The HLA-DRB3/4/5 loci are closely linked to the HLA-DRB1 gene. Mismatches in these loci occur with a frequency of about 8%–12% in otherwise 10/10 HLA-matched transplant pairs. There is preliminary evidence that these disparities may associate with increased acute graft-versus-host disease (GvHD) rates. The aim of this study was to analyze a large cohort of German patients and their donors for HLA-DRB3/4/5 compatibility and to correlate the HLA-DRB3/4/5 matching status with the outcome of unrelated hematopoietic stem cell transplantation (uHSCT). To this end, 3,410 patients and their respective donors were HLA-DRB3/4/5 and HLA-DPB1 typed by amplicon-based nextgeneration sequencing (NGS). All patients included received their first allogeneic transplant for malignant hematologic diseases between 2000 and 2014. Mismatches in the antigen recognition domain (ARD) of HLA-DRB3/4/5 genes were correlated with clinical outcome. HLA-DRB3/4/5 incompatibility was seen in 12.5% (n = 296) and 17.8% (n = 185) of the 10/10 and 9/10 HLA-matched cases, respectively. HLA-DRB3/4/5 mismatches in the ARD associated with a worse overall survival (OS), as shown in univariate (5-year OS: 46.1% vs. 39.8%, log-rank p = 0.038) and multivariate analyses [hazard ratio (HR) 1.25, 95% CI 1.02–1.54, p = 0.034] in the otherwise 10/10 HLAmatched subgroup. The worse outcome was mainly driven by a significantly higher nonrelapse mortality (HR 1.35, 95% CI 1.05–1.73, p = 0.017). In the 9/10 HLA-matched cases, the effect was not statistically significant. Our study results suggest that mismatches within the ARD of HLA-DRB3/4/5 genes significantly impact the outcome of otherwise fully matched uHSCT and support their consideration upon donor selection in the future.

De nos jours, la greffe de cellules souches hématopoïétiques (GCSH) de type allogénique est le traitement standard pour de nombreuses hémopathies malignes, et ce, en dépit des taux élevés de complications liées à la transplantation, telles que les infections, les rechutes et la maladie du greffon contre l’hôte (GVH), associées à cette procédure. Depuis les deux dernières décennies, plusieurs stratégies visant à contrôler la maladie du GVH ont émergé et incluent la déplétion partielle et la manipulation ex vivo des cellules T du greffon. En revanche, ces stratégies peuvent entrainer de sévères déficits immunitaires post-greffe. En comparaison avec les autres sources de cellules souches, une faible incidence de la maladie du GVH et de rechute est observée chez les patients recevant une greffe de sang de cordon ombilical (SC). En contrepartie, ce type de greffe engendre des retards dans la restauration immunitaire, rendant ainsi les patients plus susceptibles aux agents pathogènes. À l’inverse, une augmentation plus importante de la production de cellules T naïves, d’émigrants thymiques récents et de l’abondance des clonotypes des cellules T fut détectée chez les patients ayant reçu une GCSH dérivée du sang d’un seul cordon traité avec UM171, une molécule utilisée pour l'amplification ex vivo des CSHs dérivés de SC, relativement aux patients ayant reçu une GCSH standard. Dans ce mémoire, nous essaierons de comprendre le mécanisme moléculaire sous-jacent à la restauration immunitaire chez les patients transplantés avec un greffon de SC traité avec UM171, nous tenterons de déceler des progéniteurs lymphoïdes au sein des cellules de SC traitées avec UM171 et nous essaierons d’identifier un marqueur de surface qui pourrait être utilisé pour enrichir les progéniteurs lymphoïdes. Nos résultats démontrent la présence de deux "clusters" lymphoïdes, PCL et LMPP, qui expriment potentiellement CD10, et sont plus nombreux dans les échantillons de SC CD34+ traités avec UM171 comparativement aux contrôles. En utilisant les marqueurs de surface CD10 et CD45RA, nous avons pu identifier deux populations CD34+ (CD10medCD45RAmed/lo et CD10hiCD45RAhi) qui sont multipliées dans les cultures traitées avec UM171 relativement aux contrôles. En outre, nous démontrons également que seules les cellules CD45RA- peuvent générer les deux populations CD10+ in vitro, ce qui suggère que les cellules CD10medCD45RAmed/lo définissent une population plus primitive que les cellules CD10hiCD45RAhi. De surcroit, nous proposons que la population CD10medCD45RAmed/lo pourrait contenir des LMPPs qui se différencieront en PCLs inclus dans la population CD10hiCD45RAhi. D'un point de vue fonctionnel, nous observons un développement et une maturation des lymphocytes T nettement plus rapide pour la population CD10+CD34+ relativement à la population CD34+ totale à 3 et 6 semaines, ce qui suggère que CD10 pourrait être utilisé comme marqueur de progéniteurs lymphoïdes. Par ailleurs, nous détectons également un pourcentage plus élevé de cellules ayant un phénotype ILC dans la population CD10+CD34+ comparativement à la population CD34+ totale, ce qui suggère que la population CD10+CD34+ peut potentiellement contenir des progéniteurs lymphoïdes capables de produire différents types de cellules immunitaires. D’autre part, la population CD45RA+ produit plus rapidement des lymphocytes T contrairement à la population CD45RA-. En conséquence, nous proposons que la population CD45RA- contient des cellules primitives (ex. CSH) qui à leur tour génèrent des progéniteurs multipotents contenus dans la population CD10+CD34+ (ex. LMPP) et qui éventuellement produiront des progéniteurs à potentiel plus restreint inclus dans la population CD45RA+ (ex. CLP). En somme, ce mémoire identifie pour la première fois une population CD10+CD34+ présente dans le greffon traité avec UM171 avec un potentiel T et produisant potentiellement des ILCs. L’un des problèmes majeurs liés à la GCSH dérivée du SC est une restauration immunitaire retardée; par conséquent, optimiser l’infusion de cellules CD34- en augmentant le nombre de progéniteurs lymphoïdes pourrait considérablement aider à stimuler la restauration immunitaire chez les patients recevant une GCSH dérivée de SC. / Currently, allogeneic hematopoietic stem cell transplantation is a standard treatment for many hematological malignancies, although high rates of transplant-related complications such as infections, relapse, and GVHD has constrained the implementation of HSCT. Strategies have emerged to manage GVHD and include partial T-cell depletion and ex vivo manipulation of donor T cells, albeit they have adverse effects on post-transplantation immune recovery