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Mycetoma Control and Prevention

INTRODUCTION
Mycetoma is a slow progressing chronic inflammatory tropical disease caused by actinomycetoma bacteria and eumycetoma fungi found in soil and water1. These bacteria and fungi enter the body through open wounds or abrasions on the skin. Symptoms appear as firm painless nodules under the skin, in the subcutaneous tissue, and develop into granulose oozing sores over time1. Mycetoma lesions extend into the subcutaneous tissue and bone, leading to devastating deformities, disability, negative and surgical amputation in the most advanced cases. The majority of mycetoma cases present in the extremities, with nearly 70% of cases occurring in the feet, followed by hands (12%), legs (10%) and torso (6%). The disease is common among manual field workers who walk barefooted in rural endemic areas and have frequent direct contact with the outdoor environment. The Male to female ratio is 6:1, with most cases commonly reported in adults aged 20-40 years old1-3. The disease is endemic to tropical and subtropical regions known as the ‘mycetoma belt’ which stretch between 30°N and 15°S of the equator. Countries within the mycetoma belt include Sudan, Somalia, Senegal, India, Yemen, Mexico, Venezuela, Colombia, Brazil. and Argentina, with the majority of mycetoma cases being reported from Mexico, Sudan, and India3.
Mycetoma is not a notifiable condition, and it was only recently added to the WHO Neglected Tropical Disease List in 20164. It’s true annual incidence and prevalence rates globally are currently unknown. Because of this, there is a lack of accurate data on the number of reported cases in each country per year9. In Sudan, research conducted in mycetoma-endemic states suggest a prevalence rate of 14.5 per 1,000 individuals4. However, this is a gross underestimation, as most patients tend to present to health facilities in more advanced stages of disease. Treatment of mycetoma is long and challenging with poor outcomes. The infection is slow-progressing and painless; the incubation period varies from 3 months to 9 years5. For this reason, diagnosis and treatment of mycetoma is delayed and the number of reported cases only reflect more advanced cases of disease. There are currently no viable control or prevention programs for mycetoma. This is due to the fact that mycetoma is endemic in low-income and resource-limited countries with inadequate medical and healthcare infrastructure, surveillance systems, and diagnostic and treatment facilities9. As such, prevention at the primary level is key to reducing the incidence of mycetoma and the socio-economic dislocation caused by the disease. Prevention through synthesized antifungal microbial materials in the form of integrated products such as shoes, socks, gloves etc. can greatly minimize exposure inexpensively, thus reducing the new contracted cases of mycetoma. As a proposed solution, Sana Protective Shoes and Work Gear is cost-effective lightweight, breathable and antibacterial/antifungal functional products that will work to neutralize exposure and effectively reduce the risk of contracting mycetoma and like diseases.
ETIOLOGY OF DISEASE
Mycetoma is classified into actinomycoma, caused by a bacterial agent, and eumycetoma caused by fungus. The global incidence of actinomycetoma and eumycetoma vary by country and region9. Actinomycetoma is more prevalent in drier regions in South and Central America where eumycetoma is more common in humid regions in Africa5. This climate contributes to the breeding and survival of mycetoma agent and is characterized by short rainy seasons (4-6 months), and longer dry seasons (6-8 months) where the temperature fluctuations vary between 45-60oC to 15-18oC daily5. There are more than 56 fungal and bacterial species that cause mycetoma. The most frequent actimomycetoma cases are caused by S. Somaliensis, A. Madurae, A. pelletieri, N. Brasiliensis and N. Asteroides. Approximately 50% of all mycetoma cases are classified as eumycetoma, 70% of which are caused by Madurella mycetomatis6. The pathology of mycetoma and mode of transmission are not clearly understood. There are no known vectors or animal reservoirs for the disease6. The mycetoma agent is found primarily in soil and animal manure and can enter the body through an infected thorn prick, wood splinter, cut, or abrasion. Once the pathogen is introduced to the wound track it injects itself into the underlying skin and begins to create microcolonies of small aggregate grains called sclerotia7. As the infection spreads, grain clusters grow into hard painless nodules that secrete pus, blood and fungal grains that vary in size, color and consistency10. The infection can spread deep into the tissue and infect the underlying bone. The disease is seen to be more invasive and fast progressing among patients with malnutrition, poor general health status, immunosuppression and associated illnesses like malaria and HIV/AIDS. However, there is no definite conclusion about the relationship between a patient’s immune status and the progression of disease7.
BIOLOGY OF DISEASE
The role of the host’s immune system and effects of the mycetoma agent on immunocompromised systems are not well known. Some studies have been conducted on the role of the immune system in fighting mycetoma in vitro and in animals, but very few have been performed on humans10. The body’s innate immune system is triggered as a first line of defense in an attempt to engulf and inactivate the organism. Complement-dependent chemotaxis of polymorphonuclear leukocytes is induced once the bacteria and fungi antigens are introduced to the body10. Once the organism begins to organize itself into grains, the immune system responds in three different ways. In the first, neutrophil degranulation occurs. Neutrophils migrate from the bloodstream to the site of infection in response to chemical signals released from the mycetoma agent10. Secondly, macrophages and monocytes are called in to clear away neutrophil debris and mycetoma grains. Monocytes present to the inflammation site as a precursor of macrophages and begin phagocytic activity. Macrophages leave the bloodstream, enter the infected tissue, and attach to the pathogen. Once its attached itself to the pathogen, the macrophage engulfs the pathogen into a vesicle inside the cell where digestive enzymes break the mycetoma pathogen down10. Digestible material is used as nutrients within the phagocyte and undigestible material is removed from the cell via exocytosis. Finally, epithelioid cell granulomas are formed to protect the infection from further spread10.
Innate immunity is ineffective in stopping the infection from spreading. A study conducted in 1978 by Melendro et al, found that virulent N. Basiliensis bacteria were able to escape oxygen-dependent microbicidal activity and continue to multiply after engulfment into the phagocytic cell10. Research also suggests that mycetoma agents produce anesthetic substances, which explains why the disease is usually asymptomatic and painless. The pain characterized in the late stages of disease is related to the nerve damage associated with the tissue damage, expansion of the bone and vasoconstriction to the infected area10.
APPROACHES TO DISEASE PREVENTION AND CONTROL
The lack of a global prevention or control program for mycetoma is due to our limited understanding of the basic epidemiological characteristics of mycetoma9. Because of this, the only available methods to control and eliminate disease lie in strengthening disease advocacy and scaling up access to interventions and community-level activities through disease mapping, early case detection and treatment6. The Mycetoma Research Centre (MRC), a WHO Collaborating Centre, is a globally recognized leader in public-health management of mycetoma and the driving force behind the WHO resolution to add mycetoma to the Neglected Tropical Disease List in 20165. The MRC plays an active role in epidemiological mapping of disease, treatment and management of mycetoma cases, and the development of new field diagnostic tools6. While MRC conducts regular medical missions in endemic regions throughout the year, disease mapping for mycetoma is generally incomplete, and the number of people at risk for the disease is unknown. There is an urgent need to consolidate existing country data into a structured mycetoma database as well as conduct mapping for mycetoma where the same is required to determine its distribution.
Complete cure in mycetoma is difficult and reoccurrence of infection post-surgical treatment is common. According to the MRC, cure rates with antibiotic therapy are only 43% for actinomycetoma and 25% for eumycetoma. The average cost of treatment in Sudan can go up to as much as $330USD, while monthly income averages only $60USD. More than 50% of patients end up discontinuing treatment due to lack of progress and high costs6. At the community level, MRC conducts health education campaigns in schools, local community centers and religious institutions, as well as, hosts radio and T.V informercials and Q

