Quantitative detection of BK virus in kidney transplant recipients: a prospective validation study.

INTRODUCTION: BK virus (BKV) infection in renal transplant patients may cause kidney allograft dysfunction and graft loss. Accurate determination of BKV viral load is critical to prevent BKV-associated nephropathy (BKVAN) but the cut-off that best predicts BKVAN remains controversial. OBJECTIVE: To evaluate the performance of a commercial and an in-house qPCR test for quantitative detection of BK virus in kidney transplant recipients. METHODS: This was a prospective study with kidney transplant recipients from two large university hospitals in Brazil. Patients were screened for BKV infection every 3 months in the first year post-transplant with a commercial and an in-house real time polymerase chain reaction (qPCR) test. BKVAN was confirmed based on histopathology. The area under the curve for plasma qPCR was determined from receiver operating characteristic analysis. RESULTS: A total of 200 patients were enrolled. Fifty-eight percent were male, 19.5% had diabetes mellitus, and 82% had the kidney transplanted from a deceased donor. BKV viremia was detected in 32.5% and BKVAN was diagnosed in 8 patients (4%). BKVAN was associated with viremia of 4.1 log copies/mL, using a commercial kit. The cut-off for the in-house assay was 6.1 log copies/mL. The linearity between the commercial kit and the in-house assay was R2=0.83. CONCLUSION: Our study shows that marked variability occurs in BKV viral load when different qPCR methodologies are used. The in-house qPCR assay proved clinically useful, a cheaper option in comparison to commercial qPCR kits. There is an urgent need to make BKV standards available to the international community.

Quantitative detection of BK virus in kidney transplant recipients: a prospective validation study / Detecção quantitativa de vírus BK em receptores de transplante renal: um estudo prospectivo de validação

Abstract Introduction: BK virus (BKV) infection in renal transplant patients may cause kidney allograft dysfunction and graft loss. Accurate determination of BKV viral load is critical to prevent BKV-associated nephropathy (BKVAN) but the cut-off that best predicts BKVAN remains controversial. Objective: To evaluate the performance of a commercial and an in-house qPCR test for quantitative detection of BK virus in kidney transplant recipients. Methods: This was a prospective study with kidney transplant recipients from two large university hospitals in Brazil. Patients were screened for BKV infection every 3 months in the first year post-transplant with a commercial and an in-house real time polymerase chain reaction (qPCR) test. BKVAN was confirmed based on histopathology. The area under the curve for plasma qPCR was determined from receiver operating characteristic analysis. Results: A total of 200 patients were enrolled. Fifty-eight percent were male, 19.5% had diabetes mellitus, and 82% had the kidney transplanted from a deceased donor. BKV viremia was detected in 32.5% and BKVAN was diagnosed in 8 patients (4%). BKVAN was associated with viremia of 4.1 log copies/mL, using a commercial kit. The cut-off for the in-house assay was 6.1 log copies/mL. The linearity between the commercial kit and the in-house assay was R2=0.83. Conclusion: Our study shows that marked variability occurs in BKV viral load when different qPCR methodologies are used. The in-house qPCR assay proved clinically useful, a cheaper option in comparison to commercial qPCR kits. There is an urgent need to make BKV standards available to the international community.

