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Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, leading to the BCR::ABL1 fusion gene and hyper-proliferation of granulocytes. Tyrosine kinase inhibitors (TKIs) are effective, and minimal residual disease (MRD) monitoring is crucial. Digital PCR platforms offer increased precision compared to quantitative PCR but lack comparative studies. Eighty CML patient samples were analyzed in parallel using digital droplet PCR (ddPCR) (QXDx™ BCR-ABL %IS Kit) and chip digital PCR (cdPCR) (Dr. PCR™ BCR-ABL1 Major IS Detection Kit). Overall, qualitative and quantitative agreement was good. Sensitivity analysis showed positive percentage agreement and negative percentage agreement were both ≥90 %, and the quadratic weighted kappa index for molecular response (MR) level categorization was 0.94 (95 %CI 0.89, 0.98). MR levels subgroup analysis showed perfect categorical agreement on MR level at MR3 or above, while 35.4 % (17/48) of patient samples with MR4 or below showed discordant categorizations. Overall, Lin's concordance correlation coefficient (CCC) for the ratio of %BCR::ABL1/ABL1 converted to the International Scale (BCR::ABL1IS) was almost perfect quantitative agreement (Lin's CCC=0.99). By subgroups of MR levels, Lin's CCC showed a quantitative agreement of BCR::ABL1IS decreased as MR deepened. Both cdPCR and ddPCR demonstrated comparable performance in detecting BCR::ABL1 transcripts with high concordance in MR3 level or above. Choosing between platforms may depend on cost, workflow, and sensitivity requirements. [ABSTRACT FROM AUTHOR]

Simple Summary: SARS-CoV-2 infects humans and several animals, but infection of companion and zoo animals occurs mainly from humans through the aerosol route. Constant monitoring of the infection in animals is essential because of their close contact with humans, which increases the risk of infection and the potential for a surge of new viral variants. Therefore, it is of relevance to develop economical, fast, and effective diagnostic tests that are able to detect previous infections in multiple animal species. This study aimed to produce a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) using N and RBD proteins. The results show that this assay is able to detect antibodies against SARS-CoV-2 in serum samples from cats, dogs, guinea pigs, rats, tigers, and humans. SARS-CoV-2 infects humans and a broad spectrum of animal species, such as pets, zoo animals, and nondomestic animals. Monitoring infection in animals is important in terms of the risk of interspecies transmission and the emergence of new viral variants. Economical, fast, efficient, and sensitive diagnostic tests are needed to analyze animal infection. Double-antigen sandwich ELISA has the advantage of being multispecies and can be used for detecting infections caused by pathogens that infect several animal hosts. This study aimed to develop a double-antigen sandwich ELISA using two SARS-CoV-2 proteins, N and RBD. We compared its performance, when using these proteins separately, with an indirect ELISA and with a surrogate virus neutralization test. Positive and negative controls from a cat population (n = 31) were evaluated to compare all of the tests. After confirming that double-antigen sandwich ELISA with both RBD and N proteins had the best performance (AUC= 88%), the cutoff was adjusted using positive and negative samples from cats, humans (n = 32) and guinea pigs (n = 3). The use of samples from tigers (n = 2) and rats (n = 51) showed good agreement with the results previously obtained using the microneutralization test. Additionally, a cohort of samples from dogs with unknown infection status was evaluated. These results show that using two SARS-CoV-2 proteins in the double-antigen sandwich ELISA increases its performance and turns it into a valuable assay with which to monitor previous infection caused by SARS-CoV-2 in different animal species. [ABSTRACT FROM AUTHOR]

