Side-by-side Comparative Study of the Immunogenicity of the Intramuscular and Intradermal Rabies Post-exposure Prophylaxis Regimens in a Cohort of Suspected Rabies Virus Exposed Individuals.

All World Health Organization (WHO) pre-qualified rabies vaccines for humans are inactivated tissue culture rabies virus formulations produced for intramuscular (IM) administration. Due to costs and vaccine shortage, dose-saving intradermal (ID) administration of rabies post-exposure prophylaxis (PEP) is encouraged by WHO. This study compared the immunogenicity of the ID 2-site, 3-visit Institut Pasteur Cambodge (IPC) PEP regimen to the IM 1-site, 4-visit 4-dose Essen regimen using Verorab vaccine (Sanofi). The development of neutralizing antibodies (nAbs) and T cell response was assessed in 210 patients with a category II or III animal exposure in a rabies-endemic country. At day 28, all participants developed nAbs (≥0.5 IU/mL), irrespective of PEP scheme, age, or administration of rabies immunoglobulin. T cell response and nAb titers were similar for both PEP schemes. This study demonstrated that the 1-week ID IPC regimen is as effective as the 2-week IM 4-dose Essen regimen in inducing an anti-rabies immune response under real-life PEP.

Uncovering the endemic circulation of rabies in Cambodia.

In epidemiology, endemicity characterizes sustained pathogen circulation in a geographical area, which involves a circulation that is not being maintained by external introductions. Because it could potentially shape the design of public health interventions, there is an interest in fully uncovering the endemic pattern of a disease. Here, we use a phylogeographic approach to investigate the endemic signature of rabies virus (RABV) circulation in Cambodia. Cambodia is located in one of the most affected regions by rabies in the world, but RABV circulation between and within Southeast Asian countries remains understudied. Our analyses are based on a new comprehensive data set of 199 RABV genomes collected between 2014 and 2017 as well as previously published Southeast Asian RABV sequences. We show that most Cambodian sequences belong to a distinct clade that has been circulating almost exclusively in Cambodia. Our results thus point towards rabies circulation in Cambodia that does not rely on external introductions. We further characterize within-Cambodia RABV circulation by estimating lineage dispersal metrics that appear to be similar to other settings, and by performing landscape phylogeographic analyses to investigate environmental factors impacting the dispersal dynamic of viral lineages. The latter analyses do not lead to the identification of environmental variables that would be associated with the heterogeneity of viral lineage dispersal velocities, which calls for a better understanding of local dog ecology and further investigations of the potential drivers of RABV spread in the region. Overall, our study illustrates how phylogeographic investigations can be performed to assess and characterize viral endemicity in a context of relatively limited data.

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.

Predicting Dengue Outbreaks in Cambodia.

In Cambodia, dengue outbreaks occur each rainy season (May-October) but vary in magnitude. Using national surveillance data, we designed a tool that can predict 90% of the variance in peak magnitude by April, when typically <10% of dengue cases have been reported. This prediction may help hospitals anticipate excess patients.

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 Blood RNA Signature Detecting Severe Disease in Young Dengue Patients at Hospital Arrival.

Background: Early detection of severe dengue can improve patient care and survival. To date, no reliable single-gene biomarker exists. We hypothesized that robust multigene signatures exist. Methods: We performed a prospective study on Cambodian dengue patients aged 4 to 22 years. Peripheral blood mononuclear cells (PBMCs) were obtained at hospital admission. We analyzed 42 transcriptomic profiles of patients with secondary dengue infected with dengue serotype 1. Our novel signature discovery approach controls the number of included genes and captures nonlinear relationships between transcript concentrations and severity. We evaluated the signature on secondary cases infected with different serotypes using 2 datasets: 22 PBMC samples from additional patients in our cohort and 32 whole blood samples from an independent cohort. Results: We identified an 18-gene signature for detecting severe dengue in patients with secondary infection upon hospital admission with a sensitivity of 0.93 (95% confidence interval [CI], .82-.98), specificity of 0.67 (95% CI, .53-.80), and area under the receiver operating characteristic curve (AUC) of 0.86 (95% CI, .75-.97). At validation, the signature had empirical AUCs of 0.85 (95% CI, .69-1.00) and 0.83 (95% CI, .68-.98) for the PBMCs and whole blood datasets, respectively. Conclusions: The signature could detect severe dengue in secondary-infected patients upon hospital admission. Its genes offer new insights into the pathogenesis of severe dengue.

