Mitochondrial Disorder Diagnosis: Expert Consensus and Clinical Guidelines

Introduction

Mitochondrial disorders represent a significant category of inherited metabolic conditions, conservatively estimated to affect 1 in 5,000 individuals. These diseases arise from malfunctions within the mitochondria, the energy powerhouses of cells, due to mutations in either nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). The field of mitochondrial medicine is relatively young, having evolved over the last quarter-century. Clinicians face the ongoing challenge of diagnosing, treating, and managing these complex conditions with limited but growing evidence. A critical hurdle is the absence of highly sensitive and specific biomarkers, making definitive Mitochondrial Disorder Diagnosis often elusive, costly, and sometimes requiring invasive procedures.

Currently, there are no universally adopted, consensus-based guidelines to aid clinicians in the initial diagnosis or long-term management of mitochondrial disease. Many specialists rely on internally developed protocols, informed by theoretical frameworks, limited published recommendations, and accumulated personal experience. A recent assessment by the Mitochondrial Medicine Society highlighted considerable variations in diagnostic approaches, the breadth of testing utilized, the interpretation of results, and the evidentiary basis for establishing a mitochondrial disorder diagnosis. Inconsistencies also exist in treatment strategies and preventive care measures.

This article aims to address this gap by reviewing the existing literature on mitochondrial disease and, where possible, offering consensus-driven recommendations for effective diagnosis and patient management. To maintain conciseness, detailed background information on mitochondrial diseases, including specific testing, diagnostic modalities, and treatment approaches, will be limited. Readers seeking more in-depth foundational knowledge are directed to several comprehensive reviews and supplementary materials associated with this consensus initiative. This resource focuses on providing practical, expert-backed guidance to standardize and enhance the process of mitochondrial disorder diagnosis and care.

I. Diagnostic Approaches for Mitochondrial Disorders

A. Biochemical Testing: Biomarkers in Blood, Urine, and CSF

Most diagnostic algorithms for mitochondrial disorder diagnosis begin with the evaluation of specific mitochondrial biomarkers in readily accessible bodily fluids: blood, urine, and cerebrospinal fluid (CSF). These typically include measurements of lactate and pyruvate levels in plasma and CSF, amino acid profiles in plasma, urine, and CSF, plasma acylcarnitines, and urine organic acids.

Elevated lactate levels are a common finding in mitochondrial dysfunction. This occurs because the glycolytic pathway outpaces the mitochondria’s capacity to process pyruvate. However, the clinical utility of lactate measurements is often hampered by potential errors in sample collection and handling. For instance, venous plasma lactate can be artificially elevated if a tourniquet is applied during blood collection or if a child is distressed during the procedure. Nonetheless, a markedly elevated plasma lactate level (>3 mmol/L) in a properly collected sample is suggestive of mitochondrial dysfunction, which can stem from primary mitochondrial disease or secondary causes such as organic acidemias, other metabolic disorders, toxins, tissue ischemia, and certain other conditions.

Studies have indicated that in patients with confirmed primary mitochondrial disease, genuinely elevated lactate levels demonstrate a sensitivity ranging from 34% to 62% and a specificity between 83% and 100%. The blood lactate/pyruvate ratio is most effective in distinguishing electron transport chain (ETC) disorders from pyruvate metabolism defects, but only when lactate levels are significantly elevated. The sensitivity of this ratio is reported at 31%, with a specificity of 100%.

Elevated CSF lactate can be a valuable indicator of mitochondrial disease, especially in patients presenting with neurological symptoms. Collection artifacts are less of a concern with CSF, although it’s important to note that various brain disorders, particularly status epilepticus, can temporarily increase CSF lactate levels. Interestingly, urine lactate levels show a weaker correlation with the presence of mitochondrial disease compared to blood and CSF.

Pyruvate elevation is a particularly useful biomarker for identifying defects in enzymes directly involved in pyruvate metabolism, specifically pyruvate dehydrogenase and pyruvate carboxylase. Similar to lactate, blood pyruvate levels are susceptible to collection and handling errors, and pyruvate is also inherently unstable. One study reported a sensitivity of 75% and specificity of 87.2% for pyruvate levels in patients with primary mitochondrial disease.

Quantitative amino acid analysis in blood or CSF is a routine component of the evaluation for suspected mitochondrial disease. Dysfunction of the respiratory chain can alter the cellular redox state, leading to elevations in several amino acids, including alanine, glycine, proline, and threonine. The precise sensitivity and specificity of alanine or other amino acid elevations in mitochondrial disorder diagnosis are still under investigation. Elevations may be detected in either blood or CSF, and significant findings may only manifest during periods of clinical deterioration. Urine amino acid analysis is primarily used to assess for mitochondrial disease-associated renal tubulopathy.

Carnitine plays a crucial role in mitochondrial function, acting as a shuttle for fatty acids into mitochondria and as an acceptor of potentially toxic coenzyme A esters. It aids in restoring intramitochondrial coenzyme A and eliminating esterified intermediates. Quantification of total and free carnitine levels in blood, along with acylcarnitine profiling, helps identify primary or secondary fatty-acid oxidation defects, as well as certain primary amino and organic acidemias. While acylcarnitine testing is frequently recommended in mitochondrial disease reviews, the supporting literature is somewhat limited. This testing is generally advised due to the potential for secondary disturbances in fatty-acid oxidation in mitochondrial disease patients and the phenotypic overlap between certain mitochondrial disorders and other inborn errors of metabolism for which acylcarnitine analysis is diagnostic.

