RESUMO
The STARlet All-In-One system is a modular platform that integrates the complete molecular diagnostic workflow from nucleic acid extraction of clinical samples to PCR set-up and amplification. The platform was evaluated in comparison with laboratory developed tests (LDT) on fecal samples from patients with suspected viral gastro-enteritis. In a retrospective study, 72 positive samples were analysed, including all pathogens detected by the Seegene Allplex™ GI-virus assay, adenovirus, astrovirus, norovirus GI and GII, sapovirus, and rotavirus. Concordant results were obtained for 69 samples (96â¯%). Three discordant results were observed, one norovirus GII positive that gave an invalid result in the AIOS and two samples that were negative in the AIOS. One adenovirus positive that was subtyped as a genotype 2 virus, which is not associated with gastro-enteritis, and a sapovirus. In the prospective part of the study, 661 fecal samples were included. A total of 61 positive samples were detected, of which 60 were also detected by the AIOS. One norovirus GII positive sample (CT 35.2) was tested negative in the AIOS. Two additional sapovirus positive samples, CT 37 and 38, were detected by the AIOS but not by the LDT. The STARlet All-In-One platforms results in an automated molecular workflow with reduced hands-on time and enables running assays during out of office hours. Application of the Seegene Allplex™ GI-virus assay showed excellent concordance to the current diagnostic LDT. In a prospective comparison, only three discordant results were observed, all with CT values over 35 and therefore unlikely of clinical relevance.
Assuntos
Fezes , Gastroenterite , Técnicas de Diagnóstico Molecular , Humanos , Gastroenterite/virologia , Gastroenterite/diagnóstico , Fezes/virologia , Estudos Retrospectivos , Técnicas de Diagnóstico Molecular/métodos , Estudos Prospectivos , Pré-Escolar , Adulto , Criança , Viroses/diagnóstico , Viroses/virologia , Feminino , Automação Laboratorial/métodos , Pessoa de Meia-Idade , Masculino , Lactente , Adolescente , Adulto Jovem , Idoso , Vírus/isolamento & purificação , Vírus/genética , Vírus/classificação , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase/métodos , Norovirus/isolamento & purificação , Norovirus/genéticaRESUMO
Aims: The effect of excess glucocorticoid receptor (GR) stimulation through glucocorticoid medication or cortisol on glucose metabolism is well established. There are genetic GR variants that result in increased or decreased GR stimulation. We aimed to determine the prevalence of genetic GR variants in different ethnic groups in a cohort of patients with type 2 diabetes, and we aimed to determine their association with age of diabetes onset and metabolic and inflammation parameters. Methods: A cross-sectional analysis was performed in a multiethnic cohort (n = 602) of patients with established type 2 diabetes. Polymorphisms in the GR gene that have previously been associated with altered glucocorticoid sensitivity (TthIIII, ER22/23EK N363S, BclI and 9ß) were determined and combined into 6 haplotypes. Associations with age of diabetes onset, HbA1c, hs-CRP and lipid values were evaluated in multivariate regression models. Results: The prevalence of the SNPs of N363S and BclI was higher in Dutch than in non-Dutch patients. We observed a lower prevalence of the SNP 9ß in Dutch, South(East) Asian and Black African patients versus Turkish and Moroccan patients. We did not detect an association between SNPs and diabetes age of onset or metabolic parameters. We only found a trend for lower age of onset and higher HbA1c in patients with 1 or 2 copies of haplotype 3 (TthIIII + 9ß). Conclusions: The prevalence of genetic GR variants differs between patients of different ethnic origins. We did not find a clear association between genetic GR variants and age of diabetes onset or metabolic and inflammation parameters. This indicates that the clinical relevance of GR variants in patients with established type 2 diabetes is limited.
