ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Genotyping of rams based on melatonin receptor 1A gene polymorphisms: a tool in sire selection?

Context Several polymorphisms in the melatonin receptor 1A gene (MTNR1A ) have been related to reproductive performance in ovine. Aims To investigate the effect of the Rsa I and Mnl I polymorphisms on ram seminal quality. Methods Eighteen Rasa Aragonesa rams were genotyped for the Rsa I (C/C, C/T, T/T) and Mnl I (G/G, G/A, A/A) allelic variants of the MTNR1A gene. Individual ejaculates were analysed once a month throughout the whole year. Sperm motility, morphology, membrane integrity, levels of reactive oxygen species (ROS), phosphatidylserine (PS) inversion, DNA fragmentation and capacitation status were assessed. The effect of the season and polymorphisms on seminal quality was evaluated by mixed ANOVA. Key results Both polymorphisms had an effect on membrane integrity and viable spermatozoa with low levels of ROS and without PS translocation, and Rsa I also on motile and DNA-intact spermatozoa. An interaction between both polymorphisms was found, pointing to a negative effect on seminal quality of carrying the T or A allele in homozygosity. Differences were higher in the reproductive than in the non-reproductive season. Conclusions Mutations substituting C by T and G by A at Rsa I and Mnl I polymorphic sites, respectively, in the MTNR1A gene in rams could decrease the seminal quality. Implications Genotyping of rams based on melatonin receptor 1A could be a powerful tool in sire selection.

Evidence of the Beneficial Impact of Three Probiotic-Based Food Supplements on the Composition and Metabolic Activity of the Intestinal Microbiota in Healthy Individuals: An Ex Vivo Study.

Scientific evidence has increasingly supported the beneficial effects of probiotic-based food supplements on human intestinal health. This ex vivo study investigated the effects on the composition and metabolic activity of the intestinal microbiota of three probiotic-based food supplements, containing, respectively, (1) Bifidobacterium longum ES1, (2) Lactobacillus acidophilus NCFM®, and (3) a combination of L. acidophilus NCFM®, Lactobacillus paracasei Lpc-37™, Bifidobacterium lactis Bi-07™, and Bifidobacterium lactis Bl-04™. This study employed fecal samples from six healthy donors, inoculated in a Colon-on-a-plate® system. After 48 h of exposure or non-exposure to the food supplements, the effects were measured on the overall microbial fermentation (pH), changes in microbial metabolic activity through the production of short-chain and branched-chain fatty acids (SCFAs and BCFAs), ammonium, lactate, and microbial composition. The strongest effect on the fermentation process was observed for the combined formulation probiotics, characterized by the significant stimulation of butyrate production, a significant reduction in BCFAs and ammonium in all donors, and a significant stimulatory effect on bifidobacteria and lactobacilli growth. Our findings suggest that the combined formulation probiotics significantly impact the intestinal microbiome of the healthy individuals, showing changes in metabolic activity and microbial abundance as the health benefit endpoint.

Fine-tuning characterization of patients with interstitial pneumonia and an underlying autoimmune disease in real-world practice: We get closer with Nailfold videocapillaroscopy.

Objectives: To assess performance of interstitial pneumonia (IP) with autoimmune features (IPAF) criteria in clinical practice and describe the utility of additional workup in identifying patients with underlying connective tissue diseases (CTD). Methods: We set a retrospective study of our patients with autoimmune IP, who were allocated to CTD-IP, IPAF or undifferentiated autoimmune IP (uAIP) subgroups according to the updated classification criteria. Presence of the process-related variables comprising IPAF defining domains was scrutinized in all patients, and, when available, the results of nailfold videocapillaroscopy (NVC) were recorded. Results: Thirty nine out of 118 patients, accounting for 71% of former undifferentiated cases, fulfilled IPAF criteria. Arthritis and Raynaud's phenomenon were prevalent in this subgroup. While systemic sclerosis-specific autoantibodies were restricted to CTD-IP patients, anti-tRNA synthetase antibodies were also present in IPAF. In contrast, rheumatoid factor, anti-Ro antibodies and ANA nucleolar patterns could be found in all subgroups. Usual interstitial pneumonia (UIP) / possible UIP were the most frequently observed radiographic patterns Therefore, the presence of thoracic multicompartimental findings as also performance of open lung biopsies were useful in characterizing as IPAF those UIP cases lacking a clinical domain. Interestingly, we could observe NVC abnormalities in 54% of IPAF and 36% of uAIP tested patients, even though many of them did not report Raynaud's phenomenon. Conclusion: Besides application of IPAF criteria, distribution of IPAF defining variables along with NVC exams help identify more homogeneous phenotypic subgroups of autoimmune IP of potential relevance beyond clinical diagnosis.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas.

