AUS DEM SCHWEIZERISCHEN TROPENINSTITUT BASEL UND DER UNIVERSITÄTS-HAUTKLINIK DER ALBERT-LUDWIGS-UNIVERSITÄT FREIBURG I.BR.

EFFICACY OF TRICLOSAN SOAP AGAINST SUPERFICIAL DERMATOMYCOSES AND SCABIES - A PLACEBO- CONTROLLED STUDY AMONG 228 PRIMARY SCHOOL CHILDREN IN KILOMBERO DISTRICT, MOROGORO REGION, TANZANIA

INAUGURAL-DISSERTATION

ZUR ERLANGUNG DES MEDIZINISCHEN DOKTORGRADES DER MEDIZINISCHEN FAKULTÄT DER ALBERT-LUDWIGS-UNIVERSITÄT FREIBURG I.BR.

VORGELEGT 2005 VON ALMUTH DINKELA GEBOREN IN ROTENBURG (WÜMME)

DEKAN PROF. DR. C. PETERS

1. GUTACHTER PD DR. C. HATZ

2. GUTACHTER PROF. DR. L. BRUCKNER-TUDERMAN

JAHR DER PROMOTION 2006

STUDIENKOORDINATOR:

PD DR. MED. C. HATZ LEITENDER ARZT MEDIZIN UND DIAGNOSTIK SCHWEIZERISCHES TROPENINSTITUT UNIVERSITÄT BASEL SCHWEIZ

MITGLIEDER DES DISSERTATIONSKOMITEES:

PROF. DR. MED. L. BRUCKNER-TUDERMAN GESCHÄFTSFÜHRENDE DIREKTORIN UNIVERSITÄTS-HAUTKLINIK ALBERT-LUDWIGS-UNIVERSITÄT FREIBURG I.BR. DEUTSCHLAND

PD DR. MED. P. SCHMID-GRENDELMEIER LEITENDER ARZT DER ALLERGIESTATION DERMATOLOGISCHE KLINIK UND POLIKLINIK UNIVERSITÄTSSPITAL ZÜRICH SCHWEIZ

DR. W. BASCHONG SCIENTIFIC LIASONS & BIOLOGICAL STUDIES HOME AND PERSONAL CARE SEGMENT CIBA SPECIALTY CHEMICALS INC. SCHWEIZ

PROF. DR. PHIL. M. TANNER INSTITUTSVORSTHER SCHWEIZERISCHES TROPENINSTITUT UNIVERSITÄT BASEL SCHWEIZ

STUDIENKOORDINATOR AM IHRDC, IFAKARA/TANZANIA:

DR. B. IDINDILI

FACHARZT FÜR DERMATOLOGIE AM SFDDH, IFAKARA/TANSANIA:

DR. M. MBATA

UNTERSTÜTZUNG DER STATISTISCHEN AUSWERTUNG:

DR. T. A. SMITH, DR. P. VOUNATSOU FÜR MEINE ELTERN

“song of lawino – song of ocol”

Ring-worm sometimes eats up A little girl’s hair And this is terrible; But when hot porridge Is put on the head And a dance is held Under the sausage-fruit tree And the youths have sung

You, Ring-worm, Who is eating Duka’s hair, Here is your porridge, Then the girl’s hair Begins to grow again, And the girl is pleased.

Okot p’Bitek

LIST OF ABBREVIATIONS – IN ALPHABETICAL ORDER ...... 15

ACKNOWLEDGEMENTS – IN ALPHABETICAL ORDER ...... 16

SUMMARY ...... 17

ZUSAMMENFASSUNG...... 18

INTRODUCTION ...... 19

1. Superficial dermatomycoses ...... 19 1.1 Tinea versicolor (synonym: Pityriasis versicolor) ...... 19 1.2 Dermatophyte infections (synonym: Ringworm infections) ...... 22 1.2.1 Tinea capitis...... 22 1.2.2 Tinea corporis ...... 24 1.2.1 and 1.2.2 Diagnosis...... 24 1.2.3 Tinea pedis...... 25 1.2.5 Tinea manuum ...... 26 1.2.6 Tinea unguium ...... 26