Co-circulation of Influenza A H5, H7, and H9 Viruses and Co-infected Poultry in Live Bird Markets, Cambodia.

Longitudinal surveillance of 2 live bird markets in Cambodia revealed year-round, high co-circulation of H5, H7, and H9 influenza viruses. We detected influenza A viruses in 51.3% of ducks and 39.6% of chickens, and co-infections, mainly by H5 and H9 viruses, in 0.8% of ducks and 4.5% of chickens.

Rabies Postexposure Prophylaxis Noncompletion After Dog Bites: Estimating the Unseen to Meet the Needs of the Underserved.

Postexposure prophylaxis (PEP) prevents human rabies and is accessible in Cambodia principally in Phnom Penh, the capital. Timely, affordable access to PEP is a challenge for the mainly rural population. We aimed to identify districts independently associated with PEP noncompletion to position frontline vaccination centers. We analyzed the 2009-2013 database at the Rabies Prevention Center at the Institut Pasteur du Cambodge, Phnom Penh. Logistic regressions identified nongeographic determinants of PEP noncompletion as well as the districts that were independently associated with noncompletion after adjustment for these determinants. The influence of distance by road was estimated using a boosted regression-trees model. We computed a population attributable fraction (rabies index (RI)) for each district and developed a map of this RI distribution. A cartographic analysis based on the statistic developed by Getis and Ord identified clusters of high-RI districts. Factors independently associated with noncompletion were patients' district of residence, male sex, age 15-49 years, initial visit during rice harvest, the dog's status (culled or disappeared), and a prescribed PEP protocol requiring more than 3 PEP sessions (4 or 5). Four clusters of high-RI districts were identified using this analytical strategy, which is applicable to many vaccination or other health services. Positioning frontline PEP centers in these districts could significantly widen access to timely and adequate PEP.

Asymptomatic humans transmit dengue virus to mosquitoes.

Three-quarters of the estimated 390 million dengue virus (DENV) infections each year are clinically inapparent. People with inapparent dengue virus infections are generally considered dead-end hosts for transmission because they do not reach sufficiently high viremia levels to infect mosquitoes. Here, we show that, despite their lower average level of viremia, asymptomatic people can be infectious to mosquitoes. Moreover, at a given level of viremia, DENV-infected people with no detectable symptoms or before the onset of symptoms are significantly more infectious to mosquitoes than people with symptomatic infections. Because DENV viremic people without clinical symptoms may be exposed to more mosquitoes through their undisrupted daily routines than sick people and represent the bulk of DENV infections, our data indicate that they have the potential to contribute significantly more to virus transmission to mosquitoes than previously recognized.

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.

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.

Caring for patients with rabies in developing countries - the neglected importance of palliative care.

Although limited publications address clinical management of symptomatic patients with rabies in intensive care units, the overwhelming majority of human rabies cases occur in the rural setting of developing countries where healthcare workers are few, lack training and drugs. Based on our experience, we suggest how clinicians in resource-limited settings can make best use of essential drugs to provide assistance to patients with rabies and their families, at no risk to themselves. Comprehensive and compassionate patient management of furious rabies should aim to alleviate thirst, anxiety and epileptic fits using infusions, diazepam or midazolam and antipyretic drugs via intravenous or intrarectal routes. Although the patient is dying, respiratory failure must be avoided especially if the family, after being informed, wish to take the patient home alive for funereal rites to be observed. Healthcare staff should be trained and clinical guidelines should be updated to include palliative care for rabies in endemic countries.

