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ORIGINAL ARTICLE
Year : 2021  |  Volume : 2  |  Issue : 1  |  Page : 13-18

An analytical study to evaluate the safety of reusing of the vomited leech in leech therapy: A pilot study


1 Department of Shalya Tantra, KAHER's Shri B M K Ayurveda Mahavidyalaya, PG Studies and Research Centre, Belagavi, Karnataka, India
2 Department of Shalya Tantra, KAHER's Shri B M K Ayurveda PG Studies and Research Centre, Belagavi, Karnataka, India
3 Department of Dravyaguna, KAHER's Shri B M K Ayurveda PG Studies and Research Centre, Belagavi, Karnataka, India
4 Central Research Facility, KAHER's Shri B M K Ayurveda Mahavidyalaya, Belagavi, Karnataka, India

Date of Submission18-Feb-2021
Date of Decision28-Feb-2021
Date of Acceptance02-Mar-2021
Date of Web Publication17-Apr-2021

Correspondence Address:
Pradeep S Shindhe
Department of Shalya Tantra, KAHER's Shri B M K Ayurveda PG Studies and Research Centre, Belagavi - 590 003, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijaim.ijaim_5_21

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  Abstract 


Background: Leech therapy is a point of attraction in the medical as well as in the scientific world due to its various outstanding properties, while studying the Ayurveda and scientific literature one can find difference, i.e., discarding after single use as per modern literature, but in Ayurveda, it can be reused after 7 days of leech therapy. Studies of such kind were not carried or published in any scientific journals, so ethical approval and developing scientific methods for validating the study were necessary, so a pilot study began with ethical clearance.
Objective: The objective of the study was to evaluate the bacterial and fungal count of leech saliva and blood emesis before and after leech therapy using disc diffusion method.
Materials and Methods: Leeches were collected from the normal habitat from Mangalore region possessing the same weight and morphological characteristics as that of the Nirvisha Jalouka as mentioned in classics (Shanku-mukhi). The selected leeches were stored in well water having nonchlorinated with minimum bacterial load. The well water was selected from three geographical regions of Belagavi city, and among them, one sample was selected having minimal bacterial load. Ten patients with infectious skin diseases who were indicated for Jalaukavacharana (Vidradhi, Dushta-vrana) were selected. The experimental leech saliva was tested for total bacterial count (TBC) and total fungal count (TFC) before and on 8th day of application, along with venous blood of the patient and blood vomitus of leech after application was tested for TBC and TFC. Statistical analysis was done based on TFC and TBC before and after application.
Results: TBC and TFC of leech saliva at 8th day and before application were similar which indicates the leech gut has destroyed the bacteria or inhibited the growth of bacteria.
Conclusion: TBC and TFC help to produce evidence that after application of leech and proper vomiting will not allow the growth of bacteria. Hence, safely, it can be reused after 7 days.

Keywords: Ayurveda, haridra, infection, jalouka, leech therapy


How to cite this article:
Mane VS, Shindhe PS, Vedantam G, Pednekar HD, Killedar R. An analytical study to evaluate the safety of reusing of the vomited leech in leech therapy: A pilot study. Indian J Ayurveda lntegr Med 2021;2:13-8

How to cite this URL:
Mane VS, Shindhe PS, Vedantam G, Pednekar HD, Killedar R. An analytical study to evaluate the safety of reusing of the vomited leech in leech therapy: A pilot study. Indian J Ayurveda lntegr Med [serial online] 2021 [cited 2021 May 6];2:13-8. Available from: http://www.ijaim.com/text.asp?2021/2/1/13/313997




  Introduction Top


Leeches have been used for therapeutic purposes since the beginning of civilization. Ancient Egyptian, Indian, Greek, and Arab physicians used leeches for a wide range of diseases starting from the conventional use for bleeding to systemic ailments such as skin diseases, nervous system abnormalities, urinary and reproductive system problems, inflammation, and dental problem.[1] References of this leech therapy found since the ancient days from length and breadth of all over the world; however, the oldest reference of this therapy is seen in ”Brahatrayi.” Acharya Sushrutha elaborately described the collection, classification as Savisha (Poisonous) and Nirvisha (nonpoisonous), preservation, application, therapeutic utility, and complications of leech therapy.[2]

