Integration of High-Throughput Imaging and Multiparametric Metabolic Profiling Reveals a Mitochondrial Mechanism of Tenofovir Toxicity.

Nephrotoxicity is a major cause of kidney disease and failure in drug development, but understanding of cellular mechanisms is limited, highlighting the need for better experimental models and methodological approaches. Most nephrotoxins damage the proximal tubule (PT), causing functional impairment of solute reabsorption and systemic metabolic complications. The antiviral drug tenofovir disoproxil fumarate (TDF) is an archetypal nephrotoxin, inducing mitochondrial abnormalities and urinary solute wasting, for reasons that were previously unclear. Here, we developed an automated, high-throughput imaging pipeline to screen the effects of TDF on solute transport and mitochondrial morphology in human-derived RPTEC/TERT1 cells, and leveraged this to generate realistic models of functional toxicity. By applying multiparametric metabolic profiling-including oxygen consumption measurements, metabolomics, and transcriptomics-we elucidated a highly robust molecular fingerprint of TDF exposure. Crucially, we identified that the active metabolite inhibits complex V (ATP synthase), and that TDF treatment causes rapid, dose-dependent loss of complex V activity and expression. Moreover, we found evidence of complex V suppression in kidney biopsies from humans with TDF toxicity. Thus, we demonstrate an effective and convenient experimental approach to screen for disease relevant functional defects in kidney cells in vitro, and reveal a new paradigm for understanding the pathogenesis of a substantial cause of nephrotoxicity.

Oral step-down for Staphylococcus aureus bacteraemia: an opportunity for antimicrobial stewardship?

Objectives: Long courses of intravenous antimicrobial therapy are traditionally recommended for the treatment of methicillin sensitive Staphylococcus aureus bacteraemia (MS-SAB), but are not always completed in clinical practice. Early intravenous to oral antibiotic switch is a key component of antimicrobial stewardship. This study aimed to identify whether intravenous antibiotic duration may be safely reduced in MS-SAB. Methods: We performed a single-centre retrospective study of MS-SAB management. Successful outcome was defined as 90-day recurrence-free survival. Effect of intravenous antibiotic duration on 90-day recurrence risk was examined. Results: 281 adult cases of MS-SAB were evaluated, of which 208 (74%) had a successful outcome. 176 cases (63%) received less than 14 days of intravenous antimicrobial therapy. Very short durations of intravenous therapy were associated with increased risk of recurrence (<7 days iv, 9.8% recurrence; 7-13 days, 1.4%; ≥14 days, 2.9%; p 0.005). This effect was robust to sensitivity analysis for total antimicrobial therapy duration of 14 days. CRP reduction of at least 37% from peak value at intravenous to oral antibiotic switch was associated with decreased risk of recurrence (<37% CRP reduction, 12% recurrence; >37%, 2.0%; p 0.001). Conclusions: Oral antimicrobial switch may allow safe reductions in duration of intravenous therapy in MS-SAB.

Skeletal muscle mitochondrial dysfunction in contemporary antiretroviral therapy: a single cell analysis.

OBJECTIVE: To quantify mitochondrial function in skeletal muscle of people treated with contemporary antiretroviral therapy. DESIGN: Cross-sectional observational study. METHODS: Quantitative multiplex immunofluorescence was performed to determine mitochondrial mass and respiratory chain complex abundance in individual myofibres from tibialis anterior biopsies. Individual myofibres were captured by laser microdissection and mitochondrial DNA (mtDNA) content and large-scale deletions were measured by real-time PCR. RESULTS: Forty-five antiretroviral therapy (ART)-treated people with HIV (PWH, mean age 58 years, mean duration of ART 125 months) were compared with 15 HIV negative age-matched controls. Mitochondrial complex I (CI) deficiency was observed at higher proportional levels in PWH than negative controls ( P = 0.008). Myofibre mitochondrial mass did not differ by HIV status. No ART class was significantly associated with mitochondrial deficiency, including prior exposure to historical NRTIs (nucleoside analogue reverse transcriptase inhibitors) associated with systemic mitochondrial toxicity. To exclude an effect of untreated HIV, we also studied skeletal muscle from 13 ART-naive PWH (mean age 37). These showed negligible CI defects, as well as comparable myofibre mitochondrial mass to ART-treated PWH. Most CI-deficient myofibres contained mtDNA deletions. No mtDNA depletion was detected. CONCLUSION: Here, we show that PWH treated with contemporary ART have mitochondrial dysfunction in skeletal muscle, exceeding that expected due to age alone. Surprisingly, this was not mediated by prior exposure to mitochondrially toxic NRTIs, suggesting novel mechanisms of mitochondrial dysfunction in contemporary ART-treated PWH. These findings are relevant for better understanding successful ageing in PWH.

