The study, published in Frontiers in Veterinary Science, was motivated by a specific clinical problem: myosin heavy chain myopathy, or MYHM, a recently described genetic disorder of Quarter Horses and related breeds such as Appaloosas and American Paint Horses. The condition stems from a single non-synonymous mutation, E321G, in the MYH1 gene, which encodes the myosin-type 2X heavy chain. Horses carrying the variant allele are susceptible to non-exertional rhabdomyolysis, immune-mediated myositis, and systemic calcinosis, syndromes that can produce muscle stiffness, weakness, myoglobinuria, and acute, severe muscle wasting. The variant allele is present in roughly 6.8 percent of American Quarter Horses, with the highest prevalence among reining, working cow, and halter performance lines.

Because the exact pathogenesis of immune-mediated myositis in these horses remains unclear, researchers have looked for environmental triggers. Early evidence suggested that clinical signs may be set off by exposure to an infectious agent or by vaccination itself. Some experts now recommend avoiding vaccination of horses carrying the mutation, or restricting them to only the most essential monovalent vaccines. What remains unknown is whether the muscle trauma caused by an intramuscular injection or the immune response provoked by the vaccine is the inciting event. If muscle damage from the needle is the culprit, delivering vaccines subcutaneously might reduce the risk of triggering disease in susceptible animals—a hypothesis the new study set out to test indirectly by comparing the two routes in healthy horses.

The research team, led by Zuzanna M. Pietras of Kansas State University with colleagues from Iowa State University, Washington State University, and the Virginia Maryland College of Veterinary Medicine, enrolled 17 systemically healthy adult mares from a university research and teaching herd. Sixteen were American Quarter Horses and one was a Thoroughbred. Their mean age was 14.1 years and their mean body weight 542 kilograms. Crucially, all horses tested negative for the MYH1 mutation, meaning the study evaluated the routes in animals without the genetic susceptibility. All of the mares had been vaccinated previously with a combination vaccine including West Nile virus and tetanus toxoid, with annual spring boosters for several years—a detail that would later complicate interpretation of the immune data.

The trial used a randomized, single-blind, two-arm, two-period design. On day 0, the mares received an inactivated West Nile virus vaccine; on day 28, they received a tetanus toxoid vaccine that is approved for subcutaneous use. Nine horses in group 1 were vaccinated subcutaneously in the neck with the West Nile vaccine and intramuscularly with tetanus toxoid, while eight horses in group 2 received the reverse assignment. Both injection routes targeted the same anatomical region: intramuscular injections went into the cervical serratus ventralis muscle, and subcutaneous injections into the tissue overlying that same muscle, avoiding the cervical vertebrae, nuchal ligament, and scapula. Injection sites were clipped, cleaned with isopropyl alcohol, and marked so that a blinded observer could monitor them consistently.

Antibody responses were tracked with a battery of assays performed at Cornell University’s Animal Health Diagnostic Center. West Nile virus IgM and IgG levels were measured with isotype-specific capture enzyme-linked immunosorbent assays, reported as a positive-to-negative ratio, and IgG was additionally assessed with a serum neutralization assay. Blood was collected on days 0, 28, and 56 for West Nile virus titers and on days 28 and 56 for tetanus titers. Local reactions were assessed daily for 72 hours after each injection, with swelling measured using calipers and pain scored on a four-point scale from no pain to moderate-to-severe pain in response to manual palpation. Serum amyloid A, a sensitive acute-phase protein that rises rapidly during systemic inflammation, was measured before and 24 hours after each vaccination.

The immunological results were equivocal in an instructive way. Serum neutralization titers against West Nile virus rose significantly by 28 days after vaccination regardless of route, with a p-value of 0.007, and no differences emerged between the subcutaneous and intramuscular groups. IgM levels were low at the start and did not change, indicating that no natural West Nile virus exposure had occurred before or during the study. However, nearly all of the horses began the trial with very high baseline anti-tetanus immunoglobulin titers and West Nile virus neutralization titers, many at or near the maximum values reportable by the diagnostic laboratory—almost certainly a legacy of their years of annual boosters. Tetanus titers did not increase after revaccination, and the authors caution that the statistically significant rise in West Nile virus titers may not be biologically meaningful given the high starting points. Definitive conclusions about whether subcutaneous vaccination produces immune responses equivalent to intramuscular vaccination therefore cannot be drawn from these data.

