Nonstructural Protein 1 Antigen Research Articles

Study of association of thrombocytopenia and serological parameters in dengue fever with special reference to NS1 antigen in hilly Region of Northern India

Background: Dengue fever is an arthropod-borne disease transmitted to humans by Aedes aegypti mosquitoes and one of the leading causes of arthropod-borne viral disease in the world. Aims and Objectives: The present study aims to analyze any association between platelet count and immunochromatography -based dengue serology tests. Materials and Methods: This was a cross-sectional study conducted in Government Doon Medical College and Hospital. A total of 19,208 clinically suspected cases of dengue who reported in various outpatient departments, emergency services, and inpatient departments of our hospital between June 2023 and November 2023 were included in this study. Samples were tested by rapid immunochromatographic test for dengue non-structural protein 1 (NS1) antigen, immunoglobulin M (IgM), IgG, and platelet count was also obtained and a comparison was made. Results: Out of total 19,208 samples tested, 4876 (25.38%) samples were positive for NS1, 589 (3.06%) samples were positive for IgM. Majority of the patients were in the age group of 21–40 years 9274 (48.28%). 11,753 (61.19%) were males and 7455 (38.80%) were females. A strong correlation was seen between NS1 and thrombocytopenia where a total of 2737/4876 (n=56.13%) patients showed positivity in NS1 with low platelet counts. Conclusion: Detection of NS1 antigen helps in early diagnosis of dengue to avoid complications significantly. In confirmed dengue cases with fever, thrombocytopenia is more consistently found and can be used as a predictor to reduce the morbidity and mortality of dengue disease.

Development of a quantitative NS1 antigen enzyme-linked immunosorbent assay (ELISA) for Zika virus detection using a novel virus-specific mAb

Viruses from the Flaviviridae family, such as Dengue virus (DENV), Yellow fever virus (YFV), and Zika virus (ZIKV) are notorious global public health problems. ZIKV emergence in Polynesia and the Americas from 2013 to 2016 raised concerns as new distinguishing features set it apart from previous outbreaks, including its association with neurological complications and heightened disease severity. Virus detection is impaired as cross-reactivity to other closely related orthoflaviviruses is common among commercially available diagnostic kits. While non-structural protein 1 (NS1) has been used as an early marker of DENV and West Nile virus (WNV) infection, little is known about NS1 expression during ZIKV infection. In the present work, we developed a NS1 capture ELISA using a novel ZIKV-specific monoclonal antibody to study NS1 expression dynamics in vitro in mosquito and human cell lines. While detectable in culture supernatants, higher concentrations of NS1 were predominantly cell-associated. To our knowledge, this is the first report of NS1 detection in human cells despite viral clearance over time. Tests with human samples need to be conducted to validate the applicability of NS1 detection for diagnosis, but overall, the tools developed in this work are promising for specific detection of acute ZIKV infection.

NS1-Specific Antibody Response Facilitates the Identification of Children With Dengue and Zika in Hyperendemic Areas.

Infections by dengue virus (DENV) and Zika virus (ZIKV) have some similar symptoms and a cross-reactive immune response, although with different risk populations and outcomes. Here, we evaluated the virological characteristics and the nonstructural protein 1 (NS1)-specific antibody responses to DENV and ZIKV in children suspected of dengue in different epidemiological moments in Colombia. Viral RNA, circulating NS1 and IgM/IgG specific for DENV and ZIKV were performed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) in 301 children suspected of dengue enrolled in a hospital setting during the ZIKV epidemic and a primary healthcare setting during a DENV epidemic. For the detection of DENV and ZIKV-specific IgM, an NS1-based ELISA was validated using characterized pediatric samples. Clinical and laboratory parameters were also evaluated. DENV RNA or NS1 antigen was detected in the plasma of 62% of children, and in none, the ZIKV RNA was found. NS1-based ELISA for DENV and ZIKV IgM showed a sensitivity/specificity of 90/84% and 73/98%, respectively. Of 114 children without detectable viremia or antigenemia, 30.7%, 17.5%, 22% and 30% were IgM-DENV+, IgM-ZIKV+, IgM-DENV+ZIKV+ and IgM-DENV-ZIKV-, respectively. The ZIKV/DENV IgM-NS1 ratio allows the identification of the infecting ortho flavivirus in 88% of the children with IgM-DENV+ZIKV+, confirming a high predominance of DENV infections in the 2 pediatric settings. Overall, 88% of the children with clinical suspicion of dengue had an identifiable ortho flaviviral infection, with 80% caused by DENV, 7% by ZIKV and 0.7% classified as recent infections or coinfection, demonstrating active viral cocirculation in the pediatric population of southern Colombia. The IgM-NS1 detection improved the identification of ortho flaviviral infections in children without viremia or antigenemia, suggesting it is a helpful complementary tool for medical personnel in tropical regions with high viral cocirculation and different clinical scenes.