1.3 Scabies ...... 27

2. Information about Triclosan ...... 28 2.1 Properties ...... 29 2.2 Triclosan-containing products currently in use in Africa...... 32

METHODS...... 33

1. Study Area...... 33

2. Selection of Primary Schools ...... 34

3. Screening Examinations ...... 34

4. Admission to the Study...... 37 4.1 Inclusion Criteria ...... 37 4.2 Excludsion Criteria ...... 37

5. Case Definitions...... 38

6. Degree of Infection ...... 40

7. Randomisation...... 41

8. Follow-up Examinations...... 42

9. Evaluation of Effectiveness - Dermatomycoses ...... 43 9.1 General...... 43 9.2 Pruritus...... 43 9.3 Clinical and Microscopic Resolution...... 44 9.4 Definition of Evaluation Categories at Second Follow-up ...... 44

10. Evaluation of Effectiveness - Scabies...... 46

11. Assessment of Soap Use (Compliance) ...... 46

12. Determination of Minimal Inhibitory Concentrations (MIC’s) of Placebo Soap and of Triclosan ...... 48

13. Data Entry and Analysis...... 49

14. Additional Information...... 50

RESULTS ...... 51

1. GENERAL INFORMATION ...... 51

2. EPIDEMIOLOGY OF SUPERFICIAL DERMATOMYCOSES ...... 51

2.1 Prevalence rates ...... 51

2.2 Prevalence rates and degree of infection according to sex...... 52 2.2.1 Tinea versicolor ...... 52 2.2.2 Other superficial dermatomycotic infections ...... 53

2.3 Prevalence rates and degree of infection according to age groups ...... 54 2.3.1 Tinea versicolor ...... 54 2.3.2 Other superficial dermatomycotic infections ...... 54

3. ASSESSMENT OF THE EFFECT OF SOAP ...... 55

3.1 Assessment of the efficacy of Triclosan in soap...... 55 3.1.1 Tinea versicolor infections...... 59 3.1.2 Other superficial dermatomycotic infections: Tinea capitis, corporis and pedis infections...... 67 3.2 Determination of minimal inhibitory concentrations (MIC’s) of placebo soap and of Triclosan ...... 76

4. SCABIES...... 81 4.1 Epidemiology and Study population...... 81 4.2 Assessment of the effect of soap and the efficacy of Triclosan ...... 81

DISCUSSION: ...... 85

1. Epidemiology of superficial dermatomycoses...... 85 1.1 Tinea versicolor ...... 85 1.2 Other superficial dermatomycotic infections ...... 86

2. Assessment of the effect of soap and the efficacy of Triclosan in soap ...... 87 2.1 Superficial dermatomycoses ...... 87

3. Scabies ...... 94 3.1 Epidemiology of scabies ...... 94 3.2 Assessment of the effect of soap...... 95

4. Strength and Limitations...... 96 4.1 Physical examinations and photographic images...... 96 4.3 Sample-taking and microscopic examination ...... 97 4.4 Quality control of microscopy and additional cultivation of fungi in Zürich...... 97 4.5 Results during the first follow-up ...... 98 4.6 Characteristics of the study population...... 98 4.7 Spontaneous resolution and climatic influences ...... 98

5. Conclusions ...... 99

6. Implications...... 100

BIBLIOGRAPHY ...... 101

APPENDIX: CALCULATION OF THE SAMPLE SIZE AND ASSUMED PREVALENCE RATES...... 113

APPENDIX A: TABLES (*NUMBER OF OBSERVATIONS, ROW PERCENTAGES AND COLUMN PERCENTAGES), SYMMETRY TABLES AND FIGURES ...... 114