In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, leading to death if not promptly diagnosed and treated. In Brazil, the disease reaches all regions, and in 2020, 1,933 VL cases were reported with 9.5% lethality. Thus, an accurate diagnosis is essential to provide the appropriate treatment. Serological VL diagnosis is based mainly on immunochromatographic tests, but their performance may vary by location, and evaluation of diagnostic alternatives is necessary. In this study, we aimed to evaluate the performance of ELISA with the scantily studied recombinant antigens, K18 and KR95, comparing their performance with the already known rK28 and rK39. Sera from parasitologically confirmed symptomatic VL patients (n = 90) and healthy endemic controls (n = 90) were submitted to ELISA with rK18 and rKR95. Sensitivity (95% CI) was, respectively, 83.3% (74.2-89.7) and 95.6% (88.8-98.6), and specificity (95% CI) was 93.3% (85.9-97.2) and 97.8% (91.8-99.9). For validation of ELISA with the recombinant antigens, we included samples from 122 VL patients and 83 healthy controls collected in three regions in Brazil (Northeast, Southeast, and Midwest). When comparing the results obtained with the VL patients' samples, significantly lower sensitivity was obtained by rK18-ELISA (88.5%, 95% CI: 81.5-93.2) compared with rK28-ELISA (95.9%, 95% CI: 90.5-98.5), but the sensitivity was similar comparing rKR95-ELISA (95.1%, 95% CI: 89.5-98.0), rK28-ELISA (95.9%, 95% CI: 90.5-98.5), and rK39-ELISA (94.3%, 95% CI: 88.4-97.4). Analyzing the specificity, it was lowest with rK18-ELISA (62.7%, 95% CI: 51.9-72.3) with 83 healthy control samples. Conversely, higher and similar specificity was obtained by rKR95-ELISA (96.4%, 95% CI: 89.5-99.2), rK28-ELISA (95.2%, 95% CI: 87.9-98.5), and rK39-ELISA (95.2%, 95% CI: 87.9-98.5). There was no difference in sensitivity and specificity across localities. Cross-reactivity assessment, performed with sera of patients diagnosed with inflammatory disorders and other infectious diseases, was 34.2% with rK18-ELISA and 3.1% with rKR95-ELISA. Based on these data, we suggest using recombinant antigen KR95 in serological assays for VL diagnosis.

Impact of a DANA Event on the Thermal Response of Nectarine Trees.

This field experiment focuses on the effects of a heavy rainfall event (DANA, depresión aislada en niveles altos) that occurred on 12-14 September 2019 (DOY, Day of the year, 255-257), in southern Spain on plant water status and the thermal response of nectarine trees. Two irrigation treatments were applied during the summer-autumn postharvest period (DOY 158-329): full-irrigated (CTL) and non-irrigated (DRY). Volumetric soil water content (θv), air temperature (Ta) and canopy temperature (Tc) were monitored in real-time and the crop water stress index (CWSI) was calculated. The difference in Tc between the DRY and CTL treatments (Tc' - Tc) is proposed as a new thermal indicator. Stem water potential (Ψstem) and leaf gas exchange measurements were recorded on representative days. During the DANA event, only the Tc measured by the infrared radiometer sensors could be monitored. Therefore, the effects of the DANA forced the soil water content sensors to be switched off, which prevented Ψstem and leaf gas exchange determinations from DOY 255 to 275. Before the DANA event, withholding irrigation caused a gradual decrease in the soil and plant water status in the DRY treatment. Significant differences appeared between treatments in the studied thermal indexes. Moreover, Tc' - Tc was more sensitive than Tc - Ta in assessing nectarine water stress. The effects of the DANA reduced these differences, suggesting different baselines for the calculation of CWSI. In this respect, the relationship Tc - Ta vs. VPD improved the coefficient of determination after the DANA event in full-irrigated trees. Similar values of Ψstem and leaf gas exchange were found in both treatments after the DANA event, even though thermal indexes showed some significant differences. In addition, the strong relationship found between Tc - Ta and CWSI vs. Ψstem worsened after DANA occurred, revealing a lower sensitivity of Ψstem compared to canopy temperature to accurately assess nectarine water status in these saturated soil conditions. This research underlined the robustness of infrared thermography to continuously monitor plant water status under these extreme weather conditions.

Effect of Water Stress and Shading on Lime Yield and Quality.

