ANTIFUNGAL ACTIVITIES OF SOME SELECTED MEDICATED SOAPS AGAINST

RINGWORM (Tineacapitis).

Sola Oloruntimehin1, and Grace Gberikon (Dr)2

Abstract

Antifungal avtivities of some selected medicated soaps against ringworm (Tineacapitis) was

investigated. Samples were collected from infected children in LGEA primary school and St.

Mary’s primary school of North Bank area of Makurdi using swab sticks. Sterile swab sticks

containing samples were immediately transported to the Research Laboratory, Biological

Sciences, University of Agriculture Makurdi for analysis.Selected medicated soaps such as

Crusader, Actimed, Safeguard, Tetmosol and Dettol that contain Triclosan, Mercury (I) iodide,

and monosulfiram were used to test for their antifungal activities against ringworm in this study.

Trichophytonmentagrophytesand Trichophytonrubrumwere isolated from the samples using

Sabouraud’s Dextrose Agar medium. The zone of inhibition was conducted using agar diffusion

method. Double serial dilutions of the soaps were made at 50mg/ml, 25mg/ml and 12.5mg/ml to

check for the minimum inhibitory concentration on both T.mentagrophytes and T.rubrum.

Crusader and Tetmosol have higher zone of (30.0mm) inhibition than Safeguard and Actimed

(26.0mm) with Dettol having the least (19.0mm) inhibition capacity on T. mentagrophytes.

While on T.rubrum, Actimed has 24.0mm, Safeguard has 16.0mm, Dettol 21.0mm, Tetmosol

22.0mm and Crusader has 23.0mm zone of inhibition. Crusader and Tetmosol equally have

fungicidal activities at 12.5mg/ml and 50mg/ml respectively on both T.mentagrophytesand

T.rubrum. The control, Griseofulvin on the other hand had an average inhibition of 18.0mm and

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10.0mm on T.rubrum and T. mentagrophytes respectively. Therefore, some of these soaps can be

consider as

topical adjunct for children infected with Tineacapitis.

Introduction

Ringworm infection continues to be a problem all over the world, particularly in the developing

countries, where poor living conditions and massive un-enlightenment prevail. This infection, as

well as scabies is known to be among the “dermatoses of poverty” (Degreef etal.,2008).Although

it has no obvious fatal consequences, ringworm can be unsightly and can have far-reaching

social, economic and psychological impacts on the afflicted individual (WHO, 2006). Ringworm

is a skin infection that is caused by fungi. Some of the common places ringworm grows are the

feet (athlete’s foot), groin, scalp, face and nails. The infection gets its name from the

characteristic ring-like rash on the skin. The disease is spread through direct skin to skin contact

with person infected with ringworm or by touching an infected animal (Oyewale etal., 2015).

Barbing tools have also been reported to be an important source of ringworm infection (Enemour

etal.,2012). For instance, “barbing shop” was reported as the most frequently incriminated

probable source of ringworm disease in Anambra state of Nigeria (Emele etal.,2009). Ringworm

may also be spread through contaminated floor in locker rooms or showers or from sharing

combs, brushes, towels or clothing with an infected person. Animals, especially cats may be

carriers without having symptoms.

Tineacapitis (also known as “Herpes tonsurans”, “Ringworm of the scalp”(Enemour

etal.,2012). is a cutaneous fungal infection (dermatophytosis) of the scalp. The disease is

primarily caused by dermatophytes in Trichophyton and Microsporumgenera that invade the hair

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shaft. At least eight species of dermatophytes are associated with Tineacapitis(Peters etal.,2006).

Cases of Trichophytoninfection predominate from Central America to the United States and in

parts of Western Europe. Infections from Microsporum species are mainly in South America,

Southern and Central Europe, Africa and the Middle East (Wikipedia, 2015).

The clinical presentation is typically single or multiple patches of hair loss, sometimes

with a ‘black dot’ pattern (often with broken-off hairs), that may be accompanied by

inflammation, scaling, pustules, and itching (Oyewale etal., 2015). The rash may be reddened

and circular with disgusting and irritating appearance on infected individuals. Uncommon in

adults, Tineacapitis is predominantly seen in pre-pubertal children, more often boys than girls.

Treatment of Tineacapit is requires an oral antifungal agent; griseofulvin is the most commonly

used drug, but other newer antimycotic drugs, such asterbinafine, itraconazole, and fluconazole

have started to gain acceptance (Indira etal., 2014). FUNGI

The fungi are a large and successful group of organisms of about 90, 000 named species. They

range in size from the unicellular yeast to the large toadstools, puffballs, and stinkhorns, and

occupy a very wide range of habitats both aquatic and terrestrial (Taylor and Green, 2005). They

are also of major importance for the essential role that they play in the biosphere, and for the way

in which they have been exploited by humans for economic and medical purposes. Fungi include

the numerous moulds which grow on damp organic matter (such as bread, leather, decaying

vegetation and dead fish), the unicellular yeasts which are abundant on the sugary surfaces of

ripe fruits and many parasites of plants. They have a global distribution from polar to tropical

regions (Willey etal., 2011). Fungi are saprophytes, securing nutrient from dead organic material

by releasing degradative enzymes into the environment. They degrade complex organic materials

in the environment to simple organic and inorganic molecules. In this way, carbon, nitrogen,

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phosphorus and other critical constituents of dead organisms are released and made available for

living organisms. Many fungi are pathogenic and infect plants and animals. Over 5 000 species

attack economically valuable crops, garden plants, and many wild plants, and about 20 new

human fungal pathogens are documented each year.

