easyMAG System Research Articles

A Versatile Solution for High-Quality Uav Fluxgate Magnetic Acquisitions

We propose an unmanned aerial system fully dedicated to magnetic surveying in a variety of contexts ranging from small and shallow objects or pipelines detection to large-scale geological mapping: the Easymag system. Four major requirements are identified if one wishes to efficiently acquire quality magnetic data at such variable scales: 1- the accuracy of the magnetic acquisition must be better than the lowest expected signal according to the application (usually a few nT or lower); 2- the navigation and positioning must be precise enough to compete with ground operators with centimetre accuracy; 3- the flight flexibility must allow contrasting flight domains with topographic draping at any height; 4- the efficiency on the field must compete with existing carriers. Each of these aspects are investigated to demonstrate Terremys’ ability to perform efficient magnetic surveys. As a first case study, a drone magnetic acquisition for archaeological purposes is presented in comparison with a ground survey of the same area. Ground and drone surveys can lead to similar results. To demonstrate the ability to acquire magnetic data in different flight domains with the same system, a second case study focusing on a multiscale analysis is presented.

Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC).

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture.

Investigation of Torque Teno Virus (TTV) DNA as an immunological and virological marker in pediatric hematopoietic stem cell transplantation (HSCT) patients

BackgroundHigh viral loads are observed in Torque Teno Virus (TTV) infection after hematopoietic stem cell transplantation (HSCT). We aimed to analyze the kinetics of plasma TTV-DNA load in pediatric patients who received immunosuppressive therapy and developed infection complications in the first 100 days after HSCT. MethodsAs a patient group; 113 plasma samples taken from 33 pediatric HSCT recipients at a time interval after transplantation and as a control group; 38 plasma samples from 38 children without known chronic disease were included in the study. Viral nucleic acid isolation was performed by using the NucliSENS easyMAG (bioMerieux, France) system. A laboratory designed quantitative polymerase chain reaction process was performed on 7300 Real-Time PCR system (Applied Biosystems, CA, USA) with the amplification mixture containing primer and probe sequences for the UTR gene region. ResultsTTV-DNA was detected in all patient's samples and the median viral load was calculated as 7.67 Log10 copies/mL (range: 2.84–9.59). In the control group, the TTV-DNA median viral load was calculated as 5.51 Log10 copies/mL (range: 2.50–7.04), except for one negative sample. A significant difference was observed between the control group and the patient group in terms of TTV viral load levels. In nine patients, a median 2.15 Log10 copies/mL viral load increase was observed at 31–60 days post-transplant compared to the pre-transplant period. ConclusionTTV-DNA levels should be closely monitored to understand the immune status of the first 100 days after transplantation and the effects of treatment regimens on patients with HSCT.

Diagnostic Accuracy of a Noninvasive Test for Detection of Helicobacter pylori and Resistance to Clarithromycin in Stool by the Amplidiag H. pylori+ClariR Real-Time PCR Assay.

The noninvasive detection of Helicobacter pylori and its resistance to clarithromycin could revolutionize the management of H. pylori-infected patients by tailoring eradication treatment without any need for endoscopy when histology is not necessary. Several real-time PCR tests performed on stools have been proposed, but their performances were either poor or they were tested on too few patients to be properly evaluated. We conducted a prospective, multicenter study including 1,200 adult patients who were addressed for gastroduodenal endoscopy with gastric biopsies and who were naive for eradication treatment in order to evaluate the performance of the Amplidiag H. pylori+ClariR assay recently developed by Mobidiag (Espoo, Finland). The results of the Amplidiag H. pylori+ClariR assay performed on DNA from stools (automatic extraction with the EasyMag system [bioMérieux]) were compared with those of culture/Etest and quadruplex real-time PCRs performed on two gastric biopsy samples (from the antrum and corpus) to detect the H. pyloriglmM gene and mutations in the 23S rRNA genes conferring clarithromycin resistance. The sensitivity and specificity of the detection of H. pylori were 96.3% (95% confidence interval [CI], 92 to 98%) and 98.7% (95% CI, 97 to 99%), respectively. The positive and negative predictive values were evaluated to be 92.2% (95% CI, 92 to 98%) and 99.3% (95% CI, 98 to 99%), respectively. In this cohort, 160 patients (14.7%) were found to be infected (positive by culture and/or PCR). The sensitivity and specificity for detecting resistance to clarithromycin were 100% (95% CI, 88 to 100%) and 98.4% (95% CI, 94 to 99%), respectively.