Resumo Introdução: A infecção pelo vírus BK (BKV) em pacientes de transplante renal pode levar a disfunção do aloenxerto renal e perda do enxerto. A determinação precisa da carga viral do BKV é fundamental para prevenir a nefropatia associada ao BKV (BKVAN), mas o ponto de corte de melhor valor preditivo para BKVAN ainda é foco de debates. Objetivo: Avaliar o desempenho de um teste de qPCR comercial e outro desenvolvido internamente para detecção quantitativa de vírus BK em receptores de transplante renal. Métodos: O presente estudo prospectivo incluiu receptores de transplante renal de dois grandes hospitais universitários no Brasil. Os pacientes foram testados para infecção por BKV a cada três meses no primeiro ano pós-transplante com um teste comercial de reação em cadeia de polimerase quantitativa em tempo real (qPCR) e outro desenvolvido internamente. A presença de BKVAN foi confirmada com base na histopatologia. A área sob a curva para o qPCR plasmático foi determinada a partir da análise da característica de operação do receptor. Resultados: Um total de 200 pacientes foram incluídos. Cinquenta e oito por cento eram do sexo masculino, 19,5% tinham diabetes mellitus e 82% tiveram seus rins transplantados de doadores falecidos. Viremia de BKV foi detectada em 32,5% dos pacientes e oito (4%) foram diagnosticados com BKVAN. BKVAN foi associada a viremia de 4,1 log cópias/mL usando o kit comercial. O corte para o ensaio interno foi de 6,1 log cópias/mL. A linearidade entre o kit comercial e o ensaio interno foi R2 = 0,83. Conclusão: Nosso estudo demonstrou uma acentuada variabilidade na carga viral de BKV quando diferentes metodologias de qPCR foram utilizadas. O ensaio interno de qPCR mostrou-se clinicamente útil, além de ser uma opção menos onerosa em relação aos kits comerciais de qPCR. Há uma necessidade urgente de se definir padrões de BKV para a comunidade internacional.

Ancestry of the Brazilian TP53 c.1010G>A (p.Arg337His, R337H) Founder Mutation: Clues from Haplotyping of Short Tandem Repeats on Chromosome 17p.

Rare germline mutations in TP53 (17p13.1) cause a highly penetrant predisposition to a specific spectrum of early cancers, defining the Li-Fraumeni Syndrome (LFS). A germline mutation at codon 337 (p.Arg337His, c1010G>A) is found in about 0.3% of the population of Southern Brazil. This mutation is associated with partially penetrant LFS traits and is found in the germline of patients with early cancers of the LFS spectrum unselected for familial history. To characterize the extended haplotypes carrying the mutation, we have genotyped 9 short tandem repeats on chromosome 17p in 12 trios of Brazilian p.Arg337His carriers. Results confirm that all share a common ancestor haplotype of Caucasian/Portuguese-Iberic origin, distant in about 72-84 generations (2000 years assuming a 25 years intergenerational distance) and thus pre-dating European migration to Brazil. So far, the founder p.Arg337His haplotype has not been detected outside Brazil, with the exception of two residents of Portugal, one of them of Brazilian origin. On the other hand, increased meiotic recombination in p.Arg337His carriers may account for higher than expected haplotype diversity. Further studies comparing haplotypes in populations of Brazil and of other areas of Portuguese migration are needed to understand the historical context of this mutation in Brazil.

Effects of lifestyle modification after breast cancer treatment: a systematic review protocol.

BACKGROUND: There is no consensus in the literature regarding the effectiveness of lifestyle modification interventions, including recommendations about specific diet or exercise program for patients with breast cancer. Diet interventions and regular physical activity may reduce the risk of breast cancer and its recurrence. The primary aim of our study is to evaluate the effects of different lifestyle modification interventions (diet and physical activity) in the survival of patients with stages I to III breast cancer after treatment. METHODS/DESIGN: This review will be conducted according to the Cochrane Handbook for Systematic Reviews of Intervention and will be reported following the PRISMA statement recommendations. CENTRAL, MEDLINE and EMBASE databases will be searched for peer-reviewed literature. Randomized controlled trials of diet, exercise, or both, compared with usual care, after treatment of breast cancer stage I to III will be included in the systematic review. Two authors will independently screen titles and abstracts of studies for potential eligibility. Data will be combined using random-effect meta-analysis models with restricted maximum-likelihood as variance estimator, and will be presented as relative risk or standardized mean difference with 95% CI. The quality of evidence will be assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework and summary of findings tables will be presented for patient important outcomes. DISCUSSION: Our study may improve the current understanding of the role that lifestyle-modifiable factors can play in saving or prolonging the lives of women who have been treated for breast cancer, and also on modifying their quality of life. SYSTEMATIC REVIEW REGISTRATION: The review has been registered with PROSPERO (registration number CRD42014008743).

ESR1 rs9340799 is associated with endometriosis-related infertility and in vitro fertilization failure.

