Commercial Test Research Articles

The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

Investigation of canine parvovirus occurrence in cats with clinical signs of feline panleukopenia in Slovakia – pilot study

Abstract Introduction Feline panleukopenia is a contagious viral disease caused by the feline panleukopenia virus (FPV). A closely related pathogen is canine parvovirus (CPV), and amino acid substitutions in this virus allow it to acquire a feline host range. In feline hosts, the disease induced by CPV manifests with similar symptoms to those caused by FPV or milder ones, leading to its underdiagnosis. The aim of this study was to determine the presence of CPV type 2 (CPV-2) in cats with clinical symptoms of panleukopenia and to assess the use of commercial CPV antigen tests for the clinical diagnosis of FPV. Material and Methods Samples from 59 cats from central Slovakia were included in the study. Rectal swabs were collected and clinically tested for parvovirus infection using a commercial antigen test. Antigen-positive samples were confirmed by PCR targeting the viral VP2 gene. The sequences of the PCR products were established with the Sanger method. Results Of 59 samples, 23 were revealed to be positive for parvovirus infection by both antigen and PCR test (38.9%). Analysis with the National Center for Biotechnology Information BLASTn application showed 99.78–100% pairwise identity with FPV. The mortality rate of parvovirus-infected cats included in this study was 8.69% (2/23). Conclusion Although feline disease with CPV-2 was not confirmed, the CPV antigen test was able to detect FPV infection.

Real‐time multi‐objective speed planning ATO considering assist driving for subway

AbstractSpeed curve planning is one of the most important functions of automatic train operation (ATO). To improve the real‐time optimization capability and driver‐friendliness of the existing ATO, an extended ATO framework considering both automatic driving and assisted driving is designed. A multi‐objective optimization model based on quadratic programming is established considering energy‐saving, punctuality, and comfort. However, due to the influence of the weight of multi‐objectives, this method cannot directly obtain the speed curve satisfying the trip time constraint. Further, based on the analysis about the weight of multi‐objects, a time‐constrained quadratic programming algorithm is proposed. With the proposed method, the speed curve can be calculated in real‐time both before operations and during operations. For the former, time‐varying train mass and trip time are considered to guarantee an optimal solution. For the latter, deviations, delays, and maloperations on the way are corrected. Simulation experiments verify the solvability and real‐time performance of the proposed method. In particular, compared with the dynamic programming and (mixed‐integer linear programming) MILP method, the proposed method is more energy‐efficient and easier to be followed by the driver. In addition, a prototype is developed for commercial tests on a Beijing subway line. The relevant performance is verified in commercial tests.

Performance Assessment of Treponemal and Nontreponemal Tests for the Diagnosis of Acquired Syphilis.

There are a variety of nontreponemal test (NTT) and treponemal test (TT) kits for the serologic diagnosis of syphilis. Because of the complexity of the infection (multiple clinical stages) and the different antigens used in these kits, a systematic evaluation of the accuracy of the currently available commercial tests is warranted. Our objective was to evaluate the performance of commercially available tests for the diagnosis of syphilis infection. In this study, we analyzed one NTT (Venereal Disease Research Laboratory [VDRL] test, Wiener Laboratories, Rosario, Argentina) and two TTs (fluorescent treponemal antibody absorption [FTA-ABS] test, Euroimmun, Lübeck, Germany, and syphilis recombinant ELISA v. 4.0 test [ELISA], Wiener Laboratories, Rosario, Argentina) using a panel of 187 samples, including serum samples from 31 individuals with primary syphilis, 77 with secondary syphilis, and 79 with latent syphilis. An additional 192 samples from uninfected individuals and 323 serum samples from individuals with other diseases were included. The sensitivities of the VDRL, ELISA, and FTA-ABS tests were 97.9%, 100%, and 96.3%, respectively. The VDRL and ELISA tests showed a specificity of 100%, and the FTA-ABS test showed a specificity of 99.5%. Accuracy was 98.9% for the VDRL test, 100% for the ELISA, and 97.9% for the FTA-ABS test. For primary, secondary, and latent syphilis, the ELISA achieved a diagnostic performance of 100%, whereas the sensitivity for the VDRL and FTA-ABS tests ranged from 96.8% to 98.7% and 93.7% to 98.7%, respectively. No difference was observed when the tests were used as traditional or reverse algorithms. In general, all three tests are able to discriminate positive and negative samples for syphilis, regardless of the diagnostic algorithm.