Renal mitochondrial toxicity: effects of thymidine analogues and tenofovir disoproxil fumarate in African people with HIV.

We investigated the contributions of thymidine analogue and tenofovir disoproxil fumarate (TDF) antiretroviral therapy on renal mitochondrial toxicity in Ghanaian people with HIV (PWH). Similar levels of renal biochemical and mitochondrial dysfunction were seen, and there was no increased risk in PWH who had sequenced from thymidine analogue to TDF. However, mild renal impairment was associated with mitochondrial DNA damage in TDF but not thymidine analogue-treated PWH. These data support the continued use of TDF in resource-limited settings.

Evaluation of procalcitonin-guided antimicrobial stewardship in patients admitted to hospital with COVID-19 pneumonia.

BACKGROUND: Procalcitonin is a biomarker that may be able to identify patients with COVID-19 pneumonia who do not require antimicrobials for bacterial respiratory tract co-infections. OBJECTIVES: To evaluate the safety and effectiveness of a procalcitonin-guided algorithm in rationalizing empirical antimicrobial prescriptions in non-critically ill patients with COVID-19 pneumonia. METHODS: Retrospective, single-site, cohort study in adults hospitalized with confirmed or suspected COVID-19 pneumonia and receiving empirical antimicrobials for potential bacterial respiratory tract co-infection. Regression models were used to compare the following outcomes in patients with and without procalcitonin testing within 72 h of starting antimicrobials: antimicrobial consumption (DDD); antimicrobial duration; a composite safety outcome of death, admission to HDU/ICU or readmission to hospital within 30 days; and length of admission. Procalcitonin levels of ≤0.25 ng/L were interpreted as negatively predictive of bacterial co-infection. Effects were expressed as ratios of means (ROM) or prevalence ratios (PR) accordingly. RESULTS: 259 patients were included in the final analysis. Antimicrobial use was lower in patients who had procalcitonin measured within 72 h of starting antimicrobials: mean antimicrobial duration 4.4 versus 5.4 days, adjusted ROM 0.7 (95% CI 0.6-0.9); mean antimicrobial consumption 6.8 versus 8.4 DDD, adjusted ROM 0.7 (95% CI 0.6-0.8). Both groups had similar composite safety outcomes (adjusted PR 0.9; 95% CI 0.6-1.3) and lengths of admission (adjusted ROM 1.3; 95% CI 0.9-1.6). CONCLUSIONS: A procalcitonin-guided algorithm may allow for the safe reduction of antimicrobial usage in hospitalized non-critically ill patients with COVID-19 pneumonia.

The Isolation and Deep Sequencing of Mitochondrial DNA.

In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .

SARS-CoV-2 Testing of 11,884 Healthcare Workers at an Acute NHS Hospital Trust in England: A Retrospective Analysis.