Seroprevalence and incidence of primary dengue infection and its correlation with fetomaternal prognosis in Western Rajasthan.

Viral hemorrhagic fevers are becoming increasingly common in the tropics and subtropics. Dengue fever is currently the most important arthropod-borne viral disease because of its widespread distribution in more than 100 countries and its potential for extensive outbreaks of life-threatening disease. This study was a hospital-based cross-sectional study conducted in the Microbiology Laboratory of Maternal and Child Tertiary Care Hospital in Western Rajasthan, India, between January 2021 and December 2021. Institutional Ethical Committee permission was obtained. All patients with clinical suspicion of dengue-like illness (DLI), attending outpatient department (OPD) or inpatient department (IPD), were included in the study after obtaining their written consent. A blood sample was collected, and the Dengue Duo rapid card test was conducted for the detection of nonstructural protein 1 (NS1) antigen and immunoglobulin (Ig) M or IgG antibody estimation. All positive samples were tested for IgM enzyme-linked immunosorbent assay (ELISA) test using MAC-ELISA. Of 250 positive sample, the distribution of cases as per clinical features was as follows: all cases presented with fever (100%) followed by myalgia (24.5%), headache (16.06%), hemorrhagic manifestation (13.25%), rash (8.84%), and bleeding gums (2.01%). Thrombocytopenia was seen in 30.40% (76/250) of dengue fever cases. NS1 antigen was detected in 157 cases (62.80%) followed by IgG in 84 cases (33.60%), IgM in 77 cases (30.80%), NS1+IgG in 27 cases (10.80%), NS1 + IgM in 16 cases (6.40%), and NS1 + IgM + IgG in five cases (2%). Of 250 samples, 77 cases were IgM positive and 173 were IgM negative by the Dengue Duo card test. Among the 173 Dengue Duo IgM card negative, 131 cases (79.39%) were also detected negative by IgM ELISA and 42 cases (49.41%) were detected positive by IgM ELISA. The sensitivity was 50.59%, the specificity was 79.39%, the positive predictive value (PPV) was 55.84%, the negative predictive value (NPV) was 75.72%, and the diagnostic accuracy was 69.90%. The case fatality of the cases was 2.35%. Early diagnosis and treatment can prevent mortality in pediatric and pregnant females suffering from dengue and dengue-like illness. Facility and availability of ELISA kits should be adequate for early confirmation of suspected dengue patients by ELISA test.

Expression of recombinant dengue virus type 1 non-structural protein 1 in mammalian cells and preliminary assessment of its suitability to detect human IgG antibodies elicited by viral infection

In recent years dengue has become a rapidly growing public health problem worldwide, however, the availability of accurate and affordable diagnostic immunoassays is limited, partly due to the difficulty of producing large quantities of purified antigen. Non-structural protein 1 (NS1) has shown to be a good candidate for inclusion in diagnostic assays and for serosurveys, particularly in endemic countries as a prerequisite for vaccination. In this work the NS1 antigen derived from dengue virus type-1 (DENV1) was expressed in HEK293-T cells and purified by affinity chromatography. The recombinant protein was recovered properly folded as dimers, highly purified and with good yield (1.5 mg/L). It was applied as a serological probe in an indirect ELISA developed in this work to detect human IgG antibodies. Preliminary comparative performance values of 81.1% sensitivity and 83.0% specificity of the developed and preliminary validated iELISA, relative to a commercial kit were obtained, suggesting that the purified recombinant DENV1 NS1 antigen is suitable to detect IgG antibodies, indicative of past DENV infection.

