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Psychopathology is defined in part by its impacts on life functioning (e.g., fulfillment of daily responsibilities at work or school, in family relationships). Relations to life functioning are particularly important in the validation of culture-specific syndromes (patterns of mental health symptoms specific to a particular culture), to demonstrate that culture-specific symptom patterns do in fact represent pathology. The current study's goal was to assess the construct validity of the Cambodian Somatic Symptom and Syndrome Inventory (CSSI). The study focused on the statistically unique effects of the CSSI on life functioning (i.e., effects of the CSSI on life functioning, controlling for Western psychopathology syndromes), to determine whether the CSSI contributes information beyond standard Western measures, which would support CSSI culture-specific convergent validity. Because adolescence is a key period when psychopathology often develops, study participants were 391 high-school students in one urban and one rural area of Cambodia. Participants completed the CSSI, the Western psychopathology surveys Patient Health Questionnaire (PHQ-9) and Generalized Anxiety Disorder questionnaire (GAD-7), and life functioning measures assessing functional impairment, quality of life, and help-seeking. Results indicated strong CSSI concurrent validity (canonical correlation = 0.75) with the PHQ-9 and GAD-7, and CSSI total effects convergent validity on life functioning indicators. All CSSI statistically unique effects (controlling for the PHQ-9 and GAD-7) on life functioning measures were non-significant indicating that the CSSI, shown to be a valid assessment measure in the current study, does not add predictive information beyond standard Western measures. A key limitation that should be considered in interpretation of these results is that the life functioning measures, although reviewed by Khmer psychologists, were Western-based, thus potentially inflating relations with Western psychopathology measures.

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., (© 2022 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Background: There is growing recognition of the psychosocial burden of caregiving on relative caregivers of the mentally ill in low-and middle-income countries. Yet there remains limited formal research examining the challenges and psychosocial support needs of these relative caregivers, particularly with sensitivity to understanding caregiver experiences across diverse cultures and contexts. The purpose of this study was to study caregiver burden to inform potential intervention approaches among relative caregivers of treatment-engaged mentally ill patients in Cambodia., Methods: Participants were all relative caregivers identified through their connection to a non-governmental organization providing psychiatric care. Five focus group discussions were held with 37 participants to explore challenges experienced by relative caregivers. A total of 115 caregivers then completed a mental health assessment consisting of the Patient Health Questionnaire - 9 item scale, Generalized Anxiety Disorder - 7-item scale, and 21-item Depression, Anxiety, and Stress Scale. Internal consistencies ranged from α = .81 to .87 across scales., Results: Five themes emerged from the focus group discussions: financial burden, erratic behavior of patients, social alienation, somatic and emotional symptoms, and barriers to help-seeking. Assessment data indicated the sample was highly symptomatic. Women and those with no employment appeared to be at higher risk of distress, as were those caregivers with a biological relationship to the patient (parents, children, and siblings; relative to spouses or other relationships)., Discussion: Findings suggest a need for regular screening and greater psychosocial support for relative caregivers. It is notable that the needs identified within the current study remain prominent even as this is a treatment-engaged sample.

Rates of mental health disorders in Cambodia are markedly higher than in other low- or middle-income countries. Despite these high rates, mental healthcare resources remain scarce and mental health stigma is pervasive, particularly for vulnerable populations of young women and individuals of low socioeconomic status. To address this gap, teaching Western mental health treatments and using a mental healthcare framework are recommended within the Cambodian context. However, Western frameworks do not address cultural syndromes or idioms of distress and operate from an individualistic perspective that does not address cultural values and beliefs. The present study employs a mental health literacy framework in an exploratory analysis of rates of psychological knowledge in a nationally representative sample of Cambodian adults (N = 2,690). To address recommendations for increasing mental healthcare, we designed a survey to investigate Cambodians' knowledge about mental health constructs. Results indicated that only 18.9% of Cambodians knew about psychology, and chi-square analyses revealed that women, individuals in rural areas, and individuals with significant distress due to cultural symptoms and syndromes reported knowing about psychology significantly less than their male and non-distressed counterparts. Additionally, those who reported higher income and higher levels of education indicated significantly higher rates of psychological knowledge, as did those with clinically significant rates of PTSD, at a rate of knowledge approaching significance. Implications for this study include the need to tailor interventions and resources to vulnerable populations, to assess the fit of current recommendations for the Cambodian context, and to further emphasize the need for culturally responsive interventions that address all presentations of Cambodian distress and align with understandings of mental health within the nation.

