Rapid Confirmation Research Articles

Conventional Gel Electrophoresis and TaqMan Probes Enable Rapid Confirmation of Thousand Cankers Disease from Diagnostic Samples.

Thousand cankers disease (TCD) is caused by the fungal pathogen Geosmithia morbida and vectored by the walnut twig beetle Pityophthorus juglandis. In infected walnut and butternut (Juglans spp.) hosts and wingnut species (Pterocarya spp.) hosts, tree decline and death results in ecological disruption and economic losses. A rapid molecular detection protocol for TCD using microsatellite markers can confirm the presence of insect vector or fungal pathogen DNA, but it requires specialized expensive equipment and technical expertise. Using four different experimental approaches, capillary and conventional gel electrophoresis, and traditional polymerase chain reaction (PCR) and quantitative PCR (qPCR), we describe simplified and inexpensive processes for diagnostic confirmation of TCD. The improved and rapid detection protocols reported in this study reduce time and equipment costs associated with detection of molecular pest and pathogen DNA by (1) using conventional gel electrophoresis or TaqMan molecular probes to elucidate the detection limits for G. morbida and P. juglandis DNA and (2) identifying resources that allow visualization of positive test results for infected host plant tissue samples. Conventional gel electrophoresis and TaqMan molecular probe protocols detected presence of DNA from TCD-associated fungal and insect samples. These procedural improvements can be readily adopted by diagnostic end-users and adapted for use with other complex disease systems to enable rapid pest and pathogen detection.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Assessment of the National Veterinary Laboratory System Capacity for the Detection of Infectious Threats to Global Health Security in Benin

Introduction: The International Health Regulations 2005 require countries to establish laboratory systems for rapid and safe confirmation of public health emergencies including zoonoses. Objectives: This study assessed the national veterinary laboratory system capacity for the confirmation of infectious threats to global health security in Benin. Method: The study was descriptive, cross-sectional and evaluative. The non-probability sampling method with selection of the two existing veterinary laboratories were used. Two questionnaires, one observation grid and the Food and Agriculture Organization laboratory mapping tool-core were used. The system capacity was rated good if each laboratory met at least 80% of the assessed criteria. Otherwise, the capacity was rated insufficient. Results: The national veterinary laboratory system capacity in Benin was insufficient. Bohicon laboratory satisfied 27.2% of assessed criteria; With the laboratory mapping tool-core, Parakou laboratory had an average indicator of 50.7%. Laboratory collaboration and networking was the best performing domain with 66.7%. Only basic supplies category reached 80%. Laboratory budget, sample accession and availaible technology had the lowest scores. The veterinary laboratory system is facing shortage of staffs. Avian influenza virus is confirmed by the two laboratories; Bacillus anthracis and rift valley fever virus are only confirmed by Parakou laboratory. Ebola and Lassa fever are not confirmed. There is no collaboration between the laboratories in the country. Conclusion: The veterinary laboratories are not able to ensure timely detection of zoonoses and inform health system for preparedness. The development and implementation of the veterinary laboratory system improvement plan is essential for the global health security in Benin.

Alignment-free methods for polyploid genomes: Quick and reliable genetic distance estimation.

Polyploid genomes pose several inherent challenges to population genetic analyses. While alignment-based methods are fundamentally limited in their applicability to polyploids, alignment-free methods bypass most of these limits. We investigated the use of Mash, a k-mer analysis tool that uses the MinHash method to reduce complexity in large genomic data sets, for basic population genetic analyses of polyploid sequences. We measured the degree to which Mash correctly estimated pairwise genetic distance in simulated haploid and polyploid short-read sequences with various levels of missing data. Mash-based estimates of genetic distance were comparable to alignment-based estimates, and were less impacted by missing data. We also used Mash to analyse publicly available short-read data for three polyploid and one diploid species, then compared Mash results to published results. For both simulated and real data, Mash accurately estimated pairwise genetic differences for polyploids as well as diploids as much as 476 times faster than alignment-based methods, though we found that Mash genetic distance estimates could be biased by per-sample read depth. Mash may be a particularly useful addition to the toolkit of polyploid geneticists for rapid confirmation of alignment-based results and for basic population genetics in reference-free systems or those with only poor-quality sequence data available.

