TaqMan RT-qPCR Research Articles

Development and evaluation of polyclonal antibodies based antigen capture ELISA for detection of porcine rotavirus

Rotaviruses are rising as zoonotic viruses worldwide, causing the lethal dehydrating diarrhea in children, piglets, and other livestock of economic importance. A simple, swift, cost-effective, highly specific, and sensitive antigen-capture enzyme–linked immunosorbent assay (AC-ELISA) was developed for detection of porcine rotavirus-A (PoRVA) by employing rabbit (capture antibody) and murine polyclonal antibodies (detector antibody) produced against VP6 of PoRVA (RVA/Pig-tc/CHN/TM-a/2009/G9P23). Reactivity of the both polyclonal antibodies was confirmed by using an indirect ELISA, western-blot analysis and indirect fluorescence assay against rVP6 protein and PoRVA. The detection limit of AC-ELISA was found 50 ng/ml of PoRVA protein. The relative sensitivity and specificity of this in-house AC-ELISA were evaluated for detection of PoRVA from 295 porcine diarrhea samples, and results were compared with that of RT-PCR and TaqMan RT-qPCR. The relative sensitivity and specificity of AC-ELISA compared with those of TaqMan RT-qPCR were found as 94.4 and 99.2%, respectively, with the strong agreement (κ − 0.58) between these two techniques. Furthermore, AC-ELISA could not detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pseudo rabies virus and porcine circovirus-2. This in-house AC-ELISA efficiently detected PoRVA from clinical samples, which suggests that this technique can be used for large-scale surveillance and timely detection of rotavirus infection in the porcine farms.

Novel duplex RT-qPCR for animal rabies surveillance.

Rabies is a lethal zoonosis affecting mammals worldwide. Diagnosis of rabies follows international standard protocols, primarily relying on direct immunofluorescence (DI) followed by mouse inoculation test (MIT). WHO recommends molecular biology techniques such as RT-qPCR for replacing MIT to diagnose rabies in animal samples. Recently, a real-time PCR protocol that detects all rabies virus variants identified worldwide was validated. This assay is a pan-Lyssavirus TaqMan quantitative RT-PCR called LN34. A modified LN34 assay protocol was tested at the Paraná State Reference Laboratory (Lacen/PR) using animal samples previously tested by DI and MIT, the gold standard (GS). This method has been changed to a RT-qPCR duplex format to better fit the diagnostic routine. The new assay was called duplex LN34 and β-actin RT-qPCR. All the 88 samples evaluated using the GS test, modified pan-Lyssavirus TaqMan RT-qPCR and duplex LN34 and β-actin RT-qPCR showed 100% agreement with each other. This novel duplex RT-qPCR protocol has shown adequate diagnostic performance and may be used in research and surveillance purposes, replacing the standard MIT and ending mice use for rabies diagnosis.

Rapid Differential Detection of Japanese Encephalitis Virus and Getah Virus in Pigs or Mosquitos by a Duplex TaqMan Real-Time RT-PCR Assay.

Both JEV (Japanese encephalitis virus) and GETV (Getah virus) pose huge threats to the safety of animals and public health. Pigs and mosquitoes play a primary role in JEV and GETV transmission. However, there is no way to quickly distinguish between JEV and GETV. In this study, we established a one-step duplex TaqMan RT-qPCR for rapid identification and detection of JEV and GETV. Primers and probes located in the NS1 gene of JEV and the E2 gene of GETV that could specifically distinguish JEV from GETV were selected for duplex TaqMan RT-qPCR. In duplex real-time RT-qPCR detection, the correlation coefficients (R2) of the two viruses were higher than 0.999. The RT-qPCR assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. Detection of JEV and GETV in field mosquito and pig samples was 100 times and 10 times more sensitive than using traditional PCR, respectively. In addition, the new test took less time and could be completed in under an hour. Clinical sample testing revealed the prevalence of JEV and GETV in mosquitoes and pig herds in China. This complete duplex TaqMan RT-qPCR assay provided a fast, efficient, specific, and sensitive tool for the detection and differentiation of JEV and GETV.

