Fourth Controlled Human Infection Model (CHIM) meeting, CHIM regulatory issues, May 24, 2023.

Many aspects of Controlled Human Infection Models (CHIMs, also known as human challenge studies and human infection studies) have been discussed extensively, including Good Manufacturing Practice (GMP) production of the challenge agent, CHIM ethics, environmental safety in CHIM, recruitment, community engagement, advertising and incentives, pre-existing immunity, and clinical, immunological, and microbiological endpoints. The fourth CHIM meeting focused on regulation of CHIM studies, bringing together scientists and regulators from high-, middle-, and low-income countries, to discuss barriers and hurdles in CHIM regulation. Valuable initiatives for regulation of CHIMs have already been undertaken but further capacity building remains essential. The Wellcome Considerations document is a good starting point for further discussions.

Development and validation of a new measurement instrument to assess internship experience of medical doctors in low-income and middle-income countries.

Routine surveys are used to understand the training quality and experiences of junior doctors but there are lack of tools designed to evaluate the training experiences of interns in low-income and middle-income countries (LMICs) where working conditions and resource constraints are challenging. We describe our process developing and validating a 'medical internship experience scale' to address this gap, work involving nine LMICs that varied in geographical locations, income-level and internship training models. We used a scoping review of existing tools, content validity discussions with target populations and an expert panel, back-and-forth translations into four language versions and cognitive interviews to develop and test the tool. Using data collected from 1646 interns and junior medical doctors, we assessed factor structure and assessed its reliability and validity. Fifty items about experiences of medical internship were retained from an initial pool of 102 items. These 50 items represent 6 major factors (constructs): (1) clinical learning and supervision, (2) patient safety, (3) job satisfaction, (4) stress and burnout, (5) mental well-being, and (6) fairness and discrimination. We reflect on the process of multicountry scale development and highlight some considerations for others who may use our scale, using preliminary analyses of the 1646 responses to illustrate that the tool may produce useful data to identify priorities for action. We suggest this tool could enable LMICs to assess key metrics regarding intern straining and initial work experiences and possibly allow comparison across countries and over time, to inform better internship planning and management.

Genome evolution of dengue virus serotype 1 under selection by Wolbachia pipientis in Aedes aegypti mosquitoes.

The introgression of antiviral strains of Wolbachia into Aedes aegypti mosquito populations is a public health intervention for the control of dengue. Plausibly, dengue virus (DENV) could evolve to bypass the antiviral effects of Wolbachia and undermine this approach. Here, we established a serial-passage system to investigate the evolution of DENV in Ae. aegypti mosquitoes infected with the wMel strain of Wolbachia. Using this system, we report on virus genetic outcomes after twenty passages of serotype 1 of DENV (DENV-1). An amino acid substitution, E203K, in the DENV-1 envelope protein was more frequently detected in the consensus sequence of virus populations passaged in wMel-infected Ae. aegypti than wild-type counterparts. Positive selection at residue 203 was reproducible; it occurred in passaged virus populations from independent DENV-1-infected patients and also in a second, independent experimental system. In wild-type mosquitoes and human cells, the 203K variant was rapidly replaced by the progenitor sequence. These findings provide proof of concept that wMel-associated selection of virus populations can occur in experimental conditions. Field-based studies are needed to explore whether wMel imparts selective pressure on DENV evolution in locations where wMel is established.

The feasibility of novel point-of-care diagnostics for febrile illnesses at health centres in Southeast Asia: a mixed-methods study.