and can cause profound immunodeficiency. In comparison to other stem cell sources, a lower incidence of chronic GVHD and relapse is reported in patient receiving cord blood (CB) transplant, although they also display an enhanced susceptibility to pathogens caused by an ineffective T-cell reconstitution. In contrast, we reported a greater increase in naïve T cells production, recent thymic emigrants and T cell clonotype from 3 months to 6 and 12 months in patient transplanted with a single CB graft expanded with UM171, a small molecule used for the ex vivo expansion of CB HSCs, as compared to counterpart patients receiving unmanipulated CB graft. In this master research, we aimed to understand the molecular mechanism underlying T-cell reconstitution in patients transplanted with UM171- expanded CB graft. We tried to identify lymphoid progenitors within the highly heterogeneous CD34+ CB cells treated with UM171 using Cite-Seq, find a surface marker that can be used to enrich for early lymphoid progenitors using flow cytometry, and assess their lineage potential using artificial thymic organoids. Our results highlight the presence of two lymphoid clusters, CLP and LMPP, expressing CD10. The LMPP cluster is about 6 fold expanded in UM171-treated cells as compared to uncultured and DMSO-treated cells. Using the marker CD10 and CD45RA we identify two CD34+ populations (CD10medCD45RAmed/lo and CD10hiCD45RAhi) that are significantly expanded in CD34+ CB cells cultured in the presence of UM171 in contrast to uncultured CD34+ CB cells and DMSO-supplemented cultures. Furthermore, we demonstrate that only the CD45RA-negative cells can give rise to both CD10-positive subsets in vitro, suggesting that CD10medCD45RAmed/lo cells define a subset with a more primitive phenotype than CD10hiCD45RAhi. We propose that the CD10medCD45RAmed/lo subset could contain LMPPs that will commit to CLPs contained within the CD10hiCD45RAhi subset. Functionally, we compared T-cell development and maturation of the CD10+CD34+ subset (including CD10medCD45RAmed/lo and CD10hiCD45RAhi) to the CD45RA+CD34+, CD45RA-CD34+ and total CD34+ subsets and denote a faster T cell development and maturation at 3 and 6 weeks within the CD10-positive subset in contrast to the total CD34+ subset, suggesting that CD10 can be used as a marker of lymphoid precursors in UM171 cultures. We also observe a higher percentage of ILC-like cells within the CD10-positive subset as compared to total CD34+ cells suggesting that the CD10- positive subset potentially contains lymphoid precursors with multilineage capacity. Faster T cell development is observed for the CD45RA+ subset whereas CD45RA- cells display the slowest T cell development, hence we propose that the CD45RA- subset contains primitive cells (e.g. HSC) that segregate into lineage-biased multipotent progenitors enriched in the CD10-positive subset (e.g. LMPP) that will give rise to lineage-restricted precursors that are CD45RA+ (e.g. CLP). In sum, our findings represent the first identification of a CD10-positive population within the UM171- expanded graft that has T potential and could potentially produce ILCs. Delayed immune reconstitution is a major obstacle impeding the implementation of CB HSCT; therefore optimizing infusion of CD3+ donor cells by enriching for lymphoid precursors could help boost T-cell reconstitution following allogeneic HSCT.

The HLA-DRB3/4/5 loci are closely linked to the HLA-DRB1 gene. Mismatches in these loci occur with a frequency of about 8%–12% in otherwise 10/10 HLA-matched transplant pairs. There is preliminary evidence that these disparities may associate with increased acute graft-versus-host disease (GvHD) rates. The aim of this study was to analyze a large cohort of German patients and their donors for HLA-DRB3/4/5 compatibility and to correlate the HLA-DRB3/4/5 matching status with the outcome of unrelated hematopoietic stem cell transplantation (uHSCT). To this end, 3,410 patients and their respective donors were HLA-DRB3/4/5 and HLA-DPB1 typed by amplicon-based nextgeneration sequencing (NGS). All patients included received their first allogeneic transplant for malignant hematologic diseases between 2000 and 2014. Mismatches in the antigen recognition domain (ARD) of HLA-DRB3/4/5 genes were correlated with clinical outcome. HLA-DRB3/4/5 incompatibility was seen in 12.5% (n = 296) and 17.8% (n = 185) of the 10/10 and 9/10 HLA-matched cases, respectively. HLA-DRB3/4/5 mismatches in the ARD associated with a worse overall survival (OS), as shown in univariate (5-year OS: 46.1% vs. 39.8%, log-rank p = 0.038) and multivariate analyses [hazard ratio (HR) 1.25, 95% CI 1.02–1.54, p = 0.034] in the otherwise 10/10 HLAmatched subgroup. The worse outcome was mainly driven by a significantly higher nonrelapse mortality (HR 1.35, 95% CI 1.05–1.73, p = 0.017). In the 9/10 HLA-matched cases, the effect was not statistically significant. Our study results suggest that mismatches within the ARD of HLA-DRB3/4/5 genes significantly impact the outcome of otherwise fully matched uHSCT and support their consideration upon donor selection in the future.

Functional impairment of the bone marrow (BM) niche has been suggested as a major reason for prolonged cytopenia and secondary graft failure after allogeneic hematopoietic cell transplantation (alloHCT). Because mesenchymal stromal cells (MSCs) serve as multipotent progenitors for several niche components in the BM, they might play a key role in this process. We used collagenase digested trephine biopsies to directly quantify MSCs in 73 patients before (n = 18) and/or after alloHCT (n = 65). For the first time, we demonstrate that acute graft-versus-host disease (aGvHD, n = 39) is associated with a significant decrease in MSC numbers. MSC reduction can be observed even before the clinical onset of aGvHD (n = 10). Assessing MSCs instantly after biopsy collection revealed phenotypic and functional differences depending on the occurrence of aGvHD. These differences vanished during ex vivo expansion. The MSC endotypes observed revealed an enhanced population of donor-derived classical dendritic cells type 1 and alloreactive T cells as the causing agent for compartmental inflammation and MSC damage before clinical onset of aGvHD was ascertained. In conclusion, MSCs endotypes may constitute a predisposing conductor of alloreactivity after alloHCT preceding the clinical diagnosis of aGvHD.