252. Development and Evaluation of a Novel MultiCode Real-Time PCR Assay for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid and Induced Sputum

Background Pneumocystis jirovecii is a medically important fungal pathogen responsible for opportunistic infections in immunocompromised hosts with high morbidity and mortality. Compared with standard microscopy based assays, home-brew nucleic acid amplification tests (NAAT) have emerged as sensitive tools for the diagnosis of P. jirovecii pneumonia, but their sensitivities vary depending upon selected genetic targets. Recent studies suggest that the mitochondrial small subunit (mtSSU) is a better NAAT target given its higher copy number and stable expression in the disease process. We aimed to develop and evaluate a mtSSU-targeted MultiCode real-time PCR assay that incorporates a sample processing control (SPC) and enables detection of P. jirovecii in bronchoalveolar lavage fluid (BALF) and induced sputum.MethodsFirstly, we compared manual DNA extraction using Zymo Quick DNA kit with automated extraction using the NucliSENS easyMAG system after sample pretreatment with either FastPrep mechanical grinding or vortex-based bead beating. We then determined the mouse hepatitis virus SPC (Luminex) spike-in amount, and optimized the PCR conditions on the ABI 7500 PCR system. A new Pneumocystis mtSSU run control was generated by cloning and transforming mtSSU gene into a genetically engineered E. coli strain, and quantified with a home-brew quantitative TaqMan PCR. Lastly, the performance characteristics of the MultiCode PCR assay were determined.ResultsMechanical grinding of BALF or sputum before the easyMAG based extraction was better than the other extraction protocols as evidenced by lower CT of mtSSU or SPC. Diluted SPC added to samples before DNA extraction made its CT within 31–34. With the mtSSU run control, the limit of detection of the new assay was 80 copies/mL. No cross-reactivity was found with 9 respiratory viruses, 8 bacteria or 11 fungi. The assay has high reproducibility for three-day detection of the same sample aliquots for mtSSU (CT: 30.0–30.3; CV%: 0.5–1.6) and SPC (CT: 32.1–32.2; CV%: 0.8–2.4).ConclusionWe developed a novel MultiCode real-time PCR assay for detection of P. jirovecii in BALF and sputum, which demonstrated high analytical sensitivity, specificity and reproducibility and warrants further clinical validation.Disclosures All authors: No reported disclosures.

FIRST REPORT OF PERSIMMON CRYPTIC VIRUS AND PERSIMMON VIRUS A IN KOREA

In 2014, a total of 77 persimmon (Diospyros kaki Thunb.) trees from Korean commercial orchards were surveyed for Persimmon cryptic virus (PeCV) and Persimmon virus A (PeVA). Leaf samples were collected from symptomatic trees with necrosis (two), or mosaic and leaf malformations (one) and 74 asymptomatic trees. Total RNAs were extracted using the NucliSENS easyMAG system (bioMerieux) and subjected to RT-PCR using specific primer pairs PeCV F/PeCV R (5’-TTCCAATGGCAGACCAAGG-3’/5’-TGTGTAGGTCGGATGACG-3’; design based on GenBank accession number HE805114) and PeVAfor/PeVArev (Morelli et al., 2014). PCR products of the expected sizes (526 bp and 250 bp for PeCV and PeVA, respectively) were directly sequenced. Results of RT-PCR revealed 67 PeCV (87%) and 11 PeVA (14.3%) positive samples, including nine samples with mixed infections of PeCV and PeVA. BLASTn of consensus sequences revealed 99% nucleotide sequence identity to Italian PeCV isolate SSPI (HE805114) and 98% to Japanese PeVA isolate (AB735628) respectively. The consensus sequences were deposited in GenBank as AB968365 (PeCV) and LC177111 (PeVA). Mixed infections of both viruses were detected in one plant showing vein necrosis, and in eight asymptomatic samples. To our knowledge, this is the first report of PeCV and PeCV infection of persimmon in Korea.