Influenza A/H5N1 has circulated in Asia since 2003 and is now enzootic in many countries in that region. In Cambodia, the virus has circulated since 2004 and has intermittently infected humans. During this period, we have noted differences in the rate of infections in humans, potentially associated with the circulation of different viral clades. In particular, a reassortant clade 1.1.2 virus emerged in early 2013 and was associated with a dramatic increase in infections of humans (34 cases) until it was replaced by a clade 2.3.2.1c virus in early 2014. In contrast, only one infection of a human has been reported in the 6 years since the clade 2.3.2.1c virus became the dominant circulating virus. We selected three viruses to represent the main viral clades that have circulated in Cambodia (clade 1.1.2, clade 1.1.2 reassortant, and clade 2.3.2.1c), and we conducted experiments to assess the virulence and transmissibility of these viruses in avian (chicken, duck) and mammalian (ferret) models. Our results suggest that the clade 2.3.2.1c virus is more "avian-like," with high virulence in both ducks and chickens, but there is no evidence of aerosol transmission of the virus from ducks to ferrets. In contrast, the two clade 1 viruses were less virulent in experimentally infected and contact ducks. However, evidence of chicken-to-ferret aerosol transmission was observed for both clade 1 viruses. The transmission experiments provide insights into clade-level differences that might explain the variation in A/H5N1 infections of humans observed in Cambodia and other settings.

Avian influenza viruses (AIVs) periodically cross species barriers and infect humans. The likelihood that an AIV will evolve mammalian transmissibility depends on acquiring and selecting mutations during spillover, but data from natural infection is limited. We analyze deep sequencing data from infected humans and domestic ducks in Cambodia to examine how H5N1 viruses evolve during spillover. Overall, viral populations in both species are predominated by low-frequency (<10%) variation shaped by purifying selection and genetic drift, and half of the variants detected within-host are never detected on the H5N1 virus phylogeny. However, we do detect a subset of mutations linked to human receptor binding and replication (PB2 E627K, HA A150V, and HA Q238L) that arose in multiple, independent humans. PB2 E627K and HA A150V were also enriched along phylogenetic branches leading to human infections, suggesting that they are likely human-adaptive. Our data show that H5N1 viruses generate putative human-adapting mutations during natural spillover infection, many of which are detected at >5% frequency within-host. However, short infection times, genetic drift, and purifying selection likely restrict their ability to evolve extensively during a single infection. Applying evolutionary methods to sequence data, we reveal a detailed view of H5N1 virus adaptive potential, and develop a foundation for studying host-adaptation in other zoonotic viruses., Competing Interests: Dr. Philippe Buchy is a former Head of Virology at Institut Pasteur du Cambodge and is currently an employee of GSK Vaccines, Singapore. The other authors declare no conflict of interest.

Background: Influenza virus circulation is monitored through the Cambodian influenza-like illness (ILI) sentinel surveillance system and isolates are characterized by the National Influenza Centre (NIC). Seasonal influenza circulation has previously been characterized by year-round activity and a peak during the rainy season (June-November)., Objectives: We documented the circulation of seasonal influenza in Cambodia for 2012-2015 and investigated genetic, antigenic, and antiviral resistance characteristics of influenza isolates., Patients/methods: Respiratory samples were collected from patients presenting with influenza-like illness (ILI) at 11 hospitals throughout Cambodia. First-line screening was conducted by the National Institute of Public Health and the Armed Forces Research Institute of Medical Sciences. Confirmation of testing and genetic, antigenic and antiviral resistance characterization was conducted by Institute Pasteur in Cambodia, the NIC. Additional virus characterization was conducted by the WHO Collaborating Centre for Reference and Research on Influenza (Melbourne, Australia)., Results: Between 2012 and 2015, 1,238 influenza-positive samples were submitted to the NIC. Influenza A(H3N2) (55.3%) was the dominant subtype, followed by influenza B (30.9%; predominantly B/Yamagata-lineage) and A(H1N1)pdm09 (13.9%). Circulation of influenza viruses began earlier in 2014 and 2015 than previously described, coincident with the emergence of A(H3N2) clades 3C.2a and 3C.3a, respectively. There was high diversity in the antigenicity of A(H3N2) viruses, and to a smaller extent influenza B viruses, during this period, with some mismatches with the northern and southern hemisphere vaccine formulations. All isolates tested were susceptible to the influenza antiviral drugs oseltamivir and zanamivir., Conclusions: Seasonal and year-round co-circulation of multiple influenza types/subtypes were detected in Cambodia during 2012-2015., (© 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

