Simultaneous co-circulation of two genotypes of dengue virus serotype 3 causing a large outbreak in Sri Lanka in year 2023.

BACKGROUND: We observed a discrepancy between dengue NS1 antigen test and molecular diagnostics, with the emergence of (DENV) serotype 3 in Sri Lanka and sought to understand the cause for the rise in cases and high failure rates of molecular diagnostics. METHODS: Whole genomic sequencing was carried out in 22 DENV-3 samples. Phylogenetic and molecular clock analysis were done for genotype assignment and to understand the rate of evolution. Mutation analysis was done to understand the reasons for PCR non-detection. RESULTS: We identified two DENV-3 genotypes (I and III) co-circulating. DENV-3 genotype III strains shared a common ancestor with a sequence from India collected in 2022, while DENV-3 genotype I, was found to share a common ancestor with DENV-3 sequences from China. DENV-3 genotype III was detected by the modified CDC DENV-3 primers whereas, genotype I evaded detection due to key mutations at forward and reverse primer binding sites. We identified point mutations, C744T and A756G of the forward primer binding sites and in position G795A of the reverse primer binding sites which were not identified in DENV-3 genotype III. Furthermore, our Sri Lankan DENV-3 strains demonstrated a high root to tip ratio, compared to the previous DENV-3 sequences, indicating a high mutation rate during the points of sampling (year 2017 to 2023). CONCLUSION: The co-circulation of multiple genotypes associated with an increase in cases highlights the importance of continuous surveillance of DENVs to identify mutations resulting in non-detection by diagnostics and differences in virulence.

Antibody and memory B cell responses to the dengue virus NS1 antigen in individuals with varying severity of past infection.

To further understand the role of NS1-specific antibodies (Abs) in disease pathogenesis, we compared neutralizing antibody levels (Nabs), NS1-Ab levels, IgG antibody subclass profiles and NS1-specific memory B-cell responses (Bmems) in individuals, with varying severity of past dengue. Nabs (Neut50 titres) were assessed using the Foci Reduction Neutralization Test (FRNT) and in-house ELISAs were used to assess NS1-Abs and NS1-Ab subclasses for all four DENV serotypes in individuals with past DF (n = 22), those with past DHF (n = 14) and seronegative (SN) individuals (n = 7). B-cell ELISpot assays were used to assess NS1-specific Bmem responses. 15/22 (68.18%) individuals with past DF and 9/14 (64.29%) individuals with past DHF had heterotypic infections. Neut50 titres were found to be significantly higher for DENV1 than DENV2 (p = 0.0006) and DENV4 (p = 0.0127), in those with past DHF, whereas there was no significant difference seen in titres for different DENV serotypes in those with past DF. Overall NS1-Ab to all serotypes and NS1-specific IgG1 responses for DENV1, 2 and 4 serotypes were significantly higher in those with past DHF than individuals with past DF. Those with past DHF also had higher IgG1 than IgG3 for DENV1 and DENV3, whereas no differences were seen in those with past DF. Over 50% of those with past DF or DHF had NS1-specific Bmem responses to >2 DENV serotypes. There was no difference in the frequency of Bmem responses to any of the DENV serotypes between individuals with past DF and DHF. Although the frequency of Bmem responses to DENV1 correlated with DENV1-specific NS1-Abs levels (Spearman r = 0.35, p = 0.02), there was no correlation with other DENV serotypes. We found that those with past DF had broadly cross-reactive Nabs, while those with past DHF had higher NS1-Ab responses possibly with a different functionality profile than those with past DF. Therefore, it would be important to further evaluate the functionality of NS1-specific antibody and Bmem responses to find out the type of antibody repertoire that is associated with protection against severe disease.

Comparison of the immunogenicity of five COVID-19 vaccines in Sri Lanka.

