Lineage-Specific Real-Time RT-PCR for Yellow Fever Virus Outbreak Surveillance, Brazil.

The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting.

Predictors of chronic joint pain after Chikungunya virus infection in the INOVACHIK prospective cohort study.

BACKGROUND: Chikungunya can cause persistent chronic joint pain. Knowledge of the risk factors for disease progression is important for preventing and controlling complications. This study aimed to identify factors associated with chronic joint pain. METHODS: This prospective cohort study was conducted at a reference center in Rio de Janeiro. Men and women (aged ≥ 18 years) in the acute phase of Chikungunya were included. Clinical data and samples were collected over three months. Risk factors were evaluated using multivariate and logistic regression analyses. RESULTS: A total of 107 patients were followed up. The incidence rate of joint tenderness was 61.7 %. Female sex (adjusted odds ratio [AOR] 3.24, 95 % confidence interval [CI]:1.07-9.77), diarrhea (AOR 5.08, 95 % CI:1.55-16.67), severe joint pain (AOR 4.26, 95 % CI:1.06-17.06), and CHIKV real-time reverse transcription polymerase chain reaction positivity up to 5 days after the onset of symptoms in urine or saliva (AOR 4.56, 95 % CI:1.41-14.77) were identified as predictors of persistent chronic pain. CONCLUSIONS: In a predominantly female population, musculoskeletal symptoms are not the sole determinant of chronic pain, and careful evaluation of CHIKV detection in alternative body fluids (such as saliva and urine) during the early phase of the disease is warranted.

Serological Evidence of Exposure to Saint Louis Encephalitis and West Nile Viruses in Horses of Rio de Janeiro, Brazil.

Infections with arboviruses are reported worldwide. Saint Louis encephalitis (SLEV) and West Nile (WNV) viruses are closely related flaviviruses affecting humans and animals. SLEV has been sporadically detected in humans, and corresponding antibodies have been frequently detected in horses throughout Brazil. WNV was first reported in western Brazil over a decade ago, has been associated with neurological disorders in humans and equines and its prevalence is increasing nationwide. Herein, we investigated by molecular and serological methods the presence of SLEV and WNV in equines from Rio de Janeiro. A total of 435 serum samples were collected from healthy horses and tested for specific neutralizing antibodies by plaque reduction neutralization test (PRNT90). Additionally, samples (serum, cerebrospinal fluid, central nervous system tissue) from 72 horses, including horses with neurological disorders resulting in a fatal outcome or horses which had contact with them, were tested by real-time reverse transcription-polymerase chain reaction (RT-qPCR) for both viruses. Adopting the criterion of four-fold antibody titer difference, 165 horses (38%) presented neutralizing antibodies for flaviviruses, 89 (20.4%) for SLEV and five (1.1%) for WNV. No evidence of SLEV and WNV infection was detected by RT-qPCR and, thus, such infection could not be confirmed in the additional samples. Our findings indicate horses of Rio de Janeiro were exposed to SLEV and WNV, contributing to the current knowledge on the distribution of these viruses in Brazil.

Detection of Chikungunya virus in bodily fluids: The INOVACHIK cohort study.

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.

Chikungunya Virus Shedding in Semen: A Case Series.

BACKGROUND: Chikungunya is a viral disease that is transmitted by mosquitoes. It is characterized by an acute onset of fever and severe arthralgia. METHODS: We describe six cases of acute and post-acute chikungunya in which viral RNA was detected in semen. CONCLUSIONS: The most prolonged detection period was 56 days after illness onset. We attempted to cultivate positive semen samples, but virus isolation was unsuccessful in all cases.

Yellow fever epizootics in non-human primates, Southeast and Northeast Brazil (2017 and 2018).

BACKGROUND: Yellow fever (YF) is a severe, infectious, but non-communicable arboviral hemorrhagic disease. In the last decades, yellow fever virus (YFV) infections have been prevalent in endemic areas in Brazil, affecting human and non-human primate (NHP) populations. Monitoring of NHP infection started in 1999, and reports of epizootic diseases are considered important indicators of viral transmission, particularly in relation to the sylvatic cycle. This study presents the monitoring of YFV by real-time RT-PCR and the epidemiological findings related to the deaths of NHPs in the south-eastern states and in the north-eastern state of Bahia, during the outbreak of YF in Brazil during 2017 and 2018. METHODS: A total of 4198 samples from 2099 NHPs from south-eastern and north-eastern Brazilian states were analyzed by real-time reverse transcription polymerase chain reaction (rtRT-PCR). RESULTS: A total of 4198 samples from 2099 NHPs from south-eastern and north-eastern Brazilian states were collected between 2017 and 2018. The samples were subjected to molecular diagnostics for YFV detection using real-time reverse transcription polymerase chain reaction (rtRT-PCR) techniques. Epizootics were coincident with human YF cases. Furthermore, our results showed that the YF frequency was higher among marmosets (Callithrix sp.) than in previous reports. Viremia in species of the genus Alouatta and Callithrix differed greatly. DISCUSSION: Our results indicate a need for further investigation of the role of Callithrix spp. in the transmission cycles of YFV in Brazil. In particular, YFV transmission was observed in a region where viral circulation has not been recorded for decades and thus vaccination has not been previously recommended. CONCLUSIONS: This highlights the need to straighten epizootic surveillance and evaluate the extent of vaccination programmes in Brazil in previously considered "YFV-free" areas of the country.

Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing.

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.