Assuntos
Diabetes Mellitus Tipo 2 , Humanos , Estudos Transversais , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Etnicidade/genética , Glucocorticoides , Hemoglobinas Glicadas , Inflamação/genética , Polimorfismo de Nucleotídeo Único , Receptores de Glucocorticoides/genéticaRESUMO
PURPOSE: Fluoropyrimidines are frequently prescribed anticancer drugs. A polymorphism in the fluoropyrimidine metabolizing enzyme dihydropyrimidine dehydrogenase (DPD; ie, DPYD*2A) is strongly associated with fluoropyrimidine-induced severe and life-threatening toxicity. This study determined the feasibility, safety, and cost of DPYD*2A genotype-guided dosing. PATIENTS AND METHODS: Patients intended to be treated with fluoropyrimidine-based chemotherapy were prospectively genotyped for DPYD*2A before start of therapy. Variant allele carriers received an initial dose reduction of ≥ 50% followed by dose titration based on tolerance. Toxicity was the primary end point and was compared with historical controls (ie, DPYD*2A variant allele carriers receiving standard dose described in literature) and with DPYD*2A wild-type patients treated with the standard dose in this study. Secondary end points included a model-based cost analysis, as well as pharmacokinetic and DPD enzyme activity analyses. RESULTS: A total of 2,038 patients were prospectively screened for DPYD*2A, of whom 22 (1.1%) were heterozygous polymorphic. DPYD*2A variant allele carriers were treated with a median dose-intensity of 48% (range, 17% to 91%). The risk of grade ≥ 3 toxicity was thereby significantly reduced from 73% (95% CI, 58% to 85%) in historical controls (n = 48) to 28% (95% CI, 10% to 53%) by genotype-guided dosing (P < .001); drug-induced death was reduced from 10% to 0%. Adequate treatment of genotype-guided dosing was further demonstrated by a similar incidence of grade ≥ 3 toxicity compared with wild-type patients receiving the standard dose (23%; P = .64) and by similar systemic fluorouracil (active drug) exposure. Furthermore, average total treatment cost per patient was lower for screening (2,772 [$3,767]) than for nonscreening (2,817 [$3,828]), outweighing screening costs. CONCLUSION: DPYD*2A is strongly associated with fluoropyrimidine-induced severe and life-threatening toxicity. DPYD*2A genotype-guided dosing results in adequate systemic drug exposure and significantly improves safety of fluoropyrimidine therapy for the individual patient. On a population level, upfront genotyping seemed cost saving.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Di-Hidrouracila Desidrogenase (NADP)/genética , Técnicas de Genotipagem/métodos , Medicina de Precisão/métodos , Pirimidinas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Custos e Análise de Custo , Feminino , Testes Genéticos , Técnicas de Genotipagem/economia , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos , Medicina de Precisão/economia , Estudos Prospectivos , Pirimidinas/efeitos adversos , Adulto JovemRESUMO
BACKGROUND: Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC. METHODS: A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates. RESULTS: Both sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons. CONCLUSIONS: In this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.
Assuntos
Proteínas de Bactérias/genética , Carbapenêmicos , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/genética , Reação em Cadeia da Polimerase Multiplex , beta-Lactamases/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: This study aimed to determine the prevalence of respiratory pathogens among newborns admitted to a neonatal medium care unit (NMCU) and to identify clinical predictors. METHODS: A 1-y observational study was performed of neonates admitted to an NMCU in Amsterdam, The Netherlands. Nasopharyngeal samples were collected for the detection of respiratory viruses and bacteria by real-time PCR (RT-PCR). Cycle threshold (Ct) values were provided to estimate viral load. Predictors for the presence of study pathogens were identified. RESULTS: From October 2010 through September 2011, 334 neonates (median age 1.3 d, 53.6% male) were included. Overall, 37 respiratory pathogens were detected in 34 children (10.2%): parainfluenza-1 (n = 9), human rhinovirus (n = 7), parainfluenza-3 (n = 6), respiratory syncytial virus (RSV, n = 6), Streptococcus pneumoniae (n = 3), adenovirus (n = 2), human coronavirus (n = 2), influenza A (n = 1), and bocavirus (n = 1). Neonates with higher viral loads (Ct <35; n = 11) were more often clinically ill than those with lower viral loads (Ct ≥35; n = 23). Two variables significantly contributed to the detection of study pathogens: age (odds ratio (OR) 1.21 for each day older; 95% confidence interval 1.12-1.30) and rhinorrhea (OR 6.71; 95% confidence interval 1.54-29.21). CONCLUSION: Respiratory pathogens seem to play a role in neonates admitted to an NMCU. The influence of respiratory pathogen detection on clinical management remains to be determined.
