Direct Fluorescent Assay Research Articles

Survival outcomes correlate with the level of cell-free circulating DNA in ST-elevation myocardial infarction.

Myocardial infarction (MI) can lead to higher cellular damage, making cell-free DNA (cfDNA) a potential biomarker for assessing disease severity. The aim of this study is to evaluate survival predictions using cfDNA measurements and assess its correlation with MI. A direct fluorescence assay was employed to measure cfDNA content in the blood samples of participants. The inclusion criteria included patients who gave informed consent, suffering from ST-elevation myocardial infraction (STEMI) based on established diagnostic criteria (joint ESC/ACC guidelines), between the age of 18 and 80 years old, and had elevated troponin biomarker levels. The study included 150 patients diagnosed with STEMI and 50 healthy volunteers as controls. Serial monitoring of patients was conducted to track their postdisease status. The rate of change of cfDNA was calculated and daily measurements for 7 days were recorded. Mean levels of cfDNA were found to be 5.93 times higher in patients with STEMI compared to healthy controls, providing clear evidence of a clinical correlation between cfDNA and STEMI. Patients were further categorized based on their survival status within a 90-day period. The study observed a strong predictive relationship between the rate of change of cfDNA during daily measurements and survival outcomes. To assess its predictive capability, a receiver operating characteristics (ROC) curve analysis was performed. The ROC analysis identified an optimal cutoff value of 2.50 for cfDNA, with a sensitivity of 81.5% and specificity of 74.0% in predicting disease outcomes. This study demonstrates a robust association between cfDNA and STEMI, indicating that cfDNA levels can be a valuable early prognostic factor for patients. Serial measurements of cfDNA during early disease onset hold promise as an effective approach for predicting survival outcomes in MI patients.

Preliminary application of recombinase -aided amplification in detection of Clonorchis sinensis metacercariae in freshwater fish

To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ2 = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ2 = 11.20, P < 0.05) and fluorescent RAA assay (χ2 = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.

Circulating DNA and frequency of colorectal cancer brain metastases in a presumed high-risk group

This explorative prospective observational pilot study investigated if suggested risk factors, rectal cancer and lung metastases, could add to a relevant detection rate of asymptomatic brain metastases (BM) from colorectal cancer (CRC). Secondary, prognostic biological aspects were investigated by translational analysis of plasma samples. The study enrolled patients with rectal cancer and lung metastases. At inclusion, patients underwent a standard MRI scan of the brain. Cell-free DNA (cfDNA) level was measured by a direct fluorescence assay (DFA), and circulating tumor DNA (ctDNA) by ddPCR. BM was detected in one of twenty-nine included patients. Patients had higher cfDNA levels than healthy subjects (p < 0.01). Patients with the primary tumor in situ had higher cfDNA levels than those with resected primary tumor (p < 0.01). Patients with liver involvement had higher cfDNA levels (p = 0.12) and circulating tumor DNA levels (p = 0.01) than those without liver involvement. In conclusion, the modest incidence of BM does not justify routine MRI of the brain in this selected population. cfDNA by DFA could be a valuable tool when planning treatment and follow-up for CRC patients. Future studies should focus on identifying further characteristics and biomarkers associated with a high risk of BM, enhancing the possibility for early intervention.

Comparison of 3 Diagnostic Tests for the Detection of Giardia and Cryptosporidium spp. in Asymptomatic Dogs (Canis lupis familiaris).