To determine the antibody responses elicited by different vaccines against SARS-CoV-2, we compared antibody responses in individuals 3 months post-vaccination in those who had received different vaccines in Sri Lanka. Abs to the receptor binding domain (RBD) of the ancestral (wild type) virus (WT) as well as to variants of concern (VoCs), and ACE2 blocking Abs, were assessed in individuals vaccinated with Moderna (n = 225), Sputnik V (n = 128) or Sputnik light (n = 184) and the results were compared with previously reported data on Sinopharm and AZD1222 vaccinees. A total of 99.5% of Moderna, >94% of AZD1222 or Sputnik V and >70% of Sputnik light, >60% of Sinopharm vaccine recipients, had a positive response to ACE2 blocking antibodies. The ACE2 blocking antibody levels were highest to lowest was Moderna > Sputnik V/AZD1222 (had equal levels) > Sputnik light > Sinopharm. All Moderna recipients had antibodies to the RBD of WT, alpha and beta, while positivity rates for delta variant was 80%. The positivity rates for Sputnik V vaccinees for the WT and VoCs were higher than for AZD1222 vaccinees while those who received Sinopharm had the lowest positivity rates (<16.7%). The total antibodies to the RBD were highest for the Sputnik V and AZD1222 vaccinees. The Moderna vaccine elicited the highest ACE2 blocking antibody levels followed by Sputnik V/AZD1222, while those who received Sinopharm had the lowest levels. These findings highlight the need for further studies to understand the effects on clinical outcomes.

Kinetics of immune responses to SARS-CoV-2 proteins in individuals with varying severity of infection and following a single dose of the AZD1222.

To characterize the IgG and IgA responses to different SARS-CoV-2 proteins, we investigated the antibody responses to SARS-CoV-2 following natural infection and following a single dose of AZD1222 (Covishield), in Sri Lankan individuals. The IgG and IgA responses were assessed to S1, S2, RBD, and N proteins in patients at 4 weeks and 12 weeks since the onset of illness or following vaccination. Antibodies to the receptor-binding domain of SARS-CoV-2 wild type (WT), α, ß, and λ and ACE2 (Angiotensin Converting Enzyme 2) receptor blocking antibodies were also assessed in these cohorts. For those with mild illness and in vaccines, the IgG responses to S1, S2, RBD, and N protein increased from 4 weeks to 12 weeks, while it remained unchanged in those with moderate/severe illness. In the vaccines, IgG antibodies to the S2 subunit had the highest significant rise (P < 0.0001). Vaccines had several-fold lower IgA antibodies to all the SARS-CoV-2 proteins tested than those with natural infection. At 12 weeks, the haemagglutination test (HAT) titres were significantly lower to the α in vaccines and significantly lower in those with mild illness and in vaccines to ß and for λ. No such difference was seen in those with moderate/severe illness. Vaccines had significantly less IgA to SARS-CoV-2, but comparable IgG responses those with natural infection. However, following a single dose vaccines had reduced antibody levels to the VOCs, which further declined with time, suggesting the need to reduce the gap between the two doses, in countries experiencing outbreaks due to VOCs.

Sensitivity and specificity of two WHO approved SARS-CoV2 antigen assays in detecting patients with SARS-CoV2 infection.

BACKGROUND: SARS-CoV-2 rapid antigen (Ag) detection kits are widely used in addition to quantitative reverse transcription PCR PCR (RT-qPCR), as they are cheaper with a rapid turnaround time. As there are many concerns regarding their sensitivity and specificity, in different settings, we evaluated two WHO approved rapid Ag kits in a large cohort of Sri Lankan individuals. METHODS: Paired nasopharangeal swabs were obtained from 4786 participants for validation of the SD-Biosensor rapid Ag assay and 3325 for the Abbott rapid Ag assay, in comparison to RT-qPCR. A short questionnaire was used to record symptoms at the time of testing, and blood samples were obtained from 2721 of them for detection of SARS-CoV-2 specific antibodies. RESULTS: The overall sensitivity of the SD-Biosensor Ag kit was 36.5% and the Abbott Ag test was 50.76%. The Abbott Ag test showed specificity of 99.4% and the SD-Biosensor Ag test 97.5%. At Ct values < 25, the sensitivity was 71.3% to 76.6% for the SD-Biosensor Ag test and 77.3% to 88.9% for the Abbott Ag test. The Ct values for all genes (RdRP, S, E and N) tested with all RT-qPCR kits were significantly lower for the positive results of the Abbott Ag test compared to the SD-Biosensor test. 209 (48.04%) individuals who had antibodies gave a positive RT-qPCR result, and antibody positivity rates were higher at Ct values > 30 (46.1 to 82.9%). 32.1% of those who gave a positive result with the SD-Biosensor Ag test and 26.3% of those who gave positive results with the Abbott Ag test had SARS-CoV-2 antibodies at the time of detection. CONCLUSIONS: Both rapid Ag tests appeared to be highly sensitive in detecting individuals at lower Ct values, in a community setting in Sri Lanka, but it will be important to further establish the relationship to infectivity.