Assuntos
Infecção Hospitalar/epidemiologia , Nasofaringe/microbiologia , Nasofaringe/virologia , Infecções Respiratórias/epidemiologia , Fatores Etários , Feminino , Humanos , Recém-Nascido , Masculino , Países Baixos/epidemiologia , Razão de Chances , Cuidado Pós-Natal , Reação em Cadeia da Polimerase em Tempo Real , Carga ViralRESUMO
PURPOSE: This study aimed to determine incidence rates of novel influenza A (H1N1) infection among healthcare personnel with different exposure risks during the 2009 H1N1 pandemic. METHODS: From August 2009 until April 2010, 66 healthcare workers from a 410 bed teaching hospital in Amsterdam were monitored. The following three different exposure groups were created: a high- (n = 26), intermediate- (n = 20), and low-risk group (n = 20). Throat swabs were collected each week and analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in order to detect the H1N1 virus. Blood was drawn at study enrollment and once monthly thereafter, and serum specimens were tested with an H1N1-specific hemagglutination-inhibition serologic assay. Influenza-like signs and symptoms were assessed weekly. RESULTS: One of 26 high-risk group participants proved H1N1 positive once by RT-PCR. This corresponds to an incidence rate in the high-risk group of 5.7/1,000 person weeks (95% CI 0-17/1,000). None of the intermediate- and low-risk group participants proved H1N1 positive by RT-PCR. Significant antibody titer rises in convalescent sera were demonstrated in three participants: one was a confirmation of the case that had proved H1N1 positive by RT-PCR; the others occurred in two asymptomatic participants belonging to the low- and high-risk groups. An influenza-like illness was assumed in four participants from the high- (n = 1), intermediate- (n = 1) and low-risk (n = 2) groups; these findings were not confirmed by positive results from either diagnostic test. CONCLUSIONS: This study demonstrates a low incidence rate of influenza A (H1N1) infection among healthcare workers during the 2009 H1N1 pandemic in a setting with high hygiene standards.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana/epidemiologia , Exposição Ocupacional , Recursos Humanos em Hospital , Vigilância da População , Adulto , Análise de Variância , Feminino , Testes de Inibição da Hemaglutinação , Hospitais de Ensino , Humanos , Incidência , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Países Baixos , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
BACKGROUND: The authors assessed the impact of germline polymorphisms on clinical outcome in patients with advanced nonsmall cell lung cancer (NSCLC) who received platinum-gemcitabine (PG) chemotherapy. METHODS: In total, 137 patients with stage IIIB/IV NSCLC were included who received first-line PG chemotherapy (74% of patients received cisplatin, and 26% received carboplatin). Twenty-three germline polymorphisms that were identified in peripheral blood samples were analyzed for progression-free survival (PFS), treatment response, overall survival (OS), and toxicity. RESULTS: The median PFS was 5.8 months, the median OS was 10.2 months, and 44 patients (32%) had a partial treatment response. Carriers of the excision repair cross-complementation group 1 (ERCC1) mutant thymine (T) allele had a lower treatment response rate (29% vs 52%; P = .02), shorter PFS (adjusted hazard ratio [HR], 1.60; P = .04), and shorter OS (adjusted HR, 1.54; P = .05) compared with carriers of the wild-type cytosine/cytosine (CC) genotype. The xeroderma pigmentosum group A member 10 (XPD10) mutant adenine (A) allele (adjusted HR, 0.64; P = .04) and the x-ray cross-complementing group 1 (XRCC1) mutant guanine (G) allele (adjusted HR, 0.51; P = .02) also were independent predictors of OS. Carriers of the mutant adenosine triphosphate-dependent DNA helicase Q1 (RECQ1) C allele or the mutant cytidine deaminase (CDA) C allele were more likely to experience severe leukocytopenia (26% vs 10% [P = .03] and 28% vs 11% [P = .02], respectively) compared with wild-type genotype carriers. Patients who carried the homozygous mutant glutathione S-transferase π 1(GSTP1) GG genotype were at considerable risk for severe platinum-associated polyneuropathy (18% vs 3% in wild-type vs heterozygous mutant patients, respectively; P = .01). CONCLUSIONS: To the authors' knowledge, this is the first prospective study to date in patients with advanced NSCLC describing predictive germline polymorphisms not only for the clinical activity of PG chemotherapy (ERCC1, XPD10) but also for its toxicity (GSTP1, RECQ1, CDA). Nonplatinum-containing chemotherapy in carriers of the ERCC1 T allele or the XPD10 G allele should be studied prospectively.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Cisplatino/administração & dosagem , Desoxicitidina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Polimorfismo Genético , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Desoxicitidina/administração & dosagem , Intervalo Livre de Doença , Feminino , Células Germinativas , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Resultado do Tratamento , GencitabinaRESUMO