After detecting Giardia and Cryptosporidium infections and coinfections in 2 litters of puppies in our vivarium, our team realized that we needed a simple, quick, and economical point-of-care test for concurrent screening of asymptomatic dogs for both organisms. Periodic screening of colony dogs and of all dogs introduced into a colony can prevent the spread of Giardia and Cryptosporidium to immunologically naïve animals and help keep staff safe from these zoonotic organisms. To compare methods for diagnosing Giardia and Cryptosporidium spp. in dogs, we used a convenience sampling of feces from 2 populations of dogs; samples were tested with a lateral-flow assay (QC), a commercially available direct fluorescent assay (DFA), and an inhouse PCR test using established primers. QC results were analyzed in 2 ways: 1) relative to a reference standard that permitted comparative interpretation of DFA and PCR results; and 2) using Bayesian analysis for comparison independent of a reference standard. The QC test showed good specificity for the detection of Giardia according to both the reference standard (95%) and the Bayesian analysis (98%). Similarly, specificity of the QC for the detection of Cryptosporidium was 95% according to the reference standard and 97% according to Bayesian analysis. However, the sensitivity of the QC test was much lower for both Giardia (reference standard, 38%; Bayesian analysis, 48%) and Cryptosporidium (25% and 40%, respectively). This study demonstrates that the QC test can be used to detect both Giardia and Cryptosporidium in dogs and that positive results can be accepted with confidence, whereas negative tests should be confirmed through secondary testing methods.

Detection of human Metapneumovirus and respiratory Syncytial Virus associated with asthmatic patients using direct fluorescent assay and Real time – PCR

This study aimed to determine the incidence of human respiratory syncytial virus (hRSV) and metapneumovirus (hMPV) occurrence in asthmatic patients in Wasit province. Blood samples were collected for measure the total and differential white blood cells count (WBCs). Correlation results of total and differential WBCs count for neutrophils, lymphocytes, eosinophils and basophils count among studied groups were significant (P &lt;0.001). Enzyme linked immunosorbent assay (ELISA) technique has been applied for detection of total-IgE antibodies. Results revealed that the highest total-IgE antibodies titer in sera were significantly difference (p &lt; 0.01). A total of 80 nasopharyngeal swabs were immediately dipped in transport media and stored until using for the detection of suspected hRSV and hMPV patients group by direct fluorescent assay the results appeared 13 (16.25 %) and 11 (13.75 %) samples were given positive results for hRSV and hMPV, respectively. Results of hRSV in asthmatic patients were subjected to real time – polymerase chain reaction (RT-PCR) appeared 15 samples out of 80 samples (18.75 %) were gave positive result for this test.

Retrospective comparison of respiratory syncytial virus and metapneumovirus clinical presentation in hospitalized children.

Respiratory syncytial virus (RSV) and Human metapneumovirus (hMPV), members of Pneumoviridae family are common causes of acute respiratory tract infections (ARTI) among children. Study material includes routine nasopharyngeal samples obtained during 8-year period for hMPV and one single season for RSV in children hospitalized for ARTI between 0 and 15 years at the Center Hospitalier Universitaire (CHU) Saint Pierre in Brussels. Positive samples for RSV or hMPV identified by viral culture, lateral flow chromatography test for RSV or direct fluorescent assay for hMPV were selected retrospectively. Characteristics of children hospitalized for RSV or hMPV infections were compared. Children hospitalized for RSV infection were significantly younger and requiring more respiratory support, longer hospital stay and transfers in Pediatric intensive Care Unitsthan those hospitalized for hMPV infection. Pneumonia diagnostic and antibiotics therapies were more significantly associated with hMPV infections.In conclusion, despite their genetic similarities, RSV, and hMPV present epidemiological and clinical differences in pediatric infections. Our results should be confirmed prospectively.

Circulating cell-free DNA as predictor of pathological complete response in locally advanced rectal cancer patients undergoing preoperative chemoradiotherapy

