The formation of the new military units in the North fleet is accompanied by vaccination using Exhausted diphtheria tetanus vaccine, modified. The accination coincides with periods of a rising number of army conscripts being taken ill with community-acquired infection of respiratory tracts: acute tonsillitis, acute bronchitis and community-acquired pneumonia. We need to study is to ascertain whether there is the correlation between the periods of the increase in the number of ervicemen fallen ill with community-acquired infection of respiratory tracts and the diphtheria and tetanus vaccination. The study was carried out on the North fleet conscripts who were drawn blood samples from the ulnar vein before and after the vaccination using Exhausted diphtheria tetanus vaccine, modified. The blood was examined for the presence of antibodies to diphtheria and tetanus using direct hemagglutination test. The health status of the vaccinated conscripts was under observation for 4 months, during which acute illnesses (acute tonsillitis, acute bronchitis and community-acquired pneumonia) were registered. Serologic testing demonstrated a high rate of immunological protection against diphtheria and tetanus before vaccination. After the diphtheria and tetanus vaccination, the number of conscripts, who were taken ill in the first month, was significantly higher compared to the following months. The conscripts, who fell ill, had high antibody titers against diphtheria and tetanus in the vaccine-challenged period. Vaccination of the servicemen using Exhausted diphtheria tetanus vaccine, modified, is serologically unfounded; it leads to complications such as acute tonsillitis, acute bronchitis and community-acquired pneumonia during the vaccinechallenged period especially during the first month and less considerably during the following months. ; В Северном флоте формирование новых воинских коллективов сопровождается вакцинацией против дифтерии и столбняка АДС-М анатоксином. Это совпадает с периодами увеличения числа заболевших внебольничной инфекцией дыхательных путей: острым тонзиллитом, острым бронхитом, внебольничной пневмонией среди военнослужащих. Необходимо установить, связаны ли периоды увеличения внебольничной инфекции дыхательных путей у военнослужащих по призыву с вакцинацией АДС-М анатоксином. Исследования проводились на новобранцах Северного флота, у которых до и после введения АДС-М анатоксина брали кровь из локтевой вены. Взятый материал исследовался на наличие антител к дифтерии и столбняку методом реакции прямой гемагглютинации. Далее наблюдали за состоянием здоровья привитых в течение 4 месяцев, регистрируя острые заболевания: острый тонзиллит, острый бронхит и внебольничную пневмонию. Серологический контроль определил, что у новобранцев до вакцинации АДС-М анатоксином отмечается высокая иммунологическая защищенность против дифтерии и столбняка. После введения АДС-М анатоксина в первый месяц число заболевших призывников было достоверно выше, чем в последующие месяцы. У заболевших военнослужащих в поствакцинальном периоде внебольничной инфекцией дыхательных путей были высокие титры антител к дифтерии и к столбняку. Вакцинация АДС-М анатоксином военнослужащих серологически не обоснована; введение АДС-М анатоксина новобранцам ведет к осложненному течению поствакцинального периода, проявляясь острми заболеваниями в виде острого тонзиллита, острого бронхита, внебольничной пневмонии, более существенно в первый месяц и менее в последующие месяцы.
BACKGROUND: A high circulating concentration of interleukin 6 is associated with increased risk of coronary heart disease. Blockade of the interleukin-6 receptor (IL6R) with a monoclonal antibody (tocilizumab) licensed for treatment of rheumatoid arthritis reduces systemic and articular inflammation. However, whether IL6R blockade also reduces risk of coronary heart disease is unknown. METHODS: Applying the mendelian randomisation principle, we used single nucleotide polymorphisms (SNPs) in the gene IL6R to evaluate the likely efficacy and safety of IL6R inhibition for primary prevention of coronary heart disease. We compared genetic findings with the effects of tocilizumab reported in randomised trials in patients with rheumatoid arthritis. FINDINGS: In 40 studies including up to 133,449 individuals, an IL6R SNP (rs7529229) marking a non-synonymous IL6R variant (rs8192284; p.Asp358Ala) was associated with increased circulating log interleukin-6 concentration (increase per allele 9·45%, 95% CI 8·34-10·57) as well as reduced C-reactive protein (decrease per allele 8·35%, 95% CI 7·31-9·38) and fibrinogen concentrations (decrease per allele 0·85%, 95% CI 0·60-1·10). This pattern of effects was consistent with IL6R blockade from infusions of tocilizumab (4-8 mg/kg every 4 weeks) in patients with rheumatoid arthritis studied in randomised trials. In 25,458 coronary heart disease cases and 100,740 controls, the IL6R rs7529229 SNP was associated with a decreased odds of coronary heart disease events (per allele odds ratio 0·95, 95% CI 0·93-0·97, p=1·53×10(-5)). INTERPRETATION: On the basis of genetic evidence in human beings, IL6R signalling seems to have a causal role in development of coronary heart disease. IL6R blockade could provide a novel therapeutic approach to prevention of coronary heart disease that warrants testing in suitably powered randomised trials. Genetic studies in populations could be used more widely to help to validate and prioritise novel drug targets or to repurpose existing agents and targets for new therapeutic uses. FUNDING: UK Medical Research Council; British Heart Foundation; Rosetrees Trust; US National Heart, Lung, and