Tesis por compendio / [ES] El diagnóstico de alergias a antibióticos ß-lactámicos incluye el uso de pruebas in vivo no satisfactorias ya que consumen mucho tiempo y conllevan el riesgo de provocar una nueva reacción alérgica. Por otro lado, los métodos in vitro basados en la inmunodetección de IgE alérgeno-específica generan una información muy valiosa, confirmando o descartando la alergia y postulándose como una alternativa de diagnóstico segura. A pesar de ello, las pruebas in vitro actuales carecen de sensibilidad y exactitud, con un 81% de falsos negativos, lo que limita su uso diagnóstico. Esto ha desencadenado una creciente demanda de nuevas pruebas in vitro basadas en la inmunodetección de IgE, el biomarcador presente en suero más estudiado en alergia. Sin embargo, la falta de estándares de referencia de IgE alérgeno-específica ha imposibilitado la validación y estandarización de los métodos, lo que hace que los resultados de las distintas pruebas no sean comparables. Además, dicha falta de estándares de referencia hace que las concentraciones de IgE específica se calculen a partir de una curva de calibración de IgE total mediante una interpolación heteróloga, lo que ha demostrado no ser totalmente correcto. Por los motivos mencionados, en esta tesis doctoral se plantea como objetivo principal el diseño, obtención y caracterización de proteínas recombinantes con reactividad frente a antibióticos ß-lactámicos. Esta investigación comienza con la puesta a punto de un inmunoensayo en placa ELISA con detección quimioluminiscente para la cuantificación de IgE específica. El ensayo desarrollado permitió determinar IgE específica por debajo de 0.1 IU/mL (0.24 ng/mL), permitiendo así la identificación de pacientes alérgicos con una mayor sensibilidad, utilizando solo 25 ¿L de muestra (suero). El inmunoensayo mostró una buena sensibilidad (64.6%), así como una excelente especificidad (100%), que mejoran significativamente el carácter predictivo de las pruebas in vitro existentes. A continuación, se abordó la obtención y caracterización de proteínas recombinantes similares a las IgE específicas de los ß-lactámicos utilizando la metodología Phage Display. La primera aproximación se trata de una proteína formada por dos nanoanticuerpos que mimetiza el comportamiento de una IgE específica. De esta manera, se obtuvieron proteínas recombinantes en las que uno de los nanoanticuerpos reconoce selectivamente un determinante antigénico de un antibiótico ß-lactámico, mientras que el otro reconoce específicamente al parátopo de un anticuerpo detector anti-IgE. Estas construcciones resultaron ser estables y funcionales. El papel de estas proteínas recombinantes como calibrador homólogo se estudió determinando la concentración de IgE específica a penicilina G presente en suero mediante un ensayo quimioluminiscente. El método desarrollado duplicaba la sensibilidad clínica (66%) frente al método de referencia (28%), mientras que mantenía una especificidad clínica del 100%. Alcanzado este punto, la tesis se centró en la producción de IgE específica recombinante utilizando como material de partida el ADN de un paciente alérgico a amoxicilina y penicilina G. A partir del material genético aislado, mediante Phage Display, ingeniería de anticuerpos y métodos de expresión en células de insecto se consiguió producir una IgE específica recombinante. De esta manera, se obtuvo un producto biológico que cumple con los requisitos para su adopción como material de referencia en ensayos in vitro. Igualmente, se utilizó como calibrador homólogo para la determinación de IgE específica a amoxicilina. Se alcanzó un límite de detección de 0.05 IU/mL con el que se conseguía un aumento de la sensibilidad clínica (73%) que cuadruplicaba al método de referencia (16%), manteniendo la especificidad clínica en el 100%. / [CA] El diagnòstic d'al·lèrgies a antibiòtics ß-lactàmics inclou l'ús de proves in vivo no satisfactòries, ja que consumeixen molt de temps i comporten el risc de provocar una nova reacció al·lèrgica. D'altra banda, els mètodes in vitro basats en la immunodetecció d'IgE al·lergen-específica generen una informació molt valuosa, confirmant o descartant l'al·lèrgia i postulant-se com una alternativa de diagnòstic segura. Tanmateix, a les proves in vitro actuals hi ha una manca de sensibilitat i exactitud, amb un 81% de falsos negatius, cosa que en limita l'ús diagnòstic. Tot això ha desembocat en una demanda creixent de noves proves in vitro basades en la immunodetecció d'IgE, el biomarcador present en sèrum més estudiat en al·lèrgia. No obstant això, la manca d'estàndards de referència d'IgE al·lergen-específica ha impossibilitat la validació i estandardització dels mètodes, cosa que fa que els resultats de les diferents proves no siguen comparables. A més, aquesta manca d'estàndards de referència fa que les concentracions d'IgE específica es calculen a partir d'una corba de calibratge d'IgE total mitjançant una interpolació heteròloga, un mètode que ha demostrat no ser totalment correcte ja que no es pren en consideració la interacció entre el determinant antigènic i la IgE específica. Pels motius mencionats, en aquesta