Acute Dengue Virus Infection Research Articles

Frequency and Clinical Manifestations of Dengue in Urban Medellin, Colombia

A dengue fever surveillance study was conducted at three medical facilities located in the low-income district of San Javier in Medellin, Colombia. During March 2008 to 2009, 781 patients with fever regardless of chief complaint were recruited for acute dengue virus infection testing. Of the 781 tested, 73 (9.3%) were positive for dengue infection. Serotypes DENV-2 (77%) and -3 (23%) were detected by PCR. One patient met the diagnostic criteria for dengue hemorrhagic fever. Only 3 out of 73 (4.1%) febrile subjects testing positive for dengue infection were diagnosed with dengue fever by the treating physician. This study confirms dengue virus as an important cause of acute febrile illness in Medellin, Colombia, but it is difficult to diagnose without dengue diagnostic testing.

Chikungunya as a Cause of Acute Febrile Illness in Southern Sri Lanka

BackgroundChikungunya virus (CHIKV) re-emerged in Sri Lanka in late 2006 after a 40-year hiatus. We sought to identify and characterize acute chikungunya infection (CHIK) in patients presenting with acute undifferentiated febrile illness in unstudied rural and semi-urban southern Sri Lanka in 2007. Methodology/Principal FindingsWe enrolled febrile patients ≥ 2 years of age, collected uniform epidemiologic and clinical data, and obtained serum samples for serology, virus isolation, and real-time reverse-transcriptase PCR (RT-PCR). Serology on paired acute and convalescent samples identified acute chikungunya infection in 3.5% (28/797) patients without acute dengue virus (DENV) infection, 64.3% (18/28) of which were confirmed by viral isolation and/or real-time RT-PCR. No CHIKV/DENV co-infections were detected among 54 patients with confirmed acute DENV. Sequencing of the E1 coding region of six temporally distinct CHIKV isolates (April through October 2007) showed that all isolates posessed the E1-226A residue and were most closely related to Sri Lankan and Indian isolates from the same time period. Except for more frequent and persistent musculoskeletal symptoms, acute chikungunya infections mimicked DENV and other acute febrile illnesses. Only 12/797 (1.5%) patients had serological evidence of past chikungunya infection. Conclusions/SignificanceOur findings suggest CHIKV is a prominent cause of non-specific acute febrile illness in southern Sri Lanka.

Galectin-9 plasma levels reflect adverse hematological and immunological features in acute dengue virus infection

BackgroundDengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble β-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties. ObjectiveTo investigate plasma Gal-9 levels as a biomarker for DENV infection. Study designWe enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness. ResultsDuring the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464pg/ml; dengue fever patients (DF): 1407pg/ml; non-dengue febrile illness: 616pg/ml; healthy: 196pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P<0.01). ConclusionGal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity.

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

BackgroundEarly and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.MethodsA single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).ResultsAcute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.ConclusionThe RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.

Evaluation of a New Commercially Available Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Dengue Virus Infection

We evaluated the FDA-cleared InBios dengue virus (DENV) IgM capture enzyme-linked immunosorbent assay (ELISA) for qualitative detection of anti-DENV IgM antibodies from 79 serum samples obtained from dengue virus-infected patients or suspected dengue cases. The agreement, sensitivity, and specificity of the InBios assay compared to the gold standard in-house DENV IgM capture ELISA were 94, 92, and 94%, respectively. We conclude that the InBios DENV IgM capture ELISA can be effectively used for rapid diagnosis of acute or recent DENV infection.

