New drug candidates for osteosarcoma: Drug repurposing based on gene expression signature.

Osteosarcoma (OS) is an aggressive bone malignancy and the third most common cancer in adolescence. Since the late 1970s, OS therapy and prognosis had only modest improvements, making it appealing to explore new tools that could help ameliorate the treatment. We present a meta-analysis of the gene expression signature of primary OS, and propose small molecules that could reverse this signature. The meta-analysis was performed using GEO microarray series. We first compared gene expression from eleven primary OS against osteoblasts to obtain the differentially expressed genes (DEGs). We later filtered those DEGs by verifying which ones had a concordant direction of differential expression in a validation group of 82 OS samples versus 30 bone marrow mesenchymal stem cells (BM-MSC) samples. A final gene expression signature of 266 genes (98 up and 168 down regulated) was obtained. The L1000CDS2 engine was used for drug repurposing. The top molecules predicted to reverse the signature were afatinib (PubChem CID 10184653), BRD-K95196255 (PubChem CID 3242434), DG-041 (PubChem CID 11296282) and CA-074 Me (PubChem CID 23760717). Afatinib (Gilotrif™) is currently used for metastatic non-small-cell lung cancer with EGFR mutations, and in vitro evidence shows antineoplastic potential in OS cells. The other three molecules have reports of antineoplastic effects, but are not currently FDA-approved. Further studies are necessary to establish the potential of these drugs in OS treatment. We believe our results can be an important contribution for the investigation of new therapeutic genetic targets and for selecting new drugs to be tested for OS.

Cancer-related worry and risk perception in Brazilian individuals seeking genetic counseling for hereditary breast cancer.

In Brazil, the population in general has little knowledge about genetic risks, as well as regarding the role and importance of the Cancer Genetic Counseling (CGC). The goal of this study was to evaluate cancer-related worry and cancer risk perception during CGC sessions in Brazilian women at-risk for hereditary breast cancer. This study was performed in 264 individuals seeking CGC for hereditary breast cancer. Both cancer-affected and unaffected individuals were included. As results, individuals with and without cancer reported different motivations for seeking CGC and undergoing genetic testing. A correlation was observed between age at the first CGC session and age at which the closest relative was diagnosed with cancer. Multivariate analysis showed that educational level, cancer risk discussion within the family, and number of deaths by cancer among first-degree relatives influenced positively the cancer risk perception. In conclusion, the results of this study indicate that cancer-related worry and cancer risk perception are significant aspects of morbidity in individuals seeking CGC, whether they are cancer-affected or unaffected. CGC has an important role in health education and cancer prevention for its potential of promoting an accurate perception of the risk.

Free carnitine and branched chain amino acids are not good biomarkers in Huntington's disease / Carnitina livre e aminoácidos de cadeia ramificada não são bons biomarcadores na doença de Huntington

ABSTRACT Background: Huntington's disease (HD), caused by an expanded CAG repeat at HTT, has no treatment, and biomarkers are needed for future clinical trials. Objective: The objective of this study was to verify if free carnitine and branched chain amino acids levels behave as potential biomarkers in HD. Methods: Symptomatic and asymptomatic HD carriers and controls were recruited. Age, sex, body mass index (BMI), age of onset, disease duration, UHDRS scores, and expanded CAG tract were obtained; valine, leucine, isoleucine, and free carnitine were measured. Baseline and longitudinal analysis were performed. Results: Seventy-four symptomatic carriers, 20 asymptomatic carriers, and 22 non-carriers were included. At baseline, valine levels were reduced in symptomatic and asymptomatic HD carriers when compared to non-carriers. No difference in free carnitine or isoleucine+leucine levels were observed between groups. BMI of symptomatic individuals was lower than those of non-carriers. Valine levels correlated with BMI. Follow-up evaluation was performed in 43 symptomatic individuals. UHDRS total motor score increased 4.8 points/year on average. No significant reductions in BMI or valine were observed, whereas free carnitine and isoleucine+leucine levels increased. Conclusions: Although valine levels were lower in HD carriers and were related to BMI losses observed in pre-symptomatic individuals, none of these metabolites seem to be biomarkers for HD.