BackgroundThe watch and wait (W&W) strategy is proposed for patients with locally advanced rectal cancer (LARC) achieving clinical complete response (cCR) after neoadjuvant radiotherapy. cCR is only in partial concordance with pathological complete response (pCR) due to persisting viable tumour cells. The aim was to investigate circulating-free-deoxyribonucleic-acid (cfDNA) as a biomarker for prediction of pCR. Materials and methodsPatients treated with neoadjuvant radiotherapy for LARC, were included in a prospective biomarker study in Aarhus, Denmark from 2017 to 2020. Plasma cfDNA levels were analysed by a direct fluorescent assay (DFA). Surgical specimens were reviewed by pathologists to categorize response to cytotoxic therapy. ResultsIn total, 76 patients were included with plasma available at baseline (n = 70), mid therapy (n = 50), and end of therapy (n = 54). Higher cfDNA levels were observed in LARC patients compared with healthy subjects (p < 0.01). By ROC analysis (AUC: 0.87 (95% CI, 0.81–0.92)) the optimal cut-off was 0.71 ng/µL for differentiation between healthy subjects and LARC patients. Thirteen patients obtained pCR with a median cfDNA level of 0.57 ng/µL at end of therapy. Patients with cfDNA levels at end of therapy below the cut-off (p < 0.02) and ‘cfDNA responders’ with descending levels greater than the 75th percentile during therapy had a significantly higher chance of pCR (p < 0.01). ConclusionThis hypothesis generating study indicates that low cfDNA levels at end of treatment or ́cfDNA responderś might be associated with pCR. Quantification of cfDNA by the rapid and feasible DFA analysis could potentially facilitate personalized follow-up as a complementary tool to identify candidates for a W&W strategy.

MCL-1 interacts with MOF and BID to regulate H4K16 acetylation and homologous recombination repair.

The Participation ofmyeloid cell leukemia-1 (MCL-1), an antiapoptotic protein, in DNA repair and homologous recombination (HR) is not well understood. This study tests whether MCL-1 interacts with Males absent On First (MOF) to regulate H4K16 acetylation that promotes HR repair in response to replication stress induced by Hydroxyurea (HU) treatment. Co-immunoprecipitationof FLAG-MCL-1 from cancer cells treated with HU pulls down a complex of MCL-1, MOF and BH3-interacting domain death agonist (BID). The same complex is pulled down in cells treated with HU that express FLAG-MOF. MCL-1 regulates H4K16 acetylation during HU-induced replication stress since knockdown of MCL-1 decreases H4K16 acetylation while re-expression of MCL-1 restores H4K16 acetylation. Furthermore, knockdown of BID rescues the clonogenic survival in MCL-1 depleted cells in response to replication stress which is associated with decreased Caspase 3/7 activity compared to MCL-1 depleted cells. Cells depleted in both MCL-1 and BID display increased HR repair efficiency by direct repeats-green fluorescent protein assay and in response to HU exhibit increased ATR, Chk1, and RPA phosphorylation relative to MCL-1 depleted cells. This study uncovers that MCL-1 cooperates with MOF and regulates HR repair through H4K16 acetylation. Further, this study determines that MCL-1 and BID cooperate to regulate the crosstalk between HR repair and apoptosis.

Fluorescent enzymatic assay for direct total polyphenol determination in food-related samples

A direct and simple fluorescent assay for the total polyphenol determination based on the bioconjugate formed between the laccase enzyme (TvL from Trametes versicolor) and carbon nanodots (CD) is developed. One of the most used reactions for the determination of phenols is based on the enzymatic reaction of their oxidation to quinones. In this work, CD has been biofunctionalized with TvL (TvL-CD) and employed as a fluorescent label to follow the enzymatic reaction. The bioconjugate was formed and characterized by spectroscopy and microscopy. The optimal TvL-CD ratio was established. The reaction between the bioconjugate and a phenolic compound such as gallic acid (GA) was followed by monitoring the fluorescence bioconjugate decrease due to the quenching effect of the quinones generated in the enzymatic reaction. These studies confirm that bioconjugation does not inhibit the enzymatic activity and the fluorescence decrease during the enzymatic reaction is mainly due to an electron transfer processes. Based on these results, a new method for the quantitative determination of polyphenols measured as GA concentration is developed. The detection and quantification limit was found to be 7.4 and 25 μM, respectively. Subsequently, the method has been applied to the direct determination of GA in wine, juice, and rice leaf extracts.