Association between Magnitude of the Virus-Specific Plasmablast Response and Disease Severity in Dengue Patients

Dengue is a globally expanding disease caused by infection with dengue virus (DENV) that ranges from febrile illness to acute disease with serious complications. Secondary infection predisposes individuals to more severe disease, and B lymphocytes may play a role in this phenomenon through production of Ab that enhance infection. To better define the acute B cell response during dengue, we analyzed peripheral B cells from an adult Brazilian hospital cohort with primary and secondary DENV infections of varying clinical severity. Circulating B cells in dengue patients were proliferating, activated, and apoptotic relative to individuals with other febrile illnesses. Severe secondary DENV infection was associated with extraordinary peak plasmablast frequencies between 4 and 7 d of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average >70% of IgG-secreting cells in individuals with severe secondary DENV infection were DENV specific. Plasmablasts produced Ab that cross-reacted with heterotypic DENV serotypes, but with a 3-fold greater reactivity to DENV-3, the infecting serotype. Plasmablast frequency did not correlate with acute serum-neutralizing Ab titers to any DENV serotype regardless of severity of disease. These findings indicate that massive expansion of DENV-specific and serotype cross-reactive plasmablasts occurs in acute secondary DENV infection of adults in Brazil, which is associated with increasing disease severity.

Multiploid CD61+ Cells Are the Pre-Dominant Cell Lineage Infected during Acute Dengue Virus Infection in Bone Marrow

Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM). Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV) and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM) and humans were utilized to identify the cell lineage(s) that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.

Chikungunya infection in Malaysia: Comparison with dengue infection in adults and predictors of persistent arthralgia

BackgroundChikungunya virus (CHIKV) and dengue virus (DENV) co-circulate in areas endemic with the Aedes mosquito vectors. Both viruses cause similar illnesses which may be difficult to distinguish clinically. CHIKV is also associated with persistent arthralgia. ObjectivesTo compare and describe factors which differentiate between DENV and CHIKV infections on presentation; and to describe predictors of persistent arthralgia in CHIKV patients. Study designPatients aged >14 years diagnosed with acute CHIKV and DENV infections in Kuala Lumpur, Malaysia were retrospectively identified. Clinical and laboratory data were obtained from medical records, and compared. CHIKV patients were telephoned 15–24 months later and interviewed about persistent symptoms. Logistic regression analysis was performed. ResultsA total of 53 CHIKV and 113 DENV patients were included. CHIKV patients were older and more likely to be female. CHIKV was independently associated with arthralgia and rash, while DENV was associated with myalgia, raised aspartate transaminase, and leucopaenia. Forty CHIKV patients were followed up, with a median duration of self-reported arthralgia of 3 months (range, 0–24 months). Eighteen (45%) had persistent arthralgia beyond 4 months, for which age >40 years was an independent predictor. At 1 year, 9 (22.5%) patients had arthralgia. ConclusionIn Kuala Lumpur, selected clinical and laboratory predictors help to distinguish between DENV and CHIKV infections. Persistent arthralgia was a frequent sequel of CHIKV infection in this cohort.

Elevated levels of circulating galectin-9, a novel immune-modulator, in acute dengue virus Infection reflects disease severity (170.18)

Abstract Following a dramatic rise in incidence in recent decades, Dengue virus infection (DENV) is now a significant burden of disease in the tropics and major international public health concern. Currently no treatment or effective vaccine exists. The β-Galactoside-binding lectin, Galectin-9 (Gal-9), functions as pro-apoptotic agent and inducer of pro-inflammatory cytokines from T cells. Here, we sought to evaluate the role of Gal-9 in the pathogenesis of acute DENV in patients presenting with self-limiting dengue fever (DF) and more serious life-threatening dengue hemorrhagic fever (DHF). Blood samples were obtained from 65 patients (mean age: 23±4yrs) hospitalized with DENV from the San Lazaro Hospital, Manila, Philippines in 2010. Plasma Gal-9 was measured by an ELISA method at the critical phase (day4-5) and recovery phase (day7-8) of infection. Our results show that during the critical phase, Gal-9 levels were markedly elevated in DF (1758±1047pg/ml) and DHF(2730±1848pg/ml) compared to uninfected (363±280pg/ml) (p&amp;lt;0.05) controls. These levels of Gal-9 were the highest ever observed in humans. During the recovery phase, Gal-9 levels significantly declined (1170pg/ml and 1140pg/ml) (p&amp;lt;0.05) from peak levels in DF and DHF, respectively, but not to levels observed in uninfected donors. The above findings suggest that Gal-9 levels tracks disease severity during acute DEN, potentially mediate immuno-pathology associated with DHF, and may serve as an indicator of recovery from DENV.

