Diagnostic performance of oral swabs for non-sputum based TB diagnosis in a TB/HIV endemic setting

Objective We evaluated diagnostic performance of oral swab analysis (OSA) for tuberculosis (TB) in a high HIV/TB burden setting in Kenya. Methods In this cross-sectional study, buccal swabs and sputum were collected from 100 participants with suspected TB in outpatient clinics in Kenya at enrollment and subsequent morning visits. Buccal swabs underwent IS6110-targeted qPCR analysis. Sputum was evaluated by Xpert MTB/RIF (Xpert) and culture. Diagnostic performance of OSA for TB diagnosis was evaluated relative to a combined reference of sputum Xpert and culture. Results Among 100 participants, 54% were living with HIV (PLHIV). Twenty percent (20/100) of participants had confirmed TB (19/20 [95%] culture-positive, 17/20 [85%] Xpert-positive). Overall buccal swab sensitivity was 65.0% (95% CI 40.8–84.6%) vs. sputum Xpert/culture and 76.5% (95% CI 50.1–93.2%) vs. sputum Xpert alone. Specificity was 81.3% (95% CI 71.0–89.1%) and 81.9% (95% CI 72.0–89.5%) compared to sputum Xpert/culture and Xpert alone, respectively. Sensitivity among PLHIV (n = 54) with suspected TB was 83.3% (95% CI 35.9–99.6%) vs. sputum Xpert/culture and 100% (95% CI 47.8–100.0%) vs. sputum Xpert alone. Among participants with TB, mean OSA threshold quantitation cycle (Cq) value was lower (stronger signal) at subsequent morning compared to enrolment visit (33.4 SD ± 3.7 vs. 35.2 SD ± 2.9, p = 0.009). Conclusions In this pilot study, results confirm M. tuberculosis DNA is detectable in oral swabs including among PLHIV with fair diagnostic performance. Further work is needed to optimize OSA and evaluate its utility in diverse settings.

Diverse Single-Stranded DNA Viruses Identified in Chicken Buccal Swabs

High-throughput sequencing approaches offer the possibility to better understand the complex microbial communities associated with animals. Viral metagenomics has facilitated the discovery and identification of many known and unknown viruses that inhabit mucosal surfaces of the body and has extended our knowledge related to virus diversity. We used metagenomics sequencing of chicken buccal swab samples and identified various small DNA viruses with circular genome organization. Out of 134 putative circular viral-like circular genome sequences, 70 are cressdnaviruses and 26 are microviruses, whilst the remaining 38 most probably represent sub-genomic molecules. The cressdnaviruses found in this study belong to the Circoviridae, Genomoviridae and Smacoviridae families as well as previously described CRESS1 and naryavirus groups. Among these, genomoviruses and smacoviruses were the most prevalent across the samples. Interestingly, we also identified 26 bacteriophages that belong to the Microviridae family, whose members are known to infect enterobacteria.

Germline mutations among Polish patients with acute myeloid leukemia

Background A small but important proportion of patients (4–10 %) with AML have germline mutations. They can cause the development of AML at an earlier age, confer a higher risk of relapse or predispose to secondary leukemias, including therapy-related leukemias. The analysis of germline mutations in a patient and his/her family is also critical for the selection of suitable family donors if the patient is a candidate for hematopoietic stem cell transplantation (HSCT). Methods 103 unrelated consecutive patients with de novo AML were enrolled in the study. Control group consisted of 103 persons from the general population. We performed NGS sequencing of bone marrow cells and buccal swabs DNA of six genes: CEBPA, DDX41, ETV6, TERT, GATA2, and IDH2 to detect germline pathogenic mutations. Results In the investigated group, 49 variants were detected in six genes. 26 of them were somatic and 23 germline. Germline variants were detected in all six tested genes. Eight pathogenic germline mutations were detected in 7 AML patients, in three genes: CEBPA, ETV6, and IDH2. One patient had two pathogenic germinal mutations, one in ETV6 and one in CEBPA gene. We identified one novel pathogenic germline mutation in CEBPA gene. The difference in frequency of all pathogenic germline mutations between the tested (7.77 %) and control groups (0.97 %) was statistically significant (p = 0.046). In the tested group, the median age at AML diagnosis was 11 years lower in patients with pathogenic germline mutations than in patients without them (p = 0.028). Conclusions We showed higher frequency of CEBPA, ETV6, and IDH2 germline mutations in AML patients than in control group, which confirms the role of these mutations in the development of AML. We also showed that the median age at the onset of AML in patients with pathogenic germline mutations is significantly lower than in patients without them.