APPENDIX B: PHOTOGRAPHIC IMAGES ...... 146

APPENDIX C: 1. QUESTIONNAIRE (ENGLISH/KISWAHILI) ...... 161

Appendix D: 1. Informed Consent ...... 176

2. Information for the Parents – provided at school meeting ...... 178

3. Information for the Parents – about the skin diseases: Handouts for parents ....180

4. TREATMENT CARDS ...... 184

5. ID CARD: DISTRIBUTED TO CHILDREN AT FIRST SOAP DISTRIBUTION...... 186

15 List of abbreviations – in alphabetical order A Isolates from Bacteriological or Veterinary Institutes AD Almuth Dinkela AIDS Acquired Immunodeficiency Syndrome ATCC American Type Culture Collection BBE Benzyl Benzoate Emulsion BHI Brain Heard Infusion Agar CI Confidence Interval C.-G. Ciba Geigy CITM Centre International de Distribution de Souches (Lausanne) CM Charles Maswi DEO District Educational Officer DPT Dichlorodiphenyltrichlorethane EKBB Ethik Komitee Beider Basel ELM Epiluminescence Microscopy FDA Food and Drug Administration HHM Household Member HIV Human Immunodeficiency Virus IHRDC Ifakara Health Research and Development Centre JF Julia Ferié KCMC Kilimanjaro Christian Medical Centre KOH potassium hydroxide M Mycophil Agar M-H Müller-Hinton Agar MIC Minimal Inhibitory Concentration MM Marco Mbata NA Nutrient Agar N.A. non-applicable NCIB National Collection of Industrial Bacteria (U.K.) NCTC National Collection of Type Cultures (London) PCP Pneumocystis carinii pneumonia PAS Periodic Acid Schiff SFDDH St. Francis Designated District Hospital SMA Sabouraud Maltose Agar STI Swiss Tropical Institute, Basel 16 Acknowledgements – in alphabetical order I would like to express my deepest appreciations and gratefulness to the following persons. Without their tremendous support and assistance it would not have been possible to carry out the field study, analyse the data and write this dissertation. Werner Baschong Leena Bruckner-Tuderman Dominique Bourgau Julia Ferié Armin Gemperli Christoph Hatz Boniphace Idindili Nada Juricevic Erik Krause Charles Maswi Marco Mbata Jürg Meyer Veronika Mkope Charles Mayombana Hassan Mshinda Dietmar Ochs Antonie Roll Amanda Ross Markus Schiltknecht Peter Schmid-Grendelmeier Marcel Schnyder Thomas Smith Marcel Tanner Adriana Tami Honorathy Urassa Penelope Vounatsou Many thanks also to other staff at STI and the IHRDC and last but not least, the pupils and teachers of Lihami and Michenga Primary School. 17 SUMMARY Background: The present placebo-controlled double-blind study was performed to assess the clinical efficacy of Triclosan against selected superficial dermatomycoses and scabies. The overall effect of regular soap use was also evaluated. Methods: 820 Tanzanian primary school children were examined for the presence of skin disorders. The clinical presentation of the dermatoses was documented. Samples of suspected dermatomycoses were examined microscopically using KOH. Quality control and culture were performed, and the MIC’s of placebo soap and Triclosan against Malassezia spp. were assessed in vitro. The children with superficial dermatomycoses or scabies were selected for this trial. They received either bar soap containing Triclosan (active group) or placebo soap during a period of 8 weeks. They were followed-up after 1 and 2 months. Results: Among the 820 examined children, superficial dermatomycoses were the most common diagnoses, with a total prevalence rate of 33.9%. The prevalence rates of tinea versicolor, tinea capitis, corporis and pedis were 26.2%, 5.5%, 2.6%, 3.2% respectively. Triclosan soap was not significantly more effective against any of the selected dermatoses. Overall cure rates of tinea versicolor, tinea capitis, corporis and pedis were 28.4%, 61.1%, 56.3% and 36.8% respectively. In tinea versicolor soap use improved the overall degree of infection significantly (p = 0.000). The proportion of negative microscopic results increased from 19.0 to 51.6%. Neither the placebo nor Triclosan were efficacious against Malassezia spp. in vitro at relevant concentrations. In tinea capitis the overall degree of infection also improved significantly (p = 0.001). The addition of Triclosan yielded only slightly, non-significantly superior results. In the Triclosan group, the proportion of negative tinea capitis samples increased from 22% to 64%, whereas it decreased from 57% to 53% in the placebo group. In tinea corporis the size of the largest lesion decreased from 2.2 cm to 1.0 cm in the active group whereas it increased in the placebo group. In tinea pedis the cure rate was non-significantly higher for active than for placebo soap (86% versus 56%). The prevalence of scabies was 1.5%. All 6 children included in this study showed an overall tendency towards improvement with either soap. Conclusions: In tinea versicolor the lack of superior efficacy of Triclosan soap is consistent with the lack of in vitro efficacy of Triclosan. The overall improvement may be due to unspecific effects associated with soap use such as a reduction of the amount of superficial skin lipids. In other dermatomycoses, the overall improvement can be explained by the elimination of infectious pathogens from the integument. The in vitro activity against certain dermatophyte spp. explains the slight beneficial effect of Triclosan versus placebo soap. The lack of significance may be due to the mode of application and the common use of vaseline. The results therefore confirm the usefulness of soap, but do not justify the addition of Triclosan to treat superficial skin mycoses and scabies.