Comparison of two rapid test kits with real time polymerase chain reaction for early diagnosis of dengue in Sri Lanka.

Dengue is among the deadliest insect-borne diseases circulating in Sri Lanka. Most of the infections that are diagnosed early are manageable. However, delays in diagnosis may cause fatalities. We evaluated the dengue NS1 antigen card and NS1 SD kit for early diagnosis of dengue using samples from 116 RT-PCR-positive patients admitted within 5 days of the fever onset. RT-PCR tests were performed as standard tests. IgM and IgG ELISA tests were carried out to identify primary and secondary infections. Of the 116 patients who tested positive for dengue using PCR, 48 were positive using NS1 antigen card and 45 were positive using NS1 SD. Patients with 100 copies or higher viral load showed a higher sensitivity in both antigen card and NS1 SD. Of 34 primary infections evaluated, 23 were positive by NS1 antigen card, while the positivity was 21 by NS1 SD. Of the 30 secondary infections evaluated, 15 were positive by NS1 antigen card while 14 by NS1 SD. Our findings showed that while the rapid tests are convenient and much easier to use than PCR, they are less sensitive and need improvement. Until then, clinical diagnosis should have more emphasis on the early diagnosis of dengue.

Phenotype and functionality of follicular helper T cells in patients with acute dengue infection.

BACKGROUND: The association of functionality and phenotype of follicular helper T cells (Tfh) with dengue virus (DENV) specific antibody responses and clinical disease severity has not been well studied. METHODS: We investigated the phenotype and functionality of Tfh cells and plasmablasts in adult patients (DF = 18, DHF = 22) with acute dengue (day 4 to 8 since onset of fever) of varying severity using multiparametric flowcytometry. The properties of Tfh cells were correlated with viraemia, disease severity, plasmablast responses and DENV-specific serum antibody responses. We further evaluated the kinetics of neutralizing antibodies (Neut50) throughout the course of illness in order to evaluate their association with clinical disease severity and viraemia. RESULTS: Tfh cells (especially those producing IL-21 and co-expressing PD-1 and ICOS) were found to be significantly expanded (p < 0.0001) and highly activated in patients with DHF compared to those with DF. The frequency of Tfh cells significantly correlated with DENV-specific IgG, NS1-specific antibodies and Neut50 antibody titres in patients with DHF but not in those with DF. Although the Neut50 titres increased during the course of acute secondary DENV infection, they showed differences based on serotype. For instance, the Neut50 titres were significantly higher during the latter part of illness in patients with DF compared to DHF in DENV1 infection, while in DENV2, patients with DHF had significantly higher titres. The viral loads during early illness did not correlate with the subsequent rise in the Neut50 antibody titres during any time point of illness. CONCLUSIONS: The expansion of Tfh cells is associated with DHF and DENV-specific IgG, NS1-specific and neutralizing antibodies. Neut50 titres did not associate with disease severity or viraemia at the point of first presentation during the febrile phase, but later titres do show differential association with severity in patients with DENV1 compared to DENV2.

Regulatory T-cells in acute dengue viral infection.

