Nucleic Acid Extraction Research Articles

B-269 Plasmodium Detection: Sera, Blood, Do They All Work?

Abstract Background When diagnosing malaria, testing employs blood samples using either microscopy, the current gold standard or molecular diagnostic tests (more sensitive but significantly more expensive). In Plasmodium spp. screening programs in at-risk populations where there are not many resources available, dried blood on filter paper is used to ease transport, storage and preservation of the sample. Although Plasmodium detection studies using blood serum as a sample are available, it isn´t always as sensitive as using whole blood-based sampling, as the concentration of DNA in plasma is proportional to the level of parasitaemia. This study aims to confirm the differences in sensitivity using both samples, demonstrating the importance of choosing the right type of sample to use for diagnosis. Methods The study was carried out using the VIASURE Malaria Real Time PCR Detection Kit with sera and blood samples from 94 inhabitants from Nueva Esperanza community in the Peruvian Amazon collected between 2007 and 2020, establishing a comparative analysis between matrixes. Nucleic acid extraction was performed using the QIAamp DNA Mini Kit from QIAGEN and the VIASURE assay was performed using QuantStudio®6 Flex Real-Time PCR System thermocycler (Thermo Fisher Scientific) and the BioRad CFX96TM Real-Time PCR Detection System for analysing peripheral blood samples and the BioRad CFX96TM Real-Time PCR Detection System for analysing sera. Results From the total of samples, only 5 sera (5.31%) showed amplification of Plasmodium spp., whereas from blood samples, 30 resulted to be Plasmodium spp-positive. Therefore, 26.59% of detection is lost when serum is used as a sample, as opposed to blood. Conclusion After this analysis it could be concluded that malaria detection sensitivity is appreciably higher for blood samples than for sera samples. Thus, serum samples aren´t the best choice for Plasmodium spp. testing and whole blood-based matrixes are preferable for this diagnosis. These results reinforce the idea of the importance of the sample type when diagnosing different pathogens, as well as the relevance of a confirming comparative analysis when similar sample types are involved.

A-297 Usefulness of Non-Skin Samples in the PCR Diagnosis of Monkeypox

Abstract Background During 2022, human mpox cases were reported in several European countries. Until November 2022, in Spain, 7404 cases were detected, most of them linked to sexual intercourse. In that context, fast identification of cases and sources of infection were essential for an optimal management. Polymerase chain reaction (PCR) was the preferred laboratory test. Our objective was to evaluate the usefulness of sera and nasopharyngeal samples for MPXV detection, and its application in contact studies without skin or mucosal injuries. Methods In the Hospital Clínico Universitario of Zaragoza (Spain), 106 samples from 50 patients, 38 male and 12 female, aged between 11 to 62 years, with skin lesions suspected MPXV infection were analysed. Samples were collected between May and September 2022 in inactivating universal viral transport medium (Biocomma). Samples were obtained between 1 and 19 days of evolution. To perform the real-time PCR, the VIASURE Monkeypox Virus Real Time PCR Detection Kit and the VIASURE 48—Real Time PCR System were used. Nucleic acid extraction was performed with the automated nucleic acid extraction system MagLEAD12gC using the MagDEA®Dx SV reagents. Results Table 1 shows the results expressed in Ct values. Skin, anogenital and serum samples were the most efficient for MPXV diagnosis. Nevertheless, these data are influenced by the sample collection date. Conclusion Real-time PCR was positive in samples from all locations in 71,4% of the infected patients. Respiratory samples (pharyngeal and nasopharyngeal) were the ones that showed the highest number of false negative results, whereas there was only one skin sample and one serum sample with a negative result. This study shows that sera samples can be used in the diagnosis of MPXV infection because of ease of collection, applicability to contacts without lesions and the option of serology testing for other STI agents (HBV, HCV, HIV, Treponema pallidum…).

B-392 Standardization of Nucleic acid Extraction Method From Thyroid Fine Needle Aspiration (FNA) Cells on Stained and Fixed Slides for Next-Generation Sequencing Protocols