The aim of this study was to test the combined effect of water stress and cropping system on yield and fruit quality in Bearss lime trees. For this purpose, two irrigation treatments were applied during stage II of fruit growth: control (well irrigated, automatically managed by soil water content sensors) and stress (non-irrigated), both under open-field and shaded conditions. Soil water status was assessed by determining soil water content and plant water status by measuring stem water potential (Ψstem), stomatal conductance (gs), and net photosynthesis (Pn). Yield parameters (kg and the number of fruits per tree and fresh mass per fruit) and fruit quality were assessed on two harvest dates. In addition, on the second harvest date, the content of metabolites and nutrients in the lime juice was analyzed. The results showed that soil water deficit induced 35% lower gs values in open-field than in shaded conditions. The highest kg and the number of fruits per tree were observed in the shaded system, especially on the first harvest date. The lowest yield was observed in stressed trees grown without netting. Slightly higher fresh mass and equatorial diameter were observed in shaded fruits than in open-field fruit. Soil water deficit increased fruit total soluble solids and decreased juice content, especially in open-field trees. Shaded conditions made the lime trees more resilient to soil water deficit, which led to higher yields and better external fruit quality traits. In addition, fruit precocity was significantly higher in the shaded system.

Ultrasensitive molecular tests for Plasmodium detection: applicability in control and elimination programs and reference laboratories.

Objective: To evaluate molecular tools to detect low-level parasitemia and the five species of Plasmodium that infect humans for use in control and elimination programs, and in reference laboratories. Methods: We evaluated 145 blood samples from patients who tested positive by nested polymerase chain reaction (nPCR), from asymptomatic individuals and from the WHO Global Malaria Programme/United Kingdom National External Quality Assessment Service. Samples were assayed using the genus-specific RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) and the RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). The results from the molecular tests were compared with those from quantitative PCR (qPCR), nPCR and thick blood smear. Results: The levels of parasitemia ranged from 1 to 518 000 parasites/µL, depending on the species. Compared with nPCR, alt-S&T had a sensitivity of 100%, except for identifying P. falciparum, for which the sensitivity was 93.94%. All samples positive by alt-Gen were also positive by nPCR. When comparing alt-Gen to qPCR, the sensitivity was 100% for P. vivax, P. malariae and P. falciparum. For all Plasmodium species, the correlation between cycle threshold values of alt-S&T and alt-Gen compared with qPCR was significant (P < 0.0001, Spearman's test), with r = 0.8621 for alt-S&T and r = 0.9371 for alt-Gen. When all Plasmodium species were considered, there was a negative correlation between the level of parasitemia and real-time PCR cycle threshold values (P < 0.0001). In this study, only 2 of 28 samples from asymptomatic individuals were positive by thick blood smear; however, all 28 of these samples were positive by alt-S&T. Conclusions: The alt-Gen and alt-S&T assays are suitable for detecting submicroscopic infections for distinct epidemiological purposes, such as for use in surveys and reference laboratories, and screening in blood banks, which will contribute to global efforts to eliminate malaria.

Venous thromboembolism in pediatric patients with acute lymphoblastic leukemia under chemotherapy treatment. Risk factors and usefulness of thromboprophylaxis. Results of LAL-SEHOP-PETHEMA-2013.

INTRODUCTION: Symptomatic venous thromboembolism (VTE) is diagnosed in 3%-14% of patients during pediatric acute lymphoblastic leukemia (ALL) therapy. There are well-known risk factors, but the role of others as inherited thrombophilia is still controversial. Prophylaxis with low molecular weight heparin (LMWH) has been described, but its use is not globally accepted. METHODS: A retrospective multicentric study in ALL patients 1-18 years old following SEHOP-PETHEMA-2013 treatment guideline was performed to evaluate VTE rate, anticoagulant treatment, outcome, risk factors, and safety and usefulness of LMWH administration as primary thromboprophylaxis in children with inherited thrombophilia. RESULTS: A total of 652 patients were included in the study. VTE incidence was 8.7%. Most of the cases occurred during induction therapy associated with central venous catheter. Univariant analysis showed that family history of thrombosis, presence of mediastinal mass, high-risk treatment group, and inherited thrombophilia were statistically significant risk factors. LMWH administration seemed to decrease VTE rate in patients with inherited thrombophilia and those with T-cell ALL phenotype. CONCLUSION: Most of the VTE cases occurred in patients without inherited thrombophilia, but when it is present, the VTE risk is higher. LMWH administration was useful to decrease VTE in these patients.