AIM: To determine causative respiratory pathogens and describe epidemiological and clinical characteristics in a paediatric population with influenza-like illness during the 2009 H1N1-pandemic. METHODS: Observational study of 412 children visiting an outpatient clinic of a Dutch teaching hospital. RESULTS: From August to December 2009, 412 children were tested at the clinic; 32% proved H1N1-positive, confirmed by reverse-transcriptase-polymerase-chain-reaction (RT-PCR). Pathogens were detected in 65% of samples. Influenza A(H1N1) (n = 132), human rhinovirus (n = 55), respiratory syncytial virus (n = 45) and adenovirus (n = 34) were mostly identified. Co-infections were seen in 34 children (8.3%). Mean age was 6.8 and 4.2 years in H1N1-positive and H1N1-negative cases, respectively (p < 0.01). H1N1-positive outpatient children reported fever, cough and rhinorrhoea more frequently than their H1N1-negative counterparts. Of 72 hospitalized children, 31% proved H1N1-positive; all showed a relatively mild clinical illness. None of the children had been admitted to an intensive care unit or died. Oseltamivir treatment was initiated in 72 children and discontinued in 42 (63%) when RT-PCR results turned negative. CONCLUSION: The 2009 H1N1-pandemic showed a mild clinical course in a Dutch paediatric outpatient clinic population. Respiratory pathogens were detected in the majority of children with influenza-like illness and influenza A(H1N1) virus was identified in one-third. Testing symptomatic children during an influenza pandemic has effectively limited the use of oseltamivir.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/virologia , Pandemias , Antivirais/uso terapêutico , Criança , Pré-Escolar , Feminino , Hospitais de Ensino , Humanos , Lactente , Influenza Humana/tratamento farmacológico , Influenza Humana/epidemiologia , Masculino , Países Baixos/epidemiologia , Oseltamivir/uso terapêutico , Reação em Cadeia da Polimerase em Tempo RealRESUMO
PURPOSE: To explore the effect of dihydropyrimidine dehydrogenase (DPD) single nucleotide polymorphisms (SNP) and haplotypes on outcome of capecitabine. EXPERIMENTAL DESIGN: Germline DNA was available from 568 previously untreated patients with advanced colorectal cancer participating in the CAIRO2 trial, assigned to capecitabine, oxaliplatin, and bevacizumab ± cetuximab. The coding region of dihydropyrimidine dehydrogenase gene (DPYD) was sequenced in 45 cases with grade 3 or more capecitabine-related toxicity and in 100 randomly selected controls (cohort). Most discriminating (P < 0.1) or frequently occurring (>1%) nonsynonymous SNPs were analyzed in all 568 patients. SNPs and haplotypes were associated with toxicity, capecitabine dose modifications, and survival. RESULTS: A total of 29 SNPs were detected in the case-cohort analysis, of which 8 were analyzed in all 568 patients. Of the patients polymorphic for DPYD IVS14+1G>A, 2846A>T, and 1236G>A, 71% (5 of 7), 63% (5 of 8), and 50% (14 of 28) developed grade 3 to 4 diarrhea, respectively, compared with 24% in the overall population. All patients polymorphic for IVS14+1G>A developed any grade 3 to 4 toxicity, including one possibly capecitabine-related death. Because of toxicity, a mean capecitabine dose reduction of 50% was applied in IVS14+1G>A and 25% in 2846A>T variant allele carriers. Patients were categorized into six haplotype groups: one predicted for reduced (10%), and two for increased risks (41% and 33%) for severe diarrhea. Individual SNPs were not associated with overall survival, whereas one haplotype was associated with overall survival [HR (95% CI) = 0.57 (0.35-0.95)]. CONCLUSIONS: DPYD IVS14+1G>A and 2846A>T predict for severe toxicity to capecitabine, for which patients require dose reductions. Haplotypes assist in selecting patients at risk for toxicity to capecitabine.
Assuntos
Carcinoma/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Desoxicitidina/análogos & derivados , Di-Hidrouracila Desidrogenase (NADP)/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Fluoruracila/análogos & derivados , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos/metabolismo , Biomarcadores Tumorais/genética , Capecitabina , Carcinoma/genética , Estudos de Casos e Controles , Ensaios Clínicos Fase III como Assunto , Estudos de Coortes , Neoplasias Colorretais/genética , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/uso terapêutico , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Progressão da Doença , Relação Dose-Resposta a Droga , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Fluoruracila/uso terapêutico , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/fisiologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Retrospectivos , Resultado do TratamentoRESUMO
OBJECTIVES: The aim was to describe causative agents and clinical characteristics in adult outpatients with upper airway symptoms during the 2009 H1N1 pandemic and to evaluate case definitions that are used in clinical practice. METHODS: From August through December 2009, 964 symptomatic adult outpatients were included. RT-PCR was used to detect the following pathogens: influenza A (H1N1) and B, parainfluenza 1-4, adenovirus, respiratory syncytial virus, human rhinovirus, human metapneumovirus, human coronavirus (OC43, 229E, NL63), Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella species. The Dutch GHOR, American CDC and WHO, and British HPA case definitions were evaluated. RESULTS: A respiratory pathogen was detected in 41% of tested patient samples; influenza A (H1N1) and human rhinovirus were both detected in 16%. Clinical presentation of influenza cases was significantly more serious when compared to rhinovirus or negative-tested cases. Test characteristics were almost similar for all 4 case definitions, with an average sensitivity of 66%, specificity of 70%, positive predictive value of 34% and negative predictive value of 90%. CONCLUSIONS: Influenza A (H1N1) and human rhinovirus were the major pathogens responsible for respiratory disease. The 2009 H1N1 pandemic in Amsterdam followed a mild course. Test characteristics of 4 different clinical case definitions seemed comparable but rather useless.