Abstract Background Thyroid Fine Needle Aspiration (FNA) cytopathology is the best method for histological classification of thyroid nodules. However, between 20%–30% of thyroid nodules show indeterminate classification and are often referred for surgery due to the malignancy risk. Post-surgical histological reports indicate that 84% of these cases are benign, highlighting a high rate of unnecessary surgeries. In this sense, molecular approaches using next-generation sequencing (NGS) have been useful to confirm benignity or malignancy. As important as the choice of a powerful methodology to evaluate different genes simultaneously such as NGS, is the standardization of nucleic acid extraction methods to allow sufficient quality recovery of these molecules. Pre-analytical steps involved in FNA slide preparation include fixation and stain methods that contribute to the low quality and poor recovery of nucleic acid, representing a great challenge to NGS analysis. Thus, this work aims to describe a standardization of nucleic acid extraction method from FNA slides for NGS protocols. Methodology Nucleic acid isolation from 30 FNA samples collected between 2016–2022 were performed by Trizol or RecoverAll kits, according to the manufacturer’s instructions (ThermoScientific). Initially, cells’ slides were counted in an optical microscope, considering cell clusters >10 (equivalent to 1 counter unit). Before extractions with Trizol, the slides were submerged in xylene for 15 min. For RecoverAll extractions, the varnished slides were previously exposed to dry ice for 5 min, while the slides containing coverslip were also exposed to dry ice and submerged in xylene for 15 min. In all cases, the slides were hydrated before scraping and then extracted. Subsequently, the extraction of whole slides and split slides was tested. To split slides, one-half of the slide was separated for RNA extraction, while the remaining half was for DNA extraction. Then, the DNA/RNA were quantified using the Qubit™ 4 Fluorometer equipment (ThermoScientific) and concentrated using Amicon® Ultra filters (Millipore). Results The DNA and RNA quantifications range from 0.0720–1.77 ng/µL and 0.508–7.43 ng/µL, respectively. Among samples, 12% (DNA) and 62.5% (RNA) presented concentration below the quantification limit (too low). Four samples extracted with Trizol did not obtain recovery of nucleic acid and this methodology was discontinued. The use of Amicon® filter improved all samples’ yield. The number of thyroid cell clusters was not proportional to the amount of DNA/RNA recovered. The split slides showed worst RNA/DNA recovery, being ideal to use at least two slides for each patient since co-extraction using the RecoverAll kit was not possible. Conclusion The isolations of nucleic acid from FNA slides are essential to performing subsequent molecular tests, such as NGS. However, specific protocols for nucleic acid extraction from FNA slides are hardly found in the literature. This work showed the combination of methodologies that allowed the scraping of material from FNA slides by exposure to dry ice in samples with varnish or coverslip. The nucleic acid recovery capacity described for this type of sample indicates a way of possibilities to improve the FNA extraction methodology.

A-268 Evaluation of a Real-Time Multiplex PCR Assay for Simultaneous Detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis in Urine Specimens

Abstract Background Infections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) are among the most prevalent and treatable sexually transmitted diseases in the US. Currently, most available testing methods require patient samples to be split between CT/NG and TV assays. Here, we evaluate performance characteristics of the Seegene Novaplex STI Essential Assay, a real-time multiplex polymerase chain reaction assay for concurrent amplification and detection of multiple pathogens including CT, NG, and TV. Methods To prepare urine samples for extraction, 1 mL of each sample was centrifuged at 13 000 rpm for 15 min. The supernatant was discarded and the pellet was rehydrated in 190 µL of saline. Nucleic acid extraction was performed on a CyBio® FeliX liquid handler (Analytik Jena, Germany) using MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, MA). PCR amplification and detection were performed using Seegene Novaplex STI assay (Seegene Inc, South Korea) on a CFX96™ System (Bio-Rad Laboratories, CA). The assay utilizes Seegene MuDT™ technology that allows detection of multiple targets in a single channel without melting curve analysis. For method validation, precision studies were performed within-run and over 5 days. Limits of detection were verified by spiking known negative urine samples with CT/NG standards from Exact Diagnostics (Bio-Rad Laboratories, CA) and TV standards from ZeptoMetrix® (Antylia Scientific, IL). Method comparison was carried out by extracting and analyzing split patient urine samples against cobas® CT/NG assay (Roche Diagnostics, IL) and Solana® Trichomonas assay (Quidel, CA) performed by a reference laboratory. Additional accuracy studies were performed by spiking 20 negative patient samples with variable concentrations of the three pathogens. Results Within-run and between-day precision studies demonstrated consistent and reproducible results for all pathogens. Limit of detection was established at 10 copies/mL for CT and NG and 10 cells/mL for TV. Results of the spike-and-recovery studies were consistent with expected results for all three targets across evaluated concentrations. Patient correlation studies demonstrated 100% agreement with results obtained by the reference laboratory for both NG (32 total samples, 11 positive) and TV (22 total samples, 4 positive), and 96% agreement for CT (29 total samples, 10 positive). Conclusion The Seegene Novaplex STI Essential Assay offers a rapid and reliable method for detecting CT, NG, and TV in urine samples. The capability of the assay to detect multiple targets in a single sample well improves efficiency and turnaround time.

B-212 Application of Annexin A5Coated Beads for Collection of Extracellular Vesicles and Their Content RNA as Liquid Biopsy for Lung Cancer

Abstract Background Extracellular vesicles (EVs) carry molecules derived from cells, such as RNA. Measurement of tumor-derived extracellular RNA (exRNA) in EVs from plasma can be a promising approach for the development of blood-based cancer detection. However, the lack of an easy method for EVs separation limits its application. The aim of this study is to establish a procedure for EV collection and exRNA detection for non-small-cell lung cancer (NSCLC) diagnosis and monitoring. Methods Annexin A5-coated magnetic beads (ANX beads) were developed for EVs isolation. Next, we established a procedure for EV separation and exRNA detection from fluidic specimens using the ANX beads. Then blood samples from 22 NSCLC patients and 20 healthy controls were collected to evaluate the procedure and we detected different RNAs in the samples. Results We evaluated the ability of ANX beads in EVs separation and found that the beads could capture large and small EVs and could increase RNA concentration. Then we detected different RNAs in the samples and found that CK19 mRNA, MALAT1 lncRNA, and miR155 microRNA had good performance to differentiate NSCLC from healthy controls. Conclusion In conclusion, this assay protocol could separate EVs to enrich RNA concentration. The EVs separation employed was captured by magnetic beads which could be adopted into an automatic nucleic acid extract system to conveniently concentrate and extract nucleic acid from EVs.