The adverse-effect findings, by contrast, were unambiguous. Injection site swelling was significantly more frequent and larger after subcutaneous administration of both vaccines. After the West Nile vaccine, eight of nine subcutaneously vaccinated horses developed visible swelling, with a median area of 8,467 square millimeters, compared with three of eight intramuscularly vaccinated horses, whose median swelling was essentially zero. After tetanus toxoid, every subcutaneously vaccinated horse developed swelling, versus four of nine after intramuscular injection. Pain on palpation followed the same pattern: very mild to moderate pain was detected in six of nine subcutaneously vaccinated horses after the West Nile vaccine and in five of eight after tetanus toxoid, while no pain was detected after intramuscular West Nile vaccination and only one horse reported pain after intramuscular tetanus vaccination. Seven of the nine horses that received the subcutaneous West Nile vaccine required systemic phenylbutazone and topical diclofenac cream because of significant swelling and pain; no horse required treatment after intramuscular administration of that vaccine, and none required treatment after tetanus toxoid by either route.

Systemic reactions were mild and largely route-independent. Two horses in each group developed a self-limiting fever of 38.7 to 38.8 degrees Celsius on the day after West Nile vaccination, resolving by day 2, and no horses developed fever after tetanus toxoid. Serum amyloid A concentrations rose after West Nile vaccination regardless of route, with no detectable difference between the groups, but remained below the detection threshold of 20 micrograms per milliliter after tetanus toxoid administration in all horses, suggesting that the tetanus vaccine does not provoke a significant systemic inflammatory response. No signs of abscess formation were observed at any injection site during the monitoring period.

The authors emphasize that the study’s implications for horses with the MYH1 mutation remain uncertain. The visible inflammation after subcutaneous injection was primarily confined to subcutaneous tissues, but inflammatory changes in the underlying muscle were not critically evaluated, and serum creatine kinase activities were not measured—so it is possible that the markedly greater local inflammatory response could actually be detrimental for genetically susceptible horses rather than protective. Other limitations include the small sample size, the restriction to mares, the use of previously vaccinated animals, and the inability to assess cellular immune responses. The researchers suggest that future work should use naïve, unvaccinated horses to compare humoral responses accurately, and should examine the influenza, equine herpesvirus 4, and strangles vaccines that have been more commonly implicated in triggering immune-mediated myositis. They also note that antibody measurements might eventually serve as an alternative to routine booster vaccination in horses prone to severe vaccine reactions, provided protective thresholds for West Nile virus and tetanus in horses can be definitively established. For now, the practical message is that subcutaneous vaccination in horses, at least with these two products, comes at the cost of substantial local reactions that many owners are likely to find unacceptable.

Subject of Research: Comparison of subcutaneous and intramuscular vaccination routes for antibody responses and adverse effects in horses, in the context of MYH1-related myopathy

Article Title: Humoral immune responses and adverse effects of inactivated West Nile virus and tetanus toxoid vaccines administered via the subcutaneous or intramuscular route in horses

Article References: Pietras, Z. M., Sanz, M. G., Pinnell, E., Rangel, A., & Sellon, D. C. (2026). Humoral immune responses and adverse effects of inactivated West Nile virus and tetanus toxoid vaccines administered via the subcutaneous or intramuscular route in horses. Frontiers in Veterinary Science, 13, Article 1931255. https://doi.org/10.3389/fvets.2026.1931255

Image Credits: AI Generated

DOI: 10.3389/fvets.2026.1931255

Keywords: horses, vaccination, subcutaneous injection, intramuscular injection, West Nile virus, tetanus toxoid, myosin heavy chain myopathy, immune-mediated myositis, MYH1 mutation, antibody titers, injection site reactions, serum amyloid A

The review, led by Daniela Debone and colleagues and prospectively registered with PROSPERO, searched PubMed, Embase, and Cochrane CENTRAL from database inception to July 2025, with an update in July 2026, imposing no language or date restrictions. The team screened 4,543 records, removed 1,499 duplicates, and ultimately included 13 studies: seven randomized controlled trials, three observational cohorts, two conference abstracts, and one research letter. The studies came from Japan, Israel, Turkey, the United States, Switzerland, Brazil, Spain, India, Austria, and New Zealand, with sample sizes ranging from just 16 participants to 4,281. Because the studies varied so widely in design, dosing, populations, and outcome reporting, the authors performed a descriptive synthesis rather than a statistical meta-analysis.

