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.

Identification of differences in the magnitude and specificity of SARS-CoV-2 nucleocapsid antibody responses in naturally infected and vaccinated individuals.

As there are limited data on B-cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron, and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine. Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n = 30), WT infected (n = 30), delta infected (n = 30), omicron infected + vaccinated (n = 20) and Sinopharm (BBIBP-CorV) vaccinees (n = 30). We then investigated the sensitivity and specificity of these immunodominant regions and analyzed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses, and bat Sarbecoviruses. We identified four immunodominant regions aa 29-52, aa 155-178, aa 274-297, and aa 365-388, which were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines.

Dengue NS1 interaction with lipids alters its pathogenic effects on monocyte derived macrophages.

BACKGROUND: While dengue NS1 antigen has been shown to be associated with disease pathogenesis in some studies, it has not been linked in other studies, with the reasons remaining unclear. NS1 antigen levels in acute dengue are often associated with increased disease severity, but there has been a wide variation in results based on past dengue infection and infecting dengue virus (DENV) serotype. As NS1 engages with many host lipids, we hypothesize that the type of NS1-lipid interactions alters its pathogenicity. METHODS: Primary human monocyte derived macrophages (MDMs) were co-cultured with NS1 alone or with HDL, LDL, LPS and/or platelet activating factor (PAF) from individuals with a history of past dengue fever (DF = 8) or dengue haemorrhagic fever (DHF = 8). IL-1ß levels were measured in culture supernatants, and gene expression analysis carried out in MDMs. Monocyte subpopulations were assessed by flow cytometry. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters. Differentially expressed variables were extracted and a classifier model was developed to differentiate between past DF and DHF. RESULTS: Significantly higher levels of IL-1ß were seen in culture supernatants when NS1 was co-cultured with LDL (p = 0.01, median = 45.69 pg/ml), but lower levels when NS1 was co-cultured with HDL (p = 0.05, median = 4.617 pg/ml). MDMs of those with past DHF produced higher levels of IL-1ß when NS1 was co-cultured with PAF (p = 0.02). MDMs of individuals with past DHF, were significantly more likely to down-regulate RPLP2 gene expression when macrophages were co-cultured with either PAF alone, or NS1 combined with PAF, or NS1 combined with LDL. When NS1 was co-cultured with PAF, HDL or LDL two clusters were detected based on IL10 expression, but these did not differentiate those with past DF or DHF. CONCLUSIONS: As RPLP2 is important in DENV replication, regulating cellular stress responses and immune responses and IL-10 is associated with severe disease, it would be important to further explore how differential expression of RPLP2 and IL-10 could lead to disease pathogenesis based on NS1 and lipid interactions.

Efficacy and safety of oral ivermectin in the treatment of mild to moderate Covid-19 patients: a multi-centre double-blind randomized controlled clinical trial.

BACKGROUND: Evidence on ivermectin as a treatment for Covid-19 is controversial. A Cochrane review concluded that the efficacy and safety of ivermectin is uncertain (evidence up to April 2022) and WHO recommended its use only in the setting of clinical trials. This study aimed to assess the efficacy and safety of oral ivermectin in hospitalized patients with mild to moderate Covid-19. TRIAL DESIGN AND METHODS: A double-blind, randomized placebo-controlled clinical trial was conducted among RT-PCR-confirmed, adults, hospitalised within the first four days of symptoms. Patients received oral ivermectin 24 mg or placebo daily for five days. RT-PCR was repeated on days five and ten. Clinical progression was monitored using the World Health Organization Clinical Progression Scale. Serum ivermectin levels were measured on days three, five, and seven. The primary outcome was the difference in the viral load between day zero and ten in the two groups. RESULTS: Out of 1699 patients screened, 249 underwent randomization and 127 received ivermectin, and 122 placebo. D10 median viral load for E gene (IQR) was 2,000 copies/mL (100 - 20,500) with ivermectin (n = 80) and 4,100 copies/mL (1,000-65,600) with placebo (n = 81, p = 0.028), per protocol analysis. The difference in Log viral load between day zero and ten between ivermectin and placebo was 3.72 and 2.97 respectively (p = 0.022). There was no significant difference in the WHO clinical progression scale or the adverse effects. Ivermectin blood levels taken before or with meals were not significantly different. Only 7 and 17 patients achieved blood levels above 160ng/ML and 100ng/ML respectively and they did not achieve a significantly lower viral load. CONCLUSION: Although ivermectin resulted in statistically significant lower viral load in patients with mild to moderate Covid-19, it had no significant effect on clinical symptoms. TRIAL REGISTRATION NUMBER: SLCTR/2021/020, Sri Lanka Clinical Trials Registry. 19/07/2021.

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.

Immune Responses to a Single Dose of the AZD1222/Covishield Vaccine at 16 Weeks in Individuals in Sri Lanka.

