Proactive therapeutic monitoring of dalbavancin concentrations in the long-term management of chronic osteoarticular/periprosthetic joint infections.

Here, we describe the use of proactive therapeutic drug monitoring (TDM) to individualize the optimal timing of drug injections in 16 adult patients with chronic osteoarticular infections receiving a median of 7 injections of dalbavancin (up to 12 injections in 15 months). Dalbavancin injections were repeated at medians of 39-47 days, with infusion intervals ranging from 26 to 69 days. TDM can facilitates a precise, targeted use of dalbavancin for infections requiring prolonged treatments.

Recurrent bacteremia and endocarditis due to Staphylococcus capitis in a patient with Bowen's disease: a case report.

This case report details the management of a 79-year-old male with recurrent methicillin-resistant Staphylococcus capitis bacteremia and endocarditis. The patient's clinical journey encompassed multiple hospital admissions, with challenges in managing endocarditis, pacemaker replacements, and potential cutaneous sources of infection. The treatment regimen included intravenous antibiotic therapy during hospitalization and suppressive antibiotic treatment upon discharge, alongside a decolonization strategy for his scalp lesions.

Dalbavancin as suppressive therapy for implant-related infections: a case series with therapeutic drug monitoring and review of the literature.

Implant-related infections may need suppressive antibiotic therapy (SAT). We describe a SAT strategy using dalbavancin with therapeutic drug monitoring (TDM). This is a retrospective bicentric study of patients with implant-related infection who received dalbavancin SAT between January 2021 and September 2023. Fifteen patients were included. Median number of injections was 4 (IQR: 2-7). Median time between two reinjections was 57 days (IQR 28-82). Dalbavancin plasma concentrations were above 4 mg/L for 97.9% of dosages (93/95) and above 8 mg/L for 85% (81/95). These results support the use of dalbavancin SAT for implant-related infections.

In-label, off-label prescription, efficacy and tolerability of dalbavancin: report from a National Registry.

PURPOSE: Although dalbavancin is currently approved for the treatment of ABSSIs, several studies suggest its efficacy and tolerance as long-term therapy for other off-label indications requiring prolonged intravenous antibiotic administration. METHODS: We conducted a prospective nationwide study of dalbavancin use in real-life settings for both approved and off-label indications analysing for each case the clinical and microbiological characteristics of infection the efficacy and safety of treatments. RESULTS: During the study period (from December 2018 to July 2021), the ID specialists from 14 different centres enrolled 223 patients treated with dalbavancin [141 males (63%) and 82 females (37%); male/female ratio 1.72; mean age 59 (SD 17.2) years, (range 15-96). Most patients in the study population (136/223; 61.0%) came from community rather than health care facilities and most of them were visited in Infectious Diseases wards (93/223; 41.7%) and clinics (55/223; 24.7%) even though some patients were cured in other settings, such as surgery wards (18/223; 8.1%), orthopaedic wards (11/223; 4.9%), Emergency Rooms (7/223; 3.1%) and non-surgical other than ID wards (6/223; 2.7%). The most common ID diagnoses were osteomyelitis (44 cases/223; 19.7%; of which 29 acute and 15 chronic osteomyelitis), cellulitis (28/223; 12.5%), cutaneous abscess (23/223; 10.3%), orthopaedic prosthesis-associated infection (22/223; 9.9%), surgical site infection (20/223; 9.0%) and septic arthritis (15/223; 6.7%). CONCLUSION: In conclusion, by virtue of its PK/PD properties, dalbavancin represents a valuable option to daily in-hospital intravenous or outpatient antimicrobial regimens also for off-label indications requiring a long-term treatment of Gram-positive infections.

Experience with dalbavancin use in various gram-positive infections within Aberdeen Royal Infirmary OPAT service.

