NS1 glycoprotein detection in serum and urine as an electrochemical screening immunosensor for dengue and Zika virus.

The incidence of infection by the dengue virus (DENV) has grown dramatically, reaching 128 countries in tropical and subtropical regions worldwide, with a pattern of hyper-endemicity. DENV is a mosquito-borne disease having four serotypes, one or two circulating in epidemic outbreaks. The diagnosis of DENV is challenging mainly due to the circulation of new viruses with remarkable similarities, such as Zika (ZIKV) that may cause fetal microcephaly. DENV affects 390 million people per year, but these numbers may be higher due to the underreported and misclassified cases. Recently, the NS1 nonstructural protein has been described in serum and urine of DENV and ZIKV patients, suggesting its use as a biomarker for screening since a negative NS1 sample confirms the absence of these infections. Herein, a label-free immunosensor comprising an assembled nanostructured thin film of carbon nanotube-ethylenediamine is described. The advantage of in situ electrosynthesis of polymer film is to allow major control of thickness and conductivity, in addition to designing the reactive groups for functionalization. A quartz crystal microbalance system was used to estimate the thickness of the polymeric film obtained. The anti-NS1 monoclonal antibodies were immobilized to carbon nanotubes by covalent linkage, permitting a high stability during measurements. Analytical responses to NS1 were obtained by differential pulse voltammetry (DPV), showing a linear range from 20 to 800 ng mL-1 and reproducibility of 3.0%, with a limit of detection (LOD) of 6.8 ng mL- 1. This immunosensor was capable of detecting ZIKV and DENV NS1 in spiked urine and real serum in a clinical range.Graphical abstract.

Urinary leukotrienes and histamine in patients with varying severity of acute dengue.

BACKGROUND: Vascular leak is a hallmark of severe dengue, and high leukotriene levels have been observed in dengue mouse models, suggesting a role in disease pathogenesis. We sought to explore their role in acute dengue, by assessing levels of urinary LTE4 and urinary histamine in patients with varying severity of acute dengue. METHODS: Urinary LTE4, histamine and creatinine were measured by a quantitative ELISA, in healthy individuals (n = 19), patients with dengue fever (DF = 72) and dengue haemorrhagic fever DHF (n = 48). The kinetics of LTE4 and histamine and diurnal variations were assessed in a subset of patients. RESULTS: Urinary LTE4 levels were significantly higher (p = 0.004) in patients who proceed to develop DHF when compared to patients with DF during early illness (≤ 4 days) and during the critical phase (p = 0.02), which continued to rise in patients who developed DHF during the course of illness. However, LTE4 is unlikely to be a good biomarker as ROCs gave an AUC value of 0.67 (95% CI 0.57 and 0.76), which was nevertheless significant (p = 0.002). Urinary LTE4 levels did not associate with the degree of viraemia, infecting virus serotype and was not different in those with primary vs secondary dengue. Urinary histamine levels were significantly high in patients with acute dengue although no difference was observed between patients with DF and DHF and again did not associate with the viraemia. Interestingly, LTE4, histamine and the viral loads showed a marked diurnal variation in both patients with DF and DHF. CONCLUSIONS: Our data suggest that LTE4 could play a role in disease pathogenesis and since there are safe and effective cysteinyl leukotriene receptor blockers, it would be important to assess their efficacy in reducing dengue disease severity.

Assessing dehydration status in dengue patients using urine colourimetry and mobile phone technology.