RESUMO Introdução: A doença de Huntington (DH), causada por uma repetição CAG expandida no HTT, não possui tratamento e biomarcadores são necessários para futuros ensaios clínicos. Objetivo: Nosso objetivo foi verificar se os níveis de carnitina livre e aminoácidos de cadeia ramificada se comportam como potenciais biomarcadores na DH. Métodos: Portadores sintomáticos e assintomáticos e controles foram recrutados. Idade, sexo, índice de massa corporal (IMC), idade de início, duração da doença, escores UHDRS e trato CAG expandido foram obtidos; valina, leucina, isoleucina e carnitina livre foram medidas. Foram realizadas análises basal e longitudinal. Resultados: Setenta e quatro portadores sintomáticos, 20 portadores assintomáticos e 22 não portadores foram incluídos. No início do estudo, os níveis de valina estavam reduzidos em portadores de DH sintomáticos e assintomáticos quando comparados aos não portadores. Não foram observadas diferenças nos níveis de carnitina livre ou isoleucina + leucina entre os grupos. O IMC dos indivíduos sintomáticos foi menor que o dos não portadores. Níveis de valina correlacionaram-se com o IMC. Avaliação de acompanhamento foi realizada em 43 indivíduos sintomáticos. A pontuação do escore motor total da UHDRS aumentou 4,8 pontos/ano em média. Não foram observadas reduções significativas no IMC ou na valina, enquanto os níveis de carnitina livre e isoleucina+leucina aumentaram. Conclusões: Embora os níveis de valina tenham sido menores nos portadores de DH e estivessem relacionados às perdas de IMC observadas em indivíduos pré-sintomáticos, nenhum desses metabólitos parece ser biomarcador para a DH.

Screening and characterization of BRCA2 c.156_157insAlu in Brazil: Results from 1380 individuals from the South and Southeast.

Portuguese immigration to Brazil occurred in several waves and greatly contributed to the genetic composition of current Brazilian population. In this study, we evaluated the frequency of a Portuguese founder Alu insertion in BRCA2 exon 3 (c.156_157insAlu) among individuals fulfilling Hereditary Breast and Ovarian Cancer (HBOC) syndrome criteria in 1,380 unrelated families originated from three distinct Brazilian States. We identified the c.156_157insAlu BRCA2 mutation in nine (9/1,380; 0.65%) probands analised. In carrier probands, European ancestry had the highest proportion (80%), followed by the African (10%) and Amerindian and in most families with the rearrangement, haplotype analyses were compatible with the Portuguese ancestral haplotype. In conclusion, the present study reports a low albeit relevant frequency of the Portuguese BRCA2 founder mutation c.156_157insAlu in Brazilian patients at-risk for HBOC Brazilian population.

The germline mutational landscape of BRCA1 and BRCA2 in Brazil.

The detection of germline mutations in BRCA1 and BRCA2 is essential to the formulation of clinical management strategies, and in Brazil, there is limited access to these services, mainly due to the costs/availability of genetic testing. Aiming at the identification of recurrent mutations that could be included in a low-cost mutation panel, used as a first screening approach, we compiled the testing reports of 649 probands with pathogenic/likely pathogenic variants referred to 28 public and private health care centers distributed across 11 Brazilian States. Overall, 126 and 103 distinct mutations were identified in BRCA1 and BRCA2, respectively. Twenty-six novel variants were reported from both genes, and BRCA2 showed higher mutational heterogeneity. Some recurrent mutations were reported exclusively in certain geographic regions, suggesting a founder effect. Our findings confirm that there is significant molecular heterogeneity in these genes among Brazilian carriers, while also suggesting that this heterogeneity precludes the use of screening protocols that include recurrent mutation testing only. This is the first study to show that profiles of recurrent mutations may be unique to different Brazilian regions. These data should be explored in larger regional cohorts to determine if screening with a panel of recurrent mutations would be effective.

TP53 and CDKN1A mutation analysis in families with Li-Fraumeni and Li-Fraumeni like syndromes.

