bluuebunny/crossref_metadata_2025 huggingface dataset:
B-201 Development of a Quantitative Gas Chromatography Tandem Mass Spectrometry Method for Metabolic Profiling of Urine Organic Acids
Authors: Ptolemy A S , Young K , MacDonald C , Garrett R , Kellogg M , Peake R W
Published: September 2023

Background

Urine organic acids (UOA) analysis is an essential investigation for inborn errors of metabolism (IEMs). The vast majority of methods use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas chromatography tandem mass spectrometry (GC-MS/MS) offers greater sensitivity and selectivity, and enables the combination of qualitative screening with quantitative analysis. We describe the development and preliminary validation results of a quantitative GC-MS/MS method.

Methods

Urine creatinine concentrations were measured (CobasPro, Roche Diagnostics) and normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration standard and QC were incubated with a hydroxylamine solution followed by a liquid-liquid extraction. Further sample concentration permitted the isolated UOA to be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) with 1% chlorotrimethylsilane (TMCS) prior to injection. GC-MS/MS analysis was performed using a GC-MS-TQ8050 triple quadrupole system (Shimadzu) with a 30 m DB-5MS (Agilent J&W) capillary column (0.25 mm, ID of 0.25 µm). Split injection mode was deployed with an initial column temperature of 70°C. The mass spectrometer was operated in electron ionization (EI) and multiple reaction monitoring (MRM) modes. The linear concentration ranges of the targeted metabolites within the devised GC-MS/MS protocol were determined.

Results

The optimal temperature gradient for high-resolution UOA separations was derived and their respective retention times determined. MS/MS detection parameters were optimized to permit the identification of precursor and production ions for all (N = 38) derivatized UOA. Total analysis time was 33.8 min per injection. The achieved linear concentration ranges for each metabolite was respectively determined. Linear concentration ranges and correlation coefficients of representative UOA: 3-hydroxyisovaleric acid (range = 9 to 708 µmol/L, R2 = 0.9874); lactic acid (range = 15 to 751 µmol/L, R2 = 0.9936); 3-hydroxybutyric acid (range = 15 to 751 µmol/L, R2 = 0.9991); and 2-methyl-3-hydroxybutyric acid (range = 10 to 501 µmol/L, R2 = 0.9996). The correlation coefficients for all remaining UOA ranged from R2 = 0.9458 (4-hydroxyphenylpyruvic acid) to R2 = 0.9998 (3-methylglutanoic acid and ethylmalonic acid).

Conclusion

The devised GC-MS/MS protocol permits the unbiased identification and quantification of UOA used to investigate IEMs. The linear concentration ranges for all analytes were determined and are appropriate for monitoring these markers of inherited enzyme deficiencies. The analytical performance of this UOA GC-MS/MS method must be further validated and its diagnostic performance for selected IEMs characterized.
B-213 Colorimetric LAMP as an Effective Tool for Rapid Molecular Diagnostics
Authors: Tanner N A , Li Z , Bruce J , Moncion R A , Cunningham C V , Langhorst B W , Carlow C K
Published: September 2023

Background

LAMP has become a standard molecular diagnostic technique, and its robustness and flexibility have enabled rapid, simple tests outside of traditional laboratory settings. The need for additional diagnostic methods became urgent during the SARS-CoV-2 pandemic, and LAMP was extensively evaluated and validated for numerous testing applications. We have extended the utility of LAMP to enable colorimetric detection, multiplex assays, variant identification, and robust direct tests for point of-care and workplace surveillance testing.

Methods

We utilized fluorescent and colorimetric RT-LAMP for assay development and our workplace screening program. A simple lysis buffer was applied to whole saliva samples and added directly to our colorimetric LAMP SARS-CoV-2 tests, with more than 100 000 samples tested. For variant calling we developed novel hybridization probes with single-base sensitivity to regions of sequence changes, and we characterized more than 5000 LAMP assays internally positioning mismatches in the LAMP primer regions to ensure robustness to and tolerance of mutations.

Results

The colorimetric LAMP assay displayed a sensitivity of 97% and specificity of 100%, and during its utilization in our in-house CLIA-certified workplace screening has identified >300 unique positive cases derived from all variants of concern. 3654 blinded samples were analyzed by 3 operators and colorimetric readout was found to have a strong concordance with real-time fluorescence measurement (kappa = 0.999).

Conclusion

RT-LAMP is remarkably tolerant of sequence variation, with no individual positional mutation significantly affecting assay performance. Simple visual readout can be utilized for a reliable diagnostic indicator when sophisticated instrumentation is not available or desired.
B-224 Comparison of an Unlabeled Probe High Resolution Melting Analysis (HRMA) Assay for Factor V Leiden 1691 G > A Mutation to a TaqMan Hydrolysis Assay
Authors: Han H H , Lewis S , Hoang A , Badri K
Published: September 2023

Background

The clinically significant Factor V Leiden (FVL) mutation (1691 G > A) causes replacement of Arg with Gln, preventing activated protein C from inactivating Factor V leading to a lengthened clotting process. Individuals with the Factor II and Factor V Leiden mutations have an increased risk for venous thrombosis. The aim of this study is to compare an unlabeled probe High Resolution Melting Analysis (HRMA) assay for Factor V Leiden mutation to a TaqMan hydrolysis assay. High Resolution Melting Analysis (HRMA) is a post-PCR, homogenous, closed tube system for the detection of sequence variants. Post-PCR, the amplicons are heated gradually until the melting temperature is reached and the fluorescent dye unbinds from the amplicon and exhibits low fluorescence. A melt-curve analysis is generated that is characteristic of a particular sequence variant, therefore HRMA allows for comparison of one base changes in genetic sequences based on their differences in melting rate.