B-256 Genotype Distribution of the Hepatitis C Virus: 10 Years Data of a Clinical Laboratory in Brazil

Abstract Background Hepatitis C virus (HCV) infection is a significant health problem worldwide and is a major cause of severe liver disease including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is divided into six genotypes with several sub-genotypes depending on the geographic region, some genotypes are endemic. The purpose of this study was to describe the frequency of the HCV genotype (G) profile by gender and age using a database of a large Brazilian clinical laboratory. Methods This was a retrospective analysis from January 2012 to December 2022 based on molecular data of 3.566 isolated from clinical samples. The automated extraction and purification of nucleic acids were conducted from EDTA plasma samples of individuals suspected of HCV infection. Then, nucleic acid amplification was performed using oligonucleotide probes labeled with fluorescent dyes specific for the qualitative identification of HCV genotypes 1 to 6 and subtypes A and B of genotype 1. Results A total of 3.566 HCV genotype cases were analyzed during the period. The genotype distribution was (2.653/74.4%) G1 (48.1% G1A and 46.4% G1B), (733/ 20.5%) G3, (133/3.7%) G2, (35/0.9%) G4, (11/0.3%) G5 and (1/0.02%) G6. Twelve patients had multiple genotypes over the last ten years. At least one individual changed the genotype over a 3-year evaluation period, and 11 (0.30%) changed it twice. The genotypes G1 and G3 were predominant in all years analyzed. The prevalence of G1 and G3 was 75% and 19.6% among females and 74% and 21.2% among male patients, respectively. The analysis was conducted on individuals aged 6 to 99 years, with an overall average age of 52.6±13 years. The average age among men was 51.1 ± 12 years and among women, it was 54.7 ± 13 years. According to the age groups evaluated, genotype 1 was the most frequent, ranging from 69.3% (21 to 30 years old) to 75.8% (over 60 years old). The main subtype in the over 60 years old group was 1B. The highest frequency of genotype 2 was observed in the group over 60 years old (6.1%). The patient's symptoms, clinical history, or further diagnostic procedures were not accessible. Conclusion The study found that genotypes 1 and 3 were the most prevalent in Brazil over the ten-year period, with genotype 1 being the most frequent among all age groups. Genotype 1B was most prevalent in individuals above 60 years old. The change of genotype per individual was less than 1%, but further attention is required for potential virus tropism.

B-018 Compact all in one, Fully Automated Molecular Diagnostic System “ExiStation™ FA 96” in 100 minutes

Abstract Background Traditional PCR method’s instruments are discrete the processes of sample collection, nucleic acid(NA) extraction, and NA amplification. As human labor is required between the instruments, there is a risk of human error, infection and contamination. Biosafety facilities such as biosafety cabinet, level D protective clothing, and negative pressure facilities are required for ensuring safe testing. To improve the testing environment, the molecular diagnostic market has developed and started using automated instruments, though, which take up larger space and take longer time to test. As a result, it is necessary to develop instrument that takes up less space and reduces testing time to a minimum, so, space and time can be saved. Methods In this study, we developed an automated systematic genetic diagnostic instrument. In order to carry out the entire process within one integrated instrument, automatic mechanism of de-capping and re-capping of sample tube, sample aspiration, and sample transfer were developed. The system also includes integrated key steps of NA extraction and amplification (qPCR). We developed a primer/probe design technology that can accurately detect viruses with high accuracy in performing PCR tests using extracted genes, as well as precision temperature control algorithm and optimized real-time nucleic acid amplification using reverse transcription enzymes and gene amplification enzymes. In order to carry out all these functions within one device, we maximized user convenience by developing software for instrument operation, result analysis, and a Graphic User Interface for automation Results ExiStation™ FA 96 is largely composed of sample processing, gene extraction, and gene amplification (qPCR). (Photo to be in poster) Sample Processing automatically opens a cap of sample tube, then transfer a required quantity of sample to cartridge. While opening the cap, the barcode attached to sample tube is automatically identified and the information required for a test is entered. After sampling, the cap is automatically re-capped and placed back in the sample tube rack. When collecting samples, it measures the sample level in the tube and indirectly check the viscosity by detecting the pressure inside the tip, minimizing aspiration errors. The Extraction step performs the function of extracting genes. It consists of three modules of 32 wells, and the number of samples can even be adjusted in eight units, so the instrument can be operated flexibly upon the number of samples requested. Then, the qPCR step follows. The ExiStation™ FA 96 setup allows simultaneous testing of up to 20 targets for 94 samples in approximately 100 min. The instrument is equipped with a HEPA filter and UV lamp to maintain clean, and since the sample tubes are inserted directly on the instrument, it is no of necessity or save the costs such as of negative pressure rooms, biosafety cabinet, and level D protective clothing. Conclusion ExiStation™ FA 96 is a fully automated molecular diagnostic test instrument that can quickly and accurately process large amounts of samples, while greatly reducing user risks and shortening molecular diagnostic test time.