The physiological stakes are considerable. Vitamin B12, or cobalamin, is indispensable for DNA synthesis, methylation reactions, and the formation of myelin, the insulating sheath around nerve fibers. It also serves as a cofactor in mitochondrial energy metabolism and in the maturation of blood cells. Because the human body cannot synthesize it, cobalamin must come from animal-derived foods or fortified products. When deficiency sets in, the consequences can include megaloblastic anemia, peripheral neuropathy, cognitive impairment, and elevated cardiovascular risk. Populations at particular risk include children, vegetarians and vegans, people with pernicious anemia, patients on long-term metformin, and those who have lost part or all of their stomach.

Intramuscular injection has traditionally been favored because it bypasses the gastrointestinal tract entirely, guaranteeing systemic delivery even when gut absorption is impaired. Yet injections carry real costs: pain, the need for a healthcare professional, and repeated clinic visits. Oral supplementation has long faced skepticism, particularly in pernicious anemia, where the absence of intrinsic factor cripples active absorption. The counterargument rests on passive diffusion: roughly 1% of orally ingested cobalamin can be absorbed independent of intrinsic factor, which is why high-dose oral regimens of 1 to 2 milligrams per day can work even in malabsorptive conditions. Sublingual formulations, meanwhile, are absorbed through the highly vascularized oral mucosa, entering systemic circulation directly and avoiding hepatic first-pass metabolism.

The biochemical evidence assembled in the review is strikingly consistent in one direction: all three routes work. In the landmark trial by Kuzminski and colleagues, patients receiving 2 milligrams of oral cyanocobalamin daily actually achieved higher serum levels after four months (1,005 pg/mL) than those receiving monthly 1-milligram injections (325 pg/mL), a statistically significant difference. In contrast, the Swiss trial by Metaxas and colleagues found the opposite pattern over 28 days, with 100% of intramuscular patients normalizing their levels compared with 84.2% of oral patients, and mean concentrations of 2,796 pmol/L versus 354 pmol/L. The Spanish OB12 non-inferiority trial in older adults found essentially equivalent normalization rates at eight weeks, with a slight per-protocol edge for injections at 52 weeks that did not hold up under intention-to-treat analysis.

The sublingual route, though underrepresented with only five studies, produced some of the review’s most intriguing findings. In a large Israeli retrospective cohort of 4,281 patients, sublingual tablets produced significantly larger mean increases in serum B12 than injections (252 versus 218 ng/L) and higher odds of achieving increased levels. In a New Zealand trial of metformin-treated patients with type 2 diabetes, sublingual methylcobalamin outperformed a single hydroxocobalamin injection at three months, though the advantage vanished by six months. Head-to-head comparisons of sublingual versus oral supplementation, in Israel and India, found no meaningful differences: in the Indian trial, normalization rates were 68.4% for sublingual and 66.7% for oral after three months of 1,500 micrograms daily, with compliance above 90% in both arms.

Yet the review’s authors are careful to sound a note of caution that cuts through the numbers: higher serum B12 does not automatically mean better clinical outcomes. The magnitude of a biochemical response does not necessarily translate into greater symptom relief, neurological recovery, or quality-of-life improvement. Indeed, no study in the review demonstrated superiority of any route for long-term clinical outcomes, largely because clinical endpoints were rarely measured at all. The certainty of the evidence was further constrained by open-label designs, unadjusted confounding in the observational studies, which were judged to be at serious risk of bias, and inconsistent outcome reporting, with some trials lacking formal statistical comparisons altogether.

What the evidence does support is a shift in how route selection might be framed. When biochemical responses are broadly similar, the decision can reasonably be guided by patient preference, tolerability, and access rather than presumed efficacy differences. Patient preference data from the review are telling: in the Spanish trial, 83.4% of participants preferred oral administration, including more than two-thirds of those actually receiving injections. In the Swiss trial, 45.9% initially favored oral treatment versus 21.6% for injections, though preferences shifted after experiencing treatment. Non-invasive options also reduce reliance on healthcare infrastructure, a meaningful advantage for adherence and for resource-limited settings where repeated clinic visits impose real burdens on patients and systems alike.

The review also refines, rather than overturns, a 2024 network meta-analysis that had ranked intramuscular administration highest for biochemical efficacy while finding no statistically or clinically significant differences between routes. The new synthesis improves on that work in several ways: it was prospectively registered, searched four years more recently, applied no language restriction, focused exclusively on adults rather than pooling pediatric and adult populations, and incorporated conference abstracts and a research letter that the earlier review excluded. The authors note the review was funded by a pharmaceutical manufacturer and was undertaken to establish a shared evidence base for a subsequent Delphi consensus on B12 replacement strategies.