Methods

HRMA Assay: Blood samples were collected in EDTA tubes and DNA extracted using the Roche MagnaPure. DNA content was determined to supply 66 ng of genomic DNA to each reaction mixture. Factor Vc.1601 G > A Novallele Genotyping Assay by Canon Biosciences was used. The following primer and probe sequences were used: forward primer sequence: 5′-CCCATTATTTAGCCAGGAGA-3′, reverse primer sequence: 5′-GCCTCTGGGCTAATAGGACT-3′, unlabeled probe sequence: 5′-TTCAAGGACAAAATACCTGTATTCCTCGCCT/3AmM/3′. PCR was performed in Roche Light Cycler 480 with 11 uL volumes per reaction of 66.6 ng of DNA. Forward primer, reverse primer and probe concentration was 100 uM and a 5× volume excess amount of forward primer and probe was used. A 151 bp amplicon was generated. Three control references obtained from Coriell Institute were used including wild type (WT), mutant (MUT) and heterozygote (HET). The HRMA FVL assay was performed on a total of seventeen samples. The TaqMan hydrolysis FVL assay uses the following DNA primers and probe: forward primer: GCCTCTGGGCTAATAGGACTACTTC; reverse primer: TTTCTGAAAGGTTACTTCAAGGACAA; WT FVL probe: HEX-ACC TGT ATT CCT CGC CT-BHQ-2; MUT FVL probe: FAM-ACC TGT ATT CCT TGC CT-BHQ-2. Reactions were performed in a 20 ul volume with 50 ng of DNA per reaction. The same sets of reference DNA and 17 samples of DNA as the high-resolution melting analysis were used.

Results

For FVL, the Tm was 65.58 °C and 69.75 °C respectively for the WT and MUT. Tm for all 17 samples was 69.75 °C respectively indicating all 17 samples were WT FVL. This TaqMan FVL assay confirmed the FVL genotype of the 17 samples and reference samples.

Conclusion

Comparing the results of the unlabeled probe, HRMA FVL assay with a real-time TaqMan probe endpoint genotyping assay resulted in 100% sensitivity and 100% specificity for both assays.
B-232 Versatile, Extraction-Free Nucleic Acid Sample Preparation in 15 Seconds Via Hyperbaric Heating
Authors: Hambalek J A , Martin R L , Alvarado E , Halle P W , Davis R E , Janwari H , Kido H , Peytavi R
Published: September 2023

Nucleic acid extraction-based PCR sample prep is currently limited by long processing times, liquid transfers, kit cost, and lab storage requirements. Extraction-free approaches enable streamlined sample prep, but lack sensitivity compared to extraction-based methods. We studied several sample types to demonstrate the effectiveness of a novel 15-second Hyperbaric Heating (HBH) process to prepare nucleic acids for PCR. HBH exposes the sample to a short duration, high temperature environment to effectively lyse organisms and denature PCR inhibitors such as nucleases in seconds. Samples are introduced and sealed in a pressure-tight metallic vessel, which is then heated in seconds using magnetic induction. Within the sealed metallic vessel, the vaporization of the aqueous sample raises the internal pressure, enabling an internal temperature higher than 100°C to be achieved. Chelating agents and other proteins in HBH also protect the target nucleic acids from fragmentation. Post HBH, the sample is ready for direct PCR amplification without further dilution or extraction. Efficacy of HBH for efficient PCR amplification was tested against commercial kits. HBH was applied to viral, bacterial, eukaryotic, and fungal organisms. For SARS-CoV-2, HBH detected down to 250 viral copies/mL. Additionally, HBH outperformed commercial kits for PCR sample prep of Candida albicans and Bacillus subtilis. HBH is a generalizable sample prep technology with sensitivity comparable to that of current extraction kits. We have applied HBH to a multitude of organisms and have demonstrated that HBH can be employed as a rapid and comprehensive nucleic acid prep approach for applications that involve PCR amplification post sample prep. Future integration of HBH in devices suitable for point of care molecular testing are underway. We also envision HBH to be a useful, generalizable sample prep method for the molecular lab in applications where cost, simplicity, and speed are essential for preparing nucleic acids for amplification.
B-234 The Quality in NGS: Do You Know How Much We Lost?
Authors: Chao T , Chen P , Huang W
Published: September 2023

Background

Next generation sequencing (NGS) is a new high throughput approach for genomic sequencing and variations detection. The consumption of NGS is numerous, such as times, money and human resource, therefore, some hospitals provided the service but delivered the specimen to other laboratories. Hence, we aimed to establish a procedure to monitor the quality and integrity of report.

Method

Here we used one clinical case to elucidate the whole process of raw sequence data analysis by free resource, a website usegalaxy.org and the IGV visualization software, with flowchart and quality control (QC) items. A delivered case will be an example to show how to monitor the QC.