Tick-borne infections in the Leningrad oblast: incidence and seroprevalence

The study’s objective was to reveal trends in infections transmitted by ixodes ticks epidemic processes and the level of seroprevalence of the population to the tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Coxiella burnetii, Anaplasma phagocytophilum and Ehrlichia chaffeensis/ E. muris in the Leningrad Oblast.Materials and methods. A retrospective epidemiological analysis of the tick-bite incidence rate, the ixodic tick-borne borreliosis, tick-borne encephalitis, Q fever, human granulocytic anaplasmosis and human monocytic ehrlichiosis incidence rates, as well as vaccination against tick-borne encephalitis of the population of the Leningrad Oblast in 20112020. The official data of the tick-bite incidence rate, infections transmitted by ixodes ticks incidence rates and vaccination of the population was taken from the forms of federal statistical observation No. 2 and No. 5. Blood was sampled in 2019-2020 from randomly selected donors. Blood serum samples were examined by enzyme-linked immunosorbent assay for the presence of IgG antibodies to the pathogens of the studied infections using commercial test systems according to the manufacturer’s instructions.Results. In the Leningrad Oblast, in 2011-2020, there was a downward trend in the tick-bite incidence rate, as well as in the incidence rates of infections transmitted by ixodic ticks. The long-term average tick bite incidence rate was 369.0. The long-term average incidence rate of ixodic tick–borne borreliosis was 4.1; tick–borne encephalitis was 1.4; Q fever was 0.02. During the analyzed period, no cases of human granulocytic anaplasmosis and human monocytic ehrlichiosis were reported. The seroprevalence to tick-borne encephalitis virus was 1.7%; to B. burgdorferi s. l. – 3.7%; to C. burnetii – 1.8%; to A. phagocytophilum – 1.0%; to E. chaffeensis/ E.muris – 3.2%.Conclusion. The results obtained indicate the existence of active natural foci of infections transmitted by ixodes ticks, fairly frequent contact of the residents with these foci, and possible significant hypodiagnosis of these infections in the Leningrad Oblast.

Epidemiological assessment of hepatitis E virus infection among 1565 pregnant women in Siem Reap, Cambodia using an in-house double antigen sandwich ELISA.

This study investigated hepatitis E virus (HEV) prevalence among pregnant women in Siem Reap, Cambodia, by developing a cost-effective, user-friendly in-house enzyme-linked immunosorbent assay (ELISA) for detecting total anti-HEV immunoglobulins (Ig). The in-house ELISA was designed for large-scale screening in resource-limited settings. Its performance was benchmarked against two commercial tests: the Anti-HEV IgG EIA (Institute of Immunology, Co. Ltd) and the Anti-HEV IgG RecomLine LIA (Mikrogen). The in-house ELISA demonstrated a sensitivity of 76% and 71.4%, and a specificity of 94.1% and 98.6%, against the two commercial tests, respectively, with overall agreement rates of 92.4% and 94.3%. Among 1565 tested pregnant women, 11.6% were anti-HEV positive. Prevalence increased with age, particularly in women aged 35-40years and over 40years. No significant associations were found with education, number of children, family size, or history of blood transfusion and surgery, except for the occupation of the family head as a public officer. Of the total anti-HEV positive women, 22.7% had anti-HEV IgM, indicating recent or ongoing infection. The study concluded that the in-house ELISA is a viable option for HEV screening in regions with limited resources due to its high accuracy and cost-effectiveness. It is particularly suitable for large-scale studies and public health interventions in areas where HEV is endemic and poses a significant risk to pregnant women.

