EVALUATION AND DETERMINATION OF THE SENSITIVITY AND SPECIFICITY OF A Treponema pallidum DRIED BLOOD SPOT (DBS) METHOD FOR SEROLOGIC DIAGNOSIS OF SYPHILIS Background: Syphilis is a sexually transmitted infection (STI) caused by Treponema pallidum subspecies pallidum. Syphilis is known as the "great imitator" due to the similarity of clinical signs and symptoms to other infectious diseases. The primary diagnosis of syphilis relies on clinical findings, including the examination of treponemal lesions, and/or serologic tests. Serologic tests are divided into nontreponemal and treponemal tests. Nontreponemal tests are useful for screening, while treponemal tests are used as confirmatory tests. Methods: A total of 200 serum and DBS specimens collected from patients at the Los Angeles Municipal Sexually Transmitted Disease Clinics were tested by the DBS and enzyme immunoassay (EIA) methods. These samples were sent to the Syphilis Diagnostics Laboratory, Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia for testing. Samples were blindly evaluated by the TREP-SPOTTM DBS and the TREP- SURETM EIA methods for the detection of anti-treponemal IgG- and IgM-class antibodies. Results: The sensitivity of the DBS method was 83% (95% CI, 73.89 - 89.50) and specificity was 100% (95% CI, 95.39 - 100)). The positive predictive value and negative predictive values were 100% (95% CI, 94.48 - 100) and 85% (95% CI, 77.43 - 91.0), respectively. The efficiency of the DBS method was 91.5%. The kappa value for the agreement between the DBS method and EIA assay was 0.83 (95% CI, 0.754 - 0.906). The correlation coefficient (r2) between the anti-treponemal antibody assay results obtained from DBS and serum samples was 0.94. Conclusion: DBS is an optimal choice to be used as a screening tool for the detection of anti-treponemal antibodies for the diagnosis of syphilis. The detection of anti-treponemal antibodies (TREP-SPOTTM DBS EIA) compared favorably to the results of serum-base assay (TREP-SURETM EIA), with an overall concordance of 91.5%. Dried blood spots are technically easier to obtain and are suitable blood samples for primary health care centers.

A biomonitorização humana ou biomonitoramento (BH) é definido como a medida periódica de determinada substância química ou seu metabólito em fluidos biológicos, principalmente sangue e urina, de uma população com o objetivo de avaliar a exposição e os riscos à saúde. Tal método tem se tornado comum em países desenvolvidos, porém ainda é uma prática pouco utilizada no Brasil. Isso ocorre pela dificuldade de coleta, armazenamento e transporte das amostras, principalmente em regiões sem infraestrutura e de difícil acesso. Diante disso, alguns procedimentos alternativos de coleta de amostra vêm sendo propostos. Um destes procedimentos é o Dried Blood Spot (DBS) ou coleta e armazenamento de amostra em papel-cartão. Este método oferece uma série de vantagens sobre os procedimentos de coleta convencionais, principalmente por reduzir consideravelmente o volume de amostra coletada. Entretanto, pouco se sabe sobre a estabilidade dos analitos após a deposição da amostra no cartão e do risco de contaminação da amostra pelo substrato sólido. Além disso, procedimentos de extração dos analitos do papel, para posterior quantificação, ainda não estão totalmente estabelecidos. Neste sentido, o presente trabalho avaliou de forma sistemática o procedimento de coleta de sangue por DBS visando sua futura aplicação em programas de biomonitoramento no Brasil para determinação dos elementos químicos As, Cd, Cu, Hg, Mn, Pb, Se e Zn por espectrometria de massas com plasma indutivamente acoplado (ICP-MS). Para isso, no estudo foram utilizadas três diferentes marcas comerciais de cartão coletor: Whatman 903(TM), Munktell(TM), e DMPK-C(TM). Todas as marcas de cartão apresentaram baixas concentrações dos elementos químicos. Após a deposição da amostra no papel cartão