Although hundreds of thousands of fungal species are found in the environment, only

about 50 produce disease in humans (Willey etal.,2011). Medical mycology is the discipline that

deals with the fungi that cause human disease. These fungal diseases called mycoses are typically

divided into five groups according to the route of infection: superficial, cutaneous, subcutaneous,

systemic and opportunistic mycoses (James et

al., 2007). Superficial, cutaneous, and subcutaneous mycoses are direct contact infections of the

skin, hair, and nails. Systemic mycoses are infections that have disseminated to visceral tissues.

Most systemic mycoses are acquired by the inhalation of spores from soil in which the mould-

phase of the fungus resides. If a person inhales enough spores, an infection begins as a lung

lesion, becomes chronic, and spreads through the bloodstream to other organs (the target organ

varies with the species) (Malcolm et al., 2000). Some of the infections include blastomycosis,

coccidioidomycosis, cryptococcosis, histoplasmosis etc. Cutaneous mycoses¬¬¬¬¬¬¬¬¬- also

called dermatophytosis, ringworms, or Tineas occur worldwide and represent the most common

fungal diseases in human (Willey etal., 2011).

DERMATOPHYTES

Dermatophytes are one of the most common sources of human fungal infections. The name

dermatophye consists of two parts, namely “derm” which means skin and “phytes” which means

plants. In fact the fungi known as dermatophytes are not plants and are not confined only to the

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skin, but they can affect also hairs and nails. Consequently, the name is not correct, but it is used

because it is tradionally applied to this group of fungi and no other name was proposed

(Sheklakov etal., 1974). Annually they affect millions of individuals and are estimated to burden

the United States healthcare system to the toll of $400 million each year for treatment alone.

Examples of Dermatophytes include the fungi responsible for Tineapedis(athlete’s foot) which

has led to the immobilization of significant numbers of United States troops in recent history

(CDC, 1996). Other species are the cause of Tineacapitisa scalp infection that is a significant

pediatric health problem in urban settings. These fungi grow best on those areas of the skin that

are dark, warm and moist. The Dermatophytes are communicable and can cause chronic

infections in healthy, immune-competent individuals, and hence have adapted to evade and

maintain control of the host immune response for extended periods of time (Elson etal., 2001).

These adaptations must result from the co-evolution between Dermatophytes and their human

and other mammal hosts - evolutionary trajectories that resulted in species that span a continuum

of host specificities and mating competence (Mohamed etal., 2013). Dermatophytes are a unique

group of moulds which have the capacity to invade keratinized tissue, in man and animals,

causing cutaneous infections commonly referred to as Tineaor ‘ringworm’. They are dependent

on keratin as nutrient source as they can cause its hydrolysis. Dermatophytes affects the

keratinized tissues, for example skin, hair and nails and remain confined to the dead tissues and

do not invade the living part of the tissues (Malcolm and Michael, 2000).

These organisms colonize the keratin tissues and in response to their metabolic by products, host

experiences inflammatory reactions. They are usually restricted to the nonliving cornified layer

of the epidermis because of their inability to penetrate viable tissue of an immunocompetent host.

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Acid proteinases, elastase, keratinases and other proteinases reportedly act as virulence factors

(Garg etal., 2009).

The implicating organisms belong to 3 genera, Trichophyton,Epidermophyton and

Microsporum. Dermatophytes may be grouped into 3 categories based on host preference and

natural habitat. Anthropophilic species predominantly infect humans, geophilic species are soil

based and may infect both humans and animals, and zoophilic species generally infect non-

human mammals (Gupta etal., 2013).

Superficial mycoses are fungal infections limited to the stratum corneum and its adnexal

structures. Stratum corneum is the outermost layer of skin. Its external aspect is in contact with

the outside environment and its internal aspect is in contact with the sub-stratum corneum

environment (Malcolm and Michael, 2000). Skin is sterile at birth but soon becomes colonised

by a number of microorganisms which make up its flora. Dermatophytes are not considered part

of this flora. Dermatophyte fungi have been shown to have keratinolytic, other proteolytic and

lipolytic activity (Richardson etal., 2000). Serine proteinases (urokinase and tissue type

plasminogen activator) which are involved in extracellular protein catabolism have been found in

dermatophytes and their release by dermatophytes was suggested to play a major role in the

invasion of skin. Sulphitolysis, a process that denatures keratin non-enzymatically, has been

found during dermatophyte-induced keratinolysis and suggested as a complementary mechanism

to keratinolysis (Richardson etal., 2000). Keratinase has been partially purified and detected from

material of Tineapediscaused byTrichophytonrubrumusing immunoelectron microscopy

technique. Using a fluorescent antibody technique keratinase was also detected in biopsy from

the skin of guinea pigs infected experimentally with Trichophyton mentagrophytes (Richardson

6
et al., 1992).Disintegration of hair thought to be caused by dermatophyte enzyme digestion has

been shown in human scalp infection, experimental infection in guinea pigs and in

vitro. Thus it seems that dermatophytes have a battery of enzymes able to digest different

substrates in their habitat (Malcolm and Michael, 2000). In a research work conducted by Dr.

Richardson Malcolm and Michael Edward of the Mycology Unit, Department of Bacteriology

and Immunology, University of Helsinki, Finland in the year 2000 suggested some conditions in

the skin that favour the growth of dermatophytes while others do not. Conditions favorable for

growth of dermatophytes include:

1. The stratum corneum is an avascular tissue composed of highly specialized but deadcells.

It is distant from the body’s main defensive mechanisms.

2. The stratum corneum is well hydrated – water reaches it through eccrine sweatingand

trans-epidermal water loss. Skin temperature is cooler than body temperature; pH ranges from

5.5 to 6.7. Skin is exposed to the aerobic conditions of the atmosphere.