Testing the tester

Over the summer of 2022. I was tasked with testing the language proficiency of many potential students who were unable to access the International English Language Testing System (IELTS), either due to displacement or other adverse circumstances.  The largest cohort was that of Ukrainian students, following the Scottish government’s announcement that free education and living support would be available for those qualifying students. The second group was less homogenous.  It varied in nationality, age, background, circumstances and fell under the broad umbrella of refugees and asylum seekers. The Ukrainian students, on acceptance of academic and language qualifications would directly enter faculty as undergraduates. In contrast, the refugees and asylum seekers would join the last five weeks of a 34-week pre-sessional course and then proceed to a Widening Access Course. Completion of this, and successful settled status would then enable them to enter degree courses.  I use a reading-to-write integrated test based on an academic paper, followed by an interview of 20 – 30 minutes duration for all students who are unable to access IELTS or other recognized commercial language tests. Due to the urgency and sheer number of undergraduates needing to be tested over July/August 2022, I made the decision to use English for General Academic Purposes materials and conducted invigilated reading-to-write tests on Zoom, with individual interviews being conducted over the following days. While this format had been used successfully in the past with displaced PGT/PGR students, it quickly became apparent that it was unsuitable for the latter group of students. Here I reflect on my role and consider future test design.

Commercial Synovial Antigen Testing is Inferior to Traditional Culture for the Diagnosis of Periprosthetic Joint Infection in Patients Undergoing Revision Total Knee Arthroplasty

IntroductionDespite its limitations, a culture remains the “gold standard” for pathogen identification in patients who have periprosthetic joint infection (PJI). Recently, a synovial fluid antigen test has been introduced by a commercial entity. The purpose of this multicenter study was to determine the accuracy of the antigen test in the diagnosis of PJI. MethodsThis retrospective study identified 613 patients undergoing revision total knee arthroplasty who had undergone preoperative synovial fluid analysis. A PJI was defined using the 2018 International Consensus Meeting (ICM) criteria. Patients who had an extended period (> 180 days) from aspiration to revision procedure (n = 62), those presenting within 90 days of their index arthroplasty procedure (n = 17), and patients who had an inconclusive ICM score (n = 8) were excluded. Using receiver operator characteristic curve analyses, we examined the utility of the microbial identification (MID) antigen test and any positive culture (either preoperative or intraoperative) in the diagnosis of PJI. ResultsA total of 526 patients were included. Of these, 125 (23.8%) were ICM positive, and 401 (76.2%) were ICM negative. Culture demonstrated an AUC of 0.864, sensitivity of 75.2%, and specificity of 97.5%. On the other hand, the MID test exhibited an AUC of 0.802, sensitivity of 61.6%, and specificity of 98.8%. The AUC of culture was significantly higher than that of the MID test (P = 0.037). The MID test was positive in 41.9% of culture-negative PJI cases. We also observed a high rate of discordance (29.7%) when both culture and the MID test were positive in the ICM positive group. ConclusionSynovial fluid antigen testing does not provide additional clinical benefit when compared to traditional cultures for the diagnosis of PJI. The antigen test had low sensitivity in the diagnosis of PJI and a relatively high rate of discordance with culture.