verificou-se que a concentração dos elementos químicos manteve-se estável por um período de pelo menos 60 dias (temperatura ambiente e ao abrigo da luz). Foi otimizado o método de extração dos analitos do substrato, com melhor condição obtida após a imersão do papel (corte circular de diâmetro de 1/2´´) por 60 minutos em solução extratora (0,5% v/v HNO3 e 0,01% v/v Triton(TM) X-100) na proporção de 1:50 v/v, seguida de 10 segundos de agitação por vortex. Após a extração, a solução resultante contendo os analitos foi diretamente injetada no ICP-MS. Cabe também destacar que não foram observadas diferenças estatísticas nas concentrações dos elementos químicos com coleta de sangue da veia do antebraço (sangue venoso) ou do dedo (sangue capilar). Os resultados obtidos no presente estudo, devem contribuir para a implementação deste procedimento em análises de elementos químicos (biomonitoramento da população brasileira), principalmente considerando as dificuldades de coleta, armazenamento e transporte de amostras clínicas em nosso país por sua extensão territorial. Além disso, este procedimento pode facilitar estudos com populações vulneráveis e que vivem em áreas remotas e de difícil acesso. / Human biomonitoring or biomonitoring (BH) is defined as the measurement of a particular chemical or metabolites in biological fluids, especially blood and urine, in a population to assess the level of exposure and health risks. Human biomonitoring is a common activity in developed countries, but is still an uncommon practice in Brazil. It mainly occurs due to the difficulty of collection, storage and transport of samples, particularly in regions without infrastructure and difficult access. Therefore, some alternative procedures for sample collection have been proposed. One of the procedures is the Dried Blood Spot (DBS). This method offers distinct advantages over conventional sample collection procedures, including the reduced sample volume required for analysis. However, little is known about the stability of analytes after sample deposition on the card and the possible risk of sample contamination by the solid substrate. In addition, the extraction procedures of elements from the substrate surface before determination are not yet fully established. In this sense, the present study evaluated systematically the DBS blood collection procedure aiming at its future application in biomonitoring studies in Brazil to determine the chemical elements As, Cd, Cu, Hg, Mn, Pb, Se and Zn by inductively coupled plasma mass spectrometry (ICP-MS). For this, the study used three different brands of collecting cards: Whatman 903(TM), Munktell(TM), and DMPK-C(TM). All card brands presented low concentrations of chemical elements. After sample deposition on cardboard it was found that the concentration of chemical elements remained stable for at least 60 days (at room temperature and protected from light). It was optimized the method of analytes extraction from the substrate, with the best condition obtained after immersing the paper (circular cutting diameter of 1/2 \'\') for 60 minutes in an extraction solution containing 0,5% v/v HNO3 and 0,01% v/v Triton(TM) X-100 in the ratio 1:50 v/v, followed by 10 seconds of vortex. After extraction, the resulting solution containing the analytes was directly injected into the ICP-MS. It can also be pointed out that no statistical differences was found between the concentrations of elements determined in forearm vein blood (venous blood) and finger blood (capillary blood). Taken together, the results of the present study can contribute to the employment of the DBS procedure for the screening of chemical elements in the Brazilian population, especially considering the difficulties of collection, storage and transport of clinical specimens in our country. Moreover, studies in vulnerable populations living in remote areas and of difficult access should be simplified.

