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.

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.

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.

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.

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.

Field- and clinically derived estimates of Wolbachia-mediated blocking of dengue virus transmission potential in Aedes aegypti mosquitoes.

The wMel strain of Wolbachia can reduce the permissiveness of Aedes aegypti mosquitoes to disseminated arboviral infections. Here, we report that wMel-infected Ae. aegypti (Ho Chi Minh City background), when directly blood-fed on 141 viremic dengue patients, have lower dengue virus (DENV) transmission potential and have a longer extrinsic incubation period than their wild-type counterparts. The wMel-infected mosquitoes that are field-reared have even greater relative resistance to DENV infection when fed on patient-derived viremic blood meals. This is explained by an increased susceptibility of field-reared wild-type mosquitoes to infection than laboratory-reared counterparts. Collectively, these field- and clinically relevant findings support the continued careful field-testing of wMel introgression for the biocontrol of Ae. aegypti-born arboviruses.

Dengue.

Mortality from severe dengue is low, but the economic and resource burden on health services remains substantial in endemic settings. Unfortunately, progress towards development of effective therapeutics has been slow, despite notable advances in the understanding of disease pathogenesis and considerable investment in antiviral drug discovery. For decades antibody-dependent enhancement has been the prevalent model to explain dengue pathogenesis, but it was only recently demonstrated in vivo and in clinical studies. At present, the current mainstay of management for most symptomatic dengue patients remains careful observation and prompt but judicious use of intravenous hydration therapy for those with substantial vascular leakage. Various new promising technologies for diagnosis of dengue are currently in the pipeline. New sample-in, answer-out nucleic acid amplification technologies for point-of-care use are being developed to improve performance over current technologies, with the potential to test for multiple pathogens using a single specimen. The search for biomarkers that reliably predict development of severe dengue among symptomatic individuals is also a major focus of current research efforts. The first dengue vaccine was licensed in 2015 but its performance depends on serostatus. There is an urgent need to identify correlates of both vaccine protection and disease enhancement. A crucial assessment of vector control tools should guide a research agenda for determining the most effective interventions, and how to best combine state-of-the-art vector control with vaccination.

C-reactive protein as a potential biomarker for disease progression in dengue: a multi-country observational study.

BACKGROUND: Dengue infection can cause a wide spectrum of clinical outcomes. The severe clinical manifestations occur sufficiently late in the disease course, during day 4-6 of illness, to allow a window of opportunity for risk stratification. Markers of inflammation may be useful biomarkers. We investigated the value of C-reactive protein (CRP) measured early on illness days 1-3 to predict dengue disease outcome and the difference in CRP levels between dengue and other febrile illnesses (OFI). METHOD: We performed a nested case-control study using the clinical data and samples collected from the IDAMS-consortium multi-country study. This was a prospective multi-center observational study that enrolled almost 8000 participants presenting with a dengue-like illness to outpatient facilities in 8 countries across Asia and Latin America. Predefined severity definitions of severe and intermediate dengue were used as the primary outcomes. A total of 281 cases with severe/intermediate dengue were compared to 836 uncomplicated dengue patients as controls (ratio 1:3), and also 394 patients with OFI. RESULTS: In patients with confirmed dengue, median (interquartile range) of CRP level within the first 3 days was 30.2 mg/L (12.4-61.2 mg/L) (uncomplicated dengue, 28.6 (10.5-58.9); severe or intermediate dengue, 34.0 (17.4-71.8)). Higher CRP levels in the first 3 days of illness were associated with a higher risk of severe or intermediate outcome (OR 1.17, 95% CI 1.07-1.29), especially in children. Higher CRP levels, exceeding 30 mg/L, also associated with hospitalization (OR 1.37, 95% CI 1.14-1.64) and longer fever clearance time (HR 0.84, 95% CI 0.76-0.93), especially in adults. CRP levels in patients with dengue were higher than patients with potential viral infection but lower than patients with potential bacterial infection, resulting in a quadratic association between dengue diagnosis and CRP, with levels of approximately 30 mg/L associated with the highest risk of having dengue. CRP had a positive correlation with total white cell count and neutrophils and negative correlation with lymphocytes, but did not correlate with liver transaminases, albumin, or platelet nadir. CONCLUSIONS: In summary, CRP measured in the first 3 days of illness could be a useful biomarker for early dengue risk prediction and may assist differentiating dengue from other febrile illnesses.

Assessing the efficacy and safety of magnesium sulfate for management of autonomic nervous system dysregulation in Vietnamese children with severe hand foot and mouth disease.

BACKGROUND: Brainstem encephalitis is a serious complication of hand foot and mouth disease (HFMD) in children. Autonomic nervous system (ANS) dysregulation and hypertension may occur, sometimes progressing to cardiopulmonary failure and death. Vietnamese national guidelines recommend use of milrinone if ANS dysregulation with Stage 2 hypertension develops. We wished to investigate whether magnesium sulfate (MgSO4) improved outcomes in children with HFMD if used earlier in the evolution of the ANS dysregulation (Stage 1 hypertension). METHODS: During a regional epidemic we conducted a randomized, double-blind, placebo-controlled trial of MgSO4 in children with HFMD, ANS dysregulation and Stage 1 hypertension, at the Hospital for Tropical Diseases in Ho Chi Minh city. Study participants received an infusion of MgSO4 or matched placebo for 72 h. We also reviewed data from non-trial HFMD patients in whom milrinone failed to control hypertension, some of whom received MgSO4 as second line therapy. The primary outcome for both analyses was a composite of disease progression within 72 h - addition of milrinone (trial participants only), need for ventilation, shock, or death. RESULTS: Between June 2014 and September 2016, 14 and 12 participants received MgSO4 or placebo respectively, before the trial was stopped due to futility. Among 45 non-trial cases with poorly controlled hypertension despite high-dose milrinone, 33 received MgSO4 while 12 did not. There were no statistically significant differences in the composite outcome between the MgSO4 and the placebo/control groups in either study (adjusted relative risk (95%CI) of [6/14 (43%) vs. 6/12 (50%)], 0.84 (0.37, 1.92), p = 0.682 in the trial and [1/33 (3%) vs. 2/12 (17%)], 0.16 (0.01, 1.79), p = 0.132 in the observational cohort). The incidence of adverse events was similar between the groups. Potentially toxic magnesium levels occurred very rarely with the infusion regime used. CONCLUSION: Although we could not demonstrate efficacy in these studies, there were no safety signals associated with use of 30-50 mg/kg/hr. MgSO4 in severe HFMD. Intermittent outbreaks of HFMD are likely to continue across the region, and an adequately powered trial is still needed to evaluate use of MgSO4 in controlling hypertension in severe HFMD, potentially involving a higher dose regimen. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01940250 (Registered 22 AUG 2013). Trial sponsor: University of Oxford.