The gaps that remain are as instructive as the findings. Safety data were sparse across all routes, and none of the included studies reported adverse events for sublingual supplementation. Follow-up periods were mostly short to medium term, ranging from four weeks to 24 months. Future research, the authors argue, should include adequately powered, long-term randomized trials using standardized, patient-centered outcomes such as symptom improvement, adherence, and preference, particularly for the sublingual route. Until such trials arrive, the practical message for patients and clinicians is quietly radical: the shot in the arm that has defined B12 replacement for generations may be no better than a tablet dissolved under the tongue, and the choice may ultimately belong to the patient rather than the syringe.

Subject of Research: Comparative efficacy of oral, sublingual, and intramuscular vitamin B12 supplementation routes in adults

Article Title: Comparative efficacy of vitamin B12 supplementation routes in adults with replacement indication: a systematic review

Article References: Debone, D., Azeredo da Silva, A. F., Cordenonssi, J. T., & Cohen, R. V. (2026). Comparative efficacy of vitamin B12 supplementation routes in adults with replacement indication: a systematic review. eClinicalMedicine, 100, Article 104214. https://doi.org/10.1016/j.eclinm.2026.104214

Image Credits: AI Generated

DOI: 10.1016/j.eclinm.2026.104214

Keywords: vitamin B12, cobalamin, sublingual supplementation, intramuscular injection, oral supplementation, systematic review, vitamin B12 deficiency, pernicious anemia, metformin, patient preference, eClinicalMedicine, biochemical outcomes

The scale of the knowledge gap is difficult to overstate. Vitamin B12 deficiency affects roughly 6 to 30 percent of older adults, 10 to 30 percent of people with atrophic gastritis, and 5 to 40 percent of patients after bariatric surgery. Its consequences can be devastating and, if treatment is delayed, irreversible: megaloblastic anaemia, peripheral neuropathy, subacute combined degeneration of the spinal cord, cognitive impairment and dementia-like syndromes. Yet the entire evidence base supporting how we replace this vital vitamin rests almost exclusively on a single laboratory number—the serum B12 concentration. No randomised trial of any route has ever measured whether patients actually feel better, whether damaged nerves recover, or whether their daily functioning improves.

To build the consensus, the panel first commissioned a companion systematic review, registered prospectively, that searched PubMed, Embase and Cochrane CENTRAL from inception to July 2025, with an update in July 2026. That review identified just thirteen comparative studies of oral, sublingual and intramuscular replacement in adults, and the evidence they provided was limited, heterogeneous and at variable risk of bias. Most studies compared oral with intramuscular administration and reported broadly comparable short-term biochemical responses. The most quantitative synthesis available, a 2024 network meta-analysis of thirteen studies encompassing 4,275 participants, ranked the intramuscular route highest for biochemical efficacy using SUCRA values—a statistical metric expressing the probability that a treatment ranks among the most effective options—but found no significant difference between the two non-injectable routes. Crucially, the panel emphasised, such biochemical rankings say nothing about clinical benefit.

The consensus process itself followed the RAND/UCLA Appropriateness Method, adapted into a modified two-stage Delphi design. In the first stage, conducted electronically from August 2025, all twenty panellists voted on draft statements using a binary yes-or-no format with pre-defined agreement thresholds: statements required at least 66 percent agreement to pass, with grades rising from D through A to A-plus for unanimous approval. Statements that fell short were revised by an independent facilitator trained in consensus methodology and redistributed. The second stage brought sixteen panellists to São Paulo on October 17, 2025, where remaining statements were debated in person and re-voted anonymously using smartphone-based voting. Two patient representatives with lived experience of B12 deficiency attended as non-voting observers, helping refine the wording of statements and sharpen the focus on outcomes that matter to patients, though they did not cast votes—a limitation the authors openly acknowledge.

The results were notable for their near-total agreement. Of thirty-one graded items, twenty-two—71 percent—reached grade A-plus, meaning one hundred percent of voting panellists agreed, and the remaining nine reached grade A, representing 90 to 99.9 percent agreement. Not a single statement fell below grade A. Twenty-four statements were finalised during the electronic rounds and seven at the in-person meeting. This extraordinary consensus, the authors caution, should not be mistaken for strength of the underlying evidence; rather, it reflects shared recognition among specialists of persistent and clinically relevant gaps in knowledge that have been glossed over for decades.

