (2012) with physicians, nurses, and nurse technicians assisting children in five health care facilities. Participants reported the use and importance of dengue warning signs in pediatrics clinical practice through a structured questionnaire. Differences in the use of signs (chi-square test) and in the ranking assigned to each of them (Kruskal-Wallis) were assessed according to health care occupation and level of care (p<0.05)., Results: The final sample comprised 474 participants (97%), mean age of 37 years (standard deviation = 10.3), mainly females (83.8%), physicians (40.1%) and from tertiary care (75.1%). The majority (91%) reported using warning signs for dengue in pediatrics clinical practice. The most widely used and highly valued signs were major hemorrhages (gastrointestinal, urinary), abdominal pain, and increase in hematocrit concurrent or not with rapid decrease in platelet count. Persistent vomiting as well as other signs of plasma leakage such as respiratory distress and lethargy/restlessness were not identified as having the same degree of importance, especially by nurse technicians and in primary or secondary care., Discussion: Although most health care staff reported using dengue warning signs, it would be useful to extend the training for identifying easily recognizable signs of plasma leakage that occur regardless of bleeding., Competing Interests: The authors have declared that no competing interests exist.

Background: The Americas have suffered a dramatic epidemic of Zika since May in 2015, when Zika virus (ZIKV) was first detected in Brazil. Mosquitoes belonging to subgenus Stegomyia of Aedes, particularly Aedes aegypti, are considered the primary vectors of ZIKV. However, the rapid spread of the virus across the continent raised several concerns about the transmission dynamics, especially about potential mosquito vectors. The purpose of this work was to assess the vector competence of the house mosquito Culex quinquefasciatus from an epidemic Zika area, Rio de Janeiro, Brazil, for local circulating ZIKV isolates., Methodology/principal Findings: Culex quinquefasciatus and Ae. aegypti (positive control of ZIKV infection) from Rio de Janeiro were orally exposed to two ZIKV strains isolated from human cases from Rio de Janeiro (Rio-U1 and Rio-S1). Fully engorged mosquitoes were held in incubators at 26 ± 1°C, 12 h:12 h light:dark cycle and 70 ± 10% humidity. For each combination mosquito population-ZIKV strain, 30 specimens were examined for infection, dissemination and transmission rates, at 7, 14 and 21 days after virus exposure by analyzing body (thorax plus abdomen), head and saliva respectively. Infection rates were minimal to completely absent in all Cx. quinquefasciatus-virus combinations and were significantly high for Ae. aegypti. Moreover, dissemination and transmission were not detected in any Cx. quinquefasciatus mosquitoes whatever the incubation period and the ZIKV isolate. In contrast, Ae. aegypti ensured high viral dissemination and moderate to very high transmission., Conclusions/significance: The southern house mosquito Cx. quinquefasciatus from Rio de Janeiro was not competent to transmit local strains of ZIKV. Thus, there is no experimental evidence that Cx. quinquefasciatus likely plays a role in the ZIKV transmission. Consequently, at least in Rio, mosquito control to reduce ZIKV transmission should remain focused on Ae. aegypti., Competing Interests: The authors have declared that no competing interests exist.

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.