The modern science suggests discarding the leech after its application, i.e., leeches are killed in 70% ethyl alcohol and are disposed in bags for biological waste. To avoid cross infection,[3] but on the contrary in Ayurveda literature, it is suggested to reuse the leech on 8th day onward after making the leech to vomit properly,[4] and this raises the ethical/research question of chances of cross infection from person to person through leech. It is the era of evidence-based practice, and for global acceptance of the therapy, it is been potentiated through scientific parameters. There is no availability or published articles or work carried out in this angle, so we made an attempt to do bacterial and fungal study before and after the leech therapy and obtained ethical clearance. Protocol was developed scientifically for the study in order to justify the aim and objective by comparing pre- and postbacterial and fungal counts. The elements which were assessed to justify the study were selection of leeches, selection of patients with bacteremia, collection of water, bacterial load and fungal count of venous blood of patient, blood emesis of leech after therapy, and saliva of leech before the therapy and on 8th day.

Aim

The aim of the study was to assess the rationality of reusing the Jalouka (Leech) after 7 days of its application.

Objectives

  1. To evaluate bacterial and fungal load of leech saliva before leech therapy
  2. To evaluate bacterial and fungal load of venous blood of the patient before application
  3. To evaluate the bacterial and fungal load of blood emesis immediately after leech therapy
  4. To evaluate bacterial and fungal load of leech saliva after lag period (8th day of application)
  5. Comparison of the above four parameters to conclude the rationality about reusing the vomited leeches.



  Materials and Methods Top


The study was conducted at KLE Ayurveda Hospital and Research Centre, Shahpur, Belagavi, patients were recruited from outpatient department (OPD) and inpatient department of Department of Shalyatantra. The study was approved by the ethical committee with Ethical clearance – BMK/14/PG/ST/10.

Study design

  • Sample size: 10 subjects fulfilling inclusion criteria were registered and allotted to single group with the help of research pro forma
  • Groups: Single group
  • Stored water sample: Well water was collected from different sources, and total bacterial and fungal count was done and the water with minimal bacterial count was selected for the study
  • Leech coding: Ten selected Nirvish Jalouka as mentioned in classics (Shanku-mukhi)[5] were coded from alphabetical letter “A” to “J”
  • Saliva samples: Saliva samples of leech were collected twice, namely, before leech therapy and on 8th day after leech therapy.


Blood samples

  1. Venous blood of the patient before therapy to study total bacterial and fungal load
  2. Blood vomitus of leech immediately after leech therapy for total bacterial and fungal load.


Inclusion criteria

  • Patients with infectious skin diseases who are indicated for Jaloukavacharana like Pachaman Vidradhi (Abscess), Dushtavrana (chronic non healing ulcer)
  • Patients of either of sex and all age group.


Exclusion criteria

  • Patient on medications such as anticoagulant drugs
  • Patient on antibiotic and antitubercular therapy
  • HIV and hepatitis B virus surface antigen positive
  • Bleeding disorders.


Diagnostic criteria

The patients were assessed by venous blood analysis before the Jaloukavacharana. The diagnosis was mainly based on the clinical presentation of Pachaman Vidradhi[6] and dushtavrana[7] (mentioned below) [Table 1].
Table 1: Signs and symptoms of Pachamana Vidradhi and Dustavrana

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Method of leech application

Preoperative procedures

  • Written informed consent of the patient was taken
  • Affected part was cleaned with NS
  • The entire material (namely, Jalouka, turmeric powder, shatadhouta ghrita, and dressing trolley) was kept ready
  • Selected leech was kept in the turmeric water for 5 min for activation and then transferred to the fresh water.[8]


Operative procedures

  • Patient was made to sit/lie down comfortably on examination table
  • Jalouka was applied at the site of indication, if it unable to suck the blood, prick was taken with sterile needle made at vrana/abscess to allow the blood to ooze out
  • Leech was covered with wet cotton pad except near its mouth when it was started to suck.


Postoperative procedure

  1. Leech


    • Leeches were vomited properly by applying haridra (turmeric powder) to the mouth of leech and second bout of blood which was not contaminated with haridra was 1 ml blood was collected and sent for the TBL and total fungal count (TFC).


  2. Patient


    • Tight dressing was given at the site of leech bite and observed for 2 h for any complications.


Methodology of collection and assessment of leech saliva

  1. A glass slide was wiped with alcohol and well sterilized on blue flame and then leech kept on the glass slide at its anterior sucker and the leech saliva was collected
  2. This saliva sample was wiped with sterile gauze and the gauze then kept in the Nutrient broth to grow at 37°C for 24 h in incubator
  3. Then after 24 h 1 ml of nutrient broth was serially diluted in seven samples of prepared N.B. in the test tube and 1 ml from last test tube was added in Soyabean Casein Digest Agar and Sabouraud Dextrose Agar media by pour plate method (two plates for each with negative control) and incubated at 37°C for 5 days, and plates were observed for bacterial and fungal count at 5th day manually.