Indentification of Unknown Microorganism

Lab 5: Pure Culture Technique: Isolation of Bacteria
This experiment has two techniques which are continuous and discontinuous streaking; our laboratory instructor gave us our unknown for this experiment. We sterilized our wire loop by holding until it turns bright orange-red. We wait for it to color then streak the bacteria with a loop from the unknown and made discontinuous and continuous streaking on both plates. We covered the labeled and covered the plates with tapes on the sides. Then we incubated them at 37 for 24-48 hours in an inverted position, then we observed the plates to check if there are any well- isolated colonies.
Lab 8 – Polymerase Chain Reaction ( PCR) for DNA Amplification and Sequence Analysis DNA Extraction:
For this Lab, we turned in the heat to 100C and vortex 5% of Chelex for 60 seconds. Then we vortexed the solution for 30 more seconds and centrifuging it for 1 minute at maximum speed, which was 13,300 rpm/ 17000 g. While carefully removing the tube, we extracted 30 ul of the supernatant (contains DNA) pipette it into a 1.5 microfuge tube. Then we discard the tube with the remaining Chelex mixture. The purity was determined through a NanoDrop ND- 1000 Spectrophotometer. This was done with the help of our Lab instructor and the remaining part in lab 8.
BLAST:
Our lab Instructor, took our sequencing regarding our unknown and copied and pasted it into nucleotide option on the BLAST website. Once we blasted the sequence, it gave use many resulted for what genus our unknown belong to.
Lab 12- Physiological Characteristics: Biooxtidation, Hydrosis, and Others
For this experiment, a series of inoculations for various microorganisms was performed shown in the Table below. After the inoculations, all the cultures wee place into the incubator for 37C in preparation for the biochemical analysis. The nutrient agar plate was removed from the incubator after 24 hours and placed into a 4C cold room or refrigerator.
Inoculations
Biochemical Tests
Microorganism Inoculated
Test Reagent
1. Glucose/ Dextrose broth with Durham Tubes
· Escherichia coli
· Staphylococcus epidermidis
· Unknown #11
2. Lactose broth with Durham Tubes
· Escherichia coli
· Staphylococcus epidermidis
· Unknown #11
3. MR-VP medium (mixed acid, methyl red test)
· Escherichia coli
· Enterobacter aerogenes
· Unknown #11
Methyl Red
4. MR-VP medium (butanediol Voges-Proskauer)
· Escherichia coli
· Enterobacter aerogenes
· Unknown #11
Barritt’s Reagent A (alpha-naphthol)

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