Healthcare workers (HCWs) are known to be at increased risk of infection with SARS-CoV-2, although whether these risks are equal across all roles is uncertain. Here we report a retrospective analysis of a large real-world dataset obtained from 10 March to 6 July 2020 in an NHS Foundation Trust in England with 17,126 employees. 3,338 HCWs underwent symptomatic PCR testing (14.4% positive, 2.8% of all staff) and 11,103 HCWs underwent serological testing for SARS-CoV-2 IgG (8.4% positive, 5.5% of all staff). Seropositivity was lower than other hospital settings in England but higher than community estimates. Increased test positivity rates were observed in HCWs from BAME backgrounds and residents in areas of higher social deprivation. A multiple logistic regression model adjusting for ethnicity and social deprivation confirmed statistically significant increases in the odds of testing positive in certain occupational groups, most notably domestic services staff, nurses, and health-care assistants. PCR testing of symptomatic HCWs appeared to underestimate overall infection levels, probably due to asymptomatic seroconversion. Clinical outcomes were reassuring, with only a small minority of HCWs with COVID-19 requiring hospitalization (2.3%) or ICU management (0.7%) and with no deaths. Despite a relatively low level of HCW infection compared to other UK cohorts, there were nevertheless important differences in test positivity rates between occupational groups, robust to adjustment for demographic factors such as ethnic background and social deprivation. Quantitative and qualitative studies are needed to better understand the factors contributing to this risk. Robust informatics solutions for HCW exposure data are essential to inform occupational monitoring.

COVID-19 Management in a UK NHS Foundation Trust with a High Consequence Infectious Diseases Centre: A Retrospective Analysis.

Recent large national and international cohorts describe the baseline characteristics and outcome of hospitalised patients with COVID-19, however there is limited granularity to these reports. We aimed to provide a detailed description of a UK COVID-19 cohort, focusing on management and outcome. We performed a retrospective single-centre analysis of clinical management and 28-day outcomes of consecutive adult inpatients with SARS-CoV-2 PCR-confirmed COVID-19 from 31 January to 16 April 2020 inclusive. In total, 316 cases were identified. Most patients were elderly (median age 75) with multiple comorbidities. One quarter were admitted from residential or nursing care. Mortality was 84 out of 316 (26.6%). Most deaths occurred in patients in whom a ceiling of inpatient treatment had been determined and for whom end of life care and specialist palliative care input was provided where appropriate. No deaths occurred in patients aged under 56 years. Decisions to initiate respiratory support were individualised after consideration of patient wishes, premorbid frailty and comorbidities. In total, 59 (18%) patients were admitted to intensive care, of which 31 (10% overall cohort) required intubation. Multiple logistic regression identified associations between death and age, frailty, and disease severity, with age as the most significant factor (odds ratio 1.07 [95% CI 1.03-1.10] per year increase, p < 0.001). These findings provide important clinical context to outcome data. Mortality was associated with increasing age. Most deaths were anticipated and occurred in patients with advance decisions on ceilings of treatment.

Mitochondrial DNA Damage and Brain Aging in Human Immunodeficiency Virus.

BACKGROUND: Neurocognitive impairment (NCI) remains common in people living with human immunodeficiency virus (PLWH), despite suppressive antiretroviral therapy (ART), but the reasons remain incompletely understood. Mitochondrial dysfunction is a hallmark of aging and of neurodegenerative diseases. We hypothesized that human immunodeficiency virus (HIV) or ART may lead to mitochondrial abnormalities in the brain, thus contributing to NCI. METHODS: We studied postmortem frozen brain samples from 52 PLWH and 40 HIV-negative controls. Cellular mitochondrial DNA (mtDNA) content and levels of large-scale mtDNA deletions were measured by real-time polymerase chain reaction. Heteroplasmic mtDNA point mutations were quantified by deep sequencing (Illumina). Neurocognitive data were taken within 48 months antemortem. RESULTS: We observed a decrease in mtDNA content, an increase in the mtDNA "common deletion," and an increase in mtDNA point mutations with age (all P < .05). Each of these changes was exacerbated in HIV-positive cases compared with HIV-negative controls (all P < .05). ART exposures, including nucleoside analogue reverse transcriptase inhibitors, were not associated with changes in mtDNA. The number of mtDNA point mutations was associated with low CD4/CD8 ratio (P = .04) and with NCI (global T-score, P = .007). CONCLUSIONS: In people with predominantly advanced HIV infection, there is exacerbation of age-associated mtDNA damage. This change is driven by HIV per se rather than by ART toxicity and may contribute to NCI. These data suggest that mitochondrial dysfunction may be a mediator of adverse aging phenotypes in PLWH.

