Nonstructural Protein 1 Antigen Research Articles

Surface plasmon resonance analysis for detecting non-structural protein 1 of dengue virus in Indonesia.

Dengue is an acute febrile disease caused by dengue virus (DENV) that is transmitted by Aedes sp., which causes serious health conditions in many countries. Non-structural protein 1 (NS1) is a co-factor for the RNA replication of this virus, which represents a new strategy for the identification of dengue. Prompt and accurate laboratory diagnosis of this infection is required to assist in patient triage and management, as well as prevent the spread of this infection. In the present study, we tested the potential of surface plasmon resonance (SPR) as a diagnostic tool for dengue infections. NS1 antigen protein was used as an analyte that targets anti-NS1 antibodies, with their interaction resulting in a change in the refractive index. In comparison to currently available gold-standard detection methods [enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR)], SPR showed a similar sensitivity but greater efficiency and simplicity in terms of infection detection. Out of 26 samples collected from patients with dengue in Indonesia, SPR was able to correctly identify all 16 positively infected individuals at a lower concentration and a shorter period of time compared to ELISA and RT-PCR. This study revealed that SPR is a promising tool for DENV detection and potentially other diseases as well.

Maternal and Neonatal Outcomes during Dengue Infection Outbreak at a Tertiary National Hospital in Endemic Area of Indonesia.

BackgroundDengue infection is the most common mosquito-borne disease in Indonesia. Dengue incidence during pregnancy increases with its prevalence among women of reproductive ages. It potentially causes serious maternal and neonatal morbidity and mortality; however, its impact during a period of disease outbreak has not been described. We aimed to assess the outcomes of pregnancies affected by dengue infection during an outbreak in an endemic area of Indonesia.MethodsWe conducted a prospective observational study at Sanglah General Hospital, Bali, Indonesia from April 2016 to April 2017. All pregnant women with clinical symptoms of dengue infection who tested positive for dengue nonstructural protein 1-antigen or anti-dengue immunoglobulin M were included in the study. Clinical features and hematological and biochemical parameters were documented to assess their relationship with maternal and neonatal outcomes.ResultsOur study population consisted of 41 pregnant women. Most were multigravida (58.5%). Dengue infection without warning signs was observed in 31.7% of the cases, while 53.7% had dengue infection with warning signs, and 14.6% had dengue shock syndrome/severe dengue infection. Most pregnancies were in the third trimester (78%); five cases (12.2%) suffered clinical complications. Preterm delivery was observed in seven cases (17.1%). Most patients (75.6%) decided to seek medical treatment at a critical phase of dengue infection (days 3–6).ConclusionThe high incidence of associated morbidity and mortality should warrant clinicians in endemic regions to consider diagnoses of dengue infection when treating pregnant women with clinical signs and symptoms. Patients should be treated at centers with adequate resources for monitoring and emergency procedures. A more intensive study is needed in the future aiming to make a more comprehensive guideline.

Evaluation of a novel microfluidic immuno-magnetic agglutination assay method for detection of dengue virus NS1 antigen.

