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.

CYP2D6 Genotyping for Personalized Therapy of Tamoxifen in Indonesian Women with ER+ Breast Cancer

PurposeTamoxifen, common adjuvant therapy prescribed in estrogen receptor positive (ER+) breast cancer, is metabolized by CYP2D6 enzyme into endoxifen. The phenotypes of CYP2D6, a highly polymorphic gene, vary from ultrarapid (UM), normal (NM), intermediate (IM), and poor metabolizers (PM). Studies showed that reduced CYP2D6 activity in IMs and PMs resulted in lower endoxifen level, thereby reducing therapy efficacy. This study aims to observe the distribution of CYP2D6 profiles and their corresponding endoxifen levels in Indonesian ER+ breast cancer patients.Methods151 patients who have received tamoxifen therapy for ≥8 weeks were recruited prospectively. DNA and blood samples were collected with buccal swab and finger-prick methods, respectively. Genotyping was performed using the qPCR method while metabolites measurement was performed using HPLC-tandem MS. Patients with IM/PM CYP2D6 profile were advised to increase their tamoxifen dose or switch to aromatase inhibitor, while patients with UM or NM CYP2D6 profile remained on 20 mg daily dose. Tamoxifen metabolites levels of those given 40 mg/day of tamoxifen were measured eight weeks post dose adjustment.ResultsWe found that 40.7% of patients recruited were IM. CYP2D6*10 was the most abundant allele (28.8%) and *10/*36 was the most frequently observed diplotype (23.6%). Endoxifen levels between the NM-PM, NM-IM, and IM-PM were statistically significant, and dose increase of tamoxifen successfully increased endoxifen levels in IMs to a similar level with NMs at baseline.ConclusionIndonesian women have a relatively high proportion of IMs. The correlation between CYP2D6 genotype and phenotype was shown in the significant difference in endoxifen levels among NMs, IMs, and PMs. Dose adjustment of tamoxifen to 40 mg daily positively increased endoxifen levels in IMs to a similar level as NMs. Implementing pharmacogenomics testing of CYP2D6 on ER+ breast cancer women taking tamoxifen can potentially increase the likelihood of achieving better treatment efficacy.Trial RegistrationThe trial was retrospectively registered at ClinicalTrials.gov on 18 March 2020 with identifier NCT04312347 (accessible at: https://clinicaltrials.gov/ct2/show/NCT04312347).

First Results from the Prospective German Registry for Childhood Glaucoma: Phenotype–Genotype Association

Childhood glaucoma is a heterogeneous disease and can be associated with various genetic alterations. The aim of this study was to report first results of the phenotype–genotype relationship in a German childhood glaucoma cohort. Forty-nine eyes of 29 children diagnosed with childhood glaucoma were prospectively included in the registry. Besides medical history, non-genetic risk factor anamnesis and examination results, genetic examination report was obtained (23 cases). DNA from peripheral blood or buccal swab was used for molecular genetic analysis using a specific glaucoma gene panel. Primary endpoint was the distribution of causative genetic mutations and associated disorders. Median age was 1.8 (IQR 0.6; 3.8) years, 64% participants were female. Secondary childhood glaucoma (55%) was more common than primary childhood glaucoma (41%). In 14%, parental consanguinity was indicated. A mutation was found in all these cases, which makes consanguinity an important risk factor for genetic causes in childhood glaucoma. CYP1B1 (30%) and TEK (10%) mutations were found in primary childhood glaucoma patients. In secondary childhood glaucoma cases, alterations in CYP1B1 (25%), SOX11 (13%), FOXC1 (13%), GJA8 (13%) and LTBP2 (13%) were detected. Congenital cataract was associated with variants in FYCO1 and CRYBB3 (25% each), and one case of primary megalocornea with a CHRDL1 aberration. Novel variants of causative genetic mutations were found. Distribution of childhood glaucoma types and causative genes was comparable to previous investigated cohorts. This is the first prospective study using standardized forms to determine phenotypes and non-genetic factors in childhood glaucoma with the aim to evaluate their association with genotypes in childhood glaucoma.

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.

