Application of Dried Urine Spots for Non-Targeted Quadrupole Time-of-Flight Drug Screening.

The use of dried urine spots (DUS) can simplify sample handling, shipment and storage when compared to liquid urine samples. To prepare DUS, a small amount of urine is pipetted on a filter paper card. The subsequent drying of the specimen can prevent the post-sampling formation or degradation of substances (e.g., caused by bacteria). To evaluate the potential of DUS screening, 17 authentic urine samples, containing a broad range of substances, were extracted and analyzed on a Sciex TripleTOF® 5600+ System using a non-targeted screening and library searching approach. The screening results were compared to the analysis of the same urine sample in liquid form, using the same high-resolution liquid chromatography--quadrupole time-of-flight mass spectrometry method. More than 65 different legal and illegal drugs were successfully identified within the investigated 17 urine samples using the DUS screening approach. When compared to the analysis of liquid urine, the following compounds could not be identified: 1x ecgonine methyl ester, 1x nicotine, 1x promazine and 1x 11-nor-9-carboxy-∆9-tetrahydrocannabinol. Overall, 95.2% of the target substances that have been detected in liquid urine were identified correctly using the DUS approach. In conclusion, DUS screening offers a simple, cost-effective and easier sample handling alternative to the traditional use of liquid urine and provides the detection of the most important substances for forensic requirements. Furthermore, the DUS sample preparation can be fully automated (sample documentation, internal standard application and extraction).

Evaluation of measurable residual disease in multiple myeloma by multiparametric flow cytometry: Current paradigm, guidelines, and future applications.

Multiple myeloma (MM) is a heterogeneous group of mature B-cell diseases that are typically characterized by the presence and accumulation of abnormal plasma cells (PCs), which results in the excess production of monoclonal immunoglobulin and/or light chain found in the serum and/or urine. Multiparametric flow cytometry (MFC) is an indispensable tool to supplement the diagnosis, classification and monitoring of the disease due to its high patient applicability, excellent sensitivity and encouraging results from various clinical trials. In this regard, minimal or, more appropriately, measurable residual disease (MRD) negativity by MFC has been recognized as a powerful predictor of favourable long-term outcomes. Before flow cytometry can be effectively implemented in the clinical setting for MM MRD testing, sample preparation, panel configuration, analysis and gating strategies must be optimized to ensure accurate results. This manuscript will discuss the current consensus guidelines for flow cytometric processing of samples and reporting of results for MM MRD testing. We also discuss alternative approaches to detect plasma cells in the presence of daratumumab treatment. Finally, there is a lack of information describing the subclonal distribution of myeloma cells based on their protein expression. The advent of high-dimensional analysis may assist in following the evolution of antigen expression patterns on abnormal plasma cells in patients with relapsed/refractory disease. This in turn can help identify clonal subtypes that are more aggressive for potential informed decision. An analysis using t-SNE to identify the emergence of PCs subclones by MFC, along with the analysis of their immunophenotypic profiles are presented as a future perspective.

Dolor abdominal crónico en un niño con Blastocystis hominis: a propósito de un caso / Chronic abdominal pain in a child with Blastocystis hominis: case report

RESUMEN El Blastocystis sp. es un parásito frecuente en el humano, identificado por el laboratorio en muestras de heces fecales. Se presentó el caso de un paciente de 5 años atendido en consulta de Gastroenterología en el Hospital Pediátrico Docente Provincial Eliseo Noel Caamaño, de Matanzas, por presentar dolor abdominal, heces pastosas, náuseas y vómitos desde hacía un año. Llevó tratamiento con ranitidina, omeprazol y domperidona, sin mejoría clínica. Se realizó estudio coproparasitológico en muestras de heces fecales seriadas, con la presencia del Blastocystis hominis. Se indicó tratamiento con metronidazol, sin mejoría clínica, y posteriormente se indicó como alternativa la nitazoxanida. Se evaluó a los 15 días, sin sintomatología y con negativización de las heces fecales seriadas. Resulta frecuente el desconocimiento y la poca importancia que los profesionales sanitarios muestran ante esta infestación, aunque cada vez más se confirma la participación del parásito en manifestaciones clínicas (AU).