Differentiating Secondary Dengue Using IgG/IgM Antibody Ratio: A Cross-sectional Study

Introduction: Dengue is a viral infection with diverse clinical manifestations. Infection in most of the patients is subclinical. It may also present as undifferentiated fever, severe dengue like Dengue Haemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). Secondary dengue infection predisposes to severe dengue, a serious threat to the community as they increase morbidities and mortality. The diagnosis of secondary infection is either by Polymerase Chain Reaction (PCR) to detect the serotype or dengue IgG/IgM antibody ratio. A ratio of more than 1.1 is evidence of secondary dengue. Aim: To find out the prevalence of secondary dengue by measuring the dengue anti-IgG/IgM ratio by Enzyme Linked Immunosorbent Assay (ELISA) test. Materials and Methods: This cross-sectional observational study was conducted at Government Kilpauk Medical College, Chennai for a period of six months from July 2021 to December 2021. Sample size was 450 calculated at 95% confidence level, (CI 28.3-38.3) with 33.3% as prevalence. Blood samples were collected and Nonstructural Protein 1 (NS1) antigen assay was done. The IgG and IgM antibodies assays were done for NS1 antigen positive individuals. The IgG/IgM ratio was calculated in patients who were positive for both IgG and IgM. Statistical analysis was done with Statistical Package for Social Sciences (SPSS) version 20.0. Percentage, mean and standard deviation were calculated. Results: Total of 450 patients were included, out of which 194 were NS1 antigen positive, 106 were males and 88 females with the mean age 25±14.85 years. NS1 antigen was positive in 89.17%. Two-tailed Fisher’s-exact test showed a significant proportion of patients among NS1 positive were positive for both IgG and IgM. An IgG/IgM ratio of >1.1 was found in 62.04%. The association between IgG/IgM ratio of >1.1 and thrombocytopenia was significant [p-value=0.00504, Odd’s ratio (OD) =3.316]. Conclusion: Dengue IgG/IgM antibody ratio of more than 1.1 on the third day of symptom onset and the platelet count assist in the early diagnosis of secondary dengue thereby helps clinicians to initiate the appropriate treatment at the right time, reduce mortality rate and decrease the economic burden of the country.

Analysis of a Dengue Virus Outbreak in Rosso, Senegal 2021.

Senegal is hyperendemic for dengue. Since 2017, outbreaks have been noticed annually in many regions around the country, marked by the co-circulation of DENV1-3. On 8 October 2021, a Dengue virus outbreak in the Rosso health post (sentinel site of the syndromic surveillance network) located in the north of the country was notified to the WHO Collaborating Center for arboviruses and hemorrhagic fever viruses at Institut Pasteur de Dakar. A multidisciplinary team was then sent for epidemiological and virologic investigations. This study describes the results from investigations during an outbreak in Senegal using a rapid diagnostic test (RDT) for the combined detection of dengue virus non-structural protein 1 (NS1) and IgM/IgG. For confirmation, samples were also tested by real-time RT-PCR and IgM ELISA at the reference lab in Dakar. qRT-PCR positive samples were subjected to whole genome sequencing using nanopore technology. Virologic analysis scored 102 positives cases (RT-PCR, NS1 antigen detection and/or IgM) out of 173 enrolled patients; interestingly, virus serotyping showed that the outbreak was caused by the DENV-1, a serotype different from DENV-2 involved during the outbreak in Rosso three years earlier, indicating a serotype replacement. Nearly all field-tested NS1 positives samples were confirmed by qRT-PCR with a concordance of 92.3%. Whole genome sequencing and phylogenetic analysis of strains suggested a re-introduction in Rosso of a DENV-1 strain different to the one responsible for the outbreak in the Louga area five years before. Findings call for improved dengue virus surveillance in Senegal, with a wide deployment of DENV antigenic tests, which allow easy on-site diagnosis of suspected cases and early detection of outbreaks. This work highlights the need for continuous monitoring of circulating serotypes which is crucial for a better understanding of viral epidemiology around the country.