Objectives: Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, leading to the BCR::ABL1 fusion gene and hyper-proliferation of granulocytes. Tyrosine kinase inhibitors (TKIs) are effective, and minimal residual disease (MRD) monitoring is crucial. Digital PCR platforms offer increased precision compared to quantitative PCR but lack comparative studies., Methods: Eighty CML patient samples were analyzed in parallel using digital droplet PCR (ddPCR) (QXDx™ BCR-ABL %IS Kit) and chip digital PCR (cdPCR) (Dr. PCR™ BCR-ABL1 Major IS Detection Kit)., Results: Overall, qualitative and quantitative agreement was good. Sensitivity analysis showed positive percentage agreement and negative percentage agreement were both ≥90 %, and the quadratic weighted kappa index for molecular response (MR) level categorization was 0.94 (95 %CI 0.89, 0.98). MR levels subgroup analysis showed perfect categorical agreement on MR level at MR3 or above, while 35.4 % (17/48) of patient samples with MR4 or below showed discordant categorizations. Overall, Lin's concordance correlation coefficient (CCC) for the ratio of % BCR::ABL1 / ABL1 converted to the International Scale ( BCR::ABL1 ) was almost perfect quantitative agreement (Lin's CCC=0.99). By subgroups of MR levels, Lin's CCC showed a quantitative agreement of IS ) was almost perfect quantitative agreement (Lin's CCC=0.99). By subgroups of MR levels, Lin's CCC showed a quantitative agreement of BCR::ABL1 IS decreased as MR deepened., Conclusions: Both cdPCR and ddPCR demonstrated comparable performance in detecting BCR::ABL1 transcripts with high concordance in MR3 level or above. Choosing between platforms may depend on cost, workflow, and sensitivity requirements., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is spreading rapidly around the world. Antibody detection plays an important role in the diagnosis of COVID‐19. Here, we established a new time‐resolved fluorescence immunoassay (TRFIA) to determine COVID‐19 total antibodies. A double‐antigen sandwich TRFIA was optimized and established: recombinant nucleocapsid phosphoprotein (N protein) and spike protein (S protein) of COVID‐19 immobilized on 96‐well plates captured human COVID‐19 antibodies and then banded together with the N/S proteins labeled with europium(III) (Eu3+) chelates, and finally, time‐resolved fluorometry was used to measure the fluorescence values. We successfully established a TRFIA method for the detection of human COVID‐19 total antibodies, and the cutoff value was 2.02. There was no cross‐reactivity with the negative reference of the National Reference Panel for IgM and IgG antibodies to COVID‐19. The CV of the precision assay was 3.19%, and the assay could be stored stably for 15 days at 37°C. Compared with that of the colloidal gold method and chemiluminescence method, the sensitivity of the TRFIA method was higher, and the false positive/negative rate was lower. This established TRFIA has high sensitivity, accuracy, and specificity, which indicates that this method provides a new detection method for the high‐throughput routine diagnosis of COVID‐19. [ABSTRACT FROM AUTHOR]

This study demonstrated the terminated sialo‐sugar chains (Neu5Acα2,6Gal and Neu5Acα2,3Gal)‐mediated specificity enhancement of influenza virus and chicken red blood cell (RBC) by hemagglutination assay. These glycan chains were immobilized on the gold nanoparticle (GNP) to withhold the higher numbers. With the preliminary optimization, a clear button formation with 0.5% RBC was visualized. On the other hand, intact B/Tokio/53/99 with 750 nM hemagglutinin (HA) displayed a nice hemagglutination. The interference on the specificity of RBC and influenza virus was observed by anti‐influenza aptamer at the concentration 31 nM; however, there is no hemagglutination prevention was noticed in the presence of complementary aptamer sequences. Spiking GNP‐conjugated Neu5Acα2,6Gal or Neu5Acα2,3Gal or a mixture of these two to the reaction promoted the hemagglutination to 63‐folds higher with 12 nM virus, whereas under the same condition the heat‐inactivated viruses were lost the hemagglutination. Neuraminidases from Clostridium perfringens and Arthrobacter ureafaciens at 0.0025 neuraminidase units are able to abolish the hemagglutination. Other enzymes, Glycopeptidase F (Elizabethkingia meningoseptica) and Endoglycosidase H (Streptomyces plicatus) did not show the changes with agglutination. Obviously, sialyl‐Gal‐terminated glycan‐conjugated GNP amendment has enhanced the specificity of erythrocyte–influenza virus and able to be controlled by aptamer or neuraminidases. [ABSTRACT FROM AUTHOR]