ABSTRACT Blastocystis sp. is a frequent parasite in humans, identified in the laboratory in samples of fecal feces. The case of a 5-year-old patient is presented; he assisted the consultation of Gastroenterology in the Provincial Teaching Pediatric Hospital Eliseo Noel Caamaño in Matanzas, suffering abdominal pain, mash feces, nauseas and vomits for one year, and was treated with ranitidine, omeprazole and domperidone without clinical improvement. A coproparasitological study was carried out in serial fecal feces samples with the presence of Blastocystis hominis. Treatment with metronidazole was indicated without clinical improvement and them, as an alternative, nitazoxanide was indicated. He was evaluated at 15 days without symptoms and with negative serial fecal feces. The ignorance and the little importance that health professionals show towards this infestation are frequent, although more and more frequently it is confirmed the participation of the parasite in clinical manifestations (AU).

A Comparison of the Efficacy of Immediate Versus Delayed Frozen-Thawed Embryo Transfer on the Ongoing Pregnancy Rate After a Failed IVF Attempt: Study Protocol for a Randomized, Non-Inferiority, Parallel-Group, Controlled Trial.

Introduction: In clinical practice, the ideal time at which to perform a Frozen-thawed Embryo Transfer (FET) after a failed In-vitro Fertilization-embryo Transfer (IVF-ET) is still unclear to most practicing physicians. In addition, physicians often delay the introduction of FET due to concerns on the possible residual effects of ovarian hyperstimulation, which may interfere with the regular menstrual cycle. Moreover, given that most of the published studies on the topic are retrospective with contradictory findings, it is crucial to provide evidence-based randomized control guides for clinical practice. Methods/analysis: The study is a randomized, non-inferiority, parallel-group, controlled trial that will enroll a total of 732 women undergoing their first FET after a failed fresh embryo transfer (ET) cycle. The participants will then be randomized into two groups based on a computer-generated randomized list. The two groups include: (i) an immediate group were FET will be carried out during the first menstrual cycle after a failed fresh ET cycle and (ii) a delayed group where FET will be carried out during the second menstrual cycle after a failed fresh ET cycle. Primary outcomes will be defined as viable pregnancies with fetal heartbeats, diagnosed through pelvic ultrasonography after twelve weeks of gestation. Ethics and dissemination: The study was approved by the Ethics Committee of the Assisted Reproductive Medicine at the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (SDTCM/E-2020.2.01). In addition, written informed consent will be obtained from all the participants before the study. The results of this trial will be disseminated in a peer-reviewed journal. Discussion: Currently, there is no consensus with regard to the duration after which the effects of ovarian stimulation are observed after a failed fresh ET and the optimal time required to begin FET. Moreover, no randomized controlled trial exists that compares the ongoing pregnancy rates after immediate versus delayed FET following a failed fresh ET cycle. Therefore, it is important to conduct a well-designed randomized trial to determine whether it is necessary to delay FET for at least one menstrual cycle after the failure of fresh ET. Clinical Trial Registration: ChiCTR2000033313 (http://www.chictr.org.cn/enIndex.aspx).

Traces under nails in clinical forensic medicine: not just DNA.

When dealing with complex crimes such as rape and assault, every trace takes on an essential role. The hands are often the only means of defence and offence for the victim as well as a frequent area of contact with the environment; fingernails of a victim are a well-known possible source of DNA of the aggressor; nevertheless, they are more rarely treated as an area of interest for non-genetic material, particularly on living victims. The hyponychium, because of its physiological protective function, lends itself ideally to retaining different kinds of traces representative of an environment or various products and substrates that could shed light on the environment and objects involved in the event. We therefore tested how far this capability of the hyponychium could go by simulating the dynamics of contamination of the nail through scratching on different substrates (brick and mortar, painted wood, ivy leaves, cotton and woollen fabric, soil) and persistence of any contaminant at different time intervals. We have thus shown how these traces may remain in the living for up to 24 h after the event using inexpensive and non-destructive techniques such as the episcopic and optical microscope.

