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.

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.

Comparison of Japanese Encephalitis Force of Infection in Pigs, Poultry and Dogs in Cambodian Villages.

Japanese encephalitis virus (JEV) is the main cause of human viral encephalitis in Asia, with a mortality rate reaching 30%, mostly affecting children. The traditionally described cycle involving wild birds as reservoirs, pigs as amplifying hosts and Culex mosquitoes as vectors is questioned, with increasing evidence of a more complex multi-host system involved in areas where densities of pigs are low, such as in Cambodia. In 2018, we examined pigs, chickens, ducks and dogs from Kandal province, Cambodia, for antibody response against JEV by hemagglutination inhibition and virus neutralization assays. Forces of infection (FOI) for flaviviruses and JEV were estimated per species and per unit of body surface area (BSA). JEV seroprevalence reached 31% (95% CI: 23-41%) in pigs, 1% (95% CI: 0.1-3%) in chickens, 12% (95% CI: 7-19%) in ducks and 35% (95% CI: 28-42%) in dogs. Pigs were most likely to be infected (FOI: 0.09 per month), but the FOI was higher in ducks than in pigs for a given BSA (ratio of 0.13). Dogs had a lower FOI than ducks but a higher FOI than chickens (0.01 per month). For a given BSA, dogs were less likely to be infected than pigs (ratio of 1.9). In Cambodia, the virus may be circulating between multiple hosts. Dogs live in close contact with humans, and estimating their exposure to JEV infection could be a relevant indicator of the risk for humans to get infected, which is poorly known due to underdiagnosis. Understanding the JEV cycle and developing tools to quantify the exposure of humans is essential to adapt and support control measures for this vaccine-preventable disease.

Nipah virus circulation at human-bat interfaces, Cambodia.

OBJECTIVE: To better understand the potential risks of Nipah virus emergence in Cambodia by studying different components of the interface between humans and bats. METHODS: From 2012 to 2016, we conducted a study at two sites in Kandal and Battambang provinces where fruit bats (Pteropus lylei) roost. We combined research on: bat ecology (reproductive phenology, population dynamics and diet); human practices and perceptions (ethnographic research and a knowledge, attitude and practice study); and Nipah virus circulation in bat and human populations (virus monitoring in bat urine and anti-Nipah-virus antibody detection in human serum). FINDINGS: Our results confirmed circulation of Nipah virus in fruit bats (28 of 3930 urine samples positive by polymerase chain reaction testing). We identified clear potential routes for virus transmission to humans through local practices, including fruit consumed by bats and harvested by humans when Nipah virus is circulating, and palm juice production. Nevertheless, in the serological survey of 418 potentially exposed people, none of them were seropositive to Nipah virus. Differences in agricultural practices among the regions where Nipah virus has emerged may explain the situation in Cambodia and point to actions to limit the risks of virus transmission to humans. CONCLUSION: Human practices are key to understanding transmission risks associated with emerging infectious diseases. Social science disciplines such as anthropology need to be integrated in health programmes targeting emerging infectious diseases. As bats are hosts of major zoonotic pathogens, such integrated studies would likely also help to reduce the risk of emergence of other bat-borne diseases.

Massive Iatrogenic Outbreak of Human Immunodeficiency Virus Type 1 in Rural Cambodia, 2014-2015.

Background: In 2014-2015, 242 individuals aged 2-89 years were newly diagnosed with human immunodeficiency virus type 1 (HIV-1) in Roka, a rural commune in Cambodia. A case-control study attributed the outbreak to unsafe injections. We aimed to reconstruct the likely transmission history of the outbreak. Methods: We assessed in 209 (86.4%) HIV-infected cases the presence of hepatitis C virus (HCV) and hepatitis B virus (HBV). We identified recent infections using antibody (Ab) avidity testing for HIV and HCV. We performed amplification, sequencing, and evolutionary phylogenetic analyses of viral strains. Geographical coordinates and parenteral exposure through medical services provided by an unlicensed healthcare practitioner were obtained from 193 cases and 1499 controls during interviews. Results: Cases were coinfected with HCV (78.5%) and HBV (12.9%). We identified 79 (37.8%) recent (<130 days) HIV infections. Phylogeny of 202 HIV env C2V3 sequences showed a 198-sample CRF01_AE strains cluster, with time to most recent common ancestor (tMRCA) in September 2013 (95% highest posterior density, August 2012-July 2014), and a peak of 15 infections/day in September 2014. Three geospatial HIV hotspots were discernible in Roka and correlated with high exposure to the practitioner (P = .04). Fifty-nine of 153 (38.6%) tested cases showed recent (<180 days) HCV infections. Ninety HCV NS5B sequences formed 3 main clades, 1 containing 34 subtypes 1b with tMRCA in 2012, and 2 with 51 subtypes 6e and tMRCAs in 2002-2003. Conclusions: Unsafe injections in Cambodia most likely led to an explosive iatrogenic spreading of HIV, associated with a long-standing and more genetically diverse HCV propagation.

