CAPTURA Regional Workshop Proceedings (28-30 June 2022, Virtual).

In response to the global threat of antimicrobial resistance (AMR), the Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) project worked with microbiology laboratories, pharmacies, and local governments in South Asia and Southeast Asia to expand the volume of historical and current data available on AMR and antimicrobial use and to identify gaps in data and areas for quality improvement. When the CAPTURA project completed its country-level engagement in the first half of 2022, the consortium brought together local, regional, and global AMR stakeholders for a virtual regional workshop to review data outputs from the project and share strategies to inform national and regional efforts to combat AMR. This paper summarizes the main topics presented in the workshop held from 28 to 30 June 2022. As such, it highlights lessons learned from the project and strategies to fight AMR. Although CAPTURA has been invaluable to countries and information from the project is already being used, barriers concerning data quality and sharing remain. Regional-level initiatives should continue to build on the momentum gained from the CAPTURA project in supporting national-level surveillance and data quality improvements to inform critical decisions around planning, policies, and clinical care. Project findings have highlighted that issues with antimicrobial resistance and use are wide ranging across countries. Going forward, building on the current foundations and tailoring approaches to meet local needs and capacities will be fundamental in combatting AMR.

A high prevalence of multi-drug resistant Gram-negative bacilli in a Nepali tertiary care hospital and associated widespread distribution of Extended-Spectrum Beta-Lactamase (ESBL) and carbapenemase-encoding genes.

BACKGROUND: Multi-drug resistance (MDR) and extensive-drug resistance (XDR) associated with extended-spectrum beta-lactamases (ESBLs) and carbapenemases in Gram-negative bacteria are global public health concerns. Data on circulating antimicrobial resistance (AMR) genes in Gram-negative bacteria and their correlation with MDR and ESBL phenotypes from Nepal is scarce. METHODS: A retrospective study was performed investigating the distribution of ESBL and carbapenemase genes and their potential association with ESBL and MDR phenotypes in E. coli, Klebsiella spp., Enterobacter spp. and Acinetobacter spp. isolated in a major tertiary hospital in Kathmandu, Nepal, between 2012 and 2018. RESULTS: During this period, the hospital isolated 719 E. coli, 532 Klebsiella spp., 520 Enterobacter spp. and 382 Acinetobacter spp.; 1955/2153 (90.1%) of isolates were MDR and half (1080/2153) were ESBL producers. Upon PCR amplification, blaTEM (1281/1771; 72%), blaCTXM-1 (930/1771; 53%) and blaCTXM-8 (419/1771; 24%) were the most prevalent ESBL genes in the enteric bacilli. BlaOXA and blaOXA-51 were the most common blaOXA family genes in the enteric bacilli (918/1771; 25%) and Acinetobacter spp. (218/382; 57%) respectively. Sixteen percent (342/2153) of all isolates and 20% (357/1771) of enteric bacilli harboured blaNDM-1 and blaKPC carbapenemase genes respectively. Of enteric bacilli, Enterobacter spp. was the most frequently positive for blaKPC gene (201/337; 60%). The presence of each blaCTX-M and blaOXA were significantly associated with non-susceptibility to third generation cephalosporins (OR 14.7, p < 0.001 and OR 2.3, p < 0.05, respectively).The presence of each blaTEM, blaCTXM and blaOXA family genes were significantly associated with ESBL positivity (OR 2.96, p < 0.001; OR 14.2, p < 0.001 and OR 1.3, p < 0.05 respectively) and being MDR (OR 1.96, p < 0.001; OR 5.9, p < 0.001 and OR 2.3, p < 0.001 respectively). CONCLUSIONS: This study documents an alarming level of AMR with high prevalence of MDR ESBL- and carbapenemase-positive ESKAPE microorganisms in our clinical setting. These data suggest a scenario where the clinical management of infected patients is increasingly difficult and requires the use of last-resort antimicrobials, which in turn is likely to intensify the magnitude of global AMR crisis.

A current perspective on antimicrobial resistance in Southeast Asia.

Southeast Asia, a vibrant region that has recently undergone unprecedented economic development, is regarded as a global hotspot for the emergence and spread of antimicrobial resistance (AMR). Understanding AMR in Southeast Asia is crucial for assessing how to control AMR on an international scale. Here we (i) describe the current AMR situation in Southeast Asia, (ii) explore the mechanisms that make Southeast Asia a focal region for the emergence of AMR, and (iii) propose ways in which Southeast Asia could contribute to a global solution.

