Cytotoxicity and Antimicrobial Resistance of Aeromonas Strains Isolated from Fresh Produce and Irrigation Water.

The genus Aeromonas has received constant attention in different areas, from aquaculture and veterinary medicine to food safety, where more and more frequent isolates are occurring with increased resistance to antibiotics. The present paper studied the interaction of Aeromonas strains isolated from fresh produce and water with different eukaryotic cell types with the aim of better understanding the cytotoxic capacity of these strains. To study host-cell pathogen interactions in Aeromonas, we used HT-29, Vero, J774A.1, and primary mouse embryonic fibroblasts. These interactions were analyzed by confocal microscopy to determine the cytotoxicity of the strains. We also used Galleria mellonella larvae to test their pathogenicity in this experimental model. Our results demonstrated that two strains showed high cytotoxicity in epithelial cells, fibroblasts, and macrophages. Furthermore, these strains showed high virulence using the G. mellonella model. All strains used in this paper generally showed low levels of resistance to the different families of the antibiotics being tested. These results indicated that some strains of Aeromonas present in vegetables and water pose a potential health hazard, displaying very high in vitro and in vivo virulence. This pathogenic potential, and some recent concerning findings on antimicrobial resistance in Aeromonas, encourage further efforts in examining the precise significance of Aeromonas strains isolated from foods for human consumption.

Reappraising the need for a control CT in mild head injury patients on anticoagulation.

BACKGROUND: Head injury is a frequent reason for admission to the emergency department. In parallel, there is a growing use of anticoagulants in an increasingly aging population, which renders this particular group of trauma patients more frequent. In several countries, including Portugal, a 24-h surveillance period followed by repetition of head computed tomography (CT) is the standard procedure for these patients. However, these recommendations have not been based on studies of prevalence of intracranial hemorrhages in control head CTs, namely in this group of anticoagulated patients. This study intends to evaluate the prevalence of de novo intracranial hemorrhages in control head CTs in anticoagulated patients. METHOD: An observational study was carried out, which included patients admitted to Hospital de Braga between June 2017 and January 2018, victims of head injury and on anticoagulation therapy, whose admission head CT excluded intracranial hemorrhage. RESULTS: We collected a total of 201 patients, with a mean age of 81.6 years, and 57.5% of them were prescribed warfarin; 181 of these patients repeated the head CT 24 h later. Of these 181 patients, 3 (1.66%) exhibited intracranial hemorrhage in control CT, without surgical indication. All patients were followed up 1 month after the trauma, and there was no readmission requiring hospitalization, surgery or death. CONCLUSIONS: In conclusion, de novo intracranial hemorrhage in control head CT of anticoagulated patients is rare. We propose that these patients may be discharged if the admission CT does not reveal intracranial hemorrhage, providing that they are accompanied by a caregiver and informed about red flags.

Emergency surgery for brainstem cavernoma haemorrhage with severe neurological presentation. Is it indicated and worthwhile?

Background: Brainstem Cavernoma (BSCM) haemorrhage is a complex condition, especially when patients present rapid neurological deterioration. Traditionally, these patients were initially treated by non-interventional means. Surgery was generally reserved for cases who presented a 'benign' evolution in a subacute/delayed fashion. Timing of surgery remains controversial. Since rebleeding is frequent and carries a high mortality, many of these patients do not tolerate this approach. Urgent/emergent surgery may be indicated and lifesaving.Methods: A single center experience is reported in which an aggressive approach was used with urgent/emergency surgery carried out on patients with BSCM haemorrhage and rapid neurological deterioration, ventilatory impairment and/or coma. A review of 5 consecutive cases where urgent/emergent surgery was performed is presented. The pre-operative status, pre- and post-operative examinations, surgical approach and neurological residual deficits/outcomes are reported.Results: Four females and one male with ages ranging from 36 to 66 years with rapid neurological deterioration, ventilatory impairment and/or coma were operated between 2011 and 2018. Favourable outcomes were observed with a modified Rankin Scale varying from 1 to 4. Cranial nerve deficits as well as motor and sensitive deficits were observed but all the patients recovered cognitive integrity.Conclusions: Our small series reveals an acceptable outcome with ultra-early surgery. This approach appears to be a valid option when there is rapid neurological deterioration, respiratory impairment and/or early onset coma. However, further studies are required to elucidate the optimal strategy.