BackgroundDengue virus (DENV) is the most important arbovirus worldwide, causing infections in endemic countries and returning travellers from these areas. Rapid diagnostic tests are needed to improve patient management and monitor local transmission. The detection of DENV non-structural protein 1 (NS1) is a useful tool for the diagnosis, but the currently available methods can be time consuming or lack sensitivity. The objective of our study was to evaluate a new rapid and semi-quantitative microfluidic DENV NS1 immuno-magnetic agglutination assay based on aggregation of magnetic nanoparticles detected by an electronic reader (Virotrack Dengue Acute and Blubox, Blusense diagnostics, Copenhagen, Denmark).Methodology/Principal findingsA panel of 135 serum samples from travelers returning from dengue endemic countries was analyzed (74 DENV positive samples including the four DENV serotypes, 26 Zika virus positive samples, 25 chikungunya virus positive samples, 5 malaria positive samples and 5 negative samples). Samples were tested by three different antigen detection methods: SD Dengue NS1 Ag ELISA, SD BIOLINE Dengue Duo and ViroTrack Dengue Acute. The sensitivity observed for SD Dengue NS1 Ag ELISA, ViroTrack Dengue Acute and SD BIOLINE Dengue Duo was 97.2%, 91.1% and 68.1%, respectively. All methods showed high specificity (98.4% for ViroTrack Dengue Acute and 100% for both SD Dengue NS1 Ag ELISA and SD BIOLINE Dengue Duo). SD Dengue NS1 Ag ELISA and ViroTrack Dengue Acute only failed to detect samples positive for DENV-2.Conclusions/SignificanceViroTrack Dengue Acute is a sensitive and specific assay for DENV NS1 detection. It provides faster results than the ELISA method and a better performance than the rapid immunochromatographic tests. ViroTrack Dengue Acute could represent a valuable tool for rapid diagnosis of DENV infections in returning travellers from endemic countries.

Tick-Borne Encephalitis Virus Vaccines Contain Non-Structural Protein 1 Antigen and may Elicit NS1-Specific Antibody Responses in Vaccinated Individuals.

Vaccination against tick-borne encephalitis (TBE) is based on the use of formalin-inactivated, culture-derived whole-virus vaccines. Immune response following vaccination is primarily directed to the viral envelope (E) protein, the major viral surface antigen. In Europe, two TBE vaccines are available in adult and pediatric formulations, namely FSME-IMMUN® (Pfizer) and Encepur® (GlaxoSmithKline). Herein, we analyzed the content of these vaccines using mass spectrometry (MS). The MS analysis revealed that the Encepur vaccine contains not only proteins of the whole virus particle, but also viral non-structural protein 1 (NS1). MS analysis of the FSME-IMMUN vaccine failed due to the high content of human serum albumin used as a stabilizer in the vaccine. However, the presence of NS1 in FSME-IMMUN was confirmed by immunization of mice with six doses of this vaccine, which led to a robust anti-NS1 antibody response. NS1-specific Western blot analysis also detected anti-NS1 antibodies in sera of humans who received multiple doses of either of these two vaccines; however, most vaccinees who received ≤3 doses were negative for NS1-specific antibodies. The contribution of NS1-specific antibodies to protection against TBE was demonstrated by immunization of mice with purified NS1 antigen, which led to a significant (p < 0.01) prolongation of the mean survival time after lethal virus challenge. This indicates that stimulation of anti-NS1 immunity by the TBE vaccines may increase their protective effect.

Study of clinical and laboratory profile of dengue fever in a tertiary care hospital

Background: Dengue fever is one of the most common arboviral mediated outbreaks reported with increased prevalence over the last few years with considerable morbidity and mortality. This study was designed to study the clinical and biochemical parameters in dengue fever patients.Methods: Prospective observational study was undertaken among adult patients in a tertiary care hospital. fifty patients were studied and analysed. All patients who were NS1 (Non-Structural Protein 1) antigen or IgM dengue positive were included in the study. Clinical features, haematological and biochemical parameters were noted.Results: Of the 50 patients studied, majority were males (68%). Fever was the major symptom (100%) followed by Body ache (84%), Headache (64%), Retro-orbital pain (52%), Myalgia (48%), conjunctival injection (40%), Itching (40%), abdominal pain (36%), Bradycardia (34%), Rash (30%), pleural effusion and ascites both seen in (28%). Significant derangements in platelet (76%), leucocyte counts (84%) and serum transaminases (58%) were noted.Conclusions: Fever associated with headache, retroorbital pain, erythematous morbilliform rash, conjunctival injection and itching over palms and soles along with thrombocytopenia, leukopenia, elevated liver transaminases should prompt a clinician on the possibility of dengue infection. Platelet transfusions have little role in management of dengue patients unless patients having active bleeding secondary to thrombocytopenia due to dengue fever.

