High-purity DNA extraction from animal tissue using picking in the TRIzol-based method

TRIzol is used for the extraction of RNA, DNA and proteins from tissues or cells. Here, we present a simple picking method to extract DNA from tissues using TRIzol. Spectrophotometric analysis showed that the 260/280 and 260/230 nm optical density ratio of the picking method's DNA is ideal and better than that obtained by the classic TRIzol method. Gel electrophoresis showed that there was no RNA contamination, and the DNA had not degraded. DNA extracted by the picking method had the same performance in restriction enzyme digestion and quantitative PCR as that obtained by the traditional method. Viral DNA in the infected tissue was also obtained. This modified method facilitates various molecular biology assays.

Antibody Responses to SARS-CoV-2 in Coronavirus Diseases 2019 Patients with Different Severity

Background: More understanding of antibody responses in the SARS-CoV-2 infected population is useful for vaccine development. Aim: To investigate SARS-CoV-2 IgA and IgG among COVID-19 Thai patients with different severity. Methods: We used plasma from 118 adult patients who have confirmed SARS-CoV-2 infection and 49 patients under investigation without infection, 20 patients with other respiratory infections, and 102 healthy controls. Anti-SARS-CoV-2 IgA and IgG were performed by enzyme-linked immunosorbent assay from Euroimmun. The optical density ratio cut off for positive test was 1.1 for IgA and 0.8 for IgG. The association of antibody response with the severity of diseases and the day of symptoms was performed. Results: From Mar 10 to May 31, 2020, 289 participants were enrolled, and 384 samples were analyzed. Patients were categorized by clinical manifestations to mild (n=59), moderate (n=27) and severe (n=32). The overall sensitivity of IgA and IgG from samples collected after day 7 is 87.9% (95% CI 79.8-93.6) and 84.8% (95% CI 76.2-91.3), respectively. The severe group had a significantly higher level of specific IgA and IgG to S1 antigen compared to the mild group. All moderate to severe patients have specific IgG while 20% of the mild group did not have any IgG detected after two weeks. Interestingly, SARS-CoV-2 IgG level was significantly higher in males compared to females among the severe group (p=0.003). Conclusion: The serologic test for SARS-CoV-2 has high sensitivity after the second week after onset of illness. Serological response differs among patients with different severity and different sex.

Azimuthal-rotation sample holder for molecular orientation analysis

In this study, an azimuthal-rotation sample holder compatible with scanning transmission X-ray microscopy was developed. This holder exhibits improvement in the accuracy of rotation angles and reduces the displacement of the rotation axes during azimuthal rotation by using a crossed roller bearing. To evaluate the performance of the holder, the authors investigated the dependence of the optical density around the C K-edge absorption of π-orbital-oriented domains in natural spherical graphite on the rotational angle by using linearly horizontally and vertically polarized undulator radiation. Based on the dependence of the optical density ratio between C 1s → π* and 1s → σ* excitation on the polarization angle of the X-rays, the average two-dimensional orientation angle of the π orbital in each position in a natural spherical graphite sample was visualized.

Clinical Implications of Suspended Scattering Particles in Motion Observed by Optical Coherence Tomography Angiography

The objective of this study was to investigate the relationship between suspended scattering particles in motion (SSPiM) in optical coherence tomography angiography (OCTA) and treatment response in diabetic macular edema (DME). We retrospectively reviewed the medical records of patients diagnosed with DME who had undergone intravitreal injection. The optical density ratio (ODR) of the intraretinal cyst and the numbers of hyperreflective foci from OCT images and SSPiM from OCTA images were compared, and their association with treatment response was analyzed. Forty-five eyes from 45 patients were included in this study. Twenty-four patients were treated with anti-vascular endothelial growth factor, and 21 patients were treated with a steroid. Binary logistic regression model showed that SSPiM in OCTA images was associated with hyperreflective foci numbers (P = 0.038) and mean ODR of the intraretinal cyst (P = 0.006). Linear regression model showed that SSPiM in the inner nuclear layer was related to treatment response (P = 0.006). SSPiM on OCTA images is related to the poor structural response to treatment in DME.

Combination of Nonstructural Protein 1-Based Enzyme-Linked Immunosorbent Assays Can Detect and Distinguish Various Dengue Virus and Zika Virus Infections

ABSTRACTThe recent outbreaks of Zika virus (ZIKV) and associated birth defects in regions of dengue virus (DENV) endemicity emphasize the need for sensitive and specific serodiagnostic tests. We reported previously that enzyme-linked immunosorbent assays (ELISAs) based on the nonstructural protein 1 (NS1) of DENV serotype 1 (DENV1) and ZIKV can distinguish primary DENV1, secondary DENV, and ZIKV infections. Whether ELISAs based on NS1 proteins of other DENV serotypes can discriminate various DENV and ZIKV infections remains unknown. We herein developed DENV2, DENV3, and DENV4 NS1 IgG ELISAs to test convalescent- and postconvalescent-phase samples from reverse transcription-PCR-confirmed cases, including 25 primary DENV1, 24 primary DENV2, 10 primary DENV3, 67 secondary DENV, 36 primary West Nile virus, 38 primary ZIKV, and 35 ZIKV with previous DENV infections as well as 55 flavivirus-naive samples. Each ELISA detected primary DENV infection with a sensitivity of 100% for the same serotype and 23.8% to 100% for different serotypes. IgG ELISA using a mixture of DENV1-4 NS1 proteins detected different primary and secondary DENV infections with a sensitivity of 95.6% and specificity of 89.5%. The ZIKV NS1 IgG ELISA detected ZIKV infection with a sensitivity of 100% and specificity of 82.9%. On the basis of the relative optical density ratio, the combination of DENV1-4 and ZIKV NS1 IgG ELISAs distinguished ZIKV with previous DENV and secondary DENV infections with a sensitivity of 91.7% to 94.1% and specificity of 87.0% to 95.0%. These findings have important applications to serodiagnosis, serosurveillance, and monitoring of both DENV and ZIKV infections in regions of endemicity.