Ultrasensitive molecular tests for Plasmodium detection: applicability in control and elimination programs and reference laboratories

[ABSTRACT]. Objective. To evaluate molecular tools to detect low-level parasitemia and the five species of Plasmodium that infect humans for use in control and elimination programs, and in reference laboratories. Methods. We evaluated 145 blood samples from patients who tested positive by nested polymerase chain reaction (nPCR), from asymptomatic individuals and from the WHO Global Malaria Programme/United Kingdom National External Quality Assessment Service. Samples were assayed using the genus-specific RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) and the RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). The results from the molecular tests were compared with those from quantitative PCR (qPCR), nPCR and thick blood smear. Results. The levels of parasitemia ranged from 1 to 518 000 parasites/μL, depending on the species. Compared with nPCR, alt-S&T had a sensitivity of 100%, except for identifying P. falciparum, for which the sensitivity was 93.94%. All samples positive by alt-Gen were also positive by nPCR. When comparing alt-Gen to qPCR, the sensitivity was 100% for P. vivax, P. malariae and P. falciparum. For all Plasmodium species, the correlation between cycle threshold values of alt-S&T and alt-Gen compared with qPCR was significant (P < 0.0001, Spearman’s test), with r = 0.8621 for alt-S&T and r = 0.9371 for alt-Gen. When all Plasmodium species were considered, there was a negative correlation between the level of parasitemia and real-time PCR cycle threshold values (P < 0.0001). In this study, only 2 of 28 samples from asymptomatic individuals were positive by thick blood smear; however, all 28 of these samples were positive by alt-S&T. Conclusions. The alt-Gen and alt-S&T assays are suitable for detecting submicroscopic infections for distinct epidemiological purposes, such as for use in surveys and reference laboratories, and screening in blood banks, which will contribute to global efforts to eliminate malaria.

[RESUMEN]. Objetivo. Evaluar herramientas moleculares para detectar bajos niveles de parasitemia y las cinco especies de Plasmodium que infectan a los seres humanos, a fin de emplearlas en los programas de control y eliminación y en los laboratorios de referencia. Métodos. Se evaluaron 145 muestras de sangre de pacientes positivos por reacción en cadena de la polimerasa anidada (nPCR), de individuos asintomáticos y de muestras del Programa Mundial de Malaria de la Organización Mundial de la Salud/Servicio Nacional de Evaluación Externa de Calidad del Reino Unido. Las muestras se analizaron con el kit de PCR RealStar® Malaria 1.0 (alt-Gen; altona Diagnostics), específico para cada género, y con el kit de PCR RealStar® Malaria Screen & Type (alt-S&T; altona Diagnostics). Se compararon los resultados de las pruebas moleculares con los de la PCR cuantitativa (qPCR), la nPCR y el frotis de gota gruesa. Resultados. Los niveles de parasitemia oscilaron entre 1 y 518 000 parásitos/μl, según la especie. En comparación con la nPCR, la prueba alt-S&T tuvo una sensibilidad del 100%, excepto para la identificación de P. falciparum, para el cual la sensibilidad fue del 93,94%. Todas las muestras positivas por alt-Gen lo fueron también por nPCR. Al comparar alt-Gen con la qPCR, la sensibilidad fue del 100% para P. vivax, P. malariae y P. falciparum. Para todas las especies de Plasmodium, la correlación entre los valores del umbral de ciclo de alt-S&T y alt-Gen en comparación con la qPCR fue significativa (P < 0,0001, prueba de Spearman), con r = 0,8621 para alt-S&T y r = 0,9371 para alt-Gen. Cuando se consideraron todas las especies de Plasmodium hubo una correlación negativa entre el nivel de parasitemia y los valores de umbral de ciclo de PCR en tiempo real (P < 0,0001). En este estudio, solo 2 de las 28 muestras de individuos asintomáticos fueron positivas por frotis de gota gruesa; sin embargo, las 28 muestras fueron positivas por alt-S&T. Conclusiones. Los ensayos alt-Gen y alt-S&T son adecuados para detectar infecciones submicroscópicas con distintos fines epidemiológicos, como su uso en investigaciones y laboratorios de referencia y el cribado en bancos de sangre, lo que contribuirá a los esfuerzos mundiales para eliminar la malaria.