Results

The flow chart showed the whole steps of analysis of NGS, including evaluation genetic variants in the report from individual laboratory. We processed the FASTQ file and analyzed the quality with three parameters. An overall picture of bases showed the phred score (QV) less than 28. Distribution of sequence lengths over all sequences highlighted a prominent peak at 110–119 bp. The mean QV of reads was around 26 with low quality (QV less than 20) in some reads. We then filtered the FASTQ file and the reads with length less than 45 bp or greater than 150 bp and QV less than 20 were filtered out. The data of the BAM file mapped to the designed panel were output. There were 8252 genome regions designed over 23 chromosomes (chr). A total number of reads was 1 085 653 with the average 131.6 reads (range: 0–31034; quartiles, IQ25: 1; IQ50: 27; IQ75: 152). 7746 of designed genome regions (93.9%) had reads less than 500 (quartiles, IQ25: 0; IQ50: 20; IQ75: 118). We further used IGV to check one of mutations which showed KRAS G12V in the report. KRAS G12V (chr.12: 25245350) was read 25 times with the variant allele frequency (C to A) of 24%.

Conclusion

We showed how to monitor the NGS quality by free resource. It is important to ensure high-quality data after we received the individual laboratory reports. That may help increase the quality of patient care and clinical service.
B-239 Long-read Nanopore Sequencing is a Practicable Method of Targeted or Untargeted Microbial Contamination Detection
Authors: Suhandynata R , Maheu S , Safiari S , Bevins N
Published: September 2023

Background

Sequencing-based methods for microbe detection are poised to replace both in vivo and PCR-based detection methods due to advantages in speed, sensitivity, specificity, and multiplexing. Here we describe pilot experiments to assess the practicability of nanopore sequencing for microbe detection.

Methods

Quantitative genomic Human Adenovirus 5 or Mycoplasma hominis DNA was spiked into remnant human serum and nucleic acids were extracted. 16S rRNA PCR amplification and barcoding was performed for Mycoplasma hominis identification. Sequencing was performed with a R9.4.1 flowcell on the MinION sequencer. Basecalling and genome alignment was performed with Guppy v.6.4.2.

Results

The performance of untargeted sequencing was evaluated by spiking quantitative Adenovirus DNA at 104 genome copies per microliter. Illustrative data from a single run is presented. Replicates yielded similar results. 1 520 991 reads between 31 bases and 970 kilobases were generated. 162 reads between 700 bases and 11.3 kilobases aligned to the Adenovirus genome (Image 1).

To evaluate the performance of PCR amplified sequencing, we spiked Mycoplasma genomic DNA into serum or water. The entire 16S rRNA gene (1.5 kb) was PCR amplified and genomic DNA was spiked in at 104, 102, 10, and 0 (control) copies per microliter. A 10% signal: noise ratio cutoff yielded 100% specificity between spiked samples and negative controls.

Conclusion

Short read sequencing techniques are adequate for microbial detection but are capital intensive and prone to false positive results. We demonstrate that nanopore sequencing can generate long reads (>1.5 kb) with high-confidence microbe genome alignment. Our methods were sensitive to 104 and 10 copies per microliter utilizing untargeted and targeted methods, respectively. Further method optimization and validation will need to be performed, but these results demonstrate that nanopore sequencing is a practicable approach for microbe detection.
B-243 Utilizing Lyophilized LAMP Reagents in Rapid Molecular Assays
Authors: Alpaslan E , Zhang Y , Tanner N A , Patton G C
Published: September 2023

Background

Loop Mediated Isothermal Amplification (LAMP) has become a widely used method for detection of target nucleic acids (DNA and RNA) as it offers a robust and simple alternative to PCR. LAMP is particularly well suited to point of care (POC) applications such as COVID-19 diagnostics because only a single incubation temperature is required for nucleic acid amplification and the technology is compatible with simple detection strategies, including colorimetric or lateral flow readouts. To further increase the utility of LAMP in POC molecular diagnostic workflows, elimination of cold chain requirements for reagent shipment and storage is desired. In this study, we investigate single and multiplex detection of several viral infectious diseases using lyophilized RT-LAMP reagents.

Methods

Assays targeting SARS-CoV-2, FluA, FluB and a host control nucleic acid were evaluated using various detection methods with lyophilized RT-LAMP reagents. Compatibility was established with endpoint LAMP detection strategies, such as colorimetric dyes, and real time multiplex LAMP detection, which is increasingly finding utility in POC testing for common respiratory infections that present with similar symptoms.

Results

Assays employing lyophilized RT-LAMP reagents maintained robust detection of viral respiratory targets within 30 min at 65°C using various detection strategies. Colorimetric dyes such as eriochrome back T and calcein resulted in successful visual readout upon amplification while, one prominent colorimetric dye, hydroxy naphthol blue, was found to be incompatible with the lyophilized reagents used in the study. Additionally, simple hybridization probes permitted simultaneous detection of SARS-CoV-2, FluA and FluB in a single reaction.

Conclusion

LAMP is a robust, simple, isothermal amplification strategy that continues to enable testing beyond traditional laboratory settings. Lyophilized reagents extend the utility of LAMP in POC settings regardless of the detection strategy and will be important factors in the extension of this powerful molecular diagnostic tool to decentralized, field, and at-home testing applications.
B-253 Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing
Authors: Yang C , Wang M , Chang S , Huang C , Chang P , Chueh T
Published: September 2023

Background

High-resolution human leukocyte antigen (HLA) typing is a crucial process in transplant matching. Sequencing-based typing (SBT) has been the gold standard method used in HLA-based clinical applications. Recently, next-generation sequencing (NGS) has been adopted in clinical laboratories for HLA typing. NGS HLA typing assays cover whole or near-whole target HLA genes, allowing the detection of genetic variants outside of key exons. Multiplex amplicon-based NGS is an efficient high-resolution HLA typing method with minimal ambiguity and a high throughput. Here, we report the performance and validation of NGS for HLA typing and compare the results obtained using NGS with those of SBT to identify any discrepancies between the 2 methods.