Establishment of a duplex TaqMan RT-PCR for the differential detection of RHDV GI.1 and GI.2

BackgroundRabbit haemorrhagic disease (RHD) is a highly contagious and acute fatal hepatitis of the European rabbit (Oryctolagus cuniculus), caused by a calicivirus (genus Lagovirus). Up to 2010, all RHD viruses (RHDV) isolated belonged to one genotype. In 2010, a new genotype of RHDV (RHDV2/b, currently designated GI.2 based on phylogenetic analysis) emerged in France. The aim of this study was to develop a rapid, simple, specific and sensitive TaqMan real-time PCR assay for the classic strain of RHDV and RHDV2 detection. Specific primers and probes were designed for the VP60 gene of RHDV and RHDV2 within the conserved region of viral genome. ResultsThis study was demonstrated to be highly specific for RHDV and RHDV2, without cross-reactions with other non-targeted viruses. The detection limit of this work was 102 copies of RHDV and RHDV2, respectively. The coefficient of variation of the assay was less than 5% for both intra-assay and inter-assay. The reproducibility of method was assessed using plasmids and the coefficient of variation obtained was 0.2–3.70. Of 79 clinical samples, 68 were positive samples (86.08%), of which 60 were classic RHDV variants (75.9%), 4 were co-infected (5.06%) and 8 were RHDV2 (10.12%), those results are more sensitivity compare with conventional RT-PCR RT-PCR. ConclusionsIn conclusion, this duplex TaqMan RT-qPCR based on VP60 gene of RHDV and RHDV2 could be a valuable tool in diagnose and molecular epidemiological study of the RHDV and RHDV2.

Investigation on Natural Infection of Covert Mortality Nodavirus in Large Yellow Croaker, Larimichthys crocea

Covert mortality nodavirus (CMNV) recently became more prevalent, severely affecting farmed shrimps, and also other invertebrates and teleost fish, in the coastal areas of China. For better understanding of its prevalence and susceptibility of large yellow croaker Larimichthys crocea to CMNV, an epidemiological survey was carried out in the main producing areas (Ningbo, Zhejiang, China, and Ningde, Fujian, China) and its offshore feeding grounds in the Southern Yellow Sea. The collected cultured L. crocea individuals showed clinical symptoms such as anorexia and abnormal swimming and diagnosed for CMNV infection by using RT-nPCR assay. The positive rates for CMNV in the collected L. crocea samples from Ningbo, Ningde, and the Yellow Sea were 14.29% (4/28), 20.00% (7/35), and 16.67% (1/6), respectively. Furthermore, CMNV diagnosis using TaqMan probe-based reverse transcription quantitative PCR (TaqMan RT-qPCR) yielded positive rates of 25.00% (7/28), 22.86% (8/35), and 16.67% (1/6) respectively in the above-mentioned samples, and with a range of 12.73–3,108.33 of CMNV copy numbers/μg total RNA. Phylogenetic tree analysis based on the RNA-dependent RNA polymerase (RdRp) and capsid protein genes showed that CMNV isolates from L. crocea samples were clustered tightly with the original isolate of CMNV from Penaeus vannamei. The histopathological studies indicated that severe cellular necrosis occurred in the heart, liver, spleen, eye, and gill of naturally infected fish. Stronger positive signals for CMNV-specific probes appeared in the necrotic cells and tissues in the in situ hybridization (ISH) analysis. Transmission electron microscopy (TEM) revealed the presence of numerous 30 nm diameter CMNV-like viral particles. The purified CMNV suspension could cause a significant cytopathic effect (CPE) in epithelioma papilloma cyprini (EPC) cells and resulted in the formation of typical inclusion bodies, and also visible CMNV particles around the lysed EPC cells under the TEM. Taken together, all results of this study demonstrated that L. crocea is susceptible to CMNV. The prevalence of CMNV and its potential impacts on the wild population of L. crocea see the need for further investigations.

HIF-pathway genes prognostic for progression-free and overall survival in metastatic clear cell renal cell carcinoma (mccRCC).