Urinary organic acid analysis often reveals abnormalities in mitochondrial disease patients. Elevated levels of malate and fumarate have been found to correlate most strongly with mitochondrial disease in a retrospective study, when compared to patients with organic acidemias. Other citric acid cycle intermediates and lactate showed weaker correlations. Mild to moderate elevations of 3-methylglutaconic acid (3MG), dicarboxylic aciduria, 2-oxoadipic aciduria, 2-aminoadipic aciduria, and methylmalonic aciduria can all be observed in specific primary mitochondrial diseases. While urine organic acid analysis can detect 3MG elevations, direct quantification of 3MG in blood and urine is more reliable, especially when 3MG levels are not markedly elevated.

Elevated creatine phosphokinase and uric acid levels are commonly seen in acute rhabdomyolysis in patients with fatty-acid oxidation disorders, resulting from nucleic acid and nucleotide catabolism. Although less studied in primary mitochondrial disorders, patients can experience muscle disease, particularly in cytochrome b disease and thymidine kinase 2 deficiency, and elevations in these markers can also occur. Hematologic abnormalities, detectable via complete blood count, have been reported in some primary mitochondrial diseases, including aplastic, megaloblastic, and sideroblastic anemias, leukopenia, thrombocytopenia, and pancytopenia. Liver pathology is associated with multiple primary mitochondrial diseases, often linked to mtDNA depletion and/or general liver dysfunction, making transaminase and albumin levels diagnostically relevant. Emerging biomarkers such as FGF21 and reduced glutathione are under investigation for their potential role in mitochondrial disorder diagnosis, but require further validation.

Cerebral folate deficiency is observed in a wide range of neurological and metabolic disorders, including mitochondrial disease. It is diagnosed by measuring 5-methyltetrahydrofolate levels in CSF. Initially identified in mitochondrial disease in patients with Kearns-Sayre syndrome (KSS), cerebral folate deficiency has been further confirmed in subsequent case series involving KSS patients. It has also been identified in patients with mtDNA deletions, POLG disease, and biochemically confirmed complex I deficiency. A primary cerebral folate disorder also exists, often caused by mutations in the FOLR1 gene encoding folate receptor alpha.

Consensus Recommendations for Testing Blood, Urine, and Spinal Fluid

The initial blood evaluation for mitochondrial disorder diagnosis should include a complete blood count, creatine phosphokinase, transaminases, albumin, lactate and pyruvate, amino acids, and acylcarnitines, along with quantitative or qualitative urinary organic acids. Careful attention must be paid to proper specimen collection, especially for lactate and pyruvate measurements.
Postprandial lactate levels are more sensitive than fasting specimens and are preferred when feasible. Exercise caution against overinterpreting minor elevations in postprandial lactate.
The lactate/pyruvate ratio in blood or CSF is only valuable when the lactate level is elevated.
Quantitative 3MG measurements in plasma and urine should be obtained, when possible, in addition to urine organic acids in patients undergoing evaluation for mitochondrial disease.
Creatine phosphokinase and uric acid should be assessed in patients with muscle symptoms suspected of having mitochondrial diseases.
Urine amino acid analysis should be performed when evaluating for mitochondrial tubulopathy.
When CSF is obtained, it should be analyzed for lactate, pyruvate, amino acids, and 5-methyltetrahydrofolate levels.
Further research is necessary to validate the clinical utility of other biomarkers such as FGF21, glutathione, and CSF neopterin in mitochondrial disorder diagnosis.

B. DNA Testing and Genetic Diagnosis of Mitochondrial Diseases

Primary mitochondrial disorders are genetically heterogeneous, resulting from mutations in maternally inherited mtDNA or numerous nDNA genes. mtDNA genome sequencing and heteroplasmy analysis are now effectively performed using blood samples, although in some cases, testing other tissues from affected organs may be necessary for accurate mitochondrial disorder diagnosis. Advances in testing methodologies now allow for more sensitive detection of low-level heteroplasmy in blood, down to 5–10% and even 1–2%. Next-generation sequencing (NGS) has emerged as the gold standard for mtDNA genome sequencing, offering significantly improved reliability and sensitivity for detecting point mutations, low-level heteroplasmy, and deletions. This comprehensive approach provides a single test for accurate diagnosis of mtDNA disorders. NGS can be considered as the first-line test for comprehensive mitochondrial genome analysis in blood, urine, or tissue, depending on the clinical presentation and sample availability. Identifying a causative mitochondrial disease mutation is crucial for families, allowing them to conclude their diagnostic journey and access appropriate genetic counseling, carrier testing, and prenatal diagnostic options.

In certain cases of suspected mitochondrial disorder, preferentially testing tissues other than blood may be required for accurate mitochondrial disorder diagnosis. Urine is increasingly recognized as a valuable specimen for mtDNA genome analysis, owing to the high mtDNA content in renal epithelial cells. This is particularly relevant for MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome and its most common mutation, m.3243 A>G in MTTL1. Skeletal muscle or liver are preferred tissue sources for mtDNA genome sequencing when available, due to their high mtDNA content, dependence on mitochondrial respiration, and the possibility of harboring tissue-specific mtDNA mutations not detectable in blood.

mtDNA deletion and duplication syndromes present a spectrum of phenotypes, including KSS, chronic progressive external ophthalmoplegia, and Pearson syndrome. Traditionally, Southern blot and long-range PCR were used to detect mtDNA deletions. However, Southern blot analysis lacks the sensitivity to detect low levels of heteroplasmic deletions. Array comparative genome hybridization offers improved detection of deletions and also estimates deletion breakpoints and heteroplasmy levels. However, all these methods are being superseded by NGS of the entire mitochondrial genome, which provides sufficient depth of coverage to sensitively detect and characterize single or multiple deletions. It’s crucial to note that deletions and duplications may only be detectable in muscle or liver in many patients.