Mitochondria and ageing with HIV.

PURPOSE OF REVIEW: Some older people living with HIV (PLWH) exhibit features of unsuccessful ageing, such as frailty. Mitochondrial dysfunction is one of the best characterized ageing mechanisms. There has been recent interest in whether some people ageing with HIV may have an excess of mitochondrial dysfunction. This review aims to address this question through: analogy with ageing and chronic disease; discussion of the key unknowns; suggested ways that measures of mitochondrial dysfunction might be incorporated into HIV research studies. RECENT FINDINGS: Recent data suggest that mitochondrial dysfunction in PLWH may not be wholly a legacy effect of historical nucleoside analog reverse transcriptase inhibitor exposures. Research in the non-HIV setting has altered our understanding of the important mediators of mitochondrial dysfunction in ageing. SUMMARY: Mitochondrial dysfunction is a very plausible driver of adverse ageing phenotypes in some older PLWH. As such it may be a target for therapeutic interventions. Currently, however, there remain considerable uncertainties around the extent of this phenomenon, and its relative importance. Current studies are likely to clarify these questions over the next few years.

Current Challenges and Solutions in Research and Clinical Care of Older Persons Living with HIV: Findings Presented at the 9th International Workshop on HIV and Aging.

In the era of effective antiretroviral therapy, the number of older people with HIV (PWH) is increasing, and those aging with HIV are experiencing an increasing burden of age-associated comorbidities. Life expectancy among older PWH is approaching that of demographically comparable HIV-uninfected (HIV-) adults. With this changing demographic of PWH come new challenges for researchers and clinicians in how to identify, address, and manage the complex interplay of treated HIV infection and aging-associated factors. In response to these challenges, the annual International Workshop on HIV and Aging was initiated in 2009 as a multidisciplinary platform for scientific discourse on the research and clinical complications arising from the aging population of PWH. The multidisciplinary nature of the workshop has resulted in a wide range of topics addressed over the past 9 years, from basic mechanisms in aging and HIV pathogenesis, to epidemiology of aging within large cohorts, interventions, and implementation of clinical programs. Herein, we summarize the key topics discussed at the 9th Annual International Workshop on HIV and Aging 2018, including "inflammaging," mitochondrial dysfunction, exercise interventions, HIV-associated neurocognitive impairment, metabolic dysfunction, menopause, and polypharmacy. In addition to recent developments in research and clinical care, we discuss open questions and future research directions required to better understand the interaction of HIV and aging.

Tenofovir disoproxil fumarate-associated renal tubular dysfunction: noninvasive assessment of mitochondrial injury.

OBJECTIVE: To determine whether tenofovir disoproxil fumarate (TDF)-associated renal tubular dysfunction is associated with evidence of mitochondrial injury in urine. DESIGN: Single-centre cross-sectional observational study of HIV-positive outpatients. METHODS: Biochemistry was performed on paired serum and urine samples. Mitochondrial DNA (mtDNA) was studied by real-time PCR and long-range PCR on cellular fractions of urine. RESULTS: In total, 48 study participants were enrolled of whom half were TDF treated. Mean age was 43 years. 58% had estimated glomerular filtration rate at least 90, with no differences between ART treatment groups. Urinary phosphate wasting was common and independently associated with TDF exposure (P = 0.02). No study participants had low molecular weight proteinuria. Cellular mtDNA content in urine was heavily influenced by the cellularity of the sample. The mtDNA 'common deletion' mutation was detectable significantly more commonly in the urine of TDF exposed study participants compared with unexposed (13/22 TDF study participants (59%), 4/21 TDF (19%), P = 0.01). Common deletion levels were not associated with age, estimated glomerular filtration rate, or urinary phosphate wasting. No mtDNA measures were associated with current or nadir CD4 lymphocyte counts, duration of disease or antiretroviral therapy, or historical exposure to nucleoside analogue reverse transcriptase inhibitors with systemic mitochondrial toxicity. CONCLUSION: The presence of mtDNA mutations in the context of TDF exposure adds weight to the hypothesis that TDF-associated renal damage is at least in part mitochondrially mediated. The assessment of mtDNA markers in urine may be a feasible noninvasive investigation for TDF-treated patients.