Li-Fraumeni and Li-Fraumeni like syndromes (LFS/LFL) represent rare cancer-prone conditions associated mostly with sarcomas, breast cancer, brain tumors, and adrenocortical carcinomas. TP53 germline mutations are present in up to 80 % of families with classic Li-Fraumeni syndrome, and in 20-60 % of families with Li-Fraumeni like phenotypes. The frequency of LFS/LFL families with no TP53 mutations detected suggests the involvement of other genes in the syndrome. In this study, we searched for mutations in TP53 in 39 probands from families with criteria for LFS/LFL. We also searched for mutations in the gene encoding the main mediator of p53 in cell cycle arrest, CDKN1A/p21, in all patients with no mutations in TP53. Eight probands carried germline disease-causing mutations in TP53: six missense mutations and two partial gene deletions. No mutations in CDKN1A coding region were detected. TP53 partial deletions in our cohort represented 25 % (2/8) of the mutations found, a much higher frequency than usually reported, emphasizing the need to search for TP53 rearrangements in patients with LFS/LFL phenotypes. Two benign tumors were detected in two TP53 mutation carriers: an adrenocortical adenoma and a neurofibroma, which raises a question about the possible implication of TP53 mutations on the development of such lesions.

BRCA1 and BRCA2 rearrangements in Brazilian individuals with Hereditary Breast and Ovarian Cancer Syndrome.

Approximately 5-10% of breast cancers are caused by germline mutations in high penetrance predisposition genes. Among these, BRCA1 and BRCA2, which are associated with the Hereditary Breast and Ovarian Cancer (HBOC) syndrome, are the most frequently affected genes. Recent studies confirm that gene rearrangements, especially in BRCA1, are responsible for a significant proportion of mutations in certain populations. In this study we determined the prevalence of BRCA rearrangements in 145 unrelated Brazilian individuals at risk for HBOC syndrome who had not been previously tested for BRCA mutations. Using Multiplex Ligation-dependent Probe Amplification (MLPA) and a specific PCR-based protocol to identify a Portuguese founder BRCA2 mutation, we identified two (1,4%) individuals with germline BRCA1 rearrangements (c.547+240_5193+178del and c.4675+467_5075-990del) and three probands with the c.156_157insAlu founder BRCA2 rearrangement. Furthermore, two families with false positive MLPA results were shown to carry a deleterious point mutation at the probe binding site. This study comprises the largest Brazilian series of HBOC families tested for BRCA1 and BRCA2 rearrangements to date and includes patients from three regions of the country. The overall observed rearrangement frequency of 3.44% indicates that rearrangements are relatively uncommon in the admixed population of Brazil.

Prevalence of Café-au-Lait Spots in children with solid tumors.

Cafe-au-lait maculae (CALM) are frequently observed in humans, and usually are present as a solitary spot. Multiple CALMs are present in a smaller fraction of the population and are usually associated with other congenital anomalies as part of many syndromes. Most of these syndromes carry an increased risk of cancer development. Previous studies have indicated that minor congenital anomalies may be more prevalent in children with cancer. We investigated the prevalence of CALMs in two samples of Brazilian patients with childhood solid tumors, totaling 307 individuals. Additionally, 176 school children without diagnosis of cancer, or of a cancer predisposing syndrome, were investigated for the presence of CALMs. The prevalence of solitary CALM was similar in both study groups (18% and 19%) and also in the group of children without cancer. Multiple CALMs were more frequently observed in one of the study groups (Z = 2.1). However, when both groups were analyzed together, the significance disappeared (Z = 1.5). The additional morphological abnormalities in children with multiple CALMs were analyzed and compared to the findings observed in the literature. The nosologic entities associated with CALMs are reviewed.

Translating microRNAs into biomarkers: What is new for pediatric cancer?

Since their discovery in 2008, cell-free circulating microRNAs have been considered potential biomarkers for various conditions, including pediatric cancer. Diagnosis of pediatric cancer still relies on clinical signs, which sometimes may be non-specific or appear at later stages. Thus, there is a need for a better understanding of molecules that allow a less invasive, early and effective method of cancer diagnosis. Despite the efforts of many researches to set specific miRNAs to be routinely used as diagnostic molecules, no miR has been currently utilized so far. In this study, we review the recent discoveries on circulating miRNAs in blood of patients suffering from the following pediatric cancers: osteosarcoma, rhabdomyosarcoma, Wilms tumor, acute myeloid leukemia, acute lymphocytic leukemia, retinoblastoma and neuroblastoma. We also focus on the roles of circulating miRs in tumorigenesis pathways, the methodological approaches used to detect and quantify circulating miRs, and discuss the challenges in using them routinely as biomarkers for pediatric cancers.

Prevalence and impact of founder mutations in hereditary breast cancer in Latin America.