Total cell-free DNA measurement in metastatic colorectal cancer with a fast and easy direct fluorescent assay.

Treatment for metastatic colorectal cancer (mCRC) is focused on prolonging survival and maintaining quality of life. It is important to establish prognostic and predictive markers to avoid extended, ineffective treatment. The aim of the present study was, by a novel approach, to analyze the association between cell-free (cf)DNA levels and outcome in patients receiving systemic therapy for incurable mCRC. The study was a prospective non-interventional biomarker study for patients receiving standard of systemic treatment for mCRC. Patients with mCRC, who, according to standard guidelines, were considered for treatment with EGFR inhibitors, were included. The cfDNA levels in consecutive plasma samples were measured by a direct fluorescence assay. The study included 47 patients. Blood samples were available at baseline (n=47); prior to the third treatment cycle (n=31); the first (n=33), second (n=22) and third response evaluation during treatment (n=17); and at progression (n=22). The disease control rate was 42 and 91% in patients with high (≥75th percentile of baseline cfDNA levels) and low cfDNA levels (<75th percentile of baseline cfDNA levels), respectively (P<0.001). Median progression-free survival (PFS) was 3.8 and 8.5 months in patients with high and low cfDNA levels, respectively (hazard ratio=3.03, 95% CI 1.46-6.29, P<0.01). Median overall survival (OS) was 5.0 and 26.6 months in patients with high and low cfDNA levels, respectively (hazard ratio=3.48, 95% CI 1.44-8.44, P<0.01). In the multivariate analysis, baseline cfDNA levels remained a significant predictor of PFS and OS. In conclusion, cfDNA is a promising prognostic tool in the personalized treatment of mCRC. cfDNA levels were estimated by a simple, rapid and inexpensive method (OPTIPAL II: ClinicalTrials.gov identifier no. NCT03750175; registered November 21, 2018).

Pharmacodynamic Studies of Fluorescent Diamond Carriers of Doxorubicin in Liver Cancer Cells and Colorectal Cancer Organoids.

BackgroundWe recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs.PurposeTo generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer.MethodsFDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions.ResultsFDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence.ConclusionThe rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.

Verification of performance of a direct fluorescent assay for cell-free DNA quantification, stability according to pre-analytical storage conditions, and the effect of freeze-thawing.

A simple fluorescence-based cell-free DNA (CFD) assay has been previously developed that can directly measure nucleic acids without prior DNA extraction and amplification. However, studies on fluorescence-based CFD are lacking. In particular, there is no known information regarding the stability with regard to pre-analytical storage conditions in relation to time and temperature, or on the influence of freeze-thawing. Plasma was directly assayed to measure CFD using PicoGreen™ reagent. Standard linearity and accuracy were confirmed using salmon sperm DNA. Whole blood was left at room temperature (RT) and at 4˚C, and then plasma was separated. The CFD was also measured using thawed plasma after 1 week of freezing. As a correlation with a sperm DNA concentration, CFD demonstrated linearity over a wide range of concentrations, with a 0.998 correlation coefficient. The CFD level showed a change of up to 2.5 µg/ml according to pre-analytical storage time, and the changes were not consistent over time. The CFD values at RT after 1 h were similar to the baseline values, and the relative standard deviation was lowest under this condition. The CFD values between 4˚C and RT were similar over all time periods assessed. After freeze-thawing, the change in CFD value was reduced compared to that before freezing. The present study showed that CFD measurements using plasma processed within 1 h were optimal. Additionally, the effects of substantial changes according to storage conditions were reduced after freeze-thawing, and thus studies using stored samples is viable and relevant.