18 ZUSAMMENFASSUNG Einleitung: In der vorliegenden Placebo-Kontrollierten Doppelblindstudie wurden die klinische Effektivität von Triclosan und der Gesamteffekt regelmäßigen Seifengebrauchs gegen ausgewählte Dermatomykosen und Skabies ermittelt. Methoden: 820 tansanische Grundschüler wurden bezüglich des Vorhandenseins von Hautkrankheiten untersucht. Bei klinisch vermuteten Dermatomykosen wurden Proben von den Läsionsorten entnommen und mittels KOH mikroskopisch untersucht. Eine Qualitätskontrolle, kulturelle Anzüchtung sowie die Ermittlung der Minimal inhibitorischen Konzentrationen von Placeboseife und Triclosan gegen Malassezia spp. in vitro wurden durchgeführt. Die an oberflächlichen Dermatomykosen oder Skabies leidenden Schüler wurden für diese klinische Studie ausgewählt. Sie erhielten wöchentlich für 8 Wochen entweder eine Triclosanseife oder eine Placeboseife. Nachuntersuchungen erfolgten nach 1 und 2 Monaten. Resultate: Die Dermatomykosen T. versicolor, capitis, corporis, pedis mit Prävelenzen von 26,2%, 5,5%, 2,6%, 3,2% (Gesamt-Prävalenz: 33,9%) insgesamt die häufigsten Diagnosen. Triclosanseife war gegen keine der Dermatosen signifikant wirksamer als das Placebo. Die Gesamtheilungsraten von T. versicolor, T. capitis, corporis und pedis betrugen 28.4%, 61.1%, 56.3% und 36.8%. Der Schweregrad von T. versicolor verbesserte sich durch den Seifengebrauch (Triclosan- und Placebogruppe zusammengefasst) signifikant (p = 0.000). Der Anteil der negativen mikroskopischen Resultate erhöhte sich von 19,0 auf 51,6%. In vitro waren weder Triclosan noch Placeboseife in relevanten Konzentrationen gegen Malassezia spp. aktiv. Der Schweregrad von T. capitis verbesserte sich durch Seifengebrauch ebenfalls signifikant (p = 0,001). In der Triclosangruppe erhöhte sich der Anteil negativer mikroskopischer Resultate von 22% auf 64%, während er sich in der Placebogruppe von 57% auf 53% verringerte. Die durchschnittliche Größe der Läsionen bei T. corporis verringerte sich in der Triclosangruppe von 2.2 cm auf 1.0 cm, wohingegen er sich in der Placebogruppe vergrößerte. In der Triclosangruppe war die Heilungsrate von T. pedis höher als in der Placebogruppe, wenn auch nicht signifikant (86% versus 56%). Die Prävalenz von Skabies betrug 1,5%. Alle 6 in die Studie aufgenommen Kinder zeigten eine deutliche Verbesserung ihrer Infektionen. Schlussfolgerungen: Dass Triclosanseife bei der Behandlung von T. versicolor dem Placebo nicht überlegen war, korrespondiert mit der fehlenden in-vitro-Aktivität der Substanz. Die Gesamtverbesserung könnte auf die durch den vermehrten Seifenkonsum verringerten Hautlipide zurückzuführen sein. Die erwiesene in-vitro-Aktivität gegen Dermatophyten konnte in dieser klinischen Studie nicht signifikant bestätigt werden. Die fehlende Signifikanz könnte durch die Applikationsart und häufigen Gebrauch von Vaseline begründet sein. Die Elimination infektiöser Partikel vom Integument erklärt die Gesamtverbesserung. Die Resultate bestätigen die Nützlichkeit intensiven Seifengebrauchs, rechtfertigen aber den Zusatz von Triclosan nicht, um Dermatomykosen und Skabies zu behandeln. 19 Introduction Introduction Dermatomycoses such as dermatophyte infections and tinea versicolor are among the most common human infections in the developing world (Ayaya et al., 2001; Sunenshine, 1998). Dermatophytoses as well as scabies are known to be among the “dermatoses of poverty” (Schmeller et al., 2001; Stingl, 2001). Easy availability and affordability of treatment alone do not seem to be sufficient to reduce the prevalence rates of these dermatoses in children. It seems to be necessary that standards of living, health education and hygiene improve alongside with the provision of cheap and effective treatment schemes (Schmeller, 1998). The present study was performed in order to assess whether the antimicrobial agent “Triclosan” contained in bar soap is clinically efficacious against selected superficial dermatomycoses and scabies. The overall effect of improved hygiene and regular soap use on signs and symptoms of these diseases was also evaluated. A similar approach has, to this author’s knowledge, so far only been reported from Nigeria, where a large proportion of fungal skin infections and scabies could be cured through the application of soap (Alebiosu et al., 1994). The availability of bar soap – medicated or non-medicated – proven to be clinically effective against these common ailments would be a precious tool to treat and prevent the dermatoses that are discussed in this study. This is true not only for the study participants, but also for those individuals living in comparable settings, of which there are numerous throughout the world.