PURPOSE: Dalbavancin, approved in 2014 for Gram-positive acute bacterial skin and skin structure infections (ABSSSI), has pharmacokinetics enabling treatment with one or two doses. Dalbavancin might be useful in outpatient parenteral antibiotic therapy (OPAT) of deep-seated infections, otherwise requiring inpatient admission. We documented our experience with pragmatic dalbavancin use to assess its effectiveness for varied indications, on- and off-label, as primary or sequential consolidation therapy. METHODS: Patients prescribed dalbavancin between 1 December 2021 and 1 October 2022 were screened for demographics of age, sex, Charlson comorbidity index (CCI), allergies, pathogens, doses of dalbavancin, other antibiotics administered and surgery. Where available, infection markers were recorded. The primary outcome was a cure at the end of treatment. Secondary outcomes included any adverse events and for those with treatment failures, response to salvage antibiotics. RESULTS: Sixty-seven per cent of patients were cured. Cure rates by indication were 93% for ABSSSI, 100% for bacteraemia, 90% for acute osteomyelitis, 0% for chronic osteomyelitis, 75% for native joint septic arthritis and 33% for prosthetic joint infection. Most bone and joint infections that were not cured did not have source control, and the goal of treatment was suppressive. Successful suppression rates were greater at 48% for chronic osteomyelitis and 66% for prosthetic joint infections. Adverse events occurred in 14 of 102 patients. CONCLUSION: This report adds to clinical experience with dalbavancin for off-label indications whilst further validating its role in ABSSSI. Dalbavancin as primary therapy in deep-seated infections merits investigation in formal clinical trials.

When surgical option is not provided: a successful multidisciplinary approach to a refractory case of sternal osteomyelitis following coronary surgery.

PURPOSE: Sternal osteomyelitis is a major complication of cardiac operations performed through median sternotomy. The surgical treatment, which involves the debridement and removal of whole infected and necrotic tissue is the standard of care, although it is sometimes unachievable. This may occur, for instance, when the infectious-inflammatory process invades the anterior mediastinum and tenaciously incorporates one or more of vital anatomical structures. METHODS AND RESULTS: An inoperable case of postoperative sternal osteomyelitis that involved the right ventricle and the right coronary artery, and that was successfully treated using a nonsurgical multidisciplinary approach, is reported here. CONCLUSION: For highly selected patients with sternal osteomyelitis for whom surgery is a too risky option, an approach including the contribution of various specialists might be a viable way out.

A systematic review of dalbavancin efficacy as a sequential therapy for infective endocarditis.

INTRODUCTION: Dalbavancin is an antibiotic characterized by an extended half-life and efficacy against methicillin-resistant Staphylococci. Currently, there are only narrative reviews summarizing the evidence about the use of dalbavancin for infective endocarditis (IE), many of which are focused primarily on its use as consolidation therapy. For this reason, we conducted a systematic review to describe the clinical efficacy and the safety of dalbavancin in IE treatment. METHODS: We searched for available evidence using the MEDLINE (PubMed), Embase, Scopus, Cochrane Library and Web of Science libraries, with no restrictions regarding the publication year. The risk of bias was performed using the Cochrane ROBINS-I tool for the comparative studies and the Newcastle-Ottawa Scale for descriptive studies. RESULTS: Nine studies were included. All of them were observational. Native valve endocarditis was the most common kind of IE found in the studies' populations (128/263, 48.7%), followed by prosthetic valve endocarditis, and cardiovascular implantable electronic device-related endocarditis. Coagulase-negative Staphylococci were the most common pathogens isolated (83/269, 30.1%), followed by S. aureus, Enterococci spp and Streptococci spp. Five out of nine studies documented a clinical failure rate of less than 10%. Dalbavancin showed a favourable safety profile. Dalbavancin appears to be a promising option for the consolidation therapy of IE. However, further studies comparing dalbavancin with standard of care are needed. PROSPERO REGISTRATION NUMBER: CRD42023430032.

Unraveling novel mutation patterns and morphological variations in two dalbavancin-resistant MRSA strains in Austria using whole genome sequencing and transmission electron microscopy.

