Archived DNA Samples Research Articles

High-throughput rapid amplicon sequencing for multilocus sequence typing of Mycoplasma ovipneumoniae from archived clinical DNA samples.

Spillover events of Mycoplasma ovipneumoniae have devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread of M. ovipneumoniae between the sheep populations. Most studies involving the typing of M. ovipneumoniae have used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing. Our study aimed to develop and validate an MLST workflow for typing of M. ovipneumoniae using Nanopore Rapid Barcoding sequencing and multiplex polymerase chain reaction (PCR). We compare the workflow with Nanopore Native Barcoding library preparation and Illumina MiSeq amplicon protocols to determine the most accurate and cost-effective method for sequencing multiplex amplicons. A multiplex PCR was optimized for four housekeeping genes of M. ovipneumoniae using archived DNA samples (N = 68) from nasal swabs. Sequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time and lowest cost per sample when compared with Nanopore Native Barcoding and Illumina MiSeq methods. Our proposed workflow is a convenient and effective method for strain typing of M. ovipneumoniae and can be applied to other bacterial MLST schemes. The workflow is suitable for diagnostic settings, where reduced hands-on time, cost, and multiplexing capabilities are important.

Evaluation of ITS1 rDNA primers for the detection and identification of African trypanosomes in mammalian hosts and tsetse flies.

Although several primers targeted to the internal transcribed-spacer 1 (ITS1) of the ribosomal DNA (rDNA) have been designed to improve the detection of African trypanosomes, no study tried to compare their agreement level and ability to amplify different trypanosome species in tsetse flies and mammals in various epidemiological settings. This study was designed to fill this gap, by targeting tsetse-infested areas of Cameroon. For this, archived DNA samples reporting at-least one trypanosome species with species-specific PCR primers were reviewed. Ten sets of primers targeting different ITS1 rDNA sequences of trypanosomes were selected for assessment using single-round and nested-PCR method. Amplification rates (sensitivity) and agreement level of different ITS1 assays were compared using Cohen's-Kappa and McNemar's x2 statistic. Little agreement level (k = 0.05–0.52) were observed between different ITS1-primers PCRs detection of African trypanosome species despite significant (X2=54.3, p = 0.0001) high amplification rate 91.6 % (339/370). This sensitivity varied from quite low for T. simiae (11.9 %) and T. vivax (27.3 %) to fairly good for T. congolence (51.9 %), Trypanozoon (32.4 %) and T. theileri (40.3 %). Primers set targeting ITS1-A sequence of trypanosome species recorded the highest sensitivity (50.5 %) with fairly good agreement compared to 39.2 % for ITS1-C (k = 0.52), 32.4 % for ITS1-R (k = 0.47), 29.7 % for ITS1-N (k = 0.48) and 23.0 % for ITS1-KIN (k = 0.43) respectively. This study revealed a diversity in the sensitivity of different trypanosome species with different sets of ITS-primers enhancing the need to use the same sets of primers in different bio-ecological settings. The use of nested-PCR instead of single-round PCR enabled improvement of trypanosome infections detection in both tsetse and mammals. Among the sets of ITS1-primers tested, those designed by to amplify ITS1-A can be considered as the most appropriate for the detection of trypanosome infections in mammals and tsetse flies.

Genetic Structure of Introduced Plasmodium vivax Malaria Isolates in Greece, 2015-2019.

Greece has been malaria-free since 1974, after an intense malaria control program. However, as Greece hosts migrant populations from P. vivax malaria-endemic countries, there is a risk of introducing the disease to specific vulnerable and receptive areas of the country. Knowledge of the genetic diversity of P. vivax populations is essential for understanding the dynamics of malaria disease transmission in a given region. We used nine highly polymorphic markers to genotype 124 P. vivax-infected archived DNA samples from human blood specimens referred to the NMRL from all over Greece throughout 2015-2019. The genotypic variability of the samples studied was noted, as they comprised several unique haplotypes, indicative of the importation of a large number of different P. vivax strains in the country. However, only a few events of local transmission were recorded. Genotyping revealed and confirmed the same clusters as those identified through epidemiological investigation. In only one introduction event was the index case found. No sustained/ongoing malaria transmissions in/between the studied regions or during consecutive years or additional foci of local transmission were observed. Genotyping is an important component in assisting malaria surveillance, as it provides information concerning the patterns of introduction and the effectiveness of implemented malaria control and elimination measures.