CD8+ T Cells Can Mediate Short-Term Protection against Heterotypic Dengue Virus Reinfection in Mice.

UNLABELLED: Dengue virus (DENV) is a major public health threat worldwide. Infection with one of the four serotypes of DENV results in a transient period of protection against reinfection with all serotypes (cross-protection), followed by lifelong immunity to the infecting serotype. While a protective role for neutralizing antibody responses is well established, the contribution of T cells to reinfection is less clear, especially during heterotypic reinfection. This study investigates the role of T cells during homotypic and heterotypic DENV reinfection. Mice were sequentially infected with homotypic or heterotypic DENV serotypes, and T cell subsets were depleted before the second infection to assess the role of DENV-primed T cells during reinfection. Mice primed nonlethally with DENV were protected against reinfection with either a homotypic or heterotypic serotype 2 weeks later. Homotypic priming induced a robust neutralizing antibody response, whereas heterotypic priming elicited binding, but nonneutralizing antibodies. CD8(+) T cells were required for protection against heterotypic, but not homotypic, reinfection. These results suggest that T cells can contribute crucially to protection against heterotypic reinfection in situations where humoral responses alone may not be protective. Our findings have important implications for vaccine design, as they suggest that inducing both humoral and cellular responses during vaccination may maximize protective efficacy across all DENV serotypes. IMPORTANCE: Dengue virus is present in more than 120 countries in tropical and subtropical regions. Infection with dengue virus can be asymptomatic, but it can also progress into the potentially lethal severe dengue disease. There are four closely related dengue virus serotypes. Infection with one serotype results in a transient period of resistance against all serotypes (cross-protection), followed by lifelong resistance to the infecting serotype, but not the other ones. The duration and mechanisms of the transient cross-protection period remain elusive. This study investigates the contribution of cellular immunity to cross-protection using mouse models of DENV infection. Our results demonstrate that cellular immunity is crucial to mediate cross-protection against reinfection with a different serotype, but not for protection against reinfection with the same serotype. A better understanding of the mediators responsible for the cross-protection period is important for vaccine design, as an ideal vaccine against dengue virus should efficiently protect against all serotypes.

CD8+ T cells prevent antigen-induced antibody-dependent enhancement of dengue disease in mice.

Dengue virus (DENV) causes pathologies ranging from the febrile illness dengue fever to the potentially lethal severe dengue disease. A major risk factor for developing severe dengue disease is the presence of subprotective DENV-reactive Abs from a previous infection (or from an immune mother), which can induce Ab-dependent enhancement of infection (ADE). However, infection in the presence of subprotective anti-DENV Abs does not always result in severe disease, suggesting that other factors influence disease severity. In this study we investigated how CD8(+) T cell responses influence the outcome of Ab-mediated severe dengue disease. Mice were primed with aluminum hydroxide-adjuvanted UV-inactivated DENV prior to challenge with DENV. Priming failed to induce robust CD8(+) T cell responses, and it induced nonneutralizing Ab responses that increased disease severity upon infection. Transfer of exogenous DENV-activated CD8(+) T cells into primed mice prior to infection prevented Ab-dependent enhancement and dramatically reduced viral load. Our results suggest that in the presence of subprotective anti-DENV Abs, efficient CD8(+) T cell responses reduce the risk of Ab-mediated severe dengue disease.

Role of humoral versus cellular responses induced by a protective dengue vaccine candidate.

With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system.

Incidence of typhoid fever in Burkina Faso, Democratic Republic of the Congo, Ethiopia, Ghana, Madagascar, and Nigeria (the Severe Typhoid in Africa programme): a population-based study.