BACKGROUND: The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) strains resistant to non-beta-lactam antimicrobials poses a significant challenge in treating severe MRSA bloodstream infections. This study explores resistance development and mechanisms in MRSA isolates, especially after the first dalbavancin-resistant MRSA strain in our hospital in 2016. METHODS: This study investigated 55 MRSA bloodstream isolates (02/2015-02/2021) from the University Hospital of the Medical University of Vienna, Austria. The MICs of dalbavancin, linezolid, and daptomycin were assessed. Two isolates (16-33 and 19-362) resistant to dalbavancin were analyzed via whole-genome sequencing, with morphology evaluated using transmission electron microscopy (TEM). RESULTS: S.aureus BSI strain 19-362 had two novel missense mutations (p.I515M and p.A606D) in the pbp2 gene. Isolate 16-33 had a 534 bp deletion in the DHH domain of GdpP and a SNV in pbp2 (p.G146R). Both strains had mutations in the rpoB gene, but at different positions. TEM revealed significantly thicker cell walls in 16-33 (p < 0.05) compared to 19-362 and dalbavancin-susceptible strains. None of the MRSA isolates showed resistance to linezolid or daptomycin. CONCLUSION: In light of increasing vancomycin resistance reports, continuous surveillance is essential to comprehend the molecular mechanisms of resistance in alternative MRSA treatment options. In this work, two novel missense mutations (p.I515M and p.A606D) in the pbp2 gene were newly identified as possible causes of dalbavancin resistance.

A comprehensive study on the identification and characterization of degradation products of lipoglycopeptide Dalbavancin using LC and LC-HRMS/MS.

Dalbavancin is the second-generation approved semisynthetic lipoglycopeptide by the United States Food and Drug Administration (USFDA) for the treatment of acute bacterial skin and skin-structure infections. Unlike other lipoglycopeptides, the stability behavior of Dalbavancin was least explored, which is a prerequisite. The current study endeavors to elucidate the oxidative and hydrolytic stability behavior of Dalbavancin by exposing the drug to oxidative, acidic, and basic stress conditions. A simple liquid chromatography (LC) method was developed, where significant resolution between Dalbavancin, its homologs, and the generated degradation products was achieved. Seven degradation products were identified under acidic, basic, and oxidative stress conditions. Using liquid chromatography and high-resolution mass spectrometry (LC-HRMS), MS/MS studies, the generated degradation products were identified and characterized. Formation of isomeric degradation products was identified especially upon exposure to basic stress conditions. The mechanistic fragmentation pathway for the seven degradation products was established, and the chemical structure for the identified degradation products was elucidated. The results strongly suggest that Dalbavancin is highly susceptible to degradation under oxidative and hydrolytic stress conditions. This study provides insights into the hydrolytic and oxidative stability of Dalbavancin, which can be employed during drug development and discovery in synthesizing relatively stable analogs.

Dalbavancin for the treatment of bone and joint infections: A meta-analysis.

BACKGROUND: Bone and joint infections are challenging infectious diseases to treat and require prolonged antimicrobial treatment. Dalbavancin demonstrated promising pharmacokinetic/pharmacodynamic properties for the treatment of these infections. The objective of this meta-analysis is to compare the effectiveness of dalbavancin to standard of care (SOC) for the treatment of bone and joint infections. METHODS: Two independent authors performed a comprehensive search through the major databases up to September 2023. Interventional and observational studies that compared the clinical success of dalbavancin to SOC for the treatment of osteoarticular infections (OAI) were included. RESULTS: A total of 6 studies and 581 patients were included, 282 in dalbavancin group and 299 in SOC group. Only one study was randomized clinical trial. When the data from the 6 studies were pooled in a meta-analysis, clinical success did not differ in those who received dalbavancin versus SOC (OR = 1.55, 95 % CI = 0.95-2.55, I-squared = 15.89 %) for the treatment of OAI infections. Four studies compared the two groups in terms of hospital length of stay and demonstrated a significant shorter length of stay in dalbavancin group compared to SOC group. Treatment-emergent adverse effects were reported in up to 21.4 % of patients in the dalbavancin group and up to 36.7 % of patients in the SOC group. CONCLUSION: This meta-analysis showed that dalbavancin is as effective as SOC for the treatment of patients with OAI infections. More data are needed to validate these findings.

Use of lipoglycopeptides for moderate to severe ABSSSI in the emergency department.

The burden of acute bacterial skin and skin structure infections (ABSSSI) continue to plague the healthcare system. One approach to managing moderate-to-severe ABSSSI in low-risk patients involves use of a single dose lipoglycopeptide (LGP), dalbavancin or oritavancin, in the emergency department (ED) and discharge to home with follow-up care. Limited ED studies indicate decreased hospital stays, ED revisits, readmissions, and healthcare costs, as well as improved patient satisfaction with use of these antibiotics. However, existing literature has limitations and gaps, such as insufficient quantifiable data on patient selection criteria, outcome predictors, and risk factors leading to treatment failure. Moreover, there is lack of research on the impact of LGPs on organizational productivity, patient quality of life, and utility in indications beyond ABSSSI. This review focuses on the role of long-acting LGPs in the ED setting for select patients presenting with ABSSSI, aiming to avoid hospitalizations, expedite patient discharge, and prevent readmissions while acknowledging potential limitations of therapy. Additionally, it provides insights into strategies and considerations specifically relevant to implementing this therapeutic approach in the ED.