Epidemiological and clinical characteristics of Streptococcus tigurinus endocarditis.

BACKGROUND: Streptococcus tigurinus was recently described as a new streptococcal species within the viridans group streptococci (VGS). The objectives of the present work were to analyse the clinical and microbiological characteristics of S. tigurinus isolated from patients with bacteraemias, to determine the prevalence of S. tigurinus among VGS endocarditis in Spain, and to compare the clinical characteristics and outcomes of endocarditis caused by S. tigurinus and other VGS. METHODS: Retrospective nationwide study, performed between 2008 and 2016 in 9 Spanish hospitals from 7 different provinces comprising 237 cases of infective endocarditis. Streptococcal isolates were identified by sequencing fragments of their 16S rRNA, sodA and groEL genes. Clinical data of patients with streptococcal endocarditis were prospectively collected according to a pre-established protocol. RESULTS: Patients with endocarditis represented 7/9 (77.8%) and 26/86 (30.2%) of the bacteraemias caused by S. tigurinus and other VGS, respectively (p < 0.001), in two of the hospital participants. Among patients with streptococcal endocarditis, 12 different Streptococcus species were recognized being S. oralis, S. tigurinus and S. mitis the three more common. No relevant statistical differences were observed in the clinical characteristics and outcomes of endocarditis caused by the different VGS species. CONCLUSIONS: In this multicenter study performed in Spain, S. tigurinus showed a higher predilection for the endocardial endothelium as compared to other VGS. However, clinical characteristics and outcomes of endocarditis caused by S. tigurinus did not significantly differ from endocarditis caused by other oral streptococci.

A multicentre study investigating parameters which influence direct bacterial identification from urine.

Rapid diagnosis is one of the best ways to improve patient management and prognosis as well as to combat the development of bacterial resistance. The aim of this study was to study parameters that impact the achievement of reliable identification using a combination of flow cytometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS).The study was carried out in nine hospitals in Spain and included 1,050 urine samples with bacterial counts of 5x106 bacteria/ml. MALDI-ToF-MS-based identification was performed according to a previously described protocol. Valid identification by direct MALDI-ToF-MS was obtained in 72.8% of samples, in 80.3% of samples found to be positive by culture, 32.2% of contaminated samples, and 19.7% of negative samples. Among the positives samples with a valid identification the concordance at the species level was 97.2%. The parameters related to success of direct identification were: high bacterial count, the presence of Escherichia coli as a pathogen and rod-bacteria morphology provided by flow cytometry. The parameters related to failure were a high epithelial cell (EC) count, a high white blood cell (WBC) count and urine samples obtained from in-patients. In summary, this multicentre study confirms previously published data on the usefulness and accuracy of direct MALDI-ToF-MS-based identification of bacteria from urine samples. It seems important to evaluate not only the bacterial count, but also other parameters, such as EC and WBC counts, bacterial species and morphology, and the health care setting, to decide whether the sample is suitable for direct identification.