Genetic monitoring for effective plant conservation: An example using the threatened Saxifraga hirculus L. in Scotland

Societal Impact StatementMany mountain plants persist in small, isolated patches on the verge of extinction. Observational methods of monitoring these populations, such as recording the number of flowering stems, do not indicate the number of genetically distinct individuals, which is crucial information for conserving small populations. Here, the rate of clonal reproduction and number of genetic individuals were measured in the threatened Saxifraga hirculus in Scotland. These methods showed that population size is a poor proxy for genotype diversity and identified highly diverse small populations that may otherwise have been overlooked. This highlights the necessity of using genetic data to ensure the successful conservation of threatened plants.Summary Habitat fragmentation and loss increase the isolation of plant populations, increasing the occurrence of within population reproduction, and the potential for negative genetic effects, such as inbreeding depression and loss of genetic diversity. We use the European protected Marsh Saxifrage (Saxifraga hirculus) in Scotland as an example for declining perennial plants and the genetic resources they encapsulate. S. hirculus has declined due to agricultural intensification, drainage, industrial afforestation and grazing. The species can spread by seed or vegetatively through the production of rhizomes. Flowering is rare though due to grazing, which limits sexual reproduction and gene flow. An almost complete genetic inventory of Scottish populations was done using 11 microsatellite markers. Furthermore, archived DNA samples were used to document temporal genetic changes. We showed that clonal growth is predominant in some populations and genetic diversity (HS and allelic richness) is relatively high. However, the number of genetically distinct individuals (genets) per population is extremely low (3–34). Archived DNA samples showed that some populations consist of the same few genets with no evidence for turnover. Thus, while clonal growth may have helped the species to persist, there is limited creation of new gene combinations. Our findings highlight that reducing grazing pressure and increasing gene flow will be essential to rescue this species from its evolutionary dead end. We demonstrate the benefits of genetic monitoring for determining census population sizes and thus effective plant management and conservation. This work further sets out a strategy for moving this species towards demographic and genetic sustainability.

Molecular detection and characterization of Anaplasma spp. in cattle and sable antelope from Lusaka and North-Western provinces of Zambia

Rickettsiales of the genus Anaplasma are globally distributed tick-borne pathogens of animals and humans with complex epidemiological cycles. Anaplasmosis is an important livestock disease in Zambia but its epidemiological information is inadequate. This study aimed to detect and characterize the species of Anaplasma present in domestic and wild ruminants in Zambia with a focus on the infection risk posed by the translocation of sable antelope (Hippotragus niger) from North-Western Province to Lusaka Province. Archived DNA samples (n=100) extracted from whole blood (sable n=47, cattle n=53) were screened for Anaplasmataceae using 16S rRNA partial gene amplification followed by species confirmation using phylogenetic analysis. Out of the 100 samples, Anaplasma species were detected in 7% (4/57) of the cattle and 24% (10/43) of the sable antelope samples. Of the 14 positive samples, five were determined to be A. marginale (four from cattle and one from sable), seven were A. ovis (sable) and two were A. platys (sable). Phylogenetic analysis of the 16S rRNA partial gene sequences revealed genetic proximity between A. ovis and A. marginale, regardless of host. The detection of Anaplasma in wildlife in Zambia shows the risk of transmission of Anaplasma species associated with wildlife translocation.

Detection of Theileria orientalis Genotypes from Cattle in Kyrgyzstan.

The ikeda and chitose genotypes of Theileria orientalis, which for many years were thought to be benign, cause a disease that results in significant economic losses in the cattle industry. This study was carried out in order to determine the genotypes of T. orientalis in cattle in Kyrgyzstan, and 149 archived DNA samples known to be T. orientalis were analyzed by the PCR amplification of the major piroplasm surface protein (MPSP) gene region. Single-Strand Conformation Polymorphism (SSCP) analysis was performed to uncover the nucleotide changes in the archived DNA samples, and 15 samples showing different band profiles were subjected to sequence analysis. As a result of the sequence analysis, it was seen that the samples belonged to the buffeli and chitose A genotypes. In order to identify mixed genotypes, PCR was performed using primers specific for these genotypes, and buffeli (type 3), chitose (type 1) and buffeli+chitose were found to be positive in 26.2%, 2% and 71.8% of samples, respectively. As a result of this study, we showed the presence of buffeli (type 3) and chitose (type 1) genotypes of T. orientalis in cattle in Kyrgyzstan. Comprehensive epidemiological studies are needed to understand the clinical infections caused by the pathogenic chitose A and to determine the geographical distribution and different genotypes of T. orientalis.