O diabetes mellitus tipo 1 (DM-1) e a esclerose múltipla (EM) são doenças auto-imunes órgão-específicas, inflamatórias, mediadas por células T e B auto-reativas e caracterizadas pela destruição seletiva de células b pancreáticas produtoras de insulina e do sistema nervoso central, respectivamente. Acredita-se que a desregulação da expressão de genes reguladores da maquinaria apoptótica possa contribuir para o desenvolvimento da auto-imunidade, visto que algumas dessas moléculas participam nos processos de tolerância central e periférica de linfócitos auto-reativos. O objetivo deste projeto foi analisar a expressão de moléculas reguladoras das vias intrínseca, extrínseca e da Família de proteínas inibidoras da apoptose (IAP) em 33 indivíduos saudáveis, 15 pacientes com DM-1 e 18 com EM submetidos à terapia de imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (IAD/TACTH). As células mononucleares (CMN) foram isoladas do sangue periférico dos controles e de pacientes nos períodos pré-mobilização (pré-mob), pré-condicionamento (pré-cond), D+180, D+360, D+540 e D+720 pós-transplante. As CMN foram utilizadas para extração de RNA, síntese de cDNA, quantificação da expressão por PCR em tempo real dos genes a1, bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bid, bik, bim, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 e cIAP-2 e protéica de Bcl-2, Bcl-xL, Bak, Bim e c-FLIPL por western-blotting. Os resultados de expressão gênica foram representados por unidades relativas de expressão em medianas nas diferentes amostras. Os pacientes com DM-1 apresentaram diminuição da expressão dos genes anti-apoptóticos bcl-2 (mediana: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) e cIAP-1 (1,24; p=0,003) nas CMN dos pacientes no período pré-mob em relação aos indivíduos saudáveis (medianas: bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), enquanto a expressão de cIAP-2 (60,8; p=0,0005) estava aumentada em relação aos controles (23,3). Foi observada redução significativa na expressão dos genes pró-apoptóticos bad (0,002; p<0,0001), bax (0,01; p=0,002) e fasL (1,66; p=0,001) no período pré-mob comparada aos controles sadios (bad: 0,23; bax: 2,79; fasL: 3,56). Os níveis de RNAm de bid (0,10; p=0,001) e bok (0,72; p=0,006) estavam elevados no pré-mob em relação ao grupo controle (bid: 0,004; bok: 0,31). As moléculas bcl-2, bcl-w, bcl-xL, mcl-1, bad, bax, bok, fasL e cIAP-1 atingiram níveis de RNAm similares aos controles após o TACTH. Foi verificado que a expressão de bcl-w, cIAP-1 e noxa estava maior nos pacientes com DM-1 em remissão quando comparados àqueles em recaída. A diminuição da expressão de a1, bcl-2 e bcl-w e o aumento de fas e noxa correlacionaram-se às porcentagens de hemoglobina glicosilada, concentração de auto-anticorpos GAD65, e aos níveis séricos de peptídeo-C após o transplante. Os pacientes com EM mostraram uma expressão reduzida dos genes anti-apoptóticos bcl-w (0,11; p=0,02) e cIAP-1 (1,87; p=0,04) no pré-mob comparada aos valores dos controles (bcl-w: 0,27; cIAP-1: 7,75) e maior expressão dos genes a1 (90,8; p=0,001) e cIAP-2 (58,8; p=0,009) em relação aos controles (a1: 12,7; cIAP-2: 22,3). As moléculas pró-apoptóticas bad (0,007; p=0,01) e bax (0,0007; p=0,004) mostraram menor expressão nas CMN no período pré-mob do que nos controles (bad: 0,27; bax: 1,24). Os genes bid (20,7; p=0,004), bik (0,84; p=0,02) e bok (1,77; p=0,0001) possuíam maior expressão no período pré-mob em relação aos indivíduos sadios (bid: 2,64; bik: 0,33; bok: 0,26). Não foram observadas diferenças significativas na expressão das moléculas da via extrínseca da apoptose nos pacientes com EM (p>0,05) nos períodos avaliados. Os valores de expressão de bcl-w, bak, bax, bik, bok e cIAP-1 atingiram níveis semelhantes aos controles após o transplante. A expressão dos genes bcl-2, cIAP-1, bad e bax estava maior nos pacientes em remissão da EM quando comparados àqueles em progressão neurológica. O aumento da expressão dos genes pró-apoptóticos bax, bak e bimEL correlacionou-se inversamente aos valores de EDSS dos pacientes com EM após o TACTH. Os resultados de expressão protéica foram equivalentes aos de expressão gênica nas duas doenças, com exceção dos dados das proteínas Bcl-2 e Bim. Em conjunto, os resultados demonstraram a desregulação da expressão de várias moléculas anti- e pró-apoptóticas nas CMN dos pacientes com DM-1 e EM. Esses achados sugerem a associação de alterações nos processos de apoptose celular com o surgimento e persistência de células auto-reativas no DM-1 e EM. Os dados indicam que essas alterações, principalmente a diminuição da expressão de moléculas pró-apoptóticas, como bak e bax, possam contribuir para a patogênese do DM-1 e EM. Além disso, a terapia de IAD/TACTH foi capaz de modular a expressão da maioria dos genes anormalmente expressos nas CMN dos pacientes com DM-1 e EM, já que esses atingiram níveis de expressão similares ao grupo controle após o transplante. Esta normalização da expressão de vários genes analisados correlacionou-se com a remissão clínica da doença na maioria dos pacientes / Type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) are inflammatory, organ-specific autoimmune diseases characterized by selective destruction of insulin-producing pancreatic -cells and central nervous system, respectively, by autoreactive B and T cells. Deregulation of apoptotic machinery is supposed to contribute to self-tolerance breakdown and autoimmune diseases pathogenesis, since apoptotic molecules have an important role in B and T lymphocytes central and peripheral tolerance mechanisms. The aim of this study was to evaluate the expression of pro and anti-apoptotic molecules from intrinsic and extrinsic apoptotic pathways and IAP Family members in 33 healthy individuals, 15 T1DM and 18 MS patients submitted to high-dose immunossupression therapy followed by autologous hematopoietic stem cell transplantation (HDI/AHSCT). Peripheral blood mononuclear cells (PBMC) were isolated from controls and patients at pre-mobilization (pre-mob), pre-conditioning (pre-cond), D+180, D+360, D+540 and D+720 post-transplantation. PBMC were used for RNA extraction, cDNA synthesis, gene quantification of a1, bcl-2, bcl-w, bcl-xL, bad, bak, bax, bid, bik, bimEL, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 and cIAP-2 by Real Time PCR and Bcl-2, Bcl-xL, Bak, BimEL and c-FLIPL proteins detection by western-blotting. Results are expressed as median of relative expression units. Results from T1DM patients indicated that antiapoptotic molecules bcl-2 (median: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) and cIAP-1 (1,24; p=0,003) were downregulated at pre-mob compared with healthy controls (medians bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), while cIAP-2 (60,8; p=0,0005) gene expression was upregulated compared to healthy controls (23,3). We observed a significant decrease in proapoptotic bad (0,002; p<0,0001), bax (0,01; p=0,002) and fasL (1,66; p=0,001) genes expression in patients PBMC at pre-mob period compared to healthy subjects (bad: 0,23; bax: 2,79; fasL: 3,56). mRNA levels of bid (0.10; p=0.001) and bok (0.72; p=0.006) were elevated at pre-mob period when compared to control group (bid: 0.004; bok: 0.31). The bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bok, fasL and cIAP-1 mRNA levels reached controls levels after HDI/AHSCT. We observed that bcl-w, cIAP-1 and noxa gene expression were increased in T1DM patients in remission when compared to relapsed patients. The decreased antiapoptotic gene expression and increased in proapoptotic molecules correlated with decreased glicosilated hemoglobin percentages (Hb A1C) and anti-GAD65 antibodies and increased peptide-C levels. Results from MS patients showed decreased bcl-w (0,11; p=0,02) and cIAP-1 gene expression (1,87; p=0,04) in patients PBMC at pre-mob period compared to healthy controls (bcl-w: 0,27; cIAP-1: 7,75) and increased expression of a1 (90,8; p=0,001) and cIAP-2 (58,8; p=0,009) compared to controls (a1: 12,7; cIAP-2: 22,3). Proapoptotic molecules bad (0.007; p=0.01) and bax (0.0007; p=0.004) showed decreased gene expression at pre-mob compared to control group (bad: 0.27; bax: 1.24). bid (20.7; p=0.004), bik (0.84; p=0.01) and bok genes (1.77; p=0.0001) showed increased expression at pre-mob compared to healthy controls (bid: 2.64; bik: 0.33; bok: 0.26). Significant differences were not observed in the expression of the extrinsic pathway genes in pre-mob and healthy controls samples (p>0.05). bcl-w, bak, bax, bik, bok and cIAP-1 expression values reached healthy control values after transplantation. We observed that bcl-2, cIAP-1, bad and bax gene expression was increased in MS patients in disease remission when compared to patients with neurologic progression. Significant correlation of increased proapoptotic genes expression with decreased EDSS values in MS patients after HDI/AHSCT was observed. Results of protein quantification of apoptotic molecules in PBMC of T1DM and MS patients were similar to the gene expression results of these molecules, except for Bcl-2 and Bim proteins. Taken together, these data indicate a deregulated expression of anti- and proapoptotic genes in T1DM and MS patients PBMC. These data suggest an association of deregulated apoptosis with emergence and maintenance of autoreactive lymphocytes in analyzed patients. Based on these results, we suggest that this altered gene expression profile, mainly the decreased proapoptotic genes expression, as bak and bax, may contribute to T1DM and MS pathogenesis. Furthermore, we showed that the HDI/AHSCT therapy was able to modulate and normalize the expression of most genes abnormally expressed in T1DM and MS patients at pre-transplant period. Many analyzed genes achieved expression levels similar to healthy controls. The normalization of the expression of many evaluated genes correlated to disease remission in the majority of the patients.