Blood Institute; Du Pont Pharma; Chest, Heart and Stroke Scotland; Wellcome Trust; Coronary Thrombosis Trust; Northwick Park Institute for Medical Research; UCLH/UCL Comprehensive Medical Research Centre; US National Institute on Aging; Academy of Finland; Netherlands Organisation for Health Research and Development; SANCO; Dutch Ministry of Public Health, Welfare and Sports; World Cancer Research Fund; Agentschap NL; European Commission; Swedish Heart-Lung Foundation; Swedish Research Council; Strategic Cardiovascular Programme of the Karolinska Institutet; Stockholm County Council; US National Institute of Neurological Disorders and Stroke; MedStar Health Research Institute; GlaxoSmithKline; Dutch Kidney Foundation; US National Institutes of Health; Netherlands Interuniversity Cardiology Institute of the Netherlands; Diabetes UK; European Union Seventh Framework Programme; National Institute for Healthy Ageing; Cancer Research UK; MacArthur Foundation.
Krim-Kongo Hämorrhagisches Fieber (CCHF) ist eine beim Menschen tödlich verlaufende Erkrankung mit Letalitätsraten von 5% (Türkei) bis 80% (Volksrepublik China). Diese Variation hängt vom Kenntnisstand in der Bevölkerung über das Vorkommen dieser Infektionskrankheit, von der Qualität des Gesundheitswesens, von der Genauigkeit des Meldesystems und vom zirkulierenden Virusstamm ab. Das Krim-Kongo-Hämorrhagische‑Fieber-Virus (CCHFV) wird vorrangig durch Zecken der Gattung Hyalomma übertragen und kommt in Afrika, Asien und Europa vor. Hyalomma spp. bevorzugen im Allgemeinen ein warmes und trockenes Klima und eine weniger dichte Vegetation. Hyalomma spp.-Populationen kommen deshalb natürlicherweise nur bis zum 46. nördlichen Breitengrad vor. Weitere Infektionsquellen für den Menschen sind der Kontakt zu Blut, Körperflüssigkeiten und Gewebematerial von virämischen Tieren oder auch infektiösen Menschen; auch das Zerdrücken von CCHFV-infizierten Zecken kann zur Infektion führen. Obwohl Tiere in der Regel nach der Infektion keine klinischen Symptome zeigen, entwickeln sie einen stabilen Antikörpertiter, der noch viele Jahre nach der Infektion nachweisbar ist. Deshalb kann der CCHFV-Antikörpernachweis bei Wiederkäuern zur Feststellung von Risikogebieten verwendet werden. Am gründlichsten ist die CCHFV-Infektion in endemischen Gebieten in Europa und Kleinasien untersucht worden. Demgegenüber gibt es über das CCHFV-Vorkommen in Afrika kaum aktuelle Daten. Daher war das Hauptziel der hier vorgestellten Studien, das CCHFV-Vorkommen in verschiedenen Subsahara-Ländern (Mauretanien, Mali, Kamerun und Demokratische Republik Kongo (DR Kongo)) zu untersuchen. Hierzu wurden zunächst modifizierte serologische Tests angewendet, die zuvor zur Untersuchung von Wiederkäuerseren aus Südosteuropa entwickelt worden waren. Diese Testmethoden umfassten einen kommerziellen (Spezies-adaptierten) Enzym-gekoppelten-Immunadsorptionstest (ELISA) und einen im eigenen Labor entwickelten indirekten CCHFV‑IgG-ELISA sowie einen (Spezies-adaptierten) indirekten Immunfluoreszenz-Test (IFA). Die Anpassung dieser Tests umfasste Veränderungen in den Protokollen und bei den Grenzwerten. Die so modifizierten Tests erreichten diagnostische Sensitivitäten von 95% ‑ 98% und Spezifitäten von 98% - 100%. Da diese Tests jedoch auch in weniger gut ausgestatteten Laboren in Afrika bei tropischer Hitze verwendet werden sollten, wurde ein weiterer auch unter diesen Verhältnissen noch robuster indirekter CCHFV-IgG-ELISA für Rinder mit vergleichbarer diagnostischer Performance (Sensitivität und Spezifität jeweils 99%) entwickelt. Dieser ELISA wurde anschließend im Rahmen seroepidemiologischer Studien an Rinderseren aus Mali und Kamerun (2014 gesammelt) eingesetzt. Parallel dazu wurde ein hochsensitiver Multiplex-CCHFV-RT-qPCR-Nachweis auf der Basis von 12 Genotyp-spezifischen und 2 universalen Primern entwickelt und validiert, der darauf abzielt alle aktuell bekannten CCHFV-Stämme zu detektieren. Dieser RT-qPCR-Nachweis wurde komplettiert durch die Verwendung zweier spezifischer Carboxyfluorescein-Sonden zum direkten CCHFV-Erregergenom-Nachweis. Ein solcher universeller Nachweis war bis dato angesichts der großen Sequenz-Variationen zwischen den CCHFV-Stämmen der sechs bekannten Genotypen nicht verfügbar. Mithilfe von sechs Kalibrator-RNAs ist ferner die genaue Genotyp-spezifische Quantifizierung von CCHFV möglich. Die serologische Nachweismethoden wurden anschließend zur Untersuchung von über 3000 Wiederkäuerseren aus den in Subsahara-Afrika liegenden Ländern Mauretanien, Mali, Kamerun und DR Kongo verwendet. Wiederkäuerseren wurden als CCHFV-Antikörper-positiv erachtet, wenn sie in zwei unabhängigen ELISA-Systemen reaktive Ergebnisse erbrachten. Bei abweichenden Ergebnissen wurde eine IFA zur endgültigen Diagnose durchgeführt. Die Untersuchungen zeigen, dass CCHFV-Infektionen in allen vier Ländern (Mauretanien, Mali, Kamerun und DR Kongo) vorkommen. Dies war insbesondere für Kamerun ein überraschendes Ergebnis, da es sich um den ersten Nachweis dieser Infektionskrankheit in diesem Land handelt. Die höchsten Prävalenzraten wurden in Mauretanien, Mali und Nordkamerun nachgewiesen. Dies stimmte mit den Vegetations- und klimatischen Bedingungen und den dort jeweils vorliegenden, von Hyalomma-Zecken präferierten Habitaten überein. Umgekehrt lassen sich auch die deutlich niedrigeren Prävalenzen in Südkamerun und DR Kongo mit der (geringeren) Eignung der lokalen Vegetation und des lokalen Klimas für Hyalomma-Zecken erklären. Ggfs. steht auch die Viehdichte mit dem Vorkommen von CCHFV in einem Gebiet im direkten Zusammenhang. In Nordkamerun, wo eine deutlich höhere Viehdichte als im Süden des Landes