BACKGROUND: The decline of malaria in Southeast Asia means other causes of fever are increasingly relevant, but often undiagnosed. The objective of this study was to assess the feasibility of point-of-care tests to diagnose acute febrile illnesses in primary care settings. METHODS: A mixed-methods study was conducted at nine rural health centres in western Cambodia. Workshops introduced health workers to the STANDARD(TM) Q Dengue Duo, STANDARD(TM) Q Malaria/CRP Duo and a multiplex biosensor detecting antibodies and/or antigens of eight pathogens. Sixteen structured observation checklists assessed users' performances and nine focus group discussions explored their opinions. RESULTS: All three point-of-care tests were performed well under assessment, but sample collection was difficult for the dengue test. Respondents expressed that the diagnostics were useful and could be integrated into routine clinical care, but were not as convenient to perform as standard malaria rapid tests. Health workers recommended that the most valued point-of-care tests would directly inform clinical management (e.g. a decision to refer a patient or to provide/withhold antibiotics). CONCLUSIONS: Deployment of new point-of-care tests to health centres could be feasible and acceptable if they are user-friendly, selected for locally circulating pathogens and are accompanied by disease-specific education and simple management algorithms.

Learning meaningful latent space representations for patient risk stratification: Model development and validation for dengue and other acute febrile illness.

Background: Increased data availability has prompted the creation of clinical decision support systems. These systems utilise clinical information to enhance health care provision, both to predict the likelihood of specific clinical outcomes or evaluate the risk of further complications. However, their adoption remains low due to concerns regarding the quality of recommendations, and a lack of clarity on how results are best obtained and presented. Methods: We used autoencoders capable of reducing the dimensionality of complex datasets in order to produce a 2D representation denoted as latent space to support understanding of complex clinical data. In this output, meaningful representations of individual patient profiles are spatially mapped in an unsupervised manner according to their input clinical parameters. This technique was then applied to a large real-world clinical dataset of over 12,000 patients with an illness compatible with dengue infection in Ho Chi Minh City, Vietnam between 1999 and 2021. Dengue is a systemic viral disease which exerts significant health and economic burden worldwide, and up to 5% of hospitalised patients develop life-threatening complications. Results: The latent space produced by the selected autoencoder aligns with established clinical characteristics exhibited by patients with dengue infection, as well as features of disease progression. Similar clinical phenotypes are represented close to each other in the latent space and clustered according to outcomes broadly described by the World Health Organisation dengue guidelines. Balancing distance metrics and density metrics produced results covering most of the latent space, and improved visualisation whilst preserving utility, with similar patients grouped closer together. In this case, this balance is achieved by using the sigmoid activation function and one hidden layer with three neurons, in addition to the latent dimension layer, which produces the output (Pearson, 0.840; Spearman, 0.830; Procrustes, 0.301; GMM 0.321). Conclusion: This study demonstrates that when adequately configured, autoencoders can produce two-dimensional representations of a complex dataset that conserve the distance relationship between points. The output visualisation groups patients with clinically relevant features closely together and inherently supports user interpretability. Work is underway to incorporate these findings into an electronic clinical decision support system to guide individual patient management.

Early diagnostic indicators of dengue versus other febrile illnesses in Asia and Latin America (IDAMS study): a multicentre, prospective, observational study.