B-016 Automated Next Generation Sequencing Fluidic-Based Device for Sample to Library Preparation: A Flexible Solution for Rapid Decentralized Sequencing for Infectious Disease Diagnostics

Abstract Background In addition to the established use of Next Generation Sequencing (NGS), such as drug resistance monitoring in HIV, the recent COVID19 pandemic has shown the crucial need of NGS technologies as tools for identification of clinically relevant variants of the SARS-CoV-2 virus. Typically, in these settings, to be cost-effective most NGS analyses were conducted using larger batch sizes often resulting in long turn-around times of 2–3 weeks the need of trained personnel, and centralized testing facilities. Methods We have developed an easy to use automated fluidic-based benchtop device to bridge a perceived gap in NGS library preparation and provide a solution for decentralized testing that does not require specially trained personnel. With a batch size of 6, smaller hospitals and laboratories can perform NGS library preparation on fresh or archived samples with a fast turn-around-time of two days, facilitating earlier reporting to speed up diagnosis and treatment. The Helaxy NGS fluidic device performs sample nucleic acid extraction and a 2-step PCR in a plastic fluidic card, which is the sole consumable required for the extraction and library prep process.The sample is applied to the fluidic card by manual pipetting followed by loading of the card into the device. Nucleic acids are extracted with a combination of magnetic bead pull-down and centrifugal force in a purification chamber to give an eluate that is spun to a separate PCR chamber that uses the concept of thermal induction and cycling. Barcoding for library preparation follows within the same PCR chamber and the resulting product is transferred into another fluidic card for size selection purification, normalization and pooling to generate a library for input into compatible NGS sequencers, e.g., but not confined to, Illumina instruments. Results In order to demonstrate clinical utility we have shown that the Helaxy NGS fluidic device can successfully extract nucleic acid from at least three sample types: SARS-CoV-2 RNA from nasopharyngeal swabs, HIV RNA from plasma and 16S RNA from stools. Using in-house and commercial assays, we have generated libraries of target amplicons using the fluidic device workflow followed by sequencing with Illumina sequencers iSeq and MiSeq. Sequencing results analysed and reported by the Helaxy NGS Bioinformatics Reporting Pipeline revealed expected variants and mutations in the samples. We correctly identified all the unique mutations such as L211del and G446S in commercially available inactivated Omicron BA1 variant of the SARS-CoV-2 virus as well as characterised drug-resistant mutations such as L90M in the Protease gene and D67N and Y181C in the Reverse Transcriptase gene in commercially available inactivated HIV-1 virus. Conclusion The Helaxy NGS fluidic device can thus effectively provide an easy-to-use automated solution for sample library preparation in decentralized facilities to reduce the turn-around-time from sample collection to result reporting for clinical diagnosis and treatment.

B-388 Clinical Performance of Xpert BCR-ABL Ultra Assay for Isoform p210 Quantitation

Abstract Background Chronic myelogenous leukemia (CML) is one of the most common hematologic neoplasms. More than 95% of patients with CML have the distinctive Philadelphia chromosome (Ph1) that results from a translocation between the chromosomes 9 and 22, and consequently in the BCR::ABL1 fusion gene. This fusion gene produces a tyrosine kinase with deregulated activity that plays a key role in the development of CML. Monitoring BCR::ABL transcript levels in patients on tyrosine kinase inhibitor (TKI) therapy using real-time quantitative PCR is standard of care in the management of CML patients. The test utilizes a quantitative, real-time reverse transcription polymerase chain reaction (RT-qPCR) to measure BCR::ABL1 to ABL1 percent ratios on the International Scale (%IS), in t(9;22) positive CML patients during monitoring of treatment with TKIs. In this context, this study evaluated clinical performance of the Xpert® BCR-ABL Ultra assay for quantitation of BCR::ABL1 p210 transcripts. Methods The clinical performance of assay was evaluated by comparison to an RT-qPCR assay routinely used in our institution, which also detects and quantifies the mRNA transcripts for the p210 translocation types and uses the ABL gene as an endogenous control. In total, 30 fresh peripheral blood samples of CML patients with the level of BCR::ABL1 transcripts (international scale, IS) between >50% and undetectable were enrolled in this study. All steps of protocols were performed according to manufacturer ́s instructions. Results The comparison of two assay revealed consistente molecular response (MR) level. Linear regression analysis showed high correlation between the assays (R2 = 0.974, P < 0.0001) The slope of regression curve were not significantly different from the value of 1 (1.003; 95% CI, range 0.943–1.069). The evaluation of MR level was identical in 21/28 (75%) samples. Two samples were excluded from the study due to failure to control ABL1 (Ct > 18). The median of ABL1 copies per sample was 66 011 copies (range 20 010–236 171) for conventional RT-qPCR assay. Xpert BCR-ABL Ultra reports %IS (MR), but this test does not report ABL1 copy numbers. If the Xpert BCR-ABL Ultra test reports a positive or negative result and the ABL1 Ct value is below 18, a minimum of 32 000 ABL1 copy numbers is guaranteed in the reaction. The BCR::ABL1 negativity was concordant in 2/8 samples. Conclusion We showed that the results Xpert® BCR-ABL Ultra assay (MR) are in good correlation with the results of RT-qPCR conventional method.The Xpert® BCR-ABL Ultra assay simplifies the generation of results by integrating nucleic acid extraction, amplification, and quantification of the BCR::ABL1 p210 transcripts in peripheral blood specimens. The assay turn-around-time is approximately 3 h. This does not differentiate between b2a2 or b3a2 fusion transcripts.This assay is unable to detect BCR::ABL1 p190 or other rarer translocations.

