Epidemiology of acute respiratory viral infections in children in Vientiane, Lao People's Democratic Republic.

Respiratory infections are one of the most frequent reasons for medical consultations in children. In low resource settings such as in Lao People's Democratic Republic, knowledge gaps and the dearth of laboratory capacity to support differential diagnosis may contribute to antibiotic overuse. We studied the etiology, temporal trends, and genetic diversity of viral respiratory infections in children to provide evidence for prevention and treatment guidelines. From September 2014 to October 2015, throat swabs and nasopharyngeal aspirates from 445 children under 10 years old with symptoms of acute respiratory infection were collected at the Children Hospital in Vientiane. Rapid antigen tests were performed for influenza A and B and respiratory syncytial virus. Real-time reverse-transcription polymerase chain reactions (RT-PCRs) were performed to detect 16 viruses. Influenza infections were detected with a higher sensitivity using PCR than with the rapid antigen test. By RT-PCR screening, at least one pathogen could be identified for 71.7% of cases. Human rhinoviruses were most frequently detected (29.9%), followed by influenza A and B viruses combined (15.9%). We identify and discuss the seasonality of some of the infections. Altogether these data provide a detailed characterization of respiratory pathogens in Lao children and we provide recommendations for vaccination and further studies.

Posttranslational proteolytic processing of Leda-1/Pianp involves cleavage by MMPs, ADAM10/17 and gamma-secretase.

Leda-1/Pianp is a type I transmembrane protein expressed by CNS cells, murine melanoma cell line B16F10 and rat liver sinusoidal endothelial cells. The early steps of posttranslational modifications of Leda-1/Pianp have been described to include glycosylation and processing by proprotein convertases. Here, we comprehensively characterized the subsequent steps of proteolytic processing of Leda-1/Pianp. For this purpose specific protease inhibitors and cell lines deficient in PS1, PS2, PS1/PS2 and ADAM10/17 were deployed. Leda-1/Pianp was cleaved at numerous cleavage sites within the N-terminal extracellular domain. The sheddases involved included MMPs and ADAM10/17. Ectodomain shedding yielded C-terminal fragments (CTF) of ∼15 kDa. The CTF was further processed by the γ (gamma)-secretase complex to generate the intracellular domain (ICD) of ∼10 kDa. Although PS1 was the dominant intramembrane protease, PS2 was also able to cleave Leda-1/Pianp in the absence of PS1. Thus, Leda-1/Pianp is constitutively processed by proprotein convertases, sheddases including MMPs and ADAM10/17 and intramembrane protease γ-secretase.

Leda-1/Pianp is targeted to the basolateral plasma membrane by a distinct intracellular juxtamembrane region and modulates barrier properties and E-Cadherin processing.

Leda-1/Pianp is a type-I transmembrane protein which is sorted to the basolateral membrane domain of polarized epithelial cells. Here, we investigated trafficking mechanisms and functions of Leda-1/Pianp in MDCK and MCF-7 cells. Basolateral sorting and posttranslational modifications depended on the intracellular juxtamembrane region. Functionally, Leda-1/Pianp increased the transepithelial electrical resistance generated by a polarized cell sheet. Furthermore, resistance to junctional destabilization by tumor cells was enhanced by Leda-1/Pianp indicating increased stability and tightness of intercellular junctions. While Claudin 1 and 4 expression and activities of small GTPases were not affected, γ-Secretase-mediated cleavage of E-Cadherin was attenuated by Leda-1/Pianp. Regulation of proteolytic processing is thus a molecular mechanism by which Leda-1/Pianp can affect junctional integrity and function.

Counter-regulation of the ligand-receptor pair Leda-1/Pianp and Pilrα during the LPS-mediated immune response of murine macrophages.

Liver endothelial differentiation-associated protein-1 (Leda-1/Pianp) is a type-I-transmembrane protein that is able to bind and activate immune inhibitory receptor Pilrα. Here we show that Leda-1/Pianp is strain-specifically expressed in lymphoid organs and macrophages of Th2-prone BALB/c mice but not of Th1-prone C57BL/6J mice. LPS stimulation of BALB/c bone marrow-derived macrophages (BMM) and macrophage-like Raw 264.7 cells conversely regulated Leda-1/Pianp and Pilrα expression. Pilrα induction was caused by LPS-mediated transcriptional modulation and increased mRNA expression. On the other hand, the LPS-mediated decline of Leda-1/Pianp expression was the result of proteolytic degradation by matrix metalloproteinases. In summary, these findings demonstrate that counter-regulation of the ligand-receptor pair Leda-1/Pianp and Pilrα is part of the complex innate immune response of macrophages and its genetically determined strain-specific modulation.

