Identification and characterization of HIV positive Ethiopian elite controllers in both Africa and Israel.

OBJECTIVES: HIV elite controllers (ECs) are a unique subgroup of HIV-positive patients who are long-term virologically suppressed in the absence of antiretroviral treatment (ART). The prevalence of this subgroup is estimated to be < 1%. Various cohorts of ECs have been described in developed countries, most of which have been demographically heterogeneous. The aim of this study was to identify ECs in two large African cohorts and to estimate their prevalence in a relatively genetically homogenous population. METHODS: We screened two cohorts of HIV-positive Ethiopian patients. The first cohort resided in Mekelle, Ethiopia. The second was comprised of HIV-positive Ethiopian immigrants in Israel. In the Mekelle cohort, ART-naïve subjects with stable CD4 counts were prospectively screened using two measurements of viral load 6 months apart. Subjects were defined as ECs when both measurements were undetectable. In the Israeli cohort, subjects with consistently undetectable viral loads (mean of 17 viral load measurements/patient) and stable CD4 count > 500 cells/µL were defined as ECs. RESULTS: In the Mekelle cohort, 16 of 9515 patients (0.16%) fitted the definition of EC, whereas seven of 1160 (0.6%) in the Israeli cohort were identified as ECs (P = 0.011). CONCLUSIONS: This is the first large-scale screening for HIV-positive ECs to be performed in entirely African cohorts. The overall prevalence of ECs is within the range of that previously described in developing countries. The significant difference in prevalence between the two cohorts of similar genetic background is probably a consequence of selection bias but warrants further investigation into possible environmental factors which may underlie the EC state.

Migrant children's health problems, care needs, and inequalities: European primary care paediatricians' perspective.

BACKGROUND: Primary care paediatricians' perception of migrant children's health in Europe has not been explored before. Our aim was to examine European paediatricians' knowledge on migrant children's health problems, needs, inequalities, and barriers to access health care. METHODS: European primary care paediatricians were invited by the European Academy of Paediatrics Research in Ambulatory Setting Network country coordinators to complete a web-based survey concerning health care of migrant children. A descriptive analysis of all variables was performed. RESULTS: The survey was completed by 492 paediatricians. Sixty-three per cent of the respondents reported that the general health of migrant children is worse than that of nonmigrants, chronic diseases cited by 66% of the respondents as the most frequent health problem. Sixty-six per cent of the paediatricians reported that migrant children have different health needs compared to nonmigrant children, proper oral health care mentioned by 86% of the respondents. Cultural/linguistic factors have been reported as the most frequent barrier (90%).to access health care. However, only 37% of providers have access to professional interpreters and cultural mediators. Fifty-two per cent and 32% do not know whether one or more of the family members are undocumented and whether they are refugees/asylum seekers, respectively. Updated guidelines for care of migrant children are available for only 35% of respondents, and 80% of them have not received specific training on migrant children's care. CONCLUSIONS: European primary care paediatricians recognize migrant children as a population at risk with more frequent and specific health problems and needs, but they are often unaware of their legal state. Lack of interpreters augments the existing language barriers to access proper care and should be solved. Widespread lack of guidelines and specific providers' training should be addressed to optimize health care delivery to migrant children.

Use of electronic health records by child primary healthcare providers in Europe.

BACKGROUND: There is limited data on the use and functionality level of electronic health records (EHRs) supporting primary child health care in Europe. Our objective was to determine European primary child healthcare providers' use of EHRs, and functionality level of the systems used. METHODS: European primary care paediatricians, paediatric subspecialists and family doctors were invited by European Academy of Paediatrics Research in Ambulatory Setting Network (EAPRASnet) country coordinators to complete a web-based survey on the use of EHRs and the systems' functionalities. Binomial logistic analysis has been used to evaluate the effect of specialty and type of practice on the use of EHRs. RESULTS: The survey was completed by 679 child primary healthcare providers (response rate 53%). Five hundred and fifty four responses coming from 10 predominant countries were taken for further analysis. EHR use by respondents varied widely between countries, all electronic type use ranging between 7% and 97%. There was no significant difference in EHR use between group practice and solo practitioners, or between family doctors and primary care paediatricians. History and physical examination can be properly recorded by respondents in most countries. However, growth chart plotting capacity in some countries ranges between 22% and 50%. Vaccination recording capacity varies between 50% and 100%, and data exchange capacity with immunization databases is mostly limited, ranging between 0% and 54%. CONCLUSIONS: There is marked heterogeneity in the use and functionalities of EHRs used among child primary child healthcare providers in Europe. More importantly, lack of critical paediatric supportive functionalities like growth tracking and vaccination status has been documented in some countries. There is a need to explore the reasons for these findings, and to develop a cross European paediatric EHR standards.

