Detection Of Dengue Virus Research Articles

Establishment of Quantitative and Recovery Method for Detection of Dengue Virus in Wastewater with Noncognate Spike Control

Establishment of Quantitative and Recovery Method for Detection of Dengue Virus in Wastewater with Noncognate Spike Control

One-Pot Nitrogen-Doped Carbon Quantum Dots Synthesised from Palm Kernel Shell Coupled with Aunps as Fluorescence-Based DNA Biosensor for Dengue Virus Detection

One-Pot Nitrogen-Doped Carbon Quantum Dots Synthesised from Palm Kernel Shell Coupled with Aunps as Fluorescence-Based DNA Biosensor for Dengue Virus Detection

Magnetic Multiplex Loop Mediated Isothermal Amplification (MM-LAMP) technique for simultaneous detection of dengue and chikungunya virus

Dengue and chikungunya viruses are arthropod borne virus spread through common vector instigating infection in human. There has been an increased recognition that more attention needs to be paid to similar sympotoms caused by both of the virus as they spread in the same region at same time. It warrants need of cost effective, user friendly and rapid multiplex diagnostic technique which could simultaneously diagnose and identify between two virus diseases in resource poor setting. A magnetic multiplex loop mediated isothermal amplification (MM-LAMP) technique was developed by coupling multiplex LAMP with magnetic particle-based naked eye visualization to overcome the shortcoming of simultaneous detection of both diseases. In recent years this technology has emerged as a particularly attractive candidate as amplification reaction process completes within 45 min. The first step involves multiplexing biotin and digoxigenin coated dengue and chikungunya primers respectively in LAMP reaction followed by precipitation of the amplified DNA with polyethylene glycol (PEG) buffer and finally clumping with streptavidin and anti-digoxigenin coated magnetic particle for virus discrimination and naked eye visualization. The DNA detection limit of MM LAMP visualization was 51.65 ng/μl which is comparable to the electrophoresis base UV light visualization. The results showed potential superiority over standard methods polymerase chain reaction (PCR). This current advancement empowers multiplex LAMP utility in resource limited setting without using any of the florescent dyes, turbidimeter, or the sophisticated quantitative PCR machine etc which restrict multiplex LAMP technique to laboratorial use only. We have proposed a novel method without such limitations. This technique has potential as a point of care technique for simultaneous detection of two diseases.

Dengue NS1 antigen kit shows high sensitivity for detection of recombinant dengue virus-2 NS1 antigen spiked with Aedes aegypti mosquitoes

Dengue, caused by the dengue virus (DENV) is a significant vector-borne disease. In absence of a specific treatment and vaccine, dengue is becoming a rising threat to public health. Currently, control of dengue mainly focuses on the surveillance of the mosquito vectors. Improved surveillance methods for DENV in mosquito populations would be highly beneficial to the public health. However, current methods of DENV detection in mosquitoes requires specialized equipment and expensive reagents and highly trained personnel. As an alternative, commercially available dengue NS1 antigen ELISA kits could be used for detection of DENV infection in Aedes aegypti mosquitoes. In this study, we explored the utility of commercially available Dengue NS1 antigen kit (J. Mitra & Co. Pvt. Ltd) for the detection of recombinant dengue virus-2 (rDENV-2) NS1 protein and serum of dengue infected patient spiked with Ae. aegypti mosquito pools. The kit was found to be highly sensitive and specific towards detection of all serotypes of DENV. Further, it could detect as low as 750 femto gram rDENV-2 NS1 protein. It was also observed that rDENV-2 NS1 antigen spiked with blood-fed and unfed mosquito pools could be detected. In addition, the kit also detected dengue infected patient serum spiked with Ae. aegypti mosquito pools. Overall, the Dengue NS1 antigen kit displayed high sensitivity towards detection of recombinant as well as serum NS1 protein spiked with Ae. aegypti mosquito pools and could be considered for the dengue virus surveillance after a field evaluation in Ae. aegypti mosquitoes.

Development of a Dengue Virus Serotype-Specific Non-Structural Protein 1 Capture Immunochromatography Method.