Mitochondrial processes are implicated in plant response to biotic stress caused by viruses, actinomyces, bacteria and pests, but their function in defense against fungal invasion remains unclear. Here, we investigated the role and regulation of mitochondrial alternative oxidase (AOX) in response to black spot disease caused by the hemibiotrophic fungus Marssonina brunnea in poplar. M. brunnea inoculation induced the transcription of the AOX1a gene in the mitochondrial electron transport chain and of jasmonic acid (JA) and ethylene (ET) biosynthetic genes, with the accumulation of these phytohormones in poplar leaf, while inhibiting the transcript amount of the mitochondrial cytochrome c oxidase gene (COX6b) and genes related to salicylic acid (SA). Enhanced AOX reduced poplar susceptibility to M. brunnea with a higher ATP/ADP ratio while the repressed AOX caused the reverse effect. Exogenous JA and 1-aminocyclopropane-1-carboxylic acid (ACC, a biosynthetic precursor of ET) inhibited the transcript amount of COX6b and consequently increased the ratio of AOX pathway to total respiration. Furthermore, the transcription of CYS C1 and CYS D1 genes catalyzing cyanide metabolism was induced, while the cysteine (CYS) substrate levels reduced upon M. brunnea inoculation; exogenous JA and ACC mimicked the effect of M. brunnea infection on cysteine. Exogenous SA enhanced, while JA and ACC reduced, poplar susceptibility to M. brunnea. Moreover, inhibiting AOX completely prohibited JA- and ET-increased tolerance to M. brunnea in poplar. These observations indicate that the JA- and ET-induced mitochondrial AOX pathway triggers defense against M. brunnea in poplar. This effect probably involves cyanide. These findings deepen our understanding of plant–pathogenic fungi interactions. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) patients who have BCR-ABL T315I mutation, usually present in the advance phase of the disease with overall survival (OS) shorter than those without the mutation. This study aimed to determine the prevalence of T315I mutation amongst imatinib mesylate (IM) resistant CML patients and to compare the OS between T315I-mutated and non-T315I-mutated patients. Sixty consecutive CML patients who were treated with IM for at least 18 months and their treatment responses, were recorded. The mutation analysis was done using allele-specific oligonucleotide reverse transcriptase-polymerase chain reaction (RT-PCR) assay followed by direct sequencing technique. Forty-two patients (70%) were found to have IM-resistance. Five out of 42 patients had detectable T315I mutation. Median OS of IM-resistant T315I-mutated patients was 96 months (95% CI:54-138) compared to 84 months (95% CI:48-120) in non T315I-mutated patients, although this was found to be statistically insignificant (p = 0.43). The present study showed a higher prevalence of T315I mutation as compared to a few local studies. Median OS of T315I-mutated patients were observed to be longer than non-T315-mutated patients. Further studies encompassing larger cohort of patients are required to confirm this finding. [ABSTRACT FROM AUTHOR]

Although the majority of patients with chronic myeloid leukemia do well with treatment with tyrosine kinase inhibitors (TKIs), some patients still have inferior outcomes. There are many factors that might play a part, including the different BCR-ABL1 transcript types at baseline. The current study was performed to determine the possible impact of different transcripts on the treatment responses and outcomes of patients with chronic myeloid leukemia who are receiving TKI therapy. The authors performed a systematic literature search by using the terms "b2a2/b3a2," "e13a2/e14a2," or "transcript type." e14a2 was the more common transcript type. The majority of the studies demonstrated no significant difference regarding age, sex, leukocyte counts, and hemoglobin levels between patients with the e13a2 and e14a2 transcripts. However, in approximately one-half of the studies, the e14a2 transcript was associated with higher platelet counts. Almost no studies demonstrated a significant association between disease risk scores and transcript types. In the majority of studies, having the e14a2 transcript was associated with earlier, deeper, and higher molecular response rates. Although better event-free survival was observed in patients with the e14a2 transcript in some of the studies, the majority demonstrated that transcript type did not have an impact on progression-free and overall survival. Treatment-free remission currently is a topic of much interest, and to the authors' knowledge there are limited data with conflicting results regarding the possible effects of transcript types on the outcomes of patients after discontinuation of TKIs. Because having the e14a2 transcript appears to be related to a favorable outcome, choosing second-generation TKIs for frontline therapy might be a convenient approach in patients with chronic myeloid leukemia with the e13a2 transcript. The authors believe this finding warrants further investigation. [ABSTRACT FROM AUTHOR]