Comparison of Colistin Susceptibility Tests

Introduction: Polymyxins are important antimicrobial agents for the treatment of infections caused by Gram-negative bacteria. The susceptibility testing for polymyxins is a challenge for clinical laboratories due to the difficulty of performance, reproducibility, and accuracy of available methods. Aim: To compare the performance of the colistin susceptibility test of an automated system and a gradient test with the gold standard broth microdilution method (BMD). Materials and Methods: Multidrug-resistant isolates of Acinetobacter baumannii (n=102), Klebsiella pneumoniae (n=40), and Pseudomonas aeruginosa(n=11) were included. The VITEK 2 systems and gradient test were studied according to the manufacturer’s instructions. Broth microdilution tests were performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Commercial susceptibility testing methods were compared to BMD. Results: Rates of essential agreement of colistin test results between BMD, VITEK 2, and gradient test were 96.1% and 79.7%, respectively. The VITEK 2 and gradient test showed 95.4% and 94.8% of categorial agreement. The very major error rate of VITEK 2 was 3.2%, and the gradient test was 5.2%. The major error rate of VITEK 2 was 1.3%, and there was no major error for the gradient test. Conclusion and Suggestions: The very major error rate was higher in the gradient test (5.2%) than VITEK 2 (3.2%). Even if the very major error rate of VITEK 2 was lower, both resistance and susceptility results of VITEK 2 should be confirmed with the BMD test. Further studies for susceptibility testing are needed with a focus on the correlation of MIC’s results of different tests.

Comparison of Methods in the Serologic Diagnosis of Cystic Echinococcosis.

Cystic echinococcosis (CE) is caused by the larval form of Echinococcus granulosus. Clinical, radiologic, pathologic, and serologic findings should be evaluated together for the diagnosis of CE. The sensitivity and specificity oalf serologic tests may vary depending on the method used. In this study, we aimed to detect IgG antibodies specific to E. granulosus using indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibodies (IFA) and western blot (WB) tests. In our study, the serum samples of 74 patients sent to our laboratory with suspicion of CE were studied using two different commercial IHA tests, ELISA, IFA and WB test. The test results were evaluated along with radiological findings and histopathological examinations, the latter being the gold standard. Of all the patients, 51 (69%) were female and 23 (31%) were male. There was a statistically significant difference between males and females (χ2 = 9.7, p = 0.002). Out of 74 patients, positivity rates for Siemens IHA, Fumouze IHA, ELISA, IFA and WB test were positive as 33 (44.6%), 35 (47.3%), 43 (58.1%), 42 (56.7%) and 38 (51.3%), respectively. The sensitivity and specificity of the tests were as follows: 66.67 and 2.31% for Siemens IHA; 70.83% and 96.15% for Fumouze IHA; 85.42%, and 88.46% for ELISA; 83.33% and 88.46% for IFA; 72.92% and 88.46% for WB test. There were statistically significant differences in between all five methods (p < 0,001). While the tests with the highest specificity was Fumouze IHA, the test with the highest sensitivity was the ELISA test. It was concluded that IHA and ELISA tests were more practical in practice because of their greater applicability.

Impact of FilmArray respiratory panel test for hospitalized pediatric respiratory tract infection in Taiwan: A 3-year single-center cohort study.

Respiratory tract infections are prevalent and clinically significant in pediatric populations globally. However, pathogen testing often involves time-consuming processes, resulting in delays in diagnosis. To date, commercial testing machines, such as the FilmArray respiratory panel, have been proposed for hospitals. Therefore, this study aimed to investigate the impact of the FilmArray respiratory panel at a single center. This study utilized the medical records of our hospital to select pediatric inpatients with respiratory tract infections who underwent the FilmArray respiratory panel between September 2020 and April 2021 and those who did not undergo nucleic acid detection (a rapid test group) between September 2019 and April 2020. FilmArray is a polymerase chain reaction-based diagnostic tool. The FilmArray respiratory panel group was scheduled to recruit 150 patients (final 137 patients), whereas the rapid test group was scheduled to recruit 300 patients (final 267 patients). Differences in continuous variables between the 2 groups were analyzed using independent Student t tests. The FilmArray respiratory panel group had a longer length of inpatient days, longer duration of antibiotic use, and higher proportion of pathogens that tested positive, with significant differences than those in the rapid test group. Fever duration showed no significant difference between the 2 groups. For the polymerase chain reaction method, respiratory syncytial virus was the most commonly detected pathogen causing pneumonia, followed by human rhinovirus/enterovirus and parainfluenza virus. Mycoplasma was detected using the rapid test but not with the FilmArray respiratory panel. The FilmArray respiratory panel provides clinicians with a rapid and useful diagnostic tool. The effect was quite good for virus detection, but not for bacteria. Given its limited adoption, the tool may not aid clinicians in the diagnosis of mild cases.