Approximately 10% of all cancers are considered hereditary and are primarily caused by germline, high penetrance mutations in cancer predisposition genes. Although most cancer predisposition genes are considered molecularly heterogeneous, displaying hundreds of different disease-causing sequence alterations, founder mutations have been identified in certain populations. In some Latin American countries, founder mutations associated with increased risk of breast and other cancers have been described. This is particularly interesting considering that in most of these countries, populations are highly admixed with genetic contributions from native populations and from the in-flux of several distinct populations of immigrants. In this article, we present a review of the scientific literature on the subject and describe current data available on founder mutations described in the most common breast cancer predisposition genes: BRCA1, BRCA2 and TP53.

Polymorphisms of CDKN1A gene and risk of retinoblastoma.

UNLABELLED: Retinoblastoma (RB) is a malignant neoplasia that occurs mostly in children under 5 years. Recently, CDKN1A gene has been shown to be up-regulated in a context of loss of function of pRb. This gene encodes the p21 protein, which is the bona fide effector of p53. We hypothesized whether two putatively functional single nucleotide polymorphisms (SNPs) of CDKN1A (rs1801270 C>A and rs1059234 C>T) may influence the risk and/or survival of RB patients. We genotyped both SNPs in 141 RB patients and 120 unrelated healthy individuals. Statistical analyses consisted of chi-square (χ(2)), odds ratio (OR) and survival curves by Kaplan-Meier method. We found that patients who carry the genotype CA for rs1801270 and CT for rs1059234 were associated to an increased risk of RB [OR = 2.5, 95% confidence interval (CI) = 1.38-4.53], whereas patients with CC for both polymorphisms were associated to a lower risk of developing RB (OR = 0.43, 95% CI = 0.25-0.74). On the other hand, Kaplan-Meier curves did not show statistically significant differences in survival among the studied polymorphisms. We conclude that the minor alleles of rs1801270 and rs1059234 polymorphisms may act as risk factors for the development of RB in our sample. SUMMARY: The minor alleles of polymorphisms rs1801270 C>A and rs1059234 C>T in CDKN1A (p21) gene may act as risk factors for the development of RB; however, they do not seem to influence overall survival.

Influence of MDM2 and MDM4 on development and survival in hereditary retinoblastoma.

BACKGROUND: Retinoblastoma (RB) accounts for 3% of all childhood malignancies, with different incidences around the world. This malignancy results from loss-of-function of both RB1 alleles although other genes, like MDM2 and MDM4, have been proposed to be involved in tumor development. PROCEDURE: We genotyped rs2279744T>G and rs937283A>G in MDM2, and rs4252668T>C and rs116197192G>A in MDM4, in 104 unrelated RB patients and 104 controls. Sixty-month survival Kaplan-Meier curves and χ(2)-tests were performed for estimating the putative effect of MDM2 and MDM4 alleles on disease progression and survival of RB patients. RESULTS: MDM2 rs2279744G was significantly more frequent in controls, indicating an apparently protective effect on RB development. However, survival of patients who carried a constitutional RB1 mutation was significantly lower with rs2279744TG or GG than with rs2279744TT. Presence of rs2279744G and a constitutional RB1 mutation was sixfold more frequent in the 0-12 month age group than other age groups at onset of symptoms (P = 0.0401). MDM4 rs4252668C was present at a significantly higher frequency in controls while the frequency of MDM4 rs116197192G was significantly higher in RB patients, suggesting that this allele might increase the risk of developing RB. CONCLUSION: Our results indicate that MDM2 and MDM4 polymorphisms may influence development and/or survival in RB.

Detailed haplotype analysis at the TP53 locus in p.R337H mutation carriers in the population of Southern Brazil: evidence for a founder effect.