Estrogen receptor alpha has a central role in human fertility by regulating estrogen action in all human reproductive tissues. Leukemia inhibitory factor (LIF) expression, a cytokine critical for blastocyst implantation, is mediated by estrogen signaling, so we hypothesized that ESR1 gene polymorphisms might be candidate risk markers for endometriosis-related infertility and in vitro fertilization (IVF) failure. We included 98 infertile women with endometriosis, 115 infertile women with at least one IVF failure and also 134 fertile women as controls. TaqMan SNP assays were used for genotyping LIF (rs929271), MDM2 (rs2279744), MDM4 (rs1563828), USP7 (rs1529916), and ESR1 (rs9340799 and rs2234693) polymorphisms. The SNP ESR1 rs9340799 was associated with endometriosis-related infertility (P < 0.001) and also with IVF failure (P = 0.018). After controlling for age, infertile women with ESR1 rs9340799 GG genotype presented 4-fold increased risk of endometriosis (OR 4.67, 95% CI 1.84-11.83, P = 0.001) and 3-fold increased risk of IVF failure (OR 3.33, 95% CI 1.38-8.03, P = 0.007). Our results demonstrate an association between ESR1 rs9340799 polymorphism and infertile women with endometriosis and also with women who were submitted to IVF procedures and had no blastocyst implantation.

The TP53 fertility network.

The TP53 gene, first described in 1979, was identified as a tumor suppressor gene in 1989, when it became clear that its product, the p53 nuclear phosphoprotein, was frequently inactivated in many different forms of cancers. Nicknamed "guardian of the genome", TP53 occupies a central node in stress response networks. The p53 protein has a key role as transcription factor in limiting oncogenesis through several growth suppressive functions, such as initiating apoptosis, senescence, or cell cycle arrest. The p53 protein is directly inactivated in about 50% of all tumors as a result of somatic gene mutations or deletions, and over 80% of tumors demonstrate dysfunctional p53 signaling. Beyond the undeniable importance of p53 as a tumor suppressor, an increasing number of new functions for p53 have been reported, including its ability to regulate energy metabolism, to control autophagy, and to participate in various aspects of differentiation and development. Recently, studies on genetic variations in TP53 among different populations have led to the notion that the p53 protein might play an important role in regulating fertility. This review summarizes current knowledge on the basic functions of different genes of the TP53 family and TP53 pathway with respect to fertility. We also provide original analyses based on genomic and genotype databases, providing further insights into the possible roles of the TP53 pathway in human reproduction.

TNF -308G > a promoter polymorphism (rs1800629) and outcome from critical illness.

BACKGROUND: The susceptibility to adverse outcome from critical illness (occurrence of sepsis, septic shock, organ dysfunction/failure, and mortality) varies dramatically due to different degrees of inflammatory response. An over expression of tumor necrosis factor alpha (TNF-α) can lead to the progression of the inflammatory condition. OBJECTIVE: We assessed the relationship of the genotype distribution of -308G >A TNF-α polymorphism with regard to the development of sepsis, septic shock, higher organ dysfunction or mortality in critically ill patients. METHODS: Observational, hospital-based cohort study of 520 critically ill Caucasian patients from southern Brazil admitted to the general ICU of São Lucas Hospital, Porto Alegre, Brazil. Patients were monitored daily from the ICU admission day to hospital discharge or death, measuring SOFA score, sepsis, and septic shock occurrences. The -308G >A TNF-α SNP effect was analyzed in the entire patient group, in patients with sepsis (349/520), and in those who developed septic shock (248/520). RESULTS: The genotypic and allelic frequencies were -308GG = 0.72; -308GA = 0.27; -308AA = 0.01; -308G = 0.85; -308A = 0.15. No associations were found with sepsis, septic shock, organ dysfunction, and/or mortality rates among the TNF-α genotypes. Our results reveal that the -308G >A TNF-α SNP alone was not predictive of severe outcomes in critically ill patients. CONCLUSION: The principal novel input of this study was the larger sample size in an investigation with -308G > A TNF-α SNP. The presence of -308A allele is not associated with sepsis, septic shock, higher organ dysfunction or mortality in critically ill patients.