A biomonitorização humana ou biomonitoramento (BH) é definido como a medida periódica de determinada substância química ou seu metabólito em fluidos biológicos, principalmente sangue e urina, de uma população com o objetivo de avaliar a exposição e os riscos à saúde. Tal método tem se tornado comum em países desenvolvidos, porém ainda é uma prática pouco utilizada no Brasil. Isso ocorre pela dificuldade de coleta, armazenamento e transporte das amostras, principalmente em regiões sem infraestrutura e de difícil acesso. Diante disso, alguns procedimentos alternativos de coleta de amostra vêm sendo propostos. Um destes procedimentos é o Dried Blood Spot (DBS) ou coleta e armazenamento de amostra em papel-cartão. Este método oferece uma série de vantagens sobre os procedimentos de coleta convencionais, principalmente por reduzir consideravelmente o volume de amostra coletada. Entretanto, pouco se sabe sobre a estabilidade dos analitos após a deposição da amostra no cartão e do risco de contaminação da amostra pelo substrato sólido. Além disso, procedimentos de extração dos analitos do papel, para posterior quantificação, ainda não estão totalmente estabelecidos. Neste sentido, o presente trabalho avaliou de forma sistemática o procedimento de coleta de sangue por DBS visando sua futura aplicação em programas de biomonitoramento no Brasil para determinação dos elementos químicos As, Cd, Cu, Hg, Mn, Pb, Se e Zn por espectrometria de massas com plasma indutivamente acoplado (ICP-MS). Para isso, no estudo foram utilizadas três diferentes marcas comerciais de cartão coletor: Whatman 903(TM), Munktell(TM), e DMPK-C(TM). Todas as marcas de cartão apresentaram baixas concentrações dos elementos químicos. Após a deposição da amostra no papel cartão verificou-se que a concentração dos elementos químicos manteve-se estável por um período de pelo menos 60 dias (temperatura ambiente e ao abrigo da luz). Foi otimizado o método de extração dos analitos do substrato, com melhor condição obtida após a imersão do papel (corte circular de diâmetro de 1/2´´) por 60 minutos em solução extratora (0,5% v/v HNO3 e 0,01% v/v Triton(TM) X-100) na proporção de 1:50 v/v, seguida de 10 segundos de agitação por vortex. Após a extração, a solução resultante contendo os analitos foi diretamente injetada no ICP-MS. Cabe também destacar que não foram observadas diferenças estatísticas nas concentrações dos elementos químicos com coleta de sangue da veia do antebraço (sangue venoso) ou do dedo (sangue capilar). Os resultados obtidos no presente estudo, devem contribuir para a implementação deste procedimento em análises de elementos químicos (biomonitoramento da população brasileira), principalmente considerando as dificuldades de coleta, armazenamento e transporte de amostras clínicas em nosso país por sua extensão territorial. Além disso, este procedimento pode facilitar estudos com populações vulneráveis e que vivem em áreas remotas e de difícil acesso. / Human biomonitoring or biomonitoring (BH) is defined as the measurement of a particular chemical or metabolites in biological fluids, especially blood and urine, in a population to assess the level of exposure and health risks. Human biomonitoring is a common activity in developed countries, but is still an uncommon practice in Brazil. It mainly occurs due to the difficulty of collection, storage and transport of samples, particularly in regions without infrastructure and difficult access. Therefore, some alternative procedures for sample collection have been proposed. One of the procedures is the Dried Blood Spot (DBS). This method offers distinct advantages over conventional sample collection procedures, including the reduced sample volume required for analysis. However, little is known about the stability of analytes after sample deposition on the card and the possible risk of sample contamination by the solid substrate. In addition, the extraction procedures of elements from the substrate surface before determination are not yet fully established. In this sense, the present study evaluated systematically the DBS blood collection procedure aiming at its future application in biomonitoring studies in Brazil to determine the chemical elements As, Cd, Cu, Hg, Mn, Pb, Se and Zn by inductively coupled plasma mass spectrometry (ICP-MS). For this, the study used three different brands of collecting cards: Whatman 903(TM), Munktell(TM), and DMPK-C(TM). All card brands presented low concentrations of chemical elements. After sample deposition on cardboard it was found that the concentration of chemical elements remained stable for at least 60 days (at room temperature and protected from light). It was optimized the method of analytes extraction from the substrate, with the best condition obtained after immersing the paper (circular cutting diameter of 1/2 \'\') for 60 minutes in an extraction solution containing 0,5% v/v HNO3 and 0,01% v/v Triton(TM) X-100 in the ratio 1:50 v/v, followed by 10 seconds of vortex. After extraction, the resulting solution containing the analytes was directly injected into the ICP-MS. It can also be pointed out that no statistical differences was found between the concentrations of elements determined in forearm vein blood (venous blood) and finger blood (capillary blood). Taken together, the results of the present study can contribute to the employment of the DBS procedure for the screening of chemical elements in the Brazilian population, especially considering the difficulties of collection, storage and transport of clinical specimens in our country. Moreover, studies in vulnerable populations living in remote areas and of difficult access should be simplified.