mtDNA depletion syndromes are a clinically and genetically diverse group characterized by a significant reduction in mtDNA copy number in affected tissues. These disorders typically arise from nDNA mutations in genes involved in mitochondrial deoxynucleotide synthesis or mtDNA replication. Less frequently, germline deletions/duplications of mtDNA segments can cause mtDNA depletion. Diagnosis requires quantifying mtDNA content, typically in affected tissue, and identifying a significant decrease below the normal range for age, gender, and tissue type, when normalized to nDNA tissue content. mtDNA content is not assessed by NGS of the mtDNA genome and requires a separate quantitative real-time PCR assay.

Over 1,400 nuclear genes are directly or indirectly involved in mitochondrial function. In addition to single-gene testing, many diagnostic laboratories offer NGS-based panels targeting multiple genes. These panels range in size, from small targeted panels to larger panels encompassing hundreds or even thousands of nuclear genes. Whole-exome sequencing, clinically available since 2011, is increasingly utilized in mitochondrial disorder diagnosis. While research reports frequently describe the identification of novel pathogenic mutations in nuclear mitochondrial genes through whole-exome sequencing, clear evidence-based practice recommendations regarding the optimal use of single-gene sequencing, nuclear gene panels, or whole-exome sequencing in clinical practice are still evolving.

Consensus Recommendations for DNA Testing

Massively parallel sequencing/NGS of the mtDNA genome is the preferred methodology for mtDNA testing and should be used in suspected mitochondrial disease cases instead of limited point mutation testing.
Patients with a high clinical suspicion of mitochondrial disease due to a mtDNA mutation but negative blood testing should have mtDNA assessed in another tissue. This is crucial to avoid missing tissue-specific mutations or low heteroplasmy levels in blood. Tissue-based testing also aids in assessing the risk of multi-organ involvement and heterogeneity within families, guiding genetic counseling.
Heteroplasmy analysis in urine can be more informative and accurate than blood testing alone, particularly in MELAS cases due to the common m. 3243A>G mutation.
mtDNA deletion and duplication testing should be performed using NGS of the mtDNA genome in suspected mitochondrial disease, especially in all patients undergoing diagnostic tissue biopsy.
a. Caution is advised when interpreting a single small deletion identified by PCR-based analysis as definitively indicative of a primary mitochondrial disorder.
b. When multiple mtDNA deletions are detected, sequencing of nuclear genes involved in mtDNA biosynthesis is recommended.
When tissue specimens are obtained for mitochondrial studies, mtDNA content (copy number) testing via real-time quantitative PCR should be strongly considered for mtDNA depletion analysis, as mtDNA depletion may not be detected in blood.
a. mtDNA proliferation is a non-specific compensatory finding that can be observed in primary mitochondrial disease, secondary mitochondrial dysfunction, myopathy, hypotonia, and as a result of intense exercise.
When considering nuclear gene testing in patients with likely primary mitochondrial disease, NGS methodologies providing comprehensive coverage of known mitochondrial disease genes are preferred. Single-gene testing should generally be avoided due to phenotypic overlap caused by mutations in different genes. If known NGS gene panels fail to identify a mutation, whole-exome sequencing should be considered for comprehensive mitochondrial disorder diagnosis.

C. Tissue Pathology and Biochemical Analysis for Mitochondrial Dysfunction

Pathology

Tissue biopsy, typically muscle, has historically been considered the gold standard for mitochondrial disorder diagnosis, although its sensitivity and specificity are debated. Tissue samples are usually subjected to a range of histological, biochemical, and genetic analyses. With advancements in molecular testing, the reliance on tissue biochemical testing for primary diagnosis has lessened. However, selective tissue testing remains highly informative, especially for clinically heterogeneous conditions like mitochondrial disease. Tissue testing allows for the detection of tissue-specific mtDNA mutations or low-level heteroplasmy, quantification of mtDNA content, guidance for targeted molecular studies to cover genes of highest interest, and validation of the pathogenicity of variants of unknown significance identified in molecular tests. In patients with myopathy, muscle biopsy can also rule out other neuromuscular diseases in the differential diagnosis.

The necessity of open biopsy versus less invasive methods for tissue collection is debated. Some specialized centers are proficient in performing needle biopsies for mitochondrial testing. Open mitochondrial tissue biopsies require specialized techniques compared to routine biopsies to minimize mitochondrial damage and artifactual abnormalities. Key considerations for tissue collection and processing are summarized in Table 1.

Routine muscle histology includes hematoxylin and eosin (H&E), Gomori trichrome (for ragged red fibers), SDH (for SDH-rich or ragged blue fibers), NADH-TR (NADH-tetrazolium reductase), COX (for COX negative fibers), and combined SDH/COX staining (particularly useful for COX intermediate fibers). Other standard stains, as per the institution’s pathology department, should be routinely used to explore other myopathies in the differential diagnosis (e.g., glycogen, lipid staining). Electron microscopy (EM) is crucial for examining mitochondria for inclusions and ultrastructural abnormalities. Pediatric patients are less likely to exhibit histopathological abnormalities, and irregularities may only be evident on muscle EM, although normal results are also possible.