Four serotypes of dengue virus (DENV), type 1 to 4 (DENV-1 to DENV-4), exhibit approximately 25–40% of the difference in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from specimens followed by PCR amplification is the current standard method of DENV serotype determination. However, since this method is time-consuming, rapid detection systems are desirable. We established several mouse monoclonal antibodies directed against DENV non-structural protein 1 and integrated them into rapid DENV detection systems. We successfully developed serotype-specific immunochromatography systems for all four DENV serotypes. Each system can detect 104 copies/mL in 15 min using laboratory and clinical isolates of DENV. No cross-reaction between DENV serotypes was observed in these DENV isolates. We also confirmed that there was no cross-reaction with chikungunya, Japanese encephalitis, Sindbis, and Zika viruses. Evaluation of these systems using serum from DENV-infected individuals indicated a serotype specificity of almost 100%. These assay systems could accelerate both DENV infection diagnosis and epidemiologic studies in DENV-endemic areas.

Generation of Nanoyeast Single-Chain Variable Fragments as High-Avidity Biomaterials for Dengue Virus Detection.

Bioengineered yeast bio-nanomaterials termed nanoyeasts displaying antibody single-chain variable fragments (scFvs) against diagnostic targets are a promising alternative to monoclonal antibodies (mAbs). A potential limitation for translating nanoyeasts into diagnostic tools is batch-to-batch variability. Herein, we demonstrate a systematic approach for cost-efficient production of highly specific nanoyeasts that enabled accurate dengue virus (DENV) detection by immunoassay (2.5% CV). Yeasts bioengineered to surface express DENV-specific scFvs (up to 66% of the total cell population) were fragmented into nanoyeast fractions trialing sonication, bead beating, and high-pressure disruption methods. Nanoyeast fractions from sonication had optimal target binding, uniform particle size (±89 nm), were stable, and retained diagnostic activity for 7 days at 37 °C compared to traditional mAbs that lost activity after 1 day at 37 °C. We engineered a panel of nanoyeast scFvs targeting DENV nonstructural protein 1 (NS1): (i) specific for serotyping DENV 1-4 and (ii) cross-reactive anti-DENV scFvs that are suitable for "yes/no" diagnostic applications. We demonstrate highly specific nanoyeast scFvs for serotyping DENV. We show that nanoyeast scFvs specifically detect NS1 in simulated patient plasma with a limit of detection of 250 ng/mL, the concentration found in infected patients.

The Burden of Acute Febrile Illness Attributable to Dengue Virus Infection in Sri Lanka: A Single-Center 2-Year Prospective Cohort Study (2016–2019)

ABSTRACT.We performed a 2-year prospective cohort study to determine the incidence of dengue in Angoda, Colombo district, Sri Lanka (NCT02570152). The primary objective was to determine the incidence of acute febrile illness (AFI) because of laboratory confirmed dengue (LCD). Secondary objectives were to determine AFI incidence because of non-LCD, describe AFI symptoms, and estimate AFI incidence because of LCD by dengue virus (DENV)-type and age group. Participants from households with at least one minor and one adult (≤50 years) were enrolled and followed with scheduled weekly visits and, in case of AFI, unscheduled visits. Blood was collected for DENV detection at AFI visits, and symptoms recorded during the 7-day period following AFI onset. A total of 2,004 participants were enrolled (971 children, and 1,033 adults). A total of 55 LCD episodes were detected (overall incidence of 14.2 per 1,000 person-years). Incidence was the highest among children < 5 years (21.3 per 1,000 person-years) and 5–11 years (22.7 per 1,000 person-years), compared with adults ≥ 18 years (9.2 per 1,000 person-years). LCD was mostly (83.6%) caused by DENV-2 (n = 46), followed by DENV-1 (n = 6) and DENV-3 (n = 3). Common symptoms of LCD were headache, fatigue, myalgia, loss of appetite, and arthralgia. Incidence of AFI because of non-LCD was 47.3 per 1,000 person-years. In conclusion, this study reports the LCD incidence for a DENV-2 dominated epidemic that is comparable to the incidence of suspected dengue reported passively for 2017, one of the worst outbreaks in recent history.