Due to limited access to vaccines, many countries have only administered a single dose of the AZD1222, whereas the dosage intervals have increased ≥4 wk. We sought to investigate the immunogenicity of a single dose of vaccine at ≥16 wk postimmunization. Severe acute respiratory syndrome coronavirus 2-specific Abs in 553 individuals and Abs to the receptor-binding domain of the Wuhan virus (wild-type) and the variants of concern, angiotensin-converting enzyme 2 receptor blocking Abs ex vivo and cultured IFN-γ T cell (Homo sapiens) responses and B cell (H. sapiens) ELISPOT responses, were investigated in a subcohort. The seropositivity rates in those >70 y of age (93.7%) was not significantly different compared with other age groups (97.7-98.2; Pearson χ2 = 7.8; p = 0.05). The Ab titers (Ab index) significantly declined (p < 0.0001) with increase in age. A total of 18 of 69 (26.1%) of individuals did not have angiotensin-converting enzyme 2 receptor-blocking Abs, whereas responses to the receptor-binding domain of wild-type (p = 0.03), B.1.1.7 (p = 0.04), and B.1.617.2 (p = 0.02) were significantly lower in those who were >60 y. Ex vivo IFN-γ T cell ELISPOT responses were seen in 10 of 66 (15.1%), whereas only a few expressed CD107a. However, >85% had a high frequency of cultured IFN-γ T cell ELISPOT responses and B cell ELISPOTs. Virus-specific Abs were maintained at ≥16 wk after receiving a single dose of AZD1222, although levels were lower to variants of concern, especially in older individuals. A single dose induced a high frequency of memory T and B cell responses.

Identification of allergens in Artocarpus heterophyllus, Moringa oleifera, Trianthema portulacastrum and Syzygium samarangense.

BACKGROUND: It is clinically important to identify allergens in Artocarpus heterophyllus (jackfruit), Moringa oleifera (moringa), Trianthema portulacastrum (horse purslane) and Syzygium samarangense (rose apple). This study included 7 patients who developed anaphylaxis to jackfruit (1), moringa (2), horse purslane (3) and rose apple (1). We sought to determine allergens in the edible ripening stages of jackfruit (tender, mature, and ripened jackfruit) and seeds, edible parts of moringa (seeds, seedpod, flesh inside seedpod, and leaves), horse purslane leaves and ripened rose apple fruit. The persistence of the allergens after cooking was also investigated. METHODS: Allergens were identified by clinical history followed by a skin prick test. Protein profiles of plant/fruit crude protein extracts were determined by SDS-PAGE. Molecular weights of the allergens were determined by immunoblotting with patient sera. RESULTS: A heat-stable allergen of 114 kDa in A. heterophyllus which is shared among different ripening stages and seeds was identified. Additionally, 101 kDa allergen in boiled tender jackfruit, 86 kDa allergen in boiled seeds and 80 kDa allergen in boiled mature jackfruit were identified. Five heat-stable allergens of 14, 23, 35, 43, and 48 kDa in M. oleifera, 1 heat-stable allergen of 97 kDa in T. portulacastrum, and 4 allergens of 26, 31. 60, and 82 kDa in S. samarangense were identified. CONCLUSION: Novel IgE-sensitive proteins of A. heterophyllus, M. oleifera, T. portulacastrum and S. samarangense were identified which would be especially useful in the diagnosis of food allergies. The identified allergens can be used in Component Resolved Diagnostics (CRD).

Immune responses to Sinopharm/BBIBP-CorV in individuals in Sri Lanka.

As there are limited data of the immunogenicity of the Sinopharm/BBIBP-CorV in different populations, antibody responses against different SARS-CoV-2 variants of concern and T cell responses, we investigated the immunogenicity of the vaccine, in individuals in Sri Lanka. SARS-CoV-2-specific antibodies were measured in 282 individuals who were seronegative at baseline, and ACE2 receptor blocking antibodies, antibodies to the receptor-binding domain (RBD) of the wild-type (WT), alpha, beta and delta variants, ex vivo and cultured IFNγ ELISpot assays, intracellular cytokine secretion assays and B cell ELISpot assays were carried out in a sub cohort of the vaccinees at 4 and 6 weeks (2 weeks after the second dose). Ninety-five percent of the vaccinees seroconverted, although the seroconversion rates were significantly lower (p < 0.001) in individuals >60 years (93.3%) compared to those who were 20-39 years (98.9%); 81.25% had ACE2 receptor blocking antibodies at 6 weeks, and there was no difference in these antibody titres in vaccine sera compared to convalescent sera (p = 0.44). Vaccinees had significantly less (p < 0.0001) antibodies to the RBD of WT and alpha, although there was no difference in antibodies to the RBD of beta and delta compared to convalescent sera; 27.7% of 46.4% of vaccinees had ex vivo IFNγ and cultured ELISpot responses respectively, and IFNγ and CD107a responses were detected by flow cytometry. Sinopharm/BBIBP-CorV appeared to induce a similar level of antibody responses against ACE2 receptor, delta and beta as seen following natural infection.