  Results and Observations Top


Statistical analysis

Analysis was done on the basis of total bacterial count (TBC), and TFC of the subgroups and result was drawn on the comparison of

  1. Bacterial and fungal load of leech saliva before leech therapy [Figure 1]
  2. Bacterial and fungal load of venous blood of the patient before application [Figure 2]
  3. Bacterial and fungal load of blood emesis immediately after leech therapy [Figure 3]
  4. Bacterial and fungal load of leech saliva after lag period (8th day of application) [Figure 4].
Figure 1: Total microbial load of vomited blood of leech before application

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Figure 2: Total microbial load of vomited blood of leech after application

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Figure 3: Total microbial load of venous blood

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Figure 4: Total microbial load of vomited leech on 8th day

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The data were statistically compared in between to analyse the difference in subgroups and to conclude about reusing the leech after 7 days.

Observations

  1. Nonpoisonous leeches were selected as per the Ayurveda parameters and were well correlated with Hirudina medicinalis to confirm the safety
  2. Nonchlorinated water having minimum bacterial and fungal load was selected to store the leeches to avoid the bias related to water
  3. TBC and TFC of all leeches previous to the application are within its normal limit except leech B (normal TBC limits – 30–300 cfu/ml normal TFC limits – 10–100 cfu/ml)
  4. Escherichia coli, Staphylococcus aureus, Pseudomonas Aeruginosa, and Aeromonous are the most common organism encountered in the leech saliva
  5. Type of patients encountered throughout the study
  6. There is no significant difference in bacterial and fungal count if compared between leech saliva before application and 8th day of application
  7. TBC of venous blood of the nine patient (treated by I leech) as well as bacterial count of 8th day leech saliva of I and J leech are Nil [Graph 1] and [Table 2]
  8. There is considerable difference in bacterial and fungal count if compared in between venous blood and blood vomitus and leech saliva at 8th day of application [Graph 2] and [Table 2]
  9. Bacterial count of 8th day leech saliva of D leech is too numerous to count (TNTC) [Table 2]
  10. Fungal count of 8th day leech saliva of I and J leech is also TNTC
  11. There is statistically significant difference in the bacterial count of blood emesis and 8th day leech saliva as well as considerable difference in between fungal count of blood emesis and 8th day leech saliva which demonstrates the leech has sucked the huge amount of bacteria, but still there is a minimum bacterial load in 8th day of leech saliva which indicates its rationality to reuse [Table 3] and [Table 4].

Table 2: Results and observations based on total bacterial count and total fungal count at different stages

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Table 3: Statistical analysis in four subgroups of total bacterial count using one-way ANOVA

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Table 4: Statistical analyses in four subgroups of total fungal count using one-way ANOVA

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  Discussion Top


Literature

Evidences of leech therapy found in many countries all over the world since the ancient period of time including India which indicates its own importance. However, by careful study of Ayurveda as well as Modern literature, one can find many differences in methodology of collection, classification, storage, and application of leeches. Nowadays, modern leech therapy is not limited only up to its application, but the various researches are under taken to evaluate the efficacy of leech saliva in various diseases and conditions as the saliva of leech is the main component which makes the leech therapy special. Researches to find the efficacy of leech saliva as antimetastatic agent in cancer and antihyperglycemic agent in diabetes are the biggest hope before the researchers.[1] If leech saliva found to have antimetastatic and anti-hyperglycemic activity, it will be one of the biggest boon of leech therapy to the whole world.

Collection of water

Leeches may succumb when they stored in the sterile water and to provide a natural habitat for the survival of leeches nonchlorinated well water having minimum bacterial load (30–300 cu/mm as per the Indian Pharmacopeia) was selected to store leeches.