BACKGROUND: Improvements in the early diagnosis of dengue are urgently needed, especially in resource-limited settings where the distinction between dengue and other febrile illnesses is crucial for patient management. METHODS: In this prospective, observational study (IDAMS), we included patients aged 5 years and older with undifferentiated fever at presentation from 26 outpatient facilities in eight countries (Bangladesh, Brazil, Cambodia, El Salvador, Indonesia, Malaysia, Venezuela, and Viet Nam). We used multivariable logistic regression to investigate the association between clinical symptoms and laboratory tests with dengue versus other febrile illnesses between day 2 and day 5 after onset of fever (ie, illness days). We built a set of candidate regression models including clinical and laboratory variables to reflect the need of a comprehensive versus parsimonious approach. We assessed performance of these models via standard measures of diagnostic values. FINDINGS: Between Oct 18, 2011, and Aug 4, 2016, we recruited 7428 patients, of whom 2694 (36%) were diagnosed with laboratory-confirmed dengue and 2495 (34%) with (non-dengue) other febrile illnesses and met inclusion criteria, and were included in the analysis. 2703 (52%) of 5189 included patients were younger than 15 years, 2486 (48%) were aged 15 years or older, 2179 (42%) were female and 3010 (58%) were male. Platelet count, white blood cell count, and the change in these variables from the previous day of illness had a strong association with dengue. Cough and rhinitis had strong associations with other febrile illnesses, whereas bleeding, anorexia, and skin flush were generally associated with dengue. Model performance increased between day 2 and 5 of illness. The comprehensive model (18 clinical and laboratory predictors) had sensitivities of 0·80 to 0·87 and specificities of 0·80 to 0·91, whereas the parsimonious model (eight clinical and laboratory predictors) had sensitivities of 0·80 to 0·88 and specificities of 0·81 to 0·89. A model that includes laboratory markers that are easy to measure (eg, platelet count or white blood cell count) outperformed the models based on clinical variables only. INTERPRETATION: Our results confirm the important role of platelet and white blood cell counts in diagnosing dengue, and the importance of serial measurements over subsequent days. We successfully quantified the performance of clinical and laboratory markers covering the early period of dengue. Resulting algorithms performed better than published schemes for distinction of dengue from other febrile illnesses, and take into account the dynamic changes over time. Our results provide crucial information needed for the update of guidelines, including the Integrated Management of Childhood Illness handbook. FUNDING: EU's Seventh Framework Programme. TRANSLATIONS: For the Bangla, Bahasa Indonesia, Portuguese, Khmer, Spanish and Vietnamese translations of the abstract see Supplementary Materials section.

Burden of Postinfectious Symptoms after Acute Dengue, Vietnam.

We assessed predominantly pediatric patients in Vietnam with dengue and other febrile illness 3 months after acute illness. Among dengue patients, 47% reported >1 postacute symptom. Most resolved by 3 months, but alopecia and vision problems often persisted. Our findings provide additional evidence on postacute dengue burden and confirm children are affected.

Dengue algorithms integrated into the IMCI guidelines: An updated assessment in five Southeast-Asian countries.

BACKGROUND: Dengue is not included explicitly in the WHO Integrated Management of Childhood Illness (IMCI) algorithm. However, the assessment, classification and management of dengue has been incorporated into several IMCI country adaptations. We aimed to evaluate the dengue algorithms incorporated into IMCI guidelines and discuss the need for harmonization, including an extension of the age range for IMCI. METHODS: This study included three steps. First, we investigated dengue algorithms incorporated into five Southeast-Asian (Myanmar, Philippines, Vietnam, Indonesia, Cambodia) country IMCI guidelines through a desk-based analysis. Second, we conducted an expert survey to elicit opinions regarding the integration of dengue and extension of the age range in IMCI. Third, we compared our findings with data from a large multicentric prospective study on acute febrile illness. RESULTS: We found considerable heterogeneity between the country specific IMCI guidelines in the dengue algorithms as well as classification schemes. Most guidelines did not differentiate between diagnostic algorithms for the detection of dengue versus other febrile illness, and warning signs for progression to severe dengue. Our expert survey resulted in a consensus to further integrate dengue in IMCI and extend the age range for IMCI guidelines beyond 5 years of age. Most of the interviewees responded that their country had a stand-alone clinical guideline for dengue, which was not integrated into the IMCI approach and considered laboratory testing for dengue necessary on day three of consecutive fever. Using data from a large multicentric study of children 5-15 years of age, we could confirm that the likelihood of dengue increased with consecutive fever days. However, a significant proportion of children (36%) would be missed if laboratory testing was only offered on the third consecutive day of fever. CONCLUSIONS: This study supports the extension of the IMCI age range beyond 5 years of age as well as the inclusion of dengue relevant content in the algorithm. Because of the challenge of distinguishing dengue from other febrile illnesses, simple laboratory testing (e.g., full blood count) should be offered at an early stage during the course of the illness. Testing only children with consecutive fever over 3 days may lead to an underdiagnosis of dengue among those with acute febrile illness in children 5-15 years of age. In addition, specific laboratory testing for dengue should be made available to peripheral health facilities.

The Diagnosis of Dengue in Patients Presenting With Acute Febrile Illness Using Supervised Machine Learning and Impact of Seasonality.