Outcomes of allogeneic hematopoietic cell transplantation in adults with fusions associated with Ph-like ALL

AbstractAllogenic hematopoietic cell transplantation (alloHCT) is a well-established curative modality for acute lymphoblastic leukemia (ALL), yet large amounts of data describing alloHCT outcomes in Philadelphia (Ph)-like ALL are lacking. We retrospectively analyzed archived DNA samples from consecutive adults with B-cell Ph-negative ALL who underwent alloHCT in complete remission (CR) (n = 127) at our center between 2006 and 2020. Identification of fusions associated with Ph-like ALL was performed using cumulative results from RNA-seq, conventional cytogenetics, fluorescence in situ hybridization, and whole genome array studies. Fusions associated with Ph-like ALL were detected in 56 (44%) patients, of whom 38 were carrying CRLF2r. Compared with other non–Ph-like ALL (n = 71), patients with fusions associated with Ph-like ALL were more frequently Hispanic (P = .008), were less likely to carry high-risk cytogenetics (P < .001), and were more likely to receive blinatumomab prior to HCT (P = .019). With the median followup of 3.5 years, patients with Ph-like ALL fusions had comparable posttransplant outcomes compared with other B-cell ALL: 3-year relapse-free survival (RFS) (41% vs 44%; P = .36), overall survival (OS) (51% vs 50%; P = .59), and relapse (37% vs 31%; P = .47). In multivariable analysis, age (P = .023), disease status at the time of transplant (P < .001), and donor type (P = .015) influenced OS. RFS (primary endpoint) was significantly influenced by disease status (P < .001) and conditioning regimen intensity (P = .014). In conclusion, our data suggest that alloHCT consolidation results in similarly favorable survival outcomes in adult patients with Ph-like fusions and other high-risk B-cell ALL.

Comparison of the sensitivity, specificity, correlation and inter-assay agreement of eight diagnostic in vitro assays for the detection of African swine fever virus.

With the recent spread of African swine fever (ASF) in Europe, Asia and the Caribbean region, after being endemic for decades in Africa, PCR-based commercial kits and various master mixes are increasingly being used in addition to the Office International des Epizooties-recommended protocol from King etal. (World Organization for Animal Health). Often, the availability and cost of commercial kits or master mixes can be a limiting factor for diagnostic laboratories, in addition to the requirements for transportation and storage of temperature-sensitive reagents in remote areas. In such cases, alternatives should be ready to maximize surveillance and mining of ASF. To evaluate alternatives, we tested five commercial quantitative real-time PCR (qPCR) master mixes from Applied Biosystems, Bio-Rad, Biotechrabbit, Promega and Qiagen using the same primers and probe mix derived from the King etal.'s protocol for the sensitivity, specificity, correlation and inter-assay agreement. We further included three ad hoc molecular diagnostic kits (VetMax™ African Swine Fever Virus Detection Kit [Applied Biosystems], ID Gene African Swine Fever Duplex [ID-Vet] and Virotype ASF PCR Kit [Qiagen/Indical]). The limit of detection (LOD) was assessed for each assay. The comparative study panel comprised 83 archived DNA samples from ASF virus (ASFV) clinical samples, belonging to five different genotypes from outbreaks in 16 countries in Asia and Africa. The analytical specificity was assessed against a panel of swine pathogens. The LOD ranged from 13 to 41 gene copies per reaction; VetMax ™ African Swine Fever Virus Detection Kit from Applied Biosystems exhibited the lowest detection limit (13 gene copies per reaction) and iQ Supermix from Bio-Rad the highest detection limit (41 gene copies per reaction). Cq values obtained from the lowest dilution, in which all replicates (n=25) could still be amplified (50 gene copies per reaction), were not significantly different between kits using Kruskal-Wallis test. Inter-assay agreement was assessed using statistical test Fleiss-Kappa and was shown to be excellent in all cases. Agreement using statistical test Bland-Altman was good for samples with Cq values<25 and moderate for Cq values>25. We conclude that all the assays evaluated in this study can be used for the routine detection of ASFV.