tesi doctoral es planteja com a objectiu principal el disseny, l'obtenció i la caracterització de proteïnes recombinants amb reactivitat davant dels antibiòtics ß-lactàmics. Aquesta investigació comença amb la posada a punt d'un immunoassaig en placa ELISA amb detecció quimioluminiscent per a la quantificació d'IgE específica. L'assaig desenvolupat va permetre determinar IgE específica per baix de 0.1 IU/mL (0.24 ng/mL), el que permet la identificació de pacients al·lèrgics amb més sensibilitat, utilitzant només 25 ¿L de mostra (sèrum). L'immunoassaig mostra una bona sensibilitat (64.6%), així com una excel·lent especificitat (100%), que milloren significativament el caràcter predictiu de les proves in vitro existents. A continuació, es va abordar l'obtenció i la caracterització de proteïnes recombinants similars a les IgE específiques dels ß-lactàmics utilitzant la metodologia Phage Display. La primera aproximació és una proteïna formada per dos nanoanticossos que mimetitza el comportament d'una IgE específica. D'aquesta manera, es van obtenir proteïnes recombinants en què un dels nanoanticossos reconeix selectivament un determinant antigènic d'un antibiòtic ß-lactàmic, mentre que l'altre reconeix específicament el paràtop d'un anticòs detector anti-IgE (Omalizumab). Aquestes construccions van resultar estables i funcionals. El paper d'aquestes proteïnes recombinants com a calibrador homòleg es va estudiar determinant la concentració d'IgE específica a penicil·lina G present en sèrum mitjançant un assaig quimioluminiscent. El mètode desenvolupat duplicava la sensibilitat clínica (66%) respecte del mètode de referència (28%), mentre que mantenia una especificitat clínica del 100%. Assolit aquest punt, la tesi es va centrar en la producció d'IgE específica recombinant utilitzant com a material de partida l'ADN d'un pacient al·lèrgic a amoxicil·lina i penicil·lina G. En combinació amb la metodologia Phage Display, enginyeria d'anticossos i mètodes d'expressió en cèl·lules d'insecte, es va aconseguir produir una IgE específica recombinant. D'aquesta manera, es va obtenir un producte biològic que compleix els requisits per ser adoptat com a material de referència en assajos in vitro. Igualment, es va utilitzar com a calibrador homòleg per a la determinació d'IgE específica a amoxicil·lina. Es va assolir un límit de detecció de 0.05 IU/mL amb què s'aconseguia un augment de la sensibilitat clínica (73%) que quadriplicava al mètode de referència (16%), mantenint l'especificitat clínica al 100%. / [EN] The diagnosis of ß-lactam antibiotics allergy involves using unsatisfactory in vivo tests that are time-consuming and carry the risk of triggering a new allergic reaction. On the other hand, in vitro methods based on allergen-specific IgE immunodetection generate precious information, confirming or ruling out allergies and postulating themselves as a safe diagnostic alternative. Despite this, current in vitro tests lack sensitivity and accuracy, with 81% false negatives limiting their diagnostic use. This has led to a growing demand for new in vitro tests based on the immunodetection of IgE, the most studied biomarker in serum allergy. However, the lack of reference standards for allergen-specific IgE has made it impossible to validate and standardize methods, which means that the results of the different tests are not comparable. Furthermore, this lack of reference standards means that specific IgE concentrations are calculated from a total IgE calibration curve by heterologous interpolation, which has been shown not to be entirely correct. For the reasons mentioned above, the main objective of this doctoral thesis is to design, obtain and characterize recombinant proteins with reactivity against ß-lactam antibiotics. This research begins with developing an ELISA immunoassay with chemiluminescent detection for quantifying specific IgE. The assay developed allowed the determination of specific IgE below 0.1 IU/mL (0.24 ng/mL), thus allowing the identification of allergic patients with greater sensitivity, using only 25 µL of sample (serum). The immunoassay showed good sensitivity (64.6%) and excellent specificity (100%), significantly improving the predictive character of existing in vitro tests. Next, the obtaining and characterizing recombinant proteins similar to ß-lactam-specific IgE was approached using the Phage Display methodology. The first approach deals with a protein consisting of two single-domain antibodies that mimics the behavior of a specific IgE. In this way, recombinant proteins were obtained in which one of the nanobodies selectively recognizes an antigenic determinant of a ß-lactam antibiotic, while the other specifically recognizes the paratope of an anti-IgE detector antibody. These constructs were found to be stable and functional. The role of these recombinant proteins as a homologous calibrator was studied by determining the concentration of penicillin G-specific IgE present in serum by employing a chemiluminescent assay. The developed method doubled the clinical sensitivity (66%) compared to the reference method (28%), while maintaining a clinical specificity of 100%. At this