Background: Symptomatic dengue infection can result in a life-threatening shock syndrome and timely diagnosis is essential. Point-of-care tests for non-structural protein 1 and IgM are used widely but performance can be limited. We developed a supervised machine learning model to predict whether patients with acute febrile illnesses had a diagnosis of dengue or other febrile illnesses (OFI). The impact of seasonality on model performance over time was examined. Methods: We analysed data from a prospective observational clinical study in Vietnam. Enrolled patients presented with an acute febrile illness of <72 h duration. A gradient boosting model (XGBoost) was used to predict final diagnosis using age, sex, haematocrit, platelet, white cell, and lymphocyte count collected on enrolment. Data was randomly split 80/20% into a training and hold-out set, respectively, with the latter not used in model development. Cross-validation and hold out set testing was used, with performance over time evaluated through a rolling window approach. Results: We included 8,100 patients recruited between 16th October 2010 and 10th December 2014. In total 2,240 (27.7%) patients were diagnosed with dengue infection. The optimised model from training data had an overall median area under the receiver operator curve (AUROC) of 0.86 (interquartile range 0.84-0.86), specificity of 0.92, sensitivity of 0.56, positive predictive value of 0.73, negative predictive value (NPV) of 0.84, and Brier score of 0.13 in predicting the final diagnosis, with similar performances in hold-out set testing (AUROC of 0.86). Model performances varied significantly over time as a function of seasonality and other factors. Incorporation of a dynamic threshold which continuously learns from recent cases resulted in a more consistent performance throughout the year (NPV >90%). Conclusion: Supervised machine learning models are able to discriminate between dengue and OFI diagnoses in patients presenting with an early undifferentiated febrile illness. These models could be of clinical utility in supporting healthcare decision-making and provide passive surveillance across dengue endemic regions. Effects of seasonality and changing disease prevalence must however be taken into account-this is of significant importance given unpredictable effects of human-induced climate change and the impact on health.

The compensatory reserve index predicts recurrent shock in patients with severe dengue.

BACKGROUND: Dengue shock syndrome (DSS) is one of the major clinical phenotypes of severe dengue. It is defined by significant plasma leak, leading to intravascular volume depletion and eventually cardiovascular collapse. The compensatory reserve Index (CRI) is a new physiological parameter, derived from feature analysis of the pulse arterial waveform that tracks real-time changes in central volume. We investigated the utility of CRI to predict recurrent shock in severe dengue patients admitted to the ICU. METHODS: We performed a prospective observational study in the pediatric and adult intensive care units at the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. Patients were monitored with hourly clinical parameters and vital signs, in addition to continuous recording of the arterial waveform using pulse oximetry. The waveform data was wirelessly transmitted to a laptop where it was synchronized with the patient's clinical data. RESULTS: One hundred three patients with suspected severe dengue were recruited to this study. Sixty-three patients had the minimum required dataset for analysis. Median age was 11 years (IQR 8-14 years). CRI had a negative correlation with heart rate and moderate negative association with blood pressure. CRI was found to predict recurrent shock within 12 h of being measured (OR 2.24, 95% CI 1.54-3.26), P < 0.001). The median duration from CRI measurement to the first recurrent shock was 5.4 h (IQR 2.9-6.8). A CRI cutoff of 0.4 provided the best combination of sensitivity and specificity for predicting recurrent shock (0.66 [95% CI 0.47-0.85] and 0.86 [95% CI 0.80-0.92] respectively). CONCLUSION: CRI is a useful non-invasive method for monitoring intravascular volume status in patients with severe dengue.

Applied machine learning for the risk-stratification and clinical decision support of hospitalised patients with dengue in Vietnam.