Environmental contamination and risk factors for transmission of highly pathogenic avian influenza A(H5N1) to humans, Cambodia, 2006-2010.

BACKGROUND: Highly pathogenic avian influenza A (H5N1) virus has been of public health concern since 2003. Probable risk factors for A(H5N1) transmission to human have been demonstrated in several studies or epidemiological reports. However, transmission patterns may differ according to demographic characteristics of the population and local practices. This article aggregates these data from three studies with data collected in the previous surveys in 2006 and 2007 to further examine the risks factors associated with presence of anti-A(H5) antibodies among villagers residing within outbreak areas. METHODS: We aggregated 5-year data (2006-2010) from serology survey and matched case-control studies in Cambodia to further examine the risks factors associated with A(H5N1) infection among villagers in the outbreak areas. RESULTS: Serotesting among villagers detected 35 (1.5 % [0-2.6]) positive cases suggesting recent exposure to A(H5N1) virus. Practices associated with A(H5N1) infection among all ages were: having poultry cage or nesting area under or adjacent to the house (OR: 6.7 [1.6-28.3]; p = 0.010) and transporting poultry to market (OR: 17.6 [1.6-193.7]; p = 0.019). Practices found as risk factors for the infection among age under 20 years were swimming/bathing in ponds also accessed by domestic poultry (OR: 4.6 [1.1-19.1]; p = 0.038). Association with consuming wild birds reached borderline significance (p = 0.066). CONCLUSION: Our results suggest that swimming/bathing in contaminated pond water and close contact with poultry may present a risk of A(H5N1) transmission to human.

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.

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.

Acute lower respiratory infections in ≥ 5 year -old hospitalized patients in Cambodia, a low-income tropical country: clinical characteristics and pathogenic etiology.

BACKGROUND: Few data exist on viral and bacterial etiology of acute lower respiratory infections (ALRI) in ≥ 5 year -old persons in the tropics. METHODS: We conducted active surveillance of community-acquired ALRI in two hospitals in Cambodia, a low-income tropical country. Patients were tested for acid-fast bacilli (AFB) by direct sputum examination, other bacteria by blood and/or sputum cultures, and respiratory viruses using molecular techniques on nasopharyngeal/throat swabs. Pulmonologists reviewed clinical/laboratory data and interpreted chest X-rays (CXR) to confirm ALRI. RESULTS: Between April 2007 - December 2009, 1,904 patients aged ≥5 years were admitted with acute pneumonia (50.4%), lung sequelae-associated ALRI (24.3%), isolated pleural effusions (8.9%) or normal CXR-related ALRI (17.1%); 61 (3.2%) died during hospitalization. The two former diagnoses were predominantly due to bacterial etiologies while viral detection was more frequent in the two latter diagnoses. AFB-positive accounted for 25.6% of acute pneumonia. Of the positive cultures (16.8%), abscess-prone Gram-negative bacteria (39.6%) and Haemophilus influenzae (38.0%) were most frequent, followed by Streptococcus pneumoniae (17.7%). Of the identified viruses, the three most common viruses included rhinoviruses (49.5%), respiratory syncytial virus (17.7%) and influenza viruses (12.1%) regardless of the diagnostic groups. Wheezing was associated with viral identification (31.9% vs. 13.8%, p < 0.001) independent of age and time-to-admission. CONCLUSIONS: High frequency of H. influenzae and S. pneumoniae infections support the need for introduction of the respective vaccines in the national immunization program. Tuberculosis was frequent in patients with acute pneumonia, requiring further investigation. The relationship between respiratory viruses and wheezing merits further studies.

Characterization of soluble TLR2 and CD14 levels during acute dengue virus infection.