Evaluation of the VITEK-MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system for the identification of clinical Corynebacterium species / Evaluación del sistema VITEK-MS de espectrometría de masas MALDI-TOF para la identificación de corinebacterias de importancia clínica

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Acinetobacter baumannii in critically ill patients: Molecular epidemiology, clinical features and predictors of mortality / Acinetobacter baumannii en pacientes críticos: epidemiología molecular, características clínicas y predictores de mortalidad

INTRODUCTION: The main aim of this study was to assess changes in the epidemiology and clinical presentation of Acinetobacter baumannii over a 10-year period, as well as risk factors of mortality in infected patients. METHOD: Prospective, multicentre, hospital-based cohort studies including critically ill patients with A. baumannii isolated from any clinical sample were included. These were divided into a first period ('2000 study') (one month), and a second period ('2010 study') (two months). Molecular typing was performed by REP-PCR, PFGE and MSLT. The primary endpoint was 30-day mortality. RESULTS: In 2000 and 2010, 103 and 108 patients were included, and the incidence of A. baumanniicolonization/infection in the ICU decreased in 2010 (1.23 vs. 4.35 cases/1000 patient-days; p < 0.0001). No differences were found in the colonization rates (44.3 vs. 38.6%) or infected patients (55.7 vs. 61.4%) in both periods. Overall, 30-day mortality was similar in both periods (29.1 vs. 27.8%). The rate of pneumonia increased from 46.2 in 2000 to 64.8% in 2010 (p < 0.001). Performing MSLT, 18 different sequence types (ST) were identified (18 in 2000, 8 in 2010), but ST2 and ST79 were the predominant clones. ST2 isolates in the ICU increased from 53.4% in the year 2000 to 73.8% in 2010 (p = 0.002). In patients with A. baumanniiinfection, the multivariate analysis identified appropriate antimicrobial therapy and ST79 clonal group as protective factors for mortality. CONCLUSIONS: At 10 years of the first analysis, some variations have been observed in the epidemiology of A. baumannii in the ICU, with no changes in mortality. Epidemic ST79 clone seems to be associated with a better prognosis and adequate treatment is crucial in terms of survival

INTRODUCCIÓN: El principal objetivo fue evaluar los cambios en la epidemiología a lo largo de un periodo de 10años, así como la presentación clínica y los factores predictores de mortalidad en los pacientes críticos infectados por Acinetobacter baumannii. MÉTODO: Estudio de cohortes prospectivo y multicéntrico en el que se incluyeron pacientes críticos con A. baumannii aislado de cualquier muestra clínica. Se consideró un primer período («estudio de 2000») (un mes) y un segundo («estudio de 2010») (2 meses). La tipificación molecular se realizó mediante REP-PCR, PFGE y MSLT. La variable resultado primaria fue la mortalidad a los 30días. RESULTADOS: En 2000 y 2010 se incluyeron 103 y 108 pacientes, respectivamente, y la incidencia de colonización/infección por A. baumannii en la UCI disminuyó en 2010 respecto al 2000 (1,23 vs. 4,35 casos/1.000 pacientes-días; p < 0,0001). No se encontraron diferencias en la tasa de colonización (44,3 vs. 38,6%) o infección (55,7 vs. 61,4%) en ambos periodos. En general, la mortalidad a los30 días fue similar en ambos periodos (29,1 vs. 27,8%). La tasa de neumonía aumentó desde el 46,2% en 2000 al 64,8% en 2010 (p < 0,001). Mediante MSLT, se identificaron 18 tipos de secuencias diferentes (ST) (18 en 2000, 8 en 2010), pero ST2 y ST79 fueron los clones predominantes. La identificación de ST2 aumentó en la UCI desde el 53,4% en 2000 al 73,8% en 2010 (p = 0,002). En los pacientes infectados, el tratamiento antimicrobiano apropiado y el grupo clonal ST79 fueron factores protectores de mortalidad en el análisis multivariante. CONCLUSIONES: A los 10 años del primer análisis se han observado algunos cambios en la epidemiología de A. baumannii en la UCI, sin cambios en la mortalidad. El clon ST79 epidémico parece estar asociado con un mejor pronóstico, y el tratamiento adecuado es crucial en términos de supervivencia

Acinetobacter baumannii in critically ill patients: Molecular epidemiology, clinical features and predictors of mortality.