Quinze ans après la mise en place et l’accès élargi au traitement antirétroviral (TAR) dans les pays du Sud, la suppression virale, objectif clé de son efficacité, n’est pas atteinte chez de nombreux patients infectés par le VIH. Contrairement aux pays du Nord, où un TAR souvent plus puissant et plus robuste couplé à un suivi virologique de routine limite l’émergence de la résistance, la situation des patients vivant actuellement dans les pays du Sud est beaucoup plus préoccupante. La recherche opérationnelle menée ici vise à améliorer leur prise en charge basée sur l’approche globale de santé publique de l’OMS, et aussi leur suivi. Alors qu’elle est recommandée en routine depuis 2013, la charge virale (CV) du VIH est peu accessible et le suivi des patients reste principalement clinique voire immunologique.Dans plusieurs pays d’Afrique Centrale et de l’Ouest, nous avons documenté une multi-résistance chez presque la totalité des patients en échec virologique après une longue durée de TAR de 1ère ligne, composé de 2 inhibiteurs nucléosidiques de la transcriptase inverse (INTI, 3TC+AZT/d4T) associés à 1 inhibiteur non nucléosidique de la transcriptase inverse (INNTI, EFV/NVP). Cette résistance compromet aussi l’efficacité des INNTIs de 2ème génération, de façon importante, et aussi celle du TDF qui est actuellement recommandé en 2ème ligne. De façon plus inhabituelle, nous avons aussi rapporté que les CV élevées(>5 log10 copies/mL) étaient associées à une multi-résistance plutôt qu’à un défaut d’observance chez ces patients largement asymptomatiques. Sans véritable amélioration du suivi, au travers d’un accès élargi au test de CV permettant un diagnostic précoce de l’échec virologique, une nouvelle épidémie potentielle causée par des souches résistantes pourrait émerger en Afrique subsaharienne. Elle pourrait compromettre les efforts à réaliser pour atteindre l’objectif « 90-90-90 » (90% des infections diagnostiquées, 90% des patients diagnostiqués sous TAR, 90% des patients traités en succès virologique durable) de l’ONUSIDA et de l’OMS en 2020. En outre, notre deuxième étude menée au sein d’un hôpital de district camerounais illustre le fait que ces objectifs ambitieux seront probablement encore plus difficiles à atteindre en zones décentralisées, disposant souvent d’infrastructures de laboratoires inadaptées pour accueillir des plateformes de CV complexes.Nous avons ensuite transféré avec succès un test de CV ouvert et polyvalent universel au sein d’un laboratoire national de référence au Cameroun, capable de détecter les VIH-1 M, N, O et P, ainsi que leurs ancêtres simiens. Avec ce test RT-qPCR VIH-1/SIVcpz/SIVgor, nous avons mesuré une meilleure quantification des variants du VIH-1 M (+0.47 log10 copies/mL), comparé au test Abbott RealTime HIV-1 approuvé par la FDA. Au seuil d’échec virologique de l’OMS (1 000 copies/mL), la concordance des résultats entre les deux tests était de 95%, avec une meilleure sensibilité pour le nouveau test.Nous avons finalement cherché à savoir comment adapter ce test ouvert à une application optimale à grande échelle sur du sang séché sur papier buvard (DBS, dried blood spot en anglais), pour rendre la CV accessible en zones décentralisées, en l’absence actuelle de tests de CV simples (POC, point of care en anglais). Nous avons comparé différentes méthodes d’extraction des acides nucléiques pour réduire l’impact de l’ADN pro-viral afin d’améliorer en vain la spécificité du test sur DBS. Nous avons plutôt montré qu’avec la plupart des tests de CV par RT-qPCR, de nombreux patients pourraient être candidats pour refaire une CV de façon inappropriée (voire changer de TAR pour une 2ème ligne), ce qui induirait un surcoût programmatique.En conclusion, ces travaux de thèse ont contribué à améliorer