370 Background: Clear cell renal cell carcinoma (ccRCC) is characterized by defects in the Von Hippel-Lindau/hypoxia-inducible factor (VHL/HIF) pathway. Several FDA-approved RCC therapies target the products of HIF-response genes. HIF2A is a promising new target and drugs targeting HIF2A are undergoing clinical testing. A better understanding of HIF-related genes may reveal useful biomarkers and drug targets. Methods: Gene expression was determined from 324 archival pretreatment nephrectomy specimens available from the Cancer and Leukemia Group B (CALGB) 90206, a phase III trial of patients treated with INF-α vs. INF-α plus bevacizumab. TaqMan RT-qPCR was performed on the OpenArray platform using RNA extracted from tumor tissue macrodissected based on review of H&E staining by a genitourinary pathologist. HIF pathway genes were assessed across both treatment arms, utilizing the proportional hazards model as predictors of OS and PFS, with time from randomization to death or disease progression as endpoints. Results: A total of 28 HIF-related genes (involved either in canonical or non-canonical HIF regulation) were assessed in univariate PH. Nine of these genes were associated with OS (i.e., HIF2A , VEGFC, VEGFD, TGFA, VHL, CCND1, EGFR, EGLN3 and HSP90AA1); and 6 of them were also associated with PFS (i.e., HIF2A , TGFA, VHL, CCND1 and EGLN3, and HSP90AA1). The HIF isotypes HIF1A and HIF3A were not prognostic, likewise VEGFA and VEGFB. Prolyl hydroxylase domain (PHD) proteins efficiently hydroxylate HIFα in normoxic conditions. PHD isotypes EGLN (1-3) were evaluated and only EGLN3 was associated with OS and PFS. HIF is also regulated by non-canonical pathways that function independent of oxygen concentration. HSP90AA1 was the only non-canonical pathway gene that was prognostic, and it predicted both OS and PFS. When the 9 genes that were prognostic in univariate analysis were used in a backward stepwise multivariate cox regression, VEGFD, TGFA and EGLN3 predicted OS while HIF2A, VHL and TGFA predicted PFS. Conclusions: VHL, HIF2A (but not HIF1A and HIF3A), EGLN3 (but not EGLN1 and EGLN2) and some of the HIF-response genes were prognostic of both OS and PFS. HSP90AA1 was prognostic for OS and PFS, suggesting that the non-canonical HIF pathway also plays a role in disease progression. Future studies should consider these HIF pathway genes as potential drug targets, and as predictors of response to treatments targeting the hypoxia pathway and angiogenesis.[Table: see text]

Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus.

Tilapia lake virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in cultured tilapia worldwide. In this study, we have developed and validated a TaqMan quantitative reverse transcription PCR (RT-qPCR) assay for TiLV, targeting a conserved region within segment 10 of the genome. The RT-qPCR assay was efficient (mean ± SD: 96.71 ± 3.20%), sensitive with a limit of detection of 10 RNA viral copies per reaction, and detected TiLV strains from different geographic regions including North America, South America, Africa, and Asia. The intra- and inter-assay variability ranged over 0.18-1.41% and 0.21-2.21%, respectively. The TaqMan RT-qPCR assay did not cross-react with other RNA viruses of fish, including an orthomyxovirus, a betanodavirus, a picornavirus, and a rhabdovirus. Analysis of 91 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.7% and a diagnostic specificity of 100%. The TaqMan RT-qPCR assay also detected TiLV RNA in infected Nile tilapia liver tissue extracts following an experimental challenge study, and it successfully detected TiLV RNA in SSN-1 (E-11 clone) cell cultures displaying cytopathic effects following their inoculation with TiLV-infected tissue homogenates. Thus, the validated TaqMan RT-qPCR assay should be useful for both research and diagnostic purposes. Additionally, the TiLV qPCR assay returns the clinically relevant viral load of a sample which can assist health professionals in determining the role of TiLV during disease investigations. This RT-qPCR assay could be integrated into surveillance programs aimed at mitigating the effects of TiLVD on global tilapia production.

Circulating Cell-Free Mitochondrial DNA: A Potential Blood-Based Biomarker for Sarcopenia in Patients Undergoing Maintenance Hemodialysis.