BACKGROUND: Identifying patients at risk of dengue shock syndrome (DSS) is vital for effective healthcare delivery. This can be challenging in endemic settings because of high caseloads and limited resources. Machine learning models trained using clinical data could support decision-making in this context. METHODS: We developed supervised machine learning prediction models using pooled data from adult and paediatric patients hospitalised with dengue. Individuals from 5 prospective clinical studies in Ho Chi Minh City, Vietnam conducted between 12th April 2001 and 30th January 2018 were included. The outcome was onset of dengue shock syndrome during hospitalisation. Data underwent random stratified splitting at 80:20 ratio with the former used only for model development. Ten-fold cross-validation was used for hyperparameter optimisation and confidence intervals derived from percentile bootstrapping. Optimised models were evaluated against the hold-out set. FINDINGS: The final dataset included 4,131 patients (477 adults and 3,654 children). DSS was experienced by 222 (5.4%) of individuals. Predictors were age, sex, weight, day of illness at hospitalisation, indices of haematocrit and platelets over first 48 hours of admission and before the onset of DSS. An artificial neural network model (ANN) model had best performance with an area under receiver operator curve (AUROC) of 0.83 (95% confidence interval [CI], 0.76-0.85) in predicting DSS. When evaluated against the independent hold-out set this calibrated model exhibited an AUROC of 0.82, specificity of 0.84, sensitivity of 0.66, positive predictive value of 0.18 and negative predictive value of 0.98. INTERPRETATION: The study demonstrates additional insights can be obtained from basic healthcare data, when applied through a machine learning framework. The high negative predictive value could support interventions such as early discharge or ambulatory patient management in this population. Work is underway to incorporate these findings into an electronic clinical decision support system to guide individual patient management.

Combination of inflammatory and vascular markers in the febrile phase of dengue is associated with more severe outcomes.

Background: Early identification of severe dengue patients is important regarding patient management and resource allocation. We investigated the association of 10 biomarkers (VCAM-1, SDC-1, Ang-2, IL-8, IP-10, IL-1RA, sCD163, sTREM-1, ferritin, CRP) with the development of severe/moderate dengue (S/MD). Methods: We performed a nested case-control study from a multi-country study. A total of 281 S/MD and 556 uncomplicated dengue cases were included. Results: On days 1-3 from symptom onset, higher levels of any biomarker increased the risk of developing S/MD. When assessing together, SDC-1 and IL-1RA were stable, while IP-10 changed the association from positive to negative; others showed weaker associations. The best combinations associated with S/MD comprised IL-1RA, Ang-2, IL-8, ferritin, IP-10, and SDC-1 for children, and SDC-1, IL-8, ferritin, sTREM-1, IL-1RA, IP-10, and sCD163 for adults. Conclusions: Our findings assist the development of biomarker panels for clinical use and could improve triage and risk prediction in dengue patients. Funding: This study was supported by the EU's Seventh Framework Programme (FP7-281803 IDAMS), the WHO, and the Bill and Melinda Gates Foundation.

Diagnostic performance of anti-Zika virus IgM, IgAM and IgG ELISAs during co-circulation of Zika, dengue, and chikungunya viruses in Brazil and Venezuela.

BACKGROUND: Serological diagnosis of Zika virus (ZIKV) infection is challenging because of the antibody cross-reactivity among flaviviruses. At the same time, the role of Nucleic Acid Testing (NAT) is limited by the low proportion of symptomatic infections and the low average viral load. Here, we compared the diagnostic performance of commercially available IgM, IgAM, and IgG ELISAs in sequential samples during the ZIKV and chikungunya (CHIKV) epidemics and co-circulation of dengue virus (DENV) in Brazil and Venezuela. METHODOLOGY/PRINCIPAL FINDINGS: Acute (day of illness 1-5) and follow-up (day of illness ≥ 6) blood samples were collected from nine hundred and seven symptomatic patients enrolled in a prospective multicenter study between June 2012 and August 2016. Acute samples were tested by RT-PCR for ZIKV, DENV, and CHIKV. Acute and follow-up samples were tested for IgM, IgAM, and IgG antibodies to ZIKV using commercially available ELISAs. Among follow-up samples with a RT-PCR confirmed ZIKV infection, anti-ZIKV IgAM sensitivity was 93.5% (43/46), while IgM and IgG exhibited sensitivities of 30.3% (10/33) and 72% (18/25), respectively. An additional 24% (26/109) of ZIKV infections were detected via IgAM seroconversion in ZIKV/DENV/CHIKV RT-PCR negative patients. The specificity of anti-ZIKV IgM was estimated at 93% and that of IgAM at 85%. CONCLUSIONS/SIGNIFICANCE: Our findings exemplify the challenges of the assessment of test performance for ZIKV serological tests in the real-world setting, during co-circulation of DENV, ZIKV, and CHIKV. However, we can also demonstrate that the IgAM immunoassay exhibits superior sensitivity to detect ZIKV RT-PCR confirmed infections compared to IgG and IgM immunoassays. The IgAM assay also proves to be promising for detection of anti-ZIKV seroconversions in sequential samples, both in ZIKV PCR-positive as well as PCR-negative patients, making this a candidate assay for serological monitoring of pregnant women in future ZIKV outbreaks.

