MV140 sublingual vaccine reduces recurrent urinary tract infection in women Results from the first North American clinical experience study.

INTRODUCTION: This is the first North American clinical evidence for MV140, a novel bacterial sublingual vaccine, developed for prevention of recurrent urinary tract infection (UTI) in women. METHODS: Female subjects with ≥3 documented UTIs/year underwent three-month vaccination treatment, nine-month efficacy period, and optional three-month followup (total 15 months). Primary outcome was no clinically diagnosed UTI following vaccination (UTI-free rate). Secondary outcomes included absolute, mean, and median overall reduction in UTI compared to pre-vaccination, quality of life, global response assessment, patient satisfaction, microbiology, and safety. RESULTS: Sixty-seven subjects (mean age 56 years, range 18-80) were enrolled; 64 completed the vaccination period and at least one post-vaccination assessment. Prior to vaccination, subjects reported a mean 6.8 UTIs/year. The UTI-free rate for the nine-month efficacy period was 40.6%. Compared to the infection rate in the year prior to vaccination, the reduction was 75.3% for the nine-month efficacy period post-vaccination. At 12-month followup, 80.3% reported that they were moderately/markedly improved; 58.1% were mostly satisfied, pleased, or delighted, while mean quality of life score improved by 1.5 points. Fourteen of the adverse events in nine subjects were potentially related to the vaccine - all mild and resolved by three months. None of the 13 serious adverse events were related to vaccine. CONCLUSIONS: This first-in-North-America, prospective case series with the sublingual vaccine, MV140, adds further clinical evidence to its safety and effectiveness in reducing recurrent UTIs in women.

Next-generation sequencing of 12 obesity genes in a Portuguese cohort of patients with overweight and obesity.

We examined 12 monogenic obesity genes in 72 Portuguese individuals with overweight and obesity (class 1 and class 2), some of which with suspected genetic obesity, to identify known or unknown potential obesity variants. Genomic DNA was analyzed for variants in genes LEP, LEPR, MC4R, POMC, PCSK1, BDNF, NTRK2, SIM1, SH2B1, UCP3, GCG and ADCY3 through next generation sequencing (NGS). The impact of the rare variants was investigated in the ClinVar database and using in silico tools for prediction of pathogenicity. Four potential pathogenic missense variants were detected at the heterozygous state in five individuals: two in the ADCY3 gene, NM_004036.5:c.1153G > A (p.Val385Ile) (rs756783003) and NM_004036.5:c.1222G > A (p.Gly408Arg) (rs201606553), one in gene SH2B1, NM_001145795.1:c.127C > A (p.Arg43Ser) (rs547678855), and the fourth in gene POMC NM_000939.4:c.706C > G (p.Arg236Gly) (rs28932472), which was found in two individuals. Moreover, six rare variants near splicing sites were also identified, as well as eight rare synonymous variants. In summary, some potential pathogenic rare missense variants were identified, two of them in ADCY3 gene, the most recently identified gene as having a role in monogenic obesity. Further analysis should be performed to confirm the clinical relevance of these variants.

Variants in the new E1' cryptic exon of the VHL gene associated with congenital erythrocytosis-Description of three cases.

Congenital erythrocytosis (CE) represents a rare and heterogeneous group of hereditary disorders. The molecular basis of VHL gene mutations related to CE. Recently, Lenglet et al. reported a discovery of a novel cryptic exon in the VHL gene. Mutations in the first intronic region resulting in the creation of a cryptic exon termed E1' were found in seven families with CE and one family with VHL disease. We report three patients with prolonged CE with the aetiology being clarified several years later by sequencing of intronic region 1 of the VHL gene. This work addresses the first cases reported at the clinical level of VHL-associated CE due to the E1' cryptic exon.

Clearance and persistence of SARS-CoV-2 RNA in patients with COVID-19.