Lesson learned: Retrospective analysis of ‘missed out’ dengue NS1 positives among IgM negative population

Objective: To determine the proportion of dengue non-structural protein 1 (NS1) positives among laboratory confirmed dengue IgM negative patients.Methods: Data for 1 732 samples received from January to October 2017 at the Virus Research and Diagnostic Laboratory (VRDL) for dengue diagnosis were downloaded from the National Institute of Epidemiology server. Samples that were previously reported as IgM negative for dengue diagnosis were identified and their NS1 status was determined using ELISA. Thus, ’missed out’ NS1 positives were correlated with the duration of illness. Furthermore, an epidemic curve for the study period was constructed. The increase in positivity rate within and between the months was compared by McNemar’s and Pearson’s chi-square test, respectively.Results: The reported IgM-negatives were 813, of which, 22.5% (183) were retrospectively positive for NS1 antigen. The addition of NS1 positives revealed by this study has raised the reported positivity across the months that ranged from 8.1% to 29.6%. By analyzing the dengue positives per month and the epidemic curve, the period between January and September, 2017 was identified as non-epidemic while the epidemic started from the month of October, 2017.Conclusions: Acute dengue infection is widely confirmed by detecting NS1 antigen in serum. Missing out of NS1 positives happen due to shortened window period and such cases act as reservoir for further viral transmission. Hence, this study highly emphasizes performing all three tests for dengue diagnosis that warrants the accurate dengue proportion in India.

Identification of Dengue Serotypes using a Single Serum Specimen Algorithm in a Tertiary Care Hospital, Alappuzha, Kerala, India

Introduction: The geographic location of Alappuzha, a district in the South Indian state of Kerala, the distinct weather conditions and frequent natural calamities present a unique ecology that contributes to the prevalence of vector-borne diseases like dengue. Early dengue virus infection can be detected by using a combination of tests on a single serum specimen. Aim: To identify the dengue virus serotypes among hospitalised patients in a South Indian teaching hospital in Alappuzha, Kerala, India. Materials and Methods: Patient samples that tested positive for dengue non-structural protein-1 (NS1) antigen by ELISA were further evaluated for dengue virus RNA by real-time, multiplex reverse transcriptase Polymerase Chain Reaction (PCR) and the serotype was determined. Anonymised patient data was collected using a questionnaire as a data collection tool. The data was analysed for statistical significance. Results: Among 422 non-duplicate patient serum samples received in the Department of Microbiology, in the year 2019, 30 were positive for dengue NS1 antigen by ELISA. Dengue viral RNA was detected in 50% of the samples (15/30). DENV-3 serotype was the most prevalent (nine) followed by DENV-1 (five) and DENV-2 (one). Common presentations of the patients were fever, headache, and myalgia. No statistically significant association was found between a PCR positive result and the presence of warning signs and thrombocytopenia. Conclusion: DENV-3 was the most common serotype in the study population. Early dengue virus infection is associated with varied symptoms.

A Comprehensive Study of Dengue Epidemics and Persistence of Anti-Dengue Virus Antibodies in District Swat, Pakistan

Background: Dengue fever is one of the most common human arbovirus infections worldwide. In Pakistan, dengue initially became endemic in the big cities and then expanded to remote areas of the country. The current study reports the dengue epidemics, anti-DENV antibodies prevalence during the active and post-dengue infection, risk factors, disease symptoms, and spotting dengue infection densities in district Swat of Pakistan. Methods: Clinical signs and demographic data of dengue suspected individuals were collected at the time of screening through non-structural protein-1 antigen detection test during 2013–2015. Moreover, selected dengue confirmed individuals were screened for the presence of anti-dengue immunoglobulin (Ig) M and G during the active infection period and post-dengue infection. Results: A total of 8,770 individuals were infected with dengue in 2013 with 36 (0.41%) case fatalities, 307 in 2014 with no case fatality, and 13 in 2015 with no case fatality. The number of male and female cases were 6,139 and 2,631 in 2013, 183 and 124 in 2014, and only 10 and 3 in 2015, respectively. Among all the localities, Tehsil Babozai, an urban setting, reported the highest number of dengue patients during all the study years, that is, 7,673 (87.49% of the total cases) in 2013, 294 (95.76% of the total cases) in 2014, and 13 (100% cases) in 2015. Among 6 age groups, 21–30 years was found to be highly infected in 2013 (37.13% of all cases) and 2014 (33.55%). Furthermore, 1,231 (21.94% of all cases) had IgM antibodies and 71 (1.26%) had IgG antibodies in 2013, 78 (26% of all cases) had IgM antibodies and 7 (2.33%) had IgG antibodies in 2014, and only 4 (30.76%) patients had IgM and 0 (0%) had IgG antibodies in 2015. Furthermore, urban areas had the highest infection density in district Swat. The majority of the patients in rural areas had a traveling history to the urban areas before their illness. Conclusion: To sum up, male gender, young individuals, and those living in urban areas were at the greater risk of dengue infection.