The effects of age, sex, weight and breed on canid methylomes

Unlike genomes, which are static throughout the lifespan of an organism, DNA methylomes are dynamic. To study these dynamics we developed quantitative models that measure the effect of multiple factors on DNA methylomes including, age, sex, weight and genetics. We conducted our study in canids, which prove to be an ideal species to assess epigenetic moderators due to their extreme variability in size and well-characterized genetic structure. We collected buccal swabs from 217 canids (207 domestic dogs and 10 gray wolves) and used targeted bisulfite sequencing to measure methylomes. We also measured genotypes at over one thousand single nucleotide polymorphisms (SNPs). We found that DNA methylomes are strongly associated with age, enabling the construction of epigenetic clocks. We also show that methylomes are strongly impacted by sex, weight and sterilization status, leading to accurate predators of these factors. Methylomes are also affected by genetics and we observe multiple associations between SNP loci and methylated CpGs. Finally, we show that several factors moderate the relationship between epigenetic ages and real ages, such as body weight, which increases epigenetic aging. In conclusion, we demonstrate that the plasticity of DNA methylomes is impacted by myriad genetic and physiological factors, and that DNA methylation biomarkers are accurate predictors of age, sex and sterilization status.

Genetic Polymorphisms, Gene–Gene Interactions and Neurologic Sequelae at Two Years Follow-Up in Newborns with Hypoxic-Ischemic Encephalopathy Treated with Hypothermia

Inflammation and oxidative stress after hypoxic-ischemic brain injury may be modified by genetic variability in addition to therapeutic hypothermia. The aim of our study was to evaluate the association between the polymorphisms in genes of antioxidant and inflammatory pathways in newborns treated with therapeutic hypothermia and the development of epilepsy or CP at two years follow-up. The DNA of 55 subjects was isolated from buccal swabs. Genotyping using competitive allele-specific PCR was performed for polymorphisms in antioxidant (SOD2 rs4880, CAT rs1001179, GPX1 rs1050450) and inflammatory (NLRP3 rs35829419, CARD8 rs2043211, IL1B rs1143623, IL1B rs16944, IL1B rs10716 76, TNF rs1800629) pathways. Polymorphic CARD8 rs2043211 T allele was less frequent in patients with epilepsy, but the association was not statistically significant. The interaction between CARD8 rs2043211 and IL1B rs16944 was associated with epilepsy after HIE: CARD8 rs2043211 was associated with lower epilepsy risk, but only in carriers of two normal IL1B rs16944 alleles (ORadj = 0.03 95% CI = 0.00–0.55; padj = 0.019). Additionally, IL1B rs16944 was associated with higher epilepsy risk only in carriers of at least one polymorphic CARD8 rs2043211 (ORadj = 13.33 95% CI = 1.07–166.37; padj = 0.044). Our results suggest that gene–gene interaction in inflammation pathways might contribute to the severity of brain injury in newborns with HIE treated with therapeutic hypothermia.