Collection and Identification of Pollen from Honey Bee Colonies.

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

Systematic approach in macrophage polarization experiments: Maintaining integrity and reproducibility using flow cytometry and sample preparation.

Resolution of inflammation is an important physiological process following infection or injury. When inflammation fails to resolve, it can cause chronic inflammation, which exacerbates a myriad of diseases. Current anti-inflammatory treatment options are often inadequate to resolve inflammation, and as such, a key goal for drug discovery is to find natural products and novel compounds that can target immune resolution processes. In order to efficiently discovery new therapies, immune cell lines are often used, in conjunction with flow cytometry, to quickly and inexpensively screen potential drugs for immunomodulatory effects. However, seemingly minor or trivial differences in methodology can lead to inconsistent results across experiments and across laboratories. It was the goal of this project to examine the effects of those differences on the RAW 264.7 macrophage cell line, particularly as it relates to macrophage polarization experimentation. We found that the type of detachment method when preparing cells for flow cytometry can alter several key macrophage parameters, including markers for macrophage polarization, depending on the gating strategy used in identifying sub-populations of cells for analysis. Investigators need to incorporate best-practices in gating strategy in order to target viable cells that are not in aggregate to ensure consistent and reliable results for immunomodulatory drug discovery.

Study protocol: Whole genome sequencing Implementation in standard Diagnostics for Every cancer patient (WIDE).

BACKGROUND: 'Precision oncology' can ensure the best suitable treatment at the right time by tailoring treatment towards individual patient and comprehensive tumour characteristics. In current molecular pathology, diagnostic tests which are part of the standard of care (SOC) only cover a limited part of the spectrum of genomic changes, and often are performed in an iterative way. This occurs at the expense of valuable patient time, available tissue sample, and interferes with 'first time right' treatment decisions. Whole Genome Sequencing (WGS) captures a near complete view of genomic characteristics of a tumour in a single test. Moreover, WGS facilitates faster implementation of new treatment relevant biomarkers. At present, WGS mainly has been applied in study settings, but its performance in a routine diagnostic setting remains to be evaluated. The WIDE study aims to investigate the feasibility and validity of WGS-based diagnostics in clinical practice. METHODS: 1200 consecutive patients in a single comprehensive cancer centre with (suspicion of) a metastasized solid tumour will be enrolled with the intention to analyse tumour tissue with WGS, in parallel to SOC diagnostics. Primary endpoints are (1) feasibility of implementation of WGS-based diagnostics into routine clinical care and (2) clinical validation of WGS by comparing identification of treatment-relevant variants between WGS and SOC molecular diagnostics. Secondary endpoints entail (1) added clinical value in terms of additional treatment options and (2) cost-effectiveness of WGS compared to SOC diagnostics through a Health Technology Assessment (HTA) analysis. Furthermore, the (3) perceived impact of WGS-based diagnostics on clinical decision making will be evaluated through questionnaires. The number of patients included in (experimental) therapies initiated based on SOC or WGS diagnostics will be reported with at least 3 months follow-up. The clinical efficacy is beyond the scope of WIDE. Key performance indicators will be evaluated after every 200 patients enrolled, and procedures optimized accordingly, to continuously improve the diagnostic performance of WGS in a routine clinical setting. DISCUSSION: WIDE will yield the optimal conditions under which WGS can be implemented in a routine molecular diagnostics setting and establish the position of WGS compared to SOC diagnostics in routine clinical care.

Cultivo mediante hisopado superficial versus cultivo de la biopsia de tejidos profundos en la infección del pie diabético / Culture through superficial swab versus deep tissue biopsy in the diabetic foot infection