Although regulatory T-cells (Tregs ) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of Tregs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4+  CD25+ T-cells (FoxP3+ cells). Dengue virus (DENV) viral loads were measured by quantitative real-time polymerase chain reaction (PCR) and DENV-specific T-cell responses were measured by ex-vivo interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV-NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T-cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte-antigen 4 (CTLA-4) and Fas. While the frequency of FoxP3+  cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3+  cells did not correlate with either ex-vivo IFN-γ DENV-NS3-, NS5- or NS1-specific T-cell responses. FoxP3+  cells of patients with acute dengue were predominantly CD45RA+ FoxP3low , followed by CD45RA-FoxP3low , with only a small proportion of FoxP3+  cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T-cells, with only CCR4 only being expressed at high levels in the effector Treg population. Therefore, although FoxP3+  cells are expanded in acute dengue, they predominantly consist of naive Tregs , with poor suppressive capacity.

Patterns and causes of liver involvement in acute dengue infection.

BACKGROUND: Liver involvement in acute dengue infection is frequently observed and sometimes leads to acute liver failure, with fatal outcomes. Many factors are thought to contribute to liver dysfunction, including hypoxic injury due to decreased perfusion, direct damage by the virus and immune mediated injury. In this study, we sought to identify the pattern in the change in liver enzymes throughout the illness and its association with the degree of viraemia, onset and extent of plasma leakage and inflammatory mediators. METHODS: Serial daily blood samples were obtained from 55 adult patients with acute dengue from the time of admission to discharge and the liver function tests, viral loads and cytokines were assessed. The onset and extent of fluid leakage was measured by daily ultrasound examinations and all clinical and laboratory features were serially recorded. RESULTS: Aspartate transaminase (AST), alanine transaminase (ALT) and gamma glutamyl transferase (GGT) levels were elevated in patients with dengue infection throughout the illness. The highest AST levels were seen on day 6 of illness and both AST and GGT levels were significantly higher in patients with severe dengue (SD), when compared to those with non-severe dengue (NSD) on day 5 and 6 of illness. Three patients with SD had AST and ALT values of >1000/IU in the absence of any fluid leakage or a rise in the haematocrit (≥20 %). The peak of the AST levels and the lowest serum albumin levels were seen 24 h before the maximum fluid leakage and 24 h after the peak in viraemia. Both serum IL-10 and IL-17 levels were elevated during early illness and were significantly higher in those with SD when compared to NSD. CONCLUSION: Dengue associated liver injury appears to peak around day 6 and 7. Therefore, liver function tests done at earlier dates might not reflect the extent of liver involvement in acute infection. Since severe liver involvement can occur in the absence of fluid leakage, after the peak viraemia, and since it is associated with high IL-17 and IL-10 levels, possible immune mechanisms leading to hepatic damage should be investigated.

Dengue NS1 antigen as a marker of severe clinical disease.

BACKGROUND: Early detection of complications significantly reduces dengue associated mortality and morbidity. We set out to determine if the NS1 rapid antigen detection test could be used as a point of care test to predict severe disease. METHODS: 186 adult patients with confirmed dengue were enrolled during day 3-8 of illness. Clinical and laboratory parameters were recorded during the course of the illness and NS1 antigen levels were determined using both the Panbio dengue early ELISA (Panbio, Australia) and a NS1 rapid antigen detection kit (SD Bioline, South Korea). RESULTS: 59.1% of patients presented to hospital on day 5-6 of illness when NS1 antigen positivity was significantly (p = 0.008) associated with severe dengue (odds ratio 3.0, 95% CI 1.39 to 6.47) and the NS1 antigen levels were significantly higher (p = 0.03) in those who went on to develop shock. Serum NS1 antigen levels significantly (p < 0.0001) and inversely correlated with the total white cell counts and lymphocyte counts. The bedside NS1 test showed comparable sensitivity (97.4%) and specificity (93.7%) to the laboratory NS1 test in our setting and cohort. CONCLUSION: NS1 antigen positivity is associated with a higher risk of developing severe dengue especially when positive beyond day 5 of illness in our cohort, and while further validation studies are required, the test can therefore potentially be used as a bedside point of care test as a warning sign of severe dengue.