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.

Dengue and COVID-19: two sides of the same coin.

BACKGROUND: Many countries in Asia and Latin America are currently facing a double burden of outbreaks due to dengue and COVID-19. Here we discuss the similarities and differences between the two infections so that lessons learnt so far from studying both infections will be helpful in further understanding their immunopathogenesis and to develop therapeutic interventions. MAIN BODY: Although the entry routes of the SARS-CoV-2 and the dengue virus (DENV) are different, both infections result in a systemic infection, with some similar clinical presentations such as fever, headache, myalgia and gastrointestinal symptoms. However, while dengue is usually associated with a tendency to bleed, development of micro and macrothrombi is a hallmark of severe COVID-19. Apart from the initial similarities in the clinical presentation, there are further similarities between such as risk factors for development of severe illness, cytokine storms, endothelial dysfunction and multi-organ failure. Both infections are characterised by a delayed and impaired type I IFN response and a proinflammatory immune response. Furthermore, while high levels of potent neutralising antibodies are associated with protection, poorly neutralising and cross-reactive antibodies have been proposed to lead to immunopathology by different mechanisms, associated with an exaggerated plasmablast response. The virus specific T cell responses are also shown to be delayed in those who develop severe illness, while varying degrees of endothelial dysfunction leads to increased vascular permeability and coagulation abnormalities. CONCLUSION: While there are many similarities between dengue and SARS-CoV-2 infection, there are also key differences especially in long-term disease sequelae. Therefore, it would be important to study the parallels between the immunopathogenesis of both infections for development of more effective vaccines and therapeutic interventions.

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.

Identification of allergens in coconut milk and oil with patients sensitized to coconut milk in Sri Lanka.

BACKGROUND: Despite the low prevalence of IgE sensitivity to fresh or boiled coconut milk and coconut oil, those may contain allergens of which the clinical significance remains undetermined. This study aimed to identify and compare allergens in fresh coconut milk (FCM), boiled coconut milk (BCM), unrefined wet-processed coconut oil (WPCO), and dry-processed coconut oil (DPCO) using sera from patients with allergy to coconut milk. METHODS: The study included 18 patients with immediate hypersensitivity to coconut milk, including five who developed anaphylaxis. Sensitization was assessed by skin prick test and ImmunoCAPs using commercially available coconut extracts. Immunoblotting was performed to identify and compare allergen profiles. RESULTS: Total sIgE levels and overall IgE reactivity of patients with anaphylaxis were higher compared to patients with allergy. Twelve allergens ranging from 5 to 128 kDa including six novel allergens with 5, 12, 47, 87, 110, and 128 kDa were visualized in immunoblots with FCM. Similarly, nine allergens of 5, 12, 17, 32, 35, 47, 87, 110, and 128 kDa were detected in BCM. One allergen (110 kDa) was discerned in all four extracts. Higher IgE prevalence was detected with three allergens of 55, 87, and 110 kDa. CONCLUSIONS: Allergens of BCM and unrefined coconut oil (WPCO and DPCO) were determined for the first time. Novel allergens of 87 and 110 kDa and the 55 kDa allergen have the highest potential to be used in Component Resolved Diagnostics. Further, these findings demonstrate that, patients who have an allergy to coconut milk could also react to boiled coconut milk and unrefined coconut oil.

Natural immunogenic properties of bioinformatically predicted linear B-cell epitopes of dengue envelope and pre-membrane proteins.

BACKGROUND: The natural antibody responses to B-cell epitopes from dengue structural proteins were assessed using immune sera from people having well-defined past dengue infections with one of the four serotypes. METHOD: Based on an immune-computational analysis previously conducted, nineteen epitopes from the envelope (E) and eight epitopes from pre-membrane (prM), which were more than 50% conserved across all the four DENV serotypes, were selected. Peptides to represent these B-cell epitopes were obtained from commercially available arrays, and were subjected to enzyme linked immunosorbent assay with sera obtained from dengue seropositive healthy volunteers (DENV1 n = 12: DENV2 n = 12: DENV3 n = 12 and DENV4 n = 12), and 10 dengue seronegative healthy volunteers from Sri Lanka. The cut-off value for the positive antibody response was set by taking the mean response of a peptide to the negative sera plus three standard deviations. The peptides (N = 7) showing the broad immune responses were used to generate antibodies in three mice (Balb/c) batches. The mice antisera were then subjected to microneutralization assays against all the four DENV serotypes. An EC50 viral neutralization ≥ 40 times the serum dilution was considered as neutralizing. RESULTS: Five of the E-peptide and two prM peptides were recognised by most individuls exposed to infections with each of the four serotypes, showing a serotype cross-reactive broad antibody response. The mice immune sera against the peptides representing the five E protein epitopes neutralized all the four DENV serotypes. Two of these five epitopes are from the Domain II, whereas one of them includes the whole bc-loop region. CONCLUSION: The antibody responses of highly conserved epitopes across the serotypes, were broadly responsive with sera of all four DENV serotypes collected from individuals infected with only one DENV serotype. Weakly conserved epitopes showed rather specific antibody responses dominated by one or few serotypes.