BACKGROUND: Dengue is a systemic and dynamic disease with symptoms ranging from undifferentiated fever to dengue shock syndrome. Assessment of patients' severity of dehydration is integral to appropriate care and management. Urine colour has been shown to have a high correlation with overall assessment of hydration status. This study tests the feasibility of measuring dehydration severity in dengue fever patients by comparing urine colour captured by mobile phone cameras to established laboratory parameters. METHODOLOGY/PRINCIPAL FINDINGS: Photos of urine samples were taken in a customized photo booth, then processed using Adobe Photoshop to index urine colour into the red, green, and blue (RGB) colour space and assigned a unique RGB value. The RGB values were then correlated with patients' clinical and laboratory hydration indices using Pearson's correlation and multiple linear regression. There were strong correlations between urine osmolality and the RGB of urine colour, with r = -0.701 (red), r = -0.741 (green), and r = -0.761 (blue) (all p-value <0.05). There were strong correlations between urine specific gravity and the RGB of urine colour, with r = -0.759 (red), r = -0.785 (green), and r = -0.820 (blue) (all p-value <0.05). The blue component had the highest correlations with urine specific gravity and urine osmolality. There were moderate correlations between RGB components and serum urea, at r = -0.338 (red), -0.329 (green), -0.360 (blue). In terms of urine biochemical parameters linked to dehydration, multiple linear regression studies showed that the green colourimetry code was predictive of urine osmolality (ß coefficient -0.082, p-value <0.001) while the blue colourimetry code was predictive of urine specific gravity (ß coefficient -2,946.255, p-value 0.007). CONCLUSIONS/SIGNIFICANCE: Urine colourimetry using mobile phones was highly correlated with the hydration status of dengue patients, making it a potentially useful hydration status tool.

The detection of anti-dengue virus IgM in urine in participants enrolled in an acute febrile illness study in Puerto Rico.

BACKGROUND: Dengue is an important arboviral disease with about 100 million dengue cases per year, of which, ~5% result in severe disease. Clinical differentiation of dengue from other acute febrile illnesses (AFI) is difficult, and diagnostic blood tests are costly. We evaluated the utility of anti-DENV IgM in urine to identify dengue cases among AFI patients enrolled in a clinical study. METHODS: Between May 2012-March 2013, 1538 study participants with fever for ≤7 days were enrolled, a medical history was obtained, and serum and urine specimens were collected. Serum was tested for DENV RNA and anti-DENV IgM. Urine was tested for anti-DENV IgM, and its sensitivity and specificity to detect sera laboratory-positive dengue cases were calculated. We evaluated if urine anti-DENV IgM positivity early (≤5 days post-illness onset [DPO]) and late (6-14 DPO) in the clinical course was associated with dengue severity. RESULTS: Urine anti-DENV IgM sensitivity and specificity were 47.4% and 98.5%, respectively, when compared with serum anti-DENV IgM ELISA results, and 29.7% and 91.1% when compared with serum rRT-PCR results. There was no correlation between urine anti-DENV IgM positivity and patient sex or pre-existing chronic disease. Early in the clinical course, a significantly higher proportion of those who developed dengue with warning signs had anti-DENV IgM in their urine when compared to those without warning signs (20.4% vs. 4.3%). There was no difference in the proportion with urine anti-DENV IgM positivity between severity groups late in the clinical course. CONCLUSION: While detection of urine anti-DENV IgM lacked adequate diagnostic sensitivity, it is a highly specific marker for laboratory-positive dengue, and its presence early in the clinical course may distinguish those with more severe disease. Further assessment of urine anti-DENV IgM by DPO is warranted to determine its utility as an early diagnostic (and possibly prognostic) marker for dengue.

Case Report: A Case of Acute Intravascular Hemolysis in Dengue Fever.

Acute intravascular hemolysis (AIH) with hemoglobinuria is a rare complication of dengue viral infection. Review of the literature revealed only one report of AIH as a complication of dengue fever (DF). We report a case of AIH with massive hemoglobinuria in a 17-year-old Sri Lankan male, in the febrile phase of DF. This unusual and rare complication warranted forced diuresis, to prevent acute kidney injury. This was a departure from the recommended fluid therapy for DF.

Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya.

BACKGROUND: Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives. METHODS: Procedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration. RESULTS: The tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India. CONCLUSIONS: A kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments.

Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens.

BACKGROUND: Dengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: Serial plasma, urine and saliva samples were collected at several time-points between the day of admission to hospital until three months after the onset of fever in children with confirmed dengue disease. Quantitative RT-PCR, NS1 antigen capture and ELISA serology for anti-DENV antibody (IgG, IgM and IgA) detection were performed in parallel on the three body fluids. RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5% and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8% and 52.9%/28.6% in plasma, urine and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine. CONCLUSIONS: Although the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible.

Recovery of dengue virus from urine samples by real-time RT-PCR.