In a large national survey in Cambodia (N = 2689), the present study investigated the prominence of certain culturally salient symptoms and syndromes in the general population and among those with anxious-depressive distress (as determined by the Hopkins Symptom Checklist-25, or HSCL). Using an abbreviated Cambodian Symptom and Syndrome Addendum (CSSA), we found that the CSSA complaints were particularly elevated among those with anxious-depressive distress. Those with anxious-depressive distress had statistically greater mean scores on all the CSSA items as well as severity of endorsement analyzed by percentage: among those with HSCL caseness, 75.3% were bothered "quite a bit" or "extremely" by "thinking a lot" (vs. 27.5% without caseness); 53.8% were bothered by "standing up and feeling dizzy" (vs. 13.8%); and 45.6% by blurry vision (vs. 16.8%). In a logistic regression analysis to predict anxious-depressive distress, 51% of the variance was accounted for by five predictors: "weak heart," "thinking a lot," dizziness, " khyâl hitting up from the stomach," and sleep paralysis. Using ROC analysis, a cut-off score of 1.81 on the CSSA was optimal as a screener to indicate anxious-depressive distress, giving a sensitivity of 0.86. The study results suggest that to avoid category truncation (i.e., the omission of key complaints that are part of an assessed distress domain) when profiling anxious-depressive distress among Cambodia population that items other than those in standard psychopathology measures should be assessed such as "thinking a lot," "weak heart," "blurry vision," and "dizziness upon standing up."

Background: Reliable population-based epidemiological data of Cambodian mental health is lacking., Aims: This study identifies the prevalence of and factors associated with anxiety, depression and PTSD in Cambodian adults., Methods: A nationally representative sample of Cambodian adults (N = 2690) was interviewed utilizing the Harvard Trauma Questionnaire (assessment of PTSD symptoms) and the Hopkins Symptom Checklist-25 (assessment of symptoms of anxiety and depression)., Results: Respondents expressed high rates of anxiety (27.4%), depression (16.7%) and PTSD (7.6%). Correlations between symptoms and sociodemographic markers varied in significance. Women had significantly higher rates of mental health symptoms than men. Women who were in debt, widowed or divorced and had low levels of education were the most likely to report symptoms., Conclusions: These findings can inform Cambodian mental health policies and development strategies, especially targeting the most vulnerable groups.

Background: Our recent report demonstrates that 5.5% of Cambodian women have previously attempted suicide. Despite these high rates and critical need for intervention, research on suicide attempts in Cambodia is lacking, and life-saving information on suicide prevention is therefore unknown., Aims: This study explores factors impacting Cambodian women suicide attempts., Method: A total of 1,801 women participated in the large national survey during which 100 of these women (5.5%) reported at least one suicide attempt. Of the 100 participants 76 provided qualitative reasons for the suicide attempts. Only the 76 who provided the reasons for suicide attempt were included for analysis in this study., Results: Four major themes emerged: (1) family conflict, (2) emotional distress, (3) poverty, and (4) illness. Family conflict, emotional distress, poverty, and illness were all interrelated with each other; for example, women without money were unable to pay for treatment of otherwise treatable illnesses., Limitations: Owing to the nature of the data collection, member checking could not be conducted., Conclusion: Cumulative and intertwined personal, interpersonal, and contextual-level factors impacting suicide attempts included emotional distress, illness, family conflict, and poverty. Findings highlight points of intervention at individual, familial, and contextual levels to prevent suicide.

Trauma literature has proposed multiple theories of trauma development, maintenance, and transmission, which has led to a lack of clarity surrounding trauma in individuals, families, and communities. We investigated the impact of community-level trauma experiences on individual posttraumatic stress disorder (PTSD) symptoms using a sociointerpersonal model of PTSD (Maerker & Horn, 2013). A nationally representative sample (N = 2, 690) of Cambodian households across all regions of the country was surveyed regarding individual trauma experiences during and after the Khmer Rouge regime, symptoms of PTSD, and current stressors. Individual experiences of war trauma and current stressors were aggregated based on the district in which each individual lived. District mean and individual war trauma and current stressors were included in a multilevel model as predictors of individual levels of PTSD. Findings indicated that mean trauma experiences, β = .05, p < .001, and current stressors, β = .10, p < .001, in the district in which individuals live were positively and significantly associated with their individual PTSD symptoms. Individual war trauma, β = .02, p < .001, and current stressors, β = .08, p < .001, were also positively and significantly associated with individual PTSD symptoms. District trauma experiences accounted for 7% of the variance in individual PTSD symptoms, R 2 Level 1 = .21, R 2 Level 2 = .80. Additionally, current stressors at both the individual and district levels had a greater impact on individual PTSD symptoms than war trauma at either level of the model. Implications for policy and intervention are presented., (© 2019 International Society for Traumatic Stress Studies.)