Remote-Controlled and Pulse Pressure-Guided Fluid Treatment for Adult Patients with Viral Hemorrhagic Fevers.

Circulatory shock, caused by severe intravascular volume depletion resulting from gastrointestinal losses and profound capillary leak, is a common clinical feature of viral hemorrhagic fevers, including Ebola virus disease, Marburg hemorrhagic fever, and Lassa fever. These conditions are associated with high case fatality rates, and they carry a significant risk of infection for treating personnel. Optimized fluid therapy is the cornerstone of management of these diseases, but there are few data on the extent of fluid losses and the severity of the capillary leak in patients with VHFs, and no specific guidelines for fluid resuscitation and hemodynamic monitoring exist. We propose an innovative approach for monitoring VHF patients, in particular suited for low-resource settings, facilitating optimizing fluid therapy through remote-controlled and pulse pressure-guided fluid resuscitation. This strategy would increase the capacity for adequate supportive care, while decreasing the risk for virus transmission to health personnel.

Using NS1 Flavivirus Protein Microarray to Infer Past Infecting Dengue Virus Serotype and Number of Past Dengue Virus Infections in Vietnamese Individuals.

BACKGROUND: In recent years, researchers have had an increased focus on multiplex microarray assays, in which antibodies are measured against multiple related antigens, for use in seroepidemiological studies to infer past transmission. METHODS: We assess the performance of a flavivirus microarray assay for determining past dengue virus (DENV) infection history in a dengue-endemic setting, Vietnam. We tested the microarray on samples from 1 and 6 months postinfection from DENV-infected patients (infecting serotype was determined using reverse-transcription polymerase chain reaction during acute, past primary, and secondary infection assessed using plaque reduction neutralization tests 6 months postinfection). RESULTS: Binomial models developed to discriminate past primary from secondary infection using the protein microarray (PMA) titers had high area under the curve (0.90-0.97) and accuracy (0.84-0.86). Multinomial models developed to identify most recent past infecting serotype using PMA titers performed well in those with past primary infection (average test set: κ = 0.85, accuracy of 0.92) but not those with past secondary infection (κ = 0.24, accuracy of 0.45). CONCLUSIONS: Our results suggest that the microarray will be useful in seroepidemiological studies aimed at classifying the past infection history of individuals (past primary vs secondary and serotype of past primary infections) and thus inferring past transmission intensity of DENV in dengue-endemic settings. Future work to validate these models should be undertaken in different transmission settings and with samples later after infection.

Higher Plasma Viremia in the Febrile Phase Is Associated With Adverse Dengue Outcomes Irrespective of Infecting Serotype or Host Immune Status: An Analysis of 5642 Vietnamese Cases.