First Report of Cherry necrotic rusty mottle virus Infecting Sweet Cherry Trees in Korea.

Cherry necrotic rusty mottle virus (CNRMV), an unassigned member in the family Betaflexiviridae, has been reported in sweet cherry in North America, Europe, New Zealand, Japan, China, and Chile. The virus causes brown, angular necrotic spots, shot holes on the leaves, gum blisters, and necrosis of the bark in several cultivars (1). During the 2012 growing season, 154 sweet cherry trees were tested for the presence of CNRMV by RT-PCR. Samples were randomly collected from 11 orchards located in Gyeonggi and Gyeongsang provinces in Korea. RNA was extracted from leaves using the NucliSENS easyMAG system (bioMérieux, Boxtel, The Netherlands). The primer pair CGRMV1/2 (2) was used to amplify the coat protein region of CNRMV. Although none of the collected samples showed any notable symptoms, CNRMV PCR products of the expected size (949 bp) were obtained from three sweet cherry samples from one orchard in Gyeonggi province. The PCR products were cloned into a pGEM-T easy vector (Promega, Madison, WI) and sequenced. BLAST analyses of the three Korean sequences obtained (GenBank Accession Nos. AB822635, AB822636, and AB822637) showed 97% nucleotide sequence identity with a flowering cherry isolate from Japan (EU188439), and shared 98.8 to 99.6% nucleotide and 99.6 to 100% amino acid similarities to each other. The CNRMV positive samples were also tested for Apple chlorotic leaf spot virus (ACLSV), Cherry mottle leaf virus (CMLV), Cherry rasp leaf virus (CRLV), Cherry leafroll virus (CLRV), Cherry virus A (CVA), Little cherry virus 1 (LChV-1), Prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV) by RT-PCR. One of the three CNRMV-positive samples was also infected with CVA. To confirm CNRMV infection by wood indexing, Prunus serrulata cv. Kwanzan plants were graft-inoculated with chip buds from the CNRMV-positive sweet cherry trees. At 3 to 4 weeks post-inoculation, the Kwanzan plants showed quick decline with leaves wilting and dying; CNRMV infection of the indicators was confirmed by RT-PCR. To our knowledge, this is the first report of CNRMV infection of sweet cherry trees in Korea. Screening for CNRMV in propagation nurseries should minimize spread of this virus within Korea.

Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases

During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells. Using the NucliSENS easyMAG technique, the co-extraction of inhibitors is reduced, with an exception for whole blood, which requires supplementary extraction steps to eliminate inhibitors.

Comparative evaluation of two automated systems for nucleic acid extraction of BK virus: NucliSens easyMAG versus BioRobot MDx