Porcine circovirus-2 in Africa: Identification of continent-specific clusters and evidence of independent viral introductions from Europe, North America and Asia.

Porcine circovirus-2 (PCV-2) is associated with several disease syndromes in domestic pigs that have a significant impact on global pig production and health. Currently, little is known about the status of PCV-2 in Africa. In this study, a total of 408 archived DNA samples collected from pigs in Burkina Faso, Cameroon, Cape Verde, Ethiopia, the Democratic Republic of the Congo, Mozambique, Nigeria, Senegal, Tanzania and Zambia between 2000 and 2018 were screened by PCR for the presence of PCV-2. Positive amplicons of the gene encoding the viral capsid protein (ORF2) were sequenced to determine the genotypes circulating in each country. Four of the nine currently known genotypes of PCV-2 were identified (i.e. PCV-2a, PCV-2b, PCV-2d and PCV-2g) with more than one genotype being identified in Burkina Faso, Ethiopia, Nigeria, Mozambique, Senegal and Zambia. Additionally, a phylogeographic analysis which included 38 additional ORF2 gene sequences of PCV-2s previously identified in Mozambique, Namibia and South Africa from 2014 to 2016 and 2019 to 2020 and available in public databases, demonstrated the existence of several African-specific clusters and estimated the approximate time of introduction of PCV-2s into Africa from other continents. This is the first in-depth study of PCV-2 in Africa and it has important implications for pig production at both the small-holder and commercial farm level on the continent.

Outcomes of Allogeneic Hematopoietic Cell Transplantation in Adults with Ph-like ALL

Outcomes of Allogeneic Hematopoietic Cell Transplantation in Adults with Ph-like ALL

Microsporidia MB is found predominantly associated with Anopheles gambiae s.s and Anopheles coluzzii in Ghana

A vertically transmitted microsporidian, Microsporidia MB, with the ability to disrupt Plasmodium development was reported in Anopheles arabiensis from Kenya, East Africa. To demonstrate its range of incidence, archived DNA samples from 7575 Anopheles mosquitoes collected from Ghana were screened. MB prevalence was observed at 1.8%. An. gambiae s.s constituted 87% of positive mosquitoes while the remaining were from An. coluzzii. Both sibling species had similar positivity rates (24% and 19%; p = 0.42) despite the significantly higher number of An. gambiae s.s analysed (An. gambiae s.s = 487; An. coluzzii = 94; p = 0.0005). The microsporidian was also more prevalent in emerged adults from field-collected larvae than field-caught adults (p < 0.0001) suggestive of an efficient vertical transmission and/or horizontal transfer among larvae. This is the first report of Microsporidia MB in Anopheles mosquitoes in West Africa. It indicates possible widespread among malaria vector species and warrants investigations into the symbiont’s diversity across sub-Saharan Africa.

AML-142: Implementation of Combined DNA and RNA Sequencing in Myeloid Neoplasms

Context Identifying targetable and driver mutation and fusions in myeloid neoplasms through NGS platform. Objective The aim of this study was to optimize and validate Oncomine Myeloid Research Assay (comprising of 40 DNA target genes, 29 driver genes, and a broad fusion panel) for establishing assay sensitivity, specificity, and precision using Ion torrent PGM and implementation into clinics. Patients or other participants Archived DNA samples (n=17) with known mutations in (FLT3-ITD, CEBPA, NPM1, PTPN11, cMPL1, IDH1/2, TET, EZH2, U2AF1, SETBP1, ASLX1, TP53) and RNA samples (n=11) with FISH/RT-PCR-confirmed fusions (NUP 214, RUNX1, PML RARA BCR 1, PML RARA BCR 3, MLLT3, B14A3, B19A2) were retrieved as positive controls and negative controls for the validation. The sequences were analyzed using variant calling software (Ion Reporter Annotate variants version 5.4, 5.6, and 5.10) caller to identify variants relevant to the clinical indication. After thorough optimization and validation, this panel was used for prospective analysis in patients with suspected myeloid disorders. [AML (n=36), MDS (n=12), MPN (n=9)] AUL (n=1), Cytopenia/anaemia under evaluation (n=8) and CMML (n=3)]. Intervention FLT3-ITD by fragment analysis was performed in all cases after validation. Main outcome measures After implementation of NGS platform in myeloid neoplasms FLT3 and TET2 genes were the most common pathogenic variant in DNA sequencing and KMT2A rearrangement on RNA sequencing. Results In the four validation runs (318 chip - 4 DNA/RNA) comprising 28 known variants, all mutations/rearrangements were concordant except two cases (FLT3-ITD > 200bp and SETBP1, G870S 3.2%). For the prospective myeloid disorders (n=69), mutations were observed in 31 genes, maximum cases having FLT3 (n=8) and TET2 (n=8). The other frequently mutated genes were SRSF2 (n=7), NRAS (n=6), RUNX1 (n=6), ASXL1 (n=6), CEBPA (n=5), U2AF1 (n=4), SH2B3 (n=4), BCOR (n=4). All FLT3-ITD-positive and -negative cases were confirmed by Fragment Length Analysis. In eleven cases, fusions were detected; KMT2A (n=4), BCR-ABL (n=3), PML-RARA (bcr3) (n=1), and RUNX1T1 (n=2) and ETV6-ABL fusions Conclusions We demonstrate the validation and implementation of a combined targeted DNA and RNA sequencing for routine clinical use in myeloid malignancies.