Methods

A total of 237 samples from 2019 to July 2022 were collected from Chang Gung Memorial Hospital, including 42 samples previously subjected to SBT sequencing for 4-digit HLA analysis. NGS was performed for 5 whole genes (HLA-A/B/C/DQA1/DPA1) and 3 near-whole genes (HLA-DRB1/DQB1/DPB1; excluding exon 1 and part of intron 1) by using the AllType NGS kit (One Lambda), Illumina MiSeq platform, and Type Steam Visual NGS analysis software (One Lambda).

Results

Our validation process comprised 34 NGS runs and 237 samples. In total, 42 samples were analyzed with an overall accuracy and precision of 100% and 100% reproducibility. The overall ambiguity rate was 0% in HLA-A/B/C compared with 56.9% for SBT. Two discrepant alleles of HLA-A and DRB1 were noted in NGS and Sanger sequencing. In accordance with additional SBT exon analyses, one of the results was corrected according to the NGS results.

Conclusion

NGS HLA typing is a high-throughput approach with high accuracy and precision and a low ambiguity and is promising for use in clinical HLA laboratories.
B-256 Genotype Distribution of the Hepatitis C Virus: 10 Years Data of a Clinical Laboratory in Brazil
Authors: Nurmberger J M , de Carvalho G C , Perez B B , Borges J G , Ramadan D R , Tufik S
Published: September 2023

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-268 Validation of a New Real-Time PCR Test for the Detection of Mpox Virus on the BD MAXTM System
Authors: Marín-Royo G , Palacios-Pérez C , Aznar-García A , Llobet-Sesé L , García-Manrique B
Published: September 2023

Background

Enabling labs to rapidly respond to unexpected healthcare challenges is the kind of situations for which the BD MAX™ open system reagent suite is designed. Therefore, after the monkeypox outbreak in non-endemic countries on May 2022, development of a molecular assay for monkeypox detection on the BD MAXTM System was initiated.

Methods

VIASURE Monkeypox virus Real Time PCR Reagents for BD MAX™ System was designed for the qualitative detection of DNA from monkeypox virus in human skin lesion swabs in VTM/UTM obtained from individuals suspected of monkeypox infection.

The Limit of Detection (LoD) was determined by testing a 3-fold dilution series across 5 concentrations. The lowest concentration with 100% detection was tested with an additional 20 replicates. The preliminary LoD was confirmed with a ≥95% detection with 20 replicates. Then, a fully contrived clinical evaluation was performed. Each contrived clinical specimen was prepared using a unique natural clinical specimen matrix (lesion swabs). Twenty (20) of the clinical specimens were prepared at 1-2X LoD, ten (10) were prepared at 3-5X LoD, and thirty (30) were tested as negative samples.

Results

The lowest detectable concentration of virus at which >95% of all replicates test positive (LoD) was 288 copies/mL. The results for the contrived clinical evaluation are shown in Table 1. 19/20 (≥95%) low positive specimens (1-2xLoD) and 20/20 (100%) moderate positive specimens (3-5x LoD) were detected demonstrating the functionality of the test.

Conclusion

VIASURE Monkeypox virus Real Time PCR Reagents for BD MAX™ System provides results in a short time and requires less than one minute of hands-on time per sample due to integration of fully automated extraction and thermocycling steps.

An additional clinical testing of positive natural clinical specimens must be performed to complete evidence that this product is for the purpose and the population for which it is intended.
B-270 Validation of a New Molecular Assay for the Detection and Quantification of Hepatitis B Load in Plasma
Authors: Cortés-Portero I , Dehesa-García B , García-Murillo A , García-Manrique B
Published: September 2023

Background

Hepatitis B virus (HBV) is an enveloped DNA virus and is the most common worldwide cause of viral hepatitis in humans. It is primarily transmitted through blood or blood products. Although the diagnosis of hepatitis viral infections is usually performed with serological markers in blood, this technique has limitations and therefore the diagnosis of the disease by qPCR allows an improvement in the diagnosis of the infection, therapeutic decision making and evaluation of the response to treatment. Therefore, CerTest Biotec has developed a quantitative real-time PCR assay for the detection and quantification of hepatitis B virus in plasma samples.

Methods

Plasma-EDTA samples characterized as positive or negative and quantified in IU/ml were analyzed. The selected samples were collected from infants and adults of both sexes between September 2019 and August 2020. The origin of the population is diverse, with the majority being European. For comparative analysis, the cobas® HBV molecular assay (Roche Diagnostics) using the cobas® 8800 system was used. VIASURE analysis was performed using the following workflow: the MagDEA® Dx SV automated extraction method with magLEAD® 12gC and CFX96™ Opus Real-Time PCR Detection System (Bio-Rad).

Results

A total of 210 clinical EDTA plasma samples were tested, of which 110 were positive for hepatitis B virus and 100 were negative. The comparative analysis determined a sensitivity and specificity >99%. In addition, the positive predictive value (PPV) and negative predictive value were >99%. As for quantification, an R2 correlation value of 0.9095 was obtained.