The objective of this study was to compare the performance of two automated nucleic acid extraction systems. Specifically, the NucliSens easyMAG system (bioMerieux, Marcy l’Etoile, France), which incorporates magnetic bead technology, was compared with the BioRobot MDx system (Qiagen GmbH, Hilden, Germany), which uses a silica membrane-based method of nucleic acid extraction. Nucleic acids from the BK virus (BKV DNA) were extracted from 98 plasma and 57 urine specimens using the Real-Q BKV quantitation kit (Biosewoom, Seoul, Korea). Failed PCR was defined as negative BKV DNA results having more than 36 threshold cycles of the internal control by the manufacturer's instruction. The PCR failure rate of nucleic acids isolated from plasma samples using the MDx system was similar to that of plasma samples processed using the easyMAG system (2.0% and 3.1%, respectively). The PCR failure rate of nucleic acids isolated from urine samples using the MDx system was higher than that of urine samples processed using the easyMAG system (33.3% and 12.5%, respectively). These data suggest that the PCR inhibitors present in urine specimens are removed more efficiently by the easyMAG system. Among amplified specimens, the discordant results obtained from the two systems revealed that the BKV DNA load ranged from 2.3 log 10 copies/mL to 4.6 log 10 copies/mL. Of the 25 urine specimens that yielded BKV DNA by both extraction systems, 15 specimens (60.0%) yielded higher BKV DNA loads by the easyMAG system, indicating that the easyMAG system extracted nucleic acid more efficiently than did the MDx system. In conclusion, the easyMAG method outperformed the MDx method when used to extract BKV DNA from urine samples. Magnetic bead-based extraction methods such as the easyMAG system are therefore preferable for the quantitation of viral DNA in urine.

Rapid Semiautomated Subtyping of Influenza Virus Species during the 2009 Swine Origin Influenza A H1N1 Virus Epidemic in Milwaukee, Wisconsin

In the spring of 2009, a novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) emerged and began causing a large outbreak of illness in Milwaukee, WI. Our group at the Midwest Respiratory Virus Program laboratory developed a semiautomated real-time multiplex reverse transcription-PCR assay (Seasonal), employing the NucliSENS easyMAG system (bioMérieux, Durham, NC) and a Raider thermocycler (HandyLab Inc., Ann Arbor, MI), that typed influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) and subtyped influenza A virus into the currently circulating H1 and H3 subtypes, as well as a similar assay that identified H1 of S-OIV. The Seasonal and H1 S-OIV assays demonstrated analytical limits of detection of <50 50% tissue culture infective doses/ml and 3 to 30 input copies, respectively. Testing of the analytical specificities revealed no cross-reactivity with 41 and 26 different common organisms and demonstrated outstanding reproducibility of results. Clinical testing showed 95% sensitivity for influenza A virus and influenza B virus and 95 and 97% specificity compared to tissue culture. Comparisons of results from other molecular tests showed levels of positive agreement with the Seasonal and H1 S-OIV assay results of 99 and 100% and levels of negative agreement of 98 and 100%. This study has demonstrated the use of a semiautomated system for sensitive, specific, and rapid detection of influenza A virus, influenza B virus, and RSV and subtyping of influenza A virus into human H1 and H3 and S-OIV strains. This assay/system performed well in clinical testing of regular seasonal influenza virus subtypes and was outstanding during the 2009 Milwaukee S-OIV infection outbreak. This recent outbreak of infection with a novel influenza A (H1N1) virus also demonstrates the importance of quickly distributing information on new agents and of having rapid influenza virus subtyping assays widely available for clinical and public health decisions.

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile

Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high false-negative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5'-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity.

Rapid Detection of bla KPC Carbapenemase Genes by Real-Time PCR

Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (bla(KPC)) enzymes are among the most common beta-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of bla(KPC) genes using TaqMan chemistry. The q-PCR amplification of bla(KPC) DNA was linear over 7 log dilutions (r(2) = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for bla(KPC) genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while bla(KPC) genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for bla(KPC) genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the bla(KPC) q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to bla(KPC) detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.

Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears

Aims:Cervical cytology biobanking is a feasible concept in cervical pathology and could be an indispensable tool for fundamental and applied molecular biological research. PCR is a powerful molecular technique that...

R2199 Plasma is to be preferred above whole blood for automated nucleic acid extraction using the Nuclisens EasyMag system (Biomérieux)

R2199 Plasma is to be preferred above whole blood for automated nucleic acid extraction using the Nuclisens EasyMag system (Biomérieux)