vorzufinden ist, wurden deutlich mehr Rinder positiv auf CCHFV-spezifische Antikörper getestet als im Süden mit geringerer Rinderdichte. Eine ähnliche Korrelation wurde auch in Mali nachgewiesen. Da im Rahmen dieser Studie zum ersten Mal CCHFV-Infektionen in Kamerun detektiert werden konnten, war es wichtig, das Virus auch direkt nachzuweisen. Zu diesem Zweck wurden in einem CCHFV-hochendemischen Gebiet in Nordkamerun 109 Hyalomma-Zecken von Rindern abgesammelt und molekulardiagnostisch auf CCHFV untersucht. Hierzu wurde die hochsensitive Multiplex-SYBR–Green-CCHFV-RT-qPCR verwendet. Proben mit charakteristischer Amplifikation wurden mithilfe der Sonden-basierten Multiplex-CCHFV-RT-qPCR bestätigt. CCHFV konnte in 7 von 109 Zecken nachgewiesen werden, und die amplifizierte Genomsequenz konnte phylogenetisch als Genotyp III eingeordnet werden. Zusammenfassend kann festgehalten werden, dass Wiederkäuer auch in den Subsahara‑Ländern als ausgezeichnete Indikator-Tiere für CCHFV-Infektionen angesehen werden können und dass hierzu nun hochsensitive und -spezifische und gleichzeitig robuste ELISAs zur Verfügung stehen. Die hohe Seroprävalenz in Nordkamerun wurde durch den molekularen RT-qPCR-Nachweis des CCHFV-Genoms bestätigt. Die serologischen und molekulardiagnostischen Daten deuten auf ein weiträumiges und gleichzeitig hochgradiges Infektionsgeschehen in den Ländern Subsahara-Afrikas hin. Angesichts der vorliegenden Daten erscheint zumindest in bestimmten Regionen die Einführung von Aufklärungsaktionen für potenzielle Risikogruppen und auch der Allgemeinbevölkerung empfehlenswert. ; Crimean-Congo hemorrhagic fever (CCHF) is a fatal disease in humans with observed case fatality rates varying from 5% in Turkey to 80% in the People's Republic of China. This variability most likely depends on general awareness of the population, quality of the public healthcare system, sensitivity of the notification system and on the individual virus strain. The primary transmission route of Crimean-Congo hemorrhagic fever virus (CCHFV) is by tick bites. Therefore, the virus distribution on the African, Asian and European continent is closely linked to the main vector, ticks of the genus Hyalomma. Hyalomma spp. generally prefer a warm and dry climate with a fragmented and less dense vegetation. Hence, these ticks are not able to create a stable and self-sustaining population further north than 46° N. Other sources of infections are contact to blood, body fluids and tissues of viremic animals or human patients and by crushing infected ticks. The detection of CCHFV-specific antibodies in ruminant sera is commonly used as indicator to reveal infection risk areas also for humans. Even though animals do not show clinical signs, they develop a stable IgG antibody titer that is detectable for many years after infection. CCHFV is best investigated in endemic areas in Europe. In contrast, research on the CCHFV prevalence on the African continent was fairly neglected for many decades and up-to-date information is only available for a few African countries. Therefore, the main goal of this thesis was to investigate the CCHFV prevalence in four different countries located in sub-Saharan Africa. For this purpose, a series of serological assays recently developed for testing ruminant sera from Southeastern Europe was employed. These assays included a commercial (species adapted) enzyme linked immunosorbent assay (ELISA) and an in-house CCHFV‑IgG-ELISA as well as a commercial (species adapted) immunofluorescence assay (IFA). The adaptation of these assays included protocol and cut-off changes to reach convincing diagnostic sensitivities (95% - 98%) and specificities (98% - 100%). However, as the assays were supposed also to be run in less well equipped laboratories in Africa, a robust and highly reproducible indirect cattle in-house CCHFV-IgG-ELISA was developed. The extensive modifications in the novel CCHFV-IgG-ELISA did not only increase the robustness of the assay and simplify the procedure but also enabled testing sera under the technical standards currently available in African laboratories. Nonetheless this new assay had an excellent diagnostic sensitivity and specificity (both 99%) and was therefore used in the seroepidemiological studies in Mali and Cameroon (sera collected in 2014). In parallel, a highly specific and sensitive multiplex CCHFV RT-qPCR using 12 genotype‑specific primers, 2 universal primers and 2 carboxyfluorescein probes for the reliable detection of all currently known CCHFV strains was newly designed and validated. This assay detects synthetic and native virus sequences of all six known genotypes and six calibrator RNAs enable the genotype-specific and precise quantification of CCHFV. Serological assays were subsequently used to screen more than 3,000 ruminant sera from Mauritania, Mali, Cameroon and the Democratic Republic of the Congo (DR Congo), countries all situated in sub-Saharan Africa, and questions about the correlation of CCHFV prevalence with vegetation, climate and animal density were addressed. Ruminant sera were considered positive for CCHFV antibodies, if they were reactive in two independent ELISAs. In case of divergent results, the IFA was used for final diagnosis. This approach revealed CCHFV infections in ruminants in all four countries Mauritania, Mali, Cameroon and DR Congo, which was a surprising result especially for Cameroon as this was the first demonstration of this infectious disease in this country. Highest prevalence rates were detected in Mauritania, Mali and North Cameroon, which is consistent with the vegetation and climatic conditions and the habitat preference