Introdução: No presente estudo foram avaliados biomarcadores na ocorrência de febre em pacientes neutropênicos após transplante de células tronco hematopoiéticas (TCTH). Objetivo: O objetivo principal foi avaliar os valores séricos de biomarcadores: proteína C reativa (PCR), procalcitonina (PCT) e IL-6 (interleucina-6) que possam identificar precocemente infecção em TCTH. Outro objetivo foi fatores de risco para óbito nessa população. Métodos: Os biomarcadores foram avaliados em um estudo prospectivo que incluiu 296 pacientes neutropênicos, submetidos a TCTH autólogo ou alogênico. Os biomarcadores PCT, PCR e IL-6 foram dosados nos seguintes momentos:dia da neutropenia constatada sem febre, evento febril ou hipotermia (T < 35ºC), 24 h após a febre ou hipotermia, 72 horas após a febre ou hipotermia e febre prolongada ou seja 48 horas após a coleta no momento anterior ou na persistência da febre, cinco dias após a coleta no momento anterior. Os dados clínicos e laboratoriais, foram avaliados até a evolução para alta ou o óbito, em uma planilha Excel® 2003 e foram processados pelos programas SPSS e STATA. Os pacientes foram classificados nos seguintes grupos (I- afebril; II- febre de origem indeterminada FOI e III- febre clinica ou microbiologicamente comprovada) em relação a cada marcador estudado (PCT, PCR e IL-6). Foram feitos cálculos para estabelecer área sob a curva ROC, sensibilidade, especificidade, para avaliação da febre e óbito. Para avaliar o desfecho óbito foi realizada análise multivariada com regressão logística stepwise. Resultados: Dos 296 pacientes, 190 apresentaram febre. Duzentos e dezesseis (73%) foram submetidos a transplantes autólogos e 80 (27,0%) alogênicos. Dos 80 casos de TCTH alogênicos 74 (92,6%) eram aparentados e apenas 6 (7,4%) aparentados. Dos 80 casos alogênicos 69 (86,3%) eram fullmatch e 11(13,7%) mismatch. Em relação aos grupos já citados acima, temos a seguinte distribuição: grupo I: 106 pacientes (35,8%); grupo II: 112 pacientes (37,8%) e grupo III: 78 (26,4%). Os valores de média e mediana da IL-6 no momento afebril no grupo I em relação ao grupo II (p = 0,013), apresentando valor significativamente maiores. Os níveis da PCR no grupo I diferiram de forma significativa dos encontrados no grupo III (p < 0,05). Os grupos diferiram em relação aos níveis de IL-6 e de PCR no momento febril. O grupo II apresentou concentrações de IL-6 e de PCR significativamente menores que o grupo III. Os melhores valores de corte de PCT para os momentos de coleta: febre, 24 horas após a febre, 72 horas de febre, e febre prolongada foram respectivamente: 0,32; 0,47; 0,46 e 0,35?g/L. No momento da febre a sensibilidade foi 52,3 e a especificidade 52,6 para o diagnóstico de infecção. Os melhores valores de corte de PCR para os momentos de febre, 24 horas após, 72 horas após e febre prolongada foram, respectivamente: 79, 120, 108 e 72 mg/L. No momento da febre a sensibilidade foi 55,4 e especificidade foi 55,1. Os melhores valores de corte de IL-6 para os momentos de febre, 24 h após, 72 horas após a febre e febre prolongada foram respectivamente: 34, 32, 16 e 9 pg/mL. A sensibilidade e especificidade no momento da febre foram respectivamente: 59,8 e 59,7. Na análise dos três biomarcadores no grupo de pacientes autólogos, verifica-se que só a IL-6 apresenta valores significativos nos momentos iniciais (afebril, febre e 24 horas após a febre). Os seguintes fatores de risco independentes foram identificados na análise multivariada: doador aparentado, doador não aparentado, infecção por Gram-negativo, DHL >= 390 (UI/L), ureia >= 25 (mg/dL) e PCR >= 120 (mg/L). Conclusões: IL-6 e PCR têm associação com diagnóstico precoce de infecção clinica ou microbiologicamente confirmada em neutropenia febril após TCTH. A associação dos três marcadores não apresentou nenhuma vantagem, e não melhorou a acurácia diagnóstica. A IL-6 foi o único biomarcador significativamente associado de forma precoce com infecção quando avaliado apenas pacientes submetidos a TCTH autólogos As variáveis independentes associadas com óbito foram: transplante alogênico, infecção por Gram-negativos, DHL >= 390UI/L no momento da febre e ureia >= 25 mg/dL no momento da febre e PCR >= 120 (mg/L) / Introduction: In the present study, biomarkers were assessed in the occurrence of fever in neutropenic patients upon hematopoietic stem cell transplantation (HSCT). Objective: The main objective was to assess the serum values of biomarkers: C-reactive protein (CRP), procalcitonin (PCT) and IL-6 (interleukin-6) which can early identify infection in HSCT. Another objective was risk factors for death in that population. Methods: The biomarkers were assessed in a prospective study which comprised 296 neutropenic patients submitted to autologous or allogeneic HSCT. The biomarkers PCT, CRP and IL-6 were dosed at the following moments: day of afebrile neutropenia, febrile event or hypothermia (T < 35ºC), 24 h upon fever or hypothermia, 72 hours upon fever or hypothermia and long-standing fever, that is, 48 hours upon the last sampling or at fever persistence, five days upon the last sampling. The clinical and laboratory data were assessed up to the evolution to discharge or death, in an Excel® 2003 spreadsheet and were processed by the SPSS and STATA software. Patients were classified in the following groups (I- afebrile; II- fever of unknown origin FUO and III- clinically or microbiologically proven fever) in regard to each biomarker studied (PCT, CRP and IL-6). Calculations were made to establish the area under the ROC curve, sensitivity, specificity, for the assessment of the evolution and death. In order to assess the death outcome, a multivariate analysis with stepwise logistic regression was conducted. Results: Out of the 296 patients, 190 had fever. Two hundred and sixteen (73%) were submitted to autologous transplantations and 80 (27.0%) to allogeneic ones. Out of the 80 cases of allogeneic HSCT, 74 (92.6%) were related and only 6 (7.4%) were unrelated. Out of the 80 allogeneic cases, 69 (86.3%) were fullmatch and 11(13.7%) were mismatch. In regard to the groups mentioned above, we have the following distribution: group I: 106 patients (35.8%); group II: 112 patients (37.8%) and group III: 78 patients (26.4%). The mean and median values of IL-6 at fever onset in group I in regard to group II (p = 0.013), presenting significantly higher values. The levels of CRP in group I differed significantly from those found in group III (p < 0.05). The groups differed in regard to the levels of IL-6 and CRP at fever onset. Group II presented IL-6 and CRP concentrations significantly lower than group III. The best cut-off values of PCT for sampling: fever onset, 24 hours upon fever, 72 hours of fever, and long-standing fever were, respectively: 0.32; 0.47; 0.46 and 0.35?g/L. At fever onset, sensitivity was 52.3 and specificity 52.6 for infection diagnosis. The best cut-off values of CRP for fever onset, 24 hours upon fever, 72 hours upon fever and long-standing fever were, respectively: 79, 120, 108 and 72 mg/L. At fever onset, sensitivity was 55.4 and specificity was 55.1. The best cut-off values