3. Stratum corneum is an agreeable tissue for growth of dermatophytes because it

iscomposed of proteins, amino acids, lipids, carbohydrates and various trace elements, including

iron.

4. Over some areas of the stratum corneum there are certain anatomical considerationswhich

may enhance establishment of growth of dermatophytes. Firstly, hair on scalp may act as a

trapping device for an airborne dermatophyte infection. Secondly, the hyponychial horny layer is

covered by the distal portion of the nail plate and a groove is thus constructed which may also act

as a trapping device for dermatophyte infective particles. Thirdly, the interdigital spaces of the

toes, particularly the fourth, and the crural areas in males are naturally occluded and this may

contribute to the fact that Tineapedis in most instances starts in the toes webs and Tineacruris is

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almost exclusively a male disease. Experimentally-induced occlusion will cause the hydrated

stratum corneum to swell and develop multiple folds and allow accumulation of desquameted

corneocytes on the surface of the stratum corneum. Therefore, it is likely that occlusion increases

the surface area and nutrients available for growth of dermatophytes on stratum corneum.

Fourthly, in the pathological condition of palmoplantar hyperkeratosis, which is characterised by

a dramtic increase in the thickness of the stratum corneum, there is frequent occurrence of

dermatophytosis.

Pathogenesis

Healthy skin acts as an effective physical barrier against fungal invasion. The increased rate of

regeneration of epidermal cells in response to contact with the dermatophyte with the consequent

removal of fungus from the skin surface is another protective mechanism (Chermette and

Ferreiro, 2008). As dermatophytes cannot penetrate healthy skin, many cats are merely passive

carriers of the arthrospores or remain subclinically infected. Whether such an infection will lead

to clinical signs depends on endogenous and exogenous factors. Predisposing factors include

young age, immunosuppression (including immunosuppressive treatment), other diseases,

nutritional deficits (especially proteins and vitamin A), high temperature and high humidity

(DeBoer and Moriello, 2006). Any skin trauma resulting from increased moisture, injury by

ectoparasites or scratches due to pruritus, playing or aggressive behaviour or clipping is

important for facilitating infection. In general, poor hygiene is a predisposing factor. In

overcrowded feline groups, social stress may play a role. Thus in catteries or shelters infected

with M.canis,eradication of ringworm may be difficult (Moriello, 2004).

The prevalence of fungal flora was investigated with regard to the potential Immunosuppressive

effect of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). However, the

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higher prevalence of M.canis in FIV-infected animals compared with normal cats reported in one

survey (Mancianti etal., 1992) was not observed in another study [Sierra etal., 2000]. The

association may be related to differences in the environment rather than to the retroviral status of

the cats (Mignon and Losson, 1997). Affected hair tissues from cases of Tineacapitishave been

studied using light microscopy, scanning andtransmission electron microscopy. However, ultra

structural findings of the parasitic form of T.mentagrophytesin hair tissue have been inconclusive

and the studies have not used intact hair follicles (Malcolm etal., 2000). The pathological

changes of the affected hair structures are poorly understood. The stages by which detached hairs

are attacked by keratinophilic fungi are: (1) cuticle lifting, (2) cortical erosion, (3) production of

penetrating organs, and (4) colonisation of the medulla (Malcolm and Michael, 2000). The ability

to invade hair in vitro is a property of the keratinophilic fungi in general but various species

differ in the way this is accomplished. It has been found that the direction of invasion and the

pathological role of the fungal elements within the hair appratus are significantly different

between fungi. Experimental studies of hair penetration by dermatophytes are few and have been

restricted to cut or plucked hair and arthroconidia have not been previously used in experimental

studies of hair infection.

Transmission

Dermatophytes produce spores (conidia) that are able to infect humans and animals when they

are being contacted. Those growing in a vertebrate host normally form only (arthroconidia),

asexual spores that develop within the hyphae. In the environment (e.g., in laboratory culture),

they can also produce microconidia and macroconidia, asexual spores that develop outside the

hyphae (Ameen, 2010). Initially, the dermatophyte infects a growing hair or the stratum corneum

of the skin. These organisms do not usually invade resting hair, since the essential nutrients they

9
need for growth are absent or limited. Hyphae spread in the hairs and keratinized skin, eventually

developing infectious arthrospores (Andrews and Burns, 2008).

Anthropophilic and zoophilic dermatophytes are mainly transmitted between hosts by

arthrospores in hairs or skin scales. Other asexual or sexual spores formed by the environmental

stages may also be infectious (Lee etal., 2011). Fomites such as brushes and clippers are

important in transmission (Emele etal., 2009). Spores may remain viable in suitable

environments for up to 12-20 months, and some spores were also reported to persist for at least a

year in salt water. Certain types of spores (e.g., microconidia) might be dispersed by airborne

means (Maraki etal., 2012).

Clinical Signs

Dermatophytes generally grow only in keratinized tissues such as hair, nails and the outer layer

of skin; the fungus usually stops spreading where it contacts living cells or areas of inflammation

(Chah etal., 2012). Many dermatophytes can invade hairs as well as the skin; however, some

anthropophilic species such as E.floccosumand T.rubrum are limited to the skin. Mucus

membranes are not affected. The symptoms of dermatophytosis vary, depending on the infecting

organism, affected tissues (e.g., skin, hair or nails) and area of the body (Noble, 1998).

In unhaired (glabrous) skin, the lesions are usually characterized by inflammation that is most

severe at the edges, with erythema, scaling and occasionally blister formation. The central area

may clear, resulting in the formation of a classic “ringworm” lesion. In haired areas, the hair

become brittle and areas of alopecia may appear (Degreef, 2008). Dermatophytes acquired from

animals or the soil generally produce more inflammatory lesions than anthropophilic

dermatophytes (but not all individual cases are highly inflammatory). These infections are also

less likely to become chronic than those caused by anthropophilic organisms (Degreef, 2008).