Persistence of Dengue Virus (DENV-1, 2, 3,4) Transovarial-Transgenerational with Realtime Polymerase Chain Reaction (qPCR) in Ae. Aegypti and Ae. Albopictus (Diptera: Culicidae)

Ae. aegypti and Ae. albopictus have an important role in DHF transmission because they can simultaneously transmit the dengue virus vertically / transovarially or horizontally. This phenomenon indicates the persistence of the dengue virus by vectors. The aim of this research was to prove the persistence of the transovarial-transgenerational dengue virus (DENV-1,2,3,4) with real time polymerase chain reaction (qPCR) in Ae. aegypti and Ae. albopictus (Diptera: Culicidae). Quasi experimental design with intervention infects DENV 1-2-3-4 serotypes in Ae. aegypti and Ae. albopictus intratoracally. Research population Ae. aegypti and Ae. albopictus laboratory colony females. Dengue virus detection uses real-time polymerase chain reaction (qPCR). Transovarial detection by qPCR indicates detection of dengue virus in Ae. albopictus DENV-1 to progeny 1 (F1), DENV-2 and DENV-3 to F2, DENV-4 to F3. Next to Ae. aegypti DENV-1 to 1st progeny (F1), DENV-2 to F2, DENV-3 to F4 and DENV-4 to F3. there was no difference in MIR value (p value: 0.356) for the four serotypes in Ae. albopictus and Ae. aegypti. DENV-3 is the most persistent serotype in Ae. aegypti with 83.3% MIR and DENV-4 were the most persistent serotypes in Ae.albopictus with 100% MIR. The need to improve vector control models that focus not only on the main vector, but also other co-vectors.

Portable fiber-optic SPR platform for the detection of NS1-antigen for dengue diagnosis

Here, we present a portable, selective and cost-effective fiber-optic surface plasmon resonance (SPR) based platform for early detection of Dengue virus. NS1 protein was targeted as the biomarker of dengue. Antibody-antigen specific binding was exploited for NS1 antigen detection. The binding of antibody was assisted by a self-assembled monolayer of alkanethiols on the surface of silver-coated unclad fiber. A wavelength interrogation mode of SPR was utilized to detect NS1 antigen in the dynamic range of 0.2–2.0 μg/ml. The 40 nm thick silver coated optical fiber exhibited resonance wavelength around 500 nm and change in resonance wavelength was monitored for each attachment step on the fiber. The sensitivity at the lowest concentration of NS1 antigen was found to be 54.7 nm/(μg/ml). The limit of detection of the sensor was found to be 0.06 μg/ml, which lies in the physiological range of NS1 protein present in the infected blood, hence the present technique may provide a very early detection advantage. Real blood serum samples were also successfully tested on the set-up, confirming compatibility with the conventional methods. The presented field-deployable platform has wide applications in mass monitoring of dengue, such as during outbreaks and epidemics.

A sensitive electrochemical method for rapid detection of dengue virus by CRISPR/Cas13a-assisted catalytic hairpin assembly

Dengue fever caused by Dengue virus (DENV) infection has been widely popular, especially in tropical and subtropical areas. Rapid and sensitive diagnosis is the first priority for treatment of DENV infection. This work designed a signal amplification strategy for sensitive electrochemical detection of DENV by using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system for catalytic hairpin assembly on electrode surface. The presence of target RNA could activate the cleavage activity of the CRISPR/Cas13a system to release the blocker silenced swing arms, which then hybridized with hairpin 1 (H1) immobilized on electrode surface to expose the pre-locked toehold domain of H1 for the hybridization of ferrocene-labeled hairpin 2 (H2-Fc). Eventually, a large number of H2-Fc were captured to the electrode to produce amperometric signal for achieving signal amplification. This method showed a linear detection range from 5 fM to 50 nM with a detection limit of 0.78 fM. The proposed assay was successfully used to detect DENV type 1 in total RNA sample extracted, indicating great potential for application in early clinical diagnostic.

Conventional and point-of-care diagnostic tools for dengue virus detection and their possible treatments

Conventional and point-of-care diagnostic tools for dengue virus detection and their possible treatments

Chitosan-Integrated Single-Mode Tapered Optical Fiber DENV II E Protein Sensor

This work reports the enhancement of a single-mode tapered optical fiber (TOF) sensor for Dengue virus (DENV) II E protein using chitosan. Chitosan, an organic nanomaterial with excellent biocompatibility and abundant binding sites, was deposited on TOF as an intermediate layer between the activated taper surface and the anti-DENV II E protein antibodies. The best DENV sensing performance was attained at chitosan thickness of 17.13 nm, which was attributed to the optimum balance between the auxiliary layer thickness and penetration depth of the evanescent field. An enhanced sensitivity value of 14.19 nm/nM was achieved compared to the sensitivity obtained by standard configuration sensor. A lower dissociation constant, K <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">d</sub> of 1.00×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-10</sup> M was also acquired, which implies a higher affinity towards the targeted E protein. Findings from this work highlight the potential of organic nanomaterial such as chitosan for biosensor enhancement and further emphasize the capability of fiber optic sensors for detection of dengue virus as well as other antigens.