of IL-6 for fever onset, 24 hours upon fever, 72 hours upon fever and long-standing fever were, respectively: 34, 32, 16 and 9 pg/mL. At fever onset, sensitivity and specificity were, respectively: 59.8 and 59.7. In the analysis of the three biomarkers in the group of autologous patients, it is observed that only IL-6 presents significant values at initial moments (afebrile, fever and 24 hours upon fever). The following independent risk factors were identified in the multivariate analysis: related donor, unrelated donor, Gram-negative infection, DHL >= 390 (UI/L), urea >= 25 (mg/dL) and CRP>=120 (mg/L). Conclusions: IL-6 and CRP are associated to the early diagnosis of clinically or microbiologically confirmed infection in post-HSCT febrile neutropenia. The association of the three biomarkers did not present any advantage, nor did it improve diagnostic accuracy. IL-6 was the only biomarker significantly associated at an early stage with infection when assessed only in patients submitted to autologous HSCT. The independent variables associated with death were: allogeneic transplantation, Gram-negative infection, DHL >= 390UI/L at fever onset and urea >= 25 mg/dL at fever onset and PCR >= 120 (mg/L)

O transplante de células-tronco hematopoiéticas tem sido a melhor opção terapêutica para muitas doenças. Seu sucesso, no entanto, depende de alguns fatores que influenciam a taxa de mortalidade. A doença do enxerto-contra-hospedeiro é uma causa de mortalidade. Apresenta duas formas, a aguda e a crônica, e ambas têm linfócitos T na patofisiologia. Outra causa são as infecções, cujos patógenos variam conforme o tempo de recuperação do sistema imune. O presente estudo avaliou a recuperação linfoide T com base no timo e distinção das subpopulações e citocinas nos pacientes que desenvolveram doença do enxerto-contra-hospedeiro crônica no primeiro ano de transplante. Os pacientes foram alocados no Hospital de Transplante Amaral Carvalho (Jaú/SP) e tinham entre 18 e 60 anos de idade. No pré-transplante, foram comparados com indivíduos saudáveis, pareados em idade. Após o transplante, todos os pacientes foram acompanhados por um ano e seis meses, para observação de possíveis eventos de doença do enxerto-contra-hospedeiro e/ou infecções. A análise laboratorial foi feita com o sangue do paciente, sendo a primeira antes do transplante, seguidas por mais quatro, com intervalos de três meses. Tais avaliações laboratoriais tinham por objetivos caracterizar a função tímica por quantificação de sjTREC; as subpopulações de linfócitos T, por citometria de fluxo e a análise de citocinas, por Luminex. Foram estudados 172 indivíduos, sendo 75 do transplante alogênico, 43 do transplante autólogo, além de 54 pessoas saudáveis. Nossos resultados mostraram que os pacientes apresentaram função tímica diminuída antes mesmo da realização do transplante. A função tímica enfraquecida foi um fator de risco para doença do enxerto-contrahospedeiro crônica e a recuperação do sistema imune foi melhor nos pacientes que não apresentaram doença do enxerto-contra-hospedeiro crônica após um ano do transplante. No entanto, a função tímica não foi diferente entre os pacientes que faleceram e os que estão vivos. No geral, não houve distinção quanto à apresentação das subpopulações de linfócitos T e à produção de citocinas entre os pacientes submetidos ao transplante alogênico que desenvolveram doença do enxerto-contra-hospedeiro crônica, relativamente aos demais pacientes. Com o tempo, foi observada a recuperação gradual do compartimento linfoide T e diminuição na incidência de infecções. A taxa de infecções não influenciou a apresentação das subpopulações de linfócitos T e a produção de citocinas nos pacientes que desenvolveram doença do enxerto-contrahospedeiro crônica em relação aos demais pacientes. Como conclusão, a função tímica apresentou-se deteriorada antes da realização do transplante, porém não foi determinante para que houvesse piora na evolução clínica após o transplante. Os pacientes que desenvolveram doença do enxerto-contra-hospedeiro crônica, independentemente da incidência de infecções, apresentaram semelhança no perfil das subpopulações de linfócitos T e das citocinas, quando comparados aos demais / Hematopoietic stem cell transplantation has been the best therapeutic option for many diseases. Its success depends on some factors that influence the mortality rate. Graftversus- host disease is one cause of mortality. It has two forms, acute and chronic, and both have T lymphocytes in their pathophysiology. Other causes are infections, whose pathogens vary according to immune system recovery time. The present study evaluated the lymphoid T recuperation through the thymus and the differentiation of subpopulations and cytokines in patients who developed chronic graft-versus-host disease at the first year of transplantation. The patients were allocated at Hospital de Transplantes Amaral Carvalho (Jaú/SP). They were between 18 and 60 years old. At pre-transplant phase, patients were compared to healthy individuals, age-matched. After transplantation, they were all assisted for one year and six months, so that graft-versushost disease\'s and infections\' events could be observed. The laboratory analysis was done by blood sample; the first occurred before the transplant, followed by four more, every three months. Laboratory examinations were performed to characterize thymic function by quantification of sjTREC; T lymphocyte subpopulations, by flow cytometry, and cytokine analysis, by Luminex. A total of 172 individuals were studied: 75 allogeneic transplant patients, 43 autologous transplant patients and 54 healthy persons. Our results showed that patients presented thymic function impaired even before the transplant. The weakening of the thymic function was a risk factor for chronic graft-versus-host disease and immune system recovery was better in patients who did not develop chronic graft-versus-host disease after one year of transplantation. However, the thymic function was not different between patients who died and those who remained alive. In general, there was no distinction of the presentation of T lymphocyte subpopulations and cytokines production among patients who underwent allogeneic transplant and developed chronic graft-versus-host disease, in comparison to the other patients. Over time, a gradual recovery of the T lymphoid compartment and a decrease in the infections incidence were observed. The infection rate did not influence the presentation of the T lymphocyte subpopulations and the production of cytokines in patients who developed chronic graft-versus-host disease in comparison to the other patients. In conclusion, the thymic function was impaired before transplantation, but it was not relevant for clinical evolution worsening after transplantation. Patients who developed chronic graft-versus-host disease, regardless of the incidence of infections, showed similar profile of T lymphocytes subpopulations and cytokines, relatively to other patients