Impact of an Irrigation Dam on the Transmission and Diversity of Plasmodium falciparum in a Seasonal Malaria Transmission Area of Northern Ghana.

Water bodies such as dams are known to alter the local transmission patterns of a number of infectious diseases, especially those transmitted by insects and other arthropod vectors. The impact of an irrigation dam on submicroscopic asexual parasite carriage in individuals living in a seasonal malaria transmission area of northern Ghana was investigated. A total of 288 archived DNA samples from two cross-sectional surveys in two communities in the Bongo District of Northern Ghana were analysed. Parasite density was determined by light microscopy and PCR, and parasite diversity was assessed by genotyping of the polymorphic Plasmodium falciparum msp2 block-3 region. Submicroscopic parasitaemia was estimated as the proportional difference between positive samples identified by PCR and microscopy. Dry season submicroscopic parasite prevalence was significantly higher (71.0%, p=0.013) at the dam site compared with the nondam site (49.2%). Similarly, wet season submicroscopic parasite prevalence was significantly higher at the dam site (54.5%, p=0.008) compared with the nondam site (33.0%). There was no difference in parasite density between sites in the dry season (p=0.90) and in the wet season (p=0.85). Multiplicity of infection (MOI) based on PCR data was significantly higher at the dam site compared with the nondam site during the dry season (p < 0.0001) but similar between sites during the wet season. MOI at the nondam site was significantly higher in the wet season than in the dry season (2.49, 1.26, p < 0.0001) but similar between seasons at the dam site. Multivariate analysis showed higher odds of carrying submicroscopic parasites at the dam site in both dry season (OR = 7.46, 95% CI = 3.07–18.15) and in wet season (OR = 1.73, 95% CI = 1.04–2.86). The study findings suggest that large water bodies impact year-round carriage of submicroscopic parasites and sustain Plasmodium transmission.

Host range and geographical distribution of Babesia sp. Mymensingh.

Bovine babesiosis represents a serious threat to the cattle industry in the tropics and subtropics. Although several Babesia species infect cattle, only B. bovis, B. bigemina and B. divergens are known to cause clinical babesiosis. However, our recent study demonstrated that the newly discovered Babesia sp. Mymensingh might be a virulent species capable of causing clinical babesiosis in cattle. The objective of this study was to determine the host range and geographical distribution of Babesia sp. Mymensingh on a global scale. A total of 2,860 archived DNA samples from 2,263 cattle in Sri Lanka (n=672), the Philippines (n=408), Vietnam (n=460), Uganda (n=409), Brazil (n=164) and Argentina (n=150); 419 buffalo in Sri Lanka (n=327) and Vietnam (n=92); and 127 goats and 51 sheep in Vietnam were screened using a Babesia sp. Mymensingh-specific PCR assay. Babesia sp. Mymensingh infection was detected in cattle, buffalo, sheep and goats. Cattle of all countries surveyed in this study except Brazil were found to be infected with Babesia sp. Mymensingh. The highest positive rates were recorded in cattle from the Philippines (11.3%) and Vietnam (9.6%), followed by Argentina (4.7%), Sri Lanka (1.5%) and Uganda (1.0%). Buffalo were found to be infected with this parasite in Sri Lanka (1.2%) and Vietnam (10.9%). Unexpectedly, Babesia sp. Mymensingh was also detected in sheep (2.0%) and goats (1.3%) from Vietnam. These findings were confirmed by PCR amplicon sequencing. In conclusion, our present findings indicate that Babesia sp. Mymensingh, which infects cattle, buffalo, sheep and goats, is endemic in Asia, Africa and South America.