RESUMEN Introducción: para lograr el adecuado y precoz diagnóstico de la infección en pie diabético, es necesario la obtención de una muestra bacteriológica de calidad para la identificación del germen causal. Objetivo: identificar posibles relaciones entre los resultados obtenidos, en el cultivo realizado mediante hisopado superficial versus el obtenido mediante biopsia de los tejidos profundos en la infección del pie diabético. Materiales y métodos: se realizó un estudio explicativo observacional, longitudinal, prospectivo en el Servicio Provincial de Angiología y Cirugía Vascular del Hospital Provincial Clínico Quirúrgico Universitario "Comandante Faustino Pérez", durante un periodo de 3 años desde enero del 2016 hasta diciembre del 2018. Una selección muestral no probabilística determinó una muestra constituida por 138 extremidades en 132 pacientes con diagnóstico clínico de pie diabético infectado, que requirieron cirugía para desbridamiento de la lesión. Aceptaron ser incluidos en la investigación y para el aislamiento del germen causal fueron empleados ambos métodos de cultivo: hisopado superficial y biopsia de los tejidos profundos. Resultados: el promedio de microorganismos aislados se incrementó en relación con la severidad de la infección del pie diabético, con mayor incremento en el aislamiento hecho por el hisopado superficial. El hisopado superficial posee pobre correlación con los gérmenes aislados mediante el cultivo de la biopsia de los tejidos profundos. Conclusiones: las muestras deben ser obtenidas preferentemente por curetaje. En el diagnóstico de la infección del pie diabético es de gran utilidad, por su rapidez y concordancia con los resultados del cultivo, efectuar siempre una tinción de Gram a partir del mismo sitio (AU).

ABSTRACT Introduction: to arrive to an adequate and precocious diagnosis of the diabetic foot infection, it is necessary to obtain a qualitative bacteriological sample to identify the causing germ. Objective: to identify possible relationships between the results obtained both, in the culture made through superficial swab and the culture obtained from deep tissues biopsy in the diabetic foot infection. Materials and methods: a prospective, longitudinal, observational, explicative study was carried out in the Provincial Service of Angiology and Vascular Surgery of Provincial University Clinical Surgical Hospital "Comandante Faustino Pérez", in a period of three years, from January 2016 to December 2018. A non-probabilistic sampling choose a sample of 138 lower limbs in 132 patients with clinical diagnosis of infected diabetic foot, who required surgery for lesion debridement. They gave their consent to be included in the research; for the isolation of the casual germ were used both culture methods, superficial swab and deep tissues biopsy. Results: the average of isolated microorganism increased in relation to the severity of the diabetic food infection, with higher increase in the isolation obtained by superficial swab. The superficial swab shows poor correlation with the germ isolates by the culture the deep tissue biopsy. Conclusions: the samples should be gathered preferably by curettage. In the diagnosis of the diabetic foot infection, it is very useful, due to its speed and concordance with the culture results, to make always a Gram staining beginning from the same place (AU).

The effect of sampling height on grass pollen concentrations in different urban environments in the Helsinki Metropolitan Area, Finland.

INTRODUCTION: It is important to study potential differences in pollen concentrations between sampling heights because of diverse outdoor and indoor activity of humans (exposure) at different height levels in urban environments. Previous studies have investigated the effect of height on pollen concentrations based on just one or a few sampling points. We studied the effect of sampling height on grass pollen concentrations in several urban environments with different levels of urbanity. METHODS: This study was conducted in the Helsinki Metropolitan Area, Finland, in 2013 during the pollen season of grasses. Pollen grains were monitored in eight different points in the morning and afternoon. Rotorod-type samplers were attached on sampling poles at the heights of 1.5 meters and 4 meters. RESULTS: Grass pollen concentrations were on average higher at the height of 1.5 meters (Helsinki mean 5.24 grains / m3; Espoo mean 75.71 grains / m3) compared to the height of 4 meters (Helsinki mean 3.84 grains / m3; Espoo mean 37.42 grains / m3) with a difference of 1.40 grains / m3 (95% CI -0.21 to 3.01) in Helsinki, and 38.29 grains / m3 (7.52 to 69.07) in Espoo, although not always statistically significant. This was detected both in the morning and in the afternoon. However, in the most urban sites the levels were lower at 1.5 meters compared to 4 meters, whereas in the least urban sites the concentrations were higher at 1.5 meters. In linear regression models with interaction terms, the modifying effect of urbanity on concentration-height relation was statistically significant in both cities. The effect of urbanity on pollen concentrations at both heights was stronger in less urban Espoo. CONCLUSIONS: The present study provides evidence that height affects the abundance and distribution of grass pollen in urban environments, but this effect depends on the level of urbanity.