Objectives: BCR-ABL1 fusion transcripts with contrasting data on response to imatinib therapy have been reported from different parts of the world. Hence, the present study aimed to determine the frequencies of transcripts and their association with response to imatinib therapy in chronic myeloid leukemia (CML) patients. Methods: A total of 170 (76 follow-up and 94 imatinib-resistant) CML samples were included in the study. BCR-ABL1 fusion transcripts and expression status were analyzed in all cases using multiplex reverse transcriptase PCyR and real-time PCyR. Sanger sequencing was used for tyrosine kinase domain (TKD) mutation screening in imatinib mesylate-resistant patients. Results: Of 170 CML patients, 36.36% showed b2a2, 63.53% had b3a2, and 2.94% had b2a2 + b3a2 isoforms. Mean platelet counts and blasts were significantly lower in b2a2 carriers (P = 0.0092; P ≤ 0.0001). Patients with b2a2 transcript were found to be more in responders group (both hematological and cytogenetic), whereas b3a2 patients were more in partial responders group and death (P = 0.763; P = 0.309). In follow-up patients, mean baseline BCR-ABL1 expression levels are significantly higher in b2a2 versus b3a2 carriers (P = 0.0351). Of 94 imatinib-resistant patients, 36 (38.29%) had acquired TKD mutations. Among 36 patients, mean BCR-ABL1 levels are significantly higher in b2a2 and b2a2 + b3a2 group (P = 0.0002; P ≤ 0.0001). TKD mutation frequency was more in b3a2 (61.11%) compared to other types. With respect to follow-up status in 36 patients, 17 patients died while 19 were on imatinib higher doses or 2nd-generation tyrosine kinase inhibitors. Of 17 patients, 41.66% had b2a2 transcript and 54.54% had b3a2 transcript. Conclusion: Patients with b3a2 transcripts might be associated with poor response and worse prognosis in CML with imatinib treatment. [ABSTRACT FROM AUTHOR]

Purpose: The introduction and success of imatinib mesylate have become a paradigm shift in chronic myeloid leukemia (CML) treatment. However, despite its high efficiency, resistance to imatinib has emerged as a significant problem, which may in part be caused by pharmacogenetic variability. Three single-nucleotide polymorphisms (C1236T, G2677T/A, C3435T) and/or mRNA expression changes of ABCB1 gene were demonstrated to be associated with inter-individual variability of imatinib response in CML patients. In this study, we aimed to examine whether genetic variations and/or altered expression of ABCB1 gene may influence response to imatinib.Methods: Sixty nine CML Tunisian patients, undergoing imatinib therapy, were enrolled in this study. These were divided into two groups: responders and non-responders to imatinib. The relative transcript expression levels of ABCB1 gene and the distribution of allele and genotype frequency of ABCB1 SNPs were compared between these two categories of patients. Linkage disequilibrium tests and haplotypes analysis were also studied.Results: Our results showed that the mRNA expression level of ABCB1 gene did not differ significantly between the two categories of patients. In addition, results obtained from ABCB1 polymorphisms study and their correlation with imatinib response showed that the optimal response rate to imatinib did not differ significantly between C1236T, G2677T/A or C3435T genotypes. However, haplotype analysis showed that the 1236C-2677A-3435C haplotype was observed only in imatinib non-responders' patients suggesting that CAC haplotype was linked to higher risk of imatinib resistance.Conclusion: Furthermore, analyses of ABCB1 haplotypes should be taken into account to study the relationship between ABCB1 genotypes and imatinib efficacy. [ABSTRACT FROM AUTHOR]

Recently, point of care testing (POCT) used for diagnosis of influenza infection has a problem showing false negative diagnosis because of the low sensitivity. We would like to report detection of influenza virus A (H1N1) by an immunosensor based on electrochemiluminescence (ECL) that uses an immunoliposome encapsulating tris(2,2′-bipyridyl)ruthenium(II) complex. By using the sensor, we could detect the virus that competed with hemagglutinin (HA) peptide immobilized on self-assembled monolayers (SAMs) in immunoreaction of the antibody bound on the surface of liposome. The HA peptide was 19 mer (TGLRNGITNKVNSVIEKAA). We demonstrated great improvement of sensitivity and accuracy by introducing binary SAMs instead of mono SAMs. The binary SAMs was prepared from 3,3′-dithiodipropionic acid and 1-hexanethiol. Use of the binary SAMs enabled to increase the SAMs coverage on Au electrode; the fact was confirmed by observation of the cathodic desorption currents. By using such an electrode, first the detection method of BSA was optimized to lower ECL background signal. Then we applied the method to the detection of influenza virus. We could successfully detect the virus with higher sensitivity compared with that by POCT and ELISA. The detection range was from a concentration of 2.7 × 10 to 2.7 × 10 PFU/mL. [ABSTRACT FROM AUTHOR]