Background: In 2015, Brazil was faced with the cocirculation of three arboviruses of major public health importance. The emergence of Zika virus (ZIKV) presents new challenges to both clinicians and public health authorities. Overlapping clinical features between diseases caused by ZIKV, Dengue (DENV) and Chikungunya (CHIKV) and the lack of validated serological assays for ZIKV make accurate diagnosis difficult., Methodology / Principal Findings: The outpatient service for acute febrile illnesses in Fiocruz initiated a syndromic clinical observational study in 2007 to capture unusual presentations of DENV infections. In January 2015, an increase of cases with exanthematic disease was observed. Trained physicians evaluated the patients using a detailed case report form that included clinical assessment and laboratory investigations. The laboratory diagnostic algorithm included assays for detection of ZIKV, CHIKV and DENV. 364 suspected cases of Zika virus disease were identified based on clinical criteria between January and July 2015. Of these, 262 (71.9%) were tested and 119 (45.4%) were confirmed by the detection of ZIKV RNA. All of the samples with sequence information available clustered within the Asian genotype., Conclusions / Significance: This is the first report of a ZIKV outbreak in the state of Rio de Janeiro, based on a large number of suspected (n = 364) and laboratory confirmed cases (n = 119). We were able to demonstrate that ZIKV was circulating in Rio de Janeiro as early as January 2015. The peak of the outbreak was documented in May/June 2015. More than half of the patients reported headache, arthralgia, myalgia, non-purulent conjunctivitis, and lower back pain, consistent with the case definition of suspected ZIKV disease issued by the Pan American Health Organization (PAHO). However, fever, when present, was low-intensity and short-termed. In our opinion, pruritus, the second most common clinical sign presented by the confirmed cases, should be added to the PAHO case definition, while fever could be given less emphasis. The emergence of ZIKV as a new pathogen for Brazil in 2015 underscores the need for clinical vigilance and strong epidemiological and laboratory surveillance.

Background: The clinical definition of severe dengue fever remains a challenge for researchers in hyperendemic areas like Brazil. The ability of the traditional (1997) as well as the revised (2009) World Health Organization (WHO) dengue case classification schemes to detect severe dengue cases was evaluated in 267 children admitted to hospital with laboratory-confirmed dengue., Principal Findings: Using the traditional scheme, 28.5% of patients could not be assigned to any category, while the revised scheme categorized all patients. Intensive therapeutic interventions were used as the reference standard to evaluate the ability of both the traditional and revised schemes to detect severe dengue cases. Analyses of the classified cases (n = 183) demonstrated that the revised scheme had better sensitivity (86.8%, P<0.001), while the traditional scheme had better specificity (93.4%, P<0.001) for the detection of severe forms of dengue., Conclusions/significance: This improved sensitivity of the revised scheme allows for better case capture and increased ICU admission, which may aid pediatricians in avoiding deaths due to severe dengue among children, but, in turn, it may also result in the misclassification of the patients' condition as severe, reflected in the observed lower positive predictive value (61.6%, P<0.001) when compared with the traditional scheme (82.6%, P<0.001). The inclusion of unusual dengue manifestations in the revised scheme has not shifted the emphasis from the most important aspects of dengue disease and the major factors contributing to fatality in this study: shock with consequent organ dysfunction.

Background: Dengue is a reportable disease in Brazil; however, pregnancy has been included in the application form of the Brazilian notification information system only after 2006. To estimate the severity of maternal dengue infection, the available data that were compiled from January 2007 to December 2008 by the official surveillance information system of the city of Rio de Janeiro were reviewed., Methods and Principal Findings: During the study period, 151,604 cases of suspected dengue infection were reported. Five hundred sixty-one women in their reproductive age (15-49 years) presented with dengue infection; 99 (18.1%) pregnant and 447 (81.9%) non-pregnant women were analyzed. Dengue cases were categorized using the 1997 WHO classification system, and DHF/DSS were considered severe disease. The Mann-Whitney test was used to compare maternal age, according to gestational period, and severity of disease. A chi-square test was utilized to evaluate the differences in the proportion of dengue severity between pregnant and non-pregnant women. Univariate analysis was performed to compare outcome variables (severe dengue and non-severe dengue) and explanatory variables (pregnancy, gestational age and trimester) using the Wald test. A multivariate analysis was performed to assess the independence of statistically significant variables in the univariate analysis. A p-value<0.05 was considered statistically significant. A higher percentage of severe dengue infection among pregnant women was found, p = 0.0001. Final analysis demonstrated that pregnant women are 3.4 times more prone to developing severe dengue (OR: 3.38; CI: 2.10-5.42). Mortality among pregnant women was superior to non-pregnant women., Conclusion: Pregnant women have an increased risk of developing severe dengue infection and dying of dengue.