Conclusion

This clinical evaluation demonstrated that the VIASURE HBV q Real Time PCR Detection Kit is a good tool for the clinical diagnosis of the virus as it has good sensitivity and specificity. No false positive or false negative results were observed. Moreover, the correlation of the quantification with the reference method gives a value of R2 > 0.9. Therefore, clinical diagnosis of hepatitis B virus by real-time PCR can encompass the limitations of the current diagnostic technique by serology.
B-273 Lymphogranuloma Venereum: Validation of a Real-Time PCR Kit for Its Rapid Diagnosis
Authors: Peris M , Milagro A , Martínez N F , Algara O , Martínez-Mateos D , Dehesa-García B , García-Manrique B , Rezusta A
Published: September 2023

Background

Chlamydia trachomatis serovars L1, L2 and L3 are responsible for lymphogranuloma venereum (LGV), a sexually transmitted infection that causes ulcerated lesions and adenopathy. Following the procedures in Clinical Microbiology recommendations, rectal samples positive for C. trachomatis are processed to detect the LGV-producing serovars in order to provide appropriate treatment. Since European regulations require for CE and IVD certification that an in vitro diagnostic product must be validated by an external body, the aim of this study was to validate the VIASURE C. trachomatis (LGV) Real Time PCR Detection Kit from Certest Biotec SL, using DNAs characterized as positive or negative for LGV extracted from clinical samples previously analyzed in the Laboratory.

Methods

A total of 214 DNAs from clinical samples collected between January 2021 and November 2022 were analyzed. Based on the initial diagnosis performed with the Allplex™ Genital ulcer Assay (Seegene®), 37 samples were positive for LGV and 177 were negative. The use of all data and samples was approved by the Aragon research ethics committee (CEICA) (PI20/426). The clinical sensitivity and specificity of the kit and the positive and negative predictive values (95% CI) were calculated with MetaDisc 1.4 software.

Results

Results are summarized in Table 1.

Conclusion

The comparative analysis of the “VIASURE C. trachomatis (LGV) Real Time PCR Detection Kit” proved to be as sensitive and specific as the reference molecular assay. The main advantage is that VIASURE products feature a stabilized, ready-to-use format, reducing the number of time-consuming laboratory steps, avoiding possible contamination and allowing transport and storage at room temperature.
B-275 Retrospective Study for Mpox Detection: Validation of a Real Time PCR Kit for its Fast Diagnosis
Authors: Peris M , Milagro A , Fortuño B , Martínez-Mateos D , Gilaberte B , Dehesa-García B , García-Manrique B , Laborda L , Latorre-Millán M , Aspiroz-Sancho C
Published: September 2023

Background

In an increasingly globalised and interconnected world, a small outbreak of an infectious disease (such as monkeypox in 2022) can turn into a global health emergency. In this context, global health systems must develop effective strategies to interrupt transmission as early as possible by identifying cases and sources of infection. Since clinical diagnostic products must be externally validated to comply with current regulations, the aim of this collaborative and retrospective study was to evaluate the clinical sensitivity and specificity of the VIASURE Monkey pox Real time PCR detection kit (CerTest Biotec) for the detection of monkeypox by Real time PCR.

Methods

This is a retrospective and collaborative study conducted at the Microbiology Department of the Miguel Servet University Hospital at the request of the company Certest Biotec SL with the approvement of the Aragon research ethics committee (PI22/412; 5 Oct 2022). A total of 192 suspected MPX samples were collected in 2022–2023. RealStar Orthopoxvirus PCR kit 1.0, Altona Diagnostics and Sanger sequencing were used as reference techniques. Clinical sensitivity and specificity (95% CI) were calculated with MetaDisc 1.4 software, while overall concordance and quantitative correlation between both real-time PCR kits was checked with the statistical package SPSS v24 (IBM Corporation).

Results

All the results obtained by the kit under study coincided with the reference kit ones (being 69 of them positives for monkeypox). Therefore, clinical sensitivity and specificity values are 100%. Furthermore, the overall agreement was almost perfect (100, 98–100) as was the Spearman correlation test (rs = 0.78; P < 0.001). All positive samples were sequenced and confirmed the presence of the virus.

Conclusion

This retrospective study demonstrates the good clinical parameters and strong overall agreement of the VIASURE® Monkey pox virus Real Time PCR detection kit (CerTest Biotec) compared to both the RealStar Orthopoxvirus PCR 1.0 kit and Sanger sequencing results. In addition, the stabilised, ready-to-use format allows storage at room temperature, and facilitates transport, as it does not need to be refrigerated. This makes it environmentally friendly and reduces additional costs.
B-298 Retrospective Analysis of Fentanyl Positivity at a Large Academic Center Reveals Trends in Illicit Fentanyl Use
Authors: Berry M , Cohen L , Manthei D , Gherasim C
Published: September 2023

Background

National drug-involved overdose deaths continue to rise with deaths involving synthetic opioids (primarily fentanyl) showing the most significant upward trend. Fentanyl is a synthetic opioid with short-acting analgesic activity and a 50–100 times higher potency compared to morphine. Pharmaceutical fentanyl is typically used to treat patients with severe or chronic pain. In contrast, “illegal” fentanyl has frequently been added to other drugs including heroin, cocaine, methamphetamine, and MDMA to increase their potency and contributing to overdose deaths. In this study, we have investigated trends in fentanyl and fentanyl analogs positivity at a large academic medical center.