Flavivirus Cross-Reactivity to Dengue Nonstructural Protein 1 Antigen Detection Assays.

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses of public health relevance. Both viruses circulate in the same endemic settings and acute infections generally manifest similar symptoms. This highlights the importance of accurate diagnosis for clinical management and outbreak control. One of the commonly used acute diagnostic markers for flaviviruses is nonstructural protein 1 (NS1). However, false positives due to antigenic cross-reactivity have been reported between DENV and ZIKV infections when using DENV NS1 antigen (NS1 Ag) detection assays in acute cases. Therefore, we investigated the lowest detectable virus titres and cross-reactivity of three commercial dengue NS1 Ag rapid assays and two ELISAs for different flaviviruses. Our results showed that substantially high viral titres of ZIKV, Kunjin virus (KUNV) and yellow fever virus (YFV) are required to give false-positive results when using DENV NS1 rapid detection assays. Commercial DENV NS1 ELISAs did not react with ZIKV and YFV. In comparison, tested assays detected DENV at a significantly low virus titre. Given the relatively low viral loads reported in clinical samples, our findings suggest that commercially available dengue NS1 Ag detection assays are less likely to generate false-positive results among clinical samples in areas where multiple flaviviruses cocirculate.

Epidemiological and clinical features of dengue fever outbreak in Jiangxi Province in 2019

Objective To investigate the epidemiological and clinical characteristics of 91 cases of dengue fever outbreak in Jiangxi Province in 2019, and to strengthen the management and prevention of dengue fever. Methods The clinical data, laboratory results and etiology tests of 91 patients with dengue fever from the Ninth Hospital of Nanchang, Zhangshu People′s Hospital, Fengcheng People′s Hospital and Nanchang County People′s Hospital from July 31, 2019 to September 27, 2019 were retrospectively collected. The t test was used for continuous variables and chi-square test was used for categorical variables. Results A total of 91 dengue fever patients were reviewed. The patients age ranged from 4 to 87 years old, and the majority were adult patients. Most patients were farmers and they all had mosquito-biting history. Local cases were the major source of this outbreak. The incubation time was (5.19±2.96) days. All patients had fever over 38 ℃. Thirty-eight (41.8%) patients developed rashes, mainly distributed on the limbs and trunk, and 15 patients (16.5%) developed myalgia and fatigue. The incidence of rashes in patients admitted in September was 15.9% (7/44), which was significantly lower than 68.1% (32/47) in July and August (χ2=25.262, P<0.01). The incidence of headache in patients admitted in September was 27.3%(12/44), which was significantly lower than 78.7% (37/47) in July and August (χ2 =24.206, P<0.01). Among the 91 patients, 64 (70.3%) patients had white blood cell counts less than 4×109/L, 14(15.4%) patients had platelet counts less than 50×109/L. The positive rate of dengue non-structural protein 1 (NS1) antigen was 100.0%(37/37), the positive rates of dengue antibody IgM and IgG were 50.0%(11/22) and 13.6%(3/22), respectively. Among the 113 serum samples tested for dengue virus RNA, 106 were identified with dengue virus type-1 and two were dengue virus type-2. In 22 patients who received tests within 7 days, the positive rate of NS1 antigen was 100.0% (22/22), the IgM and IgG positive rates were 50.0%(11/22) and 13.6% (3/22), respectively, and 19 patients were infected with dengue type-1. Among the IgM antibody positive cases, the onset days before sampling was (5.09±1.64) days, longer than that of IgM antibody negative cases ((2.82±1.83) days), with statistical significance (t=3.063, P=0.007). The onset time before sampling in dengue virus RNA-positive group was (3.53±1.87) days, which was shorter than that of RNA-negative group ((6.67±0.58) days), and the difference was statistically significant (t=2.839, P=0.013). Conclusions The dengue fever outbreak cases in Jiangxi Province in 2019 have typical clinical manifestations of dengue fever. Using the NS1 antigen test, IgM and IgG antibody tests and real-time fluorescent quantitative polymerase chain reaction for dengue virus genotyping during early onset of the disease can assistant clinicians in clinical management, controlling infectious source and stopping disease transmission. Key words: Dengue; Disease outbreaks; Epidemiology; Clinical manifestations