BACKGROUND: Typhoid Fever remains a major cause of morbidity and mortality in low-income settings. The Severe Typhoid in Africa programme was designed to address regional gaps in typhoid burden data and identify populations eligible for interventions using novel typhoid conjugate vaccines. METHODS: A hybrid design, hospital-based prospective surveillance with population-based health-care utilisation surveys, was implemented in six countries in sub-Saharan Africa. Patients presenting with fever (≥37·5°C axillary or ≥38·0°C tympanic) or reporting fever for three consecutive days within the previous 7 days were invited to participate. Typhoid fever was ascertained by culture of blood collected upon enrolment. Disease incidence at the population level was estimated using a Bayesian mixture model. FINDINGS: 27 866 (33·8%) of 82 491 participants who met inclusion criteria were recruited. Blood cultures were performed for 27 544 (98·8%) of enrolled participants. Clinically significant organisms were detected in 2136 (7·7%) of these cultures, and 346 (16·2%) Salmonella enterica serovar Typhi were isolated. The overall adjusted incidence per 100 000 person-years of observation was highest in Kavuaya and Nkandu 1, Democratic Republic of the Congo (315, 95% credible interval 254-390). Overall, 46 (16·4%) of 280 tested isolates showed ciprofloxacin non-susceptibility. INTERPRETATION: High disease incidence (ie, >100 per 100 000 person-years of observation) recorded in four countries, the prevalence of typhoid hospitalisations and complicated disease, and the threat of resistant typhoid strains strengthen the need for rapid dispatch and implementation of effective typhoid conjugate vaccines along with measures designed to improve clean water, sanitation, and hygiene practices. FUNDING: The Bill & Melinda Gates Foundation.

Gamma interferon (IFN-γ) receptor restricts systemic dengue virus replication and prevents paralysis in IFN-α/ß receptor-deficient mice.

We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/ßR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/ßR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/ßR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8(+) T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8(+) T cells in preventing DENV-induced CNS disease.

Typhoid Control in an Era of Antimicrobial Resistance: Challenges and Opportunities.

Historically, typhoid control has been achieved with water and sanitation interventions. Today, in an era of rising antimicrobial resistance (AMR), two World Health Organization-prequalified vaccines are available to accelerate control in the shorter term. Meanwhile, water and sanitation interventions could be implemented in the longer term to sustainably prevent typhoid in low- and middle-income countries. This article first approaches typhoid control from a historical perspective, subsequently presents how vaccination could complement water and sanitation activities, and finally discusses the challenges and opportunities for impactful control of typhoid infection. It also addresses data blind spots and knowledge gaps to focus on for typhoid control and to ultimately progress towards elimination. This article presents a synthesis of discussions held in December 2021 during a roundtable session at the "12th International Conference on Typhoid and Other Invasive Salmonelloses".

Characterization of Typhoid Intestinal Perforation in Africa: Results From the Severe Typhoid Fever Surveillance in Africa Program.

Background: Typhoid intestinal perforation (TIP) remains the most serious complication of typhoid fever. In many countries, the diagnosis of TIP relies on intraoperative identification, as blood culture and pathology capacity remain limited. As a result, many cases of TIP may not be reported as typhoid. This study demonstrates the burden of TIP in sites in Burkina Faso, Democratic Republic of Congo (DRC), Ethiopia, Ghana, Madagascar, and Nigeria. Methods: Patients with clinical suspicion of nontraumatic intestinal perforation were enrolled and demographic details, clinical findings, surgical records, blood cultures, tissue biopsies, and peritoneal fluid were collected. Participants were then classified as having confirmed TIP, probable TIP, possible TIP, or clinical intestinal perforation based on surgical descriptions and cultures. Results: A total of 608 participants were investigated for nontraumatic intestinal perforation; 214 (35%) participants had surgically-confirmed TIP and 33 participants (5%) had culture-confirmed typhoid. The overall proportion of blood or surgical site Salmonella enterica subspecies enterica serovar Typhi positivity in surgically verified TIP cases was 10.3%. TIP was high in children aged 5-14 years in DRC, Ghana, and Nigeria. We provide evidence for correlation between monthly case counts of S. Typhi and the occurrence of intestinal perforation. Conclusions: Low S. Typhi culture positivity rates, as well as a lack of blood and tissue culture capability in many regions where typhoid remains endemic, significantly underestimate the true burden of typhoid fever. The occurrence of TIP may indicate underlying typhoid burden, particularly in countries with limited culture capability.

Nonneutralizing antibodies binding to the surface glycoprotein of lymphocytic choriomeningitis virus reduce early virus spread.

The biological relevance of nonneutralizing antibodies elicited early after infection with noncytopathic persistence-prone viruses is unclear. We demonstrate that cytotoxic T lymphocyte-deficient TgH(KL25) mice, which are transgenic for the heavy chain of the lymphocytic choriomeningitis virus (LCMV)-neutralizing monoclonal antibody KL25, mount a focused neutralizing antibody response following LCMV infection, and that this results in the emergence of neutralization escape virus variants. Further investigation revealed that some of the escape variants that arose early after infection could still bind to the selecting antibody. In contrast, no antibody binding could be detected for late isolates, indicating that binding, but nonneutralizing, antibodies exerted a selective pressure on the virus. Infection of naive TgH(KL25) mice with distinct escape viruses differing in their antibody-binding properties revealed that nonneutralizing antibodies accelerated clearance of antibody-binding virus variants in a partly complement-dependent manner. Virus variants that did not bind antibodies were not affected. We therefore conclude that nonneutralizing antibodies binding to the same antigenic site as neutralizing antibodies are biologically relevant by limiting early viral spread.