Simultaneous co-circulation of two genotypes of dengue virus serotype 3 causing a large outbreak in Sri Lanka in year 2023.

As many other countries, Sri Lanka experienced a marked rise in the number of dengue cases in 2023, with an unusual pattern of disease epidemiology. This rise coincided with the emergence of dengue virus (DENV) serotype 3 in Sri Lanka as the predominant serotype after 2009. Interestingly, a discrepancy between NS1 rapid antigen test positivity and quantitative real time PCR positivity was observed, with 50% of NS1 positive samples being negative by molecular diagnostics. Following sequencing of the DENV-3 strains in 2023, we identified two DENV-3 genotypes (I and III) co-circulating. While DENV-3 genotype III was detected by the modified CDC DENV-3 primers, genotype I evaded detection due to key mutations at forward and reverse primer binding sites. The co-circulation of multiple genotypes associated with an increase in cases highlights the importance of continuous surveillance of DENVs to identify mutations resulting in non-detection by diagnostics and differences in virulence.

Are viral loads in the febrile phase a predictive factor of dengue disease severity?

Background: As many studies have shown conflicting results regarding the extent of viraemia and clinical disease severity, we sought to investigate if viraemia during early dengue illness is associated with subsequent clinical disease severity. Methods: Realtime PCR was carried out to identify the dengue virus (DENV serotype), in 362 patients, presenting within the first 4 days of illness, from 2017 to 2022, in Colombo Sri Lanka. To characterize subsequent clinical disease severity, all patients were followed throughout their illness daily and disease severity classified according to WHO 1997 and 2009 disease classification. Results: 263 patients had DF, 99 progressed to develop DHF, and 15/99 with DHF developed shock (DSS). Although the viral loads were higher in the febrile phase in patients who progressed to develop DHF than in patients with DF this was not significant (p=0.5). Significant differences were observed in viral loads in patients infected with different DENV serotypes (p=0.0009), with lowest viral loads detected in DENV2 and the highest viral loads in DENV3. Sub-analysis for association of viraemia with disease severity for each DENV serotyped was again not significant. Although those infected with DENV2 had lower viral loads, infection with DENV2 was significantly associated with a higher risk of developing DHF (p=0.011, Odds ratio 1.9; 95% CI 1.164 to 3.078). Based on the WHO 2009 disease classification, 233 had dengue with warning signs (DWW), 114 dengue without warning signs (DWoWS), and 15 had severe dengue (SD). No significant difference was observed in the viral loads between those with SD, DWW and DWoWS (p=0.27). Conclusions: Viral loads were significantly different in the febrile phase between different DENV serotypes, and do not appear to significantly associate with subsequent clinical disease severity in a large Sri Lankan cohort.

Serum IL-10 as a marker of severe dengue infection.

BACKGROUND: Several studies have shown that serum IL-10, IFNγ and MIF are elevated in patients in severe dengue (SD) and could be used as potential biomarkers. We proceeded to determine if these cytokines could be used as biomarkers in a large cohort of adult dengue patients with varying severity of dengue infection. METHODS: Serum IL-10 levels were determined in 259 of whom 40 had severe dengue infection. Serum IFNγ and IFNα levels were done in 78 and MIF levels were done in 65 patients with acute dengue infection. Clinical features and laboratory investigations were undertaken during the febrile and critical phase. RESULTS: We found that serum IL-10 levels were significantly higher (p = 0.001) in patients with SD, when compared to those with non SD. Serum IL-10 levels significantly and inversely correlated with white cell counts (R = -0.23, p = 0.0002) and lymphocyte counts (R = -0.29, p < 0.0001) but significantly and positively correlated with aspartate tranaminase levels (R = 0.16, p = 0.01) and alanine transaminase levels (R = 0.22, p = 0.007). However, IL-10 levels did not have a good predictive value in discriminating those who were likely to develop SD (AUC = 0.66). Serum IFNγ levels were also significantly higher (p = 0.04) in patients with SD when compared to non SD. There was no difference (p = 0.34) in serum IFNα levels and serum MIF levels (p = 0.15) in patients with SD and non SD. CONCLUSION: Although serum IL-10 was significantly elevated in patients with SD it had a poor discriminatory value in identifying those with SD and non SD and therefore, is unsuitable to be used as a robust biomarker in this cohort.