BackgroundMitochondrial impairment and exaggerated inflammation are hallmarks of sarcopenia. Recently, cell-free mitochondrial DNA (cf-mtDNA) has been in the spotlight as an endogenous danger molecule that can potentially elicit inflammation. Yet, its actual impact on sarcopenia, especially in patients with maintenance hemodialysis (MHD), is still at an early stage of investigation.Material/MethodsA total of 105 MHD patients were enrolled in this study. The subjects were classified into sarcopenia group (SP) and non-sarcopenia group (NSP) according to the DXA scan and grip strength. Plasma and peripheral blood mononuclear cells (PBMCs) were separated from whole blood. Circulating cf-mtDNA (ccf-mtDNA) was detected using Taq Man RT-qPCR. Cytosolic mtDNA and inflammation- and mitophagy-related genes in PBMCs were quantitated using SYBR Green RT-qPCR. ΔΨm was analyzed using the fluorescent probe JC-1.Resultsccf-mtDNA content was significantly higher in SP group than in NSP group. Multivariate regression analysis showed a significant correlation of ccf-mtDNA with sarcopenia after adjusting for potential confounders. A similar trend of increased mtDNA was also observed in the mitochondria-free cytoplasm of PBMCs from SP patients, together with higher expression of TLR9 and IL-6 in this group. Next, using PBMCs as surrogates for mitochondria-rich cells, we found that ΔΨm was dramatically decreased in the SP group. In parallel, the mRNA levels of mitophagy-related genes Parkin and LAMP2 were increased in the SP group.ConclusionsThe results obtained demonstrated that ccf-mtDNA, as a potential driver of inflammatory component, may be involved in the pathogenesis of the MHD-related sarcopenia.

Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

Treatment With Suboptimal Dose of Benznidazole Mitigates Immune Response Molecular Pathways in Mice With Chronic Chagas Cardiomyopathy.

Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe form of Chagas disease, a neglected tropical illness caused by the protozoan Trypanosoma cruzi, and the main cause of morbimortality from cardiovascular problems in endemic areas. Although efforts have been made to understand the signaling pathways and molecular mechanisms underlying CCC, the immunological signaling pathways regulated by the etiological treatment with benznidazole (Bz) has not been reported. In experimental CCC, Bz combined with the hemorheological and immunoregulatory agent pentoxifylline (PTX) has beneficial effects on CCC. To explore the molecular mechanisms of Bz or Bz+PTX therapeutic strategies, C57BL/6 mice chronically infected with the T. cruzi Colombian strain (discrete typing unit TcI) and showing electrocardiographic abnormalities were submitted to suboptimal dose of Bz or Bz+PTX from 120 to 150 days postinfection. Electrocardiographic alterations, such as prolonged corrected QT interval and heart parasite load, were beneficially impacted by Bz and Bz+PTX. RT-qPCR TaqMan array was used to evaluate the expression of 92 genes related to the immune response in RNA extracted from heart tissues. In comparison with non-infected mice, 30 genes were upregulated, and 31 were downregulated in infected mice. Particularly, infection upregulated the cytokines IFN-γ, IL-12b, and IL-2 (126-, 44-, and 18-fold change, respectively) and the T-cell chemoattractants CCL3 and CCL5 (23- and 16-fold change, respectively). Bz therapy restored the expression of genes related to inflammatory response, cellular development, growth, and proliferation, and tissue development pathways, most probably linked to the cardiac remodeling processes inherent to CCC, thus mitigating the Th1-driven response found in vehicle-treated infected mice. The combined Bz+PTX therapy revealed pathways related to the modulation of cell death and survival, and organismal survival, supporting that this strategy may mitigate the progression of CCC. Altogether, our results contribute to the better understanding of the molecular mechanisms of the immune response in the heart tissue in chronic Chagas disease and reinforce that parasite persistence and dysregulated immune response underpin CCC severity. Therefore, Bz and Bz+PTX chemotherapies emerge as tools to interfere in these pathways aiming to improve CCC prognosis.

Non-Invasive versus Invasive Samples for Zika Virus Surveillance: A Comparative Study in New Caledonia and French Guiana in 2015-2016.