Efficacy of tenofovir and efavirenz in combination with lamivudine or emtricitabine in antiretroviral-naive patients in Europe.

BACKGROUND: The combination of tenofovir and efavirenz with either lamivudine or emtricitabine (TELE) has proved to be highly effective in clinical trials for first-line treatment of HIV-1 infection. However, limited data are available on its efficacy in routine clinical practice. METHODS: A multicentre cohort study was performed in therapy-naive patients initiating ART with TELE before July 2009. Efficacy was studied using ITT (missing or switch = failure) and on-treatment (OT) analyses. Genotypic susceptibility scores (GSSs) were determined using the Stanford HIVdb algorithm. RESULTS: Efficacy analysis of 1608 patients showed virological suppression to <50 copies/mL at 48 weeks in 91.5% (OT) and 70.6% (ITT). Almost a quarter of all patients (22.9%) had discontinued TELE at week 48, mainly due to CNS toxicity. Virological failure within 48 weeks was rarely observed (3.3%, n = 53). In multilevel, multivariate analysis, infection with subtype B (P = 0.011), baseline CD4 count <200 cells/mm³ (P < 0.001), GSS <3 (P = 0.002) and use of lamivudine (P < 0.001) were associated with a higher risk of virological failure. After exclusion of patients using co-formulated compounds, virological failure was still more often observed with lamivudine. Following virological failure, three-quarters of patients switched to a PI-based regimen with GSS <3. After 1 year of second-line therapy, viral load was suppressed to <50 copies/mL in 73.5% (OT). CONCLUSIONS: In clinical practice, treatment failure on TELE regimens is relatively frequent due to toxicity. Virological failure is rare and more often observed with lamivudine than with emtricitabine. Following virological failure on TELE, PI-based second-line therapy was often successful despite GSS <3.

HIV-1 subtype is an independent predictor of reverse transcriptase mutation K65R in HIV-1 patients treated with combination antiretroviral therapy including tenofovir.

Subtype-dependent selection of HIV-1 reverse transcriptase resistance mutation K65R was previously observed in cell culture and small clinical investigations. We compared K65R prevalence across subtypes A, B, C, F, G, and CRF02_AG separately in a cohort of 3,076 patients on combination therapy including tenofovir. K65R selection was significantly higher in HIV-1 subtype C. This could not be explained by clinical and demographic factors in multivariate analysis, suggesting subtype sequence-specific K65R pathways.

Repercussions of the COVID-19 pandemic on child and adolescent mental health: A matter of concern-A joint statement from EAP and ECPCP.

COVID-19 pandemic and the consequent rigid social distancing measures implemented, including school closures, have heavily impacted children's and adolescents' psychosocial wellbeing, and their mental health problems significantly increased. However, child and adolescent mental health were already a serious problem before the Pandemic all over the world. COVID-19 is not just a pandemic, it is a syndemic and mentally or socially disadvantaged children and adolescents are the most affected. Non-Communicable Diseases (NCDs) and previous mental health issues are an additional worsening condition. Even though many countries have responded with decisive efforts to scale-up mental health services, a more integrated and community-based approach to mental health is required. EAP and ECPCP makes recommendations to all the stakeholders to take action to promote, protect and care for the mental health of a generation.

European Academy of Paediatrics Research in Ambulatory Setting network (EAPRASnet): a multi-national general paediatric research network for better child health.