1. Superficial dermatomycoses

Fungal infections are usually divided into superficial mycoses located on the skin, hair, nails and mucosal membranes, deep or subcutaneous mycoses affecting the subcutaneous tissues and systemic mycoses primarily affecting internal organs (Svejgaard, 1986). The fungi causing superficial mycoses belong to different parts of the taxonomic system: yeasts, dermatophytes and moulds (Svejgaard, 1986). Superficial fungal infections are a common diagnosis in everyday dermatological practice. They constitute 10 – 20% of skin diseases treated in hospitals (Anezia, 1981), and are generally more prevalent in developing than in developed countries (Canizares et al., 1993).

1.1 Tinea versicolor (synonym: Pityriasis versicolor)

This skin disorder is caused by Malassezia spp., which are human saprophytes. So far eleven species have been described of which the majority are lipophilic (Gupta et al., 2003, Mirhendi et al., 2005). Studies conducted within the past six years indicate that the pathogen most frequently 20 Introduction associated with tinea versicolor may not be M. furfur but possibly M. globosa or M. sympodialis (Gupta, 2002; Gupta et al., 2001, Nakabayashi et al., 2000). Although tinea versicolor has a worldwide distribution, it is particularly common in hot and humid tropical areas (Faergemann, 1994; Hay, 1996). Overall prevalence rates in tropical zones may reach 50% (Marples, 1950; Borelli, 1991). In contrast, only 0.5% of males and 0.3% of females were found to suffer from tinea versicolor in the course of a population survey in Sweden (Hellgren et al., 1983), and 1.9% of factory workers examined in central Turkey presented with tinea versicolor lesions (Celik et al. 2003). The majority of cases occur during adolescence. This may be because of hormonal changes and/or increasing sebum production (Akpata et al., 1990). Among children tinea versicolor is generally rare, although it may be found more commonly in this age group in tropical climates (Gupta, 2002; Marples, 1950). Studies have shown variable male to female ratios, but they appear to be nearly equal. In contrast to dermatophyte infections, tinea versicolor is considered neither contagious nor is it due to poor hygiene (Sunenshine, 1998). However, since this infection has been found to be associated with malnutrition, the socio-economic status may indirectly play a role (Gupta, 2002). The organism can be found in 90-100% of healthy subjects as part of the normal skin flora (Canizares et al., 1993; Sunenshine, 1998; Schmidt, 1997; Leeming et al., 1989). Tinea versicolor occurs when the yeast converts to its mycelial form due to certain predisposing factors, which can be divided into endogenous and exogenous factors (Canizares et al., 1993). Endogenous factors include malnutrition, use of oral contraceptives, use of systemic corticosteroids or immunosuppressants, Cushing’s Syndrome, seborrhoeic dermatitis, or hyperhidrosis. According to Sunenshine, hereditary factors might play a part since a positive family