B-257 Rapid Implementation of Monkeypox Detection in a Brazilian Diagnostic Laboratory

Abstract Background Monkeypox is a double-stranded DNA virus (approximately 197 kbp) belonging to the genus Orthopoxivirus. It is responsible for sporadic outbreaks in countries in Africa. Recently, in May 2022 a patient with a recent travel history to Nigeria tested positive for monkeypox in the UK. After that, people with symptoms (usually epithelial pustular lesions) were diagnosed with the virus in several countries, such as Portugal and the United States. In Brazil, the first case was confirmed in June 2022, the patient had traveled to Spain and Portugal. It was in this context that the Technical Operational Nucleus of our laboratory, Sabin Diagnosis and Health, located in Brasilia, capital of Brazil, validated and implemented a test to detect the genetic material of Monkeypox. Thus, in this work we present the implementation of this test. Method For the detection of Monkeypox viral DNA we chose to implement a real-time PCR (qPCR) methodology. The validated samples were swabs and wound scabs. The nucleic acid extraction chosen was using the Virus Pathogen kit from (QIAGEN), ‘midi’ protocol in the qiasymphony equipment (QIAGEN). Later we validated the extraction with the Maxwell RSC Viral Total Nucleic Acid Purification Kit extraction kit (Promega). For qPCR, we selected monkeypox-specific primers and probes, which were not able to recognize other Orthopoxiviruses (such as human pox) to ensure specificity. We used RNASEP-specific primers as an endogenous extraction control. As a positive control (PC) we used a synthetic double-stranded DNA fragment with the target sequence flanked by 10 bp. The limit of detection of the test was performed using serial dilutions of the positive control. Furthermore, given the initial absence of a positive sample for validation, we designed four pairs of primers that amplified four different regions of the viral genome for sequencing by the Sanger technique. The first positive samples were confirmed by sequencing these four regions. Results The protocol was designed in the first week after the confirmation of the first case in Brazil. The first tests with the PC showed the effectiveness of the primers. After adjusting the reaction conditions to assess the specificity of the test, we tested the protocol with samples known to be positive for other viral pathogens and obtained negative results for all of them, confirming the high specificity. After that we experimentally determined the limit of detection (25 copies per reaction). On July 8, 2022, one month after the first confirmed case in Brazil in the state of São Paulo, we released the Monkeypox detection test with the methodology described above. Since then, we have performed 193 tests, of which 59 (31%) were positive, most of them (56–95%) in men. The first 10 positive results were confirmed by genetic sequencing (Sanger). Discussion The implementation of the Monkeypox detection test by our laboratory allowed a quick response for Brazilian patients who needed the test after the virus entered the country.

B-250 Simultaneous Detection of Twelve Pathogens of Sexually Transmitted Infections Using an Innovative All-in-One PCR Instrument

Abstract Background Every day, nearly 1 million people are infected with sexually transmitted infections (STI) that are transmitted through sexual contact. Early diagnosis and treatment of STI are critical in reducing their transmission, and PCR is the most sensitive method among diagnostic tests. To address this need, we have developed a new STI Real-Time PCR-based diagnostic kit, which is capable of detecting twelve STI pathogens simultaneously in up to 94 specimens at once. To evaluate the analytical performance, we used the ExistationTM FA System, which automatically performs from decapping primary sample tubes followed by nucleic acid extraction to PCR analysis within 2 h. Twelve (12) pathogens of STI are described below: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Ureaplasma urealyticum (UU), Mycoplasma genitalium (MG), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Herpes simplex virus type1 (HSV1), Herpes simplex virus type2 (HSV2), Ureaplasma parvum (UP), Treponema pallidum (TP), Candida albicans (CA), Gardnerella vaginalis (GV), from male urine and female vaginal swabs. Methods The analytical performance evaluation was performed using the new STI diagnostic kit to detect twelve pathogens of STI. The analysis was performed using culture fluids (CT, NG, UU, MG, TV, MH, HSV1, HSV2, UP, CA, GV; ATCC, USA) and TP genomic DNA (Vircell, Spain), which were spiked in urine and vaginal swab media. The limit of detection (LoD) was determined by testing multiple replicates of serial dilutions of the spiked sample around the expected LoD. The LoD was defined as the lowest dilution at which the assay detected ≥95% of 24 replicates. To evaluate precision, each sample was tested 2 times a day in duplicate per run for 5 days. To assess cross-reactivity, twenty-two bacteria and viruses related to STI were used. Results The LoD was determined 60.26 IFU/mL, 35.48 cfu/mL, 467.74 copy/mL, 45.71 cell/mL, 19.5 cells/mL, 316.23 copy/mL, 199.53 TCID50/mL, 13.18 TCID50/mL, 588.84 copy/mL, 993.25 copy/ml, 660.69 copy/mL, 389.05 copy/mL in Urine and 47.86 IFU/mL, 27.54 cfu/mL, 363.08 copy/mL, 38.90 cell/mL, 15.14 cells/mL, 245.47 copy/mL, 181.97 TCID50/mL, 8.71 TCID50/mL, 575.44 copy/mL, 741.31 copy/ml, 524.81 copy/mL, 288.40 copy/mL in vaginal swab media for CT, NG, UU, MG, TV, MH, HSV1, HSV2, UP, CA, GV, and TP, respectively. For repeatability assay, the 12 STI samples showed similar Ct value in each run, with all standard deviation (SD) were less than 2.0. In terms of cross-reactivity, there were no positive results obtained from testing bacteria and viruses using the new STI diagnostic kit. Conclusions The ExistationTM FA system is highly convenient platform for Real-Time PCR-based diagnostics. Particularly, our new STI diagnostic kit provides an accurate and efficient method for simultaneous detection of 12 STI pathogens.