Hepatic dysfunction due to mitochondrial disease is more common in pediatric patients. Liver biopsy can reveal characteristic histologic and ultrastructural features of mitochondrial hepatopathy, such as steatosis, cholestasis, disrupted architecture, and cytoplasmic crowding by atypical mitochondria with swollen cristae. Ultrastructural evaluation is recommended in unexplained cholestasis, especially when accompanied by steatosis and hepatocyte hypereosinophilia.

Consensus Recommendations for Pathology Testing

Muscle (and/or liver) biopsies should be performed in the routine analysis for mitochondrial disorder diagnosis when DNA testing is non-diagnostic.
Open biopsy is preferred for routine muscle biopsy analysis for mitochondrial disease, unless the center is experienced in obtaining adequate tissue quality and quantity via percutaneous biopsy.
The vastus lateralis is the preferred muscle biopsy site for mitochondrial disease evaluation, as most laboratories use this site to establish reference ranges.
COX, SDH, NADH-TR, and combined SDH/COX staining, along with EM, should be routinely performed in tissue analysis for mitochondrial disease. EM is strongly recommended in pediatric patients undergoing tissue biopsy due to often limited histological findings.
Mitochondrial hepatopathy may exhibit characteristic findings on liver biopsy histology.
When possible, extra tissue should be frozen to enable additional testing. Refer to Table 1 for specific considerations.

Biochemical Testing in Tissue

Functional in vitro assays in tissue, typically muscle, have been central to mitochondrial disorder diagnosis, particularly before recent genomic advancements. These assays remain vital for assessing mitochondrial function. Prior mitochondrial disease guidelines and diagnostic criteria heavily relied on biochemical studies to establish a diagnosis.

These tests evaluate various aspects of mitochondrial ETC function. Functional assays include enzyme activity measurements of individual ETC complex components, assessment of component activity, blue-native gel electrophoresis, protein component quantification within complexes and supercomplexes (via western blots and gel electrophoresis), and oxygen consumption measurements using different substrates and inhibitors. Ideally, ETC assays should be performed on the most affected tissue (e.g., muscle in exercise intolerance, heart in cardiomyopathy, liver in hepatopathy) to enhance diagnostic sensitivity for mitochondrial disorder diagnosis. Limitations of biochemical studies in tissue are important to consider, as outlined in Table 2.

Defects in coenzyme Q10 (CoQ10) synthesis can lead to treatable mitochondrial diseases. CoQ10 levels can be directly measured in muscle, lymphocytes, and fibroblasts, with muscle levels considered most sensitive for diagnosing primary CoQ10 deficiency. Low CoQ10 levels can also be secondary to other disorders.

To minimize invasive procedures like open muscle biopsy, non-invasive methods for diagnosing ETC abnormalities have been explored. A small study indicated that buccal swab analysis of complex I and complex IV showed an 80% correlation with muscle ETC analysis, but this method requires further validation before widespread clinical application for mitochondrial disorder diagnosis. Cultured fibroblast ETC activity assays are also sometimes used, but results can be normal even when muscle or liver ETC abnormalities are present, limiting their sensitivity.

Consensus Recommendations for Biochemical Testing in Tissue

Biochemical testing in tissue cannot always differentiate between primary mitochondrial disease and secondary mitochondrial dysfunction, highlighting the need for integrated diagnostic approaches for accurate mitochondrial disorder diagnosis.
When obtaining a biopsy for mitochondrial disease evaluation, ETC enzymology (spectrophotometry) of complex I–IV activities should be performed on snap-frozen tissue or freshly isolated mitochondria. Biopsy of the affected tissue is recommended whenever possible. Analyzing isolated complex III is crucial, as assessment of complex II/III and I/III alone may be insufficient for comprehensive mitochondrial disorder diagnosis.
ETC results should be interpreted using internal assay controls and normalized to marker enzymes (such as citrate synthase and/or complex II) to improve diagnostic reliability and reduce inter-laboratory variability in mitochondrial disorder diagnosis.
Fresh tissue analysis allows for functional oxidative phosphorylation/oxymetric measurements of oxygen consumption and ATP production for all five ETC complexes, which can be sufficient for diagnosing mitochondrial dysfunction. While not universally available, these tests should be considered when accessible for enhanced mitochondrial disorder diagnosis.
Various techniques for evaluating isolated mitochondria, permeabilized myofibers, immunoblot assays, and radiolabeled assays may enhance ETC abnormality detection in specialized centers. However, these require further validation as standalone diagnostic tests for mitochondrial disorder diagnosis.
Established diagnostic criteria should be used when interpreting ETC results to avoid subjective assessments of mitochondrial dysfunction and inter-physician variability in diagnoses. Caution is advised when interpreting ETC enzyme activity above 20% of the control mean. Diagnosing primary mitochondrial disease solely based on biochemical abnormalities from tissue testing should be approached with caution and integrated with other diagnostic findings for robust mitochondrial disorder diagnosis.
Significantly reduced ETC components or enzyme activity from isolated components can provide supplementary information when evaluating patients for possible mitochondrial disease, strengthening the evidence for mitochondrial disorder diagnosis.
Tissue analysis of ETC complex enzyme activities can yield false-negative results due to factors like assay timing and testing of less affected tissue. Therefore, normal ETC findings should not be the sole criterion for excluding mitochondrial dysfunction in mitochondrial disorder diagnosis.
Muscle CoQ10 levels are necessary to diagnose primary CoQ10 synthesis defects, especially when genetic studies are inconclusive. Leukocyte CoQ10 levels are insufficient for diagnosing primary CoQ10 synthesis disorders. Reduced muscle CoQ10 levels can also occur in other conditions, emphasizing the need for careful interpretation in mitochondrial disorder diagnosis.
Fibroblast ETC assays can aid in identifying mitochondrial dysfunction in some cases, but false-negative results are possible, limiting their sensitivity for mitochondrial disorder diagnosis.
Buccal swab analysis should not be a first-line mitochondrial test. More comparative studies of buccal swab ETC results with muscle ETC activity and genetically confirmed patients are needed before it can be reliably used in mitochondrial disorder diagnosis.

