Significance of a polymerase chain reaction method in the detection of Clostridioides difficile.

OBJECTIVE: Clostridioides difficile (CD) is the most common cause of nosocomial diarrhea. Detection of CD toxin in patients' faecal samples is the traditional rapid method for the diagnosis of CD infection. Various testing algorithms have been proposed: an initial screening test using a rapid test, and a confirmatory test (cytotoxicity neutralization assay, toxigenic culture, nucleic acid amplification test) for discordant results. The aim of this study was to evaluate the effectiveness of a two-step algorithm using an immunochromatographic test followed of a polymerase chain reaction (PCR). METHODS: The specimens have been tested according to the following schedule: 1) Step one: All samples were tested for detection of glutamate dehydrogenase antigen (GDH) and toxin A/B using the C. diff QUIK CHEK Complete test. All GDH and toxins positive results were considered CD positives; 2) Step two: When the results were discrepant (only GDH+ or toxins+), the samples were confirmed using the PCR test BD MAX Cdiff. All PCR positive results were considered CD positives. RESULTS: A total of 2,138 specimens were initially tested. 139 were positive for GDH and toxins. 160 discrepant results (148 GDH+ and 12 toxins+) were tested by PCR, 117 were positive (107/148 GDH+ and 10/12 toxins+). CONCLUSIONS: The implementation of a PCR method showed an increase de 117 positive results (73.1% of discrepant). Considering the sensitivity of C.diff QUIK CHEK (instructions of manufacturer), the GDH discrepant results may be false negatives, y the samples PCR and toxins positives may be real positives results.

Role of PCA3 and SelectMDx in the optimization of active surveillance in prostate cancer.

INTRODUCTION & OBJECTIVES: A not negligible percentage of patients included in active surveillance (AS) for low and very low risk prostate cancer (PCa) are reclassified in the confirmatory biopsy or have disease progression during follow-up. Our aim is to evaluate the role of PCA3 and SelectMDx, in an individual and combined way, in the prediction of pathological progression (PP) in a standard AS program. MATERIALS & METHODS: Prospective and observational study comprised of 86 patients enrolled in an AS program from 2009 to 2019, with results for PCA3 and SelectMDx previous to PCa diagnosis or during their confirmatory period. Univariate and multivariate analysis were performed to correlate PCA3 and SelectMDx scores as well as clinical and pathological variables with PP-free survival (PPFS). The most reliable cut-offs for both biomarkers in the context of AS were defined. RESULTS: SelectMDx showed statistically significant differences related to PPFS (HR 1.035, 95%CI: 1.012-1.057) (p = 0.002) with a C-index of 0.670 (95%CI: 0.529-0.810) and AUC of 0.714 (95%CI: 0.603-0.825) at 5 years. In our series, the most reliable cut-off point for SelectMDx was 5, with a sensitivity and specificity for PP of 69.8% and 67.4%, respectively. Same figure for PCA3 was 65, with a sensitivity and specificity for PP of 51.16% and 74.42%, respectively. The combination of both biomarkers did not improve the prediction of PP, C-index 0.630 (95%CI: 0.455-0.805). CONCLUSIONS: In the context of low or very low risk PCa, SelectMDx > 5 predicted 5 years PP free survival with a moderate discrimination ability outperforming PCA3. The combination of both tests did not improved outcomes.

[Epidemiology and etiology of vulvovaginal candidiasis in Spanish and immigrants' women in Fuenlabrada (Madrid)]. / Epidemiología y etiología de la candidiasis vaginal en mujeres españolas e inmigrantes en Fuenlabrada (Madrid).

OBJECTIVE: Vulvovaginal candidiasis (VVC) is a common vaginal infection. Risk factors include diabetes, antibiotic use and pregnancy. Candida albicans is the most common species identified but non-C. albicans species appear to be more commonly associated with VVC in some Asian and African countries. We had studied the distribution of Candida species in Spanish and immigrants' women residents in Spain. METHODS: Retrospective study of vaginal yeast cultures between 2015 and 2018. RESULTS: A total of 2,283 vaginal yeast cultures were collected. Candida spp. was detected in 25.7% from Spanish women and in 28.5% from immigrants (no significant differences). Immigrants have higher rates of vaginal candidiasis compared other studies in Spain. C. albicans was the most common species isolated (82.4%). CONCLUSIONS: There were no differences in vaginal candidiasis rate between Spanish and immigrants' women. Immigrants consulted proportionally more compared with the Spanish women.

Are targeted prostate biopsies ready to replace systematic prostate biopsies? / Las biopsias de próstata dirigidas ¿están listas para reemplazar las biopsias de próstata sistemáticas?

OBJECTIVES: To evaluate the efficacy and efficiency of systematic prostatic biopsy (SPB) and cognitive fusion PB (CFPB) to diagnose prostate cancer (PCa) and significant PCa (SPCa), and to analyse if CFPB could safely replace SPB. MATERIAL AND METHODS: A cohort of 314 consecutive men having PI-RADS ≥2 in a pre-biopsy 3T mp-MRI were prospectively subjected to trans-rectal ultrasound CFPB (two cores per suspicious area until a maximum of three areas) and a 12 peripheral core SPB. SPCa was considered when the WHO grade was higher than 2 (Gleason 4+3 or higher). RESULTS: PCa was diagnosed in 133 patients (42.4%), being 83 (62.4%) SPCa. SPB detected PCa in 114 men (85.7%) while CFPB in 103 (77.4%), P<.001. SPB detected SPCa in 64 men (77.1%) while CFPB in 71 (85.5%), P<.001. In 52 of the 81 men (64.2%) SPCa was detected in SPB and CFPB. In 19 men SPCa was only detected in CFPB (23.5%) while in 10, it was only detected in SPB (12.3%). 33.1 cores were needed to diagnose one PCa in SPB while 8.5 in CFPB, P<.001. 58.9 cores were needed to diagnose one SPCa in SPB, while 12.4 in CFPB, P<.001. CONCLUSIONS: CFPB are more effective and also more efficient than SPBs in detecting SPCa. However, CFPBs still can't safely replace SPBs because they are not able to detect up to 15% of SPCa.

Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development.

Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras(+/-) mice produced H-ras(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-ras did not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras(-/-) mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras(-/-) mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras(-/-)/N-ras(-/-)) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-than-expected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development.

Ras-guanine nucleotide exchange factor sos2 is dispensable for mouse growth and development.

The mammalian sos1 and sos2 genes encode highly homologous members of the Son-of-sevenless family of guanine nucleotide exchange factors. They are ubiquitously expressed and play key roles in transmission of signals initiated by surface protein tyrosine kinases that are transduced into the cell through the action of membrane-associated Ras proteins. Recent reports showed that targeted disruption of the sos1 locus results in embryonic lethality. To gain insight into the in vivo function of sos2, we disrupted its catalytic CDC25-H domain by means of gene targeting techniques. Mating among heterozygous sos2(+/-) mice produced viable sos2(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of sos2 does not interfere with embryo viability in the uterus. Adult homozygous mutant sos2(-/-) mice reached sexual maturity at the same age as their wild-type littermates, and both male and female null mutants were fertile. Histopathological analysis showed no observable differences between mutant and wild-type mice. Our results show that unlike the case for sos1, sos2 gene function is dispensable for normal mouse development, growth, and fertility.

Preliminary results of the Spanish Association of Urology National Registry in Active Surveillance for prostate cancer.

OBJECTIVES: To present a National Registry of patients with prostate cancer as monitored through active surveillance, with the intention of testing the hypothesis that cancer-specific mortality in very low-risk and low-risk patients is less than 5% at 15 years. MATERIAL AND METHODS: A multicentre observational study (AEU-PIEM/2014/0001) sponsored by the Spanish Association of Urology was conducted using their platform for multicentre studies. The clinical-pathological inclusion criteria were as follows: cT1a-cT3a, PSA ≤ 20 ng/ml, initial minimum biopsy of 10 cores, number of affected cores ≤ 3, 1st Gleason score of 3 and 2nd Gleason score ≤ 4 and a known prostate volume (in cc). A unified follow-up was not established for all recruiting centres; however, a survey was conducted that reflects the follow-up characteristics based on a number of tangible parameters that allow for their comparison. With the same philosophy of flexibility, the use of certain biomarkers and multiparametric MRI was not considered necessary for inclusion. RESULTS: We describe the Registry's characteristics and possibilities, as well as the preliminary results from the 324 patients included in its first 5 months of operation in the 15 recruiting centres. We also report the clinical-pathological variables, biomarkers, radiodiagnosis technique and quality-of-life questionnaires considered for the database, as well as the possibilities for indefinite follow-up, remaining open to any active treatment recognized in clinical guidelines. CONCLUSIONS: The AEU-PIEM/2014/0001 represents an extremely useful tool for all Spanish urologists for multicentre clinical research. The registry will undoubtedly enable the dissemination of active surveillance of our patients in a more coordinated manner, thus maintaining the advantages of optimised opportunistic screening for prostate cancer without resulting in overtreatment.

