Increased frequencies of highly activated regulatory T cells skewed to a T helper 1-like phenotype with reduced suppressive capacity in dengue patients.

The pathogenesis of dengue involves a complex interplay between the viral factor and the host immune response. A mismatch between the infecting serotype and the adaptive memory response is hypothesized to lead to exacerbated immune responses resulting in severe dengue. Here, we aim to define in detail the phenotype and function of different regulatory T cell (Treg) subsets and their association with disease severity in a cohort of acute dengue virus (DENV)-infected Cambodian children. Treg frequencies and proliferation of Tregs are increased in dengue patients compared to age-matched controls. Tregs from dengue patients are skewed to a Th1-type Treg phenotype. Interestingly, Tregs from severe dengue patients produce more interleukin-10 after in vitro stimulation compared to Tregs from classical dengue fever patients. Functionally, Tregs from dengue patients have reduced suppressive capacity, irrespective of disease severity. Taken together, these data suggest that even though Treg frequencies are increased in the blood of acute DENV-infected patients, Tregs fail to resolve inflammation and thereby could contribute to the immunopathology of dengue. IMPORTANCE: According to the World Health Organization, dengue is the fastest-spreading, epidemic-prone infectious disease. The extent of dengue virus infections increased over the years, mainly driven by globalization-including travel and trade-and environmental changes. Dengue is an immunopathology caused by an imbalanced immune response to a secondary heterotypic infection. As regulatory T cells (Tregs) are essential in maintaining immune homeostasis and dampening excessive immune activation, this study addressed the role of Tregs in dengue immunopathology. We show that Tregs from dengue patients are highly activated, skewed to a Th1-like Treg phenotype and less suppressive compared to healthy donor Tregs. Our data suggest that Tregs fail to resolve ongoing inflammation during dengue infection and hence contribute to the immunopathology of severe dengue disease. These data clarify the role of Tregs in dengue immunopathogenesis, emphasizing the need to develop T cell-based vaccines for dengue.

Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged <2 years old is efficient to stop rabies circulation in Cambodia.

Rabies control remains challenging in low and middle-income countries, mostly due to lack of financial resources, rapid turnover of dog populations and poor accessibility to dogs. Rabies is endemic in Cambodia, where no national rabies vaccination program is implemented. The objective of this study was to assess the short and long-term vaccination-induced immunity in Cambodian dogs under field conditions, and to propose optimized vaccination strategies. A cohort of 351 dogs was followed at regular time points following primary vaccination only (PV) or PV plus single booster (BV). Fluorescent antibody virus neutralization test (FAVNT) was implemented to determine the neutralizing antibody titer against rabies and an individual titer ≥0·5 IU/mL indicated protection. Bayesian modeling was used to evaluate the individual duration of protection against rabies and the efficacy of two different vaccination strategies. Overall, 61% of dogs had a protective immunity one year after PV. In dogs receiving a BV, this protective immunity remained for up to one year after the BV in 95% of dogs. According to the best Bayesian model, a PV conferred a protective immunity in 82% of dogs (95% CI: 75-91%) for a mean duration of 4.7 years, and BV induced a lifelong protective immunity. Annual PV of dogs less than one year old and systematic BV solely of dogs vaccinated the year before would allow to achieve the 70% World Health Organization recommended threshold to control rabies circulation in a dog population in three to five years of implementation depending on dog population dynamics. This vaccination strategy would save up to about a third of vaccine doses, reducing cost and time efforts of mass dog vaccination campaigns. These results can contribute to optimize rabies control measures in Cambodia moving towards the global goal of ending human death from dog-mediated rabies by 2030.

Temporal patterns of functional anti-dengue antibodies in dengue infected individuals with different disease outcome or infection history.