Development and application of a rapid scheme for detection of respiratory virus nucleic acid

This study aimed to develop a portable, accurate and easy-to-operate scheme for rapid detection of respiratory virus nucleic acid. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the effect of extraction-free respiratory virus treatment reagent (RTU) on viral nucleic acid treatment and the effect of ultra-fast fluorescence quantitative PCR instrument (FQ-8A) on nucleic acid amplification, respectively. RTU and FQ-8A were combined to develop a rapid detection scheme for respiratory virus nucleic acid, and the positive detection rate was judged by Ct value using a fluorescence quantitative PCR instrument, and the accuracy of the scheme in clinical samples detection was investigated. The results showed that RTU had comparable sensitivity to the automatic nucleic acid extraction instrument, its extraction efficiency was comparable to the other 3 extraction methods when extracting samples of different virus types, but the extraction time of RTU was less than 5 min. FQ-8A had good consistency in detection respiratory syncytial virus (RSV) and adenovirus (ADV) compared with the control instrument ABI-7500, with kappa coefficients of 0.938 (P < 0.001) and 0.887 (P < 0.001), respectively, but the amplification time was only about 0.5 h. The RTU and FQ-8A combined rapid detection scheme had a highly consistent detection rate with the conventional detection scheme, with a sensitivity of 91.70% and specificity of 100%, and a kappa coefficient was 0.944 (P < 0.001). In conclusion, by combining RTU with FQ-8A, a rapid respiratory virus nucleic acid detection scheme was developed, the whole process could be completed in 35 min. The scheme is accurate and easy-to-operate, and can provide important support for the rapid diagnosis and treatment of respiratory virus.

Research on a Magnetic Separation-Based Rapid Nucleic Acid Extraction System and Its Detection Applications.

Nucleic acid extraction represents the "first step" in molecular diagnostic experiments. The quality of this extraction serves as a fundamental prerequisite for ensuring the accuracy of nucleic acid detection. This article presents a comprehensive design scheme for a rapid automated nucleic acid extraction system based on magnetic separation. The design and implementation of the system are analyzed and investigated in-depth, focusing on the core methods, hardware control, and software control of the automated nucleic acid extraction system. Additionally, a study and evaluation were carried out concerning the nucleic acid extraction and detection aspects encompassed by the system. The results demonstrate that the temperature deviation in the lysis and elution fluids is approximately ±1 °C, the positioning accuracy of the system's movement is ±0.005 mm, the average magnetic bead recovery rate is 94.98%, and the average nucleic acid recovery rate is 91.83%. The developed automated system and manual methods are employed for sample extraction, enabling the isolation of highly pure nucleic acids from bacteria, blood, and animal tissues for RT-PCR detection. The instrument employs lysis temperatures ranging from 70-80 °C, elution temperature of 80 °C, and drying time of 5-10 min, with a total extraction time of less than 35 min for different sample types. Overall, the system yields high nucleic acid concentration and purity, exhibits stable instrument operation, good repeatability, high efficiency, and low cost. It meets the requirements of genetic-level research and is worthy of clinical promotion and usage.

Extraction-Free Detection of SARS-CoV-2 Viral RNA Using LumiraDx's RNA Star Complete Assay from Clinical Nasal Swabs Stored in a Novel Collection and Transport Medium.

Background: The rapid detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is vital for patient care. The LumiraDx™ SARS-CoV-2 RNA Star Complete (RSC) is an Emergency Use Authorization-recognized molecular test using nasal/nasopharyngeal swabs immersed in a viral/universal transport medium (VTM/UTM). However, there is a critical need for an alternative medium for point-of-care testing (POCT). This study aimed to investigate Xtract-Free (XF), a novel collection medium for transport and direct (extraction-free) use with nucleic acid tests. Methods: Using serially diluted SARS-CoV-2 viral RNA (vRNA) in a routine UTM and XF, a limit of detection (LOD) was established via an RSC test and a quantitative reverse transcription PCR (RT-qPCR). Additionally, the results obtained from a panel of 108 clinical "car-side" nasal swabs collected in XF during the coronavirus pandemic and assessed using the "gold-standard" RT-qPCR assay were compared to Lumira's RSC assay. Results: The average replicate RT-qPCR cycle threshold (CT) values for vRNA in XF and UTM were observed to be equivalent. An LOD for which five out of five replicates were detected using XF or VTM was approximately 2000 copies/mL. The nasal swabs collected in XF exhibited 93.9% positive percent agreement (sensitivity) and 100% negative percent agreement (specificity) compared to the RT-qPCR. Three specimens tested positive via an RT-qPCR were negative when tested via RSC; however, all three samples had CT values ≥ 36.4. Conclusions: XF is equivalent to VTM/UTM and is compatible for use with the RSC test. Furthermore, XF can be used directly with RT-qPCRs and rapid antigen testing without the requirement for separate nucleic acid extraction (an extraction-free process), making it ideal for cost-effective high-throughput and decentralized respiratory testing. Impact Statement: This study is the first to evaluate LumiraDx's SARS-CoV-2 RNA Star Complete assay in concert with Xtract-Free (XF), a novel collection medium containing a proprietary RNase-inactivating technology for the rapid, "extraction-free" detection of SARS-CoV-2 RNA from clinical nasal swabs. Specimens collected in XF combined with rapid LumiraDx detection provide a safe and sensitive alternative to VTM/UTM, and Molecular Transport medium (MTM) for high throughput, "extraction-free" molecular detection.