Dominant Cross-Reactive B Cell Response during Secondary Acute Dengue Virus Infection in Humans

The four serotypes of dengue virus (DENV) cause dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Severe disease has been associated with heterotypic secondary DENV infection, mediated by cross-reactive antibodies (Abs) and/or cross-reactive T cells. The role of cross-reactive immunity in mediating enhanced disease versus cross-protection against secondary heterotypic DENV infection is not well defined. A better understanding of the cross-reactive immune response in natural infections is critical for development of safe and effective tetravalent vaccines. We studied the B cell phenotype of circulating B cells in the blood of pediatric patients suspected of dengue during the 2010–2011 dengue season in Managua, Nicaragua (n = 216), which was dominated by the DENV-3 serotype. We found a markedly larger percentage of plasmablast/plasma cells (PB/PCs) circulating in DENV-positive patients as compared to patients with Other Febrile Illnesses (OFIs). The percentage of DENV-specific PB/PCs against DENV-3 represented 10% of the circulating antibody-producing cells (ASCs) in secondary DENV-3 infections. Importantly, the cross-reactive DENV-specific B cell response was higher against a heterotypic serotype, with 46% of circulating PB/PCs specific to DENV-2 and 10% specific to DENV-3 during acute infection. We also observed a higher cross-reactive DENV-specific IgG serum avidity directed against DENV-2 as compared to DENV-3 during acute infection. The neutralization capacity of the serum was broadly cross-reactive against the four DENV serotypes both during the acute phase and at 3 months post-onset of symptoms. Overall, the cross-reactive B cell immune response dominates during secondary DENV infections in humans. These results reflect our recent findings in a mouse model of DENV cross-protection. In addition, this study enabled the development of increased technical and research capacity of Nicaraguan scientists and the implementation of several new immunological assays in the field.

Unsuspected Dengue Causes Acute Febrile Illness in Rural and Semi-Urban Southern Sri Lanka

Dengue virus (DENV), a globally emerging cause of undifferentiated fever, has been documented in the heavily urbanized western coast of Sri Lanka since the 1960s. New areas of Sri Lanka are now being affected, and the reported number and severity of cases have increased. To study emerging DENV in southern Sri Lanka, we obtained epidemiologic and clinical data and acute- and convalescent-phase serum samples from patients >2 years old with febrile illness. We tested paired serum samples for DENV IgG and IgM and serotyped virus by using isolation and reverse transcription PCR. We identified acute DENV infection (serotypes 2, 3, and 4) in 54 (6.3%) of 859 patients. Only 14% of patients had clinically suspected dengue; however, 54% had serologically confirmed acute or past DENV infection. DENV is a major and largely unrecognized cause of fever in southern Sri Lanka, especially in young adults.

Risk for Emergence of Dengue and Chikungunya Virus in Israel

Risk for Emergence of Dengue and Chikungunya Virus in Israel

Rapid and Massive Virus-Specific Plasmablast Responses during Acute Dengue Virus Infection in Humans

Humoral immune responses are thought to play a major role in dengue virus-induced immunopathology; however, little is known about the plasmablasts producing these antibodies during an ongoing infection. Herein we present an analysis of plasmablast responses in patients with acute dengue virus infection. We found very potent plasmablast responses that often increased more than 1,000-fold over the baseline levels in healthy volunteers. In many patients, these responses made up as much 30% of the peripheral lymphocyte population. These responses were largely dengue virus specific and almost entirely made up of IgG-secreting cells, and plasmablasts reached very high numbers at a time after fever onset that generally coincided with the window where the most serious dengue virus-induced pathology is observed. The presence of these large, rapid, and virus-specific plasmablast responses raises the question as to whether these cells might have a role in dengue immunopathology during the ongoing infection. These findings clearly illustrate the need for a detailed understanding of the repertoire and specificity of the antibodies that these plasmablasts produce.