Metabolomic and Imaging Mass Spectrometric Assays of Labile Brain Metabolites: Critical Importance of Brain Harvest Procedures.

Metabolomic technologies including imaging mass spectrometry (IMS; also called mass spectrometry imaging, MSI, or matrix-assisted laser desorption/ionization-mass spectrometry imaging, MALDI MSI) are important methods to evaluate levels of many compounds in brain with high spatial resolution, characterize metabolic phenotypes of brain disorders, and identify disease biomarkers. ATP is central to brain energetics, and reports of its heterogeneous distribution in brain and regional differences in ATP/ADP ratios reported in IMS studies conflict with earlier studies. These discordant data were, therefore, analyzed and compared with biochemical literature that used rigorous methods to preserve labile metabolites. Unequal, very low regional ATP levels and low ATP/ADP ratios are explained by rapid metabolism during postmortem ischemia. A critical aspect of any analysis of brain components is their stability during and after tissue harvest so measured concentrations closely approximate their physiological levels in vivo. Unfortunately, the requirement for inactivation of brain enzymes by freezing or heating is not widely recognized outside the neurochemistry discipline, and procedures that do not prevent postmortem autolysis, including decapitation, brain removal/dissection, and 'snap freezing' are commonly used. Strong emphasis is placed on use of supplementary approaches to calibrate metabolite abundance in units of concentration in IMS studies and comparison of IMS results with biochemical data obtained by different methods to help identify potential artifacts.

Comparative economic analysis of soil sampling methods used in precision agriculture.

Precision agriculture is an alternative for reducing costs. This study evaluated and economically compared three sampling methods used in precision agriculture with respect to the acquisition of inputs and machines and equipment. The sampling methods used were zone management by elevation (ZME), grid sampling (GS) and sampling guided by apparent electrical conductivity of the soil (OS). Soil samples for the ZME were collected after the definition of zones according to the elevations of the plots. The sample mesh was in a georeferenced mesh of 100 x 100 m. The targeted sampling was performed after a ground proximity sensor was used to identify the apparent electrical conductivity of the soil to define the management areas. From the results of the laboratory tests, the application costs were calculated for lime, phosphorus, potassium and nitrogen to allow a comparison between the methods, volumes and costs. This approach considered the costs of depreciation, insurance, interest, operating costs, labor, maintenance and fuel. With this study, it was possible to compare the volumes of the recommended fertilizers and estimate the overall economic cost of using the technology via sensor. Taking the GS as a reference, the ZME presented as the best alternative compared to other methods.

Ultra-gentle soft robotic fingers induce minimal transcriptomic response in a fragile marine animal.

Tessler et al. demonstrate that a 'soft' robot causes less stress to a jellyfish while handling compared to a traditional 'hard' robot.

Recommendations for the diagnosis and treatment of Clostridioides difficile infection: An official clinical practice guideline of the Spanish Society of Chemotherapy (SEQ), Spanish Society of Internal Medicine (SEMI) and the working group of Postoperative Infection of the Spanish Society of Anesthesia and Reanimation (SEDAR).