Methods

Retrospective analysis of results reported over seven years (2015–2021) for untargeted urine drug screens included testing performed for acute exposure and compliance. Presence of fentanyl, fentanyl analogs, and other prescribed or illicit used drugs was determined by gas chromatography—mass spectrometry (GCMS). Overall positivity was calculated as a percentage of total reported results.

Results

Fentanyl, and fentanyl analog positivity was determined for 40 782 urine drug screen results. Overall positivity for fentanyl and fentanyl analogs increased 3.8-fold over the past seven year from 1.53% (2015) to 5.78% (2021). Co-positivity with other illicit drugs such as cocaine and heroin also increased from 7% and 8% respectively, in 2015, to 42% and 13%, in 2021. Presence of fentanyl analogs such as fluorofentanyl, despropionylfentanyl, and acetylfentayl increased in the recent years as well.

Conclusion

Our data reveals increased positivity of fentanyl and fentanyl analogs present with cocaine, heroin, or their metabolites suggesting increased use of illicit fentanyl. The high prevalence supports the inclusion of fentanyl on routine urine drug screen immunoassays, or at a minimum as a reflex with a positive opiate screen.
B-303 Comparison of Tacrolimus Concentrations Obtained By the Newly Approved Alinity i Tacrolimus Assay With a Reference Liquid-Chromatography Combined With Tandem Mass spectrometry Assay
Authors: Woodard K , Kisler T , Dasgupta A
Published: September 2023

Background

Tacrolimus belongs to the calcineurin-inhibitor class of medications and used for preventing and maintenance of organ rejection in transplant recipients alone or in combination with other medications. Due to narrow therapeutic window and significant nephrotoxicity, this drug is routinely monitored with a suggested therapeutic range of 5–15 ng/mL. Recently, Abbott Laboratories (Abbott Park, IL) received FDA approval for using tacrolimus immunoassay on the Alinity i platform. We compared tacrolimus values in 101 transplant patients using Alinity i analyzer and a reference method based on liquid chromatography combined with mass tandem spectrometry (LC-MS/MS).

Methods

Tacrolimus immunoassay on the Alinity i analyzer is a chemiluminescent microparticle immunoassay (CMIA) requiring pretreatment of whole blood specimen prior to analysis. The analytical measurement range of the assay is 2–30 ng/mL. The left-over blood specimens were sent to Eurofins-Viracor reference laboratory in Lenexa, KS for analysis using LC-MS/MS method. After liquid-liquid extraction, specimens were analyzed using ascomycin as the internal standard for the presence of tacrolimus (assay is capable of analyzing other immunosuppressants simultaneously). UPLC C-18 column (waters) was used for separation using two mobile phases (A: 2.0 mM ammonium acetate in 0.1% formic caid in water and B: 2.0 mM ammonium acetate in 0.1% formic acid in methanol). Mass spectrometer was operated in positive ionization electrospray mode (tacrolimus m/z 821.6 > 768.6, ascomycin m/z 809.756.6.

Results

The total precision of the low control was 3.6% (mean: 4.48 ng/mL; CV 0.16 ng/mL, n = 20) while the total precision of the high control was 5.1% (mean: 25.4 ng/mL, SD: 1.29 ng/mL, n = 20). The linearity of the assay (2.0–30.0 ng/mL) was verified by running various calibrators as unknown. Comparing tacrolimus values in specimens from 101 transplant patients (49 kidney, 19 bone marrow transplant, 16 liver and 15 heart transplant) using the reference method (LC-MS/MS) as the x-axis and the corresponding values obtained by the tacrolimus immunoassay using Alinity I analyzer, we observed the following regression equation: y = 0.97 x + 1.005 (n = 101, r = 0.95). For kidney transplant recipients, the regression equation was y = 0.96 + 1.20 (n = 49, r = 0.94).

Discussion

Excellent correlation between tacrolimus values obtained by the newly FDA approved tacrolimus immunoassay with the reference LC-MS/MS based assay indicates further improvement in the performance of immunoassays for therapeutic drug monitoring of immunosuppressants. This may be due to reduced cross-reactivity of metabolites as indicated in the package insert.

Conclusions

The newly approved tacrolimus immunoassay for the application on the Alinity I analyzer can be used for routine therapeutic drug monitoring of tacrolimus in clinical laboratories.
B-311 Increasing Xylazine Findings in Clinical Samples
Authors: Reinhart A
Published: September 2023

Background

Xylazine is a veterinary tranquilizer first appearing in illicit fentanyl preparations in 2019. It is believed to extend a fentanyl high and is now more commonly used as its own drug of abuse. Clinically, xylazine is of concern as it does not respond to naloxone or other antidote during overdoses. There are many forensic findings of increasing xylazine use since its original appearance, but little in the literature concerning clinical xylazine findings, possibly because xylazine is rarely screened in routine drug screening. We sought to find clinical cases with positive xylazine results from 2019–2022. Determined data included demographics (where possible to discern), sex distribution, age ranges, and frequency.

Methods

We queried our laboratory information management system (LIMS) for reported xylazine findings under expanded clinical drug screen utilizing liquid chromatography time of flight mass spectrometry (LC-TOF) for screening and liquid chromatography tandem mass spectrometry (LC-MS/MS) for confirmation, and specific xylazine testing utilizing LC-MS/MS.