Effect of the Period From COVID-19 Symptom Onset to Confirmation on Disease Duration: Quantitative Analysis of Publicly Available Patient Data.

BackgroundIn general, early intervention in disease based on early diagnosis is considered to be very important for improving health outcomes. However, there is still insufficient evidence regarding how medical care that is based on the early diagnosis of confirmed cases can affect the outcome of COVID-19 treatment.ObjectiveWe aimed to investigate the effect of the duration from the onset of clinical symptoms to confirmation of COVID-19 on the duration from the onset of symptoms to the resolution of COVID-19 (release from quarantine).MethodsFor preliminary data collection, we performed data crawling to extract data from social networks, blogs, and official websites operated by local governments. We collected data from the 4002 confirmed cases in 33 cities reported up to May 31, 2020, for whom sex and age information could be verified. Subsequently, 2494 patients with unclear symptom onset dates and 1349 patients who had not been released or had no data about their release dates were excluded. Thus, 159 patients were finally included in this study. To investigate whether rapid confirmation reduces the prevalence period, we divided the duration from symptom onset to confirmation into quartiles of ≤1, ≤3, ≤6, and ≥7 days, respectively. We investigated the duration from symptom onset to release and that from confirmation to release according to these quartiles. Furthermore, we performed multiple regression analysis to investigate the effects of rapid confirmation after symptom onset on the treatment period, duration of prevalence, and duration until release from isolation.ResultsWe performed multiple regression analysis to investigate the association between rapid confirmation after symptom onset and the total prevalence period (faster release from isolation). The time from symptom onset to confirmation showed a negative association with the time from confirmation to release (t1=−3.58; P<.001) and a positive association with the time from symptom onset to release (t1=5.86; P<.001); these associations were statistically significant.ConclusionsThe duration from COVID-19 symptom onset to confirmation date is an important variable for predicting disease prevalence, and these results support the hypothesis that a short duration of symptom onset to confirmation can reduce the time from symptom onset to release.

Ichthyosiform Erythroderma, a Multifaceted Syndromic Entity.

Ichthyosiform Erythroderma, a Multifaceted Syndromic Entity.

English

The objectives of root canal treatment are elimination of microorganisms, removal of pulpal remnants, removal of debris, and shaping of the root canal system so that it may be obturated. The most important step in endodontic therapy is canal preparation which can be achieved by accurate working length determination. Working length determines the extent of placing the instruments into the canal, it affects the degree of pain and discomfort which the patient will experience post treatment and it plays an important role in the success of the treatment if placed within correct limits. The cementodentinal junction, where the pulp tissue changes into the apical tissue, is the ideal physiologic apical limit of WL because at this point healing is supposed to be optimal, and the wound to the periapical tissues is minimal.The apical constriction is however, histological and is impossible to locate clinically or radiographically. There are several methods of determining working length which include radiographical methods, digital tactile sense, apical periodontal sensitivity, paper point method and electronic apex locators. The requirements of an ideal method for determining working length include rapid location of the apical constriction in all pulpal conditions, easy measurement, rapid periodic monitoring and confirmation, patient and clinician comfort, minimal radiation to the patient; ease of use in special patients; and cost effectiveness. To achieve the highest degree of accuracy in working length determination, a combination of several methods should be used. This article reviews the different methods to determine WL and their clinical implications. KEY WORDS Working Length, Apex Locator, Radiographic Method.

Whole-Genome Sequencing of SARS-CoV-2 from Quarantine Hotel Outbreak

Hotel quarantine for international travelers has been used to prevent coronavirus disease spread into Australia. A quarantine hotel–associated community outbreak was detected in South Australia. Real-time genomic sequencing enabled rapid confirmation tracking the outbreak to a recently returned traveler and linked 2 cases of infection in travelers at the same facility.

