American Society of Hirudotherapy

Evaluation of Methylene Blue as An Effective Antiseptic for Medicinal Leeches

Doğan S, Farzali S, Karimova B, Sağlam N (2024) · Türkiye Parazitoloji Dergisi · n=570

RCT evidence detailTrial reference
GRADE Very LowInsufficient evidence
Sample size of this trial compared with other venous-congestion-flap trialsMarquard JM 20251215Bishop JL 2023843Doğan S 2024570Troeltzsch M 2016330Kucur C 2015260Wang ZD 2022210Lehnhardt M 202196Kruer RM 201459Mozafari N 201056Merlino G 202048
This trial (highlighted) by sample size alongside other indexed venous-congestion-flap trials. Larger trials generally carry more statistical weight.

Study Profile

Design
in-vitro dose-finding study of methylene blue as an antiseptic for medicinal leeches; 570 Hirudo medicinalis specimens (Turkish leech farm, Fırat University) tested across 18 concentrations (1-512 ppm)
Sample size (n)
570
Intervention
Methylene blue antiseptic baths at concentrations ranging 1-512 ppm to determine safe and effective antiseptic concentrations for medicinal-leech husbandry
Comparator
Saline-only control bath; not a comparison to other antiseptics in this study
Primary endpoint
Lethal concentration 50% (LC50), effective concentration (EC), and lethal time 50% (LT50) for medicinal leeches
Primary result
LC50 = 60.4 ppm (95% CI 53.7-66.6); EC = 2.0 ppm (95% CI 1.8-2.2); LT50 = 213h at 32 ppm vs 18h at 512 ppm; authors conclude MB concentrations of 2-19 ppm are safe and effective for hirudotherapy clinic and leech-farm antisepsis
Follow-up duration
Up to 1486 hours (LT50 upper CI at 32 ppm)
PMID
38958415

Key Findings

  • First systematic dose-finding study of methylene blue antisepsis for medicinal leeches
  • Wide safety margin: EC50 (2 ppm) vs LC50 (60 ppm) — 30-fold therapeutic index
  • Practical operating range 2-19 ppm fits with existing aquaculture infrastructure
  • Provides leech-farm and hirudotherapy-clinic husbandry guidance
  • Indirectly relevant to patient safety via reduced bacterial bioburden on therapy leeches

Limitations

  • In-vitro / aquaculture model — does not measure clinical infection outcomes
  • Single species (Hirudo medicinalis) — generalizability to Hirudinaria or Whitmania unclear
  • Did not test methylene blue against specific pathogens (Aeromonas, Vibrio)
  • Long LT50 confidence intervals at low concentrations limit precision
  • Methylene blue residue on leech epidermis during patient application not characterized

Clinical Implications

Doğan 2024 provides actionable husbandry guidance for medicinal-leech farms and hirudotherapy clinics seeking to reduce gut-bacterial bioburden on therapy-grade leeches. For US K040187 hirudotherapy programs, the data inform potential pre-application leech-cleansing protocols but do not change patient-level antibiotic prophylaxis recommendations (still anchored on Aeromonas-active agents per Wilmer 2013, Beka 2018, Kruer 2014). The study complements Karasartova 2025 (leech microbiota and infection risk) by offering a candidate husbandry intervention.

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Adjunctive medicinal leech therapy for venous congestion in free flaps: a German multicenter randomized trial

Lehnhardt M, Daigeler A, Behr B, Schmidt SV, Wallner C (2021)

Medicinal leeches and the microsurgeon: a four-year study, clinical series and risk benefit review

Whitaker IS, Josty IC, Hawkins S, Azzopardi E, Naderi N, Graf J, Damaris L, Lineaweaver WC, Kon M (2011)

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This website provides educational information and does not constitute medical advice, diagnosis, or treatment recommendations. Medicinal leech therapy carries clinically meaningful risks and should be performed only by qualified clinicians under institutionally approved protocols. FDA 510(k) clearance for medicinal leeches is limited to specific indications; investigational and off-label discussions are labeled accordingly. For patient-specific guidance, consult a qualified healthcare provider.