BACKGROUND: In 2008, the European Academy of Paediatrics launched a paediatric-based research network - EAPRASnet (European Academy of Paediatrics Research in Ambulatory Setting network). The network has recruited primary care and general paediatricians from European and Mediterranean countries. METHODS: Every paediatrician joining the network has been asked to complete a recruitment survey. The aims of the survey were to characterize paediatrician's demographics, practice arrangements and patient's demographics, to define main incentives for research, and to learn what paediatricians view as unsolved issues that need to be studied. RESULTS: A total of 156 paediatricians from 19 countries were recruited with 144 completing the questionnaire (92%). Majority of respondents (89%) were general paediatricians for more than half of their time. Practice arrangement of 47% of paediatricians was solo practice, with 40% in group practice. Electronic medical records were being used by 72% of respondents. Over 70% of the paediatricians had more than 1000 patients under their clinical care, and patients younger than 6 years old contributed nearly half of the patient population. Areas of most interest for research were: quality of care indicators, communication with parents, obesity, attention deficit hyperactivity disorder and effective well child care. Main incentives for participation in a research project were interest in the topic (81%) and effort to improve quality of care (71%). Lack of time was the leading reported obstacle for research activity (72%). EAPRASnet is growing, and the network's structure, operation and funding are described. Methods for joining the network and the process of study development are presented. CONCLUSION: A core group of EAP general paediatricians are committed to research in their practices. The information gathered will serve for future planning of research projects in the EAPRASnet to harmonize and optimize the care given to children in the primary care setting in Europe.

No evidence of ongoing HIV replication or compartmentalization in tissues during combination antiretroviral therapy: Implications for HIV eradication.

HIV persistence during combination antiretroviral therapy (cART) is the principal obstacle to cure. Mechanisms responsible for persistence remain uncertain; infections may be maintained by persistence and clonal expansion of infected cells or by ongoing replication in anatomic locations with poor antiretroviral penetration. These mechanisms require different strategies for eradication, and determining their contributions to HIV persistence is essential. We used phylogenetic approaches to investigate, at the DNA level, HIV populations in blood, lymphoid, and other infected tissues obtained at colonoscopy or autopsy in individuals who were on cART for 8 to 16 years. We found no evidence of ongoing replication or compartmentalization of HIV; we did detect clonal expansion of infected cells that were present before cART. Long-term persistence, and not ongoing replication, is primarily responsible for maintaining HIV. HIV-infected cells present when cART is initiated represent the only identifiable source of persistence and is the appropriate focus for eradication.

Autoreactivity, dynamic tuning and selectivity.

The immune system adjusts its response to the context in which antigens, including self-antigens, are recognized. Recent observations support a conceptual framework for understanding how this may be achieved at the cellular and cell-population levels. At both levels, 'perturbations' elicit competition between excitation and de-excitation, resulting either in adaptation or in various responses. The responsiveness of individual cells is dynamically tuned, reflecting their recent experience. The tuning of T-cell activation thresholds by self-ligands facilitates positive selection and continuously regulates the level of autoreactivity in the periphery. Autoreactivity appears to be involved in regulation of the immune response, homeostasis, maintaining of the functional integrity of naïve and memory cells, and in other physiological functions.

Analysis of HIV-1 pol sequences using Bayesian Networks: implications for drug resistance.

Human Immunodeficiency Virus-1 (HIV-1) antiviral resistance is a major cause of antiviral therapy failure and compromises future treatment options. As a consequence, resistance testing is the standard of care. Because of the high degree of HIV-1 natural variation and complex interactions, the role of resistance mutations is in many cases insufficiently understood. We applied a probabilistic model, Bayesian networks, to analyze direct influences between protein residues and exposure to treatment in clinical HIV-1 protease sequences from diverse subtypes. We can determine the specific role of many resistance mutations against the protease inhibitor nelfinavir, and determine relationships between resistance mutations and polymorphisms. We can show for example that in addition to the well-known major mutations 90M and 30N for nelfinavir resistance, 88S should not be treated as 88D but instead considered as a major mutation and explain the subtype-dependent prevalence of the 30N resistance pathway.

Bayesian network analysis of resistance pathways against HIV-1 protease inhibitors.