Selection of leeches

Leeches were collected from fresh water as per the description of classics and also selected as per the ayurvedic parameters of Savisha and Nirvisha Jalouka and well correlated with Hirudina medicinalis as per the morphological structures, this may be the cause that single patient was not noticed to have any adverse effects after Jaloukavcharana (Itching, burning, pain, or fever, etc.).[2]

Selection of patients

The study is aimed to evaluate the TBC and TFC of leech therapy, so there is a need of patients having bacteremia (signs of pain and fever), so the vidradhi and dustavrana are the most common conditions seen in Shalyatantra OPD [Table 5].
Table 5: Total number of patients included in the study

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Methodology

The present study is to evaluate the safety of reuse of the vomited leeches in concerned to the bacterial transmission. Leech releases the saliva in the blood of host while sucking the blood, so the chances of cross infection are through saliva, so the saliva was taken for bacterial evaluation before and after its application. TBC and TFC of all leeches previous to the application were within its normal limit (30–300 cu/mm) except leech B, so it can be said that application of leech is ethical. There is marked depletion in the bacterial count at 8th day of leech saliva because leeches were well vomited, and this might played the crucial role to inhibit the bacterial growth. Second bout of blood vomitus of leech was collected which was noncontaminated by haridra which is used for vomiting of leech. Aeromonus hydrophilia and symbionts in the leech gut might have inhibited the foreign growth. Another study which justify that S. aureus and P. aeruginosa were found to be significantly attenuated in their ability to proliferate within the leech crop compared with the in vitro blood control, suggesting either that their growth was inhibited or that cell proliferation was balanced by death.[9]

Summary

Leech therapy is a commonly practiced treatment modality in Ayurveda, and there is a common unsolved question raised by the patients, and surgeon is that the transmission of infection after leech therapy as the leeches is reused after procedure. As it is era of evidence-based practice so to justify scientifically and practice ethically, we tried to potentiate by conducting this work as PG dissertation. The study was initiated with literature review and scientific journal review in order to carry a published scientific protocol, but such studies were not found which made us to prepare our own protocol based on scientific parameters which will justify our objectives. Total bacterial count, fungal count, and viral studies were kept as parameters initially, but the institutional ethical committee permitted only bacterial and fungal studies. Viral studies need sophisticated labs and huge financial support, so the study was limited to bacterial and fungal count.


  Conclusion Top


Rationality to reuse the leech after 7 days after making the leech to vomit can be justified primarily by the present study. Chances of contamination of leeches through water are rare if it is changed every 3rd day. Aeruginous species are multidrug-resistant species, so it can be said that leech application should be with caution in immunocompromised patient (as chances to prone to the Aerogenous Gram-negative infection is more in immune compromised patients).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Abdul Kader AM, Ghawi AM, Alaama M, Awang M. Leech therapeutic application. Indian J Pharm Sci 2013;75:127-37.  Back to cited text no. 1
    
2.
Kaviraj Ambikadutta Shastri, editor. Sushruta, Sushruta of Samhita, Sutra Sthana: Jaloukavacharna Vidhiadhyaya. Varanasi: Chaukhmba Sanskrita Sansthana; 2013. p. 56.  Back to cited text no. 2
    
3.
Mumcuoglu KY. Recommendations for the use of leeches in reconstructive plastic surgery. Evid Based Complement Alternat Med 2014;2014:1-7.  Back to cited text no. 3
    
4.
Brahmananda Tripathi, editor. Ashtangahridaya of Vagbhata, Sutra Sthana; Shastra Vidhi Adhyaya. Ch. 26., Ver. 44. Varanasi: Chaukhmba Sanskrita sansthana; 2013. p. 290-1.  Back to cited text no. 4
    
5.
Kaviraj Ambikadutta Shastri, editor. Sushruta, Sushruta of Samhita, Sutra Sthana: Jaloukavacharna Vidhiadhyaya. 14th ed. Varanasi: Chaukhmba Sanskrita Sansthana; 2003. p. 44.  Back to cited text no. 5
    
6.
Kaviraj Ambikadutta Shastri, editor. Sushruta, Sushruta of Samhita, Sutra Sthana: Amapakweshaniya Adhyaya. 14th ed. Varanasi: Chaukhmba Sanskrita Sansthana; 2003. p. 71.  Back to cited text no. 6
    
7.
Kaviraj Ambikadutta Shastri, editor. Sushruta, Sushruta of Samhita, Sutra Sthana: Vranasravavijnyaniya Adhyaya. 14th ed. Varanasi: Chaukhmba Sanskrita Sansthana; 2003. p. 65.  Back to cited text no. 7
    
8.
Kaviraj Ambikadutta Shastri, editor. Sushruta, Sushruta of Samhita, Sutra Sthana: Jaloukavacharna Vidhiadhyaya. 14th ed. Varanasi: Chaukhmba Sanskrita Sansthana; 2003. p. 45.  Back to cited text no. 8
    
9.
Indergand S, Graf J. Ingested blood contributes to the specificity of the symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech. Appl Environ Microbiol 2000;66:4735-41.  Back to cited text no. 9
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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