The Feasibility and Utility of Hair Follicle Sampling To Measure FMRP and FMR1 mRNA in Children With or Without Fragile X Syndrome

Background: Fragile X syndrome (FXS) is the most common cause inherited cause of intellectual disability in males and the most common single gene cause of autism. This X-linked disorder is caused by an expansion of a trinucleotide CGG repeat (>200 base pairs) on the promotor region of the fragile X mental retardation 1 gene (FMR1). This leads to the deficiency or absence of the encoded protein, Fragile X mental retardation protein (FMRP). FMRP has a central role in the translation of mRNAs involved in synaptic connections and plasticity. Recent studies have demonstrated the benefit of therapeutics focused on reactivation of the FMR1 locus towards improving key clinical phenotypes via restoration of FMRP and ultimately disease modification. A key step in future studies directed towards this effort is the establishment of proof of concept (POC) for FMRP reactivation in individuals with FXS. For this it is key to determine the feasibility of repeated collection of tissues or fluids to measure FMR1 and FMRP. Methods: Individuals, ages 3 to 22 years of age, with FXS and those who were typically developing participated in this single-site pilot clinical biomarker study. The repeated collection of hair follicles was compared with the collection of blood and buccal swabs for detection of FMR1 mRNA and FMRP and related molecules. Results: There were n = 15 participants, of whom 10 had a diagnosis of FXS (7.0 ± 3.56 years) and 5 were typically developing (8.2 ± 2.77 years). Absolute levels of FMRP and FMR1 mRNA were substantially higher in healthy participants compared to full mutation and mosaic FXS participants, and lowest in the FXS boys. Measurement of FMR1 and FMRP levels by any method did not show any notable variation by collection location at home versus office across the various sample collection methodologies of hair follicle, blood sample, and buccal swab. Conclusion: Findings demonstrated that repeated sampling of hair follicles in individuals with FXS, in both, home and office settings, is feasible, repeatable, and can be used for measurement of FMR1 and FMRP in longitudinal studies.

Assessment of Next Generation Sequencing Approaches for the Cytogenetic and Molecular Evaluation of Acute Myeloid Leukemia

Background: Genomic evaluation of structural and molecular alterations is essential for the classification and risk stratification of patients with acute myeloid leukemia (AML). Cytogenetics (CG) has traditionally been used for the identification of large structural or copy number changes and molecular studies for small sequence changes. Rapid technical advances and decreased sequencing (seq) costs have made whole-genome sequencing (WGS) more feasible and recent efforts have suggested it can match the analytic performance of traditional CG while providing additional prognostic information. However, the use of WGS in the clinic is still in its infancy and questions remain regarding the utility of many factors including seq depth, structural events not identifiable by traditional CG, normal samples, AML enrichment, paired RNA-seq, and single-cell DNA-seq. Methods: Baseline bone marrow (BM) samples from 10 AML patients underwent evaluation using standard clinical CG and targeted next generation seq tests. Additionally, samples were extensively characterized using: 200X WGS (bulk and enriched BM), 50X germline WGS (buccal swab and remission blood), 50X RNA-seq, targeted error-corrected (EC) DNA-seq, and single-cell DNA-seq with oligonucleotide-conjugated antibodies (scDNA-seq+ab). Results: The AML patients analyzed had a mean age of 71 (30-81) and a range of BM blast percentage by clinical flow cytometry (0.4-33%). Standard clinical assessment revealed 14 CG abnormalities and 20 mutations across all patients. 50X WGS of the bulk BM utilizing previously published conditions (PMID:33704937) successfully identified 89% of the alterations. Increasing the WGS depth to 100X recovered 2 additional abnormalities, increasing the capture rate to 94%. Enrichment of AML using the primary cell surface marker prior to WGS achieved a 94% capture rate at only 50X depth, with a depth of only 10X required for the CG abnormalities. Integration of 50X WGS on buccal swab samples reduced the large number of structural variant calls and private mutations without the need for aggressive filtering conditions required for tumor-only samples. However, this also resulted in the removal of a few true mutations due to buccal contamination with immune cells. 50X WGS on remission blood samples from 2 patients mitigated this problem. Next, we sought to determine what additional information WGS could provide that was missed by standard CG (>5Mb) and seq (>5% variant allele fraction, VAF) analyses. Firstly, 3 of the deletions reported by CG were found to be complex translocation events, resulting in different losses and gains than anticipated. Most importantly, decreasing the CNV size threshold (<5Mb) and utilizing SNP data to identify copy neutral loss of heterozygosity (LOH) identified 7 new events in bulk BM and 2 new events in enriched BM. This information provided important prognostic information, including 3 newly identified structural alternations in a patient with a CG-reported normal karyotype, multiple small deletions in critical regions (e.g. within 5q, 7q), and LOH which in combination with molecular data revealed a biallelic loss of important genes (including TP53, EZH2). Targeted EC DNA-seq was used to detect of variants <5% VAF and identified an additional 23 variants beyond clinical capture sequencing. 50X RNA-seq with integrated WGS DNA/RNA calling was able to validate the mutations and translocation fusion genes reported by 50X WGS, while also capturing an additional 4 variants found by EC DNA-seq. Finally, personalized scDNA-seq+ab provided further resolution of each patient's AML through confirmation of new structural and mutational findings, identification of mutations associated with clonal hematopoiesis versus AML, and definition of AML clonal structure (Figure 1). Conclusions: We confirmed that 50X WGS can recapitulate standard clinical CG and mutational profiling in AML patients while providing improved resolution. Deeper seq, AML pre-enrichment, and integrated RNA-seq further aided in identifying low level events. We also demonstrated that WGS can identify additional important structural events missed by CG. Finally, personalized scDNA-seq+ab was able to define clonal architecture important for prognostic and residual disease tracking. Together, these results provide a framework for future integration of WGS into the clinic and personalized patient care. Figure 1 Figure 1. Disclosures Sciambi: Mission Bio Inc.: Current Employment. Durruthy-Durruthy: Mission Bio Inc.: Current Employment. Hourigan: Sellas: Research Funding.