Recently, reverse transcription polymerase chain reaction (RT-PCR) for dengue virus (DENV) has been reported to test positive in urine samples for a longer time frame than in serum. We evaluated two RNA extraction procedures from urine and investigated the stability of DENV RNA in urine and serum up to 1 year at different storage temperatures. In addition, 24 urine samples collected from patients with a recent infection were tested with DENV real-time RT-PCR and compared to the RT-PCR results on serum. Five patients with an acute DENV infection were followed up for 6 months by RT-PCR on urine. The automated extraction method with the MagNA Pure LC 2.0 device had a higher yield of DENV RNA compared to the manual QIAGEN method, explained by the higher volume used in the former method. DENV RNA in both serum and urine was stable at room temperature up to 1 month and at 4 °C and -20 °C for at least 1 year. The detection rate by RT-PCR on urine was 50 % (4/8) until day 7, 100 % (6/6) between 1 and 3 weeks after symptom onset, and 25 % (2/8) thereafter. Generally, DENV RNA concentrations are higher in serum than in urine up till day 7, switching to lower concentrations in serum thereafter. Peak concentrations in urine are reached around day 10, and RNA becomes undetectable 3 to 4 weeks following disease onset. This diagnostic tool is of added value in clinical settings by extending the period during which DENV infections are diagnosed by RT-PCR.

Manifestaciones del dengue a nivel renal / Manifestations of dengue in the kidney

IIntroducción: el dengue es una infección con afectación multisistémica, autolimitada, con espectro clínico que varía de formas asintomáticas a graves y fatales. Hay descripciones de casos de afectación renal en todo el mundo. Objetivo: describir las manifestaciones renales por el dengue. Metodología: estudio observacional, descriptivo, prospectivo realizado en pacientes adultos con dengue grave internados en el Dpto. de Medicina Interna del Hospital Nacional (Itauguá, Paraguay) durante la epidemia del verano 2013-2013. Fueron evaluados parámetros clínicos y laboratoriales con énfasis en la función renal. Resultados: se reclutaron 135 pacientes, 69 varones y 66 mujeres, con edad media 42,7±18 años. En 37% de los casos había alguna comorbilidad. Se midió proteinuria de 24 hs en 34 sujetos, encontrándose en rango nefrótico en 8,8% y en rango elevado ( = 160 mg/día) en 67,6%. Otros hallazgos llamativos fueron la hematuria (38%) e hipertensión arterial transitoria (39%). Un solo paciente presentó alteración de urea y creatinina al alta. Conclusiones: las manifestaciones renales por el dengue grave son frecuentes, predominando la proteinuria, hematuria e hipertensión arterial, aunque son transitorias en la mayoría.

I Introduction: dengue fever is an infection with multisystem involvement, self-limiting, clinical spectrum ranging from asymptomatic to severe and fatal forms. There are descriptions of cases of renal disease worldwide. Objective: to describe renal manifestations of dengue. Methodology: observational, descriptive, prospective study in adult patients with severe dengue admitted to the Department of Internal Medicine, National Hospital (Itauguá, Paraguay) during summer 2013 to 2013 epidemic. We evaluated clinical and laboratory parameters with emphasis on renal function. Results: 135 patients, 69 men and 66 women were recruited, mean age 42.7 ± 18 years. In 37% of cases there was any comorbidity. 24 h proteinuria were measured in 34 subjects, being in nephrotic range at 8.8% and high range (= 160 mg / day) in 67.6%. Other striking findings were hematuria (38%) and transient hypertension (39%). Only one patient presented alterations in urea and creatinine at discharge. Conclusions: renal manifestations of severe dengue are common, predominantly proteinuria, hematuria and hypertension, although they are transient in most.

Assessment of microalbuminuria for early diagnosis and risk prediction in dengue infections.