Patients with coronavirus disease-2019 may be discharged based on clinical resolution of symptoms, and evidence for viral RNA clearance from the upper respiratory tract. Understanding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral clearance profile is crucial to establish a re-testing plan on discharge and ending isolation of patients. We aimed to evaluate the number of days that a patient needed to achieve undetectable levels of SARS-CoV-2 in upper respiratory tract specimens (nasopharyngeal swab and/or an oropharyngeal swab). The clearance and persistence of viral RNA was evaluated in two groups of positive patients: those who achieved two negative reverse transcription-polymerase chain reaction (RT-PCR) tests and those who kept testing positive. Patients were organized thereafter in two subgroups, mild illness patients discharged home and inpatients who had moderate to severe illness. Results from RT-PCR tests were then correlated with results from the evaluation of the immune response. The study evidenced that most patients tested positive for more than 2 weeks and that persistence of viral RNA is not necessarily associated with severe disease but may result from a weaker immune response instead.

DNA methylation age estimation in blood samples of living and deceased individuals using a multiplex SNaPshot assay.

Many studies in the forensic field have reported that analysis of DNA methylation is the most reliable method of predicting age. In a previous study, 5 CpG sites located in ELOVL2, FHL2, KLF14, C1orf132 and TRIM59 genes were tested for age prediction purposes in blood, saliva and buccal swab samples from Korean individuals using a multiplex methylation SNaPshot assay. The main goals of the present study were i) to replicate the same multiplex SNaPshot assay in blood samples from Portuguese individuals, ii) to compare DNA methylation status between two different populations and iii) to address putative differences in the methylation status between blood from living and deceased individuals. Blood samples from 59 living individuals (37 females, 22 males; aged 1-94 years-old) and from 62 deceased individuals (13 females, 49 males; aged 28-86 years-old) were evaluated. The specific primers were those previously described. Linear regression models were used to analyse relationships between methylation levels and chronological age using IBM SPSS software v.24. Our results allowed to build a final age prediction model (APM) for blood samples of living individuals with 3 CpG sites, at ELOVL2, FHL2 and C1orf132 genes, explaining 96.3% of age variation, with a mean absolute deviation (MAD) from chronological age of 4.25 years. Some differences were found in the extent of the age association in the targeted loci comparing Portuguese with Korean individuals. The final APM built for deceased individuals included 4 CpG sites, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explaining 79.3% of age variation, with a MAD of 5.36 years. Combining both sets of samples from living and deceased individuals, the most accurate APM with 4 CpGs, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explained 92.5% of variation in age, with a MAD of 4.97 years. In conclusion, our study replicated in blood samples of Portuguese living individuals a previous SNaPshot assay for age estimation. The possibility that age markers might be population specific and that postmortem changes can alter the methylation status among specific loci was suggested by our data. Our study showed the usefulness of the multiplex methylation SNaPshot assay for forensic analysis in blood samples of living and deceased individuals.

Multi-Locus Models to Address Hb F Variability in Portuguese ß-Thalassemia Carriers.

Hb F production is under the influence of major quantitative trait loci (QTL). The present study aims: i) to replicate the association with Hb F for representative genetic variants in the three major Hb F QTLs in a Portuguese sample of ß-thalassemia (ß-thal) carriers; and ii) to test different genetic multi-locus models to account for the genetic component of Hb F variation. A population sample of 79 Portuguese ß-thal carriers (39 males, 40 females), aged between 2 to 70 years old, were genotyped for polymorphisms in the locus control region (LCR)-5' hypersensitive site 4 (5'HS4) rs16912979, XmnI-HBG2 rs7482144, BCL11A rs1427407 and HMIP rs66650371, using standard biomolecular procedures. Univariate linear regression models were used to test for genetic associations with Hb F. The minor alleles of the individual variants BCL11A rs1427407 (T) (0.165), HMIP rs66650371 (3 bp del) (0.247) and XmnI-HBG2 rs7482144 (T) (0.196), were found to be significantly associated with increased levels of Hb F (p = 0.029, p = 0.002 and p = 0.0004, respectively), explaining about 6.0, 12.0 and 15.0% of Hb F variation, respectively. In a multiple linear regression approach, the three loci accounted for about 30.0% of Hb F variance. Two genetic risk scores (GRS), rationalizing the number of minor alleles into a single genetic variable, explained about 30.0 and 32.0% of the Hb F variation. In conclusion, we replicated in ß-thal carriers previously reported associations with Hb F. Multi-locus models combining three representative variants of Hb F influencing QTLs can explain a larger amount of Hb F variability.

Hereditary nonspherocytic hemolytic anemia caused by red cell glucose-6-phosphate isomerase (GPI) deficiency in two Portuguese patients: Clinical features and molecular study.

Glucose-6-phosphate isomerase (GPI) deficiency cause hereditary nonspherocytic hemolytic anemia (HNSHA) of variable severity in individuals homozygous or compound heterozygous for mutations in GPI gene. This work presents clinical features and genotypic results of two patients of Portuguese origin with GPI deficiency. The patients suffer from a mild hemolytic anemia (Hb levels ranging from 10 to 12.7g/mL) associated with macrocytosis, reticulocytosis, hyperbilirubinemia, hyperferritinemia and slight splenomegaly. Genomic DNA sequencing revealed in one patient homozygosity for a new missense mutation in exon 3, c.260G>C (p.Gly87Ala), and in the second patient compound heterozygosity for the same missense mutation (p.Gly87Ala), along with a frameshift mutation resulting from a single nucleotide deletion in exon 14, c.1238delA (p.Gln413Arg fs*24). Mutation p.Gln413Arg fs*24 is the first frameshift null mutation to be described in GPI deficiency. Molecular modeling suggests that the structural change induced by the p.Gly87Ala pathogenic variant has direct impact in the structural arrangement of the region close to the active site of the enzyme.

Chronic hemolytic anemia is associated with a new glucose-6-phosphate dehydrogenase in-frame deletion in an older woman.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked disorder, is usually observed in hemizygote males and very rarely in females. The G6PD class 1 variants, very uncommon, are associated with chronic hemolytic anemia. Here we report a Portuguese woman who suffered in her sixties from a chronic hemolytic anemia due to G6PD deficiency. Molecular studies revealed heterozygosity for an in-frame 18-bp deletion, mapping to exon 10 leading to a deletion of 6 residues, 362-367 (LNERKA), which is a novel G6PD class 1 variant, G6PD Tondela. Two of her three daughters, asymptomatic, with G6PD activity within the normal range, are heterozygous for the same deletion. The patient's leukocyte and reticulocyte mRNA studies revealed an almost exclusive expression of the mutant allele, explaining the chronic hemolytic anemia. Patient whole blood genomic DNA HUMARA assay showed a balanced pattern of X chromosome inactivation (XCI), but granulocyte DNA showed extensive skewing, harboring the mutated allele, implying that in whole blood, lymphocyte DNA, with a very long lifetime, may cover up the current high XCI skewing. This observation indicates that HUMARA assay in women should be assessed in granulocytes and not in total leukocytes.

RhD variant caused by an in-frame triplet duplication in the RHD gene.

BACKGROUND: The RHD gene is highly polymorphic and a large number of D variants have already been detected. Several mechanisms are involved in the origin of D variants. In-frame deletions, resulting in a single-amino-acid deletion, have been described associated with RhD and RhCE variants. No in-frame duplications and/or insertions have been reported in the RH genes to date. STUDY DESIGN AND METHODS: Blood samples from a Brazilian blood donor and his sister were serologically tested with routine anti-D reagents and anti-D panels (ALBAclone advanced partial D typing kit, Alba Bioscience Limited; and D-Screen, Diagast), followed by molecular biology techniques, RHD polymerase chain reaction with sequence-specific priming and sequencing. RESULTS: Samples tested negative with routine immunoglobulin M (IgM) anti-D reagents and positive with IgG anti-D, which detect weak D cells. The pattern of results with anti-D panels did not correspond to any described before. A 3-bp in-frame duplication within Exon 1 (c.75_77dupTCT), resulting in the duplication of leucine 26 (p.Leu26dup), was identified in the two samples. CONCLUSION: We report the first RhD variant associated with a 3-bp in-frame duplication in the RHD gene, predicted to be located within the RhD protein transmembrane domain that might be expected to result in a weak-D-like phenotype, concordant with serologic findings.

A novel approach to premature ejaculation: extracorporeal functional magnetic stimulation.

INTRODUCTION: Premature ejaculation (PE) is a common sexual dysfunction. Treatment ranges from behavior modification to systemic and topical pharmacological treatments. Results to date have been generally inconsistent and of limited effectiveness. New avenues of therapy are needed. AIM: To investigate the effect of extracorporeal functional magnetic stimulation (FMS) as a noninvasive treatment for men with PE. METHODS: The NeoControl System for FMS was used in the study. Baseline assessment included: history and physical, medications, hormonal assessment and intravaginal ejaculatory latency time (IVELT) by stopwatch determination. Treatment involved a first phase of five biweekly sessions (primary outcome). Men who reported an improvement in IVELT and desired to continue were given a second identical course (secondary outcome). Outcome measures included: IVELT and a global assessment questionnaire (GAQ). The responses incorporated both the patient's perception of response together with the more objective IVELT timing and were rated as: 1. Worse: no improvement in GAQ or mean IVELT for the total number of attempts; 2. Unchanged: improvement in IVELT but the patient reported no improvement; 3. Slightly improved: increase in IVELT < 100% and a reported mild improvement and 4. Better: IVELT increase of > or = 100% with GAQ indicating "moderate" to "marked" improvement. MAIN OUTCOME MEASURES: Two primary outcome measures were considered in both treatment phases, the IVELT and the GAQ. RESULTS: Fourteen men were treated. Their mean age was 43.7 years. Fifty-seven percent reported primary PE and 63% were circumcised. Hormone levels were normal in all. Baseline IVELT for the group was 60.6 seconds. All patients completed phase I. Of these, 50% reported no change in the GAQ although they recorded an increase in IVELT; 29% were categorized as slightly better and 14% as better. Eight men entered phase II. Of these, 3 (37.5%) were unchanged; 2 (25%) were slightly better and 3 (37.5%) were classified as better. The response in these last three has persisted for over 6 months post treatment. Both phases of the study showed a trend towards IVELT improvement, more evident at the end of phase II. However the differences did not reach statistical significance on either phase. Side effects were mild and non-treatment related. DISCUSSION: The use of FMS is claimed to alter the spinal centers without altering cerebral neurotransmitters. Although there were some remarkable responses, our results, are not better than the purported responses to behavioral or pharmacological methodologies. There was a clear trend in IVELT improvement; however, this didn't translate into an equivalent subjective estimation by most of the subjects. This outcome dissonance might be diminished by longer/more intense regimens of treatment. The pilot nature of the study does not permit to draw solid conclusions but stimulate the search for a new therapeutic option in PE.

Microfiltración y capacidad de penetración de los selladores de fosas y fisuras: influencia de la técnica de aplicación / Microleakage and penetration ability of pits and fissure sealants: the influence of the applying technique