D. Neuroimaging in the Diagnostic Process

Neuroimaging, including computed tomography (CT) and magnetic resonance imaging (MRI) of the brain, plays a supportive role in mitochondrial disorder diagnosis. Some diagnostic criteria protocols include neuroimaging, while others do not, reflecting the variability in its diagnostic utility across different mitochondrial disorders.

Depending on the specific mitochondrial disorder and the extent of central nervous system involvement, neuroimaging findings can range from absent to significant structural alterations. Classic neuroimaging findings in syndromic mitochondrial diseases include stroke-like lesions in a non-vascular distribution, diffuse white matter disease, and bilateral involvement of deep gray matter nuclei in the basal ganglia, midbrain, or brainstem. These “classical” changes can also be observed in non-syndromic mitochondrial diseases and other metabolic disorders. Thus, they are neither sufficiently sensitive nor specific to establish a primary mitochondrial disorder diagnosis in isolation, requiring integration with other clinical and laboratory findings. Certain mitochondrial disorders, such as KSS and MERRF, are also known to exhibit other neuroimaging abnormalities like non-specific white matter lesions, but these findings lack the sensitivity to be considered core diagnostic criteria for these syndromes. More pronounced white matter abnormalities are typically seen in MNGIE, Leigh syndrome, and mitochondrial disorders resulting from aminoacyl-tRNA synthetase defects.

Beyond qualitative changes, quantitative changes can be detected using specific acquisition sequences, proton magnetic resonance spectroscopy (MRS), and diffusion tensor imaging. MRS provides semiquantitative estimates of brain metabolites, including lactate, creatine, and N-acetyl aspartate, within defined brain regions. Diffusion tensor imaging detects and quantifies major white matter tracts. While MRS and diffusion tensor imaging changes may be observed in classic mitochondrial syndromes and non-syndromic patients, they are not specific to mitochondrial disorders and can be seen in various other metabolic or brain parenchymal disorders. Therefore, these advanced neuroimaging techniques serve as supportive tools rather than definitive diagnostic markers for mitochondrial disorder diagnosis.

Consensus Recommendations for Neuroimaging

When central nervous system involvement is suspected, brain MRI should be performed as part of the evaluation for patients with suspected mitochondrial disease. MRS findings of elevated lactate within brain parenchyma are also valuable. However, neuroimaging findings alone are not sufficient for definitive mitochondrial disorder diagnosis.
Neuroimaging can be useful in monitoring the progression of mitochondrial neurologic disease, providing objective markers of disease course and treatment response in longitudinal mitochondrial disorder diagnosis and management.
Further research is needed to clarify the role of MRS and diffusion tensor imaging in monitoring the disease course of mitochondrial disease and their utility in refining mitochondrial disorder diagnosis and prognosis.

II. Management and Treatment Guidelines for Mitochondrial Disorders

A. Acute Stroke-like Episode Management in Mitochondrial Disease

Stroke-like episodes are a hallmark feature of several mitochondrial syndromes, particularly MELAS syndrome. Emerging evidence suggests a therapeutic role for L-arginine and citrulline therapy in MELAS-related strokes. Arginine and citrulline are precursors to nitric oxide (NO). In MELAS syndrome, endothelial dysfunction due to reduced NO production and impaired smooth muscle relaxation may contribute to deficient cerebral blood flow and stroke-like episodes. Patients with MELAS often exhibit lower NO and NO metabolite levels during stroke-like episodes, along with decreased citrulline flux, de novo arginine synthesis rate, and plasma arginine and citrulline concentrations. Clinical observations indicate that administering oral and intravenous (IV) arginine to MELAS patients can alleviate symptoms associated with stroke-like episodes and reduce their severity and frequency. More recent data also suggest that arginine and citrulline supplementation can improve NO production. However, controlled studies are needed to definitively assess the beneficial effects of arginine or citrulline supplementation, determine optimal dosing regimens, and establish safety parameters for stroke-like episodes in MELAS and other mitochondrial cytopathies associated with these episodes.

Consensus Recommendations for the Treatment of Mitochondrial Stroke

Stroke-like episodes in primary mitochondrial disease typically correlate with visible abnormalities on brain MRI, aiding in differential mitochondrial disorder diagnosis and acute management.
IV arginine hydrochloride should be administered urgently in the acute setting of a stroke-like episode associated with the MELAS m.3243 A>G mutation in the MTTL1 gene. It should also be considered in stroke-like episodes associated with other primary mitochondrial cytopathies while excluding other etiologies. Patients should be reassessed after 3 days of continuous IV therapy to evaluate treatment response and guide further management in mitochondrial disorder diagnosis.
Daily oral arginine supplementation to prevent strokes should be considered in MELAS syndrome patients for long-term management and reduction of stroke recurrence in mitochondrial disorder diagnosis.
Further research is needed to clarify the role of monitoring plasma arginine and citrulline levels and the use of oral citrulline supplementation in the treatment of MELAS, optimizing therapeutic strategies in mitochondrial disorder diagnosis.