Assuntos
Assistência Ambulatorial/métodos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/epidemiologia , Pandemias , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Influenza Humana/patologia , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Prevalência , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Adulto JovemAssuntos
Calcitonina/análise , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/fisiopatologia , Precursores de Proteínas/análise , Análise Química do Sangue , Proteína C-Reativa/análise , Peptídeo Relacionado com Gene de Calcitonina , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Influenza Humana/virologia , Contagem de LeucócitosRESUMO
A wide interindividual variability in survival after cancer treatment is observed. This is attributable to many factors, including tumour and patient related factors. Genetic polymorphisms in drug metabolising enzymes and drug transporters may be one of these factors. Drug metabolising enzymes are responsible for the activation, inactivation and detoxification of many chemotherapeutic agents. Deficiencies in these enzymes may result in altered exposure (both extracellular and intracellular) to the chemotherapeutic agents, thereby influencing the efficacy of treatment. Drug transporters are important in the uptake and excretion of chemotherapeutic agents. Polymorphisms in drug transporter genes may influence the bioavailability and disposition of these agents. Studies have shown that variability in survival can (partly) be explained by polymorphisms in genes encoding drug metabolising enzymes and drug transporters. This review will discuss the role of genetic polymorphisms in drug metabolising enzymes and drug transporters in relation to survival after cancer treatment. The most important polymorphisms shown to influence survival after cancer treatment are polymorphisms in the genes encoding the phase II detoxification enzymes glutathione-S-transferases (GSTs). It appears that GSTM1 null and GSTT1 null have a clear association with longer overall survival in patients with different malignancies who are treated with substrates for these GSTs (mostly alkylating agents and platinum compounds). Genetic polymorphisms in GSTP1 and GSTA1 are also associated with an increased overall survival in patients with different malignancies. Most of the current data on the relation between treatment response and pharmacogenetics is derived from retrospective and exploratory studies. Prospective studies will be necessary.
Assuntos
Antineoplásicos/uso terapêutico , Glutationa Transferase/genética , Proteínas de Membrana Transportadoras/genética , Oxigenases de Função Mista/genética , Neoplasias/tratamento farmacológico , Polimorfismo Genético/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Humanos , Neoplasias/genética , FarmacogenéticaRESUMO
AIMS: Thiotepa is widely used in high-dose chemotherapy. Previous studies have shown relations between exposure and severe organ toxicity. Thiotepa is metabolized by cytochrome P450 and glutathione S-transferase enzymes. Polymorphisms of these enzymes may affect elimination of thiotepa and tepa, its main metabolite. The purpose of this study was to evaluate effects of known allelic variants in CYP2B6, CYP3A4, CYP3A5, GSTA1 and GSTP1 genes on pharmacokinetics of thiotepa and tepa. METHODS: White patients (n = 124) received a high-dose regimen consisting of cyclophosphamide, thiotepa and carboplatin as intravenous infusions. Genomic DNA was analysed using polymerase chain reaction and sequencing. Plasma concentrations of thiotepa and tepa were determined using validated GC and LC-MS/MS methods. Relations between allelic variants and elimination pharmacokinetic parameters were evaluated using nonlinear mixed effects modelling (nonmem). RESULTS: The polymorphisms CYP2B6 C1459T, CYP3A4*1B, CYP3A5*3, GSTA1 (C-69T, G-52A) and GSTP1 C341T had a significant effect on clearance of thiotepa or tepa. Although significant, most effects were generally not large. Clearance of thiotepa and tepa was predominantly affected by GSTP1 C341T polymorphism, which had a frequency of 9.3%. This polymorphism increased non-inducible thiotepa clearance by 52% [95% confidence interval (CI) 41, 64, P < 0.001] and decreased tepa clearance by 32% (95% CI 29, 35, P < 0.001) in heterozygous patients, which resulted in an increase in combined exposure to thiotepa and tepa of 45% in homozygous patients. CONCLUSIONS: This study indicates that the presently evaluated variant alleles explain only a small part of the substantial interindividual variability in thiotepa and tepa pharmacokinetics. Patients homozygous for the GSTP1 C341T allele may have enhanced exposure to thiotepa and tepa.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Glutationa Transferase/genética , Polimorfismo Genético , Tiotepa/farmacocinética , Trietilenofosforamida/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias da Mama/tratamento farmacológico , Carboplatina , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Ciclofosfamida , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/genética , Feminino , Glutationa S-Transferase pi/genética , Humanos , Masculino , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Oxirredutases N-Desmetilantes/genéticaRESUMO