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 In humans, dermatophytoses are referred to as “Tinea” infections, and are named

according to the area of the body involved. Infections can, however, spread from one area to

another. For example, Tineafaciei (facial dermatophytosis) in children may result from a

Tineacapitis(scalp) infection that has spread to the face (Lee etal.,2011). Mohamed etal., revealed

that ringworm of the scalp may be classified as scaly ringworm, black-dot ringworm, kerion and

favus. Infection with M.audouinii and M. canis is characterized by small-spored ectothrix hair

invasion, where the spores are surrounding the hair shaft, and the hair fluoresce green under the

U.V. light. Largespored ectothrix hair invasion is seen in case of infection with M.gypseum,T.

verrucosumandT.mentagrophytes. Infection with T.violaceum and T. tonsurans is characterized

by endothrix hair invasion. The inside of the hair may be fully filled with spores and the hair may

break and the remaining part appears as a black dot. In case of favus broad hyphae and air spaces

are seen (Mohamed etal., 2013).

Laboratory Diagnosis of Tineacapitis

Microscopic Examination and Culture

Direct microscopy, although false negative in 5 to 15% of cases in ordinary practice (Rippon

etal., 1988), is a highly efficient screening technique. Scrapings and hairs may be mounted for

direct examination in 25% KOH or NaOH mixed with 5% glycerol, heated (e.g., for 1 h at 51 to

548C) to emulsify lipids, and examined under 3400 magnification for fungal structures. Another

formulation is 20% KOH–36% dimethyl sulfoxide (Rippon etal., 1988), and two techniques for

fluorescence microscopy, the calcofluor white technique (Robinson etal.,1988) and the Congo

red technique (Sliflin etal., 1988), may be used.

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The classification of all structures seen in direct microscopy is beyond the scope of this article.

The reader is referred to several excellent texts for descriptions and photographs (Fragner etal.,

1987; Kane etal., 1980; Kwon-Chung etal., 1992; Rippon et

al., Weitzman etal., 1991).

Culture is a valuable adjunct to direct microscopy and is essential at least in all nail infections

and in any infection to be treated by systemic medication. In all cases, a medium selective against

most nondermatophytic molds and bacteria is used as a primary isolation medium.

Cycloheximide is incorporated into this medium as a semiselective agent to reduce the growth

ofnondermatophytic fungi. Sabouraud peptone-glucose agar (Emmons’ modification) amended

with cycloheximide andchloramphenicol is commonly used (Weitzman etal., 1988). It is

commercially available under various names such as Mycobiotic (Difco Laboratories, Detroit,

Mich.) and Mycosel (BBL, Becton-Dickinson, Cockeysville, Md.) agars. Dermatophyte Test

Medium (Rebell etal., 1970) is an alternative; it normally shows alkalinity generated by

dermatophyte growth as a colour change to red in its constituent phenol red indicator. Some

nonpathogenic fungi (e.g., Trichophytonterrestre), however, induce the red colour change while

some Microsporum isolates (Moriello et al., 1991) and bacterially contaminated isolates (Kane

etal., 1980) may give a false-negative reaction. Therefore, this medium is good but is not an

absolute indicator of the growth of a dermatophyte. It has the disadvantage of not allowing

visualization of colony reverse pigmentation, a character often important in identification. Some

laboratories use

cycloheximide- and antibacterial agent-amended potato

glucose or potato flake agar for primary isolation, a practice speeding the identification of

T.rubrumby rapidly inducing red pigmentation in uncontaminated, typical isolates and typical

12
isolates with relatively antibiotic-susceptible contaminants. When non dermatophytic fungi or

yeasts other than Candidaalbicansmay be etiologic agents, it is critical to use a cycloheximide-

free medium in addition to a selective dermatophyte medium incorporating this inhibitor.

Treatment of Tineacapitis

To date, no effective topical remedy against Tineacapitishas been discovered. Therapy is

systemic, although topical agents such as miconazole, clotrimazole, Whitfield’s ointment, and

selenium sulfide (Allen etat., 1982) may be used as adjuncts to eliminate the shedding of viable

inoculum from infected lesions. Griseofulvin is the long-standing drug of choice and has a

success rate of over 90% (Laude etal., 1982). It is often given as a dosage of 500 mg/day in

adults or 250 mg/day in children, administered as a fourpartdivided dose (Rippon etal., 1988).

Chronic infections may require 2 or more months of treatment. Porphyria is a counter indication.

Azoles may also be effective. Ketoconazole, however, in at least some studies has achieved

remission in only approximately 60% of patients (Conti-Diaz etal., 1984). The allylamine agent

terbinafine has proven highly effective (Villars etal., 1992), as has the triazole itraconazole

(Legendre etal., 1990). In addition to drug treatment, general sanitation measures are usually

employed to prevent recurrence and spread. Infected headgear is often boiled; infected hair is

clipped to reduce the chance of contagion, and the lesions are scrubbed daily, ideally with an

antifungal agent such as selenium sulfide (Allen etat., 1982). Kerion lesions may required

ebridement or other local care, and the use of antibacterial agents may be indicated to treat

secondary infections. Steroids such as prednisone may cause a significant decrease in

inflammation (Laude etal., 1982).

Selected Medicated Soaps

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This study focused on testing for the anti-fungal activities of five (5) different types of medicated

soaps which are: Crusader, Dettol, Safegaurd, Tetmosol, and ACTIMED.

Crusader: crusader is a medicated soap that is formulated with 1.2%

Mercuric iodide (HgI2) presents as 3% potassium mercuric iodide solution as the active

ingredient.