Due to patterns of migration, selection, and population expansion, founder effects are common among humans. In Southern Brazil, a recurrent TP53 mutation, p.R337H, is detected in families with cancer predisposition. We have used whole locus resequencing and high-density single nucleotide polymorphism (SNP) genotyping to refine TP53 locus haplotype definitions. Haplotyping of 12 unrelated p.R337H carriers using a set of 29 tag SNPs, revealed that all subjects carried the same haplotype, and presence of the mutation on this haplotype was confirmed by allele-specific PCR. The probability that this haplotype occurs independently in all index cases was of 3.1x10(-9), demonstrating a founder effect. Analysis of the patterns of 103 tumors diagnosed in 12 families showed that the presence of p.R337H is associated with multiple cancers of the Li-Fraumeni Syndrome (LFS) spectrum, with relatively low penetrance before the age of 30 but a lifetime risk comparable to classical LFS. The p.R337H families are mostly distributed along a road axis historically known as the main route used by merchants of Portuguese origin in the XVIII and XIX century. This historical circumstance and the relatively low penetrance before the age of 30 may have contributed to the maintenance of this pathogenic mutation in a large, open population.

Clinical characterization and risk profile of individuals seeking genetic counseling for hereditary breast cancer in Brazil.

Hereditary breast cancer (HBC) accounts for 5-10% of breast cancer cases and it significantly increases the lifetime risk of cancer. Our objective was to evaluate the sociodemographic variables, family history of cancer, breast cancer (BC) screening practices and the risk profile of cancer affected or asymptomatic at-risk women that undergo genetic counseling for hereditary breast cancer in public Brazilian cancer genetics services. Estimated lifetime risk of BC was calculated for asymptomatic women using the Gail and Claus models. The majority of women showed a moderate lifetime risk of developing BC, with an average risk of 19.7% and 19.9% by the Gail and Claus models, respectively. The average prior probability of carrying a BRCA1/2 gene mutation was 16.7% and overall only 32% fulfilled criteria for a hereditary breast cancer syndrome as assessed by family history. We conclude that a significant number of individuals at high-risk for HBC syndromes may not have access to the benefits of cancer genetic counseling in these centers. Contributing factors may include insufficient training of healthcare professionals, disinformation of cancer patients; difficult access to genetic testing and/or resistance in seeking such services. The identification and understanding of these barriers is essential to develop specific strategies to effectively achieve cancer risk reduction in this and other countries were clinical cancer genetics is not yet fully established.

The TP53 mutation, R337H, is associated with Li-Fraumeni and Li-Fraumeni-like syndromes in Brazilian families.

A TP53 germline mutation, R337H, has been previously described in children from southern Brazil with adrenocortical tumours but no documented familial history of other cancers. Here, we have screened for TP53 mutation 45 Brazilian unrelated individuals with family histories fulfilling the clinical definitions of Li-Fraumeni (LFS) or Li-Fraumeni-like (LFL) syndromes. Mutations were found in 13 patients (28.9%), including six (46.1%) R337H mutations, and four novel germline mutations (V173M, V197M, G244D and IVS6+1G>T). Families with the R337H mutation presented a wide spectrum of tumours, including breast cancers (30.4%), brain cancers (10.7%), soft tissue sarcomas (10.7%) and adrenocortical carcinomas (8.9%). Testing of 53 Brazilian subjects with no cancer history showed that R337H was not a common polymorphism in that population. Moreover, loss of heterozygocity with retention of the R337H allele was observed in a breast adenocarcinoma, supporting a role for this mutation in breast tumorigenesis. These results show that the TP53 R337H germline mutation predisposes to a larger spectrum of tumours, similar to the one reported for other TP53 mutations.

A molecular study of first and second RB1 mutational hits in retinoblastoma patients.

RB1 mutations accountable for biallelic inactivation are crucial events in the development of retinoblastoma because a first mutation (M1) predisposes to retinoblastoma while a second mutation (M2) is required for tumor development. Mutational analyses of this gene showed a wide spectrum of genetic alterations (single base substitutions, insertions, or deletions, as well as small and large deletions). The most frequent second hit in retinoblastoma patients is loss of heterozygosity (LOH) followed by promoter methylation. Molecular analyses of RB1 mutations were conducted in 36 patients (20 unilateral and 16 bilateral) using polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP) analysis, sequencing, and LOH analysis. Sixty-four amplified fragments showing abnormal SSCP patterns were sequenced, and mutations were confirmed in five patients (13.89%). Four mutations were located at coding regions, and a fifth one was found at an exon-intron junction. Two mutations were C-->T transitions, two were small-length deletions, and one was a G-->A transition. A total of 47.05% patients showed LOH. In one patient, the parental origin of the mutated allele was detected: the allele retained in the tumor was the paternal one. This work helps to characterize the spectrum of mutations in the Brazilian population, and to confirm that formaldehyde-fixed paraffin tissue can provide valuable information on the RB1 status in retinoblastoma patients.