Atorvastatin attenuates NS1 (Non-structural protein-1) of dengue type-2 serotype-induced expressions of matrix metalloproteinases in HL-60 cells, differentiated to neutrophils: Implications for the immunopathogenesis of dengue viral disease

BackgroundThe dengue is a vector borne viral infection in humans. Bite of mosquito infected with a dengue virus transmits the disease. The neutrophils support more to the innate immune response by switching to infected tissues and triggering immunomodulatory mechanisms including the release of proteases and host defence peptides. MethodsCell viability by MTT and trypan blue dye exclusion assay, bright field microscopy for assessment of cell morphology, cytokines measurements by ELISA, estimation of protein by Bradford assay were done. Assessments of matrix metalloproteinase genes mRNA expressions were done using real-time PCR. ResultsIn the present study, we have for the first time unveiled that, NS1 antigen of dengue type-2 serotype, induce and stimulate the neutrophils cells to express high levels of matrix metalloproteases. NS1 exposure of HL-60 cells differentiated to neutrophils affected cell morphology and in 24 h of exposure. We have demonstrated that, the NS1 antigen has induced MMP-2, MMP-14 and MMP-9 expressions in neutrophils in a 24hrs exposure time. NS1 exposure has also further upregulated MMP-1, MMP-13, and MMP-8 expressions in neutrophils in a 24hrs exposure time. Notably, treatment with atorvastatin concentrations downregulated the expression profile of the all matrix metalloprotease significantly. Importantly, NS1 antigen has significantly increased the IL-6, IL-13 release by the HL,60 cells which was reversed by atorvastatin. On the other hand, NS1 exposure enhanced the mRNA expressions of VEGF-A and VEGF-D which was reversed by atorvastatin. However, we found that, NS1 exposure reduced the mRNA expressions profile of VEGF-C, which was reversed by atorvastatin. ConclusionIn conclusion, we report that, neutrophils associated matrix metalloprotease are involved in the pathogenesis of dengue viral disease. VEGF growth factors may also be released by the neutrophils which may subsequently participate in the endothelial dysfunctions leading to dengue shock syndrome.

Predictive Analysis of Gallbladder Wall Thickness as a Marker for Bleeding Risk and Need for Transfusion in Dengue Patients.

Increased gallbladder wall thickening (GBWT) is a manifestation of increased capillary permeability caused by severe dengue. This study was carried out to link the severity of GBWT with bleeding risk and the need for transfusion. This retrospective study included all patients diagnosed with dengue infection either via dengue nonstructural protein-1 antigen or IgM antibody. Pearson's correlation, linear regression, and receiver operating characteristic curves were used for predictive analysis of GBWT with events of bleeding and need for transfusion of platelets during the hospital stay. A total of 177 participants met the inclusion criteria; the mean age was 33.17 ± 13.63 years. Mean GBWT was found to be 0.37 ± 0.15 cm, with 46.3% of patients having a thickness greater than 0.30 cm. A total of 16 patients had bleeding events, of which 7.3% had minor bleeding and 1.7% had a major bleeding event. Linear regression analysis showed that increased GBWT was associated with decreased platelet count on admission (P = 0.002) and lowest platelet counts (P = 0.004). GBWT was found to be predictive of bleeding events and transfusion of platelets at higher sensitivity and specificity than was either platelet count on admission and lowest platelet counts.

Incidence of Dengue fever, serotypes, clinical features, and laboratory markers: a case study of 2019 outbreak at district Shangla, KP, Pakistan.

Dengue is a widely spread mosquito-borne infection in humans, which in recent decades declared is public health problem globally. The dengue virus contains 4 different serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) which belong to the genus Flavivirus. A descriptive experimental study was conducted to determine the epidemiology, types of Dengue serotypes, clinical features, laboratory probe, and markers for primary diagnosis of dengue virus infection in hospitalized patients. A total of 691 suspects were diagnosed from August to October 2019 in district Shangla KP, Pakistan. Serological tests were used for nonstructural protein-1 antigen (NS1), and antibodies (immunoglobulin-M (IgM) & Immunoglobulin-G (IgG)) while real-time PCR was used to confirm the cases. The data was statistically analyzed using IBM-SPSS Statistics 20 version. The dengue virus infection was more prevalent in the male group (68.09%) than the female group (31.1%). A large number of patients were from rural areas (63.5%) while from urban areas were (36.4%), whereas Besham tehsil was found the most affected compared to other regions. The most prevalent serotype observed in our study was DENV-3 (56.60%) while DENV-4 was the least prevalent serotype (1.88%). Among the age-wise analysis of dengue-virus-infected individuals, the age group of 19-37 years (64.07%) was found the most affected group. The month-wise analysis revealed that the highest number of infections (49.8%) were recorded in September. Significant differences were noticed among blood parameters. The possible reasons for the dengue overwhelming in the study area could be less or lack of awareness particularly regarding the transmission of viral infections, improper sewage management, and no effective vector control strategies that lead the dengue outbreaks in the study population.