A aspergilose invasiva (AI) é a infecção por fungos filamentosos mais comum em pacientes imunodeprimidos, especialmente em transplantes de células tronco hematopoiético e neoplasias hematológicas. Objetivo: Geral: Estabelecer a comparação entre a dosagem de Galactomanana (GM), 1,3betaD-glucana (BDG) e dados tomográficos no diagnóstico da AI bem como seu papel no desfecho clínico. Específicos: 1. Verificar a sensibilidade e especificidade dos ensaios de Galactomanana e de 1,3betaD-glucana no soro e lavado broncoalveolar. 2. Comparar os resultados da galatomanana e 1,3betaD-glucana com os dados de imagem em pacientes com suspeita de AI. 3. Verificar a relação entre a evolução dos níveis de GM e desfecho clínico (óbito e sobrevida). Casuística, Materiais e Métodos: Realizou-se um estudo tipo coorte prospectiva, incluindo 398 sujeitos das diversas enfermarias de pacientes imunodeprimidos do HCFMUSP, sendo incluídos dois grupos de pacientes: 202(51%) AI e 198(49%) controles. Resultados: Dos casos, 18 (8,8%) tinham aspergilose provada, 28 (13,7%) provável e 158 possível (77,5%), de acordo a classificação de 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer / Mycoses Study Group). Os sujeitos submetidos ao TCTH eram 42,7%, com neoplasias hematológicas 37%, TOS 9% e outras doenças 11,3%. Os fatores de risco associados ao desenvolvimento da AI foram neutropenia, monocitopenia, uso de corticóide, presença de doença pelo citomegalovírus e rejeição ou doença do enxerto contra o hospedeiro. O fator de risco associado à evolução para o óbito foi a presença de AI. Foram observados bons desempenhos para a GM tanto no soro como no LBA com LR menores que os registrados na literatura. O melhor desempenho da GM no soro para aspergilose+provável ocorreu com LR de 0,35 com sensibilidade-S, especificidade-E, valor preditivo positivo- VPP), valor preditivo negativo-VPN) e área sob a curva-ASC de 54,4%, 73,4%, 50,8%, 76,2% e 0,64, sendo os valores superiores para aspergilose provada tanto na S, como E, VPN. No LBA os valores de S-E-VPP-VPN-ASC para GM para LR de 0,65 para aspergilose provável + provada foram 58,3%, 92,6%, 87,5%,71,4% e 0,75, sendo na aspergilose provada os valores de S, e VPN superiores. Nesta casuística, o melhor desempenho para BDG no soro apontou para uma LR de 100 pg/mL na aspergilose provável+provada, com 54,5%, 73,4%, 50,8% e 76,2%, 0,64 respectivamente para S-E-VPP-VPN-ASC. Para BDG no LBA, a LR na aspergilose provável + provada foi de 140 pg/mL, com os mesmos valores de 46,7%, 76,7%, 70%, 55,6% e 0.62, respectivamente. Conclusão: A GM no LBA e no soro foram úteis no diagnóstico da aspergilose mediante emprego de LR menores, sendo mais sensível na LBA, principalmente em estágios iniciais da forma angioinvasiva. A persistência de GM sérica foi relacionada ao óbito em relação à negativação da mesma. A proporção de concordância entre a TC e os biomarcadores no soro e no LBA variou de 0,5 a 0,6, com pequena concordância na estatística kappa. Excelente concordância foi observada entre dois radiologistas independentes, que analisaram de maneira cega as TC de sujeitos com aspergilose provada. Nesta casuística com inclusão de doenças sistêmicas e endêmicas, a BDG teve baixo desempenho diagnóstico / Invasive aspergillosis (IA) has become the leading infectious cause of death in immunocompromised hosts, particularly in subjects under SCTH and hematologic neoplasias. Objectives: General: To compare the performance of GM and BG tests in serum and bronchoalveolar lavage fluid (BAL) and computer tomography (CT) scans in the diagnosis of IA in immunocompromised hosts as well as their role in the patient outcome. Specific: 1. To analyse the sensitivity and specificity of Galactomannan and 1,3 betaD-glucan assays in the serum and bronchoalveolar lavage. 2. To compare the results of Galactomannan and 1,3betaD-glucan assays with CT scans in patients with invasive aspergilosis. 3. To analyse the relationship between the evolution of galactomannan levels and clinical outcome (death or survival). Patients, Materials and Methods: From December 2008 to March 2013, a prospective cohort of 398 patients from several wards of immunocompromised patients of Hospital das Clínicas, Faculdade de Medicina, University of São Paulo was included classified in two groups of patients: 202 (51%) with invasive aspergillosis (IA) and 198 (49%) control patients. Results: Considering 202 cases, 18(8.8%) were subjects with proven, 28(13.7%) with probable aspergillosis and 156(77.5%), with possible aspergillosis, according to 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria. The most common underlying disease were: HSCT (42.7%), hematologic malignancy (37%), SOT (9%), or other diseases (11.3%). The main risk factors associated with IA were neutropenia, monocytopenia, patients under corticosterois, presence of CMV disease, and rejection or graft versus host disease. The risk factor associated with death was the presence of invasive aspergillosis. Good performances for serum and BAL GM were registered with lower cutoffs in the present workin relationship to those found in the literature. The best cutoff for proven + probable aspergillosis for serum GM was observed at 0.35 vallue with Sensitivity-S, Specificity-Sp, Positive Predictive value-PPV), Negative Predictive Value-NPV) and AUC of 54.4%, 73.4%, 50.8%, 76.2% and 0.64; the values for proven aspergillosis alone were higher for S, Sp and NPV. On BAL tests for GM (cutoff value of 0.65) in proven+probable aspergillosis we observed 58.3%, 92.6%, 87.5%,71.4%, 0.75, respectively as S-Sp-PPV-NPVAUC; the sensitivity and VPN were higher in proven aspergillosis alone. In this work, the best performance in proven+probable aspergillosis for serum BDG showed 100 pg/ML as cutoff value, with 54.5%, 73.4%, 50.8%,76.2%, 0.64 for S-Sp-PPVNPV- AUC, respectively. For BAL- BDG, the cut off for proven+probable aspergillosis was 140 pg/mL, and we observed 46.7%, 76.7%, 70.0%, 55.,6%, 0.62, respectively for for S-Sp-PPV-NPV-AUC. Conclusion: The serum and BAL GM are useful tests for diagnosis in early stages of angioinvasive form at lower cutoffs; BAL GM is more sensitive. Agreement proportion between CT scan and each biomarker in the serum or BAL ranged from 0.5-0.6, with low ? index. Perfect ? statistic was observed for analysis of CT scan of subjects in proven aspergillosis by two independent radiologists, blinded for diagnosis. Persistence of serum GM was associated to death in relationship with its negativation. BDG test showed low performance in this work, where systemic and endemic diseases were included