Dengue rash: white islands in a sea of red

A 36-year-old Japanese woman presented to our emergency department with a 2-day history of fever, headache, eye pain, arthralgia and poor appetite after returning from Thailand. She had been sightseeing...

Evaluation of novel rapid detection kits for dengue virus NS1 antigen in Dhaka, Bangladesh, in 2017

BackgroundDengue virus (DENV) infection is one of the biggest challenges for human health in the world. In addition, a secondary DENV infection sometimes causes dengue hemorrhagic fever (DHF), which frequently leads to death. For this reason, accurate diagnosis record management is useful for prediction of DHF. Therefore, the demand for DENV rapid diagnosis tests (RDTs) is increasing because these tests are easy and rapid to use. However, commercially available RDTs often show low sensitivity for DENV and cross-reactivity against other flaviviruses, especially Zika virus (ZIKV).MethodsWe developed two types of novel DENV non-structural protein 1 (NS1) detection RDTs, designated TKK-1st and TKK-2nd kits. Specificities of the monoclonal antibodies (MAbs) used in these kits were confirmed by enzyme-linked immuno-sorbent assay (ELISA), dot blot, and western blot using recombinant NS1 proteins and synthetic peptides. For evaluation of sensitivity, specificity, and cross-reactivity of the novel DENV NS1 RDTs, we first used cultured DENV and other flaviviruses, ZIKV and Japanese encephalitis virus (JEV). We then used clinical specimens obtained in Bangladesh in 2017 for further evaluation of kit sensitivity and specificity in comparison with commercially available RDTs. In addition, RNA extracted from sera were used for viral genome sequencing and genotyping.ResultsEpitopes of three out of four MAbs used in the two novel RDTs were located in amino acid positions 100 to 122 in the NS1 protein, a region that shows low levels of homology with other flaviviruses. Our new kits showed high levels of sensitivity against various serotypes and genotypes of DENV and exhibited high levels of specificity without cross-reactivity against ZIKV and JEV. In clinical specimens, our RDTs showed sensitivities of 96.0% (145/151, TKK-1st kit) and 96.7% (146/151, TKK-2nd kit), and specificities of 98.0% (98/100, TKK-1st kit and TKK-2nd kit). On the other hand, in the case of the commercially available SD Bioline RDT, sensitivity was 83.4% (126/151) and specificity was 99.0% (99/100) against the same clinical specimens.ConclusionsOur novel DENV NS1-targeting RDTs demonstrated high levels of sensitivity and lacked cross-reactivity against ZIKV and JEV compared with commercially available RDTs.