Mercury (I) iodide is a chemical compound of mercury and iodine. It is photosensitive and

decomposes easily to mercury and mercury iodide. Mercury(I) iodide, called Protiodide, was a

very commonly used drug in the 19th century, prescribed for everything from acne to kidney

disease. It was also a treatment of choice for syphilis. It is available over the counter at any

drugstore in the world, the most common form being a concoction of protiodide, licorice,

glycerin and marshmallow.

Dettol and Safeguard: these medicated soaps are one of the most patronized soaps in the market.

Dettol is formulated with 0.6% w/w of trichlorocarbanilide, a derivative of triclocarban, as active

ingredient while Safeguard contains triclosan as active ingredient.

Structure of Triclocarban

Source: (Christian and Frank, 2005).

Triclocarban is an antibacterial agent common in personal care products like soaps and lotions as

well as in the medical field, for which it was originally developed (Chang and McDonnell, 2005).

Studies on its antibacterial qualities and mechanisms are growing. Research suggests that it is

14
similar in its mechanism to triclosan and is effective in fighting infections by targeting the

growth of bacteria such as Staphylococcusaureus. Additional research seeks to understand its

potential for causing antibacterial resistance and its effects on organismal and environmental

health (Wikipedia, 2015).Triclocarban has been used as an antibacterial and antifungal

compound since the 1960s (Orsi etal., 2010). It is commonly found in personal care products as

antimicrobials such as in soaps, lotions, deodorants, toothpaste, and plastic (Brausch et

al.,2011). About 80% of all antimicrobial bar soap sold in the United States contains triclocarban

(Orsi etal., 2010). In addition, the United States spends nearly 1 billion dollars annually on

products containing triclocarban and triclosan (Chang and McDonnell, 2005).

Mechanism of action of triclocarban:

Triclocarban is predominantly active against Gram- positive bacteria (bacteria with a thick

peptidoglycan wall). The precise mechanism of action of triclocarban is unknown, but it is shown

to be bacteriostatic, which prevents bacterial proliferation. Unlike other antibacterial compounds,

triclocarban does not interfere with the membrane. As a result, it is hypothesized that

triclocarban’s molecular mechanism resembles that of triclosan, which inhibits bacterial fatty

acid synthesis (Heath etal., 1999). By mimicking the natural substrate of the enoyl-acyl-carrier

protein reductase (ENR) enzyme, triclosan acts as a site-directed inhibitor and disrupts lipid,

phospholipid, lipopolysaccharide and lipoprotein synthesis. ENR is a highly conserved enzyme

of lipid biosynthetic pathways in bacteria, notably Gram-negative, Gram-positive and

mycobacterial species and thus is absent in humans (Shweizer etal.,2001).

Tetmosol: this soap is formulated with 5% w/w of monosulfiram as the active ingredient.

15
Monosulfiram structure

Source: (Sweetman,2009).

Monosulfiram, trade name Tetmosol, is an ectoparasiticide used in the treatment and prevention

of scabies (Sweetman, 2009). It is usually sold as a solution or medicated soap, sometimes in

combination with benzyl benzoate.

ACTIMED: this medicated soap contains triclocarban and triclosan as co-active ingredients,

though the percentages of these ingredients were not given by the producer. The mechanisms of

action and the uses of triclocarban have been discussed succinctly under Dettol and Safeguard.

Structure of Triclosan

Source: (Russell, 2004).

Triclosan, similar in its uses and mechanism of action to triclocarban is an antibacterial and

antifungal agent found in consumer products, including soaps, detergents, toys, and surgical

cleaning treatments. Its efficacy as an antimicrobial agent and the risk of bacterial resistance

16
remain controversial. Additional research seeks to understand its potential effects on organisms

and environmental health. Triclosan was used as a hospital scrub in the 1970s. Since then, it has

expanded commercially and is now prevalent in soaps, shampoos, deodorants, toothpastes, mouth

washes and cleaning supplies (Thompson etal., 2005). It is part of consumer products, including

kitchen utensils, toys, bedding, socks and trash bags (Thompson et

al., 2005). In healthcare, triclosan is used in surgical scrubs and hand washes. Use in surgical

units is effective with a minimum contact time of approximately two minutes (Brady etal., 1990;

Zafar etal., 1995). More recently, showering with 2% triclosan has become a recommended

regimen in surgical units for the decolonization of patients whose skin carries methicillin-

resistant Staphylococcusaureus (MRST) (Wikipedia, 2014).

Mechanism of action of triclosan

At high concentrations, triclosan acts as a biocide with multiple cytoplasmic and membrane

targets (Russell, 2004). However, at the lower concentrations seen in commercial products,

triclosan appears bacteriostatic, and it targets bacteria primarily by inhibiting fatty acid synthesis.

Triclosan binds to bacterialenoyl-acyl carrier protein reductase (ENR) enzyme, which is encoded

by the gene FabI. This binding increases the enzyme’s affinity for nicotinamide adenine

dinucleotide (NAD+). This results in the formation of a stable, ternary complex of ENR-NAD +-

triclosan, which is unable to participate in fatty acid synthesis (WHO, 2006). Fatty acids are

necessary for building and reproducing cell membranes. Humans do not have an ENR enzyme

and thus are not affected.

Sample Collection

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Samples used for this study were collected from 25 infected school children at different schools

in North Bank area of Makurdi with the consent of the school authorities. The site of infection

where the sample was collected was disinfected using methylated spirit. Different sterile swab

sticks was used for each child. Each sample was labeled and according to each place of

collection, the samples were labelled and packaged. The collected samples were immediately

wrapped tightly to avoid air contamination, and were brought to the Biological Research

Laboratory, Department of Biological Sciences University of Agriculture, Makurdi.