Transformation suppressor activity of C3G is independent of its CDC25-homology domain.

The guanine nucleotide releasing protein C3G was initially identified as a Crk SH3-binding protein and recently shown to exhibit exchange activity on Rap1 proteins. Overexpression in NIH3T3 cells of a full-length C3G cDNA isolated from human placenta markedly reduced the focus forming activity of cotransfected, malignantly activated, ras oncogenes (5-7-fold). C3G also had a reverting effect on sis-mediated transformation, decreasing the number of c-sis-induced foci by a factor of 5-10-fold. The observed inhibitory effect of C3G on focus-forming activity of Ras and Sis was always higher than that observed with Rap1A, a known target of C3G. The inhibition of focus formation observed in the presence of C3G was not due to toxic effects on cell viability, since transfected C3G cells exhibited the same survival and growth rates as untransfected NIH3T3 cells or cells transfected with plasmid vector alone. Surprisingly, as opposed to Rap1A, which has no effect on Raf-1 oncogene-mediated transformation, C3G also reduced dramatically (6-8-fold) the number of v-raf-induced foci in transfected NIH3T3 cells. The inhibitory effect on Raf-induced transformation suggests that C3G has other functional targets in addition to Rap1. A C3G mutant (C3G deltaCat) lacking the catalytic domain (CDC25-H) but retaining the rest of the N-terminal sequences, including the Crk-binding domain, exhibited similar ability than full length C3G to inhibit focus formation. In contrast, a C3G mutant (C3G Cat), containing the catalytic domain only but lacking the rest of the N-terminal sequences, did not have any inhibitory effect on transformation mediated by the oncogenes tested. The C3G-derived gene products overexpressed in our transfected cell lines localized to the cytoplasm and did not change the basal MAPK or JNK activity of those cell lines nor their ability to activate the kinases in response to agonists. Our results suggest that the N-terminal region of C3G, and not its catalytic domain, may be responsible for the inhibitory effects observed.

A 15 amino acid stretch close to the Grb2-binding domain defines two differentially expressed hSos1 isoforms with markedly different Grb2 binding affinity and biological activity.

We compared structure, expression and functional properties of two hSos1 cDNA isoforms (IsfI and Isf II) isolated, respectively, from human fetal brain and adult skeletal muscle libraries. IsfI and IsfII nucleotide sequences differ only by the presence in IsfII of an inframe 45 hp insertion located near the first proline-rich motif required for Grb2 binding. Some human tissues express only one isoform whereas others express different proportions of both in fetal and adult stages. In vitro binding assays and in vivo functional studies showed that MI exhibits significantly higher Grb2 binding affinity and biological activity than IsfI. These results suggest that functionally different hSos1 isoforms, with differential tissue expression and distribution, play important regulatory roles in the mechanisms controlling Ras activation in different tissues and/or developmental stages.

Expression of alternative forms of Ras exchange factors GRF and SOS1 in different human tissues and cell lines.

DNA probes and antibodies specific for different coding regions of human SOS1 and GRF genes were used to screen expression of these genes in a variety of adult and fetal human tissues and cell lines. Despite previous reports of the exclusive expression of hGRF RNA in brain, we also observed expression of this gene in various other tissues including lung and pancreas, as well as several tumor cell lines. At least three different hGRF mRNA transcripts were observed depending on the probe used, with the larger transcripts being detected by probes corresponding to the 5' end of the gene while smaller transcripts were detected by probes corresponding to the 3' end. Expression of hSOS1-related transcripts was more ubiquitous and homogeneous than with hGRF, with similar levels of specific transcripts being detected in most tissues and cell fines tested. Three to five different transcripts were detected in human tissues when using probes for the 5' end and middle regions of this gene, whereas only two were detected with probes corresponding to the 3' end. Screening of multiple human tumor cell lines showed ubiquitous expression of three specific transcripts, although the level and ratio of each of these transcripts varied widely among individual cell lines. Consistent with the variety of transcripts detected, several protein forms were also identified in Western immunoblots with antisera raised against specific domains of hSOS1 and human Ras-GRF gene products. Fluorescence in situ chromosomal hybridization suggested that, in both cases, the multiple forms arise from single chromosomal loci. The heterogeneity of hGRF and hSOS1 gene products detected (which appear to retain in most cases a functional catalytic domain), suggests that differentially expressed, alternatively spliced hSOS1 and hGRF forms may contribute to fine regulation of Ras activation in different tissues or at different stages of development.

[Cervical cysts of second branchial arch. Retrospective study of 14 cases]. / Quistes cervicales de segundo arco branquial: estudio retrospectivo de 14 casos.

Fourteen patients with lateral neck branchial cysts treated over an 18 year term were reviewed to analyze the authors' method of diagnosis and management. The possibility of an embryologic rest in the neck should therefore be kept in mind with all clinically evident neck masses. Histopathological examination revealed that cysts were lined with a stratified squamous epithelium. Computed tomography and fine-needle-aspiration currently are essential diagnostic methods in the study protocol of these lesions. Traditional surgical techniques for the management of branchial remnants rely on simple excision of the structures associated with the sinus tract, with dissection carried out in the plane between the branchial remnant and the normal anatomic structures of the neck. Initial surgery is crucial since the recurrence rate after incomplete surgery can be as high as 22%.

Papel de PCA3 y SelectMDx en la optimización de la vigilancia activa en el cáncer de próstata / Role of PCA3 and SelectMDx in the optimization of active surveillance in prostate cancer

Introducción y objetivos: Un porcentaje no despreciable de pacientes incluidos en programas de vigilancia activa (VA) para el cáncer de próstata (CaP) de bajo y muy bajo riesgo son reclasificados en la biopsia confirmatoria o desarrollan progresión de la enfermedad durante el seguimiento. Nuestro objetivo es evaluar el papel del PCA3 y el SelectMDx, de manera individual y combinada, para predecir la progresión patológica (PP) en un programa habitual de VA.Materiales y métodosEstudio prospectivo y observacional que incluyó 86 pacientes inscritos en un protocolo de VA desde 2009 hasta 2019, con resultados de PCA3 y SelectMDx previos al diagnóstico de CaP o durante su periodo de confirmación. Se realizaron análisis univariantes y multivariantes para la correlación de las puntuaciones de PCA3 y SelectMDx, así como de las variables clinicopatológicas con la supervivencia libre de progresión patológica (SLPP). Se definieron los puntos de corte más fiables para ambos biomarcadores en el contexto de VA.ResultadosSelectMDx mostró diferencias estadísticamente significativas en relación con la SLPP (HR: 1,035; IC95%: 1,012-1,057) (p=0,002) con un índiceC de 0,670 (IC95%: 0,529-0,810) y un AUC de 0,714 (IC95%: 0,603-0,825) a 5años. En nuestra serie, el punto de corte más fiable para el SelectMDx fue 5, con una sensibilidad y una especificidad para la PP del 69,8 y del 67,4%, respectivamente. El punto de corte del test PCA3 fue de 65, con una sensibilidad y una especificidad para la PP del 51,16 y del 74,42%, respectivamente. La combinación de ambos biomarcadores no mejoró la predicción de la PP, con un índiceC de 0,630 (IC95%: 0,455-0,805).ConclusionesEn el contexto del CaP de bajo o muy bajo riesgo, SelectMDx >5 predijo una supervivencia libre de PP de 5años con una capacidad de discriminación moderada, superando al PCA3. La combinación de ambos no mejoró los resultados. (AU)

Introduction and objectives: A not negligible percentage of patients included in active surveillance (AS) for low and very low risk prostate cancer (PCa) are reclassified in the confirmatory biopsy or have disease progression during follow-up. Our aim is to evaluate the role of PCA3 and SelectMDx, in an individual and combined way, in the prediction of pathological progression (PP) in a standard AS program.Materials and methodsProspective and observational study comprised of 86 patients enrolled in an AS program from 2009 to 2019, with results for PCA3 and SelectMDx previous to PCa diagnosis or during their confirmatory period. Univariate and multivariate analysis were performed to correlate PCA3 and SelectMDx scores as well as clinical and pathological variables with PP-free survival (PPFS). The most reliable cut-offs for both biomarkers in the context of AS were defined.ResultsSelectMDx showed statistically significant differences related to PPFS (HR: 1.035; 95%CI: 1.012-1.057) (P=.002) with a C-index of 0.670 (95%CI: 0.529-0.810) and AUC of 0.714 (95%CI: 0.603-0.825) at 5years. In our series, the most reliable cut-off point for SelectMDx was 5, with a sensitivity and specificity for PP of 69.8% and 67.4%, respectively. Same figure for PCA3 was 65, with a sensitivity and specificity for PP of 51.16% and 74.42%, respectively. The combination of both biomarkers did not improve the prediction of PP, C-index 0.630 (95%CI: 0.455-0.805).ConclusionsIn the context of low or very low risk PCa, SelectMDx >5 predicted 5years PP free survival with a moderate discrimination ability outperforming PCA3. The combination of both tests did not improved outcomes. (AU)

T double-stranded RNA (dsRNA) sequence reveals that T and W dsRNAs form a new RNA family in Saccharomyces cerevisiae. Identification of 23 S RNA as the single-stranded form of T dsRNA.

Some strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called T. We obtained T cDNA clones by random priming of denatured T dsRNA followed by reverse transcription. Sequence data of T show that only one strand ((+)-strand) has coding capacity for a protein with 940 amino acids which spans almost the entire length of the molecule (2.9 kilobases). Within this protein we found a sequence pattern characteristic of RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses. Although T has no homology with other dsRNAs found in S. cerevisiae, such as L-A, L-BC, M1, or W, the T-encoded protein shows a high degree of conservation with the W-encoded protein. This conservation extends beyond a region that contains the consensus sequences for RNA-dependent RNA polymerases, suggesting that both T and W are evolutionarily related. With a (+)-strand-specific probe for T we identified 23 S RNA, a new single-stranded RNA (ssRNA) species with a sedimentation coefficient of 23 S. T and 23 S RNA have the same mobility under denaturing conditions with glyoxal, suggesting that 23 S RNA is, in fact, the (+)-single-stranded RNA form of T dsRNA. 23 S RNA synthesis is induced under stress conditions such as heat shock and starvation. The relationship between T and 23 S RNA clearly resembles the one between W and its single-stranded derivative form, 20 S RNA. Thus T and W dsRNAs (and their respective single-stranded species) constitute a new RNA family in S. cerevisiae.

Molecular cloning and characterization of W double-stranded RNA, a linear molecule present in Saccharomyces cerevisiae. Identification of its single-stranded RNA form as 20 S RNA.

Most strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called W. We obtained W cDNA clones by random priming of denatured W dsRNA followed by reverse transcription. Sequence data of W shows that only one strand ((+)-strand) has coding capacity for a protein with 829 amino acids which spans almost the entire length of the molecule (2.5 kilobases). Within this protein we found a sequence pattern characteristic of RNA dependent RNA polymerases of (+)-strand and double-stranded RNA viruses. W has no homology with other dsRNAs found in S. cerevisiae, such as L-A, L-BC or M1. However, a (+)-strand-specific probe for W hybridized with 20-S RNA. Furthermore, W (+)-strands comigrated with 20 S RNA in strand separation gels. These results suggest that 20 S RNA is a (+)-single-stranded RNA form of W dsRNA itself or a closely related molecule.

Yeast viral 20 S RNA is associated with its cognate RNA-dependent RNA polymerase.

Most Saccharomyces cerevisiae strains carry in their cytoplasm 20 S RNA, a linear single-stranded RNA molecule of 2.5 kilobases in size. 20 S RNA copy number is greatly induced in stress conditions such as starvation, with up to 100,000 copies per cell. 20 S RNA has coding capacity for a protein of 91 kDa (p91) with sequences diagnostic of RNA-dependent RNA polymerases of (+) strand and double-stranded RNA viruses. We detected p91 in 20 S RNA-carrying strains with specific antisera. The amount of p91 in growing cells is higher than that of stationary cells and similar to the one in 20 S RNA-induced cells. Although 20 S RNA is not encapsidated into viral particles, p91 non-covalently forms a ribonucleoprotein complex with 20 S RNA. This suggests a role of p91 in the RNA to RNA synthesis processes required for 20 S RNA replication. Although the strain analyzed also harbors 23 S RNA, a closely related single-stranded RNA, 23 S RNA is not associated with p91 but with its putative RNA polymerase, p104. Similarly, 20 S RNA is not associated with p104 but with p91. These results suggest that 20 S RNA and 23 S RNA replicate independently using their respective cognate RNA polymerases.