TNF -308G > a promoter polymorphism (rs1800629) and outcome from critical illness

BACKGROUND: The susceptibility to adverse outcome from critical illness (occurrence of sepsis, septic shock, organ dysfunction/failure, and mortality) varies dramatically due to different degrees of inflammatory response. An over expression of tumor necrosis factor alpha (TNF-α) can lead to the progression of the inflammatory condition. OBJECTIVE: We assessed the relationship of the genotype distribution of -308G >A TNF-α polymorphism with regard to the development of sepsis, septic shock, higher organ dysfunction or mortality in critically ill patients. METHODS: Observational, hospital-based cohort study of 520 critically ill Caucasian patients from southern Brazil admitted to the general ICU of São Lucas Hospital, Porto Alegre, Brazil. Patients were monitored daily from the ICU admission day to hospital discharge or death, measuring SOFA score, sepsis, and septic shock occurrences. The -308G >A TNF-α SNP effect was analyzed in the entire patient group, in patients with sepsis (349/520), and in those who developed septic shock (248/520). RESULTS: The genotypic and allelic frequencies were -308GG = 0.72; -308GA = 0.27; -308AA = 0.01; -308G = 0.85; -308A = 0.15. No associations were found with sepsis, septic shock, organ dysfunction, and/or mortality rates among the TNF-α genotypes. Our results reveal that the -308G >A TNF-α SNP alone was not predictive of severe outcomes in critically ill patients. CONCLUSION: The principal novel input of this study was the larger sample size in an investigation with -308G > A TNF-α SNP. The presence of -308A allele is not associated with sepsis, septic shock, higher organ dysfunction or mortality in critically ill patients.

Temporal trends in acute renal dysfunction among critically ill patients according to I/D and -262A > T ACE polymorphisms.

Multiple organ failure syndrome and acute renal dysfunction share many of physiologic factors involved in their development. Recent studies correlate the susceptibility to organ dysfunction in critically ill patients with genetic inheritance. Many of them consider ACE gene could be a possible candidate to elucidate a genetic predisposition or a genetic risk factor. We aimed to examine the effects of I/D and -262A > T ACE polymorphisms in the renal function in severely ill southern Brazilians patients. A multi-organic worldwide known failure score, the SOFA (sequential organ failure assessment), was used to determine the basal health state at first day (ICU admission). Considering admission SOFA score and trend of renal function (measured by daily renal SOFA scores, with daily measure of serum creatinine and diuresis), we hypothesize that ACE polymorphisms could influence in the trend of renal function in ICU patients. A total of 153 critically ill adult patients (79 men) were included in this study. We monitored the patients daily during their entire ICU and post-ICU (hospital) stay (measured from the ICU admission day to a maximum of 224 days). We observed progression to renal failure (SOFA scores 3 and 4) in first seven days of ICU stay and need for dialysis. The general genotypic frequencies in our sample were II = 0.17; ID = 0.46; DD = 0.37 and AA = 0.30; AT = 0.55; TT = 0.15, and the allelic frequencies were I = 0.40; D = 0.60 and A = 0.56; T = 0.44. This is the first study to verify the influence of I/D and -262A > T ACE polymorphisms in acute renal dysfunction among critically ill patients. No significant association was found between genotypes or allele frequencies and the trend of the renal function. The I/D and -262A > T ACE polymorphisms have no significant impact on the trend of renal function during the first week of ICU stay, neither any influence in mortality in critically ill patients.

Análise da tendência temporal de dano renal agudo entre pacientes graves conforme polimorfi smos I/D e -262A > T da enzima conversora da angiotensina / Temporal trends in acute renal dysfunction among critically ill patients according to I/D and -262A > T ACE polymorphisms