Isolation and screening of Tineacapitisfungal species

Media preparation for the isolation of fungal species

Sabouraud’s Dextrose Agar (SDA) medium was used for the isolation of the organisms. 65g of

the medium powder is to be dissolved in 1litre of distilled water. Therefore, 32.5g per 500ml of

distilled water was prepared for 25 petri-dishes. The conical flask was covered with wad of

cotton wool, wrapped with aluminum foil. The conical flask was shaken gently to allow proper

dissolution of the medium. The medium was then autoclaved at 121 oC for 15minutes and allowed

to cool to 50oC.

Chloramphenicol was added as antibacterial agents. Aseptically, 2ml of sterile distilled water

was added to the chloramphenicol powder. It was allowed to dissolve by mixing it gently. Then,

10ml of distilled water was added and mix together. This was distributed in 2ml amounts into

appropriate sterile containers. Each 2ml volume was sufficient for 500ml of Sabouraud’s agar

medium. The final concentration is 0.4mg/ml. The chloramphenicol solution was added to the

medium and the plates were poured (Cheesbrough,2010).

Isolation of fungal species

18
Normal saline was dispensed into test tubes and were autoclaved at 121 oC for 15min. the swab

sticks were dipped each into a test tube. This was to transfer the organism into sterile medium.

Pour plate technique was used for the isolation of the organisms. Using micropipette, 20µl of

each of the sterile normal saline containing the organisms was dispensed into each corresponding

labeled petri dish. The Sabouraud’s Dextrose Agar medium was autoclaved and was allowed to

cool to 50oC. The medium was poured into the petri dishes. The poured plates were shaken at

intervals to allow even distribution of the organisms in the medium. The plates were then

incubated at room temperature (25 oC) for 5 days. After incubation, the agar plates were observed

for fungal growth (Indira, 2014).

Morphological Characterization of Fungal Isolates by Lactophenol Cotton Blue Staining.

The identification of the fungal isolates was done by its staining procedure. To study the

morphology of the isolates, thin layer of the fungal mycelia was spread on clean glass slides and

teased with needles followed by addition of a drop of lactophenol cotton blue stain on each slide.

The slides were covered with cover-slips and visualized under microscope at X40 magnification.

Biochemical Characterization of the Fungi

The culture of Tinea species were subjected to urease biochemical test for identity confirmation.

Ureas test: Urease test was conducted to differentiate Trichophytonmentagrophytes and

T.rubrumthe two species that were isolated from the samples by employing urease test medium.

4.8g of Urea base was dissolved in 190ml of distilled water. It was dissolved by heating gently.

10ml of distilled water was sterilized at 121 oC for 15 minutes. It was allowed to cool to 50 oC.

0.4g of urea concentrate was dissolved in the sterilized 10ml of distilled water. It was mixed to

19
dissolve and was added to the sterilized urea base. Phenol red was used as indicator during the

preparation of the medium (Cheesbrough,2010). The medium was allowed to cool to 50 oC before

it was poured. The medium was then dispensed in 5ml amounts into test tubes and arranged in

slant position. The medium was inoculated with the test fungi.A sterilized wire loop was used to

pick a loopful of the fungal mycelia growth. With the loopful mycelia, the medium was

inoculated by stabbing it at the centre. T.mentagrophytes, a urease positive organism shows

urease activity within 7 days and the colour of medium changed to pink. The T.rubrumisolates on

the other hand are urease negative and as a result do not produce urease enzyme (Sinki etal.,

1981).

Soap and Griseofulvin Dilution

Crusader, Dettol, Safeguard, Tetmosol and Actimed soaps were diluted in distilled water. 10g

each of the soap was cut into smaller pieces using knife and was dissolved in 50ml of distilled

water. Two-fold serial dilution of the stock dilutions was made beginning at 50mg/ml, 25mg/ml

and 12.5mg/ml concentrations as working dilutions. On the other hand, 200mg of griseofulvin

drug, served as the control, was dissolved in 10ml of distilled water to make the stock dilution.

Working dilution was made by dissolving 2ml of the solution in 8ml of distilled water to

represent 0.1µg/ml as the least concentration for control (Indira, 2014).

Determination of Anti-fungal Activity

Determination of Inhibition Capacity: The ability of the soap samples to inhibit the growth of

Trichophyton mentagrophytes and Trichophyton rubrum in vitro was determined using agar

diffusion method. 200mg/ml concentration for each of the soap was prepared as stock culture.

This was used to test for the inhibiting capacity of the soaps. Inoculum suspensions of

20
T.mentagrophytesand T.rubrumwere prepared from the five days cultures grown on Sabouraud’s

dextrose agar at 35oC. Suspensions of the fungal colonies were made by scraping the surface of

the colonies with sterile loop into 10ml of distilled water. The resulting mixture of conidia and

hyphal fragments were withdrawn and transferred to sterile tubes for 15 to 20 minutes at room

temperature to sediment the heavy particles. At the start of each experiment, pour plate technique

was done using the inoculum suspensions of the species. Cock borer was used to create wells on

the Petri dishes under aseptic condition. The plates were incubated at room temperature for five

days to check for the zone of inhibitions (Esteban etal., 2005). The diameter of the inhibition of

each of the soap was determined using mathematical rule (cm) and was recorded in millimeter.