This document gathers the opinion of a multidisciplinary forum of experts on different aspects of the diagnosis and treatment of Clostridioides difficile infection (CDI) in Spain. It has been structured around a series of questions that the attendees considered relevant and in which a consensus opinion was reached. The main messages were as follows: CDI should be suspected in patients older than 2 years of age in the presence of diarrhea, paralytic ileus and unexplained leukocytosis, even in the absence of classical risk factors. With a few exceptions, a single stool sample is sufficient for diagnosis, which can be sent to the laboratory with or without transportation media for enteropathogenic bacteria. In the absence of diarrhoea, rectal swabs may be valid. The microbiology laboratory should include C. difficile among the pathogens routinely searched in patients with diarrhoea. Laboratory tests in different order and sequence schemes include GDH detection, presence of toxins, molecular tests and toxigenic culture. Immediate determination of sensitivity to drugs such as vancomycin, metronidazole or fidaxomycin is not required. The evolution of toxin persistence is not a suitable test for follow up. Laboratory diagnosis of CDI should be rapid and results reported and interpreted to clinicians immediately. In addition to the basic support of all diarrheic episodes, CDI treatment requires the suppression of antiperistaltic agents, proton pump inhibitors and antibiotics, where possible. Oral vancomycin and fidaxomycin are the antibacterials of choice in treatment, intravenous metronidazole being restricted for patients in whom the presence of the above drugs in the intestinal lumen cannot be assured. Fecal material transplantation is the treatment of choice for patients with multiple recurrences but uncertainties persist regarding its standardization and safety. Bezlotoxumab is a monoclonal antibody to C. difficile toxin B that should be administered to patients at high risk of recurrence. Surgery is becoming less and less necessary and prevention with vaccines is under research. Probiotics have so far not been shown to be therapeutically or preventively effective. The therapeutic strategy should be based, rather than on the number of episodes, on the severity of the episodes and on their potential to recur. Some data point to the efficacy of oral vancomycin prophylaxis in patients who reccur CDI when systemic antibiotics are required again.

Simultaneous multielement analysis by ICP-MS with simple whole blood sample dilution and its application to uremic patients undergoing long-term hemodialysis.

Few studies were reported on trace elements' alterations in uremic patients undergoing long-term (>24 months) hemodialysis (HD), especially by using the whole blood as the biological fluid for the measuring purpose. Our objective was to develop an improved micro-sampling inductively coupled plasma-mass spectrometry (ICP-MS) method to determine the levels of Ca, Mg, Cu, Zn, Fe, Mn, Se and Pb in uremic patients receiving long-term HD. A ICP-MS method with a modified whole blood sample preparation procedure with small volumes was established and applied for the simultaneous quantification of the various elements in uremic patients undergoing long-term HD. 124 eligible uremic patients receiving long-term HD (75 males and 49 females) and 77 healthy subjects (54 males and 23 females) were recruited and Ca, Mg, Cu, Zn, Fe, Mn, Se and Pb levels were further determined. Our results revealed that uremic patients with HD had significantly higher blood levels of Ca, Mg, Zn and Pb and lower Cu, Fe, Se and Mn concentrations than healthy controls. In conclusion, a reproducible and reliable ICP-MS method using minimal whole blood sample volume (50 µL) with a simple dilution-based preparation procedure was successfully improved, validated and applied. Uremic patients undergoing long-term HD might be at increased risk of some essential trace elements deficiency (especially for Cu, Fe and Se) or toxic trace element excess (Pb) in respect to healthy subjects. Monitoring of blood levels and supplementation of some trace elements may be indicated in uremic patients undergoing long-term HD.

Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery.

Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.

Reactions of women underscreened for cervical cancer who received unsolicited human papillomavirus self-sampling kits.

OBJECTIVES: To evaluate experiences and reactions after receiving a mailed, unsolicited human papillomavirus self-sampling kit and identify psychosocial correlates of using kits. METHODS: Survey participants were underscreened women aged 30-64 who were mailed human papillomavirus kits as part of a pragmatic trial at Kaiser Permanente Washington, a United States integrated health care system. Six months after the mailing, we invited kit returners and non-returners to complete a web survey that measured psychosocial factors (e.g. cervical cancer/human papillomavirus knowledge, attitudes toward screening), experiences, and reactions to kits. We compared responses between kit returners and non-returners. RESULTS: Comparing 116 kit returners (272 invited) and 119 non-returners (1083 invited), we found no clinically significant differences in psychosocial factors. Overall, survey respondents showed knowledge gaps in human papillomavirus natural history (82% did not know human papillomavirus infection can clear on its own) and interpreting human papillomavirus test results (37% did not know a human papillomavirus-negative result indicates low cancer risk). Kit returners found kits convenient and easy to use (>90%). The most common reason for non-return was low confidence in ability to correctly use a kit, although many non-returners (49%) indicated that they would consider future use. Women reported low trust in human papillomavirus testing to identify women at high risk for cervical cancer (52% in returners, 42% in non-returners). CONCLUSIONS: Screening programs could improve uptake and acceptability of human papillomavirus self-sampling through outreach materials that emphasize the high efficacy of human papillomavirus testing for cervical cancer screening and educate patients about how to interpret results.