Trends in the Seroprevalence of Dengue in a Tertiary Care Hospital of North Karnataka, India

Introduction: Dengue virus of the Flaviviridae family is the causative agent of dengue fever. The Aedes aegypti mosquito is the main vector for its transmission. Though, the cases of dengue fever are mild and self-resolving, there can be fatal complications like Dengue Haemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). Aim: To study the trends in the seroprevalence of dengue in serum samples of suspected cases. Materials and Methods: The present study was a cross- sectional study which was conducted from January 2017 to December 2019, at Belagavi Institute of Medical Sciences (BIMS), Belagavi, Karnataka, India. The serum samples were collected from suspected dengue fever cases and tested by Immunoglobulin M (IgM) capture Enzyme Linked Immunosorbent Assay (ELISA), to detect IgM antibody against dengue virus and NS1 capture ELISA for dengue NS1 (nonstructural protein 1) antigen using ELISA kits manufactured by National Institute of Virology (NIV), Pune. The tests were performed according to the manufacturer’s instruction. The data obtained from the study was analysed using descriptive statistics. Results: A total of 8,992 serum samples were tested over a period of three years, of which 1,340 (14.90%) were positive for dengue infection. Among which 1,048 (78.21%) were positive for anti-dengue IgM antibodies, 109 (8.13%) were positive for NS1 antigen and 183 (13.66%) were positive for both. Most affected age group was 11-20 years and male to female ratio is 1.18:1. The seasonal peak was observed in monsoon i.e. month of June (15.52%) followed by August (12.02%). Conclusion: Seroprevalence of dengue infection being critical signifies the importance of detection of both IgM antibodies and NS1 antigen for diagnosis of dengue infection. The study also identifies younger population being at higher risk and also monsoon as the most favourable season for viral transmission in this region and highlights the importance of concerted efforts towards disease control and prevention.

Comparison of NS1 Antigen Detection by RDT and ELISA and its Concordance with RT-PCR for the Early Diagnosis of Dengue in Ananthapuramu District, Andhra Pradesh, India

Introduction: Early detection of dengue fever is important for effective clinical care and vector control. For the detection of dengue viral antigen and antibodies, several serological techniques based on the concept of immunochromatography and Enzyme-Linked Immunosorbent Assays (ELISA) are routinely utilised. The performance of these tests depends on the sensitivity and specificity. Aim: To compare Non Structural protein-1 (NS1) antigen detection by Rapid Diagnostic Tests (RDTs) and ELISA and its association with Real-Time Polymerase Chain Reaction (RT-PCR). Materials and Methods: This diagnostic cross-sectional study was done on 100 clinically suspected cases of Dengue between July-November 2021 at a tertiary care hospital in Ananthapuramu, Andhra Pradesh, India. All the sera samples were collected and subjected to NS1 antigen detection test by rapid test, NS1 ELISA, and RT-PCR, and also serotypes DENV-1, 2, 3, 4 were detected from serum sample by RT-PCR test. The results of rapid and ELISA tests were compared with RT-PCR. Data was analysed by Statistical Package for the Social Sciences (SPSS) version 16.0. Results: Out of total 100 samples, 29 samples were tested positive by NS1 rapid test, 30 samples were tested positive by NS1 ELISA, and 32 samples were tested positive by RT-PCR. The sensitivity, specificity of dengue NS1 antigen rapid test were 87.50% and 98.52%, respectively when compared to RT-PCR whereas that of NS1 ELISA were 93.75% and 100% when compared to RT-PCR. Out of 32 samples tested positive by RT-PCR, two samples were positive for DENV-1, 26 samples were positive for DENV-2, and four samples were positive were DENV-3. Conclusion: The NS1 ELISA test requires extra procedures and time. RDTs need a one-step operation that takes roughly 15-30 minutes. Despite the fact that RT-PCR and ELISA have better performance than RDTs, RDTs are more effective for early diagnosis and therapy of dengue fever in countries with limited infrastructure and in remote places.