Porous Au-seeded Ag nanorod networks conjugated with DNA aptamers for impedimetric sensing of DENV-2

An efficient nanobioconjugate has been developed as electrode coating material by self-assembling our newly reported porous Au-seeded Ag nanorod networks (Hy-Au@AgNR840-Ass) with a thiol modified DNA aptamer (HS-Apt) for the early-stage specific detection and quantification of Dengue virus (serotype 2). Characterization of the precursors (Hy-Au@AgNR840 and Hy-Au@AgNR840-Ass) for effective fabrication of the bioconjugate (Hy-Au@AgNR840-Ass-S-Apt) was performed by using microscopic (TEM and AFM), spectroscopic (Absorbance, XPS, and SERS), scattering (DLS) and diffractometric (XRD) techniques and has been detailed in our previous publication whereas the binding affinity between the bioconjugate and different serotypes of dengue is studied here using low cost electrochemical impedimetric spectroscopy (EIS). Despite the structural similarity between virion particles of different dengue serotypes, we can detect the most prevalent DENV-2 specifically by using our nonenzymatic nanobioconjugate assay for the first time through the impedimetric technique. The cross-reactivity is checked in presence of closely related serotypes like DENV-1 & DENV-3. The key achievement of this study lies in its ability in the direct detection of DENV-2 serotype without the aid of an enzyme or antibody in dengue-infected blood up to ~ 100 infectious units per mL.

An updated RT-qPCR assay for the simultaneous detection and quantification of chikungunya, dengue and zika viruses.

The real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for the detection and quantification of arboviruses, including Chikungunya, Dengue, and Zika viruses. In this study, an updated real-time RT-qPCR assay was designed and evaluated together with a synthetic positive-control chimeric RNA for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses. Amplification assays were performed to verify the construct integrity and optimal reaction/thermal cycling conditions. The analytical sensitivity of the assay was determined for each virus in single and multiplex reactions, as well as the performance in the detection and viral load quantification of experimental samples. The real-time RT-qPCR assay presented here allowed for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses and could be applied in several studies where the accurate quantification of viral genomes is required.

Non-Invasive Dengue Diagnostics-The Use of Saliva and Urine for Different Stages of the Illness.

Dengue diagnosis is largely dependent on clinical symptoms and routinely confirmed with laboratory detection of dengue virus in patient serum samples collected via phlebotomy. This presents a challenge to patients not amenable to venipuncture. Non-invasive methods of dengue diagnosis have the potential to enhance the current dengue detection algorithm. In this study, samples from dengue infected patients were collected between January 2012 until September 2012 and September 2013 until December 2013 in two different setups. Panel A samples (blood, urine, and saliva) were collected daily when the 39 patients were hospitalised and during their follow-up visits while Panel B samples (saliva) were collected from 23 patients during the acute stage of dengue. Using DENV PCR on Panel A, from day 2 to day 4 post fever onset, serum showed the best overall positivity followed by saliva and urine (100%/82.1%/67.9%). From day 5 until day 10 post fever onset, serum and urine had similar positivity (67.4%/61.2%), followed by saliva (51.3%). Beyond day 10 post fever onset, DENV was undetectable in sera, but urine and saliva showed 56.8% and 28.6% positivity, respectively. DENV in urine was detectable up until 32 days post fever. Panel B results showed overall sensitivity of 32.4%/36% (RNA/NS1) for DENV detection in saliva. Our results suggest that the urine-based detection method is useful especially for late dengue detection, where DENV is undetected in sera but still detectable in urine. This provides a potential tool for the physician to pick up new cases in an area where there is ongoing dengue transmission and subsequently prompt for intensified vector control activities.