of Hyalomma ticks. Conversely, medium to low prevalence rates were found in Southern Cameroon and the DR Congo, which was also in accordance with the suitability of local vegetation and clime for these arthropods. Moreover, cattle densities seemed also to correlate with the presence of CCHFV in an area. Most CCHFV-specific antibody positive cattle were detected in North Cameroon, where cattle density is much higher than in the South and a similar correlation was noticed for Mali. As this study revealed for the first time CCHFV infections in Cameroon, it was of crucial importance also to prove that the virus is circulating in this environment. For this purpose, 109 Hyalomma ticks were collected from infested bovines in a CCHFV highly endemic area in North Cameroon and were assayed for CCHFV genomes using a novel highly sensitive multiplex SYBR Green CCHFV RT-qPCR based on 14 different primers for the detection of all currently known CCHFV genotypes. Samples with a characteristic amplification were eventually reconfirmed using a multiplex CCHFV RT-qPCR, which had the same genotype specific primer sets and two additional CCHFV specific probes included. CCHFV was found in 7 of the 109 ticks and amplified genome sequences clustered phylogenetically into genotype III. In summary, the work presented here highlights the importance of seroprevalence studies in ruminants as indicator animals and molecular diagnostic studies in ticks to determine, whether CCHFV is circulating in sub-Saharan countries/regions. The current data obtained for Cameroon, DR Congo, Mali and Mauritania allow carrying out risk assessments for human infections and therefore help to define whether public health protection measures (e.g. raising awareness for risk groups and the broader public) should be applied.
Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to low molecular weight chemical allergens. The development of ACD requires the activation of innate immune cells, such as keratinocytes (KC), necessary for the maturation and the migration of dendritic cells (DC), which in turn are required for the activation of specific T cells. Human KC constitutively express several cytokines, including pro IL-1 alpha, pro IL-1 beta and pro IL-18. In vivo it has been demonstrated that IL-18 plays a key proximal role in the induction of allergic contact sensitization, favoring Th-1 type immune response by enhancing the secretion of pro-inflammatory mediators such as TNF-α, IL-8 and IFN-γ (Shornick et al., 1996; Wang et al., 1999, Cumberbatch et al., 2001). Toxicologists have the responsibility of identifying and characterizing the skin and respiratory allergenic potential of chemicals, and estimating the risk they pose to human health. Growing political and practical resistance to toxicity testing in animals has driven the development of animal-free methods for screening and prioritization of toxicants, including those causing allergic hypersensitivity. The purpose of this thesis was to develop an alternative in vitro test based on the keratinocytes and IL-18 to characterize the allergenic potential of low molecular weight chemicals, and to understand the molecular mechanism(s) underlying chemical allergen-induced IL-18 production. In addition to human keratinocytes cell lines (NCTC2544, HaCaT, HPKII), commercially available reconstituted human epidermis 3D-epidermal models were also used as experimental model. Due to their anatomical location and critical role in skin inflammatory and immunological reactions, the use of the KC and skin organotypic culture as a simplified in vitro model to evaluate the potential toxicity of chemicals destined for epicutaneous application is amply justified. To perform these studies 22 contact allergens, 12 photoallergens/photoirritant compounds, 3 respiratory allergens and 9 irritants chemicals were used. The choice of chemicals was dictated by the SENS-IT-IV programme as relevant and representative of the 'universe' of irritants, respiratory and contact allergens. Phototoxic chemicals were selected based on compounds used in similar published studies and reported to cause phototoxicity. Results obtained indicate that the NCTC2544 IL-18 assay is able to discriminate contact allergens and photoallergens from irritants/photoirritants and respiratory allergens. Important factors including compound solubility, chemical reactivity and metabolic activation, which may mask the potential allergenicity of some chemicals, must be considered in the development of in vitro tests. Submerged cell culture may be unfavourable for many of the respiratory sensitizers, due to chemical instability; for this reason we have tested IL-18 production also in reconstituted human epidermis, which allows application in organic solvent, i.e. acetone: olive oil. The lack of metabolic activation may be a relevant problem in case of proaptens. However, NCTC 2544 cells posses both phase I and II metabolic activation capacity (Gelardi et al., 2001), and positive results were indeed obtained with the proaptens tested (eugenol and cinnamic alcohol). A sensitivity of 87%, specificity of 95% and an accuracy of 90% was obtained (Corsini et al., 2009; Galbiati et al., 2011). In addition to being able to determine whether or not a chemical is a sensitizer (labelling) it is also equally important to determine the potency of a sensitizer (classification) in order to identify a maximum safe concentration for human exposure (risk assessment). The combination of the epidermal equivalent potency assay with the release of IL-18 lead to the development of an in vitro model able to identify contact allergens and rank them according to