Proteolytic cleavage of LEDA-1/PIANP by furin-like proprotein convertases precedes its plasma membrane localization.

Liver endothelial differentiation-associated protein-1 (LEDA-1/PIANP) is a type-I-transmembrane protein first identified by us as a putative junctional protein in liver sinusoidal endothelial cells. Others have shown that LEDA-1/PIANP binds and activates immune inhibitory receptor PILRα in trans, a process that requires sialidation of LEDA-1/PIANP. Here we show that LEDA-1/PIANP is subject to O-glycosylation and sialidation as demonstrated in brain tissue as well as in LEDA-1 expressing cell lines by using anti-LEDA-1/PIANP C-terminal antibodies. In addition, analysis of LEDA-1/PIANP processing with His-tags inserted at different positions in the extracellular domain revealed that multiple steps of proteolytic cleavage occur during maturation of the protein. Proteolytic cleavage between aa59 and aa83 preceded sorting of the protein to the plasma membrane. Deletion of aa75-79 and inhibition with Furin inhibitor I confirmed that LEDA-1/PIANP is processed by a Furin-like proprotein convertase. In summary, these findings show that Furin-like proprotein convertase-dependent processing precedes plasma membrane localization of LEDA-1/PIANP that is a pre-requisite of functional receptor-ligand interactions in vitro and in vivo.

Endothelial transdifferentiation in hepatocellular carcinoma: loss of Stabilin-2 expression in peri-tumourous liver correlates with increased survival.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a malignant tumour that is characterized by extensive vascular remodelling and responsiveness to treatment with the anti-angiogenic multikinase inhibitor sorafenib. The aim was to study endothelial remodelling in HCC. METHODS: The murine inducible albumin-SV40-large T-antigen model and two tissue microarrays (TMA) with 295 tumourous and 83 peri-tumourous samples of 296 patients with HCC were analysed for expression of liver sinusoidal endothelial cell (LSEC)-specific marker proteins, stabilin-1 and stabilin-2, LYVE-1 and CD32b. RESULTS: LSEC marker proteins were sequentially lost during HCC progression in the murine HCC model being absent from tumour nodules larger than 800 µm in diameter. Similarly, the TMA analysis of human HCCs revealed loss of all four marker proteins in the majority of tumourous tissue samples. Preservation of LYVE-1 expression showed a significant correlation with low grading (G1). In corresponding peri-tumourous liver tissue, loss of all marker proteins was seen in a minor proportion of cases (34%) while the majority of cases retained expression of at least one of the marker proteins. Loss of stabilin-2 expression in peri-tumourous liver tissue of patients with HCC was significantly less likely to occur (38%) than loss of the other marker proteins (63-95%) and it was associated with significantly longer tumour-specific (P = 0.0523) and overall (P = 0.0338) survival. Loss of stabilin-2 may enhance survival in HCC by preventing endothelial-tumour cell adhesive interactions and microvascular invasion. CONCLUSIONS: In summary, endothelial transdifferentiation is a major pathogenic event in HCC development indicating a switch from vessel co-option/intussusceptive angiogenesis to sprouting angiogenesis.

Liver sinusoidal endothelium: a microenvironment-dependent differentiation program in rat including the novel junctional protein liver endothelial differentiation-associated protein-1.

UNLABELLED: Liver sinusoidal endothelium (LSEC) is a prime example of organ-specific microvascular differentiation and functions. Disease-associated capillarization of LSEC in vivo and dedifferentiation of LSEC in vitro indicate the importance of the hepatic microenvironment. To identify the LSEC-specific molecular differentiation program in the rat we used a two-sided gene expression profiling approach comparing LSEC freshly isolated ex vivo with both lung microvascular endothelial cells (LMEC) and with LSEC cultured for 42 hours. The LSEC signature consisted of 48 genes both down-regulated in LMEC and in LSEC upon culture (fold change >7 in at least one comparison); quantitative reverse-transcription polymerase chain reaction confirmation of these genes included numerous family members and signaling pathway-associated molecules. The LSEC differentiation program comprised distinct sets of growth (Wnt2, Fzd4, 5, 9, Wls, vascular endothelial growth factors [VEGFR] 1, 2, 3, Nrp2) and transcription factors (Gata4, Lmo3, Tcfec, Maf) as well as endocytosis-related (Stabilin-1/2, Lyve1, and Ehd3) and cytoskeleton-associated molecules (Rnd3/RhoE). Specific gene induction in cultured LSEC versus freshly isolated LSEC as well as LMEC (Esm-1, Aatf) and up-regulation of gene expression to LMEC levels (CXCR4, Apelin) confirmed true transdifferentiation of LSEC in vitro. In addition, our analysis identified a novel 26-kDa single-pass transmembrane protein, liver endothelial differentiation-associated protein (Leda)-1, that was selectively expressed in all liver endothelial cells and preferentially localized to the abluminal cell surface. Upon forced overexpression in MDCK cells, Leda-1 was sorted basolaterally to E-cadherin-positive adherens junctions, suggesting functional involvement in cell adhesion and polarity. CONCLUSION: Comparative microvascular analysis in rat identified a hepatic microenvironment-dependent LSEC-specific differentiation program including the novel junctional molecule Leda-1.