B. Exercise as a Therapeutic Intervention

Numerous studies in animal models and human patients with various mitochondrial myopathies (both nDNA and mtDNA encoded) demonstrate the benefits of endurance exercise in mitochondrial disease management. Endurance training studies in mitochondrial patients have shown improvements in mitochondrial content, antioxidant enzyme activity, muscle mitochondrial enzyme activity, maximal oxygen uptake, and peripheral muscle strength. Clinical benefits include improved symptoms and reduced resting and post-exercise blood lactate levels, with sustained positive outcomes over time.

Most studies report no detrimental effects from gradually progressive exercise training, whether resistance or endurance, in patients with mitochondrial myopathy. Notably, there are no reports of elevated creatine kinase levels, negative heteroplasmic shifts, or increased musculoskeletal injuries in mitochondrial patients participating in supervised progressive exercise programs designed for physiological adaptation. Therefore, exercise represents a safe and effective therapeutic modality for improving functional capacity and quality of life in mitochondrial disease.

Consensus Recommendations for Exercise

Exercise-induced mitochondrial biogenesis is a valuable therapeutic target for improving function in patients with mitochondrial disease, enhancing cellular energy production and metabolic capacity in mitochondrial disorder diagnosis management.
Endurance exercise can increase mitochondrial enzyme activity in muscle and improve quality-of-life scores, while reducing the energy cost of daily activities in mitochondrial disease patients, leading to improved functional outcomes in mitochondrial disorder diagnosis management. Resistance exercise can enhance muscle strength and promote satellite cell recruitment in muscle fibers in mitochondrial patients, contributing to overall muscle health and function in mitochondrial disorder diagnosis management.
A combination of progressive endurance and resistance exercise is optimal for patients with mitochondrial disease and is considered safe when implemented in a supervised, progressive manner, starting with low intensity and duration and gradually increasing to achieve therapeutic benefits in mitochondrial disorder diagnosis management.
Mitochondrial patients should undergo cardiac screening prior to initiating an exercise program to ensure cardiovascular safety and tailor exercise prescriptions to individual patient profiles in mitochondrial disorder diagnosis management.
Exercise intolerance is a recognized symptom in mitochondrial disease, but deconditioned mitochondrial patients should be encouraged to engage in exercise. Physicians should actively promote adherence to exercise programs as a cornerstone of comprehensive management in mitochondrial disorder diagnosis.
High-intensity interval training has been shown to induce similar mitochondrial adaptations as endurance exercise in healthy and diabetic adults, but its effectiveness and safety in mitochondrial disease patients require further investigation before it can be widely recommended in mitochondrial disorder diagnosis management.

C. Anesthesia Considerations for Patients with Mitochondrial Disorders

Many patients with mitochondrial disease tolerate anesthesia without complications. Recent studies in small mitochondrial patient populations suggest that anesthetics are generally safe. However, reports of serious and unexpected adverse events, both during and after anesthetic exposure, including respiratory depression and white matter degeneration, underscore the need for caution. A persistent perception among physicians remains that these patients are vulnerable to decompensation during anesthesia.

Anesthetics generally affect tissues with high energy demands, and nearly every general anesthetic studied has been shown to impair mitochondrial function, particularly volatile anesthetics and propofol. Propofol and thiopental are typically tolerated when used in a limited fashion, such as during an infusion bolus. Propofol infusion syndrome susceptibility has been suggested but not definitively proven in mitochondrial disease patients.

Narcotics and muscle relaxants, frequently used in operating rooms, are generally tolerated (except possibly morphine) as they appear to have minimal impact on mitochondrial function. However, they can cause respiratory depression, necessitating careful use in mitochondrial patients who may already have hypotonia, myopathy, or altered respiratory drive. The risk of malignant hyperthermia does not seem to be elevated in mitochondrial patients.

Mitochondrial patients are often susceptible to metabolic decompensation during catabolic states, which can be triggered by anesthesia-related fasting, hypoglycemia, vomiting, hypothermia, and acidosis. Limiting preoperative fasting, providing continuous energy via IV dextrose, and closely monitoring basic chemistries are crucial perioperative strategies to mitigate metabolic risks in mitochondrial disorder diagnosis management.

Consensus Recommendations for Anesthesia

Patients with mitochondrial diseases have an increased risk of anesthesia-related complications, requiring careful preoperative assessment and tailored anesthetic management in mitochondrial disorder diagnosis.
Preoperative preparation is critical for perioperative outcomes in mitochondrial disease patients. Minimize preoperative fasting and administer glucose in perioperative IV fluids, unless contraindicated due to ketogenic diet or adverse reactions to high glucose intake, to prevent metabolic decompensation in mitochondrial disorder diagnosis management.
Exercise caution with volatile anesthetics in mitochondrial patients due to potential hypersensitivity, opting for lower concentrations and careful titration to minimize mitochondrial stress in mitochondrial disorder diagnosis management.
Use muscle relaxants cautiously in mitochondrial patients with pre-existing myopathy or decreased respiratory drive, carefully monitoring respiratory function and considering reduced dosages to avoid respiratory compromise in mitochondrial disorder diagnosis management.
Mitochondrial patients may have a higher risk of propofol infusion syndrome, so propofol use should be avoided or limited to short procedures, considering alternative anesthetic agents whenever possible to minimize risks in mitochondrial disorder diagnosis management.
Consider slow titration and adjustment of volatile and parenteral anesthetics to minimize hemodynamic changes in mitochondrial patients, ensuring stable hemodynamics throughout the perioperative period for improved safety in mitochondrial disorder diagnosis management.
Local anesthetics are generally well-tolerated in patients with mitochondrial defects and can be considered as a safer alternative when feasible, reducing systemic anesthetic exposure and associated mitochondrial risks in mitochondrial disorder diagnosis management.
There is no clear established link between malignant hyperthermia and mitochondrial disease, suggesting that standard malignant hyperthermia precautions are sufficient without additional specific measures for mitochondrial patients in mitochondrial disorder diagnosis management.