Among the key statements, the panel unanimously agreed that all three routes can effectively normalise serum B12 in most adults, but that higher oral doses may be needed in patients with atrophic gastritis or malabsorption. In patients without malabsorption, oral therapy appears comparable to injections. For pernicious anaemia—the autoimmune condition in which the stomach loses its ability to produce intrinsic factor, the protein needed to absorb B12—intramuscular administration remains the preferred route, although large oral doses can be efficacious. Perhaps most importantly, the panel agreed with 95 percent agreement that there is insufficient evidence to determine the long-term clinical superiority of any route, and unanimously that route selection should be individualised based on aetiology, patient preference, feasibility and access.

The consensus also exposed deep inconsistencies in how B12 therapy is actually delivered and monitored. Intramuscular regimens vary dramatically in frequency, dose and duration across studies, and no consensus exists on the optimal monitoring interval for serum B12, methylmalonic acid or homocysteine—the functional biomarkers that reflect cellular B12 status more faithfully than serum levels alone. Definitions of treatment response and normalisation vary so widely between studies that comparing them becomes nearly impossible. Maintenance regimens are highly variable and poorly supported by trial data. On safety, the panel found that adverse events are rarely reported and likely underrecognised, that sublingual formulations appear well tolerated but have never undergone systematic safety assessment, and that the discomfort and access requirements of injections could influence adherence, though comparative adherence data remain limited.

Certain patient groups emerged as especially poorly served by the existing evidence. After bariatric surgery, where deficiency rates reach 40 percent, data on sublingual supplementation are sparse, limited to a single small observational study, and while oral B12 may be effective post-operatively, higher doses are probably required. Older adults with cognitive impairment or frailty—a population in which deficiency is both common and potentially contributory to decline—have essentially no outcome-based evidence guiding supplementation. Patients with pernicious anaemia, who depend on lifelong replacement, are strikingly underrepresented in modern trials. The panel also noted that serum B12 alone is an uncertain guide to clinical benefit: levels rise with pharmacological dosing regardless of whether the patient improves, and the magnitude of the rise differs between cyanocobalamin and hydroxocobalamin formulations without implying any clinical advantage.

The disconnect between biochemical success and clinical evidence reflects a broader pattern in medicine, but the stakes here are unusually high because delayed or inadequate treatment can leave patients with permanent neurological damage. Current international guidelines—including those from the American Academy of Family Physicians, the British Society for Haematology, NICE and British Columbia—diverge substantially on route, dosing and monitoring, a variability the authors attribute to weaknesses in the underlying evidence rather than substantive disagreement among experts. Prescribing practices likewise vary widely across clinicians and specialities, often extrapolating from laboratory normalisation to clinical recommendation without any outcome-based trial data to justify the leap.

The consensus panel, funded by the pharmaceutical manufacturer Myralis with safeguards including anonymous voting, a pre-registered evidence base and no funder access to voting data, has set out a prioritised research agenda. Adequately powered, long-term randomised trials comparing all three routes with standardised, patient-centred endpoints—symptom resolution, neurological recovery, functional status and quality of life—should focus first on the highest-need groups: patients after bariatric surgery, older adults with cognitive impairment or frailty, and people with pernicious anaemia. The statements will be submitted to major Brazilian professional societies for endorsement, and the authors call for international validation in other health systems. Until such trials are completed, the practical message for clinicians and patients is clear: any of the three routes can correct the blood test, but choosing between them should rest on the underlying cause of deficiency, patient preference, practical feasibility and access—supported by functional biomarkers and careful clinical assessment rather than blind faith in a serum number.

Subject of Research: Comparative effectiveness of oral, sublingual and intramuscular vitamin B12 replacement routes

Article Title: Vitamin B12 replacement strategies: a Brazilian delphi consensus

Article References: Cohen, R. V., Levinson, R., Baiocchi, O., Salles, J. E. N., Cavalcante França, M., Jr, Navarro, T., Trujilho, F., Soares, J. M., Jr, Nakagawa, D., Valente, M., Barbuti, R., Spera, R. R., Moura, F., Valerio, C., Schiavon, C. A., dos Santos, F. C., Batista Dantas, A. C., Abdo, A., Steiner, M. L., … Bachour, P. (2026). Vitamin B12 replacement strategies: a Brazilian delphi consensus. eClinicalMedicine, 100, Article 104205. https://doi.org/10.1016/j.eclinm.2026.104205

Image Credits: AI Generated

DOI: 10.1016/j.eclinm.2026.104205

Keywords: vitamin B12, cobalamin deficiency, Delphi consensus, intramuscular injection, oral supplementation, sublingual, pernicious anaemia, bariatric surgery, methylmalonic acid, neurological recovery, clinical guidelines, eClinicalMedicine