history in approximately 17% was confirmed by more than one study (Sunenshine, 1998; Burke, 1961). Hafez et al. described a multifactorial inheritance, with a heridability of 22.2% in first-degree relatives (Hafez et al., 1985). Immune deficiency disorders such as HIV/AIDS are believed to predispose to more extensive infections, but Pityrosporum infections do otherwise not seem to differ from those in HIV-negative subjects (Elmets, 1994; Schechtmann et al., 1995). Exogenous factors include heat and moisture. Furthermore, lesions are most commonly found on body areas covered by clothes which results in an altered pH range (Gupta, 20020; King et al., 1978; Sunenshine, 1998). Exacerbation of the disease may also happen through the application of creams or oils to the skin (Faergemann et al., 1982; Gupta, 2002). The sites affected are usually those with the greatest density of sebaceous glands such as the upper trunk, neck and face. Lesions on the face are more common in children than in adults (Gupta, 2002; Sunenshine, 1998; aTerragni et al., 1991). Other common sites are the upper arms, but the disease 21 Introduction may also occur on the scalp (Goncalves, 1963), the penis (Daneshvar et al. 1987), the neck and the groin (Burkhart et al., 2000; Burke, 1961). Apart from tinea versicolor, Pityrosporum yeasts may also cause pityrosporum folliculitis (Bäck, et al., 1985). Besides, seborrhoeic dermatitis has been found to be associated with the colonization of this yeast in sebum-rich areas (Gupta, 2002; McGinley et al., 1975). Clinical diagnosis may be complicated due to the various forms of presentation. It is usually based on the distribution, shape and appearance of the macules and patches. The furfuraceous scales can be easily removed using the fingernails, which is called “fingernail test (Canizares et al., 1993). Microscopic examination of skin scrapings obtained from the lesions is used to confirm the diagnosis. The scales are usually placed on a microscopic slide, and 10-20% KOH solution is applied to dissolve the keratin (Gupta, 2002). Staining with i.e. 1% methylene blue (Faergemann, 1994) or ink (Dominguez-Soto, 1994) may facilitate the diagnosis (Canizares et al., 1993). Examination reveals round budding cells (spores) and hyphae or mycelia. This pattern is also referred to as “spaghetti and meatballs” (Sunenshine, 1998). Additionally, the fungus may be cultivated e.g. on Sabouraud’s agar rich in lipids. Bacterial growth and growth of moulds is inhibited by addition of chloramphenicol and cycloheximide respectively (Gupta, 2002; Enweani, 1996). The main complaint of the patient is cosmetic disturbance, but mild pruritus may be present (Gupta, 2002; Bélec, 1991). Differential diagnosis includes vitiligo, leukoderma, chloasma, dermatophytoses, seborrhoeic dermatitis, psoriasis pityriasis rosea, pityriasis alba, pityriasis rotunda, confluent and reticulate papillomatosis of Gougerot-Carteaud, secondary syphilis, pinta, leprosy and erythrasma (Gupta, 2002; Sunenshine, 1998; Canizares, 1983). