The Comet Assay as a Tool in Human Biomonitoring Studies of Environmental and Occupational Exposure to Chemicals-A Systematic Scoping Review.

Biomonitoring of human populations exposed to chemical substances that can act as potential mutagens or carcinogens, may enable the detection of damage and early disease prevention. In recent years, the comet assay has become an important tool for assessing DNA damage, both in environmental and occupational exposure contexts. To evidence the role of the comet assay in human biomonitoring, we have analysed original research studies of environmental or occupational exposure that used the comet assay in their assessments, following the PRISMA-ScR method (preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews). Groups of chemicals were designated according to a broad classification, and the results obtained from over 300 original studies (n = 123 on air pollutants, n = 14 on anaesthetics, n = 18 on antineoplastic drugs, n = 57 on heavy metals, n = 59 on pesticides, and n = 49 on solvents) showed overall higher values of DNA strand breaks in the exposed subjects in comparison with the unexposed. In summary, our systematic scoping review strengthens the relevance of the use of the comet assay in assessing DNA damage in human biomonitoring studies.

Viable Mycobacterium avium subsp. paratuberculosis Colonizes Peripheral Blood of Inflammatory Bowel Disease Patients.

Pathobionts, particularly Mycobacterium avium subsp. paratuberculosis (MAP) and Escherichia coli isolates with adherence/invasive ability (AIEC) have been associated with inflammatory bowel disease (IBD), particularly Crohn's disease (CD). This study aimed to evaluate the frequency of viable MAP and AIEC in a cohort of IBD patients. As such, MAP and E. coli cultures were established from faecal and blood samples (with a total n = 62 for each) of patients with CD (n = 18), ulcerative colitis (UC, n = 15), or liver cirrhosis (n = 7), as well as from healthy controls (HC, n = 22). Presumptive positive cultures were tested by polymerase chain reaction (PCR), for a positive confirmation of MAP or E. coli identity. E. coli-confirmed isolates were then tested for AIEC identity using adherence and invasion assays in the epithelial cell line of Caco-2 and survival and replication assays in the macrophage cell line of J774. MAP sub-culture and genome sequencing were also performed. MAP was more frequently cultured from the blood and faecal samples of patients with CD and cirrhosis. E. coli presumptive colonies were isolated from the faecal samples of most individuals, in contrast to what was registered for the blood samples. Additionally, from the confirmed E. coli isolates, only three had an AIEC-like phenotype (i.e., one CD patient and two UC patients). This study confirmed the association between MAP and CD; however, it did not find a strong association between the presence of AIEC and CD. It may be hypothesized that the presence of viable MAP in the bloodstream of CD patients contributes to disease reactivation.

DNA-based detection of Mycobacterium avium subsp. paratuberculosis in domestic and municipal water from Porto (Portugal), an area of high IBD prevalence.

Mycobacterium avium subsp. paratuberculosis (MAP) may play a role in the pathology of human inflammatory bowel disease (IBD). Previously, we found a high frequency (98% in patients with active disease) of MAP DNA detection in the blood of Portuguese Crohn's Disease patients, suggesting this cohort has high exposure to MAP organisms. Water is an important route for MAP dissemination, in this study we therefore aimed to assess MAP contamination within water sources in Porto area (the residential area of our IBD study cohort). Water and biofilms were collected in a wide variety of locations within the Porto area, including taps connected to domestic water sources and from municipal water distribution systems. Baseline samples were collected in early autumn plus further domestic water samples in early winter, to assess the effect of winter rainfall. DNA was extracted from all 131 samples and IS900-based nested PCR used to assess the frequency of MAP presence. Our results show high MAP positivity in municipal water sources (20.7% of water samples and 41.4% of biofilm samples) and even higher amongst domestic sources (30.8% of water samples and 50% of biofilm samples). MAP positivity in biofilms correlated with positivity in water samples from the same sources. A significantly higher frequency of MAP-positivity was observed during winter rains as compared with samples collected in autumn prior to the winter rainfall period (61.9% versus 30.8%). We conclude that domestic and municipal water sources of Porto region have a high burden of MAP contamination and this prevalence increases with rainfall. We hypothesize that human exposure to MAP from local water supplies is commonplace and represents a major route for MAP transmission and challenge which, if positively linked to disease pathology, may contribute to the observed high prevalence of IBD in Porto district.

Cancer Nanopharmaceuticals: Physicochemical Characterization and In Vitro/In Vivo Applications.

Physicochemical, pharmacokinetic, and biopharmaceutical characterization tools play a key role in the assessment of nanopharmaceuticals' potential imaging analysis and for site-specific delivery of anti-cancers to neoplastic cells/tissues. If diagnostic tools and therapeutic approaches are combined in one single nanoparticle, a new platform called nanotheragnostics is generated. Several analytical technologies allow us to characterize nanopharmaceuticals and nanoparticles and their properties so that they can be properly used in cancer therapy. This paper describes the role of multifunctional nanoparticles in cancer diagnosis and treatment, describing how nanotheragnostics can be useful in modern chemotherapy, and finally, the challenges associated with the commercialization of nanoparticles for cancer therapy.

Measuring Health Vulnerability: An Interdisciplinary Indicator Applied to Mainland Portugal.

Health promotion and inequality reduction are specific goals of the United Nations 2030 Agenda, which are interconnected with several dimensions of life. This work proposes a composite index SEHVI-socioeconomic health vulnerability index-to address Portuguese population socioeconomic determinants that affect health outcomes. Variables composing SEHVI are aligned with the sustainable development goals considering data and times series availability to enable progress monitoring, and variables adequacy to translate populations' life conditions affecting health outcomes. Data for 35 variables and three periods were collected from official national databases. All variables are part of one of the groups: Health determinants (social, economic, cultural, and environmental factors) and health outcomes (mortality indicators). Variables were standardized and normalized by "Distance to a reference" method and then aggregated into the SEHVI formula. Several statistical procedures for validation of SEHVI revealed the internal consistency of the index. For all municipalities, SEHVI was calculated and cartographically represented. Results were analyzed by statistical tests and compared for three years and territory typologies. SEHVI differences were found as a function of population density, suggesting inequalities of communities' life conditions and in vulnerability to health.

Year-Long Rhinovirus Infection is Influenced by Atmospheric Conditions, Outdoor Air Virus Presence, and Immune System-Related Genetic Polymorphisms.

Rhinovirus is a common picornavirus with over 150 serotypes and three species, which is responsible for half of the human common cold cases. In people with chronic respiratory conditions and elders, it may also cause life-threatening diseases. Transmission routes are not definitively established but may involve direct human-to-human and indirect transmission (surfaces and aerosols based). In the present study, year-long presence of virus was tested by qPCR in the nostrils of young healthy volunteers and indoor and outdoor air samples. Results were correlated to atmospheric conditions (meteorological and air quality parameters) and voluntaries immune system-related genetic polymorphisms (TOLLIP rs5743899, IL6 rs1800795, IL1B rs16944, TNFA rs1800629) typed by PCR-RFLP. Nasal samples showed increased frequency and viral titers of Rhinovirus in spring and autumn. No indoor air samples tested positive for Rhinovirus, whereas outdoor air samples tested positive in late autumn. Sun radiation, atmospheric SO2, and benzene levels correlated with nostrils Rhinovirus detection. Both IL6 and TOLLIP polymorphisms but not TNFA or IL1B influenced Rhinovirus detection in the nostrils of voluntaries. Taken together, the results indicate that Rhinovirus circulation is determined by environmental conditions (weather, air-borne virus, and air pollution) and genetically encoded individual variation in immunity.

A review of microbiological root canal sampling: updating an emerging picture / Uma revisão de amostragem microbiológica de canal radicular: atualizando um quadro emergente

The significance of microorganisms in root canals with regard to the aetiology of periapical infection and the need for crucial bacteria control during treatment are undeniable. In this study, we report and discuss a review of the literature on Microbiological Root Canal Sampling (MRS). The procedure is analyzed in detail, discussing its powers, limitations and the influence of sample collection procedures on the incidence of true and false positive results. Data sources: MEDLINE/PUBMED, B-On and library files of Oporto University were accessed. Selection: Papers were selected using the keywords: “root ca¬nal sampling”; “apical periodontitis”; “endodontic pathogens”; “root canal infection”; “Culture”; “molecular biology”. The references were selected under inclusion criteria such as English language, accessibility, relevance to the theme and scientific rigor. Conclusions: This review illustrated the absolute need to adhere to strict methodology procedures if valid samples are to be obtained. A combination of Culture and molecular identification approaches have confirmed the polymicrobial nature of endodontic infections with a predominance of anaerobic bacteria. Nucleic acid-based techniques provide significant additional information particularly regarding the not-yet-cultivable species of the microbial community, but greatly increase the budget of the procedure. Thus, assessment of the endodontic microflora, in the context of a polymicrobial biofilm ecosystem, and its relevance to endodontic treatments must rely in the complementariness of Culture and Metagenomics approaches as they are neither mutually exclusive nor competitive, but strongly complementary...

A importância de microrganismos em canais radiculares no que diz respeito à etiologia da infecção periapical e a necessidade de controlar bactérias durante o tratamento são incontestáveis. Neste estudo, relata-se e discute-se uma revisão da literatura sobre a amostragem microbiológica de canal radicular. O procedimento é analisado em detalhes, discutindo suas atribuições, limitações e influência de procedimentos de coleta de amostra sobre a incidência de verdadeiros e falsos resultados positivos. Fontes de dados: Foram usados MEDLINE/PubMed, B-On e arquivos da biblioteca da Universidade do Porto. Seleção: Os trabalhos foram selecionados utilizando as palavras-chave: “root canal sampling”; “apical periodontitis”; “endodontic pathogens”; “root canal infection”; “Culture”; “molecular biology”. As referências foram selecionadas de acordo com critérios de inclusão como o idioma inglês, acessibilidade, relevância para o tema e rigor científico. Conclusões: Esta revisão ilustrou a absoluta necessidade de aderir aos procedimentos metodológicos rigorosos se se pretende obter amostras válidas para análise. Uma combinação de cultura e abordagens de iden¬tificação molecular confirmaram a natureza polimicrobiana das infeções endodônticas com predominância de bactérias anaeróbias. Técnicas baseadas em ácidos nucleicos fornecem informação adicional significativa, particularmente em relação às espécies não cultiváveis da comunidade microbiana, mas aumentam muito o orçamento do procedimento. Assim, a avaliação da microflora endodôntica, no contexto de um ecossistema polimicrobiano em biofilme, e sua relevância para tratamentos endodônticos devem confiar na complementaridade entre a abordagem de cultura e de metagenômica, pois não são mutuamente exclusivas nem competitivas, mas fortemente complementares...

Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization among patients and healthcare workers in a Portuguese hospital: a pre-intervention study toward the control of MRSA.

This two-year study investigated the epidemiology of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) among patients and healthcare workers (HCWs) in two wards with a high frequency of MRSA isolation, at Hospital Geral de Santo António (HGSA), Portugal. Three point-prevalence surveys per year were carried out. A case-control approach was used to identify potential risk factors associated with MRSA carriage among patients. Incidence rates and risk factors of MRSA carriage among HCWs who were negative at the baseline observation were estimated. Prevalence of MRSA carriage among 276 patients screened was 5.1%. Admission to HGSA or attendance to the Diabetic Foot Outpatient Unit (DFOU) of HGSA within the past 12 months, and previous MRSA isolation were significant risk factors for MRSA carriage. Among HCWs (n = 126), the prevalence of MRSA carriage was 4.8% and the incidence rate was 61/1000 person-years. Nurses and nurse aids were the HCW categories with the highest risk of becoming colonized with MRSA over time (p = 0.01). One HCW chronically colonized was detected. Molecular typing revealed a clonal identity for isolates recovered from patients and HCWs of the same wards, with 88.6% of isolates belonging to the EMRSA-15 (ST22-MRSA-IV) clone.

Clinicobiological, immunophenotypic, and molecular characteristics of monoclonal CD56-/+dim chronic natural killer cell large granular lymphocytosis.

Indolent natural killer (NK) cell lymphoproliferative disorders include a heterogeneous group of patients in whom persistent expansions of mature, typically CD56(+), NK cells in the absence of any clonal marker are present in the peripheral blood. In the present study we report on the clinical, hematological, immunophenotypic, serological, and molecular features of a series of 26 patients with chronic large granular NK cell lymphocytosis, whose NK cells were either CD56(-) or expressed very low levels of CD56 (CD56(-/+dim) NK cells), in the context of an aberrant activation-related mature phenotype and proved to be monoclonal using the human androgen receptor gene polymerase chain reaction-based assay. As normal CD56(+) NK cells, CD56(-/+dim) NK cells were granzyme B(+), CD3(-), TCRalphabeta/gammadelta(-), CD5(-), CD28(-), CD11a(+bright), CD45RA(+bright), CD122(+), and CD25(-) and they showed variable and heterogeneous expression of both CD8 and CD57. Nevertheless, they displayed several unusual immunophenotypic features. Accordingly, besides being CD56(-/+dim), they were CD11b(-/+dim) (heterogeneous), CD7(-/+dim) (heterogeneous), CD2(+) (homogeneous), CD11c(+bright) (homogeneous), and CD38(-/+dim) (heterogeneous). Moreover, CD56(-/+dim) NK cells heterogeneously expressed HLA-DR. In that concerning the expression of killer receptors, CD56(-/+dim) NK cells showed bright and homogeneous CD94 expression, and dim and heterogeneous reactivity for CD161, whereas CD158a and NKB1 expression was variable. From the functional point of view, CD56(-/+dim) showed a typical Th1 pattern of cytokine production (interferon-gamma(+), tumor necrosis factor-alpha(+)). From the clinical point of view, these patients usually had an indolent clinical course, progression into a massive lymphocytosis with lung infiltration leading to death being observed in only one case. Despite this, they frequently had associated cytopenias as well as neoplastic diseases and/or viral infections. In summary, we describe a unique and homogeneous group of monoclonal chronic large granular NK cell lymphocytosis with an aberrant activation-related CD56(-/+dim)/CD11b(-/+dim) phenotype and an indolent clinical course, whose main clinical features are related to concomitant diseases.

Assessment of three rapid methods for the detection of methicillin-resistant Staphylococcus aureus.

We evaluated three rapid methods to detect methicillin-resistant Staphylococcus aureus (MRSA) and compared them with PCR amplification of mecA. A total of 103 S. aureus strains were studied by MRSA-Screen, BBL Crystal, Velogene Genomic and mecA PCR. All the methods detected the 61 MRSA strains having the mecA gene, showing 100% sensitivity and specificity. Despite the correlation between all the rapid methods and PCR, the ease of use and shorter turnaround time of MRSA-Screen were important factors leading to the selection of this method as the routine screening technique for MRSA.

Evaluación de tres métodos rápidos para la detección de Staphylococcus aureus resistente a la meticilina / Assessment of three rapid methods for the detection of methicillin-resistant Staphylococcus aureus

Se han evaluado tres métodos rápidos para detectar la presencia de Staphylococcus aureus resistente a la meticilina (SARM) y se han comparado con la amplificación del gen mecA mediante reacción en cadena de la polimerasa (PCR). Se estudiaron un total de 103 cepas de S. aureus mediante MRSA-Screen, BBL Crystal, Velogene Genomics y PCR para mecA. Con todos estos métodos se detectaron 61 cepas de SARM que presentaban el gen mecA, con una sensibilidad y especificidad del 100 por ciento.A pesar de la correlación entre todos los métodos rápidos y la PCR, la facilidad de uso y el poco tiempo que lleva a la realización de MRSA-Screen fueron factores importantes para la selección de este método como técnica sistemática de detección de SARM (AU)

Guess what: Chronic 13q14.3+/CD5-/CD23+ lymphocytic leukemia in blood and t(11;14)(q13;q32)+/CD5+/CD23- mantle cell lymphoma in lymph nodes!

We report a case of a patient with two B-cell lymphoproliferative disorders: CD5(-)/CD23(+) B-cell chronic lymphocytic leukemia and CD5(+)/CD23(-) mantle cell lymphoma. These disorders were diagnosed simultaneously based on flow cytometry, immunohistochemistry, fluorescence in situ hybridization, and polymerase chain reaction-based molecular studies. The B-cell lymphocytic leukemia clone predominated in the blood and bone marrow, whereas the mantle cell clone predominated in lymph nodes.

Detection of herpesvirus infection of the CNS: the experience of hospital Geral de Santo António.

BACKGROUND: PCR detection of CSF Herpes virus DNA is an important tool in the diagnosis of CNS infections. Use of this test has been shown to have an impact on patient management as measured by shortened patient stays, specific therapeutic intervention, reduction of empirical expensive therapy administration, all of which should translate into significant health care savings. OBJECTIVE: The present study aimed at implementing, and evaluating both clinically and analytically the performance of several commercially available PCR based assays for the detection of Herpes virus infections of the CNS. STUDY DESIGN: A total of 314 patients with suspected CNS Herpesvirus infection were investigated, between 1999 and 2001, by Stair primers PCR. Starting on January 2002, two commercially available real-time-PCR systems were implemented and tested using the Stair primers PCR assay as golden standard and three external control proficiency panels along with serial dilutions of positive clinical samples. RESULTS: Sensitivity of the assay was determined to be <200 copies per ml for HSV and <1250 copies per ml for CMV. Positive results were obtained for 17 patients (6 HSV-1, 1 HSV-2, 1 EBV, 1 CMV, 6 VZV and 2 HHV-6) whose clinical and analytical findings were consistent with the PCR results. A real-time-PCR procedure was introduced in 2002 with similar sensitivity, but a more rapid response. CONCLUSION: Conventional end-point PCR proved useful to the diagnosis of CNS herpes virus infection, with an impact on the clinical intervention. However, the use of Real-Time-PCR has greatly enhanced these advantages, making results available at a much earlier time, thus significantly reducing the need for empirical treatment.

[Glucose-6-phosphate dehydrogenase deficiency, neonatal hyperbilirubinemia and Gilbert syndrome]. / Défice de glicose-6-fosfato desidrogenase, ictericia neonatal e sindroma de Gilbert.

The aim of this work was to evaluate the influence of abnormal UDP-glucoronosyltransferase-1 (UGT1A1) gene variant, on the incidence and severity of neonatal hyperbilirubinemia, in glucose-6-phosphate dehydrogenase (G6PD) deficient newborns. The A(TA)nTAA region in the promoter of the UGT1A1 gene was analysed in 20 children with G6PD deficiency. Fourteen of these children had the African type variant (G6PDA-) and 6 had different variants (G6PDNara, G6PDGuadalajara, G6PDDurham, G6PDTomah, G6PDAveiro e G6PDNashville) related to chronic nonspherocytic haemolytic anaemia (CNSHA). The existence of a positive history of neonatal hyperbilirubinemia, as well as its severity was registered. The incidence of neonatal hyperbilirubinemia was increased in this group of children (90%) and was not associated with abnormal alleles of the UGT1A1 gene. It was not possible to assess the influence of abnormal alleles in the severity of the neonatal hyperbilirubinemia. However, these abnormal alleles did not account for the severity of jaundice in children who presented variants related to CNSHA, since 5 were treated with an exchange transfusion and none presented abnormal alleles.

Advances in the genotyping of thrombosis genetic risk factors: clinical and laboratory implications.

Since FV-Leiden polymorphism was first described in 1994, a growing number of polymorphic loci have been identified in association with increased genetic risk for thrombophilia. Often however, these risk factors have been studied in isolation of the remaining known phenotype linked polymorphisms. This fact has, at least in part, been justified by the laborious techniques traditionally used in the genotyping studies, as well as its relatively high costs. Another major problem concerning these studies has been the non-negligible incidence of dubious genotypes, resulting from the manual, labour intensive techniques applied, and their sometimes difficult to read output's. These difficulties have also hampered the widespread use of genotyping data in the clinical assessment of the genetic risk levels both in patients and their relatives, leaving some clinicians less than convinced about its clinical usefulness. Recently however, the introduction of new genetic techniques in the clinical genetics laboratory has started to change this picture. Most notably, the advent of Real-time-PCR has brought the possibility of genotyping patients and controls at a large scale, with increased specificity, automation and speed. Moreover, the use of these techniques in the clinical genetics setting has not only increased the quality of the results, but most importantly has also increased our capability of answering questions at a deeper level. Among the new questions that can now be answered without increased costs and uncertainty is the study of the association of genetic risk factors in thrombophilia. Our results show that indeed even common polymorphic loci may increase our ability to further discriminate the genetic thrombosis risk of individual patients and relatives. It must however be noted that the innovation level in the clinical genetics lab is just starting to grow. In fact we haven't even started to experience the advantages brought about by the genome program, and its massive identification of SNP's. The technology to test these is also presently being refined, and is expected to go from research to the clinical lab in the near future. Only then, can we expect to define with high certainty the combined genetic risks for such complex pathologies as the thrombophilias.