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The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 10 3 genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases. The Trioplex real-time RT-PCR assay was developed for detection of Zika virus infections in areas with dengue and chikungunya transmission. Here, Santiago et al. describe the optimization and clinical performance of the assay, showing high sensitivity for detection and differentiation of the three viruses.

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

Background In Colombia, the dengue virus (DENV) has been endemic for decades, and with the recent entry of the chikungunya virus (CHIKV) (2014) and the Zika virus (ZIKV) (2015), health systems are overloaded because the diagnosis of these three diseases is based on clinical symptoms, and the three diseases share a symptomatology of febrile syndrome. Thus, the objective of this study was to use molecular methods to identify their co-circulation as well as the prevalence of co-infections, in a cohort of patients at the Colombian-Venezuelan border. Methods A total of 157 serum samples from patients with febrile syndrome consistent with DENV were collected after informed consent and processed for the identification of DENV (conventional PCR and real-time PCR), CHIKV (conventional PCR), and ZIKV (real-time PCR). DENV-positive samples were serotyped, and some of those positive for DENV and CHIKV were sequenced. Results Eighty-two patients were positive for one or more viruses: 33 (21.02%) for DENV, 47 (29.94%) for CHIKV, and 29 (18.47%) for ZIKV. The mean age range of the infected population was statistically higher in the patients infected with ZIKV (29.72 years) than in those infected with DENV or CHIKV (21.09 years). Both co-circulation and co-infection of these three viruses was found. The prevalence of DENV/CHIKV, DENV/ZIKV, and CHIKV/ZIKV co-infection was 7.64%, 6.37%, and 5.10%, with attack rates of 14.90, 12.42, and 9.93 cases per 100,000 inhabitants, respectively. Furthermore, three patients were found to be co-infected with all three viruses (prevalence of 1.91%), with an attack rate of 4.96 cases per 100,000 inhabitants. Conclusion Our results demonstrate the simultaneous co-circulation of DENV, CHIKV, ZIKV and their co-infections at the Colombian-Venezuelan border. Moreover, it is necessary to improve the differential diagnosis in patients with acute febrile syndrome and to study the possible consequences of this epidemiological overview of the clinical outcomes of these diseases in endemic regions.

Detection of chikungunya virus (CHIKV) or viral RNA is the primary laboratory test used to diagnose infection in serum collected <6 days after onset of illness. Two real-time reverse transcription-polymerase chain reaction (RT-PCR) kits are available commercially, but validity data are limited. There are 2 commercial sources of inactivated positive-control CHIKV RNA to be used with purchased primers. The Centers for Disease Control and Prevention provides viral RNA-positive controls and primer and probe nucleotide sequences for real-time RT-PCR testing. Detection of CHIKV-specific immunoglobulin M (IgM) antibody becomes a sensitive test for samples collected approximately >5 days of illness. Commercially available CHIKV IgM-detection assays include lateral flow rapid tests, IgM antibody capture enzyme-linked immunosorbent assays (MAC-ELISAs), and indirect immunofluorescence tests. Nine commercial CHIKV IgM detection assays were evaluated at 3 reference laboratories to provide guidance to public health diagnostic laboratories on their performance parameters. Sensitivity of the rapid tests and 3 MAC-ELISAs was <50%, and thus these assays are not recommended. Three of the MAC-ELISA kits and 1 indirect immunofluorescence kit had comparable performance to the reference assays. In summary, commercial assays with performance comparable to reference assays are available for molecular and serological diagnosis of CHIKV infections.

The aim of this systematic review of prevalence is to observe and discuss the clinical manifestations of Chikungunya Virus disease in its chronic phase. To be eligible, the observational studies should accompany the individuals for at least six months. The research was conducted using electronic databases MEDLINE and EMBASE. The methodological quality was evaluated using the \"Joanna Briggs Institute's critical appraisal checklist for studies reporting prevalence data\" tool. The search has found 175 articles. The application of the inclusion criteria defined a total of 29 selected studies. From the included studies, only one did not present arthralgia as a prevalent symptom in the chronic phase. Other signs and symptoms observed were: fatigue; sleep disorders; myalgia; skin lesions; depression; digestive disorders. Because it is an often incapacitating symptom, arthralgia can affect the individuals' quality of life, with implications in their social and work life. Since the chronic phase is common in infected individuals, all levels of health care should be prepared to monitor, in the medium to long term, the patients affected by this condition.