Results

In 1928 clinical cases reported between January 2019 and January 2023, the xylazine positivity rate increased from 1.28% (2021; n = 13 positive xylazine results) to 5.05% positivity (2022; n = 29 positive xylazine results) despite a significant decrease in panels and tests with xylazine ordered (n = 1008 in 2021, n = 574 in 2022). The ages of the patients ranged 0–57 years; eleven cases concerned pediatric patients. Of cases that indicated the sex of the patient, 69% were male (n = 29). These cases were found in ten different states (AZ, CT, DE, IN, MA, NJ, OH, PA, UT, VA), with 18 incidental findings. While other drugs were present in incidental finding cases, all cases with an incidental positive xylazine result were also positive for fentanyl and norfentanyl, and several cases were also positive for fentanyl analogues, most commonly 4-ANPP, which occurred in 50% of these cases.

Conclusion

Xylazine use continues to be on the rise and should be of concern for emergency medicine doctors, poison control centers, and clinical laboratories.
B-314 Evaluation of Everolimus QMS Immunoassay on Roche Cobas c502 and Indiko Plus Analyzers
Authors: Buggs V , Song L , Metushi I
Published: September 2023

Background

Everolimus has a narrow therapeutic window and significant intra-individual variability. Rapid turn-around-time and precise results are critical for therapeutic drug monitoring and adequate dose adjustment. The Thermo Scientific Quantitiative Microsphere System (QMS®) everolimus immunoassay is the only FDA-approved immunoassay for measuring everolimus in human blood. The analytical performance of the Thermo Scientific QMS® everolimus immunoassay on Indiko Plus and Roche c502 platforms were evaluated and compared to LC-MS/MS.

Methods

Whole blood EDTA-anticoagulated patient samples were pretreated with methanol and precipitation reagent (immunoassay) or zinc sulfate/methanol (LC-MS/MS) to remove proteins before analysis. Imprecision was determined by running quality control and patient samples 20 times. Fifty transplant samples were used for method comparison. Deming regression, correlation coefficient (R2), and % bias plot analysis were performed using EP Evaluator® software.

Results

Imprecision (%CV) for QMS everolimus assay on Roche c502 was 16.7% (mean = 4.4 ng/mL) and 9.7% (mean = 10.3 ng/mL) for patient pools, and 16.5% (mean = 4.7 ng/mL), 10.3% (mean = 7.8 ng/mL), 10.7% (mean = 14.5 ng/mL) for quality control. QMS everolimus assay imprecision on Indiko Plus platform had CV of 3.1% (mean = 4.3 ng/mL), 3.3% (mean = 9.3 ng/mL), 2.8% (mean 14.3 ng/mL) for liver transplant patients and 3.3% (mean = 4.2 ng/mL), 3.3% (mean 8.3 ng/mL), 3.1 % (mean 15.6 ng/mL) for quality control. Patient comparison studies between Roche c502 vs LC-MS/MS revealed a R2 of 0.82, slope of 1.3 and %bias of −2.7%. QMS on Indiko Plus vs LC-MS/MS revealed a R2 of 0.84, slope of 0.96 and %bias of −21.9%.

Conclusions

The Indiko Plus platform had a constant negative bias vs LC-MS/MS while the Roche’s platform had lower average bias but it resulted in greater scatter in the therapeutic range (3–8 ng/mL) vs LC-MS/MS. Overall, we observed increased imprecision on the c502 analyzer.
B-334 How to Prevent the Next Wave in Overdose Crisis?—Detect Designer Benzodiazepines Using CEDIA and DRI Assays
Authors: Wu J , Ouyang A , Chua P , Ye L , Patel R
Published: September 2023

Background

Benzodiazepines are a class of drugs used for relieving symptoms of anxiety, insomnia, agitation, muscle spasms, and alcohol withdrawal. Benzodiazepines are CNS depressants enhancing the effect of GABA neurotransmitter at GABA receptors and they are well-known for drug abuse potential and drug dependence.Designer benzodiazepines are new psychoactive substances emerging in the past two decades. Designer benzodiazepines are often taken by individuals who use hallucinogenic and stimulant drugs. The drug abuse can lead to respiratory depression, coma, or death. There exists a need to develop a new homogeneous enzyme immunoassay. Existing commercial immunoassay antibodies may also cross-react with designer benzodiazepines which have chemical structure closely related to classic therapeutic benzodiazepines. Herein, we evaluated designer benzodiazepines cross-reactivity using DRI Benzodiazepines and CEDIA Benzodiazepines assays and demonstrated the potential of detecting designer benzodiazepines in urines.

Methods

DRI assay is based on competition between drug labeled with glucose-6-phosphate dehydrogenase (G6PDH) and free drug in the urine sample for a fixed amount of antibody binding sites. The enzyme activity is determined spectrophotometrically at 340 nm by measuring its ability to convert NAD to NADH. CEDIA technology is based on the bacterial enzyme β-galactosidase which has been genetically engineered into two inactive fragments, Enzyme Acceptor (EA) and Enzyme Donor (ED). The enzyme activity is then determined spectrophotometrically at 570 nm. Designer Benzodiazepines tested in this study are 3-Hydroxyphenazepam, Adinazolam, Bromazolam, Clonazolam, Cloniprazepam, Deschloroetizolam, Diclazepam, Etizolam, Flubromazepam, Flubromazolam, Flunitrazolam, Meclonazepam, N-Desmethylflunitrazepam, Nifoxipam, Nimetazepam, Nitrazolam, and Pyrazolam. Compounds were spiked into drug free urine and cross-reactivity was evaluated using DRI® Benzodiazepine Assay and CEDIA® Benzodiazepine Assays on the Beckman Coulter AU680.