Single Intravenous Dose Dalbavancin Pathway for the Treatment of Acute Bacterial Skin and Skin Structure Infections: Considerations for Emergency Department Implementation and Cost Savings.

BACKGROUND: A pathway for the treatment of acute bacterial skin and skin structure infections (ABSSSI) with a single intravenous (IV) dose of dalbavancin was previously shown to reduce hospital admissions and shorten inpatient length of stay (LOS). OBJECTIVES: To describe pathway implementation at the emergency department (ED) and evaluate cost-effectiveness of a single-dose dalbavancin administered to ED patients who would otherwise be hospitalized to receive usual care with multidose IV antibiotics. METHODS: The dalbavancin pathway was previously implemented at 11 U.S. EDs (doi:10.1111/acem.14258). Patients with ABSSSI, without an unstable comorbidity or infection complication requiring complex management, were treated with a single dose of dalbavancin. At the emergency physicians' discretion, patients were either discharged and received outpatient follow-up or were hospitalized for continued management. A decision analytic cost-effectiveness model was developed from the U.S. healthcare's perspective to evaluate costs associated with the dalbavancin pathway compared with inpatient usual care. Costs (2021 USD) were modeled over a 14-day horizon and included ED visits, drug costs, inpatient stay, and physician visits. One-way and probabilistic sensitivity analyses examined input parameter uncertainty. RESULTS: Driven largely by the per diem inpatient cost and LOS for usual care, the dalbavancin pathway was associated with savings of $5133.20 per patient and $1211.57 per hospitalization day avoided, compared with inpatient usual care. The results remained robust in sensitivity and scenario analyses. CONCLUSION: The new single-dose dalbavancin ED pathway for ABSSSI treatment, which was previously implemented at 11 U.S. EDs, offers robust cost savings compared to inpatient usual care.

Dalbavancin Use in Persons Who Use Drugs May Increase Adherence Without Increasing Cost.

Background: Dalbavancin (DAL) may obviate concerns regarding misuse of IV access in persons who use drugs (PWUD) completing treatment for infections in an outpatient setting. However, hesitancy to adopt its use exists due to the cost-prohibitive nature of DAL and perceived issues with insurance reimbursement. Our study looks to determine the financial impact of DAL use in actual, measured cost, and health care utilization, data as well as the effect on treatment completion rates. Methods: This is a retrospective cohort comparing cost information and treatment completion rates of patients who received DAL to a random sample of patients with Staphylococcus aureus bacteremia prior to the institutional availability of DAL. Results: From June 2020 to January 2022, 29 PWUD received DAL. Dalbavancin use resulted in the completion of intended duration in 19 patients (66%) compared with 11 (55%) without DAL. The contribution margin with DAL use was $7180 compared with $6655 without; this was not statistically significant (P = 0.47). Conclusion: Dalbavancin use in PWUD may increase treatment completion, with no statistically significant difference in contribution margins.

Strategies to Stabilize Dalbavancin in Aqueous Solutions; Section-1: the Effects of Metal Ions and Buffers.

PURPOSE: The effect of monovalent (Na+ and K+) and divalent (Ca2+, Mg2+, and Zn2+) metal ions combined with citrate or acetate buffers (pH 4.5) on the stability of dalbavancin in aqueous solutions was investigated. METHOD: RP-HPLC and HP-SEC were used to evaluate the stability of aqueous solutions of dalbavancin in different combinations of buffers and metal ions after four weeks of storage at 5°C and 55°C. A long-term study of formulations with divalent metal ions was conducted over six months at 5°C., 25°C and 40°C using RP-HPLC. RESULTS: All formulations in citrate buffered solutions precipitated. Dalbavancin solutions in 10 mM acetate buffer at 55°C were more stable in 10 mM CaCl2, 5 mM ZnCl2 and 10 mM MgCl2 than those containing 2 mM NaCl or 5 mM KCl, although the MgCl2 formulations precipitated slightly. No significant effect was observed for any of the divalent metal ions at 40°C for six months. CONCLUSION: Dalbavancin's stability in solution was improved by a combination of acetate and divalent metal ions at 55°C for four weeks. No effect was observed with acetate or metal ions alone, and no effect was observed after six months at 40°C suggesting that acetate and divalent metal ions together interact with dalbavancin via a thermally activated step to inhibit hydrolysis of the drug.