Rapid extraction-free detection of the R132H isocitrate dehydrogenase mutation in glioma using colorimetric peptide nucleic acid-loop mediated isothermal amplification (CPNA-LAMP).

The R132H isocitrate dehydrogenase one (IDH1) mutation is a prognostic biomarker present in a subset of gliomas and is associated with heightened survival when paired with aggressive surgical resection. In this study, we establish proof-of-principle for rapid colorimetric detection of the IDH1-R132H mutation in tumor samples in under 1 hour without the need for a nucleic acid extraction. Colorimetric peptide nucleic acid loop-mediated isothermal amplification (CPNA-LAMP) utilizes 4 conventional LAMP primers, a blocking PNA probe complementary to the wild-type sequence, and a self-annealing loop primer complementary to the single nucleotide variant to only amplify the DNA sequence containing the mutation. This assay was evaluated using IDH1-WT or IDH1-R132H mutant synthetic DNA, wild-type or IDH1-R132H mutant U87MG cell lysates, and tumor lysates from archived patient samples in which the IDH1 status was previously determined using immunohistochemistry (IHC). Reactions were performed using a hot water bath and visually interpreted as positive by a pink-to-yellow color change. Results were subsequently verified using agarose gel electrophoresis. CPNA-LAMP successfully detected the R132H single nucleotide variant, and results from tumor lysates yielded 100% concordance with IHC results, including instances when the single nucleotide variant was limited to a portion of the tumor. Importantly, when testing the tumor lysates, there were no false positive or false negative results.

Successful Confirmation of Dual Genital Herpes Co-Infection with Herpes Simplex Virus 1 and Herpes Simplex Virus 2 Using Unbiased Metagenomic Next-Generation Sequencing.

Dual co-infection with both HSV-1 and HSV-2 is rare, with few cases reported in the literature. In this case report, we describe the successful use of unbiased metagenomic next-generation sequencing (mNGS) as a rapid and alternative method for confirming dual genital herpes co-infection. Our case involves a 74-year-old woman who presented with genital lesions and initially tested positive for both HSV-1 and HSV-2 via the Luminex ARIES HSV 1&2 assay. The entire mNGS process, from nucleic acid extraction to result analysis, was completed in less than 48 h. Using mNGS, we identified mapped reads specific to either HSV-1 or HSV-2 and screened the sequences to rule out mis-genotyping by the Luminex ARIES assay. Notably, the generated sequences can reveal sequence variations within multiple gene regions, demonstrating the potential of mNGS for identifying novel HSV-1 and HSV-2 variants. Our findings suggest that mNGS can serve as a rapid and reliable alternative confirmatory method for dual genital herpes infections, providing valuable information to guide appropriate treatment options for patients. By eliminating the need for prior knowledge of causative agents, mNGS offers an unbiased approach for detecting and characterizing viral co-infections.

Simple Point-of-Care Nucleic Acid Amplification Test for Rapid SARS-CoV-2 Infection Diagnosis.

After three years of the SARS-CoV-2 pandemic, the demand for developing field-deployable point-of-care (PoC) molecular diagnostic tests has increased. Although RT-qPCR is the molecular diagnostic gold standard and is accurate, it is not readily applied to point-of-care testing (POCT). Meanwhile, rapid diagnostic kits have the disadvantage of low sensitivity. Recently, rapid isothermal nucleic acid amplification technology has emerged as an alternative for rapid diagnosis. Here, we developed a rapid SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP)-lateral flow assay (LFA) kit. This kit includes a Chelex-100/boiling nucleic acid extraction device and a one-step amplification detection apparatus capable of performing the entire process, from RNA extraction to detection, and diagnosing SARS-CoV-2 infection within 40 min without contamination. The detection limits of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 100 plaque-forming units (PFUs) mL-1 and 10-1 PFU mL-1 for RNA samples extracted using the Chelex-100/boiling nucleic acid extraction device and commercial AdvansureTM E3 system, respectively. The sensitivity and specificity of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 97.8% and 100%, respectively. Our SARS-CoV-2 RT-LAMP-LFA kit exhibited high sensitivity and specificity within 40 min without requiring laboratory instruments, suggesting that the kit could be used as a rapid POC molecular diagnostic test for SARS-CoV-2.