Introduction: Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, an abnormally shortened chromosome 22. It is the result of a reciprocal translocation of chromosomes 9 and 22, creating BCR-ABL fusion transcripts, b3a2, b2a2, and e1a2. The aim of our study was to determine the type of BCR-ABL fusion transcripts for molecular diagnosis and investigate the frequency of BCR-ABL fusion transcripts in CML patients by multiplex RT-PCR in CML.Materials and Methods: A single reaction with multiple primers multiplex PCR was used to detect and investigate the type and frequency in 200 CML patients among which 116, 33, and 51 were in CP, AP, and BC phase, respectively.Results: The study included 200 CML patients, among whom breakpoints in b3a2, b2a2 transcripts were detected in 68% and 24%, respectively, while 8% of the patients showed both b3a2/b2a2. A statistically significant difference was seen between frequency of BCR-ABL fusion transcripts and gender (P = 0.03), molecular response (P = 0.04), and hematological response (P = 0.05). However, there was no correlation found between frequencies of BCR-/ABL fusion transcripts and other clinicopathological parameters like age, type of therapy, thrombocytopenia, and white blood cell count.Conclusion: Multiplex reverse transcriptase-polymerase chain reaction is useful and saves time in the detection of BCR-ABL variants; the occurrence of these transcripts associated with CML can assist in prognosis and treatment of disease. [ABSTRACT FROM AUTHOR]

Endocrine-disrupting compounds (EDC) are chemicals responsible for disturbances in the hormonal balance of organisms. This group of chemicals includes both egzogenic and endogenic substances or their mixtures that impact functioning of natural hormones in organisms. In the available literature one can find information on the application of chromatographic and related techniques in the analysis of environmental samples for detection, identification, and quantitation of a wide spectrum of chemicals posing endocrine properties. On the other hand, more and more biotests are being developed to determine endocrine potency of environmental samples due to development of genetic engineering methods and specific detection methods of cells’ response to the action of particular chemicals of interest. This article presents revisions of the most novel methods for this potency determination with application to biological elements. [ABSTRACT FROM PUBLISHER]

Rather than the internal genome nucleic acids, the biomolecules on the surface of the influenza virus itself should be detected for a more exact and rapid point-of-care yes/no decision for influenza virus-induced infectious diseases. This work demonstrates the ultrasensitive electrical detection of the HA1 domain of hemagglutinin (HA), a representative viral surface protein of the influenza virus, using the top-down complementary metal oxide semiconductor (CMOS) processed silicon nanowire (SiNW) field-effect transistor (FET) configuration. Cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA) was employed as a probe that specifically binds both to the aldehyde self-aligned monolayer on the SiNWs and to HA1 simultaneously. CMP-NANA was serially combined with two kinds of linkers, namely 3-aminopropyltriethoxysilane and glutaraldehyde. The surface functionalization used was verified using the purification of glutathione S-transferase-tagged HA1, contact angle measurement, enzyme-linked immunosorbent assay test, and isoelectric focusing analysis. The proposed functionalized SiNW FET showed high sensitivities of the threshold voltage shift (ΔVT) ~51 mV/pH and the ΔVT = 112 mV (63 mV/decade) with an ultralow detectable range of 1 fM of target protein HA1. [ABSTRACT FROM AUTHOR]

The enzyme-linked immunosorbent assay (ELISA) has been widely used for disease surveillance and drug screening due to its relatively higher accuracy and sensitivity. Fine-tuning the ELISA is mandatory to elevate the specific detection of biomolecules at a lower abundance. Towards this end, higher molecular capture on the polystyrene (PS) ELISA surface is crucial for efficient detection, and it could be attained by immobilizing the molecules in the correct orientation. It is highly challenging to immobilize protein molecules in a well-aligned manner on an ELISA surface due to charge variations. We employed a 3-(aminopropyl) triethoxysilane (APTES)- and glutaraldehyde (GLU)-coupled PS surface chemical strategy to demonstrate the high performance with ELISA. A potassium hydroxide treatment followed by an equal ratio of 1% APTES and GLU attachment was found to be optimal, and a longer incubation with GLU favored maximum sensitivity. p24 is a vital early secreting antigen for diagnosing human immunodeficiency virus (HIV), and it has been used for efficient detection with the above chemistry. Three different procedures were followed, and they led to the improved detection of the HIV-p24 antigen at 1 nM, which is a 30-fold higher level compared to a conventional ELISA surface. The surface chemical functionalization shown here also displays a higher specificity with human serum and HIV-TAT. The above approach with the designed surface chemistry could also be recommended for disease diagnosis on other sensing surfaces involving the interaction of the probe and the analyte in heterogeneous test samples. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which discusses the case of a 29-year-old man with Von-Recklinghausens disease and malignant peripheral nerve sheath tumorin neurofibromatosis type I.