Clinical Characteristics and Outcomes of COVID-19 in HSCT Recipients

Introduction. COVID-19 pandemic has spread worldwide and reached HSCT centers in many countries. The first proven case of COVID-19 in Brazil was diagnosed in São Paulo by the end of February 2020. The Brazilian Society of Marrow Transplantation (SBTMO) promptly published local recommendations for the diagnosis and management of COVID-19 in HSCT centers. We describe the main clinical findings and outcomes of SARS CoV-2 infection and COVID-19 in 22 HSCT recipients from two Brazilian HSCT centers, followed up for at least 14 days. Methods. The detection of SARS CoV-2 infection was performed by RT-PCR in all transplant candidates before admission, and in all HSCT recipients with respiratory symptoms. Other respiratory viruses were also investigated by direct fluorescent assay (DFA) or RT-PCR. COVID-19 was managed according to the SBTMO recommendations. Results. From March to June 2020, SARS CoV-2 infection was detected in 5 asymptomatic patients (29.4%) and COVID-19 was diagnosed in 17 HSCT recipients with respiratory symptoms (7 AUTO, 6 MRD, 5 MUD and 4 HAPLO) at a median of 94 days after HSCT (range -6 to +1,850). The asymptomatic cases at diagnosis remained so during follow-up. Two asymptomatic patients were receiving the conditioning regimen when SARS CoV-2 was diagnosed. The median age at diagnosis was 39,2 (1-72) years. Five patients were children (1-12 years) and 17 were adults (19-72 years). Eleven patients (50%) had comorbidities, the most frequent being diabetes and hypertension. Fever, cough and diarrhea were the symptoms more frequently observed and occurred in 13 (76.4%), 7 (41.2%), and 5 (29.4%) of the 17 symptomatic patients, respectively. Symptoms such as anosmia and dysgeusia, which have been frequently associated with COVID-19 were not observed in this series. Eleven of the symptomatic patients (64.7%) showed altered CT or X-ray at diagnosis. Glass ground opacities were the images more frequently seen (63.6%). The median lymphocyte count was 619.5 (0-2,604) /mL. C-reactive protein and d-dimer ranged from 0.65 to 339 ng/mL and from 0.39 to 4,530 mg/L, respectively. Four (18%) HSCT recipients died (1 AUTO, 2 HAPLO, 1 MRD). COVID-19 was the cause of death in two of them. In the remaining two HAPLO HSCT recipients, KPC co-infection followed by septic shock had a major role in patients’ death. Median age of patients who died was 39 (11-59) years, and two of them had comorbidities (hypertension and chronic lung disease). Conclusions. Symptoms of COVID-19 mimic other respiratory virus infections and specific diagnosis is mandatory. Interstitial pneumonia was frequent (around 65%). Clinical outcome of SARS Cov-2 infection or COVID-19 was moderate in the majority of the patients in the present series. However, the mortality rates observed in HSCT recipients (14.3% in AUTO; 20% in ALLO) were higher than in general population. Asymptomatic cases were seen in almost 30% of the patients, suggesting that frequent screening is necessary to control transmission in HSCT centers.DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Leishmanization Using Iranian Lizard Leishmania Mixed With CpG-ODN as a Candidate Vaccine Against Experimental Murine Leishmaniasis.