RBTT-04. DGM1 MAY SERVE AS A NOVEL GENETIC BIOMARKER OF RESPONSE TO ENZASTAURIN IN GLIOBLASTOMA

Abstract BACKGROUND No significant progress has been made over the last decade in therapies available for glioblastoma (GBM) with survival remaining poor. Meaningful advances in treating this deadly malignancy may rely on precision medicine. A novel pharmacogenomic biomarker, Denovo Genomic Marker 1 (DGM1), for enzastaurin (enz) in treating lymphoma was discovered. It was evaluated to predict enz response in GBM. METHODS Biomarker discovery was performed by a genome-wide screen using DNA extracted from blood samples of a ph 3 enz lymphoma trial and confirmed in an independent ph 2 lymphoma trial. The biomarker was evaluated for its predictability in GBM using the archived DNA samples from a ph 1/2 enz GBM trial. RESULTS DGM1, a germline polymorphism on chromosome 8, was found to be highly correlated with response to enz in the two lymphoma trials. Using DNA extracted from pts blood of the single-arm ph 1/2 study with newly diagnosed GBM receiving enz added to radiation and temozolomide (tmz), we found median OS for DGM1+ pts treated with enz was 18 mon vs 12.8 mon for DGM1- pts, HR (95% CI) 0.68 (0.25, 1.81), p = 0.12. Pts receiving a mean daily dose of enz ≥ 245 mg had an OS of 19.8 mon vs 14.9 mon for pts receiving a mean daily dose of &lt; 245 mg [HR (95% CI) 0.55 (0.34, 0.90)]. CONCLUSION These data are supportive of DGM1 as a potentially predictive biomarker for enz response in both lymphoma and GBM. There is an ongoing biomarker-driven pivotal study in lymphoma. DGM1 in GBM will be further evaluated in a planned randomized ph 2b study in newly diagnosed GBM with 500 mg/day of enz in combination with tmz.

Neuronal ceroid lipofuscinosis in a German Shorthaired Pointer associated with a previously reported CLN8 nonsense variant

Two littermate German Shorthaired Pointers, a male and a female, were adopted as puppies from an animal shelter. Both puppies developed normally until approximately 11 months of age when the male began to exhibit neurological signs including ataxia, vision loss, and behavioral changes indicative of cognitive decline. These signs increased in severity over time. The female remained neurologically normal and healthy. The affected dog was euthanized at approximately 21 months of age. Autofluorescent cytoplasmic storage bodies were detected in neurons in unstained tissue sections from the cerebellum, the cerebrum, and the retina. Electron micrographs of these storage bodies showed that they were membrane bound and that most contained tightly packed aggregates of membranous whorls along with a variety of other ultrastructural features. This ultrastructure, along with the autofluorescence and the clinical signs supported a diagnosis of neuronal ceroid lipofuscinosis (NCL). Unlike earlier investigated forms of canine NCL with causal alleles in ATP13A2, TPP1, MFSD8 and CLN5 that had autofluorescent cytoplasmic storage bodies in cardiac muscle, no autofluorescence was detected in cardiac muscle from the affected German Shorthaired Pointer. A 39-fold average coverage whole genome sequence indicated that the affected German Shorthaired Pointer was homozygous for the A allele of a G > A transversion at position 30,895,648 chromosome 37. This 37:30895648G > A mutation created a CLN8 termination codon that had been previously reported to cause NCL in a mixed breed dog with Australian Shepherd and Australian Cattle Dog ancestry. This nonsense allele was heterozygous in the clinically normal female sibling, while archived DNA samples from 512 other German Shorthaired Pointers were all homozygous for the reference allele. The affected German Shorthaired Pointer and the previously diagnosed mixed breed dog with the same nonsense mutation shaired an identical homozygous haplotype that extended for 4.41 Mb at the telomeric end of chromosome 37, indicating the both dogs inherited the nonsense mutation from a common ancestor.

DGM1 may serve as a novel genetic biomarker of response to enzastaurin in glioblastoma.