Sensitive and Specific Functional Flow Cytometry Assay for Rapid Diagnosis of Heparin Induced Thrombocytopenia and Thrombosis (HIT)

Background: Reliable diagnosis of Heparin Induced Thrombocytopenia and Thrombosis (HIT) is crucial for patient management. A test that detects the presence of functional antibodies, capable of inducing platelet activation, is far superior to the commonly performed immunoassays having poor specificity. Unfortunately, the currently applied functional assays are technically difficult and require a high level of expertise not readily available in clinical laboratories, thus limiting their performance feasibility.Aims: We aimed to assess the sensitivity and the specificity of a simple, easily performed functional flow cytometric assay (FCA) to detect heparin stimulated platelet activating antibodies.Methods: Samples from patients clinically suspected of HIT were routinely tested by the PF4/H-PaGIA immunoassay (DiaMed, Switzerland), followed by the functional FCA. The capacity of the patient's serum to induce platelet activation in the presence of heparin was tested using two color flow cytometry. The FCA results, were calculated as the ratio of % activated normal donor platelets stimulated by the patient sample /% the same donor platelets activated by TRAP. Results were compared to those of the gold-standard, the radioactive serotonin-release assay (SRA). The assays results were also correlated with the HIT presentation based on the 4Ts score.Results:Samples were consecutively accrued from patients (n=650) suspected of HIT by hematologists, 99 (15.3%) were positive by H/PF4-PaGIA immunoassay and 31 (4.8%) were positive by FCA. Sample dilution of 1:32, decreased the H/PF4-PaGIA-positive results to 29 (4.5%), while the FCA-positive results (4.8%) remained consistent, with 93.1% and 94.3% sensitivity and specificity respectively. SRA was performed on 63 samples. Of the 21 SRA positive samples, 19 were positive by FCA (relative sensitivity 90.5%), and of 42 SRA negative samples, 40 were negative by FCA (relative specificity 95.2%).Patients with PaGIA+/ FCA+ showed average platelet activation ratio of 2.2+2.9 SD (n=31), compared to the average of patients negative by both tests 0.1 + 0.1 SD(n=30). The high resolution of this test is demonstrated even at the 2SD level with the average negative + 2SD (0.3) being smaller by 7.3fold than the average (2.2) of the positive patient thus facilitating easy discrimination between positive and negative values. Patients with PaGIA+/ FCA-negative were also easily resolved from the PaGIA+/FCA+. These patients with mean of 0.2 +0.2 SD, and +2SD of 0.6, had significantly smaller values than the mean of the positive patients (ratio 3.7). Overall, the FCA showed significantly higher correlation with the clinical presentation of HIT (4Ts score) performed on 182 patients, compared to the PF4/H-PaGIA (ROC-plot analysis, AUC 0.93 vs. 0.63, p<0.001). At a cut-off level of 92% sensitivity, the respective specificity of the FCA was 96% .Conclusions: The present study demonstrates that this flow cytometric functional assay is easily performed, practical for routine daily use and provides reliable, well resolved and rapid confirmation of heparin stimulated platelet activating antibodies in the patients blood. DisclosuresTomer:private: Patents & Royalties: patent pending.

Progress Toward Rubella Elimination - World Health Organization European Region, 2005-2019.