Evaluation of a new NS1 rapid diagnostic test using a single acute-phase serum panel collected during the largest dengue outbreak in Taiwan history in 2015.

Dengue virus (DENV) infection results mostly from the bites of virus-carrying Aedes mosquitoes, which results in dengue fever (DF) with or without warning signs, severe dengue, or asymptomatic infections in humans. For point-of care identification of DENV-infected patients, a rapid diagnostic test (RDT) for DENV nonstructural protein 1 (NS1) has been developed to achieve early diagnosis and timely clinical management. We evaluated the performance of a new commercially available dengue NS1 RDT AsiaGen Dengue NS1 Antigen Rapid Diagnosis Test using real-time qRT-PCR as a reference method and compared the results with SD BIOLINE Dengue NS1 Ag using a single acute-phase serum panel collected during the largest dengue outbreak in the history of Taiwan in 2015. The results suggested that the sensitivity and specificity of AsiaGen Dengue NS1 Antigen RDT (96.9% and 100%) were similar to those of SD BIOLINE Dengue NS1 RDT (100% and 100%) for detection in the acute phase of DENV-2 infection. The results suggested that the sensitivity of both RDTs was similar (95.4% ~ 100%) for the sera collected at less than or equal to three days postsymptom onset (PSO). Our results suggested that the two DENV NS1 RDTs used in this study were promising for the timely diagnosis of DENV infection during dengue outbreaks, at least for DENV-2 in areas where authorized medical laboratories are not available or medical resources are limited. However, the performance of AsiaGen DENV NS1 RDTs in the detection of primary/secondary infections and infection by serotypes of DENV other than DENV-2 requires further investigation.

DENGUE FEVER OUTBREAK IN TWIN CITIES; A TERTIARY CARE CENTER EXPERIENCE

Objective: To characterize the clinical presentations and laboratory markers (as disease predictors and markers) and severity of disease in patients admitted with dengue fever.
 Study Design: Prospective observational study.
 Place and Duration of Study: Department of Infectious Diseases, Pak Emirates Military Hospital Rawalpindi, from May to Oct 2018
 Methodology: In our study, we included the patients treated for dengue fever. Data were recorded on predesigned proforma and analyzed using Microsoft excel 365 and Social Package for Statistical Sciences version 23.
 Results: A total of 160 patients were included in this study, all were males. All cases were positive for Nonstructural Protein 1 Antigen. Symptoms seen in abundance were fever with rigors, chills, backache, retro-orbital pain, nausea and vomiting. Manual platelet count at admission ranged from 15-396 x 103 (mean 107.5 ± 6.8 x 103). Platelet count on discharge ranged from 102-577 x 103 (mean 207.9 ± 95.4 x 103). A total of 34 (21.25%) patients out of 160 confirmed and admitted dengue fever cases had Dengue Hemorrhagic Fever (Nonstructural Protein 1 Antigen positive, thrombocytopenia and leukopenia) and 2 patient went into dengue shock syndrome. The derangement in alanine aminotransferase and alkaline phosphatase levels were shown in 98 (61.2%) and 62 (31.2%) of patients. All these patients were immunoglobulin M and Nonstructural Protein 1Antigen positive.
 Conclusion: Raised serum alanine aminotransferase and alkaline phosphatase levels along with early positive Nonstructural Protein 1 Antigen is a marker of disease severity.

Performance of a New Microfluidic Dengue NS1 Immuno-magnetic Agglutination Assay for the Rapid Diagnosis of Dengue Infection in Adults