The Relationship between T1DM and the Polymorphisms of IL2RA (Rs2104286) and PTPN22 (Rs2476601) Genes among Children

Background: Type 1 diabetes mellitus (T1DM) is an autoimmune condition caused by T lymphocytes that results in the death of pancreatic cells. Protein tyrosine phosphate non-receptor type 22 (PTPN22) and interleukin 2 receptor alpha (IL2RA) polymorphisms have been discovered to have a connection to a number of autoimmune illnesses, including T1D. Objective: The purpose of this study is to clarify the significance of the polymorphisms PTPN22 (rs2476601) and IL2RA (rs2104286) in the susceptibility to T1DM in young Iraqi children. Methods: The study included 60 Iraqi children diagnosed with T1DM within the past three years and a control group of 30 healthy individuals without diabetes or autoimmune diseases. In order to conduct a molecular investigation. Five ml of venous blood from 90 participants (60 patients and 30 controls) was collected for nucleic acid extraction. Results: The PTPN22 (rs2476601) and IL2RA (rs2104286) polymorphisms were genotyped using the amplification refractory mutation system (ARMS) technique and specific primers. According to the results, PTPN22 (rs2476601) has a wild-type homozygous C/C genotype and C allele frequency of 90%, a mutant C/T genotype frequency of 10%, and no T allele. Following were the genotype frequencies for IL2RA (rs2104286): AA, AG, and GG in T1DM patients were 79%, 16%, and 4%, respectively, compared to 83%, 13%, and 3% in controls. Conclusion: The polymorphisms of PTPN22 rs2476601 and IL2RA rs2104286 did not significantly differ in their connection with type 1 diabetes. It does not appear to affect the susceptibility of Asian Iraqis to T1D.

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests.

Isothermal nucleic acid amplification tests have recently gained popularity over polymerase chain reaction (PCR), as they only require a constant temperature and significantly simplify nucleic acid amplification. Recently, numerous attempts have been made to incorporate paper microfluidics into these isothermal amplification tests. Paper microfluidics (including lateral flow strips) have been used to extract nucleic acids, amplify the target gene, and detect amplified products, all toward automating the process. We investigated the literature from 2020 to the present, i.e., since the onset of the COVID-19 pandemic, during which a significant surge in isothermal amplification tests has been observed. Paper microfluidic detection has been used extensively for recombinase polymerase amplification (RPA) and its related methods, along with loop-mediated isothermal amplification (LAMP) and rolling circle amplification (RCA). Detection was conducted primarily with colorimetric and fluorometric methods, although a few publications demonstrated flow distance- and surface-enhanced Raman spectroscopic (SERS)-based detection. A good number of publications could be found that demonstrated both amplification and detection on paper microfluidic platforms. A small number of publications could be found that showed extraction or all three procedures (i.e., fully integrated systems) on paper microfluidic platforms, necessitating the need for future work.

P20.08.B CLINICAL AND MOLECULAR ANALYSES OF A SERIES OF ADULT PILOCYTIC ASTROCYTOMA

Abstract BACKGROUND Pilocytic astrocytoma (PA) is a rare, grade 1 subtype of glioma. It is most frequent during the first two decades of life, the incidence rate decreasing with age. PAs are usually located in the cerebellum, although they have also been described in other sites. They are circumscribed, solid-cystic, with low cellularity and generally slow-growing tumours with good prognosis in the paediatric age group. PA is associated with MAPK pathway dysregulation, most often showing BRAF gene alterations. PA is very rare in older adults and is associated in some cases with a worse prognosis. In this work we set out to analyse a series of adult PA from a clinical, morphological and molecular point of view. MATERIAL AND METHODS Clinical data were collected from a group of adult patients diagnosed with PA. Nucleic acid extraction was performed by automated extraction and the samples were analysed through a 30-gene targeted panel for gliomas by using Next-Generation Sequencing. Tandem duplication causing fusion of KIAA1549 and BRAF genes (K-B fusion) was analysed by RT-PCR. RESULTS Thirty-four patients were included, with a mean age of 34.4 years (range, 18-57 years). The male/female ratio was 26/8. Mean overall survival was 150.5 months, with more than 10 years of mean follow-up. At the time of the last review, 91% (30/33) of the patients remained alive, while the remaining 9% (3/33) died. Neuroanatomic locations (31 cases) were as follows: cerebrum/lobar 23% (7), cerebellum 39% (12), brainstem 10% (3), ventricle 6% (2), spinal cord 10% (3), optic nerve 13% (4). The K-B fusion was detected in 44% of cases (8/18). Of these, 87.5% (7/8) and 12.5% (1/8) showed fusions involving exons 16:9 and 15:9, respectively. K-B positive cases had a mean age of 40 years. Analyses by NGS revealed mutations in NF1 (15%), BRAF (11%), PDGFRA (11%), EGFR (7%) and PIK3CA (7%). Other variants observed in 4% of cases involved ATRX, CIC, IDH1, KEL, KRAS, MET, MSH6, PTPN11 and RB1. No alterations were detected in 3 of the cases, suggesting that other untested driver alterations could be present. CONCLUSION In this series, no significant differences with childhood PA in the frequency of molecular alterations were observed, in contrast to other gliomas where there are strong differences in the molecular alterations between adult and paediatric tumours. The most common genetic alterations in adult PA involved the MAPK pathway, as previously reported. In contrast to other adult PA studies, the frequency of K-B fusion was similar to that of paediatric PA. Molecular alterations related to an aggressive behaviour or to other glioma types were identified, such as mutations of ATRX, PDGFRA, NF1 or PI3KCA, which can suggest other tumour types. Our results suggest the need for multilayer histological and molecular diagnosis to appropriately manage adult PA.