Mitochondrial dysfunction in aging: Much progress but many unresolved questions.

The free radical theory of aging is almost 60 years old. As mitochondria are the principle source of intracellular reactive oxygen species (ROS), this hypothesis suggested a central role for the mitochondrion in normal mammalian aging. In recent years, however, much work has questioned the importance of mitochondrial ROS in driving aging. Conversely new evidence points to other facets of mitochondrial dysfunction which may nevertheless suggest the mitochondrion retains a critical role at the center of a complex web of processes leading to cellular and organismal aging.

Deep resequencing of mitochondrial DNA.

Detecting and quantifying low-level variants in mitochondrial DNA (mtDNA) by deep resequencing can lead to important insights into the biology of mtDNA in health and disease. Massively parallel ("next-generation") sequencing is an attractive tool owing to the great depth and breadth of coverage. However, there are several important challenges to be considered when using this method, in particular: the avoidance of false discovery due to the unintended amplification of nuclear pseudogenes and the approach to delineating signal from noise at very great depths of coverage. Here we present methods for whole mtDNA genome deep sequencing (Illumina MiSeq) and short amplicon deep sequencing (Roche 454 GS-FLX).

Single-cell analysis of mitochondrial DNA.

Understanding the biology of mitochondrial DNA (mtDNA) at the single-cell level has yielded important insights into inheritance, disease, and normal aging. In nuclear gene disorders of mtDNA maintenance, neurodegeneration, and aging, different somatic mtDNA mutations exist within individual cells and may be missed by techniques applied to whole tissue DNA extract. We therefore provide a method for characterizing mtDNA within single skeletal muscle fibers. During embryogenesis, mtDNA content is subject to a tight bottleneck and this may account for differential segregation of mutant mtDNA in offspring. We also present a method to study this phenomenon by single-cell analysis of embryonic PGCs (primordial germ cells).

Use of stereotypical mutational motifs to define resolution limits for the ultra-deep resequencing of mitochondrial DNA.

Massively parallel resequencing of mitochondrial DNA (mtDNA) has led to significant advances in the study of heteroplasmic mtDNA variants in health and disease, but confident resolution of very low-level variants (<2% heteroplasmy) remains challenging due to the difficulty in distinguishing signal from noise at this depth. However, it is likely that such variants are precisely those of greatest interest in the study of somatic (acquired) mtDNA mutations. Previous approaches to this issue have included the use of controls such as phage DNA and mtDNA clones, both of which may not accurately recapitulate natural mtDNA. We have therefore explored a novel approach, taking advantage of mtDNA with a known stereotyped mutational motif (nAT > C, from patient with MNGIE, mitochondrial neurogastrointestinal encephalomyopathy) and comparing mutational pattern distribution with healthy mtDNA by ligation-mediated deep resequencing (Applied Biosystems SOLiD). We empirically derived mtDNA-mutant heteroplasmy detection limits, demonstrating that the presence of stereotypical mutational motif could be statistically validated for heteroplasmy thresholds ≥ 0.22% (P = 0.034). We therefore provide empirical evidence from biological samples that very low-level mtDNA mutants can be meaningfully resolved by massively parallel resequencing, confirming the utility of the approach for studying somatic mtDNA mutation in health and disease. Our approach could also usefully be employed in other settings to derive platform-specific deep resequencing resolution limits.