point, the thesis focused on the production of recombinant specific IgE using as starting material the DNA of a patient allergic to amoxicillin and penicillin G. in combination with the Phage Display, antibody engineering and expression methods in insect cells, a recombinant-specific IgE was produced. In this way, a biological product was obtained that meets the requirements for its adoption as reference material in in vitro assays. Likewise, it was used as a homologous calibrator for the determination of specific IgE to amoxicillin. A detection limit of 0.05 IU/mL was achieved, which increased clinical sensitivity (73%) fourfold compared to the reference method (16%), while maintaining clinical specificity at 100%. / This work was supported by CSIC 2007-348, ANII FMV 2019_156321, and ANII FMV 2018_148245. P.Q.-C. acknowledges financial support from Generalitat Valenciana through the research staff training program (GVA ACIF/2018/173). This research was also funded by Agencia Estatal de Investigación (PID2019-110713RB-I00, FEDER), program UPV-La FE 2019 (P105 VALBIOAL), and PROMETEO/ 2020/094 / Quintero Campos, P. (2022). Diseño, obtención y caracterización de proteínas recombinantes a partir de determinantes antigénicos asociadas a reacciones alérgicas a ß-lactámicos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/191429 / Compendio

The most frequent intervention performed by physicians is the writing of a prescription. Modern medicine has been remarkably effective in managing diseases. Medicines play a fundamental role in the effectiveness, efficiency and responsiveness of health care systems. However, health care expenditure is a great cause for concern and many nations around the world struggle to contain rising health care costs. Pharmaceutical benefit management programmes such as pharmacoeconomics, drug utilisation review (DUR) and disease management have emerged as control tools to ensure cost effective selection and use of medicine. These managed care instruments are often used to determine whether new strategies or interventions, such as the implementation of a managed medicine reference price list, are appropriate and have "value". The general objective of this study was to investigate the influences of the implementation of a managed medicine reference price list on the usage and cost of antibiotic medicine in the private health care sector of South Africa. The research design used in this study was retrospective, non-experimental and quantitative. The data used for the analysis were obtained over a two-year study period (1 May 2001 to 31 April 2003) from the central medicine claims database of Medschem&. Data was analysed according to prevalence, cost and original (innovator) or generic medicine items. For the purpose of this study antibiotics referred to beta-lactams (penicillins, cephalosporins and "others"), erythromycin and other macrolides, tetracyclines, sulphonamides and combinations, quinolones, chloramphenicol and aminoglycosides. The results of the empirical investigation showed the total number of medicine items claimed during the study period amounted to 49098736 medicine items having a total expenditure of R7150344897.00. There was a decrease in the prevalence of original (innovator) products during the two-year period. The prevalence of generic products increased from 25.87% to 32.47%. A total of 4092495 antibiotic medicine items were claimed with a total cost of R526309279.43 representing 7.36% (n = R7150344897.00) of all pharmaceutical products purchased during the two-year period. Original antibiotics had a prevalence of 42.32%, while generic antibiotics constituted 57.68% of all antibiotic products claimed (n = 4092495). However, original (innovator) products contributed 62.32% and generic products 37.68% to the total cost of all antibiotics claimed. It was concluded that the beta-lactam antibiotics represented 56.99% of all antibiotics claimed (n = 4092495) and contributed 52.51% to the total antibiotic expenditure (n = R526309279.43) for the two-year period. The average cost of beta-lactam items ranged between R112.88 * 69.95 and R122.18 + 81.42. The Medschema Price List (MPL) was implemented in May 2001. The aim of this reference pricing system was to allocate a ceiling price to a group of drugs, which are similar in terms of composition, clinical efficacy, safety and quality, with the ultimate goal to reduce medicine expenditure. During the year of implementation of the MPL 62.24% of beta-lactam antibiotics claimed (n = 1303464) were MPL listed. These products contributed 43.25% to the total cost of all beta-lactam antibiotics (n = R157142778.38). Medical aid companies reimbursed R61649211.86 for penicillins claimed and MPL listed. If all penicillin products were claimed at the ceiling price set by the MPL, a cost saving of 2.79% could have been achieved. Cost analysis indicated that it is possible to reduce health care costs by implementing strategies with the aim to reduce medicine cost. Further research, however, is necessary and in this regard recommendations for further research were formulated. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2005.