their potency. One other objective of this thesis was to study the signal transduction pathways involved in PPD, DNCB and citral-induced IL-18. For such purpose several inhibitors were used. To investigate the intracellular source of ROS, specific inhibitors of the three main cellular sources of ROS, namely DPI, a NADPH synthetase inhibitor; rotenone, a mitochondrial electron transport inhibitor; allopurinol, a xanthine oxidase inhibitor, were used. Z-VAD-FMK, a cell-permeant pan caspase inhibitor, that irreversible binds to the catalytic site of caspases, and a neutralizing anti-TLR4 antibody were used to investigate the role of the inflammasome and TLR4. Glycirrizic acid, a direct inhibitor of HMGB1 protein, was used to establish the role of HMGB1 as possible DAMP associated with allergen-induced IL-18. To specifically investigate the signal transduction pathway involved in PPD-induced IL-18 production selective inhibitors were used: GF109203X to inhibit PKC, PDTC and Bay 11-70-85 to inhibit NF-κB, and SB203580, as p38 MAPK inhibitor. The results obtained during this three year of research activity have clearly shown that the in vitro methods based on NCTC2544 and IL-18 production are able to discriminate contact/photocontact allergens from irritants/photoirritants and respiratory allergens. Furthermore, the combined use of the epidermis in vitro model with the IL-18 production, beside the ability to identify sensitising compounds, is able to rank them according to their potency. With respect to the molecular mechanisms behind skin sensitization I could demonstrate that different intracellular sources of ROS are triggered by different contact allergens. Allergens-induced IL-18 production is dependent upon NF-κB and p38 MAPK activation and TLR4 and inflammasome activation. Among the DAMPs, the evolutionarily conserved non-hystone chromatin-binding protein HMGB1 is released into the extracellular space following exposure to contact allergens, resulting in TLR4 activation and IL-18 neosynthesis. Even if more studies are necessary to elucidate the mechanisms that are involved in chemical allergens-induced oxidative stress, the signalling pathways activated and their role in contact allergy, data clearly indicated a pivotal role of ROS in chemical allergy. Consequently, the redox state of the cell becomes imbalanced, with the activation of several pathways, including MAPK, such as SAPK/JNK, ERK1/2 and p38, NF-κB, Akt/ASK1 or Keap1/Nrf2 pathways, resulting in a inflammatory and cytotoxic response with the production of costimulatory molecules, cytokines, chemokines, and phase 1 detoxifying enzymes. On the basis of the results obtained, the following scenario could be imagined: chemical sensitisers can induce oxidative stress owing to their elecrophilicity, which in turn activates the inflammasome and HMGB1 release (and possible other DAMPs), which can activate TLR4. Activation of TLR4 will results in NF-κB and p38 MAPK activation and in the neosynthesis of IL-18.
Field Epidemiology Training Program (FETP) program in field epidemiology adapted from the United States Centers for Disease Control and Prevention (CDC) Epidemic Intelligence Service (EIS) program and designed to assist the Ministry of Health in building or strengthening health systems and improve leadership within Public Health Emergency Management. The EFELTP provides residents a Master of Public Health in Field Epidemiology after complete two years of supervised work in applied or field epidemiology. The program has two main components: Classroom-teaching component (25%) and practical attachment or field placement component (75%). The role of public health practitioners includes ensuring effective health promotion, disease prevention and control activities by conducting surveillance on emerging public health threats and providing continues information to policy makers and public health officials. From October, 2017 to up today, I have stayed in Field Epidemiology Training Program, School of Public Health-AAU and at both EPHI and Oromia Regional Health Bureau field base. I learned a lot of public health activities during my stay. This document is compiled body of works accomplished during the two years stay at field base of the field epidemiology training program in Addis Ababa University- School of Public Health. Chapter I: We conducted two epidemiological investigation of outbreak (Malaria and Measles). We used both descriptive and Analytical epidemiology for both outbreaks to describe the pattern and magnitude of the diseases and identify associated risk factors with the outbreak. A total of 348 confirmed malaria cases with no death was identified during February to March 2018 in Darimu Woreda of Iluababora zone, Oromia region. We identify Presence of mosquito's vector/breeding sites, unprotected dam for irrigation, and similar sick patient in the house hold as independent risk factors for malaria outbreak in the woreda. Poor in detection, notification of the outbreak and implementation of larva control measures are a toll for this outbreak. we recommend strengthen malaria surveillance system, identifying potential vector breeding site, Proactive vector control, redistribution of the ITN prior to malaria season and address utilization gaps on bed net through health education. Measles outbreak in Liben woreda of Guji zone, Oromia region. we investigate from December 12 to 20, 2018. A total of 15 measles cases were identified and 3/5 tested were confirmed by measles specific IgM antibody test. Traveling history to adjacent Woreda, presence of measles cases in the house and being unvaccinated are found to be independent