Chikungunya and Dengue Fever among Hospitalized Febrile Patients in Northern Tanzania

Consecutive febrile admissions were enrolled at two hospitals in Moshi, Tanzania. Confirmed acute Chikungunya virus (CHIKV), Dengue virus (DENV), and flavivirus infection were defined as a positive polymerase chain reaction (PCR) result. Presumptive acute DENV infection was defined as a positive anti-DENV immunoglobulin M (IgM) enzyme-linked immunsorbent assay (ELISA) result, and prior flavivirus exposure was defined as a positive anti-DENV IgG ELISA result. Among 870 participants, PCR testing was performed on 700 (80.5%). Of these, 55 (7.9%) had confirmed acute CHIKV infection, whereas no participants had confirmed acute DENV or flavivirus infection. Anti-DENV IgM serologic testing was performed for 747 (85.9%) participants, and of these 71 (9.5%) had presumptive acute DENV infection. Anti-DENV IgG serologic testing was performed for 751 (86.3%) participants, and of these 80 (10.7%) had prior flavivirus exposure. CHIKV infection was more common among infants and children than adults and adolescents (odds ratio [OR] 1.9, P = 0.026) and among HIV-infected patients with severe immunosuppression (OR 10.5, P = 0.007). CHIKV infection is an important but unrecognized cause of febrile illness in northern Tanzania. DENV or other closely related flaviviruses are likely also circulating.

Evaluation and use of NS1 IgM antibody detection for acute dengue virus diagnosis: report from an outbreak investigation

The usefulness of detecting circulating non-structural protein 1 (NS1) IgM antibodies for diagnosing acute dengue virus infection was evaluated during an outbreak investigation along with other routinely used laboratory diagnostic methods. For the first time, the samples were also tested for NS1 antigen detection. NS1 IgM antibody detects all the serum samples that were positive for NS1 antigen detection within first 5 days of infection. The sensitivity of the NS1 IgM ELISA was higher when compared with RT-PCR and therefore, it could be used for early diagnosis.

Commercial dengue rapid diagnostic tests for point-of-care application: recent evaluations and future needs?

Dengue fever, dengue haemorrhagic fever, and dengue shock syndrome (DF/DHF/DSS) are tropical diseases that cause significant humanitarian and economic hardship. It is estimated that more than 2.5 billion people are at risk of infection and more than 100 countries have endemic dengue virus transmission. Laboratory tests are essential to provide an accurate diagnosis of dengue virus infection so that appropriate treatment and patient management may be administered. In many dengue endemic settings, laboratory diagnostic resources are limited and simple rapid diagnostic tests (RDTs) provide opportunities for point-of-care diagnosis. This paper addresses current issues relating to the application of commercial dengue RDTs for the diagnosis of acute dengue virus infection, recent diagnostic evaluations, and identifies future needs.

Infection of bone marrow cells by dengue virus in vivo

Abnormal bone marrow (BM) suppression is one of the hallmarks of dengue virus (DENV) infection in patients. Although the etiology remains unclear, direct viral targeting of the BM has been reasoned to be a contributing factor. The present studies were carried out in an effort to determine the potential effect of DENV infection on the cellularity of BM using a previously established nonhuman primate model of DENV-induced coagulopathy. BM aspirates were collected at various times from the infected nonhuman primate and cells were phenotypically defined and isolated using standard flow cytometry (fluorescence-activated cell sorting). These isolated cells were subjected to detection of DENV utilizing quantitative real-time reverse transcription polymerase chain reaction, electron microscopy, and immunostaining techniques. DENV RNA was detectable by quantitative real-time reverse transcription polymerase chain reaction in BM specimens and the presence of DENV-like particles within platelet was confirmed by electron microscopy. Enumeration of BM cells revealed a transient surge in cellularity at day 1, followed by a gradual decline from days 2 to 10 post infection. Detailed phenotypic studies showed similar kinetics in the frequencies of CD41(+)CD61(+) cells, regardless of CD34 and CD45 expression. The CD61(+) cells were not only the predominant cells that stained for DENV antigen but fluorescence-activated cell sorting-assisted isolation of CD61(+) cells from the BM were shown to contain infectious DENV by coculture with Vero cells. These data support the view that intravenous infection of nonhuman primate with DENV leads to direct infection of the BM, which is likely to be a contributing factor for transient cell suppression in the peripheral blood characteristic of acute DENV infection.