Dengue (DENV) infections are a public health concern worldwide and thus early diagnosis is important to ensure appropriate clinical management. The rapid diagnostic test (RDT) targets nonstructural protein 1 (NS1) detection and is the main tool used for diagnostic purpose. In this study, we evaluated the performance of a new rapid and semi-quantitative microfluidic DENV NS1 immuno-magnetic agglutination assay or IMA (ViroTrack Dengue Acute, BluSense Diagnostics, Copenhagen, Denmark). We studied 233 subjects confirmed to have DENV infection (by a real-time reverse transcriptase polymerase chain reaction) and 200 control samples were taken from patients with confirmed diagnoses of other febrile illnesses, in Thailand. Samples were tested using the NS1 antigen (Ag) detection methods: in-house NS1 Ag ELISA (ELISA), SD BIOLINE Dengue NS1 Ag RDT (ICT), and ViroTrack Dengue Acute (IMA). Sensitivities of these tests were 86.3%, 78.9%, and 85.5%, respectively. All tests showed high specificity (100%, 99%, and 97% for ELISA, ICT, and IMA, respectively). The sensitivities of both RDTs were affected by the low sensitivity to DENV-2 and DENV-4. NS1 Ag was detected in every patient on day 1 and day 2 after onset of illness by ELISA and IMA with a decline in detection rates over time after day 6 of illness. NS1 detection rate using ICT decreased from 100% on day 1 of illness to 98.6% on day 2 after onset of illness. By day 6, the detection rate was 45.9%. Thus, IMA performed better than ICT for early and rapid diagnosis of DENV infections in endemic countries.

Highly sensitive detection of dengue biomarker using streptavidin-conjugated quantum dots

A highly sensitive immunosensor using streptavidin-conjugated quantum dots (QDs/SA) was developed to detect dengue biomarker of non-structural protein 1 (NS1) at very low concentration, so that it can probe dengue infection even in the early stage. The QDs/SA were first bound to biotinylated NS1 antibody (Ab) and the QDs/SA-Ab conjugates were then used to detect the NS1 antigen (Ag) in the Ag concentration range of 1 pM to 120 nM. The formation of QDs/SA-Ab and QDs/SA-Ab-Ag conjugates was confirmed by the measurements of field emission scanning electron microscopy (FF-SEM), field emission transmission electron microscopy (FE-TEM), dynamic light scattering (DLS), and zeta-potential. Fluorescence emission spectra of QDs/SA-Ab-Ag conjugates showed that the magnitude of fluorescence quenching was linearly proportional to the NS1 Ag concentration and it nicely followed the Stern–Volmer (SV) equation in phosphate buffer solution. However, in human plasma serum solution, the fluorescence quenching behavior was negatively deviated from the SV equation presumably due to interference by the serum component biomolecules, and it was well explained by the Lehrer equation. These results suggest that the current approach is promising because it is highly sensitive, fast, simple, and convenient, and thus it has a potential of application for point-of-care.

Engineered NS1 for Sensitive, Specific Zika Virus Diagnosis from Patient Serology.

Dengue virus (DENV) and Zika virus (ZIKV) belong to the Flaviviridae family of viruses spread by Aedes aegypti mosquitoes in tropical and subtropical areas. Accurate diagnostic tests to differentiate the 2 infections are necessary for patient management and disease control. Using characterized ZIKV and DENV patient plasma in a blind manner, we validated an ELISA and a rapid immunochromatographic test for ZIKV detection. We engineered the ZIKV nonstructural protein 1 (NS1) for sensitive serologic detection with low cross reactivity against dengue and developed monoclonal antibodies specific for the ZIKV NS1 antigen. As expected, the serologic assays performed better with convalescent than acute plasma samples; the sensitivity ranged from 71% to 88%, depending on the performance of individual tests (IgM/IgG/NS1). Although serologic tests were generally less sensitive with acute samples, our ZIKV NS1 antibodies were able to complement the serologic tests to achieve greater sensitivity for detecting early infections.

Trend of Dengue Virus Infection with Seasonal Variation at HIMS, Varanasi, Uttar Pradesh, India