2023 Background: Despite countless clinical trials being conducted, little has changed over the last decade in the chemotherapies available for glioblastoma (GBM) with survival remaining poor. Meaningful advances in treating this deadly malignancy may rely on precision medicine. We discovered a novel pharmacogenomic biomarker for enzastaurin (enz) in treating lymphoma (lymph). We evaluated if this biomarker can be used to predict enz response in GBM. Methods: Biomarker discovery was performed by a genome-wide screen using DNA extracted from blood samples from a ph 3 enz lymph trial and confirmed in an independent ph 2 enz lymph trial. The biomarker was then evaluated for its predictability in GBM using the archived DNA samples from a prior ph 1/2 enz GBM trial. Results: A novel biomarker, Denovo Genomic Marker 1 (DGM1), a germline polymorphism on chromosome 8, was found to be highly correlated with response to enz in the two lymph trials. Using DNA extracted from blood of pts from the single-arm ph 1/2 study of newly diagnosed GBM receiving enz added to radiation and temozolomide (tmz), we found median OS for DGM1+ pts treated with enz was 18 mon vs 12.8 mon for DGM1- pts, HR (95% CI) 0.68 (0.25, 1.81), p = 0.12. In addition, we found pts in the GBM study receiving a mean daily dose of enz ≥ 245 mg had an OS of 19.8 mon vs 14.9 mon for pts receiving a mean daily dose of &lt; 245 mg [HR (95% CI) 0.55 (0.34, 0.90)]; enz 500 mg/day was used in the lymph studies. Conclusions: These data are supportive of DGM1 as a potentially predictive biomarker for enz response in both lymph and GBM. There is an ongoing biomarker-driven pivotal ph 3 study in lymph at 500 mg/day, and DGM1 in GBM will be further evaluated in a planned randomized ph 2b study in newly diagnosed GBM with 500 mg/day of enz in combination with tmz.

Epidemiological survey of Anaplasma marginale in cattle and buffalo in Sri Lanka.

Bovine anaplasmosis caused by Anaplasma marginale represents a serious threat to cattle farming worldwide, especially in the tropics and subtropics. In the present study, archived DNA samples from the blood of cattle (n=437) in the Nuwara Eliya, Galle, Ampara, Polonnaruwa, and Jaffna districts and buffalo (n=327) in the Galle, Polonnaruwa, Mannar, and Mullaitivu districts in Sri Lanka, were screened for A. marginale using a major surface protein 5 (msp5) gene-based PCR assay. The findings showed that 32.7 and 57.5% of cattle and buffalo, respectively, were A. marginale-positive. The rate of positivity differed significantly among geographical regions. In conclusion, the high rates of A. marginale infection in cattle and buffalo highlight the importance of effective control measures in Sri Lanka.

Slow parasite clearance, absent K13-propeller gene polymorphisms and adequate artesunate levels among patients with malaria: A pilot study from southern India.

Artemisinin-based combination therapy (ACT) is widely used in India and many generic preparations are available. Delayed response has been reported, suggesting inadequate response to artesunate (AS) or genotypic resistance. We designed a prospective observational study to assess the therapeutic response, elaborate pharmacokinetics of AS and identify Plasmodium falciparum kelch 13 (pfk13) propeller gene polymorphisms among hospitalized Indian patients with severe malaria. We collected blood samples from adult patients with severe P. falciparum or mixed (P. falciparum and P. vivax) malaria on ACT. We calculated the parasite clearance (CL) half-life using the Worldwide Antimalarial Resistance Network (WWARN) online parasite clearance estimator (PCE). We used the liquid chromatography tandem mass spectrophoto-metry method for simultaneous quantification of AS and dihydroartemisinin. We genotyped longitudinally archived DNA samples obtained pre-treatment (day 0) to study the point mutations in the pfk13 propeller domain. A total of 54 patients with malaria were included, with a majority fulfilling the definitions of severe malaria. The median parasite CL slope half-life was estimated to be 6.44 hours (interquartile range 4.79-10.24). AS pharmacokinetics, assessed in 17 patients, were found to be similar in the groups with rapid (<48 hours) and slow CL (>48 hours) of parasites. No known mutations associated with artemisinin resistance in Southeast Asia were observed in our study participants. Slow parasite CL was seen with a high parasite burden without genotypic evidence of AS resistance. There is a need to standardize definitions of therapeutic efficacy of AS in cases of severe malaria.