Background: Our recent report demonstrates that 5.5% of Cambodian women have previously attempted suicide. Despite these high rates and critical need for intervention, research on suicide attempts in Cambodia is lacking, and life-saving information on suicide prevention is therefore unknown., Aims: This study explores factors impacting Cambodian women suicide attempts., Method: A total of 1,801 women participated in the large national survey during which 100 of these women (5.5%) reported at least one suicide attempt. Of the 100 participants 76 provided qualitative reasons for the suicide attempts. Only the 76 who provided the reasons for suicide attempt were included for analysis in this study., Results: Four major themes emerged: (1) family conflict, (2) emotional distress, (3) poverty, and (4) illness. Family conflict, emotional distress, poverty, and illness were all interrelated with each other; for example, women without money were unable to pay for treatment of otherwise treatable illnesses., Limitations: Owing to the nature of the data collection, member checking could not be conducted., Conclusion: Cumulative and intertwined personal, interpersonal, and contextual-level factors impacting suicide attempts included emotional distress, illness, family conflict, and poverty. Findings highlight points of intervention at individual, familial, and contextual levels to prevent suicide.

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.

Global suicide rates are steadily increasing, and suicide completions in Asia outnumber those in Western countries. Young females are especially at risk, with higher rates of completion and lack of suicide support because of familial and cultural stigma and constraints. Lack of infrastructure to systematically record suicide deaths and attempts makes studying suicide in low- and middle-income countries challenging. Given the critical public health need for suicide intervention and prevention, research on suicide is crucial. The present study adds to the lack of information regarding suicide in Cambodia by exploring reports of attempted suicide by women from a nationally representative sample of Cambodian women (N = 1813). In a series of logistic regression models, findings indicate that a culturally salient measure of Cambodian syndromes, symptoms of depression, and posttraumatic stress disorder contributed to increased odds of attempting to commit suicide. Implications for policymakers and interventionists within Cambodia and Asian contexts are discussed.

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.

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.

Surveillance for avian influenza viruses (AIVs) in poultry and environmental samples was conducted in four live-bird markets in Cambodia from January through November 2013. Through real-time RT-PCR testing, AIVs were detected in 45% of 1048 samples collected throughout the year. Detection rates ranged from 32% and 18% in duck and chicken swabs, respectively, to 75% in carcass wash water samples. Influenza A/H5N1 virus was detected in 79% of samples positive for influenza A virus and 35% of all samples collected. Sequence analysis of full-length haemagglutinin (HA) and neuraminidase (NA) genes from A/H5N1 viruses, and full-genome analysis of six representative isolates, revealed that the clade 1.1.2 reassortant virus associated with Cambodian human cases during 2013 was the only A/H5N1 virus detected during the year. However, multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HA and NA genes revealed co-circulation of at least nine low pathogenic AIVs from HA1, HA2, HA3, HA4, HA6, HA7, HA9, HA10 and HA11 subtypes. Four repeated serological surveys were conducted throughout the year in a cohort of 125 poultry workers. Serological testing found an overall prevalence of 4.5% and 1.8% for antibodies to A/H5N1 and A/H9N2, respectively. Seroconversion rates of 3.7 and 0.9 cases per 1000 person-months participation were detected for A/H5N1 and A/H9N2, respectively. Peak AIV circulation was associated with the Lunar New Year festival. Knowledge of periods of increased circulation of avian influenza in markets should inform intervention measures such as market cleaning and closures to reduce risk of human infections and emergence of novel AIVs.

Little is known about circulation of influenza and other respiratory viruses in remote populations along the Thai-Cambodia border in western Cambodia. We screened 586 outpatients (median age 5, range 1-77) presenting with influenza-like-illness (ILI) at 4 sentinel sites in western Cambodia between May 2010 and December 2012. Real-time reverse transcriptase (rRT) PCR for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ILI-specimens negative for influenza were cultured, followed by rRT-PCR for enterovirus and rhinovirus (EV/RV) and EV71. Influenza was found in 168 cases (29%) and occurred almost exclusively in the rainy season from June to November. Isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in Cambodia. Influenza vaccination coverage was low (<20%). Western Cambodian H1N1(2009) isolate genomes were more closely related to 10 earlier Cambodia isolates (94.4% genome conservation) than to 13 Thai isolates (75.9% genome conservation), despite sharing the majority of the amino acid changes with the Thai references. Most genes showed signatures of purifying selection. Viral culture detected only adenovirus (5.7%) and parainfluenza virus (3.8%), while non-polio enteroviruses (10.3%) were detected among 164 culture-negative samples including coxsackievirus A4, A6, A8, A9, A12, B3, B4 and echovirus E6 and E9 using nested RT-PCR methods. A single specimen of EV71 was found. Despite proximity to Thailand, influenza epidemiology of these western Cambodian isolates followed patterns observed elsewhere in Cambodia, continuing to support current vaccine and treatment recommendations from the Cambodian National Influenza Center. Amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