Clinical, neuroimaging and cytogenetic findings in 20 patients with corpus callosum dysgenesis

Twenty children with corpus callosum agenesis or hypoplasia were evaluated under a standardized investigation protocol. Psychomotor retardation, seizures, and craniofacial anomalies were the most prominent findings. There were three cases of chromosomal anomalies, all of them representing trisomy of chromosome 8

Síndrome de Noonan: Estudo clínico-genético de 30 pacientes / Noonan syndrome: clinical-genetic study of 30 patients

A síndrome de Noonan (SN), entidade genética de herança autossômica dominante, subdiagnosticada em funçäo da expressividade extremamente variável do quadro clínico, é possivelmente uma das síndromes mendelianas mais freqüentes. Em um estudo prospectivo foram avaliados 30 pacientes a partir de um protocolo de investigaçäo clínico-laboratorial. Entre os critérios clínicos descritos na SN os mais importantes para o diagnóstico foram: ptose palpebral, micrognatia, fendas anti-down, raiz nasal deprimida, base nasal larga, hipertelorismo mamilar, criptorquidia e ptergium colli. Retardo mental era presente ou questionável em 14 pacientes. Os achados laboratoriais mais freqüentes foram: cardiopatia (predominando estenose pulmonar e comunicaçäo inter-atrial), anomalias renais, atraso de idade óssea, anomalias de coluna cervical, alteraçöes oftalmológicas, alteraçöes de audiçäo e coagulograma alterado. Apenas três pacientes (10 porcento) apresentaram displasia linfática, sugerindo possivelmente uma mortalidade aumentada neste grupo. Dois pacientes têm a reconhecida associaçäo da SN com neurofibromatose. Dois casos familiais foram confirmados e há nove casos familiais suspeitos. A idade paterna avançada nos casos näo familiais seria compatível com a mutaçäo nova para gen autossômico dominante. Os dados apresentados ressaltam a importância da sistematizaçäo do protocolo de investigaçäo e acompanhamento, visando a antecipaçäo e prevençäo de complicaçöes, bem como subsídios para o aconselhamento genético

Aspectos da contaminaçäo ambiental, resistência aos antimicrobianos e disseminaçäo dos bacilos Gram-negativos na infecçäo hospitalar / Environmental contamination, resistance to antimicrobials and dissemination of Gram-negative bacillus in infection

Os autores, no presente trabalho, analisam amostras da flora gram-negativa hospitalar, obtidas através de culturas feitas de 25 pias de enfermarias da I.S.C.M.P.A. A partir da cultura e identificaçäo dos germes, foram realizados testes de sensibilidade in vitro a dezesseis antibióticos. Dentre as 25 amostras cultivadas, em três näo houve crescimento bacteriano. Das 22 restantes, foram identificados quatro grupos: Pseudomonas, Klebsiella-Enterobacter, outros Coliformes e Proteus. Os padröes dos antibiogramas realizados demonstraram que a cefotaxima, cefalosporina de terceira geraçäo, foi o que apresentou maior índice de sensibilidade aos germes testados (60%), seguido da amicacina (30%), kanamicina (23,3%), tobramicina (23,3%), etc. enquanto que antimicrobianos como a cefaloridina apresentaram índice de 3,3% e a carbenicilina mostrou-se ineficaz contra todos os germes testados. Estes dados foram comparados com os resultados de antibiogramas de germes isolados de 59 pacientes com infecçäo hospitalar por bacilos gram-negativos. Os achados foram relacionados com a literatura, sendo também abordados aspectos relativos à contaminaçäo ambiental, resistência aos antimicrobianos e disseminaçäo de bacilos gram-negativos. Concluiu-se que a resistencia bacteriana é uma evidência inquestionável no ambiente hospitalar. Säo discutidas e propostas várias medidas visando o controle da infecçäo nosocomial. Quanto à alta resistência encontrada nos antibiogramas dos germes isolados de pacientes, serve de alerta a todos os profissionais de saúde para que o uso de antimicrobianos, bem como a assepsia das mäos e do ambiente, sejam os mais criteriosos possíveis para evitarmos que nos próximos anos tenhamos esgotado o arsenal terapêutico para doenças infecciosas adquiridas no hospital