Detection of colonisation by extended-spectrum beta-lactamase or carbapenemase producing Enterobacterales from frozen stool specimens.

OBJECTIVE: To determine the impact of pre-culture ultra-low temperature (ULT, - 80 °C) storage of human stool specimens on recovery of Extended-Spectrum Beta-Lactamase (ESBL) or Carbapenemase (CPM) producing Enterobacterales. RESULTS: Twenty stool specimens from a community-based household colonisation study in Cambodia were cultured fresh and after 4-5 days and ~ 6 months of ULT storage (as a slurry in tryptone soya broth-10% glycerol). Presumptive ESBL- and CPM-Escherichia coli isolates were detected in 19/20 (95%) and 1/20 (5%) freshly cultured specimens, respectively. The specimens yielded identical results when re-cultured after ULT storage at both time points. Detection of presumptive ESBL- and CPM-Klebsiella / Enterobacter / Citrobacter group was less frequent and slightly less stable over time. Comparison of antimicrobial susceptibility test profiles between pairs of E. coli and K. pneumoniae isolates from the two frozen culture time points revealed concordance in only 13/28 (46%) pairs, indicating likely colonisation by multiple strains. In conclusion, ULT storage of human stool specimens prior to culture appears to be an acceptable method for managing laboratory workflow in culture-based ESBL / CPM Enterobacterales colonisation studies in high prevalence settings.

Prevalence of MDR organism (MDRO) carriage in children and their household members in Siem Reap Province, Cambodia.

BACKGROUND: The rising incidence of infections caused by MDR organisms (MDROs) poses a significant public health threat. However, little has been reported regarding community MDRO carriage in low- and middle-income countries. METHODS: We conducted a cross-sectional study in Siem Reap, Cambodia comparing hospital-associated households, in which an index child (age: 2-14 years) had been hospitalized for at least 48 h in the preceding 2-4 weeks, with matched community households on the same street, in which no other child had a recent history of hospitalization. Participants were interviewed using a survey questionnaire and tested for carriage of MRSA, ESBL-producing Enterobacterales (ESBL-E) and carbapenemase-producing Enterobacterales (CPE) by culture followed by antibiotic susceptibility testing. We used logistic regression analysis to analyse associations between collected variables and MDRO carriage. RESULTS: Forty-two pairs of households including 376 participants with 376 nasal swabs and 290 stool specimens were included in final analysis. MRSA was isolated from 26 specimens (6.9%). ESBL-producing Escherichia coli was detected in 269 specimens (92.8%) whereas ESBL-producing Klebsiella pneumoniae was isolated from 128 specimens (44.1%), of which 123 (42.4%) were co-colonized with ESBL-producing E. coli. Six (2.1%) specimens tested positive for CPE (4 E. coli and 2 K. pneumoniae). The prevalence ratios for MRSA, ESBL-producing E. coli and ESBL-producing K. pneumoniae carriage did not differ significantly in hospital-associated households and hospitalized children compared with their counterparts. CONCLUSIONS: The high prevalence of ESBL-E across both household types suggests that MDRO reservoirs are common in the community. Ongoing genomic analyses will help to understand the epidemiology and course of MDRO spread.

Effect of community active case-finding strategies for detection of tuberculosis in Cambodia: study protocol for a pragmatic cluster randomized controlled trial.