Implications of Measles Inclusion by Commercial Syndromic Polymerase Chain Reaction Panels - United States, May 2022-April 2023.

Syndromic polymerase chain reaction (PCR) panels are used to test for pathogens that can cause rash illnesses, including measles. Rash illnesses have infectious and noninfectious causes, and approximately 5% of persons experience a rash 7-10 days after receipt of a measles, mumps, and rubella (MMR) vaccine. MMR vaccine includes live attenuated measles virus, which is detectable by PCR tests. No evidence exists of person-to-person transmission of measles vaccine virus, and illness does not typically result among immunocompetent persons. During September 2022-January 2023, the Tennessee Department of Health received two reports of measles detected by syndromic PCR panels. Both reports involved children (aged 1 and 6 years) without known risk factors for measles, who were evaluated for rash that occurred 11-13 days after routine MMR vaccination. After public health responses in Tennessee determined that both PCR panels had detected measles vaccine virus, six state health departments collaborated to assess the frequency and characteristics of persons receiving a positive measles PCR panel test result in the United States. Information was retrospectively collected from a commercial laboratory testing for measles in syndromic multiplex PCR panels. During May 2022-April 2023, among 1,548 syndromic PCR panels, 17 (1.1%) returned positive test results for measles virus. Among 14 persons who received a positive test result and for whom vaccination and case investigation information were available, all had received MMR vaccine a median of 12 days before specimen collection, and none had known risk factors for acquiring measles. All positive PCR results were attributed to detection of measles vaccine virus. Increased awareness among health care providers about potential measles detection by PCR after vaccination is needed. Any detection of measles virus by syndromic PCR testing should be immediately reported to public health agencies, which can use measles vaccination history and assessment of risk factors to determine the appropriate public health response. If a person recently received MMR vaccine and has no risk factors for acquiring measles, additional public health response is likely unnecessary.

Seasonal variation of mycotoxin levels in poultry feeds and feed ingredients in Oyo State, Nigeria.

Mycotoxins pose a major problem to poultry production as a result of feed contamination which has deleterious consequences such as production losses and human health risks. A total of 158 chicken feed samples were randomly collected from 46 consenting poultry farms in Oyo State throughout the wet season (April-October; 91 samples) and the dry season (November-March; 67 samples), including compounded feed (n = 129) and feed ingredients (n = 29). Samples were promptly transported to the laboratory in sterile plastic vials for lateral flow assay for mycotoxins using six different commercial mycotoxin test kits each for aflatoxin B1, zearalenone, deoxynivalenol, ochratoxin A, fumonisin, and T-2 toxin/HT-2 toxin. Summary values of mycotoxin levels (µg/kg) in the feedstuffs were represented as frequency or median (and range). Fisher exact or Mann-Whitney U tests were carried out where appropriate at α = 0.05. Every sample contained at least four mycotoxins. Aflatoxins and fumonisin co-occurred in 80% of the samples. Aflatoxin and fumonisin concentrations were above the permissible limits in 32.9% and 18.4% respectively in feedstuff sampled in the dry season while the values were 17.1% and 6.3% respectively during the wet season. Among feed ingredients, peanut cake and maize had the highest median concentration of aflatoxin and fumonisin, respectively. Median aflatoxin concentration in the feedstuff was significantly higher than the permissible limit irrespective of season. There is a need to frequently monitor mycotoxin levels of feed and feed ingredients and improve storage system for feed ingredients in order to reduce the risk associated with high mycotoxin intake in poultry.

Detection of circulatory E. granulosus-derived cell-free DNA in the plasma and urine of human cystic echinococcosis using an in-house PCR: a potential promising diagnostic biomarker.