Introduction: Dengue is the most common mosquito borne arboviral infection in India, and it has become endemic in India with consistent episodes. Dengue is an acute febrile illness caused by Dengue Virus (DENV-1, DENV-2, DENV-3, DENV-4), an arthropod of family Flaviviridae, transmitted through the bite of female Aedes aegypti mosquitoes. Although, dengue is more prevalent in humans but also it may sometimes be seen in monkeys. The only animals to be affected when bitten by a mosquito, infected with dengue fever are monkeys other animals do not carry or spread dengue. Patient presents with hyperthermia, headache, severe joint and muscle pain, fatigue and skin rashes. Neurological manifestation in sever dengue results from multiorgan dysfunction due to cerebral edema, haemorrhage due to vascular leak, cerebral hypoperfusion, and electrolyte disturbances. Aim: To study serological markers with association of platelet count and trend of DENV infection with seasonal variation. Materials and Methods: The present retrospective study was conducted for a period of two years in Heritage Institute of Medical Sciences (HIMS), Varanasi, Uttar Pradesh, India, from January 2017 to December 2018. Blood samples were collected for 2140 suspected dengue patients. Out of the serologically positive cases, serological confirmation and platelet count was done for dengue infection. Data was presented as percentages. Results: Total 2140 suspected cases were admitted and out of them 199 (9.3%) were found to be seropositive for dengue infection. Non structural protein 1 (NS1) positive cases out of which total seropositive cases were reported to be 127 (63.81%) and 147 (73.86%) cases were positive for NS1 antigen either alone or along with antibody. Out of the total 199 positive cases thrombocytopenia was observed in 126 (63.31%) cases. Positive cases which were under 15 years of age were reported to be 92 (46.23%). Conclusion: The study concludes that NS1 antigen and IgM- IgG antibody consideration in the diagnosis of dengue infection builds the opportunity of early diagnosis so as to keep away the complications significantly.

Co-circulation of all the Four Serotype of Dengue Virus in Endemic Region of Saurashtra, Gujarat during 2019-2020 Season

Introduction: Dengue has rapidly emerged as a vector - borne viral disease in recent years and also endemic in all continents. The agent of dengue, i.e., dengue viruses, are categorised under the genus Flavivirus, with the four dengue virus serotypes: designated as DENV-1, DENV-2, DENV-3 and DENV-4. These all four serotypes are in circulation either singly, or more than one at the same time. Aim: To study the epidemiological update of dengue with circulating serotype and co-infection in Saurashtra region, Gujarat, India. Materials and Methods: A cross-sectional study was conducted during January 2019 to December 2020 and total samples received were 12,563 which were clinically suspected dengue samples case. After receiving blood samples, serum was separated and proceeded for Dengue NS1Ag (nonstructural protein 1 antigen), and Dengue IgM Ab (Immunoglobulin M antibody). After serological confirmation, 151 samples from different geographical area were selected for Dengue specific Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) for serotyping. results: Total 4069 (32%) had confirmed dengue positive by Enzyme Linked Immunosorbent Assay (ELISA). The ratio of male cases was higher than female, and in age group 21-35 year (47%). Seasonal trend showed a gradual increase in positivity from June with high peak in October. Circulation of all the four serotypes in area, higher monotypic infection by DENV-1 serotype (41%), followed by DENV-4, DENV-2 and DENV-3. Co-infection of different serotypes were also found. conclusion: The present study concluded that all four serotypes circulate with predominant being DENV-1 type and co-infection of different serotypes in the saurashtra region. Dengue mainly affected adult male population, and seasonal peak during monsoon and post-monsoon period.

Early Diagnosis and Early Management Proposal in Dengue Infection: New Normal to Applied Strategy for COVID-19 Infection

Dengue infection may lead to various sequels, such as undifferentiated fever (UF), dengue fever (DF), or dengue hemorrhagic fever (DHF), progressive illness, and death. It also causes significant economic burdens that are associated with health-care costs and loss of labor. Usually, the treatment of dengue infection is supportive therapy and not the specific treatment for it, while it is often clinically difficult to predict whether dengue-infected patients will progress to severe diseases or not. We here propose integrated management strategies for early dengue virus (DENV) infection based on our 5-year experience in early management of dengue infection, literature reviews, and research studies over the last 10 years. This proposal consists of 4 aspects: (1) rehydration with oral rehydration therapy, (2) reduction of the pro-inflammatory cytokines, including TNF-α and IL-6 with oral cytokine inhibitors i.e., pentoxifylline and doxycycline, (3) inhibition of DENV replication, viral load reduction, nonstructural protein 1 antigen (NS1Ag) clearance with doxycycline and ivermectin, (4) restoration of the immune function with vitamin D and zinc supplementation. The major benefit of these drugs and supplements is that they are already approved by regulatory bodies, affordable, and clinically safe. They result in better clinical and laboratory outcomes, including in a reduction in hospitalization and cost of treatment. Another benefit is that it may use this strategy for other virus-induced hypercytokinemia such as coronavirus disease (COVID-19) and Ebola.