Interpretation of Human Immunodeficiency Virus 1 (HIV-1) genotypic drug resistance is still a major challenge in the follow-up of antiviral therapy in infected patients. Because of the high degree of HIV-1 natural variation, complex interactions and stochastic behaviour of evolution, the role of resistance mutations is in many cases not well understood. Using Bayesian network learning of HIV-1 sequence data from diverse subtypes (A, B, C, F and G), we could determine the specific role of many resistance mutations against the protease inhibitors (PIs) nelfinavir (NFV), indinavir (IDV), and saquinavir (SQV). Such networks visualize relationships between treatment, selection of resistance mutations and presence of polymorphisms in a graphical way. The analysis identified 30N, 88S, and 90M for nelfinavir, 90M for saquinavir, and 82A/T and 46I/L for indinavir as most probable major resistance mutations. Moreover we found striking similarities for the role of many mutations against all of these drugs. For example, for all three inhibitors, we found that the novel mutation 89I was minor and associated with mutations at positions 90 and 71. Bayesian network learning provides an autonomous method to gain insight in the role of resistance mutations and the influence of HIV-1 natural variation. We successfully applied the method to three protease inhibitors. The analysis shows differences with current knowledge especially concerning resistance development in several non-B subtypes.

Translation of vascular endothelial growth factor mRNA by internal ribosome entry: implications for translation under hypoxia.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic growth factor that promotes compensatory angiogenesis in circumstances of oxygen shortage. The requirement for translational regulation of VEGF is imposed by the cumbersome structure of the 5' untranslated region (5'UTR), which is incompatible with efficient translation by ribosomal scanning, and by the physiologic requirement for maximal VEGF production under conditions of hypoxia, where overall protein synthesis is compromised. Using bicistronic reporter gene constructs, we show that the 1,014-bp 5'UTR of VEGF contains a functional internal ribosome entry site (IRES). Efficient cap-independent translation is maintained under hypoxia, thereby securing efficient production of VEGF even under unfavorable stress conditions. To identify sequences within the 5'UTR required for maximal IRES activity, deletion mutants were analyzed. Elimination of the majority (851 nucleotides) of internal 5'UTR sequences not only maintained full IRES activity but also generated a significantly more potent IRES. Activity of the 163-bp long "improved" IRES element was abrogated, however, following substitution of a few bases near the 5' terminus as well as substitutions close to the translation start codon. Both the full-length 5'UTR and its truncated version function as translational enhancers in the context of a monocistronic mRNA.

Natural killer cells and their relationship to T-cells: hypothesis on the role of T-cell receptor gene rearrangement on the course of adaptive differentiation.

We propose that the differentiation of NK cells and the differentiation of T-cells are intimately interrelated, although mature effector cells of each type usually can be distinguished from each other. The divergence in their characteristics may be initiated upon rearrangement of the genes for the T-cell receptor, with a subsequent inverse relationship between the expression of T-cell receptors and NK cell receptors. However, an essential element of our hypothesis is that the differentiation of these cells is partially adaptive rather than rigidly preprogrammed. This concept is considerably more compatible with the phenotypic plasticity which has been exhibited by cultured cells in general and by T-cells and LGL in particular. We suggest that the nature of the self environment has a major influence on the direction of development of precursor cells, both by controlling the ratio between the rates of proliferation and differentiation at each stage of maturation and by inducing quantitative or qualitative changes in the pattern of gene expression. As maturation proceeds, the degree of plasticity probably decreases, possibly due to inheritable epigenetic changes in the genome. Our hypothesis accommodates most if not all of the available experimental data on the phenotypic, genetic, and functional interrelationships between NK cells and T-cells. In particular, it accounts for the extensive and controversial data on cultured cell lines with varying degrees of similarity to T-cells and to NK cells. In addition, our model emphasizes the inherent limitations in utilizing such data from cell lines as the basis for drawing conclusions on the properties of cells developing under physiological conditions. Most importantly, our hypothesis leads to a series of experimentally testable predictions, which should provide considerably greater insight into the ontogeny of NK cells and their relationship to the T-cell lineage.

Structural organization of the murine c-kit proto-oncogene.