Background and Objectives: The live non-pathogenic Leishmania tarantolae has recently provided a promising approach as an effective vaccine candidate against experimental leishmaniasis (ILL). Here, we evaluated the immunoprotective potential of the live Iranian Lizard Leishmania mixed with CpG adjuvant against L. major infection in BALB/c mice.Methods: Four groups of female BALB/c mice were included in the study. The first and second groups received PBS and CpG, respectively. The immunized groups received 2 × 105 ILL promastigotes and the CpG-mixed ILL (ILL+CpG). Injections were performed subcutaneously in the right footpad. Three weeks later, all mice were challenged with 2 × 105 metacyclic promastigotes of Leishmania majorEGFP; inoculation was done in the left footpad. The measurement of footpad swelling and in vivo fluorescent imaging were used to evaluate disease progress during infection course. Eight weeks after challenge, all mice were sacrificed and the cytokines levels (IFN-γ, IL-4, and IL-10) and sera antibodies concentrations (IgG2a and IgG1) using ELISA assay, nitric oxide production using Griess assay, and arginase activity in cultured splenocytes, were measured. In addition, direct fluorescent microscopy analysis and qPCR assay were used to quantify the splenic parasite burden.Result: The results showed that mice immunized with ILL+CpG were protected against the development of the dermal lesion. Moreover, they showed a significant reduction in the parasite load, in comparison to the control groups. The observed protection was associated with higher production of IFN-γ, as well as a reduction in IL-4 level. Additionally, the results demonstrated that arginase activity was decreased in ILL+CpG group compared to other groups.Conclusion: Immunization using ILL+CpG induces a protective immunity; indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.

Development and Evaluation of a Fully Automated Molecular Assay Targeting the Mitochondrial Small Subunit rRNA Gene for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid Specimens

The fungal pathogen Pneumocystis jirovecii causes Pneumocystis pneumonia. Although the mitochondrial large subunit rRNA gene (mtLSU) is commonly used as a PCR target, a mitochondrial small subunit rRNA gene (mtSSU)-targeted MultiCode PCR assay was developed on the fully automated ARIES platform for detection of P. jirovecii in bronchoalveolar lavage fluid specimens in 2.5hours. The assay showed a limit of detection of 800 copies/mL (approximately equal to 22 organisms/mL), with no cross-reactivity with other respiratory pathogens. Compared with the reference Pneumocystis-specific direct fluorescent antibody assay (DFA) and mtLSU-targeted PCR assay, the new assay demonstrated sensitivity of 96.9% (31/32) and specificity of 94.6% (139/147) in detecting P.jirovecii in 180 clinical bronchoalveolar lavage fluid specimens. This assay was concordant with all DFA-positive samples and all but one mtLSU PCR-positive sample, and detected eight positive samples that were negative by DFA and mtLSU PCR. Receiver operating characteristic curve analysis revealed an area under the curve of 0.98 and a threshold cycle (CT) cutoff of 39.1 with sensitivity of 90.9% and specificity of 99.3%. The detection of 39.1 <CT<40.0 indicates the presence of a low load of the organism and needs further determination of either colonization or probable/possible Pneumocystis pneumonia. Overall, the new assay demonstrates excellent analytical and clinical performance and may be more sensitive than mtLSU PCR target for the detection of P. jirovecii.

Prognostic and predictive value of circulating DNA for hepatic arterial infusion of chemotherapy for patients with colorectal cancer liver metastases.

Hepatic arterial infusion (HAI) of chemotherapy is an experimental treatment option for patients with colorectal cancer liver metastases (CRCLM). The current study aimed to investigate the predictive and prognostic value of cell free DNA (cfDNA) in patients with CRCLM receiving HAI with oxaliplatin and systemic capecitabine. Plasma samples from 62 patients were investigated who were included into a single arm phase II study investigating HAI treatment for patients with CRCLM. The clinical outcome of the trial has been presented previously. In brief, treatment consisted of intrahepatic infusion of oxaliplatin 100 mg/m2 every second week with concomitant oral capecitabine 3,500 mg/m2 every second week for up to 12 cycles. Blood samples were drawn at baseline and follow-up and plasma was analyzed for cell free DNA using a direct fluorescent assay. The baseline level of plasma cfDNA was 0.92 ng/µl (95% CI 0.84-1.00). Patients with a baseline value of cfDNA above the 75th quartile had a median overall survival of 2.4 years (95% CI 0.7-2.8), compared with 3.9 years (95% CI 2.8-5.9) for patients below the 75th quartile (P=0.02). The baseline level of cfDNA was significantly lower (0.91 ng/µl, 95% CI 0.76-0.98) in patients who achieved an objective response compared to non-responders (1.79 ng/µl; 95% CI 0.99-2.57; P=0.02). The current study demonstrated a possible prognostic and predictive value of cfDNA for patients with CRCLM undergoing HAI with oxaliplatin and concomitant capecitabine.