A aspergilose invasiva (AI) é a infecção por fungos filamentosos mais comum em pacientes imunodeprimidos, especialmente em transplantes de células tronco hematopoiético e neoplasias hematológicas. Objetivo: Geral: Estabelecer a comparação entre a dosagem de Galactomanana (GM), 1,3betaD-glucana (BDG) e dados tomográficos no diagnóstico da AI bem como seu papel no desfecho clínico. Específicos: 1. Verificar a sensibilidade e especificidade dos ensaios de Galactomanana e de 1,3betaD-glucana no soro e lavado broncoalveolar. 2. Comparar os resultados da galatomanana e 1,3betaD-glucana com os dados de imagem em pacientes com suspeita de AI. 3. Verificar a relação entre a evolução dos níveis de GM e desfecho clínico (óbito e sobrevida). Casuística, Materiais e Métodos: Realizou-se um estudo tipo coorte prospectiva, incluindo 398 sujeitos das diversas enfermarias de pacientes imunodeprimidos do HCFMUSP, sendo incluídos dois grupos de pacientes: 202(51%) AI e 198(49%) controles. Resultados: Dos casos, 18 (8,8%) tinham aspergilose provada, 28 (13,7%) provável e 158 possível (77,5%), de acordo a classificação de 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer / Mycoses Study Group). Os sujeitos submetidos ao TCTH eram 42,7%, com neoplasias hematológicas 37%, TOS 9% e outras doenças 11,3%. Os fatores de risco associados ao desenvolvimento da AI foram neutropenia, monocitopenia, uso de corticóide, presença de doença pelo citomegalovírus e rejeição ou doença do enxerto contra o hospedeiro. O fator de risco associado à evolução para o óbito foi a presença de AI. Foram observados bons desempenhos para a GM tanto no soro como no LBA com LR menores que os registrados na literatura. O melhor desempenho da GM no soro para aspergilose+provável ocorreu com LR de 0,35 com sensibilidade-S, especificidade-E, valor preditivo positivo- VPP), valor preditivo negativo-VPN) e área sob a curva-ASC de 54,4%, 73,4%, 50,8%, 76,2% e 0,64, sendo os valores superiores para aspergilose provada tanto na S, como E, VPN. No LBA os valores de S-E-VPP-VPN-ASC para GM para LR de 0,65 para aspergilose provável + provada foram 58,3%, 92,6%, 87,5%,71,4% e 0,75, sendo na aspergilose provada os valores de S, e VPN superiores. Nesta casuística, o melhor desempenho para BDG no soro apontou para uma LR de 100 pg/mL na aspergilose provável+provada, com 54,5%, 73,4%, 50,8% e 76,2%, 0,64 respectivamente para S-E-VPP-VPN-ASC. Para BDG no LBA, a LR na aspergilose provável + provada foi de 140 pg/mL, com os mesmos valores de 46,7%, 76,7%, 70%, 55,6% e 0.62, respectivamente. Conclusão: A GM no LBA e no soro foram úteis no diagnóstico da aspergilose mediante emprego de LR menores, sendo mais sensível na LBA, principalmente em estágios iniciais da forma angioinvasiva. A persistência de GM sérica foi relacionada ao óbito em relação à negativação da mesma. A proporção de concordância entre a TC e os biomarcadores no soro e no LBA variou de 0,5 a 0,6, com pequena concordância na estatística kappa. Excelente concordância foi observada entre dois radiologistas independentes, que analisaram de maneira cega as TC de sujeitos com aspergilose provada. Nesta casuística com inclusão de doenças sistêmicas e endêmicas, a BDG teve baixo desempenho diagnóstico / Invasive aspergillosis (IA) has become the leading infectious cause of death in immunocompromised hosts, particularly in subjects under SCTH and hematologic neoplasias. Objectives: General: To compare the performance of GM and BG tests in serum and bronchoalveolar lavage fluid (BAL) and computer tomography (CT) scans in the diagnosis of IA in immunocompromised hosts as well as their role in the patient outcome. Specific: 1. To analyse the sensitivity and specificity of Galactomannan and 1,3 betaD-glucan assays in the serum and bronchoalveolar lavage. 2. To compare the results of Galactomannan and 1,3betaD-glucan assays with CT scans in patients with invasive aspergilosis. 3. To analyse the relationship between the evolution of galactomannan levels and clinical outcome (death or survival). Patients, Materials and Methods: From December 2008 to March 2013, a prospective cohort of 398 patients from several wards of immunocompromised patients of Hospital das Clínicas, Faculdade de Medicina, University of São Paulo was included classified in two groups of patients: 202 (51%) with invasive aspergillosis (IA) and 198 (49%) control patients. Results: Considering 202 cases, 18(8.8%) were subjects with proven, 28(13.7%) with probable aspergillosis and 156(77.5%), with possible aspergillosis, according to 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria. The most common underlying disease were: HSCT (42.7%), hematologic malignancy (37%), SOT (9%), or other diseases (11.3%). The main risk factors associated with IA were neutropenia, monocytopenia, patients under corticosterois, presence of CMV disease, and rejection or graft versus host disease. The risk factor associated with death was the presence of invasive aspergillosis. Good performances for serum and BAL GM were registered with lower cutoffs in the present workin relationship to those found in the literature. The best cutoff for proven + probable aspergillosis for serum GM was observed at 0.35 vallue with Sensitivity-S, Specificity-Sp, Positive Predictive value-PPV), Negative Predictive Value-NPV) and AUC of 54.4%, 73.4%, 50.8%, 76.2% and 0.64; the values for proven aspergillosis alone were higher for S, Sp and NPV. On BAL tests for GM (cutoff value of 0.65) in proven+probable aspergillosis we observed 58.3%, 92.6%, 87.5%,71.4%, 0.75, respectively as S-Sp-PPV-NPVAUC; the sensitivity and VPN were higher in proven aspergillosis alone. In this work, the best performance in proven+probable aspergillosis for serum BDG showed 100 pg/ML as cutoff value, with 54.5%, 73.4%, 50.8%,76.2%, 0.64 for S-Sp-PPVNPV- AUC, respectively. For BAL- BDG, the cut off for proven+probable aspergillosis was 140 pg/mL, and we observed 46.7%, 76.7%, 70.0%, 55.,6%, 0.62, respectively for for S-Sp-PPV-NPV-AUC. Conclusion: The serum and BAL GM are useful tests for diagnosis in early stages of angioinvasive form at lower cutoffs; BAL GM is more sensitive. Agreement proportion between CT scan and each biomarker in the serum or BAL ranged from 0.5-0.6, with low ? index. Perfect ? statistic was observed for analysis of CT scan of subjects in proven aspergillosis by two independent radiologists, blinded for diagnosis. Persistence of serum GM was associated to death in relationship with its negativation. BDG test showed low performance in this work, where systemic and endemic diseases were included

Introdução: No presente estudo foram avaliados biomarcadores na ocorrência de febre em pacientes neutropênicos após transplante de células tronco hematopoiéticas (TCTH). Objetivo: O objetivo principal foi avaliar os valores séricos de biomarcadores: proteína C reativa (PCR), procalcitonina (PCT) e IL-6 (interleucina-6) que possam identificar precocemente infecção em TCTH. Outro objetivo foi fatores de risco para óbito nessa população. Métodos: Os biomarcadores foram avaliados em um estudo prospectivo que incluiu 296 pacientes neutropênicos, submetidos a TCTH autólogo ou alogênico. Os biomarcadores PCT, PCR e IL-6 foram dosados nos seguintes momentos:dia da neutropenia constatada sem febre, evento febril ou hipotermia (T < 35ºC), 24 h após a febre ou hipotermia, 72 horas após a febre ou hipotermia e febre prolongada ou seja 48 horas após a coleta no momento anterior ou na persistência da febre, cinco dias após a coleta no momento anterior. Os dados clínicos e laboratoriais, foram avaliados até a evolução para alta ou o óbito, em uma planilha Excel® 2003 e foram processados pelos programas SPSS e STATA. Os pacientes foram classificados nos seguintes grupos (I- afebril; II- febre de origem indeterminada