[RESUMO]. Objectivo. Avaliar ferramentas moleculares para detectar parasitemia de baixo nível e as cinco espécies de Plasmodium que infectam humanos, para utilização em programas de controlo e eliminação e em laboratórios de referência. Métodos. Avaliámos 145 amostras de sangue de doentes que testaram positivo por reacção em cadeia da polimerase aninhada (nPCR), de indivíduos assintomáticos, e do Programa Global de Paludismo da Organização Mundial de Saúde/Serviço Nacional de Avaliação da Qualidade Externa do Reino Unido. As amostras foram ensaiadas utilizando o RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) e o RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). Os resultados dos testes moleculares foram comparados com os resultados da PCR quantitativa (qPCR), nPCR e exame da gota espessa. Resultados. Os níveis de parasitemia variaram de 1 a 518 000 parasitas/μL, dependendo da espécie. Em comparação com a nPCR, alt-S&T tinha uma sensibilidade de 100%, excepto na identificação de P. falciparum, para a qual a sensibilidade era de 93,94%. Todas as amostras positivas por alt-Gen foram também positivas por nPCR. Ao comparar alt-Gen com qPCR, a sensibilidade foi de 100% para P. vivax, P. malariae e P. falciparum. Para todas as espécies Plasmodium, a correlação entre os valores limiares de ciclo de alt-S&T e alt-Gen comparados com qPCR foi significativa (P < 0,0001, teste de Spearman), com r = 0,8621 para alt- S&T e r = 0,9371 para alt-Gen. Quando todas as espécies de Plasmodium foram consideradas, houve uma correlação negativa entre o nível de parasitemia e os valores limiares do ciclo de PCR em tempo real (P < 0,0001). Neste estudo, apenas 2 de 28 amostras de indivíduos assintomáticos foram positivas por exame da gota espessa; no entanto, todas estas 28 amostras foram positivas por alt-S&T. Conclusões. Os ensaios alt-Gen e alt-S&T são adequados para a detecção de infecções submicroscópicas para fins epidemiológicos distintos, tais como para utilização em inquéritos e laboratórios de referência e o rastreio em bancos de sangue, o que contribuirá para os esforços globais de eliminação da malária.

Antimicrobial effects of a new brushing solution concept on a multispecies in vitro biofilm model growing on titanium surfaces.

OBJECTIVES: To evaluate the antibiofilm and antibacterial effects of a new brushing solution concept, in a validated peri-implant biofilm model. MATERIALS AND METHODS: A multispecies in vitro biofilm model, including Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum, was used. To evaluate the antibiofilm capacity, titanium discs (Ti-SLA) were immersed in 1 ml of the tested solution (one tablet dissolved in warm water) for 2 min, prior and every 24 h during a 3-day biofilm development. Negative (water) and positive (0.12% chlorhexidine/0.05% cetylpyridinium chloride mouth rinse) controls treated discs were run in parallel. To evaluate the antibacterial effects, planktonic cells and 72-h biofilms on sterile Ti-SLA discs were exposed (2 min) to the mentioned treatments. Biofilm structure was analysed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Bacterial load was measured by quantitative polymerase chain reaction and by culture in planktonic cells. RESULTS: The tested product showed antibiofilm effects, impacting on the 48-h and 72-h biofilm thickness and significantly reducing viability of all bacterial species, except A. actinomycetemcomitans. Antibacterial effects were observed against the six bacterial species in planktonic state and in 72-h biofilms, especially for F. nucleatum and A. actinomycetemcomitans. CONCLUSION: The tested brushing solution demonstrated antibacterial and antibiofilm properties, mainly against the peri-implant pathogens included in the validated in vitro biofilm model used.

Ultrasensitive molecular tests for Plasmodium detection: applicability in control and elimination programs and reference laboratories / Pruebas moleculares ultrasensibles para la detección de Plasmodium: aplicabilidad en los programas de control y eliminación y en los laboratorios de referencia / Testes moleculares ultra-sensíveis para detecção de Plasmodium: aplicabilidade em programas de controlo e eliminação e laboratórios de referencia

ABSTRACT Objective. To evaluate molecular tools to detect low-level parasitemia and the five species of Plasmodium that infect humans for use in control and elimination programs, and in reference laboratories. Methods. We evaluated 145 blood samples from patients who tested positive by nested polymerase chain reaction (nPCR), from asymptomatic individuals and from the WHO Global Malaria Programme/United Kingdom National External Quality Assessment Service. Samples were assayed using the genus-specific RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) and the RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). The results from the molecular tests were compared with those from quantitative PCR (qPCR), nPCR and thick blood smear. Results. The levels of parasitemia ranged from 1 to 518 000 parasites/µL, depending on the species. Compared with nPCR, alt-S&T had a sensitivity of 100%, except for identifying P. falciparum, for which the sensitivity was 93.94%. All samples positive by alt-Gen were also positive by nPCR. When comparing alt-Gen to qPCR, the sensitivity was 100% for P. vivax, P. malariae and P. falciparum. For all Plasmodium species, the correlation between cycle threshold values of alt-S&T and alt-Gen compared with qPCR was significant (P < 0.0001, Spearman's test), with r = 0.8621 for alt-S&T and r = 0.9371 for alt-Gen. When all Plasmodium species were considered, there was a negative correlation between the level of parasitemia and real-time PCR cycle threshold values (P < 0.0001). In this study, only 2 of 28 samples from asymptomatic individuals were positive by thick blood smear; however, all 28 of these samples were positive by alt-S&T. Conclusions. The alt-Gen and alt-S&T assays are suitable for detecting submicroscopic infections for distinct epidemiological purposes, such as for use in surveys and reference laboratories, and screening in blood banks, which will contribute to global efforts to eliminate malaria.