The most frequent intervention performed by physicians is the writing of a prescription. Modern medicine has been remarkably effective in managing diseases. Medicines play a fundamental role in the effectiveness, efficiency and responsiveness of health care systems. However, health care expenditure is a great cause for concern and many nations around the world struggle to contain rising health care costs. Pharmaceutical benefit management programmes such as pharmacoeconomics, drug utilisation review (DUR) and disease management have emerged as control tools to ensure cost effective selection and use of medicine. These managed care instruments are often used to determine whether new strategies or interventions, such as the implementation of a managed medicine reference price list, are appropriate and have "value". The general objective of this study was to investigate the influences of the implementation of a managed medicine reference price list on the usage and cost of antibiotic medicine in the private health care sector of South Africa. The research design used in this study was retrospective, non-experimental and quantitative. The data used for the analysis were obtained over a two-year study period (1 May 2001 to 31 April 2003) from the central medicine claims database of Medschem&. Data was analysed according to prevalence, cost and original (innovator) or generic medicine items. For the purpose of this study antibiotics referred to beta-lactams (penicillins, cephalosporins and "others"), erythromycin and other macrolides, tetracyclines, sulphonamides and combinations, quinolones, chloramphenicol and aminoglycosides. The results of the empirical investigation showed the total number of medicine items claimed during the study period amounted to 49098736 medicine items having a total expenditure of R7150344897.00. There was a decrease in the prevalence of original (innovator) products during the two-year period. The prevalence of generic products increased from 25.87% to 32.47%. A total of 4092495 antibiotic medicine items were claimed with a total cost of R526309279.43 representing 7.36% (n = R7150344897.00) of all pharmaceutical products purchased during the two-year period. Original antibiotics had a prevalence of 42.32%, while generic antibiotics constituted 57.68% of all antibiotic products claimed (n = 4092495). However, original (innovator) products contributed 62.32% and generic products 37.68% to the total cost of all antibiotics claimed. It was concluded that the beta-lactam antibiotics represented 56.99% of all antibiotics claimed (n = 4092495) and contributed 52.51% to the total antibiotic expenditure (n = R526309279.43) for the two-year period. The average cost of beta-lactam items ranged between R112.88 * 69.95 and R122.18 + 81.42. The Medschema Price List (MPL) was implemented in May 2001. The aim of this reference pricing system was to allocate a ceiling price to a group of drugs, which are similar in terms of composition, clinical efficacy, safety and quality, with the ultimate goal to reduce medicine expenditure. During the year of implementation of the MPL 62.24% of beta-lactam antibiotics claimed (n = 1303464) were MPL listed. These products contributed 43.25% to the total cost of all beta-lactam antibiotics (n = R157142778.38). Medical aid companies reimbursed R61649211.86 for penicillins claimed and MPL listed. If all penicillin products were claimed at the ceiling price set by the MPL, a cost saving of 2.79% could have been achieved. Cost analysis indicated that it is possible to reduce health care costs by implementing strategies with the aim to reduce medicine cost. Further research, however, is necessary and in this regard recommendations for further research were formulated. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2005.