FOI e III- febre clinica ou microbiologicamente comprovada) em relação a cada marcador estudado (PCT, PCR e IL-6). Foram feitos cálculos para estabelecer área sob a curva ROC, sensibilidade, especificidade, para avaliação da febre e óbito. Para avaliar o desfecho óbito foi realizada análise multivariada com regressão logística stepwise. Resultados: Dos 296 pacientes, 190 apresentaram febre. Duzentos e dezesseis (73%) foram submetidos a transplantes autólogos e 80 (27,0%) alogênicos. Dos 80 casos de TCTH alogênicos 74 (92,6%) eram aparentados e apenas 6 (7,4%) aparentados. Dos 80 casos alogênicos 69 (86,3%) eram fullmatch e 11(13,7%) mismatch. Em relação aos grupos já citados acima, temos a seguinte distribuição: grupo I: 106 pacientes (35,8%); grupo II: 112 pacientes (37,8%) e grupo III: 78 (26,4%). Os valores de média e mediana da IL-6 no momento afebril no grupo I em relação ao grupo II (p = 0,013), apresentando valor significativamente maiores. Os níveis da PCR no grupo I diferiram de forma significativa dos encontrados no grupo III (p < 0,05). Os grupos diferiram em relação aos níveis de IL-6 e de PCR no momento febril. O grupo II apresentou concentrações de IL-6 e de PCR significativamente menores que o grupo III. Os melhores valores de corte de PCT para os momentos de coleta: febre, 24 horas após a febre, 72 horas de febre, e febre prolongada foram respectivamente: 0,32; 0,47; 0,46 e 0,35?g/L. No momento da febre a sensibilidade foi 52,3 e a especificidade 52,6 para o diagnóstico de infecção. Os melhores valores de corte de PCR para os momentos de febre, 24 horas após, 72 horas após e febre prolongada foram, respectivamente: 79, 120, 108 e 72 mg/L. No momento da febre a sensibilidade foi 55,4 e especificidade foi 55,1. Os melhores valores de corte de IL-6 para os momentos de febre, 24 h após, 72 horas após a febre e febre prolongada foram respectivamente: 34, 32, 16 e 9 pg/mL. A sensibilidade e especificidade no momento da febre foram respectivamente: 59,8 e 59,7. Na análise dos três biomarcadores no grupo de pacientes autólogos, verifica-se que só a IL-6 apresenta valores significativos nos momentos iniciais (afebril, febre e 24 horas após a febre). Os seguintes fatores de risco independentes foram identificados na análise multivariada: doador aparentado, doador não aparentado, infecção por Gram-negativo, DHL >= 390 (UI/L), ureia >= 25 (mg/dL) e PCR >= 120 (mg/L). Conclusões: IL-6 e PCR têm associação com diagnóstico precoce de infecção clinica ou microbiologicamente confirmada em neutropenia febril após TCTH. A associação dos três marcadores não apresentou nenhuma vantagem, e não melhorou a acurácia diagnóstica. A IL-6 foi o único biomarcador significativamente associado de forma precoce com infecção quando avaliado apenas pacientes submetidos a TCTH autólogos As variáveis independentes associadas com óbito foram: transplante alogênico, infecção por Gram-negativos, DHL >= 390UI/L no momento da febre e ureia >= 25 mg/dL no momento da febre e PCR >= 120 (mg/L) / Introduction: In the present study, biomarkers were assessed in the occurrence of fever in neutropenic patients upon hematopoietic stem cell transplantation (HSCT). Objective: The main objective was to assess the serum values of biomarkers: C-reactive protein (CRP), procalcitonin (PCT) and IL-6 (interleukin-6) which can early identify infection in HSCT. Another objective was risk factors for death in that population. Methods: The biomarkers were assessed in a prospective study which comprised 296 neutropenic patients submitted to autologous or allogeneic HSCT. The biomarkers PCT, CRP and IL-6 were dosed at the following moments: day of afebrile neutropenia, febrile event or hypothermia (T < 35ºC), 24 h upon fever or hypothermia, 72 hours upon fever or hypothermia and long-standing fever, that is, 48 hours upon the last sampling or at fever persistence, five days upon the last sampling. The clinical and laboratory data were assessed up to the evolution to discharge or death, in an Excel® 2003 spreadsheet and were processed by the SPSS and STATA software. Patients were classified in the following groups (I- afebrile; II- fever of unknown origin FUO and III- clinically or microbiologically proven fever) in regard to each biomarker studied (PCT, CRP and IL-6). Calculations were made to establish the area under the ROC curve, sensitivity, specificity, for the assessment of the evolution and death. In order to assess the death outcome, a multivariate analysis with stepwise logistic regression was conducted. Results: Out of the 296 patients, 190 had fever. Two hundred and sixteen (73%) were submitted to autologous transplantations and 80 (27.0%) to allogeneic ones. Out of the 80 cases of allogeneic HSCT, 74 (92.6%) were related and only 6 (7.4%) were unrelated. Out of the 80 allogeneic cases, 69 (86.3%) were fullmatch and 11(13.7%) were mismatch. In regard to the groups mentioned above, we have the following distribution: group I: 106 patients (35.8%); group II: 112 patients (37.8%) and group III: 78 patients (26.4%). The mean and median values of IL-6 at fever onset in group I in regard to group II (p = 0.013), presenting significantly higher values. The levels of CRP in group I differed significantly from those found in group III (p < 0.05). The groups differed in regard to the levels of IL-6 and CRP at fever onset. Group II presented IL-6 and CRP concentrations significantly lower than group III. The best cut-off values of PCT for sampling: fever onset, 24 hours upon fever, 72 hours of fever, and long-standing fever were, respectively: 0.32; 0.47; 0.46 and 0.35?g/L. At fever onset, sensitivity was 52.3 and specificity 52.6 for infection diagnosis. The best cut-off values of CRP for fever onset, 24 hours upon fever, 72 hours upon fever and long-standing fever were, respectively: 79, 120, 108 and 72 mg/L. At fever onset, sensitivity was 55.4 and specificity was 55.1. The best cut-off values of IL-6 for fever onset, 24 hours upon fever, 72 hours upon fever and long-standing fever were, respectively: 34, 32, 16 and 9 pg/mL. At fever onset, sensitivity and specificity were, respectively: 59.8 and 59.7. In the analysis of the three biomarkers in the group of autologous patients, it is observed that only IL-6 presents significant values at initial moments (afebrile, fever and 24 hours upon fever). The following independent risk factors were identified in the multivariate analysis: related donor, unrelated donor, Gram-negative infection, DHL >= 390 (UI/L), urea >= 25 (mg/dL) and CRP>=120 (mg/L). Conclusions: IL-6 and CRP are associated to the early diagnosis of clinically or microbiologically confirmed infection in post-HSCT febrile neutropenia. The association of the three biomarkers did not present any advantage, nor did it improve diagnostic accuracy. IL-6 was the only biomarker significantly associated at an early stage with infection when assessed only in patients submitted to autologous HSCT. The independent variables associated with death were: allogeneic transplantation, Gram-negative infection, DHL >= 390UI/L at fever onset and urea >= 25 mg/dL at fever onset and PCR >= 120 (mg/L)