Adipokine levels and their association with clinical disease severity in patients with dengue.

Adipokines have not been studied in acute dengue, despite their emerging role in inducing and regulating inflammation. Therefore, we sought to identify adipokine levels in patients with varying severities of acute dengue to understand their role in disease pathogenesis. We determined the levels of leptin, resistin, omentin, adiponectin, as well as IFNß, and NS1 using quantitative ELISA in patients with dengue fever (DF = 49) and dengue haemorrhagic fever (DHF = 22) at admission (febrile phase) and at the time of discharge (recovery phase). The viral loads and serotypes of all samples were quantified using quantitative real-time RT-PCR. Resistin levels (p = 0.04) and omentin (p = 0.006) levels were significantly higher in patients who developed DHF. Omentin levels in the febrile phase also correlated with the AST (Spearman's r = 0.38, p = 0.001) and ALT levels (Spearman's r = 0.24, p = 0.04); as well as serum leptin levels with both AST (Spearman's r = 0.27, p = 0.02) and ALT (Spearman's r = 0.28, p = 0.02). Serum adiponectin levels in the febrile phase did not correlate with any of the other adipokines or with liver enzymes, but inversely correlated with CRP levels (Spearman's r = -0.31, p = 0.008). Although not significant (p = 0.14) serum IFNß levels were lower in the febrile phase in those who progressed to develop DHF (median 0, IQR 0 to 39.4 pg/ml), compared to those who had DF (median 37.1, IQR 0 to 65.6 pg.ml). The data suggest that adipokines are likely to play a role in the pathogenesis of dengue, which should be further explored for the potential to be used as prognostic markers and as therapeutic targets.

Adipokine levels and their association with clinical disease severity in patients with dengue.

Adipokines have not been studied in acute dengue, despite their emerging role in inducing and regulating inflammation. Therefore, we sought to identify adipokine levels in patients with varying severities of acute dengue to understand their role in disease pathogenesis. We determined the levels of leptin, resistin, omentin, adiponectin, as well as IFNß, and NS1 using quantitative ELISA in patients with dengue fever (DF=49) and dengue haemorrhagic fever (DHF=22) at admission (febrile phase) and at the time of discharge (recovery phase). The viral loads and serotypes of all samples were quantified using quantitative real-time RT-PCR. Resistin levels (p =0.04) and omentin (p=0.006) levels were significantly higher in patients who developed DHF. Omentin levels in the febrile phase also correlated with the AST (Spearman's r=0.38, p=0.001) and ALT levels (Spearman's r=0.24, p=0.04); as well as serum leptin levels with both AST (Spearman's r=0.27, p=0.02) and ALT (Spearman's r=0.28, p=0.02). Serum adiponectin levels in the febrile phase did not correlate with any of the other adipokines or with liver enzymes, but inversely correlated with CRP levels (Spearman's r=-0.31, p=0.008). Although not significant (p=0.14) serum IFNß levels were lower in the febrile phase in those who progressed to develop DHF (median 0, IQR 0 to 39.4 pg/ml), compared to those who had DF (median 37.1, IQR 0 to 65.6 pg.ml). The data suggest that adipokines are likely to play a role in the pathogenesis of dengue, which should be further explored for the potential to be used as prognostic markers and as therapeutic targets.

Dengue NS1 induces phospholipase A2 enzyme activity, prostaglandins, and inflammatory cytokines in monocytes.