Alpers- and MNGIE-like disease with disturbed CSF folate transport and an unusual mode of genetic transmission of POLG mutations: a case report

We report about a family with three of five siblings affected by a variable remitting-relapsing disease with epileptic seizures, coma and abdominal crises, and lethal outcome in all. In the youngest son and in one of his deceased brothers we identified two disease causing compound heterozygous POLG mutations. One of these was inherited from the mother, but the other was absent in the father`s blood, saliva, buccal swab and hair bulbs although his paternity was proven genetically. Thus, we assume germline mosaicism for this mutation in the father. Very low 5-methyltetrahydrofolate (5-MTHF) and absence of folate receptor-alpha was repeatedly found in the CSF of the youngest brother indicating a secondary cerebral folate transport deficiency. Folinic acid supplementation over 18 months resulted in some improvement of the neurological condition; however, it did not prevent progression of the systemic disease.

Pathogenic oral bacteria in hospitalised patients with dysphagia: The silent epidemic

Background: Aspiration pneumonia is a serious and fatal complication of dysphagia, secondary to the ingestion of bacteria-laden secretions. However, no studies have documented the oral hygiene features present in patients who present with dysphagia.Objectives: The purpose of this study was to describe the oral hygiene problems of adults admitted to a sub-acute rehabilitation hospital and who presented with dysphagia.Methods: A descriptive, cross-sectional survey was conducted, during which 40 participants – 57.5% (n = 23) male and 42.5% (n = 17) female – underwent a clinical swallow evaluation using the Mann Assessment of Swallowing Ability (MASA) augmented with cervical auscultation (CA) and pulse oximetry (PO), an oral hygiene assessment using an adapted version of the Oral Health Assessment Tool (OHAT), followed by microbiology laboratory analysis of buccal swab samples to detect bacteria not considered part of the normal oral flora.Results: Results indicated that poor oral hygiene status was a common feature amongst all participants who presented with dysphagia. The most prevalent oral hygiene issues were related to abnormalities concerning saliva (60%), oral cleanliness (82.5%), the tongue (80%) and the use of dentures (71.4%). A high prevalence, 62.5% (n = 25), of opportunistic bacteria was found. The most commonly occurring bacteria groups were: (1) Candida albicans (47.5%) and (2) respiratory pathogens (37.5%) such as Klebsiella pneumoniae and Staphylococcus aureus.Conclusion: Persons with dysphagia have poor oral hygiene which creates favourable environments for bacteria to flourish and increases the prevalence of pathogenic oral bacteria associated with the development of aspiration pneumonia. The management of oral health issues for persons with dysphagia should receive greater attention during hospitalisation.