Exploring NICU nurses' views of a novel genetic point-of-care test identifying neonates at risk of antibiotic-induced ototoxicity: A qualitative study.

AIM: To explore the views of neonatal intensive care nursing staff on the deliverability of a novel genetic point-of-care test detecting a genetic variant associated with antibiotic-induced ototoxicity. DESIGN: An interpretive, descriptive, qualitative interview study. METHODS: Data were collected using semi-structured interviews undertaken between January and November 2020. Participants were neonatal intensive care nursing staff taking part in the Pharmacogenetics to Avoid Loss of Hearing trial. RESULTS: Thematic analysis resulted in four themes: perceived clinical utility; the golden hour; point-of-care device; training and support. Recommendations were made to streamline the protocol and ongoing training and support were considered key to incorporating the test into routine care. CONCLUSION: Exploring the views of nurses involved in the delivery of the point-of-care test was essential in its implementation. By the study endpoint, all participants could see the value of routine clinical introduction of the point-of care test. IMPLICATIONS FOR THE PROFESSION AND/OR PATIENT CARE: Nurses are in a key position to support the delivery of point-of-care genetic testing into mainstream settings. This study has implications for the successful integration of other genetic point-of-care tests in acute healthcare settings. IMPACT: The study will help to tailor the training and support required for routine deployment of the genetic point-of-care test. The study has relevance for nurses involved in the development and delivery of genetic point-of-care tests in other acute hospital settings. REPORTING METHOD: This qualitative study adheres to the Standards for Reporting Qualitative Research EQUATOR guidelines and utilizes COREQ and SRQR checklists. PATIENT OR PUBLIC CONTRIBUTION: All staff working on the participating neonatal intensive care units were trained to use the genetic point-of-care test. All inpatients on the participating units were eligible to have testing via the point-of-care test. The Pharmacogenetics to Avoid Loss of Hearing Patient and Public Involvement and Engagement group provided valuable feedback. TRIAL AND PROTOCOL REGISTRATION: Registered within the University of Manchester. Ethics approval reference numbers: IRAS: 253102 REC reference: 19/NW/0400. Also registered with the ISRCTN ref: ISRCTN13704894.

Rapid Point-of-Care Genotyping to Avoid Aminoglycoside-Induced Ototoxicity in Neonatal Intensive Care.

Importance: Aminoglycosides are commonly prescribed antibiotics used for the treatment of neonatal sepsis. The MT-RNR1 m.1555A>G variant predisposes to profound aminoglycoside-induced ototoxicity (AIO). Current genotyping approaches take several days, which is unfeasible in acute settings. Objective: To develop a rapid point-of-care test (POCT) for the m.1555A>G variant before implementation of this technology in the acute neonatal setting to guide antibiotic prescribing and avoid AIO. Design, Setting, and Participants: This pragmatic prospective implementation trial recruited neonates admitted to 2 large neonatal intensive care units between January 6, 2020, and November 30, 2020, in the UK. Interventions: Neonates were tested for the m.1555A>G variant via the rapid POCT on admission to the neonatal intensive care unit. Main Outcomes and Measures: The primary outcome assessed the proportion of neonates successfully tested for the variant of all infants prescribed antibiotics. Secondary outcomes measured whether implementation was negatively associated with routine clinical practice and the performance of the system. The study was statistically powered to detect a significant difference between time to antibiotic administration before and after implementation of the MT-RNR1 POCT. Results: A total of 751 neonates were recruited and had a median (range) age of 2.5 (0-198) days. The MT-RNR1 POCT was able to genotype the m.1555A>G variant in 26 minutes. Preclinical validation demonstrated a 100% sensitivity (95% CI, 93.9%-100.0%) and specificity (95% CI, 98.5%-100.0%). Three participants with the m.1555A>G variant were identified, all of whom avoided aminoglycoside antibiotics. Overall, 424 infants (80.6%) receiving antibiotics were successfully tested for the variant, and the mean time to antibiotics was equivalent to previous practice. Conclusions and Relevance: The MT-RNR1 POCT was integrated without disrupting normal clinical practice, and genotype was used to guide antibiotic prescription and avoid AIO. This approach identified the m.1555A>G variant in a practice-changing time frame, and wide adoption could significantly reduce the burden of AIO.

Pharmacogenetics to Avoid Loss of Hearing (PALOH) trial: a protocol for a prospective observational implementation trial.

INTRODUCTION: In conjunction with a beta-lactam, aminoglycosides are the first-choice antibiotic for empirical treatment of sepsis in the neonatal period. The m.1555A>G variant predisposes to ototoxicity after aminoglycoside administration and has a prevalence of 1 in 500. Current genetic testing can take over 24 hours, an unacceptable delay in the acute setting. This prospective-observational trial will implement a rapid point of care test (POCT), facilitating tailored antibiotic prescribing to avoid hearing loss. METHODS AND ANALYSIS: The genedrive POCT can detect the m.1555A>G variant in 26 min from buccal swab. This system will be integrated into the clinical pathways at two large UK neonatal centres over a minimum 6-month period. The primary outcome is the number of neonates successfully tested for the variant out of all babies prescribed antibiotics. As a secondary outcome, clinical timings will be compared with data collected prior to implementation, measuring the impact on routine practice. ETHICS AND DISSEMINATION: Approval for the trial was granted by the Research Ethics Committee (REC) and Human Research Authority in August 2019. Results will be published in full on completion of the study. TRIAL REGISTRATION NUMBER: ISRCTN13704894. PROTOCOL VERSION: V 1.3.

Development of a rapid field-applicable molecular diagnostic for knockdown resistance (kdr) markers in An. gambiae.