INTRODUCTION: The main aim of this study was to assess changes in the epidemiology and clinical presentation of Acinetobacter baumannii over a 10-year period, as well as risk factors of mortality in infected patients. METHOD: Prospective, multicentre, hospital-based cohort studies including critically ill patients with A. baumannii isolated from any clinical sample were included. These were divided into a first period ("2000 study") (one month), and a second period ("2010 study") (two months). Molecular typing was performed by REP-PCR, PFGE and MSLT. The primary endpoint was 30-day mortality. RESULTS: In 2000 and 2010, 103 and 108 patients were included, and the incidence of A. baumannii colonization/infection in the ICU decreased in 2010 (1.23 vs. 4.35 cases/1000 patient-days; p<0.0001). No differences were found in the colonization rates (44.3 vs. 38.6%) or infected patients (55.7 vs. 61.4%) in both periods. Overall, 30-day mortality was similar in both periods (29.1 vs. 27.8%). The rate of pneumonia increased from 46.2 in 2000 to 64.8% in 2010 (p<0.001). Performing MSLT, 18 different sequence types (ST) were identified (18 in 2000, 8 in 2010), but ST2 and ST79 were the predominant clones. ST2 isolates in the ICU increased from 53.4% in the year 2000 to 73.8% in 2010 (p=0.002). In patients with A. baumannii infection, the multivariate analysis identified appropriate antimicrobial therapy and ST79 clonal group as protective factors for mortality. CONCLUSIONS: At 10 years of the first analysis, some variations have been observed in the epidemiology of A. baumannii in the ICU, with no changes in mortality. Epidemic ST79 clone seems to be associated with a better prognosis and adequate treatment is crucial in terms of survival.

Sternal wound infection caused by Gordonia bronchialis: identification by MALDI-TOF MS.

INTRODUCTION: Gordonia spp. infections are uncommon. However, a few clinical cases have been reported in the literature, particularly those involving immunocompromised hosts. Advanced microbiology diagnosis techniques, such as matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), have been recently introduced in clinical microbiology laboratories in order to improve microbial identification, resulting in better patient management. CASE PRESENTATION: Here, we present a new clinical case of persistent wound infection caused by Gordonia bronchialis in a 64-year-old woman after a mitral valve replacement, using two MALDI-TOF-based systems for identifying this micro-organism. CONCLUSION: Both MALDI-TOF systems were able to identify Gordonia spp.; thus, providing a useful tool that overcomes the current limitations of phenotypic identification associated with this micro-organism. Although the technique validation deserves additional verification, our study provides guidance about MALDI-TOF as a fast and easy method for Gordonia spp. identification.

Epidemiologic and clinical impact of Acinetobacter baumannii colonization and infection: a reappraisal.

Acinetobacter baumannii is one of the most important antibiotic-resistant nosocomial bacteria. We investigated changes in the clinical and molecular epidemiology of A. baumannii over a 10-year period. We compared the data from 2 prospective multicenter cohort studies in Spain, one performed in 2000 (183 patients) and one in 2010 (246 patients), which included consecutive patients infected or colonized by A. baumannii. Molecular typing was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). The incidence density of A. baumannii colonization or infection increased significantly from 0.14 in 2000 to 0.52 in 2010 in medical services (p < 0.001). The number of non-nosocomial health care-associated cases increased from 1.2% to 14.2%, respectively (p < 0.001). Previous exposure to carbapenems increased in 2010 (16.9% in 2000 vs 27.3% in 2010, p = 0.03). The drugs most frequently used for definitive treatment of patients with infections were carbapenems in 2000 (45%) and colistin in 2010 (50.3%). There was molecular-typing evidence of an increase in the frequency of A. baumannii acquisition in non-intensive care unit wards in 2010 (7.6% in 2000 vs 19.2% in 2010, p = 0.01). By MSLT, the ST2 clonal group predominated and increased in 2010. This epidemic clonal group was more frequently resistant to imipenem and was associated with an increased risk of sepsis, although not with severe sepsis or mortality. Some significant changes were noted in the epidemiology of A. baumannii, which is increasingly affecting patients admitted to conventional wards and is also the cause of non-nosocomial health care-associated infections. Epidemic clones seem to combine antimicrobial resistance and the ability to spread, while maintaining their clinical virulence.

Characterization of plasmids carrying the blaOXA-24/40 carbapenemase gene and the genes encoding the AbkA/AbkB proteins of a toxin/antitoxin system.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAb) is a major source of nosocomial infections in Spain associated with the production of OXA-58-like or OXA-24/40-like ß-lactamase enzymes. We analysed the plasmids carrying the bla(OXA-24/40)-like gene in CRAb isolates obtained a decade apart. METHODS: The presence of ß-lactamases was screened for by PCR (metallo-ß-lactamases, carbapenem-hydrolysing class D ß-lactamases, GES and KPC) in 101 CRAb isolates obtained in two multicentre studies (GEIH/REIPI-Ab-2000 and GEIH/REIPI-Ab-2010; n = 493 Acinetobacter spp). We analysed the distribution and characterization of the plasmids carrying the bla(OXA-24/40)-like gene and sequenced two plasmids, AbATCC223p (2000) and AbATCC329p (2010) from A. baumannii ATCC 17978 transformants. RESULTS: Acquisition of the bla(OXA-24/40)-like gene was the main mechanism underlying resistance to carbapenems (48.7% in 2000 compared with 51.6% in 2010). This gene was mainly isolated in ST2 A. baumannii strains in both studies, although some novel STs (ST79 and ST80) appeared in 2010. The gene was located in plasmids (8-12 kbp) associated with the repAci2 or repAci2/repGR12 types. The sequences of AbATCC223p (8840 bp) and AbATCC329p (8842 bp) plasmids were similar, particularly regarding the presence of the genes encoding the AbkA/AbkB proteins associated with the toxin/antitoxin system. Moreover, the abkA/abkB gene sequences (>96% identity) were also located in plasmids harbouring the bla(OXA-58)-like gene. CONCLUSIONS: The action of OXA-24/40 and OXA-58 ß-lactamase-like enzymes represents the main mechanism underlying resistance to carbapenems in Spain in the last decade. AbkA/AbkB proteins in the toxin/antitoxin system may be involved in the successful dissemination of plasmids carrying the bla(OXA-24/40)-like gene, and probably also the bla(OXA-58)-like gene, thus contributing to the plasmid stability.

Recurrent respiratory infection caused by Bordetella bronchiseptica in an immunocompetent infant.

Bordetella bronchiseptica rarely infects immunocompetent humans. We report an unusual case of recurrent pertussis-like syndrome caused by B. bronchiseptica in a 7-month-old immunocompetent boy. Molecular analysis demonstrated that the isolates from the child and mother were identical.

Phenotypic and molecular characterization of Arcanobacterium haemolyticum isolated from clinical samples.

The aim of this study was to assess the phenotypic and genotypic diversity of 56 Arcanobacterium haemolyticum isolates isolated from 51 patients attending primary health care centres and emergency units in the health area of Santander (Cantabria, northern Spain). Phenotypic characterization was based on morphological, biochemical, and antigenic tests. Species identification was confirmed by amplification and sequencing of the 16S rDNA gene. Antimicrobial susceptibility testing was determined by microdilution following the Clinical and Laboratory Standards Institute recommendations for coryneform bacteria. Genetic diversity was evaluated using BOX-PCR and pulsed-field gel electrophoresis. Eighty percent of the isolates had an identical BOX-PCR pattern, suggesting the spread of a single clone. The present report provides extensive information on the phenotypic and genotypic characterization of A. haemolyticum.

Impact of EGFR genetic variants on glioma risk and patient outcome.

BACKGROUND: The epidermal growth factor receptor (EGFR) regulates important cellular processes and is frequently implicated in human tumors. Three EGFR polymorphisms have been described as having a transcriptional regulatory function: two single-nucleotide polymorphisms in the essential promoter region, -216G/T and -191C/A, and a polymorphic (CA)(n) microsatellite sequence in intron 1. We aimed to elucidate the roles of these EGFR polymorphisms in glioma susceptibility and prognosis. METHODS: We conducted a case-control study with 196 patients with glioma and 168 cancer-free controls. Unconditional multivariate logistic regression models were used to calculate ORs and 95% confidence intervals. A Cox regression model was used to evaluate associations with patient survival. False-positive report probabilities were also assessed. RESULTS: None of the EGFR -216G/T variants was significantly associated with glioma risk. The -191C/A genotype was associated with higher risk for glioma when the (CA)(n) alleles were classified as short for ≤16 or ≤17 repeats. Independently of the (CA)(n) repeat cutoff point used, shorter (CA)(n) repeat variants were significantly associated with increased risk for glioma, particularly glioblastoma and oligodendroglioma. In all tested models with different (CA)(n) cutoff points, only -191C/A genotype was consistently associated with improved survival of patients with glioblastoma. CONCLUSIONS: Our findings implicate EGFR -191C/A and the (CA)(n) repeat polymorphisms as risk factors for gliomas, and suggest -191C/A as a prognostic marker in glioblastoma. IMPACT: Our data support a role of these EGFR polymorphisms in determining glioma susceptibility, with potential relevance for molecularly based stratification of patients with glioblastoma for individualized therapies.

Pharmacological versus microvascular decompression approaches for the treatment of trigeminal neuralgia: clinical outcomes and direct costs.

In idiopathic trigeminal neuralgia (TN) the neuroimaging evaluation is usually normal, but in some cases a vascular compression of trigeminal nerve root is present. Although the latter condition may be referred to surgery, drug therapy is usually the first approach to control pain. This study compared the clinical outcome and direct costs of (1) a traditional treatment (carbamazepine [CBZ] in monotherapy [CBZ protocol]), (2) the association of gabapentin (GBP) and analgesic block of trigger-points with ropivacaine (ROP) (GBP+ROP protocol), and (3) a common TN surgery, microvascular decompression of the trigeminal nerve (MVD protocol). Sixty-two TN patients were randomly treated during 4 weeks (CBZ [n = 23] and GBP+ROP [n = 17] protocols) from cases of idiopathic TN, or selected for MVD surgery (n = 22) due to intractable pain. Direct medical cost estimates were determined by the price of drugs in 2008 and the hospital costs. Pain was evaluated using the Numerical Rating Scale (NRS) and number of pain crises; the Hospital Anxiety and Depression Scale, Sickness Impact Profile, and satisfaction with treatment and hospital team were evaluated. Assessments were performed at day 0 and 6 months after the beginning of treatment. All protocols showed a clinical improvement of pain control at month 6. The GBP+ROP protocol was the least expensive treatment, whereas surgery was the most expensive. With time, however, GBP+ROP tended to be the most and MVD the least expensive. No sequelae resulted in any patient after drug therapies, while after MDV surgery several patients showed important side effects. Data reinforce that, (1) TN patients should be carefully evaluated before choosing therapy for pain control, (2) different pharmacological approaches are available to initiate pain control at low costs, and (3) criteria for surgical interventions should be clearly defined due to important side effects, with the initial higher costs being strongly reduced with time.