les connaissances sur l’efficacité réelle des programmes et services de TAR, ainsi que sur l’utilisation d’outils de suivi virologique adaptés aux patients vivant avec le VIH dans les pays du Sud. / Fifteen years after the introduction and expanded access to antiretroviral therapy (ART) in resource limited countries (RLC), viral suppression, a key objective of its effectiveness is not achieved in many patients infected with HIV. Unlike developed countries, where often more powerful and robust ART coupled with a routine virological monitoring limit the emergence of resistance, the current situation of RLC is more worrying. Operational research conducted here aims to improve care and monitoring of patients living in these countries, and receiving ART according to the WHO public health approach. While recommended routinely since 2013, HIV viral load (VL) is rarely available and monitoring remains primarily clinical or immunological.In several countries in Central Africa and West, we documented high rates of HIV drug resistance (HIVDR) (99%) and multi-drug resistance (97%) in patients with virologic failure after long-term first-line ART, consisting of 2 nucleoside reverse transcriptase inhibitors (NRTI, 3TC+AZT/d4T) associated with 1 non-NRTI (NNRTI, EFV/NVP). This multi-drug resistance also reduces significantly the effectiveness of second-generation NNRTIs and of the NRTI, TDF, which is now currently recommended in 2nd line by the WHO. More unusually, we also reported that high VLs (> 5 log 10 copies / ml) are associated with multi-resistance rather than an insufficient adherence in these patients who were largely asymptomatic. Without improvement of patient monitoring, through expanded access to VL testing to enable early diagnosis of virological failure, a potential new epidemic caused by HIVDR strains could emerge in sub-Saharan Africa. This could jeopardize efforts to achieve the "90-90-90" objective (90% of diagnosed infections, 90% of diagnosed patients on ART, 90% of patients treated durable virologic success) of UNAIDS and WHO in 2020. Moreover, our second study in a district hospital in Cameroon illustrates that these ambitious targets will probably be even more difficult to achieve in decentralized areas, often having inadequate laboratory infrastructure to acquire complex VL platforms.We then successfully transferred an open and polyvalent universal VL assay in a national reference laboratory in Cameroon. This assay can detect HIV-1 group M, N, O and P and their simian ancestors. With this RT-qPCR HIV-1/SIVcpz/SIVgor assay, HIV-1 M variants were better quantified (+0.47 log10 copies / mL) compared to Abbott RealTime HIV-1 assay which is approved by the FDA. At the VL threshold of virological failure defined by the WHO (1000 copies/mL), the correlation of the results between the two tests was 95%, with a higher sensitivity for the new test.We finally explored how to adapt this open test at optimal large-scale application for dried blood spot (DBS), to make VL testing available in decentralized areas, given the current lack of point of care test for VL. We compared different extraction methods of nucleic acids that could potentially reduce the contribution of the pro-viral DNA present in DBS, with the aim to improve their specificity without success. Instead, we have shown that with most of the RT-qPCR VL assays, many patients would be inappropriately eligible to perform a repeated VL testing (or switched to a costly 2nd line ART), which increase significantly costs of national programs.In conclusion, the studies from this thesis have contributed to improve current knowledge about the effectiveness of ART programs and services and also on the use of virological monitoring tools adapted to patients living with HIV in RLCs.