Minimum Inhibitory and Fungicidal Concentration: the concentrations: 50mg/ml, 25mg/ml and

12.5mg/ml were used to determine the minimum concentration at which these organisms can be

inhibited. The three concentrations were mixed with prepared Sabouraud’s Dextrose Agar

medium respectively. The medium containing each concentration was dispensed in 20ml to

sterile bottles and were autoclaved at 121 oC for 15min. After autoclaving, the media were poured

into labeled Petri dishes according to each of the soaps for the different concentrations. Under

aseptic condition, a sterilized wire loop was used to pick the fungal suspension to make spot

inoculation. The Petri dishes were then incubated at room temperature for five days. Two plates

without any soap diffusion were inoculated with test organisms as control to check for the

minimum inhibitory concentration of the soaps. The concentration at which there was no fungal

colony growth at all was the minimum fungicidal concentration of that soap.

RESULTS

21
The samples collected from the infected children were inoculated of SDA medium and two

species, Trichophytonmentagrophytesand Trichophytonrubrumwere isolated in the ratio 8:2. The

number of Trichophyton mentagrophytes was 20 while that of Trichophytonrubrumis 4 with one

plate showed no growth (Table 1). The biochemical test was done to differentiate the two species

using urease agar. The T. mentagrophyteschanged the colour of the medium to pink while

T.rubrum,the urease

negative did produce colour change.

Urease Biochemical Test for T.mentagrophytesand T.rubrum

Bottle A is the urease positive result of T.mentagrophytesthat changes the colour to pink while

bottle B indicate urease result for T.rubrumwhich has no colour change.

The zones of inhibition of the soaps were done and were compared with that of the antifungal

drug Griseofulvin. On T.mentagrophytes, Actimed has 19.0mm zone of inhibition, Safeguard

26.0mm, Dettol 26.0mm, Tetmosol 30.0mm and Crusader 30.0mm with the control, Griseofulvin

haven an average inhibition of 18.0mm (Table 2). While on T.rubrum, Actimed has 24.0mm,

22
Safeguard 16.0mm, Dettol 21.0mm, Tetmosol 22.0mm and Crusader 23.0mm. Griseofulvin has

an average (10.0mm) zone of inhibition (Table 3). Crusader and Actimed have their minimum

inhibitory concentrations on T. mentagrophytesat 12.5mg/ml while Tetmosol was at 25mg/ml

and Dettol and Safeguard were at 50mg/ml. On T.rubrum, Crusader and Tetmosol inhibited at

12.5mg/ml and 25mg/ml respectively. Actimed, Safeguard and Dettol all have their minimum

inbition at 50mg/ml (Table 4). The treatment means of the soaps and Griseofulvin on T.

mentagrophytes were the same using ANOVA data analysis (Appendix 1) while the treatment

means on T.rubrumwere not the same (Appendix 2). The ANOVA result also shows that the

treatment means of the soaps both on T. mentagrophytesand T.rubrumare the same. The soaps

produced better inhibition on T. rubumthan Griseofulvin (Table 3). The difference in inhibitions

might be due to other components of the soaps that have effect on the organisms.

23
 Reverse colony Microscopic Species

Table 1: Morphological Characteristics colour macroconidia

of T.mentagrophytes and T.rubrum (Undersurface using

Number ofColony appearance view). Lactophenol.
infected on Wine Cigar-shaped T.rubrum
pupils SDA (Top view) with thick
1 White and downy rough walls

- - -

2 No growth Yellow Cigar-shaped with T.mentagrophytes

3 White and downy thin walls

Yellow Cigar-shaped with T.mentagrophytes

4 White and downy
thin walls

5 White and downy Yellow Cigar-shaped with T.mentagrophytes

thin walls
6 and
Brown to tan Cigar-shaped with T.mentagrophytes
Buff powdery
thin walls
7 and
Buff powdery
Brown to tan Cigar-shaped with T.mentagrophytes
8 White to withbuff
thin walls
age.
Yellow Cigar-shaped T.rubrum
9 and
with thick
Buff powdery
rough walls
10 and
Buff powdery Brown to tan Cigar-shaped with T.mentagrophytes

11 and thin walls

Buff powdery
Brown to tan Cigar-shaped with T.mentagrophytes

thin walls
24
Brown to tan Cigar-shaped with T.mentagrophytes

thin walls
Isolated from Infected Pupils Attending LGEA and ST. Mary’s Primary Schools of North Bank

Area, Makurdi.

25
12 Buff and Brown to tan Cigar-shaped with T.mentagrophytes

powdery thin walls

13 White and downy Yellow Cigar-shaped with T.mentagrophytes

thin walls

14 White to buff with Yellow Cigar-shaped T.rubrum

age with thick

rough walls

15 White and downy Yellow Cigar-shaped with T.mentagrophytes

thin walls

16 Buff and Brown to tan Cigar-shaped with T.mentagrophytes

powdery thin walls

17 Buff and Brown to tan Cigar-shaped with T.mentagrophytes

powdery thin walls

18 Buff and Brown to tan Cigar-shaped with T.mentagrophytes

powdery thin walls

19 Buff and Brown to tan Cigar-shaped with T.mentagrophytes

powdery thin walls

20 Buff and Brown to tan Cigar-shaped with T.mentagrophytes

powdery thin walls

21 White to buff with Yellow Cigar-shaped T.rubrum

age with thick

rough walls

26
 22 White and downy Yellow Cigar-shaped with T.mentagrophytes

thin walls

23 Buff and Brown to tan Cigar-shaped with T.mentagrophytes

powdery thin walls

24 White and downy Yellow Cigar-shaped with T.mentagrophytes

thin walls

25 Buff and Brown to tan Cigar-shaped with T.mentagrophytes

powdery thin walls

Table 2: Zones of inhibition by the soaps at 200mg/ml and the control (Griseofulvin) at 0.1µg/ml

on T.mentagrophyte.

Soaps Zones of inhibition with soaps Zones of inhibition with

(mm). Griseofolvin (mm).