INTRODUCTION: Dengue virus (DENV) NS1 is a non-structural secretory protein associated with severe disease and known to cause vascular leak leading to dengue haemorrhagic fever (DHF). As phospholipases A2 (PLA2) enzymes, platelet activating factor, and leukotrienes are elevated in dengue, we sought to investigate whether NS1 potentially contributes to disease pathogenesis by inducing PLA2s. METHODS: THP-1 cells and primary human monocytes of healthy adults (n = 6) were co-cultured with DENV1 NS1, LPS and media alone. The latter two were used as positive and negative controls. The cell culture supernatants and lysates were harvested at 12 and 24 h and the activity of secretory and cytoplasmic PLA2, prostaglandins (PGE2 and PGD2) were measured by ELISA and cytokines levels were measured using a magnetic Luminex assay. Expression of PLA2G4A, PLA2G2A, PLA2G5, PLA2G10, PLA2G7, GAPDH, NLRP3 and DDX58 genes were assessed using quantitative RT-PCR. RESULTS: cPLA2 (p = 0.005), sPLA2 (p = 0.04), PGE2 metabolite (p = 0.02) and PGD2 metabolite (p = 0.04) levels were significantly higher at 12 h in monocytes co-cultured with NS1. Levels of IP-10 (p = 0.005) and IL-10 (p = 0.009) was significantly higher at 24 h, whereas IFNα level was significantly higher (p = 0.013) only at 12 h. IL-1ß (p = 0.028 and p = 0.031) and TNFα (p = 0.007 and p = 0.011) showed significantly higher levels at both time points. At 12 h significant upregulation of PLA2G4A (p < 0.0001) was seen, whereas PLA2G7 (p = <0.0001), NLRP3 (p = 0.0009) and DDX58 (p = 0.0056) were significantly downregulated. This pattern changed at 24 h with PLA2G4A (p = 0.0069) showing a marked downregulation and PLA2G7, DDX58 and NLRP3 showing an upregulation, although not significant. CONCLUSION: Dengue NS1 induces the production of PLA2 enzymes, prostaglandins and inflammatory cytokines from primary human monocytes, which could play a role in vascular leak in dengue.

Epidemiological and virological factors determining dengue transmission in Sri Lanka during the COVID-19 pandemic.

With the onset of the COVID-19 pandemic in early 2020 there was a drastic reduction in the number of dengue cases in Sri Lanka, with an increase towards the end of 2021. We sought to study the contribution of virological factors, human mobility, school closure and mosquito factors in affecting these changes in dengue transmission in Sri Lanka during this time. To understand the reasons for the differences in the dengue case numbers in 2020 to 2021 compared to previous years, we determined the association between the case numbers in Colombo (which has continuously reported the highest number of cases) with school closures, stringency index, changes in dengue virus (DENV) serotypes and vector densities. There was a 79.4% drop in dengue cases from 2019 to 2020 in Colombo. A significant negative correlation was seen with the number of cases and school closures (Spearman's r = -0.4732, p <0.0001) and a negative correlation, which was not significant, between the stringency index and case numbers (Spearman's r = -0.3755 p = 0.0587). There was no change in the circulating DENV serotypes with DENV2 remaining the most prevalent serotype by early 2022 (65%), similar to the frequencies observed by end of 2019. The Aedes aegypti premise and container indices showed positive but insignificant correlations with dengue case numbers (Spearman r = 0.8827, p = 0.93). Lockdown measures, especially school closures seemed to have had a significant impact on the number of dengue cases, while the vector indices had a limited effect.

Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR.

As different SARS-CoV-2 variants emerge and with the continuous evolvement of sub lineages of the delta variant, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, due to limited resources as many resource poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a two-step commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies' (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using Illumina generated <5%. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. This difference results in a base error probability of 1 in 10 by the ONT and 1 in 1000 for Illumina sequencing platform. Sub-consensus single nucleotide variations (SNV) are highly correlated between both platforms (R2 = 0.79) while indels appear to have a weaker correlation (R2 = 0.13). Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number or reads, generated less ambiguous bases and was significantly less expensive than Illumina sequencing technology.