This dissertation thesis is focused on development and optimization of high- performance liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS) methods, and its utility for diagnosis of inherited metabolic diseases. The first thematic part describes a comprehensive laboratory approach to diagnostics of patients with hereditary xanthinuria by determination of specific markers and enzyme activity. For this purpose HPLC method with diode array detection for measurement of hypoxanthine, xanthine, allopurinol and oxypurinol in urine and plasma and HPLC method with fluorimetric detection for analysis of pterin and isoxanthopterin in plasma were employed. These methods were successfully applied in clinical practice to ascertain two patients with hereditary xanthinuria type I. The second thematic part aims at developing and clinical application of new LC-MS/MS method for simultaneous determination of total homocysteine (tHcy), methionine (Met) and cystathionine (Cysta) in dried blood spots (DBS) and plasma. The results demonstrated the clinical utility of this method for detection of patients with homocystinuria and possibility to distinguish between defects in the remethylation and transsulfuration pathways of homocysteine metabolism. Due to ease of DBS collection and sample transportation...

Submitted by Verônica Esteves (vevenesteves@gmail.com) on 2017-09-27T17:47:16Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) ACURÁCIA DA CREATININA DOSADA EM GOTA DE SANGUE SECA EM PAPEL DE FILTRO PARA RASTREAMENTO DA DOENÇA RENAL CRÔNICA.pdf: 840099 bytes, checksum: e1a6a3188e8d5575446207666d1aac97 (MD5) / Approved for entry into archive by Verônica Esteves (vevenesteves@gmail.com) on 2017-09-27T17:51:13Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) ACURÁCIA DA CREATININA DOSADA EM GOTA DE SANGUE SECA EM PAPEL DE FILTRO PARA RASTREAMENTO DA DOENÇA RENAL CRÔNICA.pdf: 840099 bytes, checksum: e1a6a3188e8d5575446207666d1aac97 (MD5) / Made available in DSpace on 2017-09-27T17:51:13Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) ACURÁCIA DA CREATININA DOSADA EM GOTA DE SANGUE SECA EM PAPEL DE FILTRO PARA RASTREAMENTO DA DOENÇA RENAL CRÔNICA.pdf: 840099 bytes, checksum: e1a6a3188e8d5575446207666d1aac97 (MD5) Previous issue date: 2015 / Consultório / A identificação precoce da doença renal crônica (DRC) em populações de risco é preconizada devido à elevada morbidade e mortalidade dessa patologia. O rastreamento é normalmente feito utilizando testes de baixo custo e pouco invasivos, como a detecção de albumina na urina ou a dosagem da creatinina sérica. Apresentamos nesse estudo uma forma alternativa inovadora e de simples execução para dosar a creatinina. Cento e seis pessoas com alto risco de desenvolverem DRC foram envolvidas. A idade dos participantes foi de 57 ± 12 anos, 78 (73,5%) eram do sexo feminino, 43 eram brancos (40,5%), 36 eram pardos (34%) e 27 negros (25,5%). Setenta e seis por cento alegaram serem hipertensos, 30% já sabiam ter diabetes, 37% disseram ter história familiar para DRC e 22% eram tabagistas. O índice de massa corpórea (IMC) foi de 29,5 ± 6,9 kg/m2, a pressão arterial sistólica foi de 125 mmHg (intervalo interquartil [IQR] 120-140 mmHg) e a pressão arterial diastólica de 80 mmHg (IQR 70-80 mmHg). A validade do teste feito com papel filtro foi calculada considerando como a variável de interesse uma taxa de filtração glomerular estimada menor que 60/ml/min/1,73m2, calculado pelas equações MDRD e CKD-EPI. Desta maneira, a sensibilidade do PF foi de 96% na equação MDRD e de 94% na CKD-EPI. O valor preditivo positivo foi de 96% para ambas equações. A acurácia mostrou um desempenho de 92% na MDRD e de 90% na CKD-EPI. O método de Bland e Altman mostrou que os valores de creatinina entre os dois testes estão numa faixa relativamente estreita (+0.68 mg/dL a -0.55 mg/dL) para um desvio padrão de ±1.96. Concluímos que a dosagem da creatinina em papel filtro é um teste pouco invasivo e apresentou um bom desempenho para rastrear a DRC. / Chronic kidney disease (CKD) screening is advisable in populations with risk factors for chronic kidney disease due to its high morbidity and mortality. It´s usually performed by sampling blood for creatinine and urine for albuminuria. Here we present an innovative and simpler method, by measuring