RESUMEN Objetivo. Evaluar herramientas moleculares para detectar bajos niveles de parasitemia y las cinco especies de Plasmodium que infectan a los seres humanos, a fin de emplearlas en los programas de control y eliminación y en los laboratorios de referencia. Métodos. Se evaluaron 145 muestras de sangre de pacientes positivos por reacción en cadena de la polimerasa anidada (nPCR), de individuos asintomáticos y de muestras del Programa Mundial de Malaria de la Organización Mundial de la Salud/Servicio Nacional de Evaluación Externa de Calidad del Reino Unido. Las muestras se analizaron con el kit de PCR RealStar® Malaria 1.0 (alt-Gen; altona Diagnostics), específico para cada género, y con el kit de PCR RealStar® Malaria Screen & Type (alt-S&T; altona Diagnostics). Se compararon los resultados de las pruebas moleculares con los de la PCR cuantitativa (qPCR), la nPCR y el frotis de gota gruesa. Resultados. Los niveles de parasitemia oscilaron entre 1 y 518 000 parásitos/µl, según la especie. En comparación con la nPCR, la prueba alt-S&T tuvo una sensibilidad del 100%, excepto para la identificación de P. falciparum, para el cual la sensibilidad fue del 93,94%. Todas las muestras positivas por alt-Gen lo fueron también por nPCR. Al comparar alt-Gen con la qPCR, la sensibilidad fue del 100% para P. vivax, P. malariae y P. falciparum. Para todas las especies de Plasmodium, la correlación entre los valores del umbral de ciclo de alt-S&T y alt-Gen en comparación con la qPCR fue significativa (P < 0,0001, prueba de Spearman), con r = 0,8621 para alt-S&T y r = 0,9371 para alt-Gen. Cuando se consideraron todas las especies de Plasmodium hubo una correlación negativa entre el nivel de parasitemia y los valores de umbral de ciclo de PCR en tiempo real (P < 0,0001). En este estudio, solo 2 de las 28 muestras de individuos asintomáticos fueron positivas por frotis de gota gruesa; sin embargo, las 28 muestras fueron positivas por alt-S&T. Conclusiones. Los ensayos alt-Gen y alt-S&T son adecuados para detectar infecciones submicroscópicas con distintos fines epidemiológicos, como su uso en investigaciones y laboratorios de referencia y el cribado en bancos de sangre, lo que contribuirá a los esfuerzos mundiales para eliminar la malaria.

RESUMO Objectivo. Avaliar ferramentas moleculares para detectar parasitemia de baixo nível e as cinco espécies de Plasmodium que infectam humanos, para utilização em programas de controlo e eliminação e em laboratórios de referência. Métodos. Avaliámos 145 amostras de sangue de doentes que testaram positivo por reacção em cadeia da polimerase aninhada (nPCR), de indivíduos assintomáticos, e do Programa Global de Paludismo da Organização Mundial de Saúde/Serviço Nacional de Avaliação da Qualidade Externa do Reino Unido. As amostras foram ensaiadas utilizando o RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) e o RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). Os resultados dos testes moleculares foram comparados com os resultados da PCR quantitativa (qPCR), nPCR e exame da gota espessa. Resultados. Os níveis de parasitemia variaram de 1 a 518 000 parasitas/µL, dependendo da espécie. Em comparação com a nPCR, alt-S&T tinha uma sensibilidade de 100%, excepto na identificação de P. falciparum, para a qual a sensibilidade era de 93,94%. Todas as amostras positivas por alt-Gen foram também positivas por nPCR. Ao comparar alt-Gen com qPCR, a sensibilidade foi de 100% para P. vivax, P. malariae e P. falciparum. Para todas as espécies Plasmodium, a correlação entre os valores limiares de ciclo de alt-S&T e alt-Gen comparados com qPCR foi significativa (P < 0,0001, teste de Spearman), com r = 0,8621 para alt-S&T e r = 0,9371 para alt-Gen. Quando todas as espécies de Plasmodium foram consideradas, houve uma correlação negativa entre o nível de parasitemia e os valores limiares do ciclo de PCR em tempo real (P < 0,0001). Neste estudo, apenas 2 de 28 amostras de indivíduos assintomáticos foram positivas por exame da gota espessa; no entanto, todas estas 28 amostras foram positivas por alt-S&T. Conclusões. Os ensaios alt-Gen e alt-S&T são adequados para a detecção de infecções submicroscópicas para fins epidemiológicos distintos, tais como para utilização em inquéritos e laboratórios de referência e o rastreio em bancos de sangue, o que contribuirá para os esforços globais de eliminação da malária.