Efficacy of rupatadine in reducing the incidence of dengue haemorrhagic fever in patients with acute dengue: A randomised, double blind, placebo-controlled trial.

BACKGROUND: Rupatadine was previously shown to reduce endothelial dysfunction in vitro, reduced vascular leak in dengue mouse models and to reduce the extent of pleural effusions and thrombocytopenia in patients with acute dengue. Therefore, we sought to determine the efficacy of rupatadine in reducing the incidence of dengue haemorrhagic fever (DHF) in patients with acute dengue. METHODS AND FINDINGS: A phase 2, randomised, double blind, placebo controlled clinical trial was carried out in patients with acute dengue in Sri Lanka in an outpatient setting. Patients with ≤3 days since the onset of illness were either recruited to the treatment arm of oral rupatadine 40mg for 5 days (n = 123) or the placebo arm (n = 126). Clinical and laboratory features were measured daily to assess development of DHF and other complications. 12 (9.7%) patients developed DHF in the treatment arm compared to 22 (17.5%) who were on the placebo although this was not significant (p = 0.09, relative risk 0.68, 95% CI 0.41 to 1.08). Rupatadine also significantly reduced (p = 0.01) the proportion of patients with platelet counts <50,000 cells/mm3 and significantly reduced (p = 0.04) persisting vomiting, headache and hepatic tenderness (p<0.0001) in patients. There was a significant difference in the duration of illness (p = 0.0002) although the proportion of individuals who required hospital admission in both treatment arms. Only 2 patients on rupatadine and 3 patients on the placebo developed shock, while bleeding manifestations were seen in 6 patients on rupatadine and 7 patients on the placebo. CONCLUSIONS: Rupatadine appeared to be safe and well tolerated and showed a trend towards a reducing proportion of patients with acute dengue who developed DHF. Its usefulness when used in combination with other treatment modalities should be explored. TRIAL REGISTRATION: International Clinical Trials Registration Platform: SLCTR/2017/024.

Kinetics of immune responses to the AZD1222/Covishield vaccine with varying dose intervals in Sri Lankan individuals.

BACKGROUND: To understand the kinetics of immune responses with different dosing gaps of the AZD1222 vaccine, we compared antibody and T cell responses in two cohorts with two different dosing gaps. METHODS: Antibodies to the SARS-CoV-2 virus were assessed in 297 individuals with a dosing gap of 12 weeks, sampled 12 weeks post second dose (cohort 1) and in 77 individuals with a median dosing gap of 21.4 weeks (cohort 2) sampled 6 weeks post second dose. ACE2-blocking antibodies (ACE2-blocking Abs), antibodies to the receptor-binding domain (RBD) of  variants of concern (VOC), and ex vivo T cell responses were assessed in a subcohort. RESULTS: All individuals (100%) had SARS-CoV-2-specific total antibodies and 94.2% of cohort 1 and 97.1% of cohort 2 had ACE2-blocking Abs. There was no difference in antibody titers or positivity rates in different age groups in both cohorts. The ACE2-blocking Abs (p < .0001) and antibodies to the RBD of the VOCs were significantly higher in cohort 2 compared to cohort 1. 41.2% to 65.8% of different age groups gave a positive response by the hemagglutination assay to the RBD of the ancestral virus and VOCs in cohort 1, while 53.6%-90% gave a positive response in cohort 2. 17/57 (29.8%) of cohort 1 and 17/29 (58.6%) of cohort 2 had ex vivo interferon (IFN)γ ELISpot responses above the positive threshold. The ACE2-blocking antibodies (Spearman's r = .46, p = .008) and ex vivo IFNγ responses (Spearman's r = .71, p < .0001) at 12 weeks post first dose, significantly correlated with levels 12 weeks post second dose. CONCLUSIONS: Both dosing schedules resulted in high antibody and T cell responses post vaccination, although those with a longer dosing gap had a higher magnitude of responses, possibly as immune responses were measured 6 weeks post second dose compared to 12 weeks post second dose.