The diagnostic tool for identifying cystic echinococcosis (CE) patients at an early stage is currently lacking. However, circulatory cell-free DNA (cfDNA) has shown potential as a biomarker for parasitic infections and could be used for diagnosing CE. The plasma and urine samples were collected from 39 patients with confirmed CE through imaging and histopathological techniques. All plasma samples were tested for anti-echinococcal antibodies using a commercial ELISA test. Total plasma and urine cfDNA were extracted and an in-house PCR assay was developed to detect E. granulosus specific cfDNA in the samples of CE patients. Out of the 39 patients, 30 tested positive for E. granulosus using serology, with a sensitivity of 76.9%. Moreover, the detection rates for the cfDNA were 79.5% in plasma samples and 58.97% in urine samples using the 80bp COX1 gene. The plasma-based PCR and serology test showed the highest agreement (Kappa = 0.53). Plasma-based PCR has been found to be a reliable diagnostic tool for identifying CE patients at different cyst stages. It offers validity, speed, and sufficient sensitivity, making it an alternative to serology in diagnosing CE in endemic areas.

“First alert” for Drinking Water Quality: Commercial Water Quality Testing Kits for Community-Engaged Research

“First alert” for Drinking Water Quality: Commercial Water Quality Testing Kits for Community-Engaged Research

Abstract 5655: Association between tumor genomic variants and the long term post surgical recurrence as detected by a tumor informed ctDNA assay

Abstract Background: While surgical resection of early stage cancers is often curative, a number of patients will experience disease recurrence. Given the genomic heterogeneity of cancer, we hypothesized that different driver mutations present in a patient’s tumor may influence whether and when recurrence occurs. To investigate this, the association between the presence of driver mutations and rates of molecular residual disease (MRD) detection by circulating tumor DNA (ctDNA) was evaluated in patients with stage I-III colorectal cancer (CRC). Methods: This was a retrospective evaluation of patients who underwent commercial ctDNA testing (SignateraTM) following curative surgical resection for stage I-III CRC from 2019 to February 2023. For this analysis, patients who were ctDNA-negative post-surgery (2-12 weeks) and had ctDNA testing performed between 6-24 months after surgery were included. Patients were stratified based on the presence or absence of mutations in driver genes and the associated percentage of patients with ctDNA-positivity over time was calculated. Results: Of 11,436 patients with stage I-III CRC, 45.7% (5,230) had ctDNA testing performed between 2-12 weeks post-surgery. During this window, 81% (4,239/5,230) had a negative post-surgical ctDNA test result available. For this analysis, 78.4% (3,323/4,239), 46.7% (1,979/4,239), 24% (1,017/4,239) and 8.2% (348/4,239) had follow-up ctDNA testing performed for at least 6, 12, 18, and 24 months post-surgery, respectively. Certain driver mutations were associated with increased risk of post-surgical ctDNA-positivity rate. In patients with stage I CRC, APC mutations were associated with elevated rates of ctDNA positivity at 12 months [(APC-mutated: 13% (11/85) vs. APC-wildtype 3% (1/31); p=0.06)] and 18 months [(APC-mutated: 38% (12/32) vs. APC-wildtype: 6% (1/18); p=0.02)]. Interestingly, similar findings were not observed in stage II or stage III CRC. Similarly in patients with stage III CRC, SMAD4 mutations exhibited a substantially increased probability of ctDNA-positivity at 6 months [(SMAD4-mutated: 8% (15/194) vs. wildtype: 4% (54/1267) p=0.09)], 12 months [(SMAD4-mutated: 25% (32/129) vs. wildtype: 12.4% (102/817); p=0.0005)], and 24 months [(SMAD4-mutated: 69% (41/59) vs. wildtype: 53% (149/279); p=0.02)]. Conclusions: These findings point to several genomic alterations that may underlie cancer recurrence after a long period of undetectable disease with a differential impact across different stages. This may allow, in the future, for a personalized surveillance strategy based on driver mutations and stage of the disease. Correlation of ctDNA positivity with clinical outcomes based on driver mutational status may inform to what extent tumor-specific characteristics drive patient outcomes following a successful surgery. Citation Format: Noah Friedman, Megan Richters, J. Bryce Ortiz, Robert Burns, Sara L. Bristow, Minetta C. Liu, Alexey Aleshin, Adham Jurdi, Helio A. Costa. Association between tumor genomic variants and the long term post surgical recurrence as detected by a tumor informed ctDNA assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5655.