Electrochemical biosensing of DENV nucleic acid amplified with triplet nanostructure-mediated dendritic hybridization chain reaction

A universal and highly sensitive electrochemical biosensing strategy was developed by using a triplet nanostructure-mediated dendritic hybridization chain reaction (HCR) for the analysis of Dengue virus (DENV) nucleic acid. A locked dsDNA was firstly recognized by the target DENV nucleic acid fragment to release the initiator, which was then captured by a capture DNA modified gold electrode to initiate the HCR in the presence of biotin labeled substrates A and B, and two helpers. After the recognition reactions of the captured initiator with substrate A and then helper 1, one DNA structure was formed to react with substrate B, which produced a triplet nanostructure to trigger the dendritic HCR by the toehold of the hybridization structure. After the dendritic product was bound with avidin labeled horseradish peroxidase (avidin-HRP), an amperometric signal could be obtained to achieve ultrasensitive electrochemical detection of DENV. The non-enzymatic isothermal amplification was driven only by entropy. The stable three-dimensional dendritic nanostructure with rigid double helix could assure the excellent performance of the designed electrochemical biosensing strategy. The proposed method showed a detection range of 1.6–1000 pM with a detection limit of 188 fM and ability to distinguish single-base mutation. By changing the recognition sequence of the initiator, the detection of different DENV nucleic acid fragments could be achieved with the same performance. Thus, this method had good extendability for other nucleic acids, providing a promising candidate for nucleic acid detection in early clinical diagnosis.

PAMAM-Graphene Oxide-Integrated Microfiber Sensor for Label-Free Dengue II E Protein Detection

We present a polyamidoamine-graphene oxide (PAMAM-GO) integrated microfiber sensor for the detection of Dengue virus II E protein (DENV II E protein). The microfiber was fabricated by heating a region of a single-mode optical fiber and pulling both ends at constant speed to achieve a diameter of 12 μm from 125 μm. The narrowing of the optical fiber diameter induced evanescent waves on the surface of the microfiber that interacted with the antibody/antigen complex adhered on the surface of the microfiber. The intensity of the interaction corresponds to the concentration of the antigen and can be quantified as a wavelength shift to the transmission spectrum. With the integration of PAMAM-GO, the sensor managed to achieve a sensitivity of 13.25 ± 0.2 nm/nM which is double the value achieved with a standard microfiber configuration. The overall performance of the sensor has potential towards improving dengue diagnostics today.

Advanced Lyophilised Loop Mediated Isothermal Amplification (L-LAMP) based point of care technique for the detection of dengue virus

Dengue virus infects millions of the people globally each year and its diagnosis remains a challenge. Conventionally used diagnostic methods are complex and time consuming. LAMP technique is a potential alternative for diagnosis of dengue virus. The benefits of LAMP are its ease and ability, as it does not require an expensive equipment and results are effortlessly visualized by the naked eye. However, it does not aid as point of care technique owing to need of contamination free area, deep freezer for chemical storage and primer self amplification. Each small modification in LAMP method bring it towards an ideal point of care technique. An advanced lyophilized loop mediated isothermal amplification (L-LAMP) was developed in which the dye was dried on the cap and reaction reagents was lyophilized at the bottom of the tube to overcome the common hurdles of LAMP technique. The technique was able to diagnose disease within 35 min with 4U of Bst polymerase. The least concentration of dye required was 1000×.Result given by the seminested reverse transcriptase polymerase chain reaction (RT-PCR) and L-LAMP with enzyme linked immuno sorbent assay (ELISA) were compared using Chi square test. The L-LAMP showed 100 % specificity and 92 % sensitivity with respect ELISA and was found better than RT-PCR which showed 100 % specificity and 88 % sensitivity. There was no cross reactivity of primers with other disease like malaria caused by Plasmodium falciparum and P. vivax and with viral disease chikungunya. L-LAMP has dynamic potential as point of care technique.

Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens

Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several “in-house” components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.

Sensitive and Prolonged Detection of Dengue Virus RNA in Whole Blood.

Molecular detection of dengue virus (DENV) RNA from serum or plasma provides an accurate acute-phase diagnostic (< 7 days after symptom onset). Detection may be prolonged in whole blood, although data are limited. We tested for DENV by real-time reverse transcription-PCR in 345 paired acute-phase plasma and whole blood samples from individuals with a Flavivirus-like illness in southwestern Guatemala. In 18/18 cases with detectable DENV RNA in plasma, whole blood samples were positive and yielded similar cycle threshold values. In seven individuals with convalescent samples obtained 2-3 weeks later, DENV RNA remained detectable in whole blood but not plasma. In three additional cases, DENV RNA was only detectable in whole blood at the acute visit. In two cases, whole blood detection was linked to a virologically confirmed DENV infection 6-11 weeks earlier. Whole blood DENV RNA detection is sensitive for acute dengue infection and may remain positive for weeks to months.