Cytokine Gene Polymorphisms Are Associated with Disease Response to Blinatumomab in Patients with B-Cell Acute Lymphoblastic Leukemia

Blinatumomab (BT), a CD19/CD3 bispecific T-cell engager (BiTE) antibody recently approved for treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), has resulted in a 40-50% complete response (CR)/CR with incomplete count recovery (CRi) rate and frequent cytokine release syndrome (CRS) events. While extent of disease burden has been identified as a key predictor of disease response, there have been no studies to date evaluating the impact of single nucleotide polymorphisms (SNPs) in cytokine genes on disease response or CRS after treatment with BT. Here, we evaluated the possible association between cytokine SNPs, disease response, and/or CRS in r/r ALL patients with receiving BT.A total of 82 patients (consecutive case-series) received BT between 2012 and 2017 at City of Hope. Of these, 68 patients had archived DNA samples available for study and analysis. Genotyping was done using Illumina's Omni 5-4 bead array, which interrogates more than 4.2 million SNPs. Array processing was done following manufacturer's recommendations and arrays were scanned by Illumina's iScan instrument. Beeline software and Genome Studio (Illumina) were used for analysis of scanned images and to generate the genotyping calls and quality control reports, respectively. Our analysis focused on the following cytokine genes of interest (known to be associated with autoimmune diseases/graft-versus-host disease): IL-1B, IL-2, IL-6, IL-7R, IL-10, IL-11, IL-12, IL-17A, IL-23R, TNF-α, TGF-β, and IFN-γ. Two modes of inheritance (dominant and recessive) were considered whenever appropriate. Univariate analyses of SNPs, response and CRS were performed using Fisher's exact test. P values were not adjusted for multiple hypothesis testing due to the exploratory nature of the analyses.In our cohort, median age of patients at BT treatment was 34 years old (range: 7-85), with 32 females (39%) and 50 males (61%). Patients' race/ethnicity included 49 Hispanic (60%), 20 non-Hispanic Caucasians (24%), 9 Asians (11%), and 3 African Americans (3%). All patients had primary refractory (n=16) or relapsed disease status (1st relapse: n=38, 2nd or subsequent relapse: n=27) with median lines of prior therapy of 2 (range: 0-5). Cytogenetic abnormalities included Ph+ALL in 11 patients, while 50 patients had other cytogenetic abnormalities and 15 had normal cytogenetics. Bone marrow blasts of >50% at BT treatment was detected in 45 patients, and 22 patients had a history of or active extra-medullary involvement. After BT treatment, 37 patients (45%) achieved CR/CRi with a median duration of 9.5 months (range: 1-37), 54 patients (66%) experienced CRS (grade 1: n=35, grade 2: n=14, and grade 3: n=5), and 9 patients developed neurotoxicity. Lastly, the peak C-reactive protein level (median/range) was 17 mg/L (range: 1-392).By univariate analysis, disease response (CR/CRi) to BT was significantly associated with the following cytokine SNPs: IL2 rs2069762 [48.3% (T/T-G/T: n=60) Vs. 0% (G/G; n=7), p=0.016], IL17A rs3819024 [30.8% (A/A; n=39) Vs. 58.6% (A/G-G/G; n=29), p=0.027], and IL17A rs4711998 [54.5% (A/A: n=33) Vs. 29.4% (A/G-G/G: n=34), p=0.049]. (Table 1) These three cytokine SNPs and two additional SNPs with p-values between 0.05 and 0.1 (IL7R rs1494555, IL17A rs8193036) were evaluated by multivariable analysis, adjusted for >50% bone marrow blasts, which was associated with lower rate of CR/CRi (33% vs. 63% with <50%, p=0.02). By multivariable analysis, IL17A rs3819024 remained significant while IL17A rs4711998 was at borderline significance (p=0.06). (IL2 rs2069762 could not be tested due to no CR/CRi occurrence). None of the cytokine SNPs analyzed were associated with CRS (0 vs. any grade, or grades 0-1 vs. 2-3).In conclusion, to our knowledge this is the first study to demonstrate a possible association between treatment response to BT and cytokine genetic polymorphisms. Our hypothesis-generating data suggest a potential role of IL17 and IL2 in BT response and justify a larger confirmatory study, which may lead to personalized BT immunotherapy for B-ALL. [Display omitted] DisclosuresStein:Amgen Inc.: Speakers Bureau; Celgene: Speakers Bureau. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.