Antibiotics Prescription by Spanish General Practitioners in Primary Dental Care.

The aim of this study was to analyze the antibiotics prescription habits, both prophylactically and therapeutically, of Spanish general dental practitioners in the management of endodontic infections in primary care. Two hundred Spanish general dental practitioners were asked to respond to a survey on indications for antibiotics prescription in the treatment of endodontic infections, being 190 general dentists (95%) included in the study. Data were analyzed using descriptive statistics and the chi-square test. The average duration of antibiotics therapy was 6.5 ± 1.0 days. In patients without medical allergies, most of them (97%) selected amoxicillin as the antibiotic of the first choice, alone (51.1%) or associated with clavulanic acid (45.8%); in patients with penicillin allergies, the drug of choice was clindamycin 300 mg (70%). For cases of symptomatic irreversible pulpitis, 44% of the respondents prescribed antibiotics, in the scenario of prophylactic antibiotic prescription, up to 27% of the general dentists prescribe according to non-current guidelines (1 g 1 h before or 1 g 1 h before and 1 g 1 h after) in non-indicated cases (16% in patients taking oral bisphosphonates). It is necessary to improve the antibiotic prescription habits of Spanish general dentists in endodontics.

In Vitro Anti-Biofilm and Antibacterial Properties of Streptococcus downii sp. nov.

The aim of this study was to evaluate the potential anti-biofilm and antibacterial activities of Streptococcus downii sp. nov. To test anti-biofilm properties, Streptococcus mutans, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans were grown in a biofilm model in the presence or not of S. downii sp. nov. for up to 120 h. For the potential antibacterial activity, 24 h-biofilms were exposed to S. downii sp. nov for 24 and 48 h. Biofilms structures and bacterial viability were studied by microscopy, and the effect in bacterial load by quantitative polymerase chain reaction. A generalized linear model was constructed, and results were considered as statistically significant at p < 0.05. The presence of S. downii sp. nov. during biofilm development did not affect the structure of the community, but an anti-biofilm effect against S. mutans was observed (p < 0.001, after 96 and 120 h). For antibacterial activity, after 24 h of exposure to S. downii sp. nov., counts of S. mutans (p = 0.019) and A. actinomycetemcomitans (p = 0.020) were significantly reduced in well-structured biofilms. Although moderate, anti-biofilm and antibacterial activities of S. downii sp. nov. against oral bacteria, including some periodontal pathogens, were demonstrated in an in vitro biofilm model.

Relevance of Biofilm Models in Periodontal Research: From Static to Dynamic Systems.

Microbial biofilm modeling has improved in sophistication and scope, although only a limited number of standardized protocols are available. This review presents an example of a biofilm model, along with its evolution and application in studying periodontal and peri-implant diseases. In 2011, the ETEP (Etiology and Therapy of Periodontal and Peri-Implant Diseases) research group at the University Complutense of Madrid developed an in vitro biofilm static model using representative bacteria from the subgingival microbiota, demonstrating a pattern of bacterial colonization and maturation similar to in vivo subgingival biofilms. When the model and its methodology were standardized, the ETEP research group employed the validated in vitro biofilm model for testing in different applications. The evolution of this model is described in this manuscript, from the mere observation of biofilm growth and maturation on static models on hydroxyapatite or titanium discs, to the evaluation of the impact of dental implant surface composition and micro-structure using the dynamic biofilm model. This evolution was based on reproducing the ideal microenvironmental conditions for bacterial growth within a bioreactor and reaching the target surfaces using the fluid dynamics mimicking the salivary flow. The development of this relevant biofilm model has become a powerful tool to study the essential processes that regulate the formation and maturation of these important microbial communities, as well as their behavior when exposed to different antimicrobial compounds.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection.