Heterotypic secondary dengue virus (DENV) infection is a risk factor for the development of severe disease. To assess the contribution of the developing polyclonal humoral immune response to the course of acute infection, we have determined anti-DENV IgG titers, neutralizing antibodies, percentages of antibodies binding to DENV-infected cells and antibody­dependent enhancement (ADE) to the infecting serotype in DENV-infected Cambodian children (n = 58), ranging from asymptomatic dengue to severe disease. The results showed that ADE titers are highest against the infecting serotype during heterotypic secondary DENV-2 infection. Moreover, IgG titers, neutralizing antibodies and ADE titers against the infecting serotype peak at D10 and are maintained until D60 after laboratory-confirmed secondary DENV infection. Anti-DENV IgG titers and the magnitude of the functional antibody response were higher in secondary DENV-infected patients compared to primary infected patients. No differences in antibody titers, neutralizing or enhancing antibodies could be observed between asymptomatic or hospitalized patients between 6 and 8 days after laboratory-confirmed DENV-1 infection. However, at this time point, the level of IgG bound to DENV-infected cells was associated with disease severity in hospitalized patients. Taken together, our data offer insights for more comprehensive interpretation of antibody response profile to natural infection and its correlation to disease outcome.

Robust and Functional Immune Memory Up to 9 Months After SARS-CoV-2 Infection: A Southeast Asian Longitudinal Cohort.

The duration of humoral and cellular immune memory following SARS-CoV-2 infection in populations in least developed countries remains understudied but is key to overcome the current SARS-CoV-2 pandemic. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for Spike (S)-binding and neutralizing antibodies and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-S antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immune memory was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased antibody-dependent cellular cytotoxicity (ADCC) and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immune memory. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection and in the absence of re-infection.

Robust and Functional Immunity up to 9 months after SARS-CoV-2 infection: a Southeast Asian longitudinal cohort

Assessing the duration of humoral and cellular immunity remains key to overcome the current SARS-CoV-2 pandemic, especially in understudied populations in least developed countries. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for humoral immune response to the viral spike protein and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-spike (S) antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immunity was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased ADCC and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immunity. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection in the absence of re-infection. One sentence summaryFunctional immune memory to SARS-CoV-2, consisting of polyfunctional antibodies, memory B cells and memory T cells are maintained up to nine months in a South-East Asian cohort in the absence of re-infection.

Large scale dog population demography, dog management and bite risk factors analysis: A crucial step towards rabies control in Cambodia.

Cambodia is a rabid-endemic country. However, data on dog population characteristics are lacking, and there is no national dog vaccination program. We implemented the first extensive door-to-door longitudinal survey in 2 Cambodian provinces, namely Kandal and Battambang, to estimate dog population demographic parameters, identify dog ownership determinants, analyze dog management practices and estimate the yearly cumulative bite incidence and associated factors. During the first session, more than 5000 dogs were recorded and identified. Data on families, dogs and cats characteristics, as well as the number of bites experienced the year before in the family, were recorded. One year later, a second session was performed in both provinces to record missing dogs and the reasons for missing. Age-specific survival rates of the dog populations were computed using Kaplan-Meier estimates. Ownership determinants and bite risk factors were identified using a negative binomial regression model. Dog trade and dog meat consumption were often reported. We estimated high dog-to-human ratios (1:3.8 in Kandal, and 1:3.3 in Battambang). The mean age of dog populations was 26.4 months in Kandal against 24.3 in Battambang, with a survival rate of 52% at 24 months in Kandal (34% only in Battambang). They were no feral dogs, but the large majority of recorded dogs were free roaming. In both provinces, the number of dogs significantly increased in families with children younger than 15, and when the head of the family was a male. The estimated yearly cumulative bite incidences were 2.3 and 3.1% in Kandal and Battambang provinces respectively, and are among the highest in the world. Our survey provides valuable data to focus information programs, parametrize transmission models and identify efficient vaccination strategies to control rabies in Cambodia in the future.

Impaired Antibody-Independent Immune Response of B Cells in Patients With Acute Dengue Infection.