Background: Despite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny., Study Design: We document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09., Results: Complete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID50 of 10(4)/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia., Conclusions: It is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Human infection with the avian influenza A H5N1 virus results in disease with a high fatality rate, against which antiviral treatments have limited efficacy. We aimed to investigate the safety, pharmacokinetics, and therapeutic potential of specific polyclonal immunoglobulin equine F(ab')₂ fragments raised against influenza A/Vietnam/1194/2004 virus (H5N1 subtype) in healthy volunteers., Methods: We did a randomised, double-blind, placebo-controlled, single-centre phase 1 study. In stage 1 (one infusion) and stage 2 (five infusions) of the trial, we randomly assigned healthy male volunteers to receive once-daily intravenous infusions of 0·85 U/kg body weight of F(ab')₂ or once-daily saline placebo. Randomisation was done centrally, with one block of four patients and one block for substitutes (three actives, one placebo) in stage 1, and two blocks of six patients (five actives and one placebo) and the same block for substitutes in stage 2. The primary objective was assessment of the clinical and laboratory safety of F(ab')₂, which was monitored for 22 days in the group that received one dose (assessments on days 0-2, 4, 8, 15, and 22) and 33 days in the group that received five doses (days 0-6, 8, 10, 12, 19, 26, and 33). A final post-study safety assessment was done at 120 days. We also assessed pharmacokinetic outcomes, and assayed haemagglutination and seroneutralisation activity. Analysis was done according to intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT02295813., Findings: We enrolled 16 healthy Asian men between Sept 28 and Dec 28, 2012, and randomly assigned 13 to one or five doses of F(ab')₂ and three to placebo. F(ab')₂ was well tolerated, and no deaths or serious adverse events occurred. Three patients had mild adverse events (one each of blepharospasm, sinusitis, and pyrexia). The pyrexia (38°C) was regarded as probably related to the infusion, and resolved after 37 min. Our laboratory assessments of blood and urine samples and physical examinations of heart rate, electrocardiogram readings, and weight showed no clinically significant safety issues. Mean peak plasma concentrations were 19·3 μg/mL (SD 3·5) with the one dose schedule and 23·0 μg/mL (4·5) with the five-dose schedule. F(ab')₂ were still detectable in plasma on average up to 5 days after five doses. Haemagglutination inhibition was only increased after the third dose, but in-vitro seroneutralisation activity was transiently increased after each of the five doses to concentrations regarded as clinically beneficial in infected patients., Interpretation: F(ab')₂ showed good safety, tolerability, and therapeutic potential for managing of H5N1 exposed patients., Funding: Fab'entech., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: The Cambodian National Influenza Center (NIC) monitored and characterized circulating influenza strains from 2009 to 2011., Methodology/principal Findings: Sentinel and study sites collected nasopharyngeal specimens for diagnostic detection, virus isolation, antigenic characterization, sequencing and antiviral susceptibility analysis from patients who fulfilled case definitions for influenza-like illness, acute lower respiratory infections and event-based surveillance. Each year in Cambodia, influenza viruses were detected mainly from June to November, during the rainy season. Antigenic analysis show that A/H1N1pdm09 isolates belonged to the A/California/7/2009-like group. Circulating A/H3N2 strains were A/Brisbane/10/2007-like in 2009 before drifting to A/Perth/16/2009-like in 2010 and 2011. The Cambodian influenza B isolates from 2009 to 2011 all belonged to the B/Victoria lineage represented by the vaccine strains B/Brisbane/60/2008 and B/Malaysia/2506/2004. Sequences of the M2 gene obtained from representative 2009-2011 A/H3N2 and A/H1N1pdm09 strains all contained the S31N mutation associated with adamantanes resistance except for one A/H1N1pdm09 strain isolated in 2011 that lacked this mutation. No reduction in the susceptibility to neuraminidase inhibitors was observed among the influenza viruses circulating from 2009 to 2011. Phylogenetic analysis revealed that A/H3N2 strains clustered each year to a distinct group while most A/H1N1pdm09 isolates belonged to the S203T clade., Conclusions/significance: In Cambodia, from 2009 to 2011, influenza activity occurred throughout the year with peak seasonality during the rainy season from June to November. Seasonal influenza epidemics were due to multiple genetically distinct viruses, even though all of the isolates were antigenically similar to the reference vaccine strains. The drug susceptibility profile of Cambodian influenza strains revealed that neuraminidase inhibitors would be the drug of choice for influenza treatment and chemoprophylaxis in Cambodia, as adamantanes are no longer expected to be effective.