Horseradish peroxidase-labeled rabbit anti-non-structural protein 1 of dengue virus-2 for the diagnosis of dengue virus infections

BACKGROUND Early diagnosis of dengue virus (DENV) infection is essential for patient management and disease control. Detection of the antigen non-structural protein 1 (NS1) has been proven to provide early diagnosis of DENV infection. Thus, commercial NS1 antigen detection assays have been increasingly used and are becoming thetool of choice among clinicians to confirm DENV infection in Indonesia.&#x0D; METHODS To obtain anti-NS1 DENV antibody, NS1 protein (90 µg/ml) from the collection of the Department of Microbiology, Faculty of Medicine, Universitas Indonesia was injected into a rabbit. The anti-NS1 antibody from the rabbit was then labeled with horseradish peroxidase (HRP) using the periodate oxidation method. Sera were tested by enzyme-linked immunosorbent assay (ELISA) to detect NS1 from DENV-infected patients.&#x0D; RESULTS Serially diluted antibody labeled with HRP tested using the direct ELISA method showed the highest absorbance value at a 1:100 dilution (Mean [SD] = 1.35 [0.35]); even at a dilution as high as 1:3,200 (0.22 [0.15]), antibody labeled with HRP was able to detect the NS1 protein, although the absorbance value did not differ greatly from that of the negative control (0.13 [0.01]).&#x0D; CONCLUSIONS In an attempt to develop an NS1-based diagnostic test, polyclonal anti-NS1 DENV antibody was successfully produced as a diagnostic assay to determine the presence of DENV NS1 antigen in patients’ sera.

Nonstructural protein-1 (NS1) of dengue virus type-2 differentially stimulate expressions of matrix metalloproteinases in monocytes: protective effect of paracetamol

BackgroundDengue fever is a re-emerging viral disease and affects millions of population worldwide. Monocytes are involved in dengue viral disease, however, their exact role is still not clear. In the present study, we investigated, the effect of NS1 antigen of dengue virus and paracetamol on THP-1 monocytes associated to expressions of matrix metalloproteinases (MMPs) and cytokine release. MethodsAssessment of cell morphology by bright field microscopy, cell viability by MTT assay, protein estimation by Bradford reagent were done in cells exposed to NS1 antigen in the presence and absence of paracetamol. Cytokines estimations were done by ELISA. Expression profile of matrix metalloproteinase genes was done using real-time PCR and reverse-transcriptase PCR. ResultsNS1 exposure of THP-1 monocytes cells, changed their cell morphology and activated them for release of proteins in 24 h. Expressions of MMP-2, MMP-8, MMP-9 and MMP-14 genes were upregulated by NS1 exposure. Further, exposure of NS1 to THP-1 monocytes cells increased expression profile of MMP-10 and MMP-13 genes to a lesser extent. Treatment with paracetamol (1 mg/ml and 2 mg/ml), significantly down-regulated the expression profile of MMP-2, MMP-8, MMP-9 and 14 in dose dependent manner. NS1 exposure also increased the release of cytokines IL-4, IL-6, and IL-10 but decreased the release of TNF-α and IL-15. Interestingly, paracetamol reversed NS1 induced changes in the release of these cytokine in dose dependent manner. ConclusionMonocytes mediated expression of MMPs participates in the development of dengue pathogenesis in the severe cases of disease and paracetamol may have a protective effect in dengue viral disease.

Antibody responses to tick-borne encephalitis virus non-structural protein 1 and whole virus antigen–a new tool in the assessment of suspected vaccine failure patients

ABSTRACTWe report a new tool for improved serological diagnostics in suspected tick-borne encephalitis (TBE) vaccine failure cases. Due to an increase in the incidence of disease as well as the number of vaccinees, specific and simplified diagnostic methods are needed. Antibody responses to TBE-virus (TBEV) non-structural protein 1 (NS1) are detectable post TBEV infection but not post vaccination. We have used samples from 14 previously confirmed Swedish TBEV vaccine failure patients to study antibody responses against NS1 and whole virus antigens, respectively. Our conclusion is that the detection of antibodies directed to TBEV NS1 antigen is a useful tool to considerably simplify and improve the quality in investigations regarding suspected TBEV infection in vaccinated patients.