Prognostic value of MGMT promoter methylation in glioblastoma patients treated with temozolomide-based chemoradiation: a Portuguese multicentre study.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The identification of novel molecular prognostic markers of GBM has recently been an area of great interest in neuro-oncology. The methylation status of the MGMT gene promoter is currently a promising molecular prognostic marker, but some controversial data have precluded its clinical use. We analyzed MGMT methylation by methylation-specific PCR in 90 GBM patients from four Portuguese hospitals, uniformly treated with radiotherapy combined with concomitant and adjuvant temozolomide (Stupp protocol). The Kaplan-Meier method was used to construct survival curves, and the log-rank test and a Cox-regression model were used to analyze patient survival. The methylation status of MGMT was successfully determined in 89% (80/90) of the tumors. The frequency of tumoral MGMT promoter methylation was 47.5%. The median overall survivals (OSs) were 16 months (95% CI 12.2-19.8) and 13 months (95% CI 13.3-18.7) for patients whose tumors had a methylated or unmethylated MGMT, respectively. Univariate and multivariate analyses did not show any statistically significant association between MGMT methylation status and patient OS (P=0.583 by the log-rank test; P=0.617 by the Cox-regression test) or progression-free survival (P=0.775 by the log-rank test; P=0.691 by the Cox-regression test). None of the patient clinical features were significantly correlated with survival. This is the first study to report the frequency of MGMT methylation among Portuguese GBM patients. Our data did not show statistically significant associations between MGMT promoter methylation and the outcome of GBM patients treated with temozolomide. Additional robust prospective studies are warranted to clarify whether the MGMT status should be used in clinical decisions.

Selection of extreme phenotypes: the role of clinical observation in translational research.

Systematic collection of phenotypes and their correlation with molecular data has been proposed as a useful method to advance in the study of disease. Although some databases for animal species are being developed, progress in humans is slow, probably due to the multifactorial origin of many human diseases and to the intricacy of accurately classifying phenotypes, among other factors. An alternative approach has been to identify and to study individuals or families with very characteristic, clinically relevant phenotypes. This strategy has shown increased efficiency to identify the molecular features underlying such phenotypes. While on most occasions the subjects selected for these studies presented harmful phenotypes, a few studies have been performed in individuals with very favourable phenotypes. The consistent results achieved suggest that it seems logical to further develop this strategy as a methodology to study human disease, including cancer. The identification and the study with high-throughput techniques of individuals showing a markedly decreased risk of developing cancer or of cancer patients presenting either an unusually favourable prognosis or striking responses following a specific treatment, might be promising ways to maximize the yield of this approach and to reveal the molecular causes that explain those phenotypes and thus highlight useful therapeutic targets. This manuscript reviews the current status of selection of extreme phenotypes in cancer research and provides directions for future development of this methodology.

The relationship between homocysteine and genes of folate-related enzymes in migraine patients.

BACKGROUND: It has been suggested that homocysteine (Hcy) and the 5'-10'-methylenetetrahydrofolate reductase (MTHFR) C677T variant are implicated in the pathogenesis of migraine. Homocysteine has the potential to damage endothelium and accelerate atherosclerosis. Genetic factors such as the MTHFR C677T polymorphism, and other polymorphisms in folate-related genes associated with high homocysteine levels, may contribute to increasing this vascular risk. RESULTS: We recruited 427 migraine patients (199 without aura [MO]; 228 with aura [MA]), and 310 controls in a neurologic clinic. Hcy levels and 6 polymorphisms corresponding to 6 folate-related genes, including the MTHFR C677T variant, were determined in all migraine participants and in a subset of 155 controls. We found higher sex-adjusted Hcy levels in MA (mean: 11.02 microM) than MO patients (9.86 microM; P = .005 for the difference). Hcy levels higher than 12.0 microM doubled the risk for MA (OR = 2.145; 95% confidence intervals [CI] = 1.3-3.4; P = .001), and those higher than 15.0 microM incurred a 6-fold increase (OR = 5.95; 95% CI = 2.1-20.0, P < .001). The number of MTHFR 677T alleles was the best genetic predictor of Hcy levels (r(2) = 0.06; P = 6.2e-6; corrected for genetic variants analyzed) and this effect remained significant after correction for other confounding factors. Using multi-dimensionality reduction approaches, we observed significant epigenetic interaction among some of the folate-related genetic variants to predict higher Hcy levels, and also among higher Hcy levels and folate-related genetic variants to predict the end-diagnosis of MA only among migraineurs. In controls, Hcy levels and the number of MTHFR 677T alleles were found to be intermediate between those observed in MA and MO patients. CONCLUSION: Our results suggest that MA patients have higher Hcy levels. We also observed complex epigenetic interaction among folate-related enzymes, sex, and Hcy levels predicting MA phenotype. Nevertheless, genetic factors explained only a minor proportion of the variance for both Hcy plasma levels and for predicting MA phenotype. Determination of MTHFR C677T polymorphisms and Hcy levels may be useful to identify patients with a high risk of suffering from MA.