Aim: There is still no specific therapy for infection with the highly pathogenic avian influenza A virus (HPAI) H5N1, which caused 39 human cases with a 64% fatality rate in 2013., Materials & Methods: We prepared highly purified specific equine polyclonal immunoglobulin fragments (F(ab')2) against H5N1 and tested them for efficacy in vitro and with different administration schedules in H5N1-challenged BALB/c mice., Results: in vitro, F(ab')2 neutralized 21 different H5N1 strains from different areas, representative of 11 different clades and sub-clades and 9 years of evolution of the virus. In vivo mouse experiments identified that the most efficient administration protocol consists of five consecutive daily injections after infection; 10 mg/kg giving a 60% increase in survival., Conclusion: These data demonstrate the ability of anti-H5N1 F(ab')2 to markedly reduce the mortality and morbidity associated with infection of mice with HPAI H5N1 virus, and their potential for human therapy.

Background: Little is known about post-infectious pulmonary sequelae in countries like Cambodia where tuberculosis is hyper-endemic and childhood pulmonary infections are highly frequent. We describe the characteristics of hospitalized Cambodian patients presenting with community-acquired acute lower respiratory infections (ALRI) on post-infectious pulmonary sequelae (ALRIPS)., Methods: Between 2007 and 2010, inpatients ≥15 years with ALRI were prospectively recruited. Clinical, biological, radiological and microbiological data were collected. Chest radiographs were re-interpreted by experts to compare patients with ALRIPS, on previously healthy lungs (ALRIHL) and active pulmonary tuberculosis (TB). Patients without chest radiograph abnormality or with abnormality suggestive as other chronic respiratory diseases were excluded from this analysis., Results: Among the 2351 inpatients with community-acquired ALRI, 1800 were eligible: 426 (18%) ALRIPS, 878 (37%) ALRIHL and 496 (21%) TB. ALRIPS patients had less frequent fever than other ALRI (p < 0.001) and more productive cough than ALRIHL (p < 0.001). Streptococcus pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa accounted for 83% of ALRIPS group positive cultures. H. influenzae and P. aeruginosa were significantly associated with ALRIPS compared with ALRIHL. Treatment was appropriate in 58% of ALRIPS patients. Finally, 79% of ALRIPS were not recognized by local clinicians. In-hospital mortality was low (1%) but probably underestimated in the ALRIPS group., Conclusion: ALRIPS remains often misdiagnosed as TB with inappropriate treatment in low-income countries. Better-targeted training programs would help reduce the morbidity burden and financial costs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A novel method for applying high-throughput docking to challenging metalloenzyme targets is described. The method utilizes information-based virtual transformation of library carboxylates to hydroxamic acids prior to docking, followed by compound acquisition, one-pot (two steps) chemical synthesis and in vitro screening. In two experiments targeting the botulinum neurotoxin serotype A metalloprotease light chain, hit rates of 32% and 18% were observed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: This study was conducted from 2006 to 2010 and investigated the seroprevalence of influenza A viruses in Cambodian pigs, including human H1N1, H3N2, 2009 pandemic H1N1 (A(H1N1)pdm09), and highly pathogenic avian H5N1 influenza A viruses., Methods: A total of 1147 sera obtained from pigs in Cambodia were tested by haemagglutination inhibition (HI) assays for antibody to human influenza A viruses along with both HI and microneutralization (MN) tests to assess immunological responses to H5N1 virus. The results were compared by year, age, and province., Results: Antibodies against a human influenza A virus were detected in 14·9% of samples. A(H1N1)pdm09 virus were dominant over the study period (23·1%), followed by those to human H1N1 (17·3%) and H3N2 subtypes (9·9%). No pigs were serologically positive for avian H5 influenza viruses. The seroprevalence of human H1N1 and H3N2 influenza viruses peaked in 2008, while that of A(H1N1)pdm09 reached a peak in 2010. No significant differences in seroprevalence to human influenza subtypes were observed in different age groups., Conclusions: Cambodian pigs were exposed to human strains of influenza A viruses either prior to or during this study. The implications of these high prevalence rates imply human-to-swine influenza virus transmission in Cambodia. Although pigs are mostly raised in small non-commercial farms, our preliminary results provide evidence of sustained human influenza virus circulation in pig populations in Cambodia., (© 2012 Blackwell Publishing Ltd.)