PURPOSE: High-dose chemotherapy with cyclophosphamide, thiotepa and carboplatin (CTC) has been developed as a possible curative treatment modality in several solid tumours. However, a large interindividual variability in toxicity is encountered in high-dose chemotherapy. A priori identification of patients at risk for toxicity could be an attractive prospect. Genotyping of genes encoding drug-metabolising enzymes might provide such a tool. EXPERIMENTAL DESIGN: We assessed 16 selected polymorphisms in nine genes (CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1) of putative relevance in CTC metabolism using polymerase chain reaction and DNA sequencing in 113 patients who were treated with high-dose chemotherapy regimens based on CTC. RESULTS: Patients heterozygous for the ALDH3A1*2 allele (allelic frequency 21.2%) had an increased risk of haemorrhagic cystitis when compared with patients with wild-type alleles [5/38 vs. 1/70; odds ratio (OR): 11.95, 95% confidence interval (CI): 1.18-120.56; P=0.04]. Furthermore, patients heterozygous for the ALDH1A1*2 allele (allelic frequency 5.8%) had an increased risk of liver toxicity when compared with patients with wild-type alleles (6/13 vs. 19/99; OR: 5.13, 95% CI: 1.30-20.30; P=0.02). No other relations reached significance. CONCLUSION: Patients heterozygous for the ALDH3A1*2 and ALDH1A1*2 allele have an increased risk of haemorrhagic cystitis and liver toxicity, respectively, compared with patients with wild-type alleles when treated with a high-dose chemotherapy combination of CTC. Pharmacogenetic approaches can identify patients who are at risk of experiencing toxic side effects in high-dose chemotherapy.
Assuntos
Carboplatina/efeitos adversos , Ciclofosfamida/efeitos adversos , Inativação Metabólica/genética , Neoplasias/tratamento farmacológico , Polimorfismo de Nucleotídeo Único/genética , Tiotepa/efeitos adversos , Adolescente , Adulto , Carboplatina/administração & dosagem , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Ciclofosfamida/administração & dosagem , Ciclofosfamida/farmacologia , Ciclofosfamida/uso terapêutico , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Tiotepa/administração & dosagem , Tiotepa/farmacologia , Tiotepa/uso terapêuticoRESUMO
PURPOSE: The anticancer agent, cyclophosphamide, is metabolized by cytochrome P450 (CYP), glutathione S-transferase (GST) and aldehyde dehydrogenase (ALDH) enzymes. Polymorphisms of these enzymes may affect the pharmacokinetics of cyclophosphamide and thereby its toxicity and efficacy. The purpose of this study was to evaluate the effects of known allelic variants in the CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1 genes on the pharmacokinetics of the anticancer agent, cyclophosphamide, and its active metabolite 4-hydroxycyclophosphamide. EXPERIMENTAL DESIGN: A cohort of 124 Caucasian patients received a high-dose chemotherapy combination consisting of cyclophosphamide (4-6 g/m2), thiotepa (320-480 mg/m2) and carboplatin (area under the curve 13-20 mg x min/ml) as intravenous infusions over 4 consecutive days. Genomic DNA was analysed using PCR and sequencing. Liquid chromatography-tandem mass spectrometry was used to measure plasma concentrations of cyclophosphamide and 4-hydroxycyclophosphamide. The relationship between allelic variants and the elimination pharmacokinetic parameters noninducible cyclophosphamide clearance (CL(nonind)), inducible cyclophosphamide clearance (CL(ind)) and elimination rate constant of 4-hydroxycyclophosphamide (k(4OHCP)) were evaluated using nonlinear mixed effects modelling. RESULTS: The interindividual variability in the noninducible cyclophosphamide clearance, inducible cyclophosphamide clearance and 4-hydroxycyclophosphamide clearance was 23, 27 and 31%, respectively. No effect of the allelic variants investigated on the clearance of cyclophosphamide or 4-hydroxycyclophosphamide could be demonstrated. CONCLUSION: This study indicates that the presently evaluated variant alleles in the CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1 genes do not explain the interindividual variability in cyclophosphamide and 4-hydroxycyclophosphamide pharmacokinetics and are, probably, not the cause of the observed variability in toxicity.