Dengue and chikungunya are arboviral diseases with overlapping clinical characteristics. Dengue virus (DENV) is endemic in Colombia, and in 2014/2015, the chikungunya virus (CHIKV) caused an epidemic that resulted in over 350,000 cases. Since then, both viruses have been actively co-circulating. The early and accurate identification of pediatric infection caused by DENV or CHIKV is essential for proper medical management. Given that subsequent infections and co-infections with DENV and CHIKV have been reported, virological and immunological factors may influence their clinical outcomes. Here, we analyzed the viremia, antigenemia, and virus-specific antibody responses in hospitalized children suspected of having dengue during the peak of CHIKV infections in Colombia. Ninety-one children with a clinical diagnosis of dengue were included in the peak of the CHIKV epidemic (December 2014 to May 2015) at a reference healthcare center in Huila, south of Colombia. Multiplexed RT-qPCR for DENV, CHIKV, and ZIKV was performed, and DENV antigenemia was evaluated using an ELISA for the NS1 antigen. Commercial capture or in-house indirect NS1-based ELISAs were used to assess circulating DENV and CHIKV-IgM and IgG. Clinical and laboratory characteristics were analyzed during hospitalization, and convalescent follow-up was conducted for a fraction of children. DENV and CHIKV monoinfections were confirmed in 54% and 12% of children, respectively, with the expected virus-specific seroconversion in recovery. Overlapping infections occurred in 22% of the children, while 12% showed no detectable DENV or CHIKV infections. Abdominal pain, vomiting, hepatomegaly, and thrombocytopenia were common findings associated with DENV, while arthralgia and rash characterized CHIKV monoinfections. One fatal secondary DENV-3 monoinfection was registered, and DENV infection dominated the symptoms of overlapping infections without producing different clinical outcomes compared to monoinfections. Thirty-eight percent of children were seropositive for CHIKV-IgG, indicating a significant burden of CHIKV infection in the pediatric population shortly after its introduction in Colombia. The previous virus-specific IgG serostatus did not impact the clinical outcome of the current heterotypic arboviral infection. The pediatric population in southern Colombia was rapidly exposed to CHIKV infections during the first months following its arrival, with up to 12% of hospitalized children suspected of having dengue experiencing CHIKV monoinfection, supporting that complex and dynamic epidemiological patterns may lead to delayed or missed diagnoses. The overlapping infections of DENV and CHIKV were frequent and did not lead to worse clinical or fatal outcomes.

The explosive pandemic of Zika virus infection in South and Central America is the most recent of four unexpected arrivals of important arthropod-borne viral diseases in the Western Hemisphere over the past 20 years. Is this an important new disease-emergence pattern? The explosive pandemic of Zika virus infection occurring throughout South America, Central America, and the Caribbean (see map) and potentially threatening the United States is the most recent of four unexpected arrivals of important arthropod-borne viral diseases in the Western Hemisphere over the past 20 years. It follows dengue, which entered this hemisphere stealthily over decades and then more aggressively in the 1990s; West Nile virus, which emerged in 1999; and chikungunya, which emerged in 2013. Are the successive migrations of these viruses unrelated, or do they reflect important new patterns of disease emergence? Furthermore, are there secondary health consequences . . .