Genetic DNA Identification from Bone Remains in Kinship Analysis Using Automate Extraction System

The first ever human identification through DNA analysis was done in the year 1987. Since then, this test has been used, not only in the ruling of civil and juridical cases, but also for human identification of missing persons and mass disaster victims. In this chapter we will present the usefulness of genetic DNA testing of skeletonized remains for human identification, by using automate DNA extraction from three different human bone types: tooth, femur and petrous pyramid. For each case, we obtained saliva samples on buccal swabs from relatives. After the bones were washed and cleaned, Bead Balls Mill Mix 20 (Tehtnica Domel, Slovenia), was used to obtain the bone powder. The DNA extraction from bone samples was performed on the automate Maxwell RSC 48 Instrument (Promega, USA), using the Maxwell FSC DNA IQ Casework Kit (Promega, USA). Power Quant System (Promega, USA) was used for DNA quantification of the samples. The DNA samples were amplified on a Pro Flex PCR System (Thermo Fischer, USA), using the Global Filer PCR Amplification Kit (Applied Biosystems, USA). PCR products were run on a 3500 Genetic Analyzer (Thermo Fischer, USA). Data analysis was performed by Gene Mapper 1.4. Considering that these cases involved DNA extraction from teeth, bones and old human remains, automate system was felt to be the best option to reduce handling errors and increase the possibilities of obtaining good quality DNA.

Rapid Isothermal Amplification for the Buccal Detection SARS-CoV-2 in the Context of Out-Patient COVID-19 Screening

A commercially available isothermal amplification of SARS-CoV-2 RNA was applied to self-collected saliva samples using dry dental cotton rolls, which were held in the mouth for two minutes. Of 212 tests, isothermal amplification yielded three (0.14%) invalid results, 120 (56.6%) positive results and 89 (42%) negative results. Compared to reference RT-PCR assays routinely performed simultaneously on nasopharyngeal swabs, excluding the three invalid isothermal amplification assays and one RT-PCR invalid assay, these figures indicated that 119/123 (96.7%) samples were positive in both methods and 85/85 samples were negative in both methods. Four positive buccal swabs which were missed by the isothermal amplification, exhibited Ct values of 26–34 in reference RT-PCR assays. Positive isothermal amplification detection was achieved in less than 10 min. Supervision of the self-sampling procedure was key to achieve these performances. These data support the proposal to use the protocol reported in this paper, including supervised buccal self-sampling, to screen people suspected of having COVID-19 at the point of care.

Identification of genetic markers associated with ibrutinib-related cardiovascular toxicity.

7526 Background: Cardiovascular side effects (CVSEs: atrial fibrillation, hypertension, etc.) are common in patients with chronic lymphocytic leukemia (CLL) treated with ibrutinib and often lead to dose reductions or discontinuation. However, the etiology of ibrutinib related CVSEs has not been elucidated. This study sought to interrogate the association between ibrutinib related CVSEs and polymorphisms in genes of the Bruton Tyrosine Kinase (BTK) signaling pathway (identified through Ingenuity Pathway Analysis) Methods: Newly diagnosed and relapsed patients with CLL who underwent treatment with ibrutinib between December 2019 and November 2020 at Levine Cancer Institute were identified. Buccal swabs were collected through an IRB approved specimen collection protocol. Data extraction included: demographics, CLL stage, cytogenetics, previous treatments, ibrutinib start dates and dose, drug related SEs, and other medications. DNA isolated from buccal swabs was genotyped for 40 single nucleotide polymorphisms (SNPs) in GATA4, SGK1, KCNQ1, KCNA4, NPPA and SCN5A genes using a custom NGS panel. Logistic regression analysis evaluated the association between SNPs and CVSEs. Results: In 50 evaluable patients, the median age was 71 years (range:48-90) and 50% received frontline ibrutinib monotherapy. CVSEs occurred in 20% of patients (n=10). In univariate analysis, 4 SNPs in 3 genes were significantly associated with CVSEs (Table). Because the genes were in the same pathway, a genetic risk score was developed which indicated that patients with at least 2 SNPs had a 12-fold increase in risk of CVSEs (Table). Conclusions: Our findings provide insights into the genetic determinants of ibrutinib related CVSEs. If replicated in a larger study, this will facilitate utility of pharmacogenetic testing (for GATA4, KCNQ1 and KCNA5 polymorphisms ) as a clinical tool to individualize ibrutinib treatment.[Table: see text]