Actimed 19.0 20.0

Safeguard 26.0 18.0

Dettol 26.0 18.0

Tetmosol 30.0 18.0

Crusader 30.0 18.0

Table 3: Zones of inhibition by the soaps at 200mg/ml and the control (Griseofulvin) at 0.1µg/ml

on T.rubrum.

Soaps Zones of inhibition with soaps Zones of inhibition with

(mm). Griseofolvin (mm).

Actimed 24.0 14.0

27
 Safeguard 16.0 11.0

Dettol 21.0 10.0

Tetmosol 22.0 10.0

Crusader 23.0 9.0

50mg/ml 25mg/ml 12.5mg/ml 25mg/ml 12.5mg/ml

Actimed + + + + - -

Table 4: MIC- minimum inhibitory concentration of soap samples on T.mentagrophyte and

Soaps T.mentagrophytes T.rubrum

50mg/ml
T.rubrum

Safeguard + - - + - -

Dettol + - - + - -

Tetmosol + + - + + Crusader + + + +

+ +

The (+) indicates inhibition of the organism while (-) indicates resistance of the organism.

Table 5: MFC- minimum fungicidal concentration of soap samples on T. mentagrophytesand

T.rubrum

Soaps T.mentagrophytes T.rubrum

28
 50mg/ml 25mg/ml 12.5mg/ml 50mg/ml 25mg/ml 12.5mg/ml

Actimed + - - - - -

Safeguard - - - - - -

Dettol - - - - - -

Tetmosol + - - + - -

Crusader + + + + + +

The (+) indicates total fungicidal of the organism at that concentration while (-) indicates the

organism’s resistance.

DISCUSSION

The soap samples show different inhibiting capacities on Trichophytonmentagrophytes and

Trichophyton rubrum respectively. ACTIMED, Safeguard and Dettol all have Triclosan as the

active ingredient. Therefore their ability to inhibit Trichophytonin vitro agrees with the work

done by Jones etal., 2000 that showed that Triclosan has in vitro effectiveness against a wide

range of dermatophyte species. The result agrees with the study conducted in Zürich by Jones

etal., 2000 that among the implicating agent for dermatophyte that could be identified through

cultures, Triclosan was proven to be effective against T. mentagrophytes and T. tonsurans with

minimum inhibitory concentrations ranging from 1 to 10ppm. A research was conducted among

primary school children in Kilombero District, Tanzania by Peters etal.,2006 which had similar

sample source with this study. This suggests that the morphology and the biochemistry of the

pathogens might be similar. In the study it was reported that Triclosan was able to decrease the

29
growth circumference of Tineacapitisfrom 2.9cm to 2.4cm among the school the children.

Therefore, these active soaps could be potent against Tineacapitis.

Tetmosol and Crusader have Monosulfiram and Mercury (I) iodide as active ingredient

respectively. These two soaps showed better inhibition and fungicidal activities than those

containing Triclosan. Tetmosol have 30.0mm and 22.0mm zones of inhibition on

T.mentagrophytesand T.rubrumrespectively while Crusader has 30.0mm and 23.0mm zones of

inhibition on T.mentagrophytesand T.rubrumrespectively. This could be as a result of the

chemicals which had better control on the organisms.

Minimum inhibitory concentration of the soap samples was done using 50mg/ml, 25mg/ml and

12.5mg/ml for the soap samples. After 5 days incubation, Actimed and Crusader showed

minimum inhibitory concentration at 12.5mg/ml, Tetmosol at 25mg/ml while Safeguard and

Dettol each showed minimum inhibition at 50mg/ml on T. mentagrophytes. On T.rubrum,the

minimum inhibitory concentration was at 50mg/ml for Actimed, Safeguard and Dettol. Tetmosol

has its minimum inhibition at 25mg/ml while Crusader shows minimum inhibition at 12.5mg/ml.

This result shows that though T.rubrum is a slow growing organism but seems to be more

resistant than T. mentagrophytes.

The fungicidal activities of the soap samples were determined using the above concentration.

Actimed and Tetmosol showed fungicidal activities at 50mg/ml and Crusader at 12.5mg/ml while

Safeguard and Dettol do not show fungicidal activity at any of the concentration on

T.mentagrophytes. No fungicidal was seen on T.rubrum for Actimed, Safeguard and Dettol but

Tetmosol and Crusader have fungicidal activities at 50mg/ml and 12.5mg/ml concentrations

respectively. Because of these in vitro findings, one might expect a more pronounced

30
mycological effectiveness of Crusader and Tetmosol against these pathogens. Though topical

treatment of Tineacapitishave been debated and doubt not to have been capable of eliminating

hyphae in the deeper part of the follicle or penetrate hair shafts according to Chan et al., 2004.

However, these findings will agree with Weitzman etal.,1996 recommendation that topical agents

may be used as adjuncts to eliminate viable material from the lesions and thus prevent further

spreading and particularly, he suggested that an effective antifungal soap available for masses

might fulfil this purpose better than any other sort of topical treatment, which will only be used

by patients who are aware of their disease. With these soaps, there is hope that asymptomatic

carrier could be “treated” effectively and this will prevent the spread of ringworm infection to

susceptible individuals.

CONCLUSION

Antifungal susceptibility testing has indeed come of age. The use of effective medicated soaps as

adjunct for topical treatment of Tineacapitiscould be encouraged. The results and findings from

this study showed that Crusader and Tetmosol may be better options as topical treatment agents

for Tineacapitisas they produce fungicidal activities at different concentrations. Though the use

of Crusader may be restricted to adult only, as it is consider being too strong for children use.

However adult carriers may still find it helpful in managing the infection spread.

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