BACKGROUND: Dengue is the most important arboviral infection of humans. Following an initial febrile period, a small proportion of infected patients develop a vasculopathy, with children at particular risk for severe vascular leakage and shock. Differentiation between dengue and other common childhood illnesses is difficult during the early febrile phase, and risk prediction for development of shock is poor. The presence of microalbuminuria is recognized as a useful early predictor for subsequent complications in a number of other disorders with vascular involvement. Significant proteinuria occurs in association with dengue shock syndrome and it is possible that early-phase microalbuminuria may be helpful both for diagnosis of dengue and for identification of patients likely to develop severe disease. METHODOLOGY/PRINCIPAL FINDINGS: We measured formal urine albumin to creatinine ratios (UACRs) in daily samples obtained from a large cohort of children with suspected dengue recruited at two outpatient clinics in Ho Chi Minh City, Vietnam. Although UACRs were increased in the 465 confirmed dengue patients, with a significant time trend showing peak values around the critical period for dengue-associated plasma leakage, urine albumin excretion was also increased in the comparison group of 391 patients with other febrile illnesses (OFI). The dengue patients generally had higher UACRs than the OFI patients, but microalbuminuria, using the conventional cutoff of 30 mg albumin/g creatinine discriminated poorly between the two diagnostic groups in the early febrile phase. Secondly UACRs did not prove useful in predicting either development of warning signs for severe dengue or need for hospitalization. CONCLUSION/SIGNIFICANCE: Low-level albuminuria is common, even in relatively mild dengue infections, but is also present in many OFIs. Simple point-of-care UACR tests are unlikely to be useful for early diagnosis or risk prediction in dengue endemic areas.

Severe falciparum malaria with dengue coinfection complicated by rhabdomyolysis and acute kidney injury: an unusual case with myoglobinemia, myoglobinuria but normal serum creatine kinase.

BACKGROUND: Acute kidney injury (AKI) is a complication of severe malaria, and rhabdomyolysis with myoglobinuria is an uncommon cause. We report an unusual case of severe falciparum malaria with dengue coinfection complicated by AKI due to myoglobinemia and myoglobinuria while maintaining a normal creatine kinase (CK). CASE PRESENTATION: A 49-year old Indonesian man presented with fever, chills, and rigors with generalized myalgia and was diagnosed with falciparum malaria based on a positive blood smear. This was complicated by rhabdomyolysis with raised serum and urine myoglobin but normal CK. Despite rapid clearance of the parasitemia with intravenous artesunate and aggressive hydration maintaining good urine output, his myoglobinuria and acidosis worsened, progressing to uremia requiring renal replacement therapy. High-flux hemodiafiltration effectively cleared his serum and urine myoglobin with recovery of renal function. Further evaluation revealed evidence of dengue coinfection and past infection with murine typhus. CONCLUSION: In patients with severe falciparum malaria, the absence of raised CK alone does not exclude a diagnosis of rhabdomyolysis. Raised serum and urine myoglobin levels could lead to AKI and should be monitored. In the event of myoglobin-induced AKI requiring dialysis, clinicians may consider using high-flux hemodiafiltration instead of conventional hemodialysis for more effective myoglobin removal. In Southeast Asia, potential endemic coinfections that can also cause or worsen rhabdomyolysis, such as dengue, rickettsiosis and leptospirosis, should be considered.

Electrolyte disturbances and abnormal urine analysis in children with dengue infection.

Serum electrolytes and urine analysis results were retrospectively reviewed in children with either dengue fever (DF) or dengue hemorrhagic fever (DHF). Children who had positive serology for dengue infection and serum electrolytes determined before starting intravenous fluid were included in the study. During the years 2004-2007, 73 DF patients, age 9.29 +/- 3.62 years, and 77 DHF patients, age 10.04 +/- 3.64 years were enrolled in the study. The patients were admitted to the hospital on average on days 4.12 +/- 1.1 and 4.25 +/- 1.4 of febrile illness for DF and DHF, respectively. The prevalence of hyponatremia in patients with DF was 61% and DHF was 72% (p = 0.149). The mean serum sodium levels in patients with DF and DHF were 133.5 +/- 3.52 and 133.5 +/- 3.20 mEq/l (p = 0.938), respectively. The prevalence of hyponatremia in patients with mild (grade I), moderate (grade II) and severe (grade III-IV) DHF were 70, 77, and 78% (p = 0.729), respectively, and the mean serum sodium levels were 134.1 +/- 3.05, 132.9 +/- 3.33, and 132.5 +/- 3.28 (p = 0.189), respectively. The prevalence of hypokalemia in patients with DF was 14% and 17% in patients with DHF (p = 0.588). A high urine specific gravity reflecting dehydration was found in 63% of patients with DF and 60% of patients with DHF (p = 0.77). The prevalences of hematuria in patients with DF and DHF were 18% and 27% (p = 0.182), respectively and proteinuria were 15% and 27% (p = 0.072), respectively. The prevalences of hematuria and proteinuria were not different among patients with mild, moderate and severe DHF. No patients had gross hematuria or developed acute renal failure requiring dialysis. Mild hyponatremia is a common electrolyte disturbance and renal involvement is mild in patients with DF and DHF.