D. Managing Illness and Catabolic Stress

Literature on managing mitochondrial disease patients in acute settings is limited. Diseased mitochondria produce more reactive oxygen species and are prone to energy-deficient states. Catabolic stressors necessitate increased cellular energy generation from protein, carbohydrate, and fat stores. Catabolism is induced by physiologic stressors like fasting, fever, illness, trauma, or surgery. Due to reduced cellular reserves, mitochondrial patients are more susceptible to entering catabolic states, generating more toxic metabolites and reactive oxygen species. These stresses can cause cell injury and worsen baseline symptoms or trigger new ones. Management recommendations are based on established protocols for other inborn errors of metabolism during vulnerable periods. Treatments include IV dextrose for anabolic support, avoiding prolonged fasting, and minimizing exposure to substances that impair mitochondrial function. Proactive management during illness is crucial to prevent metabolic decompensation and symptom exacerbation in mitochondrial disease.

Consensus Recommendations for Treatment During an Acute Illness

Specific patient management decisions, including hospitalization, require clinical judgment and should be case-specific. Decisions should reflect individual patient presentation, the etiology of acute decompensation, and the pathophysiology of the underlying mitochondrial disorder to optimize care in mitochondrial disorder diagnosis management.
Patients with mitochondrial disease should carry an emergency care plan detailing their disorder and providing management recommendations, enabling rapid and appropriate interventions during acute illnesses and potential emergencies in mitochondrial disorder diagnosis management.
Mitochondrial patients should wear a Medic Alert bracelet to ensure prompt recognition of their condition by medical personnel during emergencies, facilitating informed and timely care in mitochondrial disorder diagnosis management.
Mitochondrial patients should take precautions to prevent catabolism, especially during medical stressors, including avoiding prolonged fasting and receiving dextrose-containing IV fluids before, during, and after procedures and surgeries. (Dextrose should be limited or avoided in suspected or confirmed pyruvate metabolism disorders, ketogenic diet adherence, or adverse reactions to high glucose), tailoring metabolic support to specific patient needs in mitochondrial disorder diagnosis management.
Acute evaluations of mitochondrial patients should include routine chemistries, glucose, transaminases, and lactate. Further testing should be guided by clinical indications, considering the potential for cardiac and neurologic decompensation in these patients, ensuring comprehensive assessment in mitochondrial disorder diagnosis management.
Acute decompensation treatment should include dextrose-containing IV fluids, discontinuing potentially toxic medications, and correcting metabolic derangements. (Limit or avoid dextrose in pyruvate metabolism disorders, ketogenic diet, or adverse glucose response). IV fluid rates should be clinically determined. Outpatient mitochondrial therapies should be continued when possible, providing consistent and supportive care in mitochondrial disorder diagnosis management.
Lipids can be used when needed in mitochondrial patients, even with secondary fatty-acid oxidation dysfunction, ensuring adequate caloric intake and metabolic support during acute illness in mitochondrial disorder diagnosis management.
The following medications should be avoided or used cautiously in mitochondrial disease patients: valproic acid, statins, metformin, high-dose acetaminophen, and selected antibiotics (aminoglycosides, linezolid, tetracycline, azithromycin, erythromycin), minimizing potential drug-induced mitochondrial toxicity and decompensation in mitochondrial disorder diagnosis management.
Repeat neuroimaging should be considered for any mitochondrial patient with acute changes in neurologic status to promptly assess for stroke-like episodes or other CNS complications requiring immediate intervention in mitochondrial disorder diagnosis management.

E. Vitamin and Xenobiotic Therapies

Multiple vitamins and cofactors are used in mitochondrial disease treatment, but therapies are not standardized, and treatment variations are common. Despite the rationale for using these agents, few trials have evaluated their clinical effects. Consensus is lacking on which agents to use, although CoQ10, L-carnitine, creatine, α-lipoic acid (ALA), and certain B-vitamins are frequently prescribed. A Cochrane review found limited evidence supporting any vitamin or cofactor intervention, highlighting the need for more rigorous clinical trials to establish efficacy and guidelines for vitamin and supplement use in mitochondrial disorder diagnosis management.

CoQ10, in various forms, is the most widely used supplement in mitochondrial disease and acts as an antioxidant with diverse functions. Evidence supporting CoQ10 use beyond primary CoQ10 deficiency is limited, with few placebo-controlled studies. Most supportive data are anecdotal, from open-label treatments, or combination cofactor therapies, with minimal benefit reported for CoQ10 alone.

ALA is also commonly used in mitochondrial disease therapy, but no controlled clinical trial has assessed its monotherapy efficacy. One randomized study used ALA with creatine and CoQ10. Case reports describe ALA use with other therapies (ubiquinone, riboflavin, creatine, vitamin E) with some reported clinical benefits.

B-vitamins, alone or as multivitamins (e.g., vitamin B50), are commonly recommended in clinical practice, but no randomized trials have explored their efficacy. Riboflavin use has shown clinical and biochemical improvements in small, open-label studies. Riboflavin as part of a vitamin combination showed no clear clinical benefit in an open-label study of 16 children.