The Rise of Imported Dengue Infections in Victoria, Australia, 2010–2016

Dengue notifications have increased dramatically over the past seven years in Victoria, Australia—a trend which has been seen nationally and reflects increased cases internationally. We reviewed the epidemiology of dengue among Victorian travellers, changes in diagnostic methods and describe the burden placed on local health systems resulting from this disease of public health importance. Cases of dengue notified to the Department of Health and Human Services in Victoria, Australia, between 1 January 2010 and 31 December 2016 were included in this review. Demographic, clinical, diagnostic methods, and risk factor data were examined using descriptive epidemiological analyses. Cases of dengue increased on average by 22% per year, with a total of 2187 cases (5.5 cases/100,000 population) notified over the 7-year reporting period. The most frequently reported country of acquisition was Indonesia (45%) followed by Thailand (14%). The use of multiple diagnostic methods, including the non-structural protein 1 antigen (NS1Ag) detection test, increased over time. The median time between onset of illness and diagnosis diminished from 9 days (IQR: 2–15) in 2010 to 4 days (IQR: 2–7) in 2016. Proportionally more cases were discharged directly from emergency departments in recent years (10% in 2010 to 28% in 2016, p < 0.001).The increasing incidence of dengue in Australia is reflective of its growing prominence as a travel medicine problem in western countries. For travellers with non-severe dengue, the improved timeliness of dengue diagnostics allows for consideration of best practice ambulatory management approaches as used in endemic areas.

Immunofluorescence–based biosensor for the determination of dengue virus NS1 in clinical samples

The sharp increase in incidence of dengue infection has necessitated the development of methods for the rapid diagnosis of this deadly disease. Here we report the design and development of a reliable, sensitive, and specific optical immunosensor for the detection of the dengue nonstructural protein 1 (NS1) biomarker in clinical samples obtained during early stages of infection. The present optical NS1 immunosensor comprises a biosensing surface consisting of specific monoclonal NS1 antibody for immunofluorescence-based NS1 antigen determination using fluorescein isothiocyanate (FITC) conjugated to IgG antibody. The linear range of the optical immunosensor was from 15−500ngmL−1, with coefficient of determination (R2) of 0.92, high reproducibility (the relative standard deviation obtained was 2%), good stability for 21days at 4°C, and low detection limit (LOD) at 15ngmL−1. Furthermore, the optical immunosensor was capable of detecting NS1 analytes in plasma specimens from patients infected with the dengue virus, with low cross−reaction with plasma specimens containing the Japanese encephalitis virus (JEV) and Zika virus. No studies have been performed on the reproducibility and cross-reactivity regarding NS1 specificity, which is thus a limitation for optical NS1 immunosensors. In contrast, the present study addressed these limitations carefully where these two important experiments were conducted to showcase the robustness of our newly developed optical-based fluorescence immunosensor, which can be practically used for direct NS1 determination in any untreated clinical sample.

Comparison of nonstructural protein-1 antigen detection by rapid and enzyme-linked immunosorbent assay test and its correlation with polymerase chain reaction for early diagnosis of dengue.

INTRODUCTION:Early diagnosis of dengue is important for appropriate clinical management and vector control. Different serological tests based on the principle of immunochromatography and enzyme-linked immunosorbent assay (ELISA) are commonly used for detection of antigen and antibodies of dengue virus. The performance of these tests depends on the sensitivity and specificity. Hence, the study was undertaken to compare nonstructural protein-1 (NS1) antigen detection by rapid and ELISA with real-time polymerase chain reaction (RT-PCR) for diagnosis of dengue.MATERIALS AND METHODS:Prospective laboratory study was carried out on sera samples (n = 200) from clinically suspected cases of dengue. The sera samples were subjected for NS1 antigen detection test by rapid test, NS1 ELISA, and RT-PCR. The results of rapid and ELISA tests were compared with real Time PCR.RESULTS:The sensitivity, specificity, positive, and negative predictive value of rapid dengue NS1 antigen test were 81.5%, 66.7%, 78.2%, and 71.1%, respectively whereas that of NS1 ELISA were 89.9%, 100%, 100%, and 94%, respectively. Concordance of Rapid NS1 and NS1 ELISA with PCR was 75.5% and 94%.DISCUSSION AND CONCLUSION:NS1 antigen ELISA can be implemented in diagnostic laboratories for diagnosis of dengue in the acute phase of illness. The test also has great potential value for use in epidemic situations, as it could facilitate the early screening of patients and limit disease expansion.