In 2005, the Regional Committee of the World Health Organization (WHO) European Region (EUR) passed a resolution calling for the regional elimination of measles, rubella, and congenital rubella syndrome (CRS) (1). In 2010, all 53 countries in EUR* reaffirmed their commitment to eliminating measles, rubella, and CRS (2); this goal was included in the European Vaccine Action Plan 2015-2020 (3,4). Rubella, which typically manifests as a mild febrile rash illness, is the leading vaccine-preventable cause of birth defects. Rubella infection during pregnancy can result in miscarriage, fetal death, or a constellation of malformations known as CRS, which usually includes one or more visual, auditory, or cardiac defects (5). The WHO-recommended measles and rubella elimination strategies in EUR include 1) achieving and maintaining ≥95% coverage with 2 doses of measles- and rubella-containing vaccine (MRCV) through routine immunization services; 2) providing measles and rubella vaccination opportunities, including supplementary immunization activities (SIAs), to populations susceptible to measles or rubella; 3) strengthening surveillance by conducting case investigations and confirming suspected cases and outbreaks with laboratory results; and 4) improving the availability and use of evidence to clearly communicate the benefits and risks of preventing these diseases through vaccination to health professionals and the public (6). This report updates a previous report and describes progress toward rubella and CRS elimination in EUR during 2005-2019 (7). In 2000, estimated coverage with the first dose of a rubella-containing vaccine (RCV1) in EUR was 60%, and 621,039 rubella cases were reported (incidence = 716.9 per 1 million population). During 2005-2019, estimated regional coverage with RCV1 was 93%-95%, and in 2019, 31 (58%) countries achieved ≥95% coverage with the RCV1. During 2005-2019, approximately 38 million persons received an RCV during SIAs in 20 (37%) countries. Rubella incidence declined by >99%, from 234.9 cases per 1 million population (206,359 cases) in 2005 to 0.67 cases per 1 million population (620 cases) by 2019. CRS cases declined by 50%, from 16 cases in 2005 to eight cases in 2019. For rubella and CRS elimination in EUR to be achieved and maintained, measures are needed to strengthen immunization programs by ensuring high coverage with an RCV in every district of each country, offering supplementary rubella vaccination to susceptible adults, maintaining high-quality surveillance for rapid case detection and confirmation, and ensuring effective outbreak preparedness and response.

Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing.

Infectious diseases represent one of the major challenges to sustainable aquaculture production. Rapid, accurate diagnosis and genotyping of emerging pathogens during early‐suspected disease cases is critical to facilitate timely response to deploy adequate control measures and prevent or reduce spread. Currently, most laboratories use PCR to amplify partial pathogen genomic regions, occasionally combined with sequencing of PCR amplicon(s) using conventional Sanger sequencing services for confirmatory diagnosis. The main limitation of this approach is the lengthy turnaround time. Here, we report an innovative approach using a previously developed specific PCR assay for pathogen diagnosis combined with a new Oxford Nanopore Technologies (ONT)‐based amplicon sequencing method for pathogen genotyping. Using fish clinical samples, we applied this approach for the rapid confirmation of PCR amplicon sequences identity and genotyping of tilapia lake virus (TiLV), a disease‐causing virus affecting tilapia aquaculture globally. The consensus sequences obtained after polishing exhibit strikingly high identity to references derived by Illumina and Sanger methods (99.83%–100%). This study suggests that ONT‐based amplicon sequencing is a promising platform to deploy in regional aquatic animal health diagnostic laboratories in low‐ and medium‐income countries, for fast identification and genotyping of emerging infectious pathogens from field samples within a single day.

NOFSal-MP10: A single hypervariable STR 12-plex for accurate verification of triploidy in Atlantic salmon (Salmo salar L.)

An increasing interest in production of sterile fish in aquaculture requires rapid, accurate and efficient testing for certification of triploid eggs prior to sale. Further, validation of triploidy can be beneficial, and even required, for accrediting methods of triploid production. A PCR-genotyping approach using a single megaplex of 12 hypervariable STR markers shows accurate and highly repeatable results enabling verification of ploidy in a single test. The NOFSal-MP10 panel contains 12 STR markers mapped to 9 chromosomes with an average of 21.4 alleles per marker for a combined total of 257 alleles based on genotyped samples to-date. The hypervariable nature of these 12 STRs leads to a large probability for three uniquely sized alleles to be observed at each marker, thus providing a rapid confirmation of ploidy based on the count of allele fragments per marker. Further, as a PCR amplification step is involved, this method is robust to DNA quality and quantity, making it suitable for very early determination of ploidy, as early as the eyed-egg stage. Repeat genotyping of positive control diploid Atlantic salmon over two different capillary electrophoretic instruments in different laboratories and with multiple laboratory personnel proves the panel's robustness to scoring errors with an overall allelic error rate of 0.3% and a false-positive triploid assignment rate of zero. Genotyping of DNA from 1238 eggs and larvae from 18 independent triploid production batches over three years confirmed triploidy in 98% of samples based on a semi-strict criterion of three unique alleles at one or more loci, and 95% based on a strict criterion of three unique alleles at two or more loci.