Assuntos
Aldeído Desidrogenase/genética , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacocinética , Sistema Enzimático do Citocromo P-450/genética , Glutationa Transferase/genética , Polimorfismo Genético , Adolescente , Adulto , Família Aldeído Desidrogenase 1 , Hidrocarboneto de Aril Hidroxilases/genética , Sequência de Bases , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estudos de Coortes , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A/genética , Primers do DNA/genética , Feminino , Glutationa S-Transferase pi/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Oxirredutases N-Desmetilantes/genética , Farmacogenética , Retinal DesidrogenaseRESUMO
BACKGROUND: HIV-infected women are at increased risk for cervical dysplasia. Cervical dysplasia is caused by persistent infections with certain types of human papillomavirus (HPV). Conventional testing for genital HPV infections requires cervical cytology. A non-invasive screening method by detection of HPV DNA in urine samples is preferable but is not a routine practice. OBJECTIVES: To investigate the prevalence and concordance of HPV in paired urine and cervical smear samples and cytological results of Pap smears in HIV-infected women. STUDY DESIGN: Paired urine and cervical smear samples were collected from 27 HIV-infected women. RESULTS: The HPV prevalence in urine and cervical smear samples was 81.5% and 51.9%, respectively (p = 0.01). The concordance for HPV positivity and negativity between urine and cervical smear samples is 71%. Seven women (25.9%) had an abnormal cervical smear of Pap II or higher. In all urine samples from these cases HPV DNA was detected. CONCLUSION: In the present study we show that the HPV prevalence in urine and cervical smear samples of HIV-infected women is high and HPV test results are highly concordant. Therefore, urine samples can be used as medium for HPV testing. HPV testing in urine samples is a simple, reliable, non-invasive screening method.
Assuntos
Colo do Útero/virologia , Infecções por HIV/complicações , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Urina/virologia , Adulto , DNA Viral/análise , DNA Viral/isolamento & purificação , Feminino , Humanos , Teste de Papanicolaou , Papillomaviridae/classificação , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Prevalência , Esfregaço VaginalRESUMO
OBJECTIVE: Epstein-Barr virus (EBV)-positive lymphomas in HIV carriers are paralleled by elevated EBV-DNA loads in the circulation. Approximately 20% of asymptomatic HIV carriers also show elevated circulating EBV-DNA loads. We aimed to characterize the nature of this EBV DNA and to determine the transcriptional phenotype of EBV in blood, in relation to serological parameters. DESIGN: A total of 197 random asymptomatic HIV carriers, representing 2% of the Dutch HIV-positive population, were sampled for blood, peripheral blood mononuclear cells and plasma. In addition, 39 EBV-DNA carriers were sampled twice, with a 5-year interval. METHODS: EBV-DNA loads were determined by LightCycler-based real-time polymerase chain reaction (PCR). EBV transcription was studied by nucleic acid sequence-based amplification and reverse transcriptase PCR. IgA and IgG antibodies to EBV antigens EBNA1 and VCA-p18 were quantified by synthetic peptide-based enzyme-linked immunosorbent assay. RESULTS: : Elevated EBV-DNA loads were found in whole blood of 19.3% of the tested HIV population, which were persistent in 82%. Plasma samples were EBV-DNA negative and circulating EBV DNA could be attributed to the B-cell compartment. Transcription of only LMP2 and (non-translated) transcripts from the BamHI-A region of the EBV genome was found, whereas EBNA1, LMP1 and lytic EBV transcripts were absent despite high cellular EBV-DNA loads. IgA-reactivity to VCA-p18 was seen in 69%. IgG to VCA-p18 was significantly higher in high EBV-DNA load carriers. CONCLUSION: Asymptomatic HIV carriers show aberrant EBV persistence in the circulation, characterized by elevated, B-cell-associated EBV-DNA loads. EBV transcription is restricted, arguing for EBV gene shutdown in circulating EBV-carrying B cells. Increased IgA and IgG reactive to VCA-p18 is indicative of increased lytic EBV replication, possibly occurring at mucosal lymphoid sites but not in the circulation.