risk factor for this outbreak. Religious exemption is identified as major factor for being not vaccinated, which is opposed the >100% vaccine coverage report of the woreda. Target measles vaccination with vitamin A supplementation amongst under five, Suspension of public collection from suspected measles cases rumour reported, health education on religious area, strengthen cold chain management and furthers study on vaccine acceptance and associated factor with sufficient sample size were recomended. Chapter II: We conduct Five-year (2013-2017) malaria data analysis at south west shoa zone and describe by person, place and time. Malaria cases in the zone were decreased by 80.5 percent by 2017 compared to the baseline year of 2013. The zone was in line with achieve high level National malaria strategy plan. Burden of malaria cases was still high among three Woredas. Peak malaria case between September and December. We recommend ITN's distribution and IRS for respective high malaria endemic woreda and scale up of malaria prevention and control intervention prior to the respective period and harmonizing HMIS and PHEM system at all reporting level in order to generating reliable and quality data. Chapter III: We conducted Evaluation of surveillance system from February 01-18, 2019 in Bale zone. (N=43): Measles surveillance was selected and assessed. The system in place found to be simple, flexible and stable in operating well without interruption and helpful in case detection but not useful (fail) to meet objectives of surveillance for action and low in representativeness and acceptable. We recommend widen the surveillance chain among private health facilities, upgrading data management to electronic at all level. Chapter IV: We conducted description of Health profile in Gindeberet Woreda, West Shoa Zone, Oromia region February 10 to 30, 2018. we found Acute febrile illness, pneumonia and acute upper respiratory tract infection were leading causes of adult morbidity and very low TB and HIV case detection. We recommend, targeted HIV counseling and testing. Chapter V: We prepared scientific manuscript for peer reviewed journals on Malaria outbreak investigation in Darimu woreda, Iluababora Zone 2018. The manuscript was prepared according to Ethiopian journal of health development authors guideline. Chapter VI: We prepared two abstracts for submission to scientific conference: - 1. Measles Outbreak Investigation in pocket area of Liben Woreda, Guji Zone, Oromia Region, Ethiopia- December, 2018. 2. Malaria Outbreak Investigation Darimu Woreda of Iluababora Zone, Oromia Region, Ethiopia, March 2018. Chapter VII: We conduct Narrative Summary of Disaster situation report (Meher Assessment) at three Zone of Agro pastoral zone of Oromia Region (Guji, West Guji and Borana) from November 22 to December 12, 2019. There were increased malnutrition case, because of double burden effect of high influx of IDPs and drought in all accessed zones. With this junction, there is emergency nutrition intervention/supply stock out in West Guji and Borana Zone. We recommended the RHB and FMOH should fill the gaps/shortage of nutrition supplies, emergency drug and ensure capacity for timely response. Chapter VIII: Epidemiological research project was prepared on ITNS Utilization and associated factors among settler's population in Darimu woreda of Iluababora Zone, Oromia Region 2019. Community based cross sectional study will be conducted from April to May/2019. Multi-stage sampling technique will be used to get study subjects. Sample size will be determined by using Epi info by using 80% ITNs utilization from pervious study. Total 541 house hold will be assessed in this study and 37,966.96 ETB estimated budget requiring. Chapter IX: Training was given for 59 Health professionals working at Woreda and Health facilities of two zones from December 22-25, 2018. The training was organized by Oromia regional health bureau with collaboration of WHO at Ambo Town. The training was addressed overview of PHEM System, Public Health Emergency Preparedness, Epidemiology of 20 Notifiable Diseases, Early Warning Prevention, Health Emergency Response and Recovery. The training was supported by practical demonstration and group work presentation. Lack of printed training manual is a challenge, so we recommend training preparation should de include all necessary format and manual for trainers. Also I participate in different trainings and conferences in different places, namely: - I have attended AFENET Scientific conference at Addis Ababa June 2018. I have attended the training of Disaster Medical Assistance Team as central DMAT member at Bishoftu town from January 21-27,2019. I participate on Regional semi-annual PHEM review at Adama from February 1114/2019 and Public Health emergency preparedness and response on public mass-gathering at Kulib-Gebril Celebration December 2018. Other additional output: I conduct Six Regional public health emergency Weekly bulletin. I including only one of weekly bulletin in this document. Weekly bulletin results were disseminated for all Zones, Administrative Towns, Regional PHEM staff and different stakeholders including governmental and non-governmental organization on weekly bases.