An accurate, rapid, and sensitive method for simultaneous determination of four typical heterocyclic amines in roasted pork patties: Application in the study of inhibitory effects of astaxanthin.

Heterocyclic aromatic amines, as a group of mutagenic and carcinogenic compounds, have gained worldwide concern. In this study, an accurate, rapid, and sensitive confirmation and quantification method of four major heterocyclic aromatic amines in roasted pork was developed based on Q-Orbitrap along with Quick, Easy, Cheap, Effective, Rugged, and Safe extraction. The limit of detections and limit of quantitations were found to be 0.2-1.2 μg/kg and 0.6-3.5μg/kg, respectively, revealing the high sensitivity of this method. Obtained results showed recoveries ranging from 78.1 to 97.4%, depending on the different heterocyclic aromatic amines and spiked levels. Precision was in the range of 2.6-4.5% for four heterocyclic aromatic amines at different levels. In addition, the developed method had been applied to investigate the inhibitory effects of astaxanthin on the above-mentioned heterocyclic aromatic amines in roasted pork. The amount of astaxanthin with the best inhibitory effects was 7.5mg (0.0375%), which led to significant reduction in heterocyclic aromatic amines levels over 50%.

Rapid diagnostic tests for the detection of recent dengue infections: An evaluation of six kits on clinical specimens.

IntroductionEarly and rapid confirmation of dengue infections strengthens disease surveillance program and are critical to the success of vector control measures. Rapid diagnostics tests (RDTs) are increasingly used to confirm recent dengue infections due to their ease of use and short turnaround time for results. Several studies undertaken in dengue-endemic Southeast Asia have reported the performance of RDTs against enzyme-linked immunosorbent assay (ELISA), reverse transcriptase polymerase chain reaction (RT-PCR) and virus isolation methods. However, few studies have compared multiple RDTs for the detection of dengue NS1 antigen and IgM antibody in a single combo cassette. We evaluated six RDTs in Singapore for their utility in routine clinical testing to detect recent dengue infections.MethodsThe evaluation comprised two phases. The first phase sought to determine each RDT’s specificity to dengue NS1 and IgM using zika and chikungunya virus supernatant and zika convalescent samples. RDTs that cross-reacted with zika or chikungunya were not further tested in phase 2. The second phase sought to determine the sensitivity and specificity of the remaining RDTs to dengue NS1 and IgM using pre-characterised dengue specimens and non-dengue/chikungunya febrile clinical specimens.ResultsNone of the RDTs cross-reacted with zika IgM in Phase 1. Truquick and Quickprofile cross reacted with zika and chikungunya viruses and were not evaluated thereafter. Standard Q had the highest dengue NS1 and IgM sensitivity at 87.0% and 84.3% respectively whereas Bioline (68.5%) and Multisure (58.3%) had the lowest dengue NS1 and IgM sensitivity respectively. Combining dengue NS1/IgM detection results greatly improved the RDT ability to detect recent dengue infection; Standard Q had the highest sensitivity at 99.1% while Multisure had the lowest at 92.6%. All the RDTs were highly specific for dengue NS1 and IgM (96.7% to 100%). All the RDTs had high positive predictive values (98.4% to 100%) for NS1, IgM and combined NS1/IgM parameters whereas Standard Q had the highest negative predictive values at 68.2% (NS1), 63.8% (IgM) and 96.8% (NS1/IgM). For the RDTs, detection of NS1 declined from acute to convalescent phase of illness whereas IgM detection rate gradually increased over time.ConclusionIn our study, several RDTs were evaluated for their diagnostic accuracy and capability in detecting recent dengue infection. Standard Q demonstrated a high degree of diagnostic accuracy and capability in the detection of NS1 and IgM biomarkers. RDTs can provide rapid and accurate confirmation of recent dengue infections and augment dengue surveillance and control programmes. Further studies are required to assess the usefulness of these RDTs in other epidemiology settings.