Measurement of circulating free DNA in squamous cell carcinoma of the anus and relation to risk factors and recurrence

BackgroundMeasuring circulating-free-deoxyribonucleic-acid (cfDNA) has created a new framework for personalized treatment in oncology. The aim of this study was to analyze the relation between cfDNA and risk factors and outcome in squamous cell carcinoma of the anus (SCCA). MethodsPatients treated with radiotherapy for localized SCCA were included in Aarhus, Denmark from 2016 to 2019. Serum samples from baseline, during and after therapy, were measured for the level of cfDNA in ng per µL by a direct fluorescent assay. ResultsEighty patients were included. Samples were available at baseline (n = 73) mid-therapy (n = 74), end-therapy (n = 67) and one-year follow-up (1Y) (n = 29). P16-positivity was found in 89% (n = 55). The median level of cfDNA was higher for P16 negative tumors (1.48) compared with the P16 positive tumors (0.90, P = 0.04). Data showed a correlation between baseline cfDNA levels and Gross Tumor Volume (R2 = 0.13, P < 0.01), and increasing levels with increasing T-stage (T1 = 0.80, T2 = 0.94, T3 = 1.11, T4 = 1.3). Higher cfDNA levels were observed in patients with poor performance status (P < 0.01). The cfDNA level decreased from baseline to mid-therapy (0.92–0.78, P < 0.01) and from baseline to 1Y (0.92–0.71, P < 0.01). Baseline levels for patients with treatment failure (n = 8) were above the 25th percentile (p = 0.05) which translates into difference in disease free survival. ConclusionResults indicate an association between baseline cfDNA levels and risk factors in SCCA and a low baseline level correlates to lower risk of treatment failure. Findings contribute with new knowledge of the biological role of cfDNA in SCCA and holds potential knowledge for personalized treatment of SCCA.

Molecular evidence of bovine herpesvirus 1 and 5 in cattle with suspected rabies in Rio Grande do Sul state, Brazil

Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples.

In silico maturation of affinity and selectivity of DNA aptamers against aflatoxin B1 for biosensor development

A high affinity and selectivity DNA aptamer for aflatoxin B1 (AFB1) was designed through Genetic Algorithm (GA) based in silico maturation (ISM) strategy. The sequence of a known AFB1 aptamer (Patent: PCT/CA2010/001292, Apt1) applied as a probe in many aptasensors was modified using seven GA rounds to generate an initial library and three different generations of ss DNA oligonucleotides as new candidate aptamers. Molecular docking methodology was used to screen and analyze the best aptamer–AFB1 complexes. Also, a new pipeline was proposed to faithfully predict the tertiary structure of all single stranded DNA sequences. By the second generation, aptamer Apt1 sequence was optimized in the local search space and five aptamers including F20, g12, C52, C32 and H1 were identified as the best aptamers for AFB1. The selected aptamers were applied as probes in an unmodified gold nanoparticles-based aptasensor to evaluate their binding affinity to AFB1 and their selectivity against other mycotoxins (aflatoxins B2, G1, G2, M1, ochratoxin A and zearalenone). In addition, a novel direct fluorescent anisotropy aptamer assay was developed to confirm the binding interaction of the selected aptamers over AFB1. The ISM allowed the identification of an aptamer, F20, with up to 9.4 and 2 fold improvement in affinity and selectivity compared to the parent aptamer, respectively.

Nanobody\u2011horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

BackgroundAntibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV.ResultsIn the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni–NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests.ConclusionsFor the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed immunoassays eliminate the use of commercial secondary antibodies and shorten detection time. Meanwhile, both assays display great developmental prospect for further commercial production and application.