Results

14 out of 17 (82%) tested designer benzodiazepines showed high cross-reactivity (>100%) in DRI benzodiazepine assay. 12 out of 17 (71%) tested designer benzodiazepines showed high cross-reactivity (>100%) in CEDIA assays with 200 ng/mL cutoff and 300 ng/mL cutoff. Compared to NPS-benzodiazepines trend data, this study demonstrated that DRI and CEDIA benzodiazepine assays can detect more than 50% (10 out of 19) designer benzodiazepines over 2020 Q1 to 2022 Q4 in the United States.

Conclusion

DRI and CEDIA Benzodiazepine Assays showed high cross reactivity of most seventeen designer benzodiazepines. The results provide potential tools for detect emerging designer benzodiazepines. Note: This was preliminary data that has not been reviewed by any regulatory authorities/cleared by FDA.
B-335 Rapid Quantitative Clozapine Measurements at the Point-of-Care Using Capillary Blood
Authors: Christenson R H , Hilaire M R , Salamone S J , Kelly D L
Published: September 2023

Background

Clozapine is the most effective medication for treatment-resistant schizophrenia, and is endorsed in professional guidelines. Therapeutic drug monitoring (TDM) is recommended for Clozapine due to high pharmacokinetic variability and risk of adverse drug reactions. The effectiveness of TDM for managing clozapine patients is limited by the lack of immediate results.

Methods

A novel immunoassay, the MyCare Insite Clozapine Test (MCI), was developed that uses 20 µL of capillary blood (CP) and delivers 7-minute quantitative results for a portable point-of-care (POC) device. To validate the MCI system, matching CP and serum samples were collected from 113 patients at two sites. CP was tested immediately with MCI and compared to serum immunoassay results on a Beckman-Coulter AU480 analyzer. Precision was evaluated using three whole blood spiked samples and two assay controls over five days with five replicates per day.

Results

The figure shows a correlation plot evaluated by Passing-Bablok regression of the POC MCI (y-axis) and the Beckman analyzer (x-axis). The regression line was y = 1.05*x + 1.4 ng/L, r = 0.927, indicating good agreement. Precision studies demonstrated that repeatability was ≤11% and within-lab precision was ≤13%.

Conclusion

Quantitative Clozapine results are available in 7 min using CP in a portable POC device. A rapid POC alternative can achieve timely patient results in many testing environments. Rapid Clozapine testing has the potential for expanding utilization of this important drug by providing actionable POC results.
B-339 A Clinical Research LC-MS/MS Method for the Analysis of Antidepressants in Plasma
Authors: Balloch S , Calton L , Hammond G
Published: September 2023

Background

Depression is common globally, with an estimated 3.8% of the population affected. The condition can impact individuals’ ability to function in work, social and family settings. Many antidepressant drugs are currently prescribed, encompassing selective serotonin reuptake inhibitors (SSRIs), serotonin-nonresponsive reuptake inhibitors (SNRIs) and tetracyclic antidepressants (TeCAs). However pharmacokinetic and drug interactions are known, therefore a reliable quantitative clinical research method may play a role in researching the effects of their administration. Waters has developed a clinical research method for the following antidepressants in plasma; citalopram, desmethylfluoxetine, duloxetine, fluoxetine, fluvoxamine, O-desmethylvenlafaxine, sertraline and venlafaxine (10–1000 ng/mL); mirtazapine (5–500 ng/mL) and trazadone (30–3000 ng/mL).

Methods

Matrix matched calibrators and QCs were prepared using in-house stocks and pooled plasma. Samples (50 µL) were treated with internal standard in acetonitrile. A water/methanol/ammonium acetate gradient was used with a Waters™ XSelect™ Premier HSS T3 column on a Waters ACQUITY™ UPLC™ I-Class followed by detection on a Xevo® TQD mass spectrometer in a 5 min run.

Results

No system carryover was observed following analysis of plasma samples containing the highest concentration calibrators. Analytical sensitivity investigations indicated precise quantification (≤20% CV, ≤17.6% bias) at concentrations equal to or lower than the lowest concentration calibrator. Total precision and repeatability were assessed (3 pools, 5 replicates, 5 days; n = 25) and determined to be ≤10.0% RSD. Linearity experiments determined the method provided first or second order fits over the ranges analyzed; additionally, each run met acceptance criteria (coefficient of correlation ≥ 0.995, determined concentrations of calibrators ±15% of nominal, ±20% in the case of the lowest calibrator). Post-column infusion experiments demonstrated analytes eluted in regions free of major ion suppression or enhancement. Evaluation of matrix effects at low and high concentrations indicated compensation by the internal standard. Addition of high concentrations of several endogenous and exogenous materials did not affect quantification.

Conclusion

This quantitative method for clinical research demonstrates very good precision with minimal matrix effects and allows for the multiplexing of a panel of antidepressants in plasma in a short run time. For Research Use Only. Not for Use in Diagnostic Procedures.