In Cambodia, the first detection of HPAI H5N1 virus in birds occurred in January 2004 and since then there have been 33 outbreaks in poultry while 21 human cases were reported. The origin and dynamics of these epizootics in Cambodia remain unclear. In this work we used a range of bioinformatics methods to analyze the Cambodian virus sequences together with those from neighboring countries. Six HA lineages belonging to clades 1 and 1.1 were identified since 2004. Lineage 1 shares an ancestor with viruses from Thailand and disappeared after 2005, to be replaced by lineage 2 originating from Vietnam and then by lineage 3. The highly adapted lineage 4 was seen only in Cambodia. Lineage 5 is circulating both in Vietnam and Cambodia since 2008 and was probably introduced in Cambodia through unregistered transboundary poultry trade. Lineage 6 is endemic to Cambodia since 2010 and could be classified as a new clade according to WHO/OIE/FAO criteria for H5N1 virus nomenclature. We propose to name it clade 1.1A. There is a direct filiation of lineages 2 to 6 with a temporal evolution and geographic differentiation for lineages 4 and 6. By the end of 2011, two lineages, i.e. lineages 5 and 6, with different transmission paths cocirculate in Cambodia. The presence of lineage 6 only in Cambodia suggests the existence of a transmission specific to this country whereas the presence of lineage 5 in both Cambodia and Vietnam indicates a distinct way of circulation of infected poultry., (Copyright © 2012 Elsevier B.V. All rights reserved.)

First identified in 2001, human metapneumovirus (HMPV) is a novel pathogen and causative agent of acute respiratory tract infection. Re-infection with HMPV is common, and currently there is no available vaccine against HMPV infection. Two genotypes of HMPV have been identified, A and B, both of which can be divided further into at least two distinct sub-genotypes. Here we report the results of the first study to investigate the genetic variability of HMPV strains circulating within Cambodia. The overall incidence of HMPV infection amongst an all-ages population of patients hospitalised with ALRI in Cambodia during 3 consecutive years, between 2007 and 2009, was 1.7%. The incidence of HMPV infection was highest amongst children less than 5 years of age, with pneumonia or bronchopneumonia the most frequent clinical diagnoses across all age groups. The incidence of HMPV infection varied annually. As anticipated, genetic diversity was low amongst the conserved F gene sequences but very high amongst G gene sequences, some strains sharing as little as 56.3% and 34.2% homology at the nucleotide and amino acid levels, respectively. Simultaneous co-circulation of strains belonging to the HMPV sub-genotypes B1, B2 and lineage A2b, amongst patients recruited at 2 geographically distinct provincial hospitals, was detected. Sub-genotype B2 strains were responsible for the majority of the infections detected, and a significant (p=0.013) association between infection with lineage A2b strains and disease severity was observed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Human bocavirus (HBoV) is a novel parvovirus that is associated with respiratory and gastrointestinal tract disease., Objectives: To investigate the prevalence and genetic diversity of HBoV amongst hospitalized patients with acute lower respiratory infection (ALRI) in Cambodia., Study Design:   Samples were collected from 2773 patients of all ages hospitalised with symptoms of ALRI between 2007 and 2009. All samples were screened by multiplex RT-PCR/PCR for 18 respiratory viruses. All samples positive for HBoV were sequenced and included in this study., Results: Of the samples tested, 43 (1·5%) were positive for HBoV. The incidence of HBoV did not vary between the consecutive seasons investigated, and HBoV infections were detected year-round. The incidence of HBoV infection was highest in patients aged < 2 years, with pneumonia or bronchopneumonia the most common clinical diagnosis, regardless of age. A total of 19 patients (44%) were co-infected with HBoV and an additional respiratory pathogen. All isolates were classified as HBoV type 1 (HBoV-1). High conservation between Cambodian NP1 and V1V2 gene sequences was observed., Conclusions: Human bocavirus infection can result in serious illness, however is frequently detected in the context of viral co-infection. Specific studies are required to further understand the true pathogenesis of HBoV in the context of severe respiratory illness., (© 2012 Blackwell Publishing Ltd.)

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.

Human respiratory syncytial virus (HRSV) is the leading cause of hospitalization of children aged <5 years due to respiratory illness in industrialized countries, and pneumonia is the leading cause of mortality among children aged <5 years worldwide. Although HRSV was first identified in 1956, a preventative vaccine has yet to be developed. Here we report the results of the first study to investigate the circulation and genetic diversity of HRSV in Cambodia among an all-ages population over 5 consecutive years. The incidences of HRSV infection among all-ages outpatient and hospitalized populations were equivalent, at 9.5% and 8.2%, respectively. Infection was most prevalent among children aged <5 years, with bronchiolitis being the most frequently observed clinical syndrome in the same age group. Circulation of HRSV was seasonal, typically coinciding with the rainy season between July and November annually. Strains belonging to HRSV groups A and B were detected with equivalent frequencies; however, we observed a potentially biennial shift in the predominant circulating HRSV genotype. The majority of HRSV group B strains belonged to the recently described BA genotype, with the exception of 10 strains classified as belonging to a novel HRSV group B genotype, SAB4, first reported here.

Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small molecule furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addition of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochemical cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Molecular modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-Lys-Arg-CH(2)Cl. The compounds presented here are bona fide synthetic small molecule furin inhibitors that exhibit potency in the nanomolar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furin-dependent diseases.

The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.

To avoid detection and targeting by the immune system, the plague-causing bacterium Yersinia pestis uses a type III secretion system to deliver a set of inhibitory proteins into the cytoplasm of immune cells. One of these proteins is an exceptionally active tyrosine phosphatase termed YopH, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. Because Y. pestis strains lacking YopH are avirulent, we set out to develop small molecule inhibitors for YopH. We used a novel and cost-effective approach, in which leads from a chemical library screening were analyzed and computationally docked into the crystal structure of YopH. This resulted in the identification of a series of novel YopH inhibitors with nanomolar Ki values, as well as the structural basis for inhibition. Our inhibitors lack the polar phosphate-mimicking moiety of rationally designed tyrosine phosphatase inhibitors, and they readily entered live cells and rescued them from YopH-induced tyrosine dephosphorylation, signaling paralysis, and cell death. These inhibitors may become useful for treating the lethal infection by Y. pestis.

Bid is a key member of the Bcl-2 family proteins involved in the control of the apoptotic cascade in cells, leading to cell death. Uncontrolled cell death is associated with several human pathologies, such as neurodegenerative diseases and ischemic injuries. Therefore, Bid represents a potential yet unexplored and challenging target for strategies aimed at the development of therapeutic agents. Here we show that a multidisciplinary NMR-based approach that we named SAR by ILOEs (structure activity relationships by interligand nuclear Overhauser effect) allowed us to rationally design a series of 4-phenylsulfanyl-phenylamine derivatives that are capable of occupying a deep hydrophobic crevice on the surface of Bid. These compounds represent the first antiapoptotic small molecules targeting a Bcl-2 protein as shown by their ability to inhibit tBid-induced SMAC release, caspase-3 activation, and cell death.

Epidemiological data and in vitro studies on cancer chemoprevention by tea polyphenols have gained attention recently from the scientific community, nutritionists, the pharmaceutical industry, and the public. Despite the several efforts made recently to elucidate the molecular basis for the anticancer activity of these natural products, little correlation has been found thus far between the putative protein targets of compounds found in tea extracts and levels found in plasma after tea consumption. Here, by using a combination of nuclear magnetic resonance binding assays, fluorescence polarization assay, and computational docking studies, we found that certain green tea catechins and black tea theaflavins are very potent inhibitors (K(i) in the nanomolar range) of the antiapoptotic Bcl-2-family proteins, Bcl-x(L) and Bcl-2. These data suggest a strong link between the anticancer activities of these tea polyphenols and their inhibition of a crucial antiapoptotic pathway, which is implicated in the development of many human malignancies.

Among the most promising chemopreventive agents, certain natural polyphenols have recently received a great deal of attention because of their demonstrated inhibitory activity against tumorigenesis. In view of their anticancer properties, these compounds also hold great promise as potential chemotherapeutic agents. However, to translate these chemopreventive agents into chemotherapeutic compounds, their exact mechanisms of action must be delineated. By using a multidisciplinary approach guided by modern nuclear magnetic resonance spectroscopy techniques, fluorescence polarization displacement assays, and cell-based assays, we have begun to unravel the mechanisms of actions of certain polyphenols such as Gossypol (a compound from cotton seed extracts) and Purpurogallin (a natural compound extracted from Quercus sp. nutgall) and their derivatives. Our findings suggest that these natural products bind and antagonize the antiapoptotic effects of B-cell lymphocyte/leukemia-2 (Bcl-2) family proteins such as Bcl-x(L). Our in vitro and in vivo data not only open a window of opportunities for the development of novel cancer treatments with these compounds but also provide structural information that can be used for the design and development of novel and more effective analogues.

A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar. Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first. Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar. These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function.

Cationomycin, isolated from Actinomadura azurea belongs to a large family of carboxylic polyether antibiotics, transporting monovalent cations through membranes by a mobile carrier mechanism, leading globally to an H+, M+ exchange. In this report the cation transporting properties of cationomycin were characterized in large unilamellar vesicles (LUVs) by 23Na- and 39K-NMR. Kinetic studies showed that cationomycin transported potassium more rapidly than sodium, and the more stable complex was formed with potassium at the water/membrane interface. The transport rate constants measured for cationomycin were compared with those obtained for monensin. Cationomycin transports Na+ more slowly than monensin and has a lower stability complex with Na+ because of the lower formation rate for the complex on the membrane surface. Our results show that transport selectivity of cationomycin is in favour of K+ versus Na+ while the reverse situation is observed for monensin. The relationships between the ionophore properties of cationomycin and monensin with their biological activities are discussed.