Dengue virus infection results in a broad spectrum of diseases ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Hitherto, there is no consensus biomarker for the prediction of severe dengue disease in patients. Yet, early identification of patients who progress to severe dengue is pivotal for better clinical management. We have recently reported that an increased frequency of classical (CD14 ++CD16-) monocytes with sustained high TLR2 expression in acutely infected dengue patients correlates with severe dengue development. Here, we hypothesized that the relatively lower TLR2 and CD14 expression in mild dengue patients is due to the shedding of their soluble forms (sTLR2 and sCD14) and that these could be used as indicators of disease progression. Therefore, using commercial sandwich ELISAs, we evaluated the release of sTLR2 and sCD14 by peripheral blood mononuclear cells (PBMCs) in response to in vitro dengue virus (DENV) infection and assessed their levels in acute-phase plasma of 109 dengue patients. We show that while both sTLR2 and sCD14 are released by PBMCs in response to DENV infection in vitro, their co-circulation in an acute phase of the disease is not always apparent. In fact, sTLR2 was found only in 20% of patients irrespective of disease status. In contrast, sCD14 levels were detected in all patients and were significantly elevated in DF patients when compared to DHF patients and age-matched healthy donors. Altogether, our results suggest that sCD14 may help in identifying patients at risk of severe dengue at hospital admittance.

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia.

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1 week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats.

Genomic epidemiology of SARS-CoV-2 in Cambodia, January 2020 to February 2021.

The first case of coronavirus disease 2019 (COVID-19) in Cambodia was confirmed on 27 January 2020 in a traveller from Wuhan. Cambodia subsequently implemented strict travel restrictions, and although intermittent cases were reported during the first year of the COVID-19 pandemic, no apparent widespread community transmission was detected. Investigating the routes of severe acute respiratory coronavirus 2 (SARS-CoV-2) introduction into the country was critical for evaluating the implementation of public health interventions and assessing the effectiveness of social control measures. Genomic sequencing technologies have enabled rapid detection and monitoring of emerging variants of SARS-CoV-2. Here, we detected 478 confirmed COVID-19 cases in Cambodia between 27 January 2020 and 14 February 2021, 81.3 per cent in imported cases. Among them, fifty-four SARS-CoV-2 genomes were sequenced and analysed along with representative global lineages. Despite the low number of confirmed cases, we found a high diversity of Cambodian viruses that belonged to at least seventeen distinct PANGO lineages. Phylogenetic inference of SARS-CoV-2 revealed that the genetic diversity of Cambodian viruses resulted from multiple independent introductions from diverse regions, predominantly, Eastern Asia, Europe, and Southeast Asia. Most cases were quickly isolated, limiting community spread, although there was an A.23.1 variant cluster in Phnom Penh in November 2020 that resulted in a small-scale local transmission. The overall low incidence of COVID-19 infections suggests that Cambodia's early containment strategies, including travel restrictions, aggressive testing and strict quarantine measures, were effective in preventing large community outbreaks of COVID-19.

Household clustering supports a novel chemoprophylaxis trial design for a mosquito-borne viral disease.

Infections because of chikungunya and other mosquito-borne viruses, such as dengue and Zika, represent an area of significant unmet medical need. There are currently no approved medicines for prophylaxis or treatment of these diseases, and the development and implementation of vaccines against these viruses have proved problematic. Although antiviral molecules with treatment and prophylactic potential against the chikungunya virus have been identified, no successful field trials have been reported. Chemoprophylaxis may be attractive for unvaccinated at-risk populations; however, performing a successful chemoprophylaxis trial during a chikungunya outbreak will require a clearly identifiable at-risk population. We propose the application of a household transmission model as used in testing drugs against respiratory viruses. Current evidence on household clustering of chikungunya and other Aedes mosquito-borne viral infections is supportive. We suggest that this model may improve prophylaxis trial feasibility and focus research and future treatment on a population likely to benefit.