CD4+ T cells are not required for the induction of dengue virus-specific CD8+ T cell or antibody responses but contribute to protection after vaccination.

The contribution of T cells to the host response to dengue virus (DENV) infection is not well understood. We previously demonstrated a protective role for CD8(+) T cells during primary DENV infection using a mouse-passaged DENV strain and IFN-α/ßR(-/-) C57BL/6 mice, which are susceptible to DENV infection. In this study, we examine the role of CD4(+) T cells during primary DENV infection. Four I-A(b)-restricted epitopes derived from three of the nonstructural DENV proteins were identified. CD4(+) T cells expanded and were activated after DENV infection, with peak activation occurring on day 7. The DENV-specific CD4(+) T cells expressed intracellular IFN-γ, TNF, IL-2, and CD40L, and killed peptide-pulsed target cells in vivo. Surprisingly, depletion of CD4(+) T cells before DENV infection had no effect on viral loads. Consistent with this observation, CD4(+) T cell depletion did not affect the DENV-specific IgG or IgM Ab titers or their neutralizing activity, or the DENV-specific CD8(+) T cell response. However, immunization with the CD4(+) T cell epitopes before infection resulted in significantly lower viral loads. Thus, we conclude that whereas CD4(+) T cells are not required for controlling primary DENV infection, their induction by immunization can contribute to viral clearance. These findings suggest inducing anti-DENV CD4(+) T cell responses by vaccination may be beneficial.

Serology as a Tool to Assess Infectious Disease Landscapes and Guide Public Health Policy.

Understanding the local burden and epidemiology of infectious diseases is crucial to guide public health policy and prioritize interventions. Typically, infectious disease surveillance relies on capturing clinical cases within a healthcare system, classifying cases by etiology and enumerating cases over a period of time. Disease burden is often then extrapolated to the general population. Serology (i.e., examining serum for the presence of pathogen-specific antibodies) has long been used to inform about individuals past exposure and immunity to specific pathogens. However, it has been underutilized as a tool to evaluate the infectious disease burden landscape at the population level and guide public health decisions. In this review, we outline how serology provides a powerful tool to complement case-based surveillance for determining disease burden and epidemiology of infectious diseases, highlighting its benefits and limitations. We describe the current serology-based technologies and illustrate their use with examples from both the pre- and post- COVID-19-pandemic context. In particular, we review the challenges to and opportunities in implementing serological surveillance in low- and middle-income countries (LMICs), which bear the brunt of the global infectious disease burden. Finally, we discuss the relevance of serology data for public health decision-making and describe scenarios in which this data could be used, either independently or in conjunction with case-based surveillance. We conclude that public health systems would greatly benefit from the inclusion of serology to supplement and strengthen existing case-based infectious disease surveillance strategies.

Estimating typhoid incidence from community-based serosurveys: a multicohort study.

BACKGROUND: The incidence of enteric fever, an invasive bacterial infection caused by typhoidal Salmonellae (Salmonella enterica serovars Typhi and Paratyphi), is largely unknown in regions without blood culture surveillance. The aim of this study was to evaluate whether new diagnostic serological markers for typhoidal Salmonella can reliably estimate population-level incidence. METHODS: We collected longitudinal blood samples from patients with blood culture-confirmed enteric fever enrolled from surveillance studies in Bangladesh, Nepal, Pakistan, and Ghana between 2016 and 2021 and conducted cross-sectional serosurveys in the catchment areas of each surveillance site. We used ELISAs to measure quantitative IgA and IgG antibody responses to hemolysin E and S Typhi lipopolysaccharide. We used Bayesian hierarchical models to fit two-phase power-function decay models to the longitudinal antibody responses among enteric fever cases and used the joint distributions of the peak antibody titres and decay rate to estimate population-level incidence rates from cross-sectional serosurveys. FINDINGS: The longitudinal antibody kinetics for all antigen-isotypes were similar across countries and did not vary by clinical severity. The seroincidence of typhoidal Salmonella infection among children younger than 5 years ranged between 58·5 per 100 person-years (95% CI 42·1-81·4) in Dhaka, Bangladesh, to 6·6 per 100 person-years (4·3-9·9) in Kavrepalanchok, Nepal, and followed the same rank order as clinical incidence estimates. INTERPRETATION: The approach described here has the potential to expand the geographical scope of typhoidal Salmonella surveillance and generate incidence estimates that are comparable across geographical regions and time. FUNDING: Bill & Melinda Gates Foundation. TRANSLATIONS: For the Nepali, Bengali and Urdu translations of the abstract see Supplementary Materials section.

A protective role for dengue virus-specific CD8+ T cells.

Infection with one of the four serotypes of dengue virus (DENV1-4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8(+) T cells in the immune response to primary DENV infection. S221 did not replicate well in wild-type mice, but did induce a CD8(+) T cell response, whereas viral replication and a robust CD8(+) T cell response were observed after infection of IFN-alpha/betaR(-/-) mice. Depletion of CD8(+) T cells from IFN-alpha/betaR(-/-) mice before infection resulted in significantly higher viral loads compared with undepleted mice. Mapping the specificity of the CD8(+) T cell response led to the identification of 12 epitopes derived from 6 of the 10 DENV proteins, with a similar immunodominance hierarchy observed in wild-type and IFN-alpha/betaR(-/-) mice. DENV-specific CD8(+) T cells produced IFN-gamma, TNF-alpha, expressed cell surface CD107a, and exhibited cytotoxic activity in vivo. Finally, immunization with four of the immunodominant CD8(+) T cell epitopes enhanced viral clearance. Collectively, our results reveal an important role for CD8(+) T cells in the host defense against DENV and demonstrate that the anti-DENV CD8(+) T cell response can be enhanced by immunization, providing rationale for designing DENV-specific vaccines that induce cell-mediated immunity.

Systemic inflammation, innate immunity and pathogenesis after Zika virus infection in cynomolgus macaques are modulated by strain-specificity within the Asian lineage.

Zika virus (ZIKV) is an emerging arbovirus with recent global expansion. Historically, ZIKV infections with Asian lineages have been associated with mild disease such as rash and fever. However, recent Asian sub-lineages have caused outbreaks in the South Pacific and Latin America with increased prevalence of neurological disorders in infants and adults. Asian sub-lineage differences may partially explain the range of disease severity observed. However, the effect of Asian sub-lineage differences on pathogenesis remains poorly characterized. Current study conducts a head-to-head comparison of three Asian sub-lineages that are representative of the circulating ancestral mild Asian strain (ZIKV-SG), the 2007 epidemic French Polynesian strain (ZIKV-FP), and the 2013 epidemic Brazil strain (ZIKV-Brazil) in adult Cynomolgus macaques. Animals infected intervenously or subcutaneously with either of the three clinical isolates showed sub-lineage-specific differences in viral pathogenesis, early innate immune responses and systemic inflammation. Despite the lack of neurological symptoms in infected animals, the epidemiologically neurotropic ZIKV sub-lineages (ZIKV-Brazil and/or ZIKV-FP) were associated with more sustained viral replication, higher systemic inflammation (i.e. higher levels of TNFα, MCP-1, IL15 and G-CSF) and greater percentage of CD14+ monocytes and dendritic cells in blood. Multidimensional analysis showed clustering of ZIKV-SG away from ZIKV-Brazil and ZIKV-FP, further confirming sub-lineage differences in the measured parameters. These findings highlight greater systemic inflammation and monocyte recruitment as possible risk factors of adult ZIKV disease observed during the 2007 FP and 2013 Brazil epidemics. Future studies should explore the use of anti-inflammatory therapeutics as early treatment to prevent ZIKV-associated disease in adults.

Antibody effector analysis of prime versus prime-boost immunizations with a recombinant measles-vectored chikungunya virus vaccine.

Chikungunya is a mosquito-borne disease that causes periodic but explosive epidemics of acute disease throughout the tropical world. Vaccine development against chikungunya virus (CHIKV) has been hampered by an inability to conduct efficacy trials due to the unpredictability of CHIKV outbreaks. Therefore, immune correlates are being explored to gain inference into vaccine-induced protection. This study is an in-depth serological characterization of Fab- and Fc-mediated antibody responses in selected phase II clinical trial participants following immunization with the recombinant measles-vectored CHIKV vaccine, MV-CHIK. Antibody comparisons were conducted between participants who received prime and those who received prime-boost vaccine regimens. MV-CHIK vaccination elicited potent Fab-mediated antibody responses (such as CHIKV-specific IgG, neutralization, and avidity), including dominant IgG3 responses, which translated into strong antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. At 1 month, prime-boost immunization led to significantly greater responses in every measured Fab and Fc antibody parameter. Interestingly, prime-boost-elicited antibodies decreased rapidly over time, until at 6 months both vaccine regimens displayed similar antibody profiles. Nonetheless, antibody avidity and antibody-dependent cellular phagocytosis remained significantly greater following boost immunization. Our observations suggest that a prime-boost administration of MV-CHIK will be more appropriate for CHIKV-endemic regions, while a prime-only regimen may be sufficient for travel purposes or outbreak situations.

Vaccination against SARS-CoV-2 and disease enhancement - knowns and unknowns.

INTRODUCTION: The world is currently fighting a COVID-19 pandemic, perhaps the most disruptive infectious disease outbreak since the 1918 Spanish influenza. Governments have taken drastic measures to curb the spread of SARS-CoV-2, and the development of safe and efficacious vaccine candidates is being accelerated. The possibility of vaccine-mediated disease enhancement with coronavirus vaccines has been flagged as a potential safety concern, and, despite the urgent need, should be thoroughly assessed as vaccines against SARS-CoV-2 are being tested. AREA COVERED: We review the in vivo evidence suggesting a theoretical risk of disease enhancement after vaccination with SARS-CoV and MERS-CoV vaccine candidates. We also identify knowledge gaps that need to be filled to maximize the chance of developing a safe vaccine and minimize the risk of encountering disease enhancement in vaccinated individuals after exposure to SARS-CoV-2. EXPERT OPINION: We compile and propose avenues to investigate the risk of vaccine-mediated disease enhancement both during pre-clinical and early clinical development. While the pressing need for a vaccine against COVID-19 (and future epidemic coronaviruses) cannot be ignored, we advocate to keep safety at the center of the debate. Protecting individuals with effective and safe vaccines should be a priority, even during extraordinary times like the COVID-19 pandemic.

Disentangling etiologies of CNS infections in Singapore using multiple correspondence analysis and random forest.

Central nervous system (CNS) infections cause substantial morbidity and mortality worldwide, with mounting concern about new and emerging neurologic infections. Stratifying etiologies based on initial clinical and laboratory data would facilitate etiology-based treatment rather than relying on empirical treatment. Here, we report the epidemiology and clinical outcomes of patients with CNS infections from a prospective surveillance study that took place between 2013 and 2016 in Singapore. Using multiple correspondence analysis and random forest, we analyzed the link between clinical presentation, laboratory results, outcome and etiology. Of 199 patients, etiology was identified as infectious in 110 (55.3%, 95%-CI 48.3-62.0), immune-mediated in 10 (5.0%, 95%-CI 2.8-9.0), and unknown in 79 patients (39.7%, 95%-CI 33.2-46.6). The initial presenting clinical features were associated with the prognosis at 2 weeks, while laboratory-related parameters were related to the etiology of CNS disease. The parameters measured were helpful to stratify etiologies in broad categories, but were not able to discriminate completely between all the etiologies. Our results suggest that while prognosis of CNS is clearly related to the initial clinical presentation, pinpointing etiology remains challenging. Bio-computational methods which identify patterns in complex datasets may help to supplement CNS infection diagnostic and prognostic decisions.

A mouse-passaged dengue virus strain with reduced affinity for heparan sulfate causes severe disease in mice by establishing increased systemic viral loads.

The four serotypes of dengue virus (DENV1 to DENV4) cause extensive morbidity and mortality. A major obstacle to studying disease pathogenesis and developing therapies has been the lack of a small-animal model. We previously reported isolation of a DENV2 strain, obtained by passaging a clinical isolate between mosquito cells and mice, that caused severe DENV disease in mice and contained multiple mutations, including many in domain II of the envelope (E) protein. Here, we describe a recombinant virus, differing from the non-mouse-passaged virus by two mutations in the E protein, that induces vascular leakage and tumor necrosis factor alpha (TNF-alpha)-mediated lethality, while the non-mouse-passaged virus causes paralysis. This recombinant virus has a weaker affinity for heparan sulfate, resulting in an increased serum half-life, higher systemic viral loads, and high levels of TNF-alpha in the serum of infected mice. These results exemplify the role of the E protein in modulating virion clearance and connect the effect of clearance on the systemic viral loads responsible for severe disease manifestations.