Implementation of HPV-based Cervical Cancer Screening Combined with Self-sampling Using a Midwifery Network Across Rural Greece: The GRECOSELF Study.

Self-sampling for human papillomavirus (HPV) testing is an alternative to physician sampling particularly for cervical cancer screening nonattenders. The GRECOSELF study is a nationwide observational cross-sectional study aiming to suggest a way to implement HPV-DNA testing in conjunction with self-sampling for cervical cancer screening in Greece, utilizing a midwifery network. Women residing in remote areas of Greece were approached by midwives, of a nationwide network, and were provided with a self-collection kit (dry swab) for cervicovaginal sampling and asked to answer a questionnaire about their cervical cancer screening history. Each sample was tested for high-risk (hr) HPV with the Cobas HPV test. HrHPV-Positive women were referred to undergo colposcopy and, if needed, treatment according to colposcopy/biopsy results. Between May 2016 and November 2018, 13,111 women were recruited. Of these, 12,787 women gave valid answers in the study questionnaire and had valid HPV-DNA results; hrHPV prevalence was 8.3%; high-grade cervical/vaginal disease or cancer prevalence was 0.6%. HrHPV positivity rate decreased with age from 20.7% for women aged 25-29 years to 5.1% for women aged 50-60 years. Positive predictive value for hrHPV testing and for HPV16/18 genotyping ranged from 5.0% to 11.6% and from 11.8% to 27.0%, respectively, in different age groups. Compliance to colposcopy referral rate ranged from 68.6% (for women 25-29) to 76.3% (for women 40-49). For women residing in remote areas of Greece, the detection of hrHPV DNA with the Cobas HPV test, on self-collected cervicovaginal samples using dry cotton swabs, which are provided by visiting midwives, is a promising method for cervical cancer secondary prevention.

Management of Mycobacterium avium subsp. paratuberculosis in dairy farms: Selection and evaluation of different DNA extraction methods from bovine and buffaloes milk and colostrum for the establishment of a safe colostrum farm bank.

The aim of this study was to develop and validate different innovative DNA extraction methods to detect Mycobacterium avium subsp. paratuberculosis (MAP) DNA from bovine and buffalo colostrum. Paratuberculosis is a chronic inflammatory infection of domestic and wild animals, especially ruminants, caused by MAP. The primary route of disease transmission is feces, but MAP can also be excreted in milk and colostrum. In 2015, the Italian Ministry of Health has issued a voluntary control plan of MAP in order to allow risk-based certification of bovine and buffaloes farms. In addition to the annual diagnostic screening and to the clinical surveillance of animals the plan includes the adoption of biosecurity and management measures to progressively mitigate the incidence of MAP. To achieve this goal it is crucial to ensure the accuracy of the methods used to detect the presence of MAP in bovine and buffaloes milk and colostrum, in order to: (1) support a "safe colostrum farm-bank" set-up and thus prevent the main within-farm MAP transmission route and (2) to allow the MAP-free certification of milk products for export purposes. To achieve these goals, seven different DNA extraction protocols were identified from bibliography, out of which three methods were finally selected after the adoption of an evaluation procedure aimed at assessing the efficiency of extraction of DNA, the purity of DNA and the adaptability of the DNA amplification: NucleoSpin® Food Kit (Macherey-Nagel), NucleoSpin® Food Kit (Macherey-Nagel) combined with the magnetic beads, and QIAamp Cador Pathogen Mini kit (QIAGEN). In particular, the NucleoSpin® Food Kit (Macherey-Nagel) and the QIAamp Cador Pathogen Mini kit (QIAGEN) were tested on bovine and buffalo colostrum, showing a LOD between 4 × 104 (2.6 × 106  cfu/ml) and 4.08 (26.7 cfu/ml) IS900 target copies and a LOD between 5.3 × 105 (4.1 × 106  cfu/ml) and 53 (4.1 × 103  cfu/ml) IS900 target copies, respectively.