학위논문(박사)--서울대학교 대학원 :농업생명과학대학 농생명공학부,2020. 2. 최영진. ; 현대사회에서 인간의 평균수명은 지속적으로 증가하고 있으며, 이에 따른 행복과 건강에 대한 관심이 크게 증가하고 있다. 전세계적인 이러한 현상은 자연스럽게 진단기술의 개발을 촉진시켰다. 특히, 바이오센서는 현장 진단용 기술로 큰 기대를 받고 있다. 분석물을 생체 인식 시스템을 이용하여 분석하는 장치인 바이오센서는 일반적으로 감지기, 변환기, 신호 분석 시스템 세 가지 요소로 구성된다. 바이오센서의 목표는 생물학적인 감지를 높은 민감도와 선택도로 구현하는 것이다. 의료 및 현장검사(point-of-care testing, POCT) 용을 시작으로 발전한 바이오센서의 진단기술은 식품안전, 군사, 환경 모니터링 등의 다양한 분야로 확대·응용되었다. 식품안전 분야에서의 바이오센서의 개발은 큰 어려움이 존재하는데, 그 이유로는 식품이 가지는 매우 다양하고 복잡한 matrix 때문이다. 즉, 식품은 종류도 다양하며, 그 안의 구성 matrix도 매우 복잡·다양해서 바이오센서의 작동을 어렵게 한다. 따라서, 식품 안전분야의 바이오센서의 개발은 식품에 대한 이해를 토대로 연구개발되어야 한다. 즉, 식품안전분야에서는 전처리 과정이 적으며, 식품의 matrix에 영향을 많이 받지 않으면서, 많은 양의 샘플을 처리할 수 있는 현장적용이 가능한 바이오센서의 개발이 필요하다. 또한, 현장 적용에 최적화되기 위해서는 장치의 소형화, 자동화, 간편화 등이 동반되어야 한다. 금 나노입자의 응집을 이용한 비색반응은 육안으로 신호분석을 할 수 있어서 특별한 분석장비가 필요하지 않다는 이유로 현장적용에 적합한 기술로 평가받고있다. 하지만 이 반응은 약 1010 ea/mL이상의 농도의 금 나노입자를 이용해야만 하는 점과 분석물의 감지와 신호분석이 동시에 일어나는 특성이 맞물려서 민감도가 좋지 않다는 한계점이 존재한다. 이에 따라서, 이중기능링커(bi-functional linker), 동일한 의미인 스위치어블 링커(switchable linker)를 사용한 새로운 금 나노입자의 응집 시스템은 금 나노입자의 응집과 분석물과의 반응을 서로 독립시켜서 이러한 문제점을 해결함으로써, 현장 적용에 적합한 바이오센서로서 대두되었다. 본 연구에서는 이중기능링커를 활용한 금 나노입자의 응집반응으로 토마토에서 살모넬라 균(Salomonella Typhimurium)을 간단한 조작으로 45분 이내에 10 cells/mL 이하로 검출할 수 있음을 보임으로서 식품산업현장에 적용가능성을 입증하였다. 또한, 1/10 희석한 serum에서 단백질 바이오마커인 prostate-specific antigen을 100 fg/mL의 수준까지 검출 가능함을 보임으로서 다양한 분석물에 적용할 수 있음을 보였다. 마지막으로, 3-way valve chamber와 주사기 필터를 적용한 소형화된 검출 장치를 통하여 살모넬라 균(Salomonella Typhimurium)을 검출함으로서, 이중기능링커의 금 나노입자 응집반응 시스템이 현장에 적용할 수 있음을 보였다. 물론 본 전략이 식품현장에서 사용하기 위해서는 자동화, 대량화, 안정성 등의 문제를 해결해야 할 것이다. 하지만, 간편하고, 민감하며, 매우 빠르게 분석물을 진단할 수 있다는 점에서 현장적용의 잠재력이 높다고 판단된다. ; The average human life span is continuously increasing as are efforts worldwide to improve health and happiness, which has spurred the development of diagnostic technologies. In particular, great advances are being made in biosensors that enable on-site diagnosis. Biosensors, i.e., devices that examine analytes using biometric systems, typically consist of three components: detectors, transducers, and signal analysis systems. The goal for biosensors has evolved to enable biological sensing with high sensitivity and selectivity. The use of biosensor diagnostic technology developed for medical and point-of-care testing has been expanded and is now applied in various fields, including food safety, the military, and environmental monitoring. The development of biosensors in the field of food safety presents significant challenges because of the very diverse and complex matrices that characterize food. In other words, foods come in many varieties, and their composition matrices are complex and diverse, which makes it difficult for biosensors to operate. Therefore, biosensors in the field of food safety must be researched and developed based on our current understanding of food. Such a biosensor must require minimal pretreatment and be able to process a large number of samples without being affected by the food matrix. In addition, to be optimized for field application, the device must be miniature in size, automated, and simple to use. Colorimetric methods that employ the strategy of gold nanoparticle (Au NP) aggregation have been determined to be suitable for field application because they can perform signal analysis using only the naked eye and require no specialized analytical equipment. However, these methods are limited by their poor sensitivity for the following reasons: First, color can be distinguished by the naked eye only when using Au NPs at a concentration of about 1011 ea/mL or more. Second, in general, Au NPs facilitate both detection and signal analysis. As a result, high concentrations of Au NPs require high concentrations of analytes for signal analysis. Accordingly, a novel Au NP aggregation system is proposed that uses a bi-functional linker (BL), which has the same function as a switchable linker, and thus solves this problem by separating the signal analysis step from the analyte detection step by the aggregation of Au NPs. In addition, a BL-based assay, which has advantages such as simple operation and requiring no washing step, has emerged as a biosensor suitable for application in the food industry. In this study, the aggregation strategy of using Au NPs as a BL showed that Salmonella Typhimurium could be detected in tomatoes at concentrations of less than 10 cells/400 μL within 45 minutes through simple manipulation. This indicates that this strategy is applicable to the food industry. In addition, prostate-specific antigen, a protein biomarker, was detected at a concentration of 100 fg/mL in a serum diluted to 1/10, which indicates that this strategy could be applied to various analytes. Finally, by detecting Salmonella Typhimurium using a miniaturized detection device with a 3-way valve chamber and a syringe filter, the BL-based assay could be applied in the field. Of course, to qualify this strategy for use in the food field, issues related to automation, sample bulk-up, and stability must be addressed. However, the potential for field application is high due to this strategy's simplicity, sensitivity, and rapid diagnosis of analytes. ; Chapter I. Introduction: 1 I-1. Biosensors 2 I-1-1. Background of biosensors 2 I-1-2. Biosensors in the food field 5 I-1-3. Biosensors for on-site detection 5 I-2. Colorimetric assay in the food safety field 8 I-2-1. The methods for the identification of food-borne pathogens 7 I-2-2. Au NP-based colorimetric biosensing strategy 7 I-2-3. Au NP-based colorimetric biosensing in the food safety 11 I-3. Bi-functional linker-based assay 16 I-4. References 17 Chapter II. A bi-functional linker based immunoassay for ultrasensitive visible detection of Salmonella in tomatoes 20 II-1. Introduction 21 II-2. Materials and Methods 24 II-2-1. Chemicals, reagents, and instruments 24 II-2-2. Preparation of streptavidin-coated Au NPs 25 II-2-3. Bacterial strain and culture conditions 25 II-2-4. Artificial inoculation of tomato samples with Salmonella 26 II-2-5. Comparision of homogenization methods 27 II-2-6. Time for large-scale aggregation