Objetivo: este estudio evaluó la capacidad de penetración y sellado de un sellador de fisras convencional, una resina fluida y un ionómero de vidrio en función del tipo de acondicionamiento (grabado ácido sólo, grabado ácido y adhesivo, adhesivo autograbador) y la preparación de la fisura (realización o no de ameloplastía). Metodología: este estudio evaluó la capacidad de penetración y sellado de varios selladores de fisuras. Setenta terceros molares sanos se dividieron en tres grupos en función de la resina utilizada. Cada grupo se subdividió a su vez en dos de acuerdo a la preparación de la fisura y cada subgrupo se dividió en tres de acuerdo al tipo de acondicionamiento. Los dientes se termociclaron en agua (250 ciclos entre 5ºC y 55ºC) y se colocaron en solución de fucsina al 0,5 por ciento durante 24 hs. Se midió la capacidad de penetración y la microfiltración en milímetros. Resultados: se encontró que el grupo que presentó valores más altos de penetración fue el grupo ameloplastía-Prime&BOnd-Tetric Flow con una media de 1,31 mm, siendo este dato estadísticamente significativo. En cuanto a la microfiltración, no se encontraron diferencias estadísticamente significativas entre los grupos de estudio. Conclusión: se puede concluir que el tipo de sellador, la aplicación del adhesivo y la realización de ameloplastía influyen en la capacidad de penetración.

Microfiltración y capacidad de penetración de los selladores de fosas y fisuras: influencia de la técnica de aplicación / Microleakage and penetration ability of pits and fissure sealants: the influence of the applying technique

Objetivo: este estudio evaluó la capacidad de penetración y sellado de un sellador de fisras convencional, una resina fluida y un ionómero de vidrio en función del tipo de acondicionamiento (grabado ácido sólo, grabado ácido y adhesivo, adhesivo autograbador) y la preparación de la fisura (realización o no de ameloplastía). Metodología: este estudio evaluó la capacidad de penetración y sellado de varios selladores de fisuras. Setenta terceros molares sanos se dividieron en tres grupos en función de la resina utilizada. Cada grupo se subdividió a su vez en dos de acuerdo a la preparación de la fisura y cada subgrupo se dividió en tres de acuerdo al tipo de acondicionamiento. Los dientes se termociclaron en agua (250 ciclos entre 5ºC y 55ºC) y se colocaron en solución de fucsina al 0,5 por ciento durante 24 hs. Se midió la capacidad de penetración y la microfiltración en milímetros. Resultados: se encontró que el grupo que presentó valores más altos de penetración fue el grupo ameloplastía-Prime&BOnd-Tetric Flow con una media de 1,31 mm, siendo este dato estadísticamente significativo. En cuanto a la microfiltración, no se encontraron diferencias estadísticamente significativas entre los grupos de estudio. Conclusión: se puede concluir que el tipo de sellador, la aplicación del adhesivo y la realización de ameloplastía influyen en la capacidad de penetración.(AU)

Prenatal determination of the fetal RhD blood group by multiplex PCR: a 7-year Portuguese experience.

OBJECTIVES: This is a retrospective study to evaluate the efficacy and accuracy of the multiplex polymerase chain reaction (PCR) amplification, for early detection of fetuses at risk for hemolytic disease, in the population living in Portugal, and to characterize the RhD-negative individuals at serologic and molecular level. METHODS: 2030 uncultured amniotic fluid samples and 2012 blood samples from the respective RhD-negative pregnant women were studied by multiplex PCR of intron 3/intron 4, exon 7 and 3'UTR. Amniocentesis was performed for a variety of medical indications. For quality control, serologic RhD blood groups were determined in the cord blood, after birth. RESULTS: 1361 fetal amniotic samples were RhD-positive (67%), 669 were RhD-negative. The average time for diagnosis was 2 days for uncultured amniocytes and the molecular versus serologic RhD typing (n = 809) had 99.5% concordance. Among the 2012 serologic RhD-negative mothers, 26 had an RhD-positive allele. CONCLUSION: The multiplex PCR amplification used in this study was a rapid and accurate method to determine the RhD blood type in the population living in Portugal, being a great tool for management of pregnancies with fetuses at risk for alloimmune hemolytic disease. In this population, 1.3% of the serologic RhD-negative women have an RHD-positive allele.