BACKGROUND: The spread of insecticide resistance (IR) is a major threat to vector control programmes for mosquito-borne diseases. Early detection of IR using diagnostic markers could help inform these programmes, especially in remote locations where gathering reliable bioassay data is challenging. Most current molecular tests for genetic IR markers are only suitable for use in well-equipped laboratory settings. There is an unmet need for field-applicable diagnostics. METHODS: A single-cartridge test was designed to detect key IR mutations in the major African vector of malaria, Anopheles gambiae. Developed on the portable, rapid, point-of-care compatible PCR platform - Genedrive® (genedrive® plc), the test comprises two assays which target single nucleotide polymorphisms (SNPs) in the voltage gated sodium channel (VGSC) gene that exert interactive effects on knockdown resistance (kdr): L1014F, L1014S and N1575Y. RESULTS: Distinct melt peaks were observed for each allele at each locus. Preliminary validation of these assays using a test panel of 70 An. gambiae samples showed complete agreement of our assays with the widely-used TaqMan assays, achieving a sensitivity and specificity of 100%. CONCLUSION: Here we show the development of an insecticide resistance detection assay for use on the Genedrive® platform that has the potential to be the first field-applicable diagnostic for kdr.

Development and clinical validation of the Genedrive point-of-care test for qualitative detection of hepatitis C virus.

OBJECTIVE: Recently approved direct acting antivirals provide transformative therapies for chronic hepatitis C virus (HCV) infection. The major clinical challenge remains to identify the undiagnosed patients worldwide, many of whom live in low-income and middle-income countries, where access to nucleic acid testing remains limited. The aim of this study was to develop and validate a point-of-care (PoC) assay for the qualitative detection of HCV RNA. DESIGN: We developed a PoC assay for the qualitative detection of HCV RNA on the PCR Genedrive instrument. We validated the Genedrive HCV assay through a case-control study comparing results with those obtained with the Abbott RealTime HCV test. RESULTS: The PoC assay identified all major HCV genotypes, with a limit of detection of 2362 IU/mL (95% CI 1966 to 2788). Using 422 patients chronically infected with HCV and 503 controls negative for anti-HCV and HCV RNA, the Genedrive HCV assay showed 98.6% sensitivity (95% CI 96.9% to 99.5%) and 100% specificity (95% CI 99.3% to 100%) to detect HCV. In addition, melting peak ratiometric analysis demonstrated proof-of-principle for semiquantification of HCV. The test was further validated in a real clinical setting in a resource-limited country. CONCLUSION: We report a rapid, simple, portable and accurate PoC molecular test for HCV, with sensitivity and specificity that fulfils the recent FIND/WHO Target Product Profile for HCV decentralised testing in low-income and middle-income countries. This Genedrive HCV assay may positively impact the continuum of HCV care from screening to cure by supporting real-time treatment decisions. TRIAL REGISTRATION NUMBER: NCT02992184 .

An in vitro diagnostic certified point of care single nucleotide test for IL28B polymorphisms.

Numerous genetic polymorphisms have been identified as associated with disease or treatment outcome, but the routine implementation of genotyping into actionable medical care remains limited. Point-of-care (PoC) technologies enable rapid and real-time treatment decisions, with great potential for extending molecular diagnostic approaches to settings with limited medical infrastructure (e.g., CLIA certified diagnostic laboratories). With respect to resource-limited settings, there is a need for simple devices to implement biomarker guided treatment strategies. One relevant example is chronic hepatitis C infection, for which several treatment options are now approved. Single nucleotide polymorphisms (SNPs) in the IL-28B / IFNL3 locus have been well described to predict both spontaneous clearance and response to interferon based therapies. We utilized the Genedrive® platform to develop an assay for the SNP rs12979860 variants (CC, CT and TT). The assay utilizes a hybrid thermal engine, permitting rapid heating and cooling, enabling an amplification based assay with genetic variants reported using endpoint differential melting cure analysis in less than 60 minutes. We validated this assay using non-invasive buccal swab sampling in a prospective study of 246 chronic HCV patients, achieving 100% sensitivity and 100% specificity (95% exact CI: 98.8-100%)) in 50 minutes as compared to conventional lab based PCR testing. Our results provide proof of concept that precision medicine is feasible in resource-limited settings, offering the first CE-IVD (in vitro diagnostics) validated PoC SNP test. We propose that IL-28B genotyping may be useful for directing patients towards lower cost therapies, and rationing use of costly direct antivirals for use in those individuals showing genetic risk.

BCR-ABL alters the proliferation and differentiation response of multipotent hematopoietic cells to stem cell factor.

Chronic myeloid leukaemia (CML), a hematopoietic stem cell disorder is characterized by the expression of BCR-ABL. To investigate the effects of BCR-ABL on multipotent hematopoietic cells, a temperature sensitive BCR-ABL tyrosine kinase was expressed in the cell line, FDCP-Mix. BCR-ABL mediated an increase in c-kit expression that correlated with an enhanced mitogenic response to SCF. This was not observed in the absence of Bcr-Abl kinase activity or presence of the BCR-ABL inhibitor STI571, which also inhibits c-kit. When cultured in a combination of SCF plus G-CSF the FDCP-Mix cells undergo neutrophilic differentiation over a 7-10 day period. When BCR-ABL was active there was a marked inhibition of cell maturation compared to control cells in which BCR-ABL was either inactive or not present. However, BCR-ABL did not block differentiation as the cells eventually undergo terminal maturation. These data argue that BCR-ABL is directly responsible for the enhanced response to SCF reported in CML progenitor cells. Furthermore, although the primary effect of STI571 is via direct inhibition of BCR-ABL, STI571 additionally reduces the enhanced response to SCF. Thus there are two sites of STI571 action of potential importance in Bcr-Abl expressing cells.