In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2

Human Cathelicidin antimicrobial peptide LL-37 is known to have antiviral activity against many viruses. In the present study, we investigated the in-vitro effect of LL-37 on dengue virus type 2 (DENV-2) infection and replication in Vero E6 cells. To study the effect of pretreatment of virus or cells with LL-37, the virus was pretreated with different concentrations of LL-37 (2.5μM–15μM) or scrambled (Scr) LL-37(5μM–15μM) and used for infection or the cells were first treated with LL-37 and infected. To study the effect of LL-37 post infection (PI), the cells were infected first followed by addition of LL-37 to the culture medium 24h after infection. In all conditions, after the incubation, the culture supernatant was assessed for viral RNA copy number by real time RT-PCR, infectious virus particles by focus forming unit assay (FFU) and non structural protein 1 (NS1) antigen levels by ELISA. Percentage of infection was assessed using immunoflourescence assay (IFA). The results revealed that pretreatment of virus with 10–15μM LL-37 significantly reduced its infectivity as compared to virus control (P<0.0001). Moreover, pretreatment of virus with 10–15μM LL-37 significantly reduced the levels of viral genomic RNA and NS1 antigen (P<0.0001). Treatment of virus with 10–15μM LL-37 resulted in two to three log reduction of mean log10 FFU/ml as compared to virus control (P<0.0001). Treatment of the virus with scrambled LL-37 had no effect on percentage of infection and viral load as compared to virus control cultures (P>0.05). Pretreatment of cells before infection or addition of LL-37 to the culture 24h PI had no effect on viral load. Molecular docking studies revealed possible binding of LL-37 to both the units of DENV envelope (E) protein dimer. Together, the in-vitro experiments and in-silico analyses suggest that LL-37 inhibits DENV-2 at the stage of entry into the cells by binding to the E protein. The results might have implications for prophylaxis against DENV infections and need further in-vivo studies.

External quality assessment of dengue and chikungunya diagnostics in the Asia Pacific region, 2015

ObjectiveTo conduct an external quality assessment (EQA) of dengue and chikungunya diagnostics among national-level public health laboratories in the Asia Pacific region following the first round of EQA for dengue diagnostics in 2013.MethodsTwenty-four national-level public health laboratories performed routine diagnostic assays on a proficiency testing panel consisting of two modules. Module A contained serum samples spiked with cultured dengue virus (DENV) or chikungunya virus (CHIKV) for the detection of nucleic acid and DENV non-structural protein 1 (NS1) antigen. Module B contained human serum samples for the detection of anti-DENV antibodies.ResultsAmong 20 laboratories testing Module A, 17 (85%) correctly detected DENV RNA by reverse transcription polymerase chain reaction (RT–PCR), 18 (90%) correctly determined serotype and 19 (95%) correctly identified CHIKV by RT–PCR. Ten of 15 (66.7%) laboratories performing NS1 antigen assays obtained the correct results. In Module B, 18/23 (78.3%) and 20/20 (100%) of laboratories correctly detected anti-DENV IgM and IgG, respectively. Detection of acute/recent DENV infection by both molecular (RT–PCR) and serological methods (IgM) was available in 19/24 (79.2%) participating laboratories.DiscussionAccurate laboratory testing is a critical component of dengue and chikungunya surveillance and control. This second round of EQA reveals good proficiency in molecular and serological diagnostics of these diseases in the Asia Pacific region. Further comprehensive diagnostic testing, including testing for Zika virus, should comprise future iterations of the EQA.