From Smartphone Lateral Flow Immunoassay Screening to Direct MS Analysis: Development and Validation of a Semi-Quantitative Direct Analysis in Real-Time Mass Spectrometric (DART-MS) Approach to the Analysis of Deoxynivalenol.

In current food safety monitoring, lateral flow immunoassays (LFIAs) are widely used for rapid food contaminant screening. Recent advances include smartphone readouts, offering semi-quantitative analysis of LFIAs with time, location, and data transfer in case of on-site testing. Following the screening, the next step in the EU regulations is confirmation by, e.g., liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this work, using direct analysis in real time ambient ionization and triple quadrupole MS/MS (DART-QqQ-MS/MS), we achieved rapid confirmation of the identity of the substance(s) causing the LFIA result. In the workflow proposed, an individual performs the (on-site) smartphone LFIA screening, and when the result is suspect, an identification LFIA (ID-LFIA) strip is developed with the same sample extract. The ID-LFIA can be dissociated and rapidly analyzed in a control laboratory with DART-QqQ-MS/MS. The ID-LFIA consists of multiple lines of monoclonal antibodies against the mycotoxin deoxynivalenol, acting as a bioaffinity trap. The ID-LFIA/DART-QqQ-MS/MS approach has been developed and validated, along with the screening smartphone LFIA, and has demonstrated its applicability by analyzing incurred and spiked samples. The developed approach has been critically compared with our previous direct electrospray ionization MS method and was found to provide highly complementary information on the total deoxynivalenol contamination in the sample.

Nosocomial Pneumonia in the Era of Multidrug-Resistance: Updates in Diagnosis and Management.

Nosocomial pneumonia (NP), including hospital-acquired pneumonia in non-intubated patients and ventilator-associated pneumonia, is one of the most frequent hospital-acquired infections, especially in the intensive care unit. NP has a significant impact on morbidity, mortality and health care costs, especially when the implicated pathogens are multidrug-resistant ones. This narrative review aims to critically review what is new in the field of NP, specifically, diagnosis and antibiotic treatment. Regarding novel imaging modalities, the current role of lung ultrasound and low radiation computed tomography are discussed, while regarding etiological diagnosis, recent developments in rapid microbiological confirmation, such as syndromic rapid multiplex Polymerase Chain Reaction panels are presented and compared with conventional cultures. Additionally, the volatile compounds/electronic nose, a promising diagnostic tool for the future is briefly presented. With respect to NP management, antibiotics approved for the indication of NP during the last decade are discussed, namely, ceftobiprole medocaril, telavancin, ceftolozane/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam.

Single-lead ECGs (AliveCor) are a feasible, cost-effective and safer alternative to 12-lead ECGs in community diagnosis and monitoring of atrial fibrillation

BackgroundCommunity management of atrial fibrillation (AF) often requires the use of electrocardiographic (ECG) investigation. Patients discharged following treatment of AF with fast ventricular response (fast AF) can require numerous ECGs...