Chikungunya, caused by a mosquito-borne alphavirus, has emerged as a major public health concern due to its expanding geographical range and debilitating health outcomes in affected populations. Although research on chikungunya is growing, variations in the clinical presentation between age groups remain poorly understood. Understanding these variations is crucial for appropriate diagnoses, treatments, and timely prevention efforts. Therefore, this study aims to assess the clinical presentation of chikungunya across age groups and identify age-related differences and similarities. We conducted a systematic literature review (17-09-2024) using Embase, MEDLINE, and gray literature, including a risk of bias assessment (Downs and Black, National Institute of Health). Results were descriptively presented. A total of 101 studies reported clinical outcomes in three age groups: children (<18 years), adults (18-64 years), and the elderly (≥65 years), with fourteen directly comparing age groups. The findings highlight substantial differences in disease presentation. Hospitalization and mortality rates were highest among the elderly and younger children, while faster recovery was reported for older children compared to other age groups. Symptom presentation also varied by age, with rash and headache being more common in adolescents and adults than in younger children and the elderly. Fever was more prevalent among adults and the elderly compared with children. Arthralgia was less commonly reported in children and the elderly than in adults, warranting attention for potential misdiagnosis due to atypical disease presentation. Lastly, the occurrence of chronic chikungunya symptoms appears to increase with age. Yet, the heterogeneity in reporting and scarcity of articles describing clinical features of children and the elderly limit definitive conclusions. Our findings highlight the age-related variations in chikungunya disease presentation, reinforcing the need for tailored diagnostics, clinical management, and prevention strategies in all age groups. By identifying these differences, our study helps guide future research and public health policies aimed at reducing the impact of chikungunya worldwide.

Background. Beyond the acute illness phase, chikungunya constitutes a public health problem given its chronic disease phase, which may include long-term arthralgia, arthritis, fatigue, and depression. Currently, there is no consensus on how to define chikungunya chronicity. Methods. A comprehensive cross-sectional survey was performed in Curaçao in June and July 2015 to evaluate 304 adult laboratory-confirmed chikungunya patients 3–16 months after diagnosis. We developed a novel tool, the Curaçao Long-Term Chikungunya Sequelae (CLTCS) score, to classify chronic chikungunya disease and estimate its burden regarding disease duration, clinical presentation, and impact on quality of life. Results. Disease persistence was estimated to be 79% one month after symptom onset and 64% after 400 days. Chikungunya persistence was characterized by higher proportions of arthralgia, weakness, myalgia, and age 41–60 years. Individuals were classified as \"highly affected,\" \"mildly affected,\" and \"recovered.\" \"Highly affected\" disease status was associated with clinical complaints (arthralgia, weakness, loss of vitality, and being diabetic) and major decreases in quality-of-life scores. Conclusions. In the Caribbean, a high proportion of chikungunya patients remains chronically affected. We propose the CLTCS as a suitable score to easily and rapidly classify the severity of chikungunya chronic disease and to assess the need for symptom-alleviating treatment.

Pathogens causing acute fever, with the exception of malaria, remain largely unidentified in sub-Saharan Africa, given the local unavailability of diagnostic tests and the broad differential diagnosis. We conducted a cross-sectional study including outpatient acute undifferentiated fever in both children and adults, between November 2015 and June 2016 in Kinshasa, Democratic Republic of Congo. Serological and molecular diagnostic tests for selected arboviral infections were performed on blood, including PCR, NS1-RDT, ELISA and IFA for acute, and ELISA and IFA for past infections. Investigation among 342 patients, aged 2 to 68 years (mean age of 21 years), with acute undifferentiated fever (having no clear focus of infection) revealed 19 (8.1%) acute dengue-caused by DENV-1 and/or DENV-2 -and 2 (0.9%) acute chikungunya infections. Furthermore, 30.2% and 26.4% of participants had been infected in the past with dengue and chikungunya, respectively. We found no evidence of acute Zika nor yellow fever virus infections. 45.3% of patients tested positive on malaria Rapid Diagnostic Test, 87.7% received antimalarial treatment and 64.3% received antibacterial treatment. Chikungunya outbreaks have been reported in the study area in the past, so the high seroprevalence is not surprising. However, scarce evidence exists on dengue transmission in Kinshasa and based on our data, circulation is more important than previously reported. Furthermore, our study shows that the prescription of antibiotics, both antibacterial and antimalarial drugs, is rampant. Studies like this one, elucidating the causes of acute fever, may lead to a more considerate and rigorous use of antibiotics. This will not only stem the ever-increasing problem of antimicrobial resistance, but will-ultimately and hopefully-improve the clinical care of outpatients in low-resource settings. ClinicalTrials.gov NCT02656862.