Zika virus, an arbovirus responsible for major outbreaks, can cause serious health issues, such as neurological diseases. In the present study, different types of samples (serum, saliva, and urine), collected in 2015–2016 in New Caledonia and French Guiana from 53 patients presenting symptoms and clinical signs triggered by arbovirus infections, were analyzed using a recently developed, and in-house validated, 4-plex RT-qPCR TaqMan method for simultaneous detection and discrimination of the Zika and Chikungunya viruses. Subsequently, statistical analyses were performed in order to potentially establish recommendations regarding the choice of samples type to use for an efficient and early stage Zika infection diagnosis. On this basis, the use of only urine samples presented the highest probability to detect viral RNA from Zika virus. Moreover, such a probability was improved using both urine and saliva samples. Consequently, the added value of non-invasive samples, associated with a higher acceptance level for collection among patients, instead of serum samples, for the detection of Zika infections was illustrated.

In vitro production and multiplex quantification of 2',3'-cyclic phosphate-containing 5'-tRNA half molecules.

RNA cleavages by many ribonucleases generate RNA molecules that contain a 2',3'-cyclic phosphate (cP) at their 3'-termini, and many cP-containing RNAs (cP-RNAs) are expressed as functional molecules in cells and tissues. 5'-tRNA half molecules are representative examples of functional cP-RNAs, playing important roles in various biological processes. We here show in vitro production of cP-containing 5'-tRNA half molecules that is able to prepare abundant synthetic cP-RNAs enough for functional analyses. Furthermore, we report a multiplex TaqMan RT-qPCR method which can simultaneously quantify multiple cP-containing 5'-tRNA half species. The method enabled us to efficiently quantify 5'-tRNA halves using samples with limited amounts, such as human plasma samples, revealing drastic enhancement of 5'-tRNA half levels at approximately 1,000-fold in patients infected with Mycobacterium tuberculosis. These in vitro production and multiplex quantification methods can be applied to any cP-RNAs, and they provide cost-effective, in-house techniques to accelerate expressional and functional characterizations of 5'-tRNA halves and other cP-RNAs.

A one-enzyme RT-qPCR Assay for SARS-CoV-2, and Procedures for Reagent Production

Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed an RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson et al., 2016). Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use – (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent RTX. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs and begin to develop their own assays, within whatever regulatory framework exists for them.

Determining the prevalence and density of asymptomatic Plasmodium falciparum infections in low and high transmission settings in Western Kenya

Background/Objective: Malaria remains a global health burden as 2018 saw 228 million cases worldwide. Pre-elimination efforts aim to decrease disease transmission, which depends on development of asexual parasites to sexual gametocytes within the human host. To better determine the relationship between asexual parasites and gametocytes, we aim to quantify both stages within the same blood sample by developing a multiplex RT-qPCR assay. We hypothesize that gametocytemia decreases linearly with asexual parasitemia at higher parasitemias but increases at lower parasitemia to improve transmission probability. 
 
 Methods: We conducted a cross-sectional survey of healthy participants aged 0 to 92 years at four sites in Western Kenya (Ajigo, Webuye, Kipsamoite, Kapsisywa) with varying levels of transmission to determine prevalence of asymptomatic P. falciparum blood-stage infections. Parasite density was quantified by light microscopy and by quantitative PCR of genomic DNA isolated from dried blood spots using varATS primers. For varATS-positive samples, we isolated RNA from whole-blood and performed stage-specific quantification by multiplex Taqman RT-qPCR targeting PfPIESP2 (asexual), Pfs25 (female), and Pfs230p (male). 
 
 Results: Among all participants, 127 of 1355 (9.37%) were positive for asymptomatic P. falciparum infection by varATS-qPCR. Using varATS positivity as the standard, multiplex RT-qPCR targeting PfPIESP2 yielded a sensitivity of 78%. Across sites, overall parasite prevalence and gametocyte prevalence were highest in Ajigo (42.1%; 36%) followed by Webuye (10.5%; 9%) and Kipsamoite/Kapsisywa (0.873%; 0.1%). Pfs25-quantified gametocytemia decreased with decreasing varATS-quantified asexual parasitemia; however, the data poorly fit a linear model (R2 = 0.088). 
 
 Conclusions: PfPIESP2 detected 78% of varATS-positive samples, suggesting it may be poorly sensitive at detecting low-density blood-stage infections. Assay design may improve with a more sensitive gene target and further optimization. Based on quantification results, the relationship between gametocytemia and asexual parasitemia appears direct but non-linear. Further work is necessary to better model the relationship between stage-specific parasitemia levels.