Bulk tank milk ELISA to detect IgG1 prevalence and clustering to determine spatial distribution and risk factors of Fasciola hepatica-infected herds in Mexico

Fasciola hepatica is a helminth parasite that causes huge economic losses to the livestock industry worldwide. Fasciolosis is an emerging foodborne zoonotic disease that affects both humans and grazing animals. This study investigated the associations between climatic/environmental factors (derived from satellite data) and management factors affecting the spatial distribution of this liver fluke in cattle herds across different climate zones in three Mexican states. A bulk-tank milk (BTM) IgG1 enzyme-linked immunosorbent assay (ELISA) test was used to detect F. hepatica infection levels of 717 cattle herds between January and April 2015. Management data were collected from the farms by questionnaire. The parasite's overall herd prevalence and mean optical density ratio (ODR) were 62.76% and 0.67, respectively. The presence of clustered F. hepatica infections was studied using the spatial scan statistic. Three marked clusters in the spatial distribution of the parasite were observed. Logistic regression was used to test three models of potential statistical association from the ELISA results using climatic, environmental and management variables. The final model based on climatic/environmental and management variables included the following factors: rainfall, elevation, proportion of grazed grass in the diet, contact with other herds, herd size, parasite control use and education level as significant predictors. Geostatistical kriging was applied to generate a risk map for the presence of parasites in dairy herds in Mexico. In conclusion, the spatial distribution of F. hepatica in Mexican cattle herds is influenced by multifactorial effects and should be considered in developing regionally adapted control measures.

Optical Density of Subretinal Fluid as a Predictive Biomarker for Eyes With Chronic Central Serous Retinopathy Treated With Eplerenone

Purpose: To investigate how the optical density ratio (ODR) of subretinal fluid (SRF) obtained by optical coherence tomography (OCT) in eyes with chronic central serous retinopathy (CSR), defined as symptoms >3 months, treated with eplerenone correlates with treatment response and visual outcomes. Methods: This retrospective, single-center observational study included patients with chronic CSR treated with eplerenone who had at least 6 weeks of follow-up. Eyes with poor-quality OCT scans, shallow SRF precluding sampling of optical density (OD), and macular pathology other than CSR were excluded. Optical density of the vitreous and SRF was measured using ImageJ and used to calculate ODR (ODR = ODSRF/ODVIT). Peak SRF height, foveal SRF height, central macular thickness (CMT), and Snellen visual acuity (VA) were measured. The Mann-Whitney U test was used to compare continuous variables. Pearson correlation coefficient was used to determine the statistical significance of applied linear regressions. Results: Twenty-three eyes of 23 patients met the inclusion criteria. Six (26%) eyes had complete resolution of foveal SRF (resolvers) with eplerenone treatment, and the remainder of patients had worsening or incomplete resolution (nonresolvers). Resolvers had significantly lower baseline logarithm of the minimum angle of resolution (logMAR) VA ( P < .01) than nonresolvers. There was a positive linear correlation between ODR and logMAR VA at baseline ( R2 = 0.208; P = .03) and follow-up ( R2 = 0.206; P = .03). A significant correlation between ODR and percentage change in CMT was found ( R2 = 0.263; P = .01). Linear correlations were not found between ODR and age, peak SRF height, and foveal SRF height (all P > .125). Conclusion: Lower baseline ODR was associated with improved percentage change in CMT and VA in patients treated with eplerenone for chronic CSR. Optical density ratio may represent a novel prognostic biomarker that may help determine which eyes with chronic CSR may respond better to eplerenone.

Modelling the spatial distribution of Fasciola hepatica in dairy cattle in Europe

A harmonized sampling approach in combination with spatial modelling is required to update current knowledge of fasciolosis in dairy cattle in Europe. Within the scope of the EU project GLOWORM, samples from 3,359 randomly selected farms in 849 municipalities in Belgium, Germany, Ireland, Poland and Sweden were collected and their infection status assessed using an indirect bulk tank milk (BTM) enzyme-linked immunosorbent assay (ELISA). Dairy farms were considered exposed when the optical density ratio (ODR) exceeded the 0.3 cut-off. Two ensemble-modelling techniques, Random Forests (RF) and Boosted Regression Trees (BRT), were used to obtain the spatial distribution of the probability of exposure to <em>Fasciola hepatica</em> using remotely sensed environmental variables (1-km spatial resolution) and interpolated values from meteorological stations as predictors. The median ODRs amounted to 0.31, 0.12, 0.54, 0.25 and 0.44 for Belgium, Germany, Ireland, Poland and southern Sweden, respectively. Using the 0.3 threshold, 571 municipalities were categorized as positive and 429 as negative. RF was seen as capable of predicting the spatial distribution of exposure with an area under the receiver operation characteristic (ROC) curve (AUC) of 0.83 (0.96 for BRT). Both models identified rainfall and temperature as the most important factors for probability of exposure. Areas of high and low exposure were identified by both models, with BRT better at discriminating between low-probability and high-probability exposure; this model may therefore be more useful in practise. Given a harmonized sampling strategy, it should be possible to generate robust spatial models for fasciolosis in dairy cattle in Europe to be used as input for temporal models and for the detection of deviations in baseline probability. Further research is required for model output in areas outside the eco-climatic range investigated.