BACKGROUND: One of the generally accepted constructs of dengue pathogenesis is that clinical disease severity is at least partially dependent upon plasma viremia, yet data on plasma viremia in primary versus secondary infections and in relation to clinically relevant endpoints remain limited and contradictory. METHODS: Using a large database comprising detailed clinical and laboratory characterization of Vietnamese participants enrolled in a series of research studies executed over a 15-year period, we explored relationships between plasma viremia measured by reverse transcription-polymerase chain reaction and 3 clinically relevant endpoints-severe dengue, plasma leakage, and hospitalization-in the dengue-confirmed cases. All 4 dengue serotypes and both primary and secondary infections were well represented. In our logistic regression models we allowed for a nonlinear effect of viremia and for associations between viremia and outcome to differ by age, serotype, host immune status, and illness day at study enrollment. RESULTS: Among 5642 dengue-confirmed cases we identified 259 (4.6%) severe dengue cases, 701 (12.4%) patients with plasma leakage, and 1441 of 4008 (40.0%) patients recruited in outpatient settings who were subsequently hospitalized. From the early febrile phase onwards, higher viremia increased the risk of developing all 3 endpoints, but effect sizes were modest (ORs ranging from 1.12-1.27 per 1-log increase) compared with the effects of a secondary immune response (ORs, 1.67-7.76). The associations were consistent across age, serotype, and immune status groups, and in the various sensitivity and subgroup analyses we undertook. CONCLUSIONS: Higher plasma viremia is associated with increased dengue severity, regardless of serotype or immune status.

Visual and Biochemical Evidence of Glycocalyx Disruption in Human Dengue Infection, and Association With Plasma Leakage Severity.

Background: Dengue is the most common arboviral infection globally; a minority of patients develop shock due to profound plasma leak through a disrupted endothelial barrier. Understanding of the pathophysiology underlying plasma leak is incomplete, but emerging evidence indicates a key role for degradation of the endothelial glycocalyx. Methods: We conducted an observational study in Vietnam to evaluate the sublingual microcirculation using sidestream darkfield imaging in (1) outpatients with confirmed dengue (2) patients hospitalized with dengue and (3) outpatients with other febrile illness (OFI). We estimated the glycocalyx degradation by measuring the perfused boundary region (PBR hf) and an overall microvascular health score (MVHS) with the software application GlycoCheckTM at enrolment, 48 h later and hospital discharge/defervescence. We measured plasma syndecan1 and endocan at the same time-points. We compared PBR hf, MVHS, syndecan1 and endocan, between (1) outpatients with confirmed dengue vs. OFI and (2) patients with dengue subdivided by clinical severity of plasma leak. Results: We included 75 patients with dengue (41 outpatients, 15 inpatients, 19 in intensive care) and 12 outpatients with OFI. Images from 45 patients were analyzed using GlycoCheckTM. There was no significant difference in PBR hf or MVHS between outpatients with dengue and OFI. Median plasma syndecan1 was not significantly different in outpatients with dengue vs. OFI, while median plasma endocan was significantly lower among patients with dengue vs. OFI during the critical phase. In patients with dengue, PBR hf was higher in patients with Grade 2 vs. Grade 0 plasma leakage during the critical phase (PBR hf 1.96 vs. 1.36 µm for Grade 2 vs. Grade 0 plasma leakage on days 4-6, respectively, p < 0.001). Median levels of plasma syndecan1 and endocan were higher in Grade 2 vs. Grade 0 plasma leakage, especially during the critical phase (Syndecan1 2,613.8 vs. 125.9 ng/ml for Grade 2 vs. Grade 0 plasma leakage on days 4-6, respectively, p < 0.001, and endocan 3.21 vs. 0.16 ng/ml for Grade 2 vs. Grade 0 plasma leakage on days 4-6, respectively). Conclusions: We present the first human in vivo evidence of glycocalyx disruption in dengue, with worse visual glycocalyx damage and higher plasma degradation products associated with more severe plasma leak.

Assessing the vertical transmission potential of dengue virus in field-reared Aedes aegypti using patient-derived blood meals in Ho Chi Minh City, Vietnam.