One-Enzyme Reverse Transcription qPCR Using Taq DNA Polymerase.

Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCRs. We demonstrate the utility of Taq-alone RT-qPCRs by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/μL of input viral genomic RNA.

Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P=.009), decreased expression of injury biomarker NGAL (P=.012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P=.019, medulla P=.001), downregulation of interleukin (IL)-1β (P=.050), and upregulation of IL-10 (P<.047) and Indolamine-2, 3-dioxygenase (P=.050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P<.001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

MiR375-3p Distinguishes Low-Grade Neuroendocrine From Non-neuroendocrine Lung Tumors in FFPE Samples.

Lung cancer is still one of the leading cause of death worldwide. The clinical variability of lung cancer is high and drives treatment decision. In this context, correct discrimination of pulmonary neuroendocrine tumors is still of critical relevance. The spectrum of neuroendocrine tumors is various, and each type has molecular and phenotypical differences. In order to advance in the discrimination of neuroendocrine from non-neuroendocrine lung tumors, we tested a series of 95 surgically resected and formalin-fixed paraffin embedded lung cancer tissues, and we analyzed the expression of miR205-5p and miR375-3p via TaqMan RT-qPCR. Via a robust mathematical approach, we excluded technical outliers increasing the data reproducibility. We found that miR375-3p levels are higher in low-grade neuroendocrine lung tumor samples compared to non-neuroendocrine lung tumors. However, miR375-3p is not able to distinguish among different types of neuroendocrine lung tumors. In this work, we provide a new molecular marker for distinguishing non-neuroendocrine from low-grade neuroendocrine lung tumors samples establishing an easy miRNA score to be used in clinical settings, enabling the pathologist to classify more accurately lung tumors biopsies, which may be ambiguously cataloged in routine examination.

Sapovirus detection and quantification in fecal samples from allogeneic hematopoietic stem cell transplant recipients.

Sapovirus are important agents of acute gastroenteritis (AGE) and they are associated with outbreaks and sporadic cases worldwide. They infect people of all ages, but mainly children, the elderly and immunocompromised individuals are affected. The aim of this study was investigate sapovirus and to determine viral loads in fecal samples from patient undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fecal samples were submitted to extraction of the genetic material using a commercial kit, and RT-qPCR TaqMan was used for sapovirus screening and determination of viral loads, using a standard curve with serial dilutions of a recombinant plasmid. Positive samples were sequence by Sanger method. Sapovirus was detected in one patient, 5.3% (1/19). Viral excretion lasted for 16days. Viral load varied from 1.73 × 106 to 8.97 × 106GC/g. One of the positive samples was characterized as GI.1 genotype. This is the first study to determine sapovirus loads in samples from allo-HSCT and to identify GI.1 genotype in immunocompromised patients.

Pathogenicity of porcine reproductive and respiratory syndrome virus (ORF5 RFLP 1-7-4 viruses) in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and respiratory problems in piglets. PRRSV infection leads to substantial pig mortality and causing huge economic losses so that disease outbreaks caused by the new PRRSV strain from other regions have caused great concern in China. In this study, we analysed the pathogenicity of the novel ORF5 RFLP 1-7-4-like PRRSV strain, named PRRSV-ZDXYL-China-2018-1 in pigs. The viral challenge test showed that PRRSV-ZDXYL-China-2018-1 infection can cause persistent fever, moderate dyspnoea, serum viraemia and interstitial pneumonia in piglets. The levels of viral loads in serum and PRRSV-specific antigen were also detected in lung tissues were used one-step Taq-Man RT-qPCR and Immunohistochemistry, respectively. At 28dpi, the level of specific antibodies was increased among infected piglets. Importantly, the new virus appeared be a moderately virulent isolate with pathogenicity compared to HP-PRRSV strain LQ (JXA1-like strain). Histological examination revealed severe monocyte haemorrhage and interstitial pneumonia associated with monocyte infiltration in the lung tissue of pigs infected with PRRSV-ZDXYL-China-2018-1 and LQ-JXA1 strains. Immunohistochemistry (IHC) results showed positive brown-red epithelial cells and macrophages in pig lungs. Therefore, it is critical to establish an effective strategy to control the spread of PRRSV in China.