No clinical trials have investigated L-carnitine for mitochondrial disease treatment, although it’s commonly used in this population. L-Carnitine is also used for neurometabolic disorders, including organic acidemias and some fatty-acid oxidation disorders, but randomized controlled trials are lacking even in these conditions. L-Carnitine has been used in case studies, often combined with other vitamins.

Patients with mitochondrial disease, especially mtDNA deletion syndromes like KSS, may have low CSF 5-methyltetrahydrofolate. Folinic acid supplementation is common in mitochondrial patients with neurologic signs or symptoms. However, no clinical trials have evaluated folinic acid therapy for mitochondrial disease, highlighting a significant gap in evidence-based treatment guidelines for mitochondrial disorder diagnosis management.

Many other compounds have been reviewed, with data available in online summaries. Consensus is that further research is needed for these compounds to establish their role in mitochondrial disease therapy and inform evidence-based guidelines for mitochondrial disorder diagnosis management.

Consensus Recommendations for Vitamin and Xenobiotic Use

CoQ10 should be offered to most patients diagnosed with mitochondrial disease, not exclusively for primary CoQ10 deficiency, as a supportive therapy to potentially enhance mitochondrial function in mitochondrial disorder diagnosis management.
a. Reduced CoQ10 (ubiquinol) is the most bioavailable form, requiring appropriate dose adjustments when used, optimizing absorption and efficacy in mitochondrial disorder diagnosis management.
b. Plasma and/or leukocyte CoQ10 levels are helpful in monitoring absorption and treatment adherence. Plasma levels are more variable and less reflective of tissue levels, guiding dosage adjustments and assessing compliance in mitochondrial disorder diagnosis management.
ALA and riboflavin should be offered to mitochondrial disease patients as potential adjunctive therapies, based on some limited evidence and mechanistic rationale, within a comprehensive management plan in mitochondrial disorder diagnosis.
Folinic acid should be considered for mitochondrial disease patients with central nervous system manifestations and routinely administered to those with documented CSF deficiency or conditions associated with deficiency, addressing potential cerebral folate deficiency in mitochondrial disorder diagnosis management.
L-Carnitine should be administered to mitochondrial disease patients with documented deficiency, with levels monitored during therapy to ensure appropriate supplementation and avoid potential imbalances in carnitine homeostasis in mitochondrial disorder diagnosis management.
When initiating supplement therapy, introduce one supplement at a time when possible, considering the patient’s clinical status to monitor individual responses and potential adverse effects, allowing for personalized and safer supplement regimens in mitochondrial disorder diagnosis management.
There is no evidence to support adjusting a person’s diet based solely on ETC results. Dietary recommendations should be guided by overall nutritional needs and specific metabolic considerations in mitochondrial disorder diagnosis management.
Goal levels for most vitamin therapies used in mitochondrial disease are not yet established. Prudently, replace deficiency states and monitor clinical responses to guide individualized vitamin supplementation strategies in mitochondrial disorder diagnosis management.

SUMMARY

Evidence-based clinical protocols are the ideal for diagnostic and medical management recommendations. However, for mitochondrial disease, sufficient data for such recommendations are lacking. The Delphi process enabled our group to reach consensus-based recommendations to guide clinicians evaluating and treating mitochondrial disease patients, offering practical guidance in the context of limited evidence for mitochondrial disorder diagnosis and management.

This document is not intended to replace clinical judgment and cannot apply to every individual case or condition. It does not address potential inconsistencies in diagnostic laboratory interpretations of metabolite changes. Clinicians should consult the literature and mitochondrial disease researchers for updated information. These recommendations should be superseded by clinical trials or high-quality evidence as they emerge. In the interim, we hope these consensus recommendations help standardize the evaluation, diagnosis, and care of patients with suspected or confirmed mitochondrial disease, improving consistency and quality of care in mitochondrial disorder diagnosis and management.

While mitochondrial diseases are collectively common, individual etiologies or subtypes are relatively rare, making large-scale clinical trials challenging. The North American Mitochondrial Disease Consortium is fostering improved understanding of natural history and individual differences through large cohort studies. Continued research is essential to better understand these patients and develop ideal evidence-based clinical care protocols, advancing the field of mitochondrial disorder diagnosis and treatment. The Mitochondrial Medicine Society remains committed to evaluating patient and physician needs and advancing education, research, and collaboration in the field, driving progress in mitochondrial disorder diagnosis and mitochondrial medicine.

Footnotes

DISCLOSURE

G.M.E. has received unrestricted research gift funds from Edison Pharmaceuticals. M.J.F. is a consultant for Mitokyne, research collaborator for Cardero, and a research grant awardee of RAPTOR Pharmaceuticals, and is on the Scientific and Medical Advisory Board for the United Mitochondrial Disease Foundation. A.G. is a consultant for Stealth Peptides. A.L.G. is a consultant for GeneDx. S.P. conducts research for Edison Pharmaceuticals and the North American Mitochondrial Disease Consortium (NAMDC), and is on the Scientific and Medical Advisory Board for the United Mitochondrial Disease Foundation. R.S. conducts research studies for Edison Pharmaceuticals and is part of the NAMDC. F.S. works for a medical college department that owns a Mitochondrial Diagnostic Laboratory and conducts research studies for Edison and Raptor Pharmaceuticals. M.T. is president and CEO of Exerkine, which develops mitochondrial therapies, and receives speaker honoraria from Prevention Genetics and Genzyme.