BACKGROUND: Cambodia has made notable progress in the fight against tuberculosis (TB). However, these gains are impeded by a significant proportion of undiagnosed cases. To effectively reach people with TB, active case-finding (ACF) strategies have been adopted by countries affected by the epidemic, including Cambodia, alongside passive case finding (PCF). Despite increased efforts to improve case detection, approximately 40% of TB cases in Cambodia remained undiagnosed in 2018. In Cambodia, several community-based TB ACF modalities have been implemented, but their effectiveness has yet to be systematically assessed. METHODS: This pragmatic cluster randomized controlled trial will be conducted between December 2019 and June 2021. We will randomize eight operational districts (clusters) in seven provinces (Kampong Cham, Kampong Thom, Prey Veng, Thbong Khmum, Kampong Chhnang, Kandal, and Kampong Speu) to either the control group (PCF) or the intervention groups (ACF using a seed-and-recruit model, ACF targeting household and neighborhood contacts, and ACF targeting persons aged ≥ 55 years using mobile screening units). The primary endpoints will be TB case notification rates, additionality, and cumulative yield of TB cases. The secondary endpoints include treatment outcomes, the number needed to screen to find one TB case, and cost-effectiveness outcome measures. We will analyze the primary and secondary endpoints by intention to treat. We will compare cluster and individual-level characteristics using Student's t test and hierarchical or mixed-effect models to estimate the ratio of these means. The incremental cost-effectiveness ratio per disability-adjusted life year averted will also be considered as a benchmark to determine whether the interventions are cost-effective. DISCUSSION: This study will build an evidence base to inform future scale-up, implementation, and sustainability of ACF strategies in Cambodia and other similar settings. Implementation of this study will also complement TB control strategies in Cambodia by conducting ACF in operational districts without active interventions to find TB cases currently. Those who are ill and might have TB will be promptly screened, diagnosed, and linked to care. Early diagnosis and treatment initiation will also benefit their community by interrupting transmission and prevent further infections. The experience gained from this project will inform future attempts in conducting pragmatic trials in low-resource settings. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04094350. Registered on 18 September 2019.

Patients' and healthcare providers' perspectives of diabetes management in Cambodia: a qualitative study.

OBJECTIVE: This study aimed to explore the challenges encountered by patients and healthcare providers and opportunities for improvement in managing diabetes mellitus (DM) in a low- and middle-income country (LMIC) facing a rise in DM prevalence. DESIGN: Qualitative cross-sectional study. SETTING: Urban, semiurban, and rural areas in Cambodia. PARTICIPANTS: Thirty health service providers and fifty-nine adult DM patients. RESULTS: Most of the 59 DM patients reported having developed DM complications when they first sought treatment. The biggest challenges for the patients were geographical barriers, diet control, and shortage of medication supply. The healthcare staff expressed concerns about their limited knowledge and lack of confidence to treat diabetes, limited availability of diabetes care services, inadequate laboratory services, shortage of staff, poor patients' compliance, and insufficient medication supplies. Both healthcare staff and patients urged an expansion of diabetes services in Cambodia and prioritisation of diabetes care in a manner similar to communicable disease control programmes of the recent past. CONCLUSIONS: Currently, the Cambodian healthcare system has very limited capacity to provide quality care for chronic diseases. As a consequence, many patients are either left untreated or have interrupted care due to several barriers including financial, geographical, and lack of knowledge and skills. A more comprehensive and multipronged approach is urgently needed to improve DM care, which would require a collaborative effort from government, external funding agencies, private sector, and communities.

Spatial and temporal projections of the prevalence of active tuberculosis in Cambodia.

INTRODUCTION: Cambodia is among the 30 highest burden of tuberculosis (TB) countries. Active TB prevalence has been estimated using nationally representative multistage sampling that represents urban, rural and remote parts of the country, but the prevalence in non-sampled communes remains unknown. This study uses geospatial Bayesian statistics to estimate point prevalence across Cambodia, and demographic modelling that accounts for secular trends in fertility, mortality, urbanisation and prevalence rates to project the future burden of active TB. METHODS: A Bayesian hierarchical model was developed for the 2011 National Tuberculosis Prevalence survey to estimate the differential effect of age, sex and geographic stratum on active TB prevalence; these estimates were then married with high-resolution geographic information system layers to project prevalence across Cambodia. Future TB projections under alternative scenarios were then derived by interfacing these estimates with an individual-based demographic model. RESULTS: Strong differences in risk by age and sex, together with geographically varying population structures, yielded the first estimated prevalence map at a 1 km scale. The projected number of active TB cases within the catchment area of each existing government healthcare facility was derived, together with projections to the year 2030 under three scenarios: no future improvement, c ontinual r eduction and GDP projection. CONCLUSION: Synthesis of health and geographic data allows likely disease rates to be mapped at a high resolution to facilitate resource planning, while demographic modelling allows scenarios to be projected, demonstrating the need for the acceleration of control efforts to achieve a substantive impact on the future burden of TB in Cambodia.

Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids.

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, ß-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.