Elevated Levels of Cell-Free Circulating DNA in Patients with Acute Dengue Virus Infection

BackgroundApoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue.Methods and FindingsA direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome.ConclusionsCirculating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.

Dengue Virus Immunoglobulin M Detection in a Reference Laboratory Setting during the 2010 Dengue Virus Outbreak on Caribbean Islands

A large outbreak of dengue virus (DV) infections occurred on Caribbean islands during 2010, with cases peaking during the second half of the year. In conjunction with the outbreak, we observed an unprecedented spike in the number of sera submitted for DV antibody testing between June and December 2010, with a concomitant increase in the number of IgM-positive specimens, indicative of acute DV infection. Analysis of the place of residence of the IgM-positive patients identified from June to December of 2010 revealed that 58.1% were residents of Caribbean islands (Puerto Rico and the U.S. Virgin Islands), whereas 40.6% were residents of the U.S. mainland or Hawaii. The U.S. residents represented 42 states plus the District of Columbia, but most (53%) were from just 3 states (California, Florida, and New York). In comparison to the Caribbean IgM-positive patient group, the U.S. IgM-positive patient group contained proportionately more adults 21 to 60 years old and fewer individuals <21 years old. These findings indicate that the 2010 Caribbean DV outbreak affected many U.S. residents (mostly adults, presumably travelers) from diverse geographic areas and emphasize the potential for a viremic DV-infected returning traveler to spark a local DV outbreak by introducing DV into a community with competent mosquito vectors.

Long-term persistence of clinical symptoms in dengue-infected persons and its association with immunological disorders

The acute manifestations of dengue are well known. The clinical symptoms that present during the convalescent phase of infection are less well characterized, but may be autoimmune-based. This study was undertaken to determine the prevalence of persistent clinical symptoms among individuals infected during the 2006 Cuban epidemic and to evaluate the immunological and genetic factors associated with their occurrence. In 2008, clinical data and blood samples were collected from a random sample of adult individuals diagnosed during the 2006 epidemic with dengue fever (DF, n=68), dengue hemorrhagic fever (DHF, n=29), or an asymptomatic infection (AI, n=42). The presence of persistent symptoms was evaluated in all individuals and a psychological assessment was performed. IgG titers and the Fc receptor (FcR) were also evaluated. The following autoimmune markers were assessed in a subset (n=26) of symptomatic individuals: complement factors C3/C4, rheumatoid factor (RF), C-reactive protein (CRP), antinuclear antibodies (ANA), and immune complex (IC). Over half (55/97) the individuals with a prior of diagnosis of DF or DHF had persistent clinical symptoms in the 2 years following infection. The sequelae were unrelated to the initial diagnosis and were more common among women (44/55). No symptoms were reported in the AI group and all study participants had normal mental and cognitive function. Persistent clinical symptoms were associated with HH polymorphic variant (p=0.027) and high IgG titer (p=0.041). Autoimmune marker alterations were common (20/26) in the subset of symptomatic individuals evaluated. Clinical sequelae after recovery from an acute dengue virus infection are common in the 2 years following infection. The results obtained in this study suggest that persistent symptoms are associated with alterations in some immunological parameters and FcγRIIa gene polymorphism. This could suggest an autoimmune-based disturbance.