Abstract 3814: Molecular background conditioning by temozolomide in neuroendocrine tumors

Abstract Temozolomide (TMZ)-based regimens represent a valid therapeutic option in well-differentiated neuroendocrine tumors (NETs G2 and G3). As an alkylating agent, it increases DNA alteration by altering guanine residues, which either triggers cell death or results in novel missense mutations especially in MGMT deficient cells. Here, we describe an exploratory case series of TMZ-treated NETs cases that underwent NGS analysis at the University Hospital of Verona. Methods. We included 32 patients (16 G2 and 16 G3). NGS was performed before TMZ treatment for 22 cases (54%), after for 19 (46%) and in both for 4 cases (13%). We performed NGS on tissue samples for 73% of assays and via liquid biopsy for the remaining 27%. For tissue NGS, in 77% of samples we used an in-house panel encompassing 174 genes and in 23% commercial test interrogating at least 500 genes. Only ACMG/AMP class IV-V molecular alterations were considered for further analysis. Main Results From 2021 we performed NGS analysis on 32 NETs (G2 and G3) treated with TMZ-based regimes. Most cases (28 pf 32, 84.4%) were pancreatic NETs. Mean Ki-67 at baseline was 24.5% (range 3-60). The mean number treatments prior to TMZ were 1.4 and 0.5 between CAPTEM and subsequent NGS. The top five altered genes at baseline were MEN1 (32%), TP53 (14%), DAXX (14%), PTEN (9%) and SMAD4 (9%). None of the patients showed a high TMB (&amp;gt;15Mut/Mb) at baseline, while 26% of post-TMZ samples displayed that feature. Mean TMB at baseline and after treatment was 6,51 (range 1-10.6) and 22,9 mut/Mb (range 1-216) respectively. Alterations in DDR systems genes, including ATM, ATR, CHEK2, BRCA1, BRCA2, MLH1, MLH2, PMS2 MLH6 and MUTYH, were found in 8 patients (47%) after TMZ exposure; only one DDR-specific alteration (MUTYH) was found at baseline. DDR defects were enriched in patients exposed to higher numbers of treatments. The increase in mean TMB and enrichment in mutations in the DDR system is also confirmed when analysing the 6 cases having both pre- and post-TMZ treatment samples. Among other pathways, cell cycle deregulating genes (CDKN1A, CDKN2B, CDKN1B, CDK12, CDK6, RB1) were altered in 2 (11%) and 5 (29%) pre- and post-TMZ NGS samples respectively, the mTOR pathway (MTOR, NF1, PIK3CA, AKT, TSC1, TSC2, PTEN) in 2 (11%) and 8 (47%), and the MAPK pathway (KRAS and BRAF) in 1 (5%) and 2 (12%). Conclusions. The wide and effective use of temozolomide in neuroendocrine tumors, together with the potential therapeutic implications of DDR defects make this topic of great interest. The DDR pathway was one of the most affected by alterations in post-TMZ samples, although the genotoxic agent caused a general increase in the rate of somatic mutation. The status of DDR systems, either germinal or somatic, may represent an important selection and/or stratification parameter for future immunotherapy clinical studies also in neuroendocrine tumours provided that the analysis is carried out in a systematic, comprehensive and longitudinal way. Citation Format: Elena Trevisani, Anna Reni, Irene Torresan, Alice Rossi, Michele Borghesani, Silvia Nicolini, Claudio Luchini, Luca Landoni, Davide Melisi, Aldo Scarpa, Michele Milella, Andrea Mafficini, Sara Cingarlini. Molecular background conditioning by temozolomide in neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3814.