Low Circulation of Zika Virus, Cambodia, 2007-2016.

We describe a retrospective study on circulation of Zika virus in Cambodia during 2007-2016 among patients with dengue-like symptoms and Aedes aegypti mosquitoes. Our findings suggest that Zika virus in Cambodia belongs to the Asia genotype, is endemic, has low prevalence, and has had low-level impact on public health.

Risk factors for hepatitis C transmission in HIV patients, Hepacam study, ANRS 12267 Cambodia.

In 2009, we conducted a case-control study to explore the routes of HCV transmission in people living with HIV/AIDS (PLHIV) in Cambodia. Cases were HCV/HIV co-infected patients (who tested RT-PCR positive for HCV-RNA or had confirmed presence of HCV antibodies) (n = 44). Controls were HIV mono-infected patients, with no HCV antibodies (n = 160). They were recruited among the PLHIV presenting at one national reference centre of HIV/AIDS. Multivariate analysis showed that factors associated with the co-infection were the age older than 50 years (OR 5.4, 95 % confidence interval (CI) 1.5-19.6), the exposure to multiple parenteral infusions before the year 2000 (OR 3.4, 95 % CI 1.5-7.6), to surgery (OR 2.6, 95 % CI 1.2-5.7) and to fibroscopy (OR 2.4, 95 % CI 1.0-5.7). These results show the need to implement HCV screening in PLHIV, to support the implementation of national infection control guidelines, and to reinforce public awareness on the risks linked to parenteral medications.

Natural polymorphisms of HIV-1 CRF01_AE integrase coding region in ARV-naïve individuals in Cambodia, Thailand and Vietnam: an ANRS AC12 working group study.

The HIV integrase enzyme is essential for the HIV life cycle as it mediates integration of HIV-1 proviral DNA into the infected cell's genome. Recently, the development of drugs capable of inhibiting integrase has provided major new options for HIV-infected, treatment-experienced patients with multidrug resistant virus, as well treatment-naïve patients. More than 40 amino acid substitutions within integrase have been described as associated mostly with resistance of HIV B-subtypes to currently available integrase inhibitors (INIs). We have analyzed the natural polymorphisms of the integrase coding region in 87 antiretroviral-naïve subjects (32 from Cambodia, 37 from Thailand and 18 from Vietnam) infected with CRF01_AE virus, the predominant HIV-1 strain circulating in Southeast Asia. The 864bp integrase coding region was sequenced using the ANRS consensus sequencing technique from plasma samples, and amino acid results were interpreted for drug resistance according to the ANRS (Updated July 2009, version 18) and Stanford algorithms (Version November 6, 2009). Alignment of the 87 amino acid sequences against the 2004 Los Alamos HIV-1 clade B consensus sequence showed that overall, 119 of 288 (41.3%) amino acid positions presented at least one polymorphism each. Substitutions found in >60% of study subjects occurred at: K14, A21, V31, S39, I72, T112, T124, T125, G134, I135, K136, D167, V201, L234 and S283. Also, new amino acid substitutions of as yet unknown significance were identified: E152K/H, S153F/L, N155I and E157G. None of the known integrase resistance mutations were observed, except E157Q found in one Cambodian subject (1.1%, CI 95% 0.02-6.3%). The clinical impact of this substitution on resistance of B and nonB-viruses to the licensed INI raltegravir is unclear. If this substitution is confirmed to compromise the virologic response to raltegravir, further studies will be needed to better assess the prevalence of this substitution among CRF01_AE virus.

Effect of groundwater iron and phosphate on the efficacy of arsenic removal by iron-amended BioSand filters.

Naturally occurring arsenic in groundwater in Cambodia is a serious health concern. This study tested the efficacy of a BioSand filter amended with iron nails, Kanchan filter, as a household water treatment option with three natural arsenic-bearing groundwater sources of varying compositions and spiked with lab cultured E. coli and MS2. The effectiveness of arsenic and pathogen removal was not constant over time and was highly dependent on the influent composition. The filter was relatively ineffective in treating arsenic contaminated groundwater and effluent arsenic concentrations were between 74 and 2206 microg L(-1), which is higher than accepted drinkng water standards. The overall average arsenic removal was 39.4, 74.9, and 45.4%, respectively, and the extent of arsenic removal was not related to the influent arsenic concentration. The main reasons for poor arsenic removal was due to the combination of high influent P (> 0.5 mg L(-1)) and low Fe (< 5 mg L(-1)) concentrations and that the added iron nails were largely ineffective due to insufficient contact time withthe water. The findings suggest that such amended filters should not be widely deployed until improvements are made to address the consistency and efficacy of treatment In addition, the filter poses some potential health risk associated with the production of elevated nitrate levels in the effluent within the filter, possibly due to nitrification and high levels of ammonia in the groundwater.