BACKGROUND: Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE: We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS: Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS: For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS: For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Análisis de la demanda de detección de SARS-CoV-2 en un área de salud de España / Analysis of the demand for detection of SARS-CoV-2 in a health area of Spain

INTRODUCCIÓN: Desde el descubrimiento del virus SARSCoV-2 la técnica de reacción en cadena de la polimerasa (RT-PCR) se ha convertido en el método fundamental para el diagnóstico de la enfermedad en su fase aguda. El objetivo es describir la serie basada en la demanda de determinaciones de RT-PCR recibidas en un Servicio de Microbiología en un hospital de tercer nivel de referencia durante tres meses desde el inicio de la epidemia por SARS-CoV-2. MATERIAL Y MÉTODOS: Se realizó un análisis retrospectivo del total de las RT-PCR solicitadas en el servicio de microbiología analizado desde el 25 de febrero de 2020 al 26 de mayo de 2020 (90 días). Se agruparon por semanas epidemiológicas y servicio peticionario. Se realizó un análisis descriptivo por edad, género y número de solicitudes por paciente. Se consideró significativo un nivel de confianza del 95% (p < 0.05). RESULTADOS: Se recibieron un total de 27.106 de solicitudes que correspondían a 22.037 pacientes. Edad mediana 53,7 (RIC 40,9-71,7) años, mujeres: 61,3%. Proporción de pacientes con alguna RT-PCR positiva: 14%. Del total de peticiones de RT-PCR fueron positivas 3.710. La rentabilidad máxima fue la semana epidemiológica 13, con un 39,0%. El servicio peticionario que más RT-PCR ha solicitado de forma global ha sido atención primaria con 15.953 solicitudes. Pacientes con 3 o más RT-PCR: 565, de ellos, 19 pacientes presentaron un resultado positivo tras haber sido negativos. CONCLUSIONES: Las solicitudes han ido aumentando en función de la evolución de la epidemia. La RT-PCR posee un elevado rendimiento diagnóstico en las fases de mayor contagiosidad y/o transmisibilidad del virus

INTRODUCTION: Since the discovery of the SARS-CoV-2 virus, the polymerase chain reaction technique (RT-PCR) has become the fundamental method for diagnosing the disease in its acute phase. The objective is to describe the demand-based series of RT-PCR determinations received at a Microbiology Service at a third-level reference hospital for a health area for three months spanning from the onset of the epidemic by SARS-CoV-2. METHODS: A retrospective analysis of the total of the RT-PCR requested in the Microbiology Service analyzed from 02/25/2020 to 05/26/2020 (90 days) has been carried out. They have been grouped by epidemiological weeks and by the petitioner service. A descriptive analysis was carried out by age, gender and number of requests for each patient. In the tests carried out, a confidence level of 95% (p <0.05) was considered significant. RESULTS: A total of 27,106 requests was received corresponding to 22,037 patients. Median age 53.7 (RIC 40.9-71.7) years, women: 61.3%. Proportion of patients with any positive RT-PCR: 14%. Of the total requests for RT-PCR, positive 3,710. Week 13 had the highest diagnosis performance (39.0%). The primary care has been the service thar has made the most requests (15,953). Patients with 3 or more RT-PCR: 565, of them, 19 patients had a positive result after previously having a negative one. CONCLUSIONS: Requests have been increasing depending on the evolution of the epidemic. The RT-PCR has a high diagnostic performance in the phases of highest contagiousness and / or transmissibility of the virus

Smoking and Radiolucent Periapical Lesions in Root Filled Teeth: Systematic Review and Meta-Analysis.

AIM: This systematic review and meta-analysis aimed to investigate the association between smoking habits and the prevalence of radiolucent periapical lesions (RPLs) in root-filled teeth (RFT). METHODS: The Population, Intervention, Comparison, and Outcome (PICO) question was: in adult patients who have RFT, does the absence or presence of a smoking habit affect the prevalence of RPLs associated with RFT? Systematic MEDLINE/PubMed, Wiley Online Database, Web of Science, Scopus, and PRISMA protocol were used to evaluate and present the results. Studies comparing smokers with control non-smoker subjects, including RFT, and providing data on the prevalence of RFT with RPLs, were included. The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system was used for certainty in the evidence. The risk of bias was assessed according to Cochrane Collaboration common scheme for bias and ROBINS-I tool. Cumulative meta-analysis was performed with a random effects model. PROSPERO registration code: CRD42020165279. RESULTS: Four studies reported data on inclusion criteria, representing data from 9257 root-filled teeth-4465 from non-smokers and 4792 from smoker patients. The meta-analysis provided an odds ratio indicating a significant association between smoking and higher prevalence of root filled teeth with radiolucent periapical lesions (OR = 1.16; 95% CI = 1.07-1.26; p = 0.0004). The certainty of the literature assessment was moderate per GRADE. The ROBINS-I tool classified three studies as low risk of bias, and the fourth as moderate risk of bias. CONCLUSIONS: Moderate, quality scientific evidence indicates a weak but significant relationship between smoking and the prevalence of RPLs in RFT. Smoking can be considered a negative prognostic factor for the outcome of root canal treatment. Endodontic providers should be aware of the relationship between smoking and persistent apical periodontitis, assessed as RPLs, in RFT.