creatinine in a dry blood spot on filter paper. One-hundred and six individuals at high risk for CKD were enrolled. Mean age was 57±12 years, 74% were female, 40% white, and 60% non-white. Seventy-six percent were hypertensive, 30% diabetic, 37% had family history of CKD, and 22% of smoking. The BMI was 29.5 ± 6.9 kg/m2, median systolic blood pressure was 125 mmHg (IQR 120-140 mmHg) and median diastolic blood pressure was 80 mmHg (IQR 70-80 mmHg). Adopting CKD with the found of a e_GFR60/ml/min/1,73m2 for MDRD and CKD-EPI equation, the sensitivity was 96% for MDRD and 94% in CKD-EPI. The positive predictive value was 96% for both equations. The accuracy was 92% in MDRD and 90% for CKD-EPI. . A Bland and Altman analysis showed a relatively narrow range of creatinine values differences (+0.68 mg/dL to -0.55 mg/dL) inside the ±1.96 SD, without systematic differences. Accordingly, the minimally invasive dry blood spot creatinine measurement disclosed good performance, and may be useful as a screening tool for CKD.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease that causes a disorder of steroidogenesis in the adrenal cortex. This disease is a part of a panel of diseases searched in preclinical nationwide neonatal screening. The methodology is based on measuring the concentration of 17-hydroxyprogesterone (17-OHP) in a dried blood spot using fluorescence immunoassay (FIA). However, this determination is not entirely specific and generates a high rate of false positive results (up to 4.3 %). In this diploma thesis the LC-MS / MS method was developed. This method measures selected steroid hormones involved in cortisol metabolism with respect to the diagnosis of CAH disease. The method was validated and applied to clinical samples, it identified CAH patients from negative controls and significantly reduced the false positivity of neonatal screening results. Compared to the FIA results, the LC-MS / MS method reduced false positivity up to 50 % by evaluating the concentration of 17-OHP. Moreover, by extending the diagnostic algorithm with other measured markers, the reduction was enhanced up to 98%. The developed method is also applicable for the measurement of serum and plasma samples, respectively, and has become a part of the confirmation tests for suspected CAH screening findings. Key...

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Un nouveau règlement (CE) n° 1901/2006 établi par le Parlement européen et le Conseil de l’UE, relatif aux médicaments à usage pédiatrique, vise à améliorer la santé et la qualité de vie des enfants en Europe, en garantissant que les nouveaux médicaments pédiatriques et les médicaments déjà commercialisés seront pleinement adaptés à leurs besoins spécifiques. Ce règlement prévoit de nouvelles obligations pour l'industrie pharmaceutique, assorties de récompenses et d'incitations. Dans ce contexte, un plan d’investigation pédiatrique a été proposé pour l’ivabradine dans plusieurs sous-groupes de la population pédiatrique dans le traitement de l’insuffisance cardiaque chronique. L’ivabradine est une molécule déjà commercialisée chez l’adulte dans la prise en charge de l’angor, et de l’insuffisance cardiaque. Un premier travail a été d’aider au design de cette étude pédiatrique : évaluer la formulation pédiatrique, aider au choix de la dose initiale à administrer chez l’enfant, choisir le protocole de prélèvements et conseiller la méthode de prélèvements. Pour évaluer la formulation pédiatrique, une étude a été conduite pour déterminer la biodisponibilité relative de la formulation pédiatrique par rapport aux comprimés utilisés chez l’adulte. Une biodisponibilité relative similaire a été retrouvée entre les deux formulations. Une approche physiologique (PBPK « Physiollogically based PharmacoKineticsmodel ») a été utilisé pour prédire la dose initiale à administrer et pour proposer un protocole de prélèvements PK. La méthode DBS (Dried blood spot) consistant à collecter à chaque temps de prélèvement une goutte de sang (au pli du coude ou au bout du doigt) a été recommandée. La première dose à administrer chez l’enfant peut être également être déterminée par des modèles de population développés chez l’adulte et adaptés à l’enfant grâce à l’allométrie et à l’ajout de fonctions de maturation. Cette approche