BACKGROUND: Dengue viruses (DENV) can be transmitted from an adult female Aedes aegypti mosquito through the germ line to the progeny; however, there is uncertainty if this occurs at a frequency that is epidemiologically significant. We measured vertical transmission of DENV from field-reared Ae. aegypti to their F1 progeny after feeding upon blood from dengue patients. We also examined the transmission potential of F1 females. METHODS: We examined the frequency of vertical transmission in field-reared mosquitoes, who fed upon blood from acutely viremic dengue patients, and the capacity for vertically infected females to subsequently transmit virus horizontally, in two sets of experiments: (i) compared vertical transmission frequency of field-reared Ae. aegypti and Ae. albopictus, in individual progeny; and (ii) in pooled progeny derived from field- and laboratory-reared Ae. aegypti. RESULTS: Of 41 DENV-infected and isofemaled females who laid eggs, only a single female (2.43%) transmitted virus to one of the F1 progeny, but this F1 female did not have detectable virus in the saliva when 14 days-old. We complemented this initial study by testing for vertical transmission in another 460 field-reared females and > 900 laboratory-reared counterparts but failed to provide any further evidence of vertical virus transmission. CONCLUSIONS: In summary, these results using field-reared mosquitoes and viremic blood from dengue cases suggest that vertical transmission is uncommon. Field-based studies that build on these observations are needed to better define the contribution of vertical DENV transmission to dengue epidemiology.

Dengue virus induces PCSK9 expression to alter antiviral responses and disease outcomes.

Dengue virus (DENV) infection requires cholesterol as a proviral factor, although statin treatment did not show antiviral efficacy in patients with dengue. Here, we show that DENV infection manipulated cholesterol metabolism in cells residing in low-oxygen microenvironments (hypoxia) such as in the liver, spleen, and lymph nodes. DENV infection induced expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), which reduces low-density lipoprotein receptor (LDLR) recycling and hence cholesterol uptake. We found that, whereas LDLR uptake would have distributed cholesterol throughout the various cell compartments, de novo cholesterol synthesis enriched this lipid in the endoplasmic reticulum (ER). With cholesterol enrichment in the ER, ER-resident STING and type I IFN (IFN) activation was repressed during DENV infection. Our in vitro findings were further supported by the detection of elevated plasma PCSK9 levels in patients with dengue with high viremia and increased severity of plasma leakage. Our findings therefore suggest that PCSK9 plays a hitherto unrecognized role in dengue pathogenesis and that PCSK9 inhibitors could be a suitable host-directed treatment for patients with dengue.

Multiple Wolbachia strains provide comparative levels of protection against dengue virus infection in Aedes aegypti.

The insect bacterium Wolbachia pipientis is being introgressed into Aedes aegypti populations as an intervention against the transmission of medically important arboviruses. Here we compare Ae. aegypti mosquitoes infected with wMelCS or wAlbB to the widely used wMel Wolbachia strain on an Australian nuclear genetic background for their susceptibility to infection by dengue virus (DENV) genotypes spanning all four serotypes. All Wolbachia-infected mosquitoes were more resistant to intrathoracic DENV challenge than their wildtype counterparts. Blocking of DENV replication was greatest by wMelCS. Conversely, wAlbB-infected mosquitoes were more susceptible to whole body infection than wMel and wMelCS. We extended these findings via mosquito oral feeding experiments, using viremic blood from 36 acute, hospitalised dengue cases in Vietnam, additionally including wMel and wildtype mosquitoes on a Vietnamese nuclear genetic background. As above, wAlbB was less effective at blocking DENV replication in the abdomen compared to wMel and wMelCS. The transmission potential of all Wolbachia-infected mosquito lines (measured by the presence/absence of infectious DENV in mosquito saliva) after 14 days, was significantly reduced compared to their wildtype counterparts, and lowest for wMelCS and wAlbB. These data support the use of wAlbB and wMelCS strains for introgression field trials and the biocontrol of DENV transmission. Furthermore, despite observing significant differences in transmission potential between wildtype mosquitoes from Australia and Vietnam, no difference was observed between wMel-infected mosquitoes from each background suggesting that Wolbachia may override any underlying variation in DENV transmission potential.