Different patterns of oxidized lipid products in plasma and urine of dengue fever, stroke, and Parkinson's disease patients: cautions in the use of biomarkers of oxidative stress.

Many products of lipid oxidation have been associated with human diseases. These include F2-isoprostanes (F2-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), and cholesterol oxidation products (COPs). Here we present measurements of F2-IsoPs, HETEs, COPs, and arachidonate in single plasma samples of patients with acute (dengue fever and ischemic stroke) and chronic (Parkinson's) diseases, and in age-matched study controls. Urine samples were collected for F2-IsoPs analysis. Our analysis demonstrated elevated F2-IsoPs levels in ischemic stroke, HETEs in Parkinson's disease, dengue fever, and ischemic stroke, and COPs in Parkinson's disease and dengue fever patients, as compared with those in age-matched study controls. Strong but complex correlations were observed between levels of certain oxidized lipid products and age. The relations between various oxidized lipids and dengue fever, stroke, and Parkinson's disease are discussed in relation to the selection and application of biomarkers of oxidative lipid damage, in particular the need for corrections for age and lipid levels.

Confirmation of dengue virus infection by detection of dengue virus type 1 genome in urine and saliva but not in plasma.

We successfully detected dengue virus (DENV) genome in urine and saliva but not in plasma samples from a Japanese dengue fever patient. The results of the present study suggest that detection of DENV genome in urine and saliva can be an effective diagnostic method, particularly for children with viral hemorrhage.

Serum and urine sodium levels in dengue patients.

We evaluated serum and urine sodium levels in children with dengue infections. Children with acute febrile illness admitted to Bhumibol Adulyadej Hospital in Bangkok from January 1999 to January 2000 were enrolled. Serum and urine sodium levels were measured before initiating intravenous fluid therapy. Two milliliters of blood were obtained on admission and before discharge to test for anti-dengue virus antibody using the enzyme-linked immunosorbent assay technique. Hyponatremia was defined as a serum sodium level <130 mEq/l and depletion of circulatory volume was defined as a urine sodium level < 20 mEq/l. Out of 93 enrolled patients, 49 were categorized as dengue patients and 44 were as non-dengue patients. Six dengue patients developed shock whereas 43 patients did not. The mean serum sodium level was significantly lower in dengue patients compared to non-dengue patients (p-value < 0.0001). Hyponatremia was 9.7 times more common in dengue patients. Among dengue patients, the mean serum sodium level was significantly lower in shock patients compared to non-shock patients (p-value = 0.003). However, the prevalence of hyponatremia was not different between the two groups. The mean urine sodium level was significantly lower in dengue patients compared to non-dengue patients (p-value < 0.0001). A urine sodium level < or = 20 mEq/l was 8.1 times more common in dengue patients. Among dengue patients, the mean urine sodium level was not significantly different between shock and non-shock patients. In shock patients, a urine sodium level < or = 20 mEq/l was 7.6 times more common.

Rhabdomyolysis associated with dengue virus infection.

We describe 2 patients who developed rhabdomyolysis due to dengue virus infection. The first patient recovered with no sequelae, but the second developed multiple organ failure and died. Rhabdomyolysis is not well described as a complication of dengue virus infection and is probably underrecognized. All patients with severe dengue virus infection should undergo urinalysis, and serum creatinine kinase levels should be measured if urinalysis reveals heme.

Histamine content in 24-hour urine in patients with dengue haemorrhagic fever.

Twenty-four hour urinary histamine in 12 patients with DHF compared to 12 normal subjects in the comparable age and sex were studied. The results revealed significantly increased urinary excretion in patients with DHF than in normal subjects in both free and total forms. This finding suggests that histamine may be one, if not all, of the mediators released during the course of the disease, especially in the severe cases. Histamine may play an important role for the leakage of intravascular fluid to the various serous spaces resulting in hypovolemia and shock.