using st Au NPs and b-Ab 28 II-2-7. Selectivity of b-Ab as a BL 28 II-2-8. BL-based immunoassay 29 II-2-9. Validation test 32 II-2-10. Statistical Analysis 32 II-3. Results and Discussion 34 II-3-1. The principle of BL-based immunoassay 34 II-3-2. Optimization of the conditions for the BL-based immunoassay 35 II-3-2-1. The range of BL concentration 35 II-3-2-2. The reaction time for creating large-scale aggregates 38 II-3-2-3. Selectivity 40 II-3-3. Detection of Salmonella using the BL-based immunoassay 42 II-3-4. The use of an BL-based assay with contaminated tomato samples 45 II-4. References 48 II-5. Appendix: Optimization of BL-based assay 50 II-5-1. Evaluation of the degree of shift in REVC 50 II-5-2. Detection of a single bacterium 51 II-5-3. Confirmation of the effectiveness of homogenization 52 II-5-4. Quantification of Color Change by Precipitation 52 Chapter III. Colorimetric bi-functional linker based bioassay for ultrasensitive detection of prostate-specific antigen as a protein target 53 III-1. Introduction 54 III-2. Materials and Methods 57 III-2-1. Chemicals, reagents, and instruments 57 III-2-2. Nanoparticle fabrication 58 III-2-3. Au NP surface functionalization 58 III-2-4. Detection of streptavidin and PSA using BL-based assay 61 III-2-5. Selectivity of b-antibody as a BL 61 III-2-6. Enzyme-Linked Immunosorbent Assay (ELISA) 62 III-2-7. Statistical Analysis 63 III-3. Results and Discussion 64 III-3-1. BL-based assay scheme: characteristics of the BL and its construction 64 III-3-2. Determination of the concentration of Au NPs 68 III-3-3. Ultra-sensitive performance of the immunoassay 71 III-3-4. Selectivity of the BL-based immunoassay 75 III-3-5. Detection of PSA in serum 77 III-3-6. Comparison of colorimetric biosensors and ELISA method 79 III-3-7. Sensitivity difference with respect to the BL design 79 III-4. References 81 III-5. Appendix: Optimization of BL-based assay for detection of protein targets 84 III-5-1. Optimization of degree of REVC shift 84 III-5-2. Optimization of shift in REVC differences 85 III-5-3. Shift in REVC of the BL-based assay for detecting streptavidin 86 III-5-4. Schematic representation of the switching off process 87 Chapter IV. Development of a portable lab-on-a-valve device for the primary diagnostic fields based on gold nanoparticle aggregation induced by bi-functional linker 88 IV-1. Introduction 89 IV-2. Materials and Methods 92 IV-2-1. Chemicals, reagents, and instruments 92 IV-2-2. Preparation of colloidal streptavidin-coated Au NPs 93 IV-2-3. Fabrication of the portable kit 95 IV-2-4. Optimization of the syringe filter 97 IV-2-5. Detection procedure 99 IV-2-6. Optimization of secondary reaction time for filteration 100 IV-2-7. Statistical Analysis 100 IV-3. Results and Discussion 101 IV-3-1. Optimization of 3-way valve chamber (3-VC) 101 IV-3-2. The BL-based immunosensing mechanism by 3-VC 101 IV-3-3. System stability of pH and salt conditions 101 IV-3-4. Determination of reaction time (first reaction time) for targets to crosslink with bi-functional linkers 105 IV-3-5. Determination of reaction time (second reaction time) for producing the REVC signal using the filter 107 IV-3-6. Evaluation experiments 109 IV-4. References 110 국문 초록 112 ; Doctor
The World Bank initiated a review of HIV prevention among injection drug users in Thailand, with the objective of providing technical assistance to strengthen national capacity to develop state-of-the-art injecting drug use harm reduction interventions. Thailand has received international recognition for its successful interventions to reduce the transmission of HIV among female sex workers and military recruits. It is looked upon as a role model for HIV education and awareness campaigns that include the extensive promotion and wide acceptance of condoms as an HIV prevention strategy. Thailand has the most progressive and comprehensive antiretroviral program in the region with a reported coverage of over 80 percent of eligible individuals. In 2001, it embarked on a progressive universal health care program that provides free access to a wide array of health care diagnostics and therapeutics for the people of Thailand. With these impressive achievements, it is remarkable how poorly Thailand has responded to the HIV epidemic among injection drug users (IDUs). From available data, it appears that the HIV prevalence rates among IDUs have remained high and stagnant over the last decade. Failure to provide effective interventions to reduce HIV transmission among drug users has resulted in unnecessary suffering, and for many, HIV-related death. Continued inaction threatens to undermine successful HIV prevention efforts in the country through ongoing HIV transmission among injection drug users and their sexual partners. The current focus on enforcement and punishment, along with the reliance on compulsory drug treatment centers, has done little to control drug use in Thailand. The unintended consequence of this approach has been to push drug users into precarious and dangerous environments that have directly led to risky drug using patterns and persistently high rates of HIV transmission. Adopting a harm reduction approach to deal with injection drug use could have a major impact on reducing HIV transmission as well as engaging drug users into better health care and effective drug treatment. This will require strong leadership in key government Ministries and related agencies so that the central stakeholders can roll out harm reduction programs. Thailand has the potential to greatly reduce the transmission of HIV among injection drug users and become a regional leader in harm reduction.