Strategies To Stabilize Dalbavancin in Aqueous Solutions; Section 3: The Effects of 2 Hydroxypropyl-ß-Cyclodextrin and Phosphate Buffer with and without Divalent Metal Ions.

PURPOSE: New formulations of the glycopeptide drug dalbavancin containing 2-hydroxpropyl-ß-cyclodextrin (2HPßCD) with or without divalent metal ions in phosphate buffer (pH 7.0) were tested to evaluate whether these excipients influence the aqueous solution stability of dalbavancin. METHOD: Recovery of dalbavancin from phosphate buffered solutions at pH 7.0 with different concentrations of 2HPßCD and a divalent metal ion (Ca2+, Mg2+, or Zn2+) was evaluated by RP-HPLC and HP-SEC after four weeks of storage at 5°C and 55°C. A long-term study of formulations with 2HPßCD and Mg2+ was carried out over six months at 5°C, 25°C, and 40°C using RP-HPLC. RESULTS: Dalbavancin solutions with either 5.5 mM or 55 mM 2HPßCD were significantly more stable with Mg2+ than with the other divalent metal ions, both at 55°C for four weeks and at 40°C for six months. Dalbavancin was found to be more stable in aqueous solutions at a concentration of 1 mg/mL than at 20 mg/mL with 2HPßCD and Mg2+ at 40°C for six months. CONCLUSION: The results suggest that 2HPßCD forms an inclusion complex with dalbavancin that slows the formation of the major degradant, mannosyl aglycone (MAG). The effect of 2HPßCD is increased in the presence of Mg2+ and phosphate at pH 7.0, and the complex is more stable at a dalbavancin concentration of 1 mg/mL than at 20 mg/mL. These observations point towards the possibility of formulating a dalbavancin injection solution with a long shelf life at room temperature and physiological pH.

Ototoxicity associated with extended dalbavancin treatment for a shoulder prosthetic joint infection.

BACKGROUND: Dalbavancin is a lipoglycopeptide antibiotic approved for treatment of skin and soft tissue infections, administered as a single or two-dose treatment. The extended half-life, good penetration into bone and synovial fluid, and bactericidal activity against gram-positive bacteria, including those in biofilm, make dalbavancin an appealing choice for treatment of bone and joint infections in outpatient settings. However, we present a rare case of ototoxicity associated with off-label extended dalbavancin treatment of a prosthetic joint infection. CASE PRESENTATION: A 55-year-old man with a prosthetic joint infection of the shoulder underwent off-label extended dalbavancin treatment, receiving a cumulative dose of 2500 mg. The patient experienced a gradual onset of hearing loss following the first dose, leading to a diagnosis of bilateral sensorineural hearing loss that persisted 1 year after dalbavancin was discontinued. CONCLUSIONS: This case report highlights the importance of exercising caution when administering dalbavancin beyond approved dosing guidelines, and emphasizes the need for vigilance regarding the potential for ototoxicity.

Experience with dalbavancin for long-term antimicrobial suppression of left ventricular assist device infections.

BACKGROUND: Left ventricular assist devices (LVAD) are a common strategy for management of end-stage heart failure. LVADs carry a risk of infection of the implanted device components, and skin flora are commonly implicated. Long-term antibiotics may be needed for management of deep device infection or recurrent superficial infections. In appropriately selected patients, dalbavancin can be a feasible option given its extended dosing interval. METHODS: This is a retrospective, single-center review of patients presenting with an LVAD infection between January 2011 and November 2022, where management included the use of dalbavancin. Data regarding LVAD placement, details of index infection, dalbavancin use and outcomes was obtained from chart review, and documented in a RedCap database. RESULTS: The mean time from LVAD placement to index infection was 131.6 weeks (standard deviation 87.2 weeks). The most common targeted organism was Corynebacterium striatum in six of 10 patients. Index infection presented as deep driveline infection in four patients and recurrent superficial driveline infection in three patients. Five patients had a concurrent bloodstream infection. Dalbavancin was discontinued in two patients due to breakthrough infection, with one patient requiring surgical intervention. No drug-related adverse events were noted. CONCLUSION: Dalbavancin is an attractive option in the management of long-term LVAD infection in patients for whom alternative oral or parenteral antibiotics are not a feasible option. Additional studies are needed to determine the optimal dosing of dalbavancin in this setting, and to study adverse events and long-term outcomes of dalbavancin.