Multilocus analyses reveal involvement of the ESR1, ESR2, and FSHR genes in migraine.

OBJECTIVE: Female hormone genes have been investigated in migraine in recent years. Research in this field has been controversial, especially in regard to ESR1 gene findings. None of the reports have yet to approach the problem from a multigenic point of view. METHODS: We investigated 5 polymorphisms implicated in female hormone metabolism (FSHR, CYP19A1, ESR1, NRIP1, and ESR2) in a cohort of 730 subjects matched for age and sex. The effect of gene-gene interaction was assessed using the set association approach, and the corresponding haplotypes were studied with PM Plus software. To corroborate initial results, we analyzed the selected markers using a cohort of 134 families in which 168 trios were suitable for transmission-disequilibrium test (TDT) analysis under the migraine with aura (MA) phenotype. RESULTS: A total of 356 consecutive migraine patients (198 with MA [76% females] and 158 migraine without aura [MO, 74% females], and 374 matched controls [71% females]) were genotyped. In the 2-point analyses, the ESR1 and ESR2 polymorphisms showed nominal association under MA/MO phenotype, and this association was higher with the FSHR polymorphism in MA females (P = .004, uncorrected). Using the SUMSTAT program, we observed ESR2-ESR1-FSHR significant gene-gene interaction, suggesting association with the MA/MO phenotype (P = .005; P = .003 in females), and with MA alone (P = .021; P = .030 for females).We corroborated that ESR2-ESR1-FSHR haplotypes interacted for migraine under a model-free hypothesis (empirical P = .010 for the whole sample; P = .001 for females), and the association was stronger for the MA phenotype alone (empirical P = 5.0e-4, under the heterogeneity model; P = .001 for females). These results were corroborated using family-based association approaches. We observed nominal association for ESR2 and ESR1 (P = .031 and .034, respectively) in the TDT study, and significant association for ESR1 using family-based association test statistics. Haplotype-TDT analyses showed further significant gene-gene interaction for ESR1-ESR2 (global P = .009), ESR2-FSHR (global P = .011), and nominally significant interaction for ESR2-ESR1-FSHR genes (global P = .037). CONCLUSION: We found significant association of female hormone metabolism polymorphisms under the perspective of multigene approach.

Lack of association of endothelial nitric oxide synthase polymorphisms and migraine.

OBJECTIVE: The aim of this study was to evaluate if 2 functional endothelial nitric oxide synthase (eNOS) gene polymorphisms might be risk factors for migraine. BACKGROUND: Nitric oxide synthase promotes the synthesis of nitric oxide (NO). NO is a potent vasodilator and mediates several processes involved in migraine pathophysiology. Only one study has suggested an association with migraine with aura. METHODS: We performed a sex- and age-matched case-control study using 2 eNOS polymorphisms (rs1800779 and rs1799983), which are in linkage disequilibrium. Genotypes were obtained with allele-specific probes in a real-time polymerase chain reaction assay. Genotypic and allelic distributions were compared with chi(2) method. We also estimated the reconstructed haplotypes and calculated ORs for individual haplotypes. RESULTS: A total of 337 migraine patients (188 with aura) and 341 healthy controls were recruited. We found no significant differences in the distribution of genotypes and alleles for either polymorphism among clinical subgroups. Neither rs1800779 nor rs1799983 polymorphisms increased the risk for suffering from migraine aura. CONCLUSIONS: As others have previously reported, we failed to demonstrate genetic association of the eNOS gene with migraine.