Rapid screening and confirmation of 415 pesticide residues in red cabbages by liquid chromatography-quadrupole-time of flight-mass spectrometry

应用液相色谱-四极杆-飞行时间质谱(LC-QTOF/MS)建立了一次进样可同时对紫甘蓝中415种农药残留进行快速筛查和准确确证的分析方法。实验采用1%(v/v)醋酸乙腈溶液提取,无水硫酸镁和氯化钠进行盐析,ZORBAX SB-C18色谱柱(100 mm×2.1 mm, 3.5 μm)分离,以0.1%(v/v)甲酸水溶液(含5 mmol/L乙酸铵)-乙腈为二元流动相进行梯度洗脱,应用LC-QTOF/MS在电喷雾电离、全离子MS/MS(All Ions MS/MS)扫描正模式下进行检测,基质匹配外标法定量分析。通过优化全自动MS/MS采集模式(Auto MS/MS)和全离子MS/MS采集模式下的不同参数,得到每种采集模式下的最佳条件。然后在2种不同采集模式的最佳条件下对比,最终选取All Ions MS/MS采集模式。实验结果表明,采用所建立的分析方法可以准确定性和定量筛查紫甘蓝中415种农药残留,所有415种农药在各自的范围内线性相关系数(r2)均大于0.990,其中411种农药的筛查限(SDL)≤5 μg/kg, 413种农药的定量限(LOQ)≤10 μg/kg。在1倍、2倍和10倍LOQ添加水平下,农药的回收率分别为65.7%~118.4%、72.0%~118.8%和70.2%~111.2%,相对标准偏差分别为0.9%~19.7%、0.2%~19.9%和0.6%~19.9%。将该方法应用于2019年欧盟能力验证项目的紫甘蓝样品中未知农药残留筛查方法和定量方法考核样的检测,所有添加农药均被准确定性筛查和定量检测,没有假阳性和假阴性。结果表明,该方法快速、准确、可靠,适用于对紫甘蓝中多种农药残留的高通量定性筛查和准确定量,可以扩展到其他果蔬基质中多农残的高通量筛查。

Development of a Diagnostic Assay for Race Differentiation of Podosphaera macularis.

Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

Rapid confirmation of five non-typhoidal Salmonella (NTS) serotypes using a low-cost portable-DIY OpenPCR© thermocycler

Rapid confirmation of five non-typhoidal Salmonella (NTS) serotypes using a low-cost portable-DIY OpenPCR© thermocycler

605 Universal urine drug screening upon admission to labor and delivery - phase 2 study

Illicit drug use or narcotic prescription use during pregnancy is a growing epidemic and can affect the neonate. The benefit of universal urine drug screening (UDS) in pregnancy is controversial. Previously, we reported a 47% false positive (FP) rate when implementing universal UDS in pregnant women admitted to Labor and Delivery (L&D). Gas chromatography was used for confirmation with a turnaround time (TT) of one week, carrying a legal and emotional burden on parents. In 2019 our hospital implemented an inhouse confirmation with mass spectrometry (MS) with TT of 24 hours. This phase 2 study aims to examine the FP, true positive (TP) rates and “new catches” after establishing a more rapid confirmation test. This is a retrospective cohort study of all pregnant women admitted to L&D between 10/2019 and 9/2020. All positive UDS tests were reflexed for inhouse confirmation via MS except for fentanyl which was sent to an outside lab with a TT of one week. For some mothers the infant’s meconium was sent for drug screening. Positive results were determined to be TP or FP based on MS results. New catches were defined as TP results in mothers who did not admit to drug use. A total of 2395 deliveries occurred during this time period of which 2122 (88.6%) had available UDS results. Sixty women (60/2122, 2.8%) had a positive UDS for at least one drug with a total of 69 positive drugs. Of 63 positive tests with available confirmation, 32 (32/63, 50.7%) were TP and 31 (31/63, 49.2%) were FP. Of the TP results, 11 babies were “new catches” (11/32, 34.3%), 16 babies developed neonatal abstinence syndrome (NAS) (16/32, 50%) and five babies were discharged to the Administration of Child Services (ACS) (5/32, 15.6%). Seven babies with NAS (7/16, 43.7%) required treatment with morphine. ACS was not called for any of the FP cases. In our phase 2 study with rapid UDS confirmation, the FP rate was similar but there was a higher percentage of new catches. This approach may improve the efficiency of universal UDS since actions are taken only for confirmed results.