Dalbavancin Boosts the Ability of Neutrophils to Fight Methicillin-Resistant Staphylococcus aureus.

Polymorphonuclear leukocytes (PMNs) are the most important cell type involved in the early nonspecific host response to bacterial pathogens. Staphylococcus aureus has evolved mechanisms to evade immune responses that contribute to its persistence in PMNs, and acquired resistance to several antimicrobials. Additionally, methicillin-resistant S. aureus (MRSA) is one of the most common causes of acute bacterial skin and skin-structure infections (ABSSSIs). Dalbavancin (DBV), a lipoglycopeptide, is indicated for the treatment of ABSSSIs, and has a broad spectrum of action against most microorganisms. Here, we sought to determine the effect of DBV on the neutrophil killing of MRSA and its potential immunomodulating activity. Our results revealed that DBV boosts MRSA killing by acting on both bacteria and PMNs. DBV pre-treatment of PMNs did not change the respiratory burst or degranulation, while an increased trend in neutrophil extracellular traps-associated elastase and in the production of TNFα and CXCL8 was revealed. In parallel, DBV caused a delay in the apoptosis of MRSA-infected neutrophils. In conclusion, we demonstrated a cooperative effect between the antimicrobial properties of PMNs and DBV, thus owing to their immunomodulatory activity. In the choice of the treatment management of serious S. aureus infections, DBV should be considered as an outstanding option since it reinforces PMNs pathogen clearance capability by exerting its effect directly, not only on MRSA but also on neutrophils.

Effective use of a two-dose regimen of dalbavancin to treat prosthetic joint infections and spinal hardware infections.

PURPOSE: Dalbavancin is an attractive antibiotic for the treatment of Gram-positive musculoskeletal infections given its long half-life and prolonged duration in cortical bones. For certain patient populations compliance with antibiotic regimens can be problematic. Therefore, the purpose of this study was to assess the effectiveness, tolerance, and compliance of treating prosthetic joint and spinal hardware infections with a unique two-dose regimen of dalbavancin. METHODS: Identification of patients that had prosthetic joint infections and spinal hardware infections from January 1, 2017, through December 31, 2021, that had received a two-dose regimen of dalbavancin for these infections was conducted. Patient demographics, infection recurrence, compliance and adverse drug reactions to the two-dose regimen of dalbavancin were recorded. Furthermore, preserved clinical isolates from these infections were assessed for susceptibility to dalbavancin with microbroth dilutions. RESULTS: All patients were fully compliant with the two dose dalbavancin regimen and no patient had any adverse reactions to the two-dose dalbavancin regimen. Thirteen of fifteen patients (85.7%) have not had any recurrence of their infections and all preserved clinical isolates showed susceptibility to dalbavancin. DISCUSSION: The two-dose regimen of dalbavancin is an effective and attractive option in treating prosthetic joint and spinal hardware infections to forgo long term central venous access and ensure compliance. However, the use of rifampin and suppression antibiotics still needs to be considered when treating these infections. Nonetheless this study supports that a two-dose dalbavancin regimen is a viable alternative in certain clinical settings and consideration for a randomized controlled clinical trial should be entertained to prove its non-inferiority to conventional treatments.

Emergence of Dalbavancin, Vancomycin, and Daptomycin Nonsusceptible Staphylococcus aureus in a Patient Treated With Dalbavancin: Case Report and Isolate Characterization.

A patient with end-stage renal disease received 2 doses of dalbavancin for methicillin-resistant Staphylococcus aureus (MRSA) arteriovenous fistula infection and presented 5 weeks later with infective endocarditis secondary to vancomycin, daptomycin, and dalbavancin nonsusceptible MRSA. Resistance was associated with walK and scrA mutations, reduced long-chain lipid content, and reduced membrane fluidity.