a été comparée au PBPK dans le cas de l’ivabradine et des résultats similaires ont été obtenus. Un deuxième travail a été réalisé après que l’étude clinique ait été conduite dans la population pédiatrique. L’étude a été menée chez 116 enfants (74 enfants recevant l’ivabradine, 42 recevant le placebo) âgés de 6 mois à 18 ans et les données ont été analysées. Tout d’abord, une relation a été établie entre les concentrations d’ivabradine plasmatiques et les concentrations d’ivabradine mesurées dans le sang total. Puis, afin de décrire les concentrations d’ivabradine et de son métabolite, un modèle de population prenant en compte l’effet de l’âge et du poids a été développé. En comparant les expositions plasmatiques, une dose par kilogramme plus élevée aurait été nécessaire chez les patients les plus jeunes pour atteindre un niveau d’exposition similaire aux patients plus âgés. Enfin, il a été monté que la relation PK/PD qui avait développé chez l’adulte était conservée dans la population pédiatrique. / New legislation governing the development and authorization of medicines for use in children was introduced in the European Union (EU) in January 2007. This Regulation aims to facilitate the development and accessibility of medicinal products for use in the paediatric population, to ensure that medicinal products used to treat the paediatric population are subject to ethical research of high quality and are appropriately authorised for use in the paediatric population, and to improve the information available on the use of medicinal products in the various paediatric populations. Several rewards and incentives for the development of paediatric medicines for children are available in the European Union (EU). In compliance with the paediatric European regulation, a study will be conducted in paediatric patients with CHF with the objective to determine the efficacious and safe dose of ivabradine, a compound already marketed in adults, and to assess its efficacy and safety in children over 1 year old. A first work was to help design a paediatric study for ivabradine focusing on: the paediatric formulation evaluation, the doses to be administered, the sampling design and the sampling technique. A study was conducted in order to assess the relative bioavailability (Frel) of the paediatric formulation and a similar Frel was observed between the paediatric formulation and the adult marketed tablet. PBPK modelling was used to predict initial doses to be administered in the paediatric study and to select the most appropriate sample time collections. The dried blood spot (DBS) technique was recommended in the clinical trial in children. A secondary objective was to perform a comparison of the prediction of ivabradine pharmacokinetics (PK) in children using a physiologically-based pharmacokinetic (PBPK) approach and allometric scaling of a population pharmacokinetic (PPK) model. Simulations obtained by both the PBPK approach and allometric scaling of a PPK model were compared a posteriori to the paediatric study observations. Both PPK and PBPK approaches allowed an adequate prediction of the PK of ivabradine and its metabolite in children. The second work was done after the study conduction in the paediatric population. The study was performed in 116 children (74 received ivabradine, 42 received the placebo) aged from 6 months to less than 18 years old and data were analysed. The relationship between blood and plasma concentrations was described using linear mixed effect models. In order to describe ivabradine and its metabolite blood concentrations in children, a joint population PK model was developed taking into account weight & age effects on PK parameters. Plasma exposure comparison indicated that higher dose/kg were necessary to achieve a similar exposure between younger and older children. The PK/PD relationship in adult patients is conserved in children.

Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders (“conditions”) then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40–50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, both in terms of methodology (modernised) and with regard to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. We can only accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate action by working together.