An Evaluation of a SARS-CoV-2 Pseudovirus Neutralization Test and A Comparison to a SARS-CoV-2 Surrogate Virus Neutralization Test in a COVID-19 Long-Term Follow-Up Cohort

Background: The detection of neutralizing anti-SARS-CoV-2 antibodies is important since they represent the subset of antibodies able to prevent the virus to invade human cells. The aim of this study is to evaluate the clinical performances of an in-house pseudovirus neutralization test (pVNT) versus a commercial surrogate neutralization test (sVNT). Material and Methods: A total of 114 RT-PCR positives samples from 75 COVID-19 patients were analyzed using a pVNT and an sVNT technique. Fifty-six pre-pandemic samples were also analyzed to assess the specificity of the two techniques. An analysis of the repeatability and the reproducibility of the pVNT was also performed. Results: A coefficient of variation (CV) of 10.27% for the repeatability of the pVNT was computed. For the reproducibility test, CVs ranged from 16.12% for low NAbs titer to 6.40% for high NAbs titer. Regarding the clinical sensitivity, 90 RT-PCR positive samples out of 114 were positive with the pVNT (78.94%), and 97 were positive with the sVNT (84.21%). About the clinical specificity, all 56 pre-pandemic samples were negative in both techniques. When comparing the sVNT to the pVNT, the specificity and sensibility were 66.67% (95%CI: 47.81–85.53%) and 98.88% (95%CI: 96.72–99.99%), respectively. Conclusions: The results obtained with the automated sVNT technique are consistent with those obtained with the pVNT technique developed in-house. The results of the various repeatability and reproducibility tests demonstrate the good robustness of the fully manual pVNT technique.

Non-Invasive Predictive Biomarkers for Immune-Related Adverse Events Due to Immune Checkpoint Inhibitors.

Immune-related adverse events (irAEs) are the most common complication of immune checkpoint inhibitor (ICI) therapy. With the widespread use of ICIs in patients with solid tumors, up to 40% of the patients develop irAEs within five months of treatment, and 11% develop severe irAEs requiring interventions. A predictive test for irAEs would be a crucial tool for monitoring for complications during and after ICI therapy. We performed an extensive review of potential predictive biomarkers for irAEs in patients who received ICI therapy. Currently, only thyroid-stimulating hormone is utilized in common clinical practice. This is due to the unavailability of commercial tests and unclear predictive values from various studies. Given the lack of single strong predictive biomarkers, some novel approaches using composite scores using genomic, transcriptomics, cytokine levels, or clinical parameters appear appealing. Still, these have yet to be validated and incorporated into clinical practice. Further research conducted to validate the models before implementing them into real-world settings will be of the utmost importance for irAE prediction.

Reflections on the Aim of the DEMPE Concept in Contemporary Transfer Pricing: Delineation, Non-Recognition and Valuation

Within the contemporary transfer pricing debate, one may identify a question that is no less elusive than it is central, yet relatively unaddressed: can the DEMPE model be used for delineation and non-recognition purposes? In other words, whether the DEMPE model comes into play only after having delineated the transaction and passed the commercial rationality test or whether it can be relied upon for the purpose of delineating and recognizing a transaction. The question is crucial because the answer directly determines the extent to which DEMPE can be used in an intercompany transaction involving intangibles. Addressing the proposed research question also raises issues about the changes that have been recently incorporated into the OECD Guidelines. The present study is structured in the following manner: section 1. introduces the topic setting the ground for the discussion, section 2. provides some preliminary considerations before approaching the research question, section 3. highlights the evolution of the non-recognition, section 4. focuses on the primary question to be answered as the focal point of the study, and section 5. provides a conclusion.