Assuntos
Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Soropositividade para HIV/virologia , Herpesvirus Humano 4/fisiologia , Imunoglobulina A/sangue , Linfoma de Células B/virologia , Adulto , Antígenos Virais/imunologia , Autorradiografia , Proteínas do Capsídeo/imunologia , DNA Viral/sangue , Infecções por Vírus Epstein-Barr/imunologia , Soropositividade para HIV/imunologia , Herpesvirus Humano 4/genética , Humanos , Linfoma de Células B/imunologia , Transcrição Gênica , Carga Viral , Latência ViralRESUMO
BACKGROUND: Deficiency of dihydropyrimidine dehydrogenase (DPD) has been linked to severe or lethal fluorouracil (FU)-related toxicity. The most prominent mutation in the DPYD gene is the IVS14+1G>A mutation, which causes skipping of exon 14 in the messenger RNA (mRNA) and results in DPD enzyme deficiency. Several methods have been described to detect this mutation, but all are labor intensive and low throughput. OBJECTIVE Our aim was to develop a high-throughput real-time PCR assay to screen patients for the IVS14+1G>A mutation. METHODS: Primers and probes were developed and several reaction conditions were tested. In total, 165 individuals were screened for this mutation, with DNA sequencing as a reference method. RESULTS: Results of the real-time PCR assay and DNA sequencing were 100% identical. In total, eight heterozygous individuals were identified, of which six were patients with severe FU-related toxicity after FU or capecitabine treatment and two were healthy volunteers. CONCLUSION: This new real-time PCR assay with a high throughput is particularly suitable for large-scale screening for the IVS14+1G>A mutation in patients selected for treatment with fluoropyrimidines in order to prevent severe FU-related toxicity.
Assuntos
Análise Mutacional de DNA/métodos , Di-Hidrouracila Desidrogenase (NADP)/genética , Fluoruracila/toxicidade , Mutação , Frequência do Gene , Humanos , Estrutura Terciária de Proteína/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de TempoRESUMO
Cytochrome P450 (CYP) 2D6 is one of the most important enzymes involved in the metabolism of drugs. Multiple, clinically relevant, genetic variants of this gene have been identified and, among them, a gene deletion as well as multiplications of the gene. These large structural mutations in CYP2D6 occur at a relatively high frequency in several populations. Genotyping of CYP2D6 could therefore be applied to individualize drug therapy to improve therapeutic efficacy and decrease adverse effects in patients. However, a prerequisite for the pharmacogenetic screening of CYP2D6 in a clinical setting is the development of fast, reliable and cost-effective techniques for the routine genotyping of patients. In the case of CYP2D6, besides the general problems that arise in the detection of large gene deletions and multiplications, the presence of two highly homologous pseudogenes, CYP2D7 and CYP2D8, forms an extra challenge. This review provides an overview of the techniques that have been described to detect the CYP2D6 gene deletion and multiplication: Southern-blotting RFLP, long-template PCR, and real-time PCR. Of these techniques, real-time PCR is the only technique giving quantitative information about the exact copy number of the gene. Considering all of the other advantages of this method over other methods, such as cost-effectiveness and suitability for high throughput screening, real-time PCR is the most promising method for the genotyping of large structural alterations in the CYP2D6 gene in a routine clinical setting.
Assuntos
Citocromo P-450 CYP2D6/genética , Deleção de Genes , Duplicação Gênica , Farmacogenética/métodos , Animais , Genótipo , Humanos , Farmacogenética/economia , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos TestesRESUMO
Amyloid beta is an in vitro substrate for P-glycoprotein (P-gp), an efflux pump at the blood brain barrier (BBB). The Multi Drug Resistance (ABCB1) gene, encoding for P-gp, is highly polymorphic and this may result in a changed function of P-gp and may possibly interfere with the pathogenesis of Alzheimer's disease. This study investigates to what extent ABCB1 Single Nucleotide Polymorphisms (SNPs; C1236T in exon 12, G2677T/A in exon 21 and C3435T in exon 26) and inferred haplotypes exist in an elderly population and if these SNPs and haplotypes differ between patients with dementia and age-matched non-demented control patients. ABCB1 genotype, allele and haplotype frequencies were neither significantly different between patients with dementia and age-matched controls, nor between subgroups of different types of dementia nor age-matched controls. This study shows ABCB1 genotype frequencies to be comparable with described younger populations. To our knowledge this is the first study on ABCB1 genotypes in dementia. ABCB1 genotypes are presently not useful as a biomarker for dementia, as they were not significantly different between demented patients and age-matched control subjects.