PD39-03 HYPOXIA-INDUCIBLE FACTOR (HIF) PATHWAY GENES PREDICT OVERALL SURVIVAL FOR METASTATIC CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Advanced (including Drug Therapy) II (PD39)1 Apr 2020PD39-03 HYPOXIA-INDUCIBLE FACTOR (HIF) PATHWAY GENES PREDICT OVERALL SURVIVAL FOR METASTATIC CLEAR CELL RENAL CELL CARCINOMA Paige K Kuhlmann*, MD Shengchen Su, Sungyong You, PhD Devin Patel, MD Brian Rini, MD Susan Halabi, Eric J Small, andMD Hyung L KimMD Paige K Kuhlmann*Paige K Kuhlmann* More articles by this author , Shengchen SuShengchen Su More articles by this author , Sungyong YouSungyong You More articles by this author , Devin PatelDevin Patel More articles by this author , Brian RiniBrian Rini More articles by this author , Susan HalabiSusan Halabi More articles by this author , Eric J SmallEric J Small More articles by this author , and Hyung L KimHyung L Kim More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000918.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prognosis for metastatic RCC (mRCC) is variable. Aberrant HIF signaling is associated with progression of RCC. Using primary tumor tissue from Cancer and Leukemia Group B (CALGB) 90206, we assessed HIF pathway genes as predictors of overall survival (OS) in mRCC. METHODS: Gene expression was determined from 324 archival pretreatment nephrectomy specimens available from a phase III trial of 732 patients treated with interferon alpha (INF) vs. INF plus bevacizumab. TaqMan RT-qPCR was performed on the OpenArray platform using RNA extracted from tumor tissue macrodissected based on review of H&E staining by a genitourinary pathologist. Normalized gene expressions were centered and scaled across samples with means set at 0 and standard deviations set at 1 (z-score). HIF pathway genes were assessed in univariable and multivariable cox proportional hazard models as predictors of OS. The Cancer Genome Atlas (TCGA) RCC dataset was used for validation of the multivariable prognostic model. RESULTS: A total of 17 HIF pathway genes were assessed in univariable analysis, and 5 of these genes were associated with OS. Expression of VHL predicted OS (p=0.006). HIF2a (p=0.044) but not HIF1a (p=0.716) predicted OS. VEGF-D (p=0.026), but not VEGF-A (p=0.425), VEGF-B (p=0.440), or VEGF-C (p=0.135), was associated with OS. TGFA (p=0.014) and CCND1 (p=0.039) also exhibit significant association with OS. For illustration, patients were dichotomized into high and low risk groups based on mean gene expression score, and Kaplan-Meier plots were generated (Figure). In multivariable analysis, VEGF-D, TGFA and VHL were significant predictors of OS with HR of 1.49 [95% CI 1.16-1.94] between high and low risk groups (p=0.002). The model had a 5-year c-index of 0.62. When MSKCC clinical risk factors were added to this model, the 5-year c-index remained unchanged at 0.62, with HR=1.77 [95% CI 1.36-2.29] between high and low risk groups (p<0.0001). TCGA data does not allow MSKCC risk score to be calculated. Our multimarker model had a 5-year c-index of 0.56 when applied to the TCGA RCC dataset with HR=1.48 [95% CI 1.06-2.06] between high and low risk groups (p=0.022). CONCLUSIONS: The HIF pathway genes associated with OS include CCND1, VEGF-D, HIF2a, TGFA, and VHL. In a multivariable model, VEGF-D, TGFA and VHL were independent predictors of OS. Future studies should consider HIF pathway genes and multivariable models based on these genes as predictors of response to treatments targeting the hypoxia pathway and angiogenesis. Source of Funding: Supported by Award Numbers R01CA133072, Homer and Gloria Harvey Family Endowed Chair, and Lauhlere Family Fund © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e808-e809 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Paige K Kuhlmann* More articles by this author Shengchen Su More articles by this author Sungyong You More articles by this author Devin Patel More articles by this author Brian Rini More articles by this author Susan Halabi More articles by this author Eric J Small More articles by this author Hyung L Kim More articles by this author Expand All Advertisement PDF downloadLoading ...