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.

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.

Dengue NS1 interaction with lipids alters its pathogenic effects on monocyte derived macrophages.

Background: While dengue NS1 antigen has been shown to be associated with disease pathogenesis in some studies, it has not been linked in other studies, with the reasons remaining unclear. NS1 antigen levels in acute dengue are often associated with increased disease severity, but there have been a wide variation in results based on past dengue infection and infecting dengue virus (DENV) serotype. As NS1 engages with many host lipids, we hypothesize that the type of NS1-lipid interactions alters its pathogenicity. Methods: Primary human monocyte derived macrophages (MDMs) were co-cultured with NS1 alone or with HDL, LDL, LPS and/or platelet activating factor (PAF) from individuals with a history of past dengue fever (DF=8) or dengue haemorrhagic fever (DHF=8). IL-1ß levels were measured in culture supernatants, and gene expression analysis carried out in MDMs. Monocyte subpopulations were assessed by flow cytometry. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters. Differentially expressed variables were extracted and a classifier model was developed to differentiate between past DF and DHF. Results: Significantly higher levels of IL-1ß were seen in culture supernatants when NS1 was co-cultured with LDL (p=0.01), but with lower levels with HDL (p=0.05). MDMs of those past DHF produced more IL-1ß when NS1 with PAF (p=0.02). MDMs of individuals with past DHF, were significantly more likely to down-regulate RPLP2 gene expression when macrophages were co-cultured with either PAF alone, or NS1 combined with PAF, or NS1 combined with LDL. When NS1 was co-cultured with PAF, HDL or LDL two clusters were detected based on IL10 expression, but these did not differentiate those with past DF or DHF. Conclusions: As RPLP2 is important in DENV replication and in regulating cellular stress responses and immune responses and IL-10 is associated with severe disease, it would be important to further explore how differential expression of RPLP2 and IL-10 could lead to disease pathogenesis based on NS1 and lipid interactions.

Genomic Surveillance and Evolutionary Dynamics of Influenza A Virus in Sri Lanka.

Background Influenza A has been named as a priority pathogen by the WHO due to the potential to cause pandemics. Genomic sequencing of influenza strains is important to understand the evolution of the influenza strains and also to select the appropriate influenza vaccines to be used in the different influenza seasons in Sri Lanka. Therefore, we sought to understand the molecular epidemiology of the influenza viruses in the Western Province of Sri Lanka, including mutational analysis to investigate the evolutionary dynamics. Methods A total of 349 individuals presenting with fever and respiratory symptoms were enrolled in this study from November 2022 to May 2024. Nasopharyngeal and oropharyngeal specimens were collected and screened using quantitative PCR to detect Influenza A, Influenza B, and SARS-CoV-2. Subtyping and genomic sequencing was carried out on influenza A strains using Oxford Nanopore Technology. Results Influenza A was detected in 49 (14%) patients, influenza B in 20 (5.7%) and SARS-CoV-2 in 41 (11.7%). Co-infections were observed in five participants. The phylogenetic analysis assigned the H1N1 HA gene sequences within the 6B.1A.5a.2a clade. The HA gene of the H1N1 sequences in 2023 were assigned as belonging to the subclades C.1, C.1.2, and C.1.8, while the 2024 sequences were assigned to subclades C.1.8 and C.1.9. The H3N2 sequences from 2023 were assigned to the 3C.2a1b.2a.2a.1b clade and subclade G.1.1.2, while the 2024 sequences were assigned to the 3C.2a1b.2a.2a.3a.1 clade and subclade J.2. The K54Q, A186T, Q189E, E224A, R259K, K308R, I418V, and X215A amino acid substitutions were seen in the H1N1 in the 2023 and 2024 sequences. The 2024 H1N1 sequences additionally exhibited further substitutions, such as V47I, I96T, T120A, A139D, G339X, K156X, and T278S. Conclusion In this first study using genomic sequencing to characterize the influenza A strains in Sri Lanka, which showed different influenza A viruses circulating in an 18-month period. As the Sri Lankan strains also had certain mutations of unknown significance, it would be important to continue detailed surveillance of the influenza strains in Sri Lanka to choose the most suitable vaccines for the population and the timing of vaccine administration.

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.