Dengue is a mosquito-borne viral disease caused by dengue virus (DENV). The disease is endemic to more than 100 countries with 390 million dengue infections per year. Humoral immune responses during primary and secondary DENV infections are well-investigated. However, the impact of DENV infection on B cell subsets and their antibody-independent functions are not well-documented. Through this study, we aimed to define the distribution of B cell subsets in the acute phase of DENV infection and characterize the effect of DENV infection on B cell functions such as differentiation into memory and plasma cells and cytokine production. In our cohort of Cambodian children, we observed decreased percentages of CD24hiCD38hi B cells and CD27- naïve B cells within the CD19 population and increased percentages of CD27+CD38hiCD138+ plasma cells as early as 4 days post appearance of fever in patients with severe dengue compared to patients with mild disease. Lower percentages of CD19+CD24hiCD38hi B cells in DENV-infected patients were associated with decreased concentrations of soluble CD40L in patient plasma and decreased platelet counts in these patients. In addition, CD19+CD24hiCD38hi and CD19+CD27- B cells from DENV-infected patients did not produce IL-10 or TNF-α upon stimulation in vitro, suggesting their contribution to an altered immune response during DENV infection. In addition, CD19+CD27- naïve B cells isolated from dengue patients were refractory to TLR/anti-IgM stimulation in vitro, which correlated to the increased expression of inhibitory Fcγ receptors (FcγR) CD32 and LILRB1 on CD19+CD27- naïve B cells from DENV-infected patients. Collectively, our results indicate that a defective B cell response in dengue patients may contribute to the pathogenesis of dengue during the early phase of infection.

Asymptomatic Dengue Virus Infections, Cambodia, 2012-2013.

We investigated dengue virus (DENV) and asymptomatic DENV infections in rural villages of Kampong Cham Province, Cambodia, during 2012 and 2013. We conducted perifocal investigations in and around households for 149 DENV index cases identified through hospital and village surveillance. We tested participants 0.5-30 years of age by using nonstructural 1 rapid tests and confirmed DENV infections using quantitative reverse transcription PCR or nonstructural 1-capture ELISA. We used multivariable Poisson regressions to explore links between participants' DENV infection status and household characteristics. Of 7,960 study participants, 346 (4.4%) were infected with DENV, among whom 302 (87.3%) were <15 years of age and 225 (65.0%) were <9 years of age. We identified 26 (7.5%) participants with strictly asymptomatic DENV infection at diagnosis and during follow-up. We linked symptomatic DENV infection status to familial relationships with index cases. During the 2-year study, we saw fewer asymptomatic DENV infections than expected based on the literature.

A multi-country study of the economic burden of dengue fever based on patient-specific field surveys in Burkina Faso, Kenya, and Cambodia.

BACKGROUND: Dengue fever is a rapidly growing public health problem in many parts of the tropics and sub-tropics in the world. While there are existing studies on the economic burden of dengue fever in some of dengue-endemic countries, cost components are often not standardized, making cross-country comparisons challenging. Furthermore, no such studies have been available in Africa. METHODS/PRINCIPAL FINDINGS: A patient-specific survey questionnaire was developed and applied in Burkina Faso, Kenya, and Cambodia in a standardized format. Multiple interviews were carried out in order to capture the entire cost incurred during the period of dengue illness. Both private (patient's out-of-pocket) and public (non-private) expenditure were accessed to understand how the economic burden of dengue is distributed between private and non-private payers. A substantial number of dengue-confirmed patients were identified in all three countries: 414 in Burkina Faso, 149 in Kenya, and 254 in Cambodia. The average cost of illness for dengue fever was $26 (95% CI $23-$29) and $134 (95% CI $119-$152) per inpatient in Burkina Faso and Cambodia, respectively. In the case of outpatients, the average economic burden per episode was $13 (95% CI $23-$29) in Burkina Faso and $23 (95% CI $19-$28) in Kenya. Compared to Cambodia, public contributions were trivial in Burkina Faso and Kenya, reflecting that a majority of medical costs had to be directly borne by patients in the two countries. CONCLUSIONS/SIGNIFICANCE: The cost of illness for dengue fever is significant in the three countries. In particular, the current study sheds light on the potential economic burden of the disease in Burkina Faso and Kenya where existing evidence is sparse in the context of dengue fever, and underscores the need to achieve Universal Health Coverage. Given the availability of the current (CYD-TDV) and second-generation dengue vaccines in the near future, our study outcomes can be used to guide decision makers in setting health policy priorities.

Assessing the performance of remotely-sensed flooding indicators and their potential contribution to early warning for leptospirosis in Cambodia.

Remote sensing can contribute to early warning for diseases with environmental drivers, such as flooding for leptospirosis. In this study we assessed whether and which remotely-sensed flooding indicator could be used in Cambodia to study any disease for which flooding has already been identified as an important driver, using leptospirosis as a case study. The performance of six potential flooding indicators was assessed by ground truthing. The Modified Normalized Difference Water Index (MNDWI) was used to estimate the Risk Ratio (RR) of being infected by leptospirosis when exposed to floods it detected, in particular during the rainy season. Chi-square tests were also calculated. Another variable-the time elapsed since the first flooding of the year-was created using MNDWI values and was also included as explanatory variable in a generalized linear model (GLM) and in a boosted regression tree model (BRT) of leptospirosis infections, along with other explanatory variables. Interestingly, MNDWI thresholds for both detecting water and predicting the risk of leptospirosis seroconversion were independently evaluated at -0.3. Value of MNDWI greater than -0.3 was significantly related to leptospirosis infection (RR = 1.61 [1.10-1.52]; χ2 = 5.64, p-value = 0.02, especially during the rainy season (RR = 2.03 [1.25-3.28]; χ2 = 8.15, p-value = 0.004). Time since the first flooding of the year was a significant risk factor in our GLM model (p-value = 0.042). These results suggest that MNDWI may be useful as a risk indicator in an early warning remote sensing tool for flood-driven diseases like leptospirosis in South East Asia.

Seroprevalence and Transmission of Human Influenza A(H5N1) Virus before and after Virus Reassortment, Cambodia, 2006-2014.

Thirty-five human influenza A(H5N1) cases were reported in Cambodia during 2013-2014 after emergence of a clade 1.1.2 reassortant virus. We tested 881 villagers and found 2 cases of pauci- or asymptomatic infection. Seroprevalence after emergence of the reassortant strain (0.2%) was lower than the aggregate seroprevalence of 1.3% reported in earlier studies.

Long-Lasting Immune Protection and Other Epidemiological Findings after Chikungunya Emergence in a Cambodian Rural Community, April 2012.

The East/Central/South African genotype of Chikungunya virus with the E1-A226V mutation emerged in 2011 in Cambodia and spread in 2012. An outbreak of 190 cases was documented in Trapeang Roka, a rural village. We surveyed 425 village residents within 3-4 weeks after the outbreak, and determined the sensitivity and specificity of case definitions and factors associated with infection by CHIKV. Self-reported clinical presentation consisted mostly of fever, rash and arthralgia. The presence of all three clinical signs or symptoms was identified as the most sensitive (67%) and specific (84%) self-reported diagnostic clinical indicator compared to biological confirmation by MAC-ELISA or RT-PCR used as a reference. Having an indoor occupation was associated with lower odds of infection compared with people who remained at home (adjOR 0.32, 95%CI 0.12-0.82). In contrast with findings from outbreaks in other settings, persons aged above 40 years were less at risk of CHIKV infection, likely reflecting immune protection acquired when Chikungunya circulated in Cambodia before the Khmer Rouge regime in 1975. In view of the very particular history of Cambodia, our epidemiological data from Trapeang Roka are the first to support the persistence of CHIKV antibodies over a period of 40 years.