The murine Kit receptor gene on chromosome 5 has been found to be frequently involved in germline mutations and rearrangements, leading to a characteristic set of severe developmental defects, known as the W phenotype. Here we describe the structure of the murine c-kit gene, based on restriction analysis of genomic phage clones and sequence determination of exon-intron boundaries. The Kit-coding region is distributed over 21 exons, most of which have sizes that range between 100 and 200 base pairs. The 3' non-translated sequence and the 3' end of the coding region form a single large exon, which encompasses 2.3 kb and is flanked by polyadenylation signals. The entire region spans a genomic distance of at least 70 kb. Though the exonic demarcations of c-kit show remarkable similarity to those of the human c-fms gene (which encodes the highly related colony-stimulating factor 1 receptor), no correlation could be found between the sizes of introns that separate homologous exon pairs. The data suggest that evolutionary pressures were confined to the conservation of structures of coding exons, whereas flanking regions were subject to large changes, owing to insertions and deletions. Finally, the analysis of the Kit genomic structure reveals that the inherited mutations of the Kit gene that have been reported thus far occur at various dispersed positions within the gene. Hence, the entire gene appears to have as yet unknown features which cause it to be frequently subject to mutations in murine germline tissues.

Cloning and characterization of the SmIMP25 integral membrane protein of the parasitic helminth Schistosoma mansoni.

The cDNA and genomic clones encoding a 25 kDa integral membrane protein, termed SmIMP25, were isolated from Schistosoma mansoni. The 2.2 kb SmIMP25 mRNA was found in all developmental stages of the parasite tested: miracidium, sporocyst, cercaria and adult worm. The SmIMP25 gene is at least 16 kb long and it is split by four introns ranging in size from 36 bp to > or = 9 kb. Excluding the introns, the gene and the cDNA show 100% sequence identity. The cDNA has an open reading frame encoding a protein 223 amino acids long. The predicted sequence reveals a distinct hydrophobic domain of 20 amino acids located 12 residues from the carboxyl-terminal end. The properties of this domain (marked hydrophobicity, size, flanking by charged residues and C-terminal location) are typical of the transmembrane segments of integral membrane proteins. The presence of three potential N-glycosylation sites is also consistent with membrane proteins that are often glycosylated at the extracellular domain. Accordingly we propose that SmIMP25 is an integral membrane protein in which residues 1-191 are extracellular, residues 192-211 comprise the hydrophobic domain that spans the membrane, and residues 212-223 are intracellular. The SmIMP25 was synthesized as a fusion protein in bacteria and antibodies were elicited in rabbits. Antibodies against SmIMP25 specifically precipitated a 25 kDa protein from cell-free products programmed by schistosome mRNA, in agreement with the size of the protein predicted from the cDNA sequence. Immunofluorescence studies showed SmIMP25 on the surface of the parasite. Surface molecules expressed at the host-parasite interface are likely to provide information on host parasite relationship and may serve as targets for protective immunity.

Drug-resistant HIV infection among drug-naive patients in Israel.

BACKGROUND: In Israel, <0.06% of the general population is infected with human immunodeficiency virus (HIV), with a much higher prevalence among specific groups. These groups are distinguished demographically by risk behavior category and by virus subtype. We investigated transmission of drug resistance within groups to assess the impact of these factors. METHODS: Plasma samples from >15% of all patients with new diagnoses of HIV infection were randomly collected between June 1999 and June 2003. Sequences from 176 drug-naive patients included 20 of subtype A, 20 of subtype AE, 2 of subtype AC, 29 of subtype B, 100 of subtype C, and 5 of subtype F. RESULTS: Major drug resistance mutations (protease: L90M; reverse transcriptase: M41L, K103N, V106M, M184V, Y181S, G190A, L210W, T215Y/F, and K219R) were detected in 1 subject with A subtype, 3 with subtype B, and 9 with subtype C. In addition, 1 subject with A subtypes, 2 with subtype B, and 10 with subtype C had secondary mutations (protease: M46I; reverse transcriptase: A98G, K101Q, and V108I). Only 1 patient had mutations associated with >1 class of drugs. Among subjects who contracted HIV infection in Israel, 16 of 56 (1 of 7 with subtypes A or AE, 4 of 17 with subtype B, and 11 of 32 with subtype C; P=.7-1.0) carried resistant virus--a significantly higher proportion (P<.001) than in subjects infected in other countries (10 of 120 infected). CONCLUSIONS: Drug-resistant virus was detected in 14.8% of patients with new diagnoses of HIV infection but in 28.6% of patients known to have been infected in Israel. The implications include a need for pretreatment resistance testing and for better programs aimed at prevention of transmission, directed particularly at patients. We did not find significant differences in transmission of resistant virus between those infected with subtypes B and C, despite the different demographic background. A conclusive analysis and interpretation should await a more extensive study.