A síndrome de disfunção de múltiplos órgãos e sistemas (DMOS) e a disfunção renal aguda compartilham muitos dos fatores fisiológicos envolvidos em seu desenvolvimento. Estudos recentes correlacionam suscetibilidades individuais, determinadas geneticamente, à disfunção de órgãos em pacientes criticamente enfermos, situação em que o gene da enzima conversora da angiotensina (ECA) poderia ser um candidato para elucidar predisposição ou risco genético. Nosso objetivo foi examinar os efeitos da presença de dois polimorfismos, I/D e -262A > T, na disfunção renal em pacientes agudamente graves do Sul do Brasil. O escore SOFA (sequential organ failure assessment) à admissão e a tendência da função renal (medida pelo escore renal diário do SOFA) foram determinados em pacientes de unidade de terapia intensiva (UTI). Um total de 153 pacientes adultos (79 homens) foi incluído no estudo. Houve monitoração diária da função renal durante toda a permanência na UTI e também pós-UTI. Observou-se a progressão para insuficiência renal (SOFA 3 e 4) nos primeiros sete dias de internação em UTI, bem como necessidade de diálise. As frequências genotípicas gerais em nossa amostra foram II = 0,17; ID = 0,46; DD = 0,37; e AA = 0,30; AT = 0,55; TT = 0,15; e as frequências alélicas foram I = 0,40, D = 0,60; e A = 0,56; T = 0,44. Este é o primeiro estudo para verificar a influência de polimorfismos I/D e -262A > T da ECA em disfunção renal aguda em pacientes críticos. Nenhuma associação significativa foi encontrada entre os genótipos ou as frequências alélicas e a evolução da função renal. Os polimorfismos I/D e -262A > T da ECA não têm impacto significativo sobre a evolução da função renal durante a primeira semana de internação na UTI nem exercem qualquer influência sobre a mortalidade em pacientes graves.

Multiple organ failure syndrome and acute renal dysfunction share many of physiologic factors involved in their development. Recent studies correlate the susceptibility to organ dysfunction in critically ill patients with genetic inheritance. Many of them consider ACE gene could be a possible candidate to elucidate a genetic predisposition or a genetic risk factor. We aimed to examine the effects of I/D and -262A > T ACE polymorphisms in the renal function in severely ill southern Brazilians patients. A multi-organic worldwide known failure score, the SOFA (sequential organ failure assessment), was used to determine the basal health state at first day (ICU admission). Considering admission SOFA score and trend of renal function (measured by daily renal SOFA scores, with daily measure of serum creatinine and diuresis), we hypothesize that ACE polymorphisms could influence in the trend of renal function in ICU patients. A total of 153 critically ill adult patients (79 men) were included in this study. We monitored the patients daily during their entire ICU and post-ICU (hospital) stay (measured from the ICU admission day to a maximum of 224 days). We observed progression to renal failure (SOFA scores 3 and 4) in first seven days of ICU stay and need for dialysis. The general genotypic frequencies in our sample were II = 0.17; ID = 0.46; DD = 0.37 and AA = 0.30; AT = 0.55; TT = 0.15, and the allelic frequencies were I = 0.40; D = 0.60 and A = 0.56; T = 0.44. This is the first study to verify the influence of I/D and -262A > T ACE polymorphisms in acute renal dysfunction among critically ill patients. No significant association was found between genotypes or allele frequencies and the trend of the renal function. The I/D and -262A > T ACE polymorphisms have no significant impact on the trend of renal function during the first week of ICU stay, neither any influence in mortality in critically ill patients.

CD14 expression in the first 24h of sepsis: effect of -260C>T CD14 SNP.

Sepsis is defined as systemic inflammation caused by infection. The membrane bound CD14 (mCD14) or the soluble form (sCD14) play a crucial role facing Gram-negative and Gram-positive sepsis since they are pattern recognition receptors of the innate immune response enabling cells to produce inflammatory cytokines against bacterial infections. A -260C>T single nucleotide polymorphism (SNP) was detected in the promoter modulating the CD14 gene expression. We hypothesized that the CD14 expression depends of the genetic inheritance of -260C>T CD14 SNP and it is modulated by sepsis condition. We investigated human CD14 expression on early sepsis diagnosis (in vivo) and after LPS stimulation (in vitro), and determined the -260C>T CD14 SNP. We found that TT homozygotes showed higher mCD14 density (p = 0.0207), but not different sCD14 levels when compared to the CT+CC genotypes. Monocyte mCD14 density and sCD14 serum levels in our sample of early 14 septic patients were significantly higher than normal 30 controls (p<0.0001). Our results suggest that the -260TT CD14 genotype is associated with higher monocyte mCD14, but not sCD14 expression, and that in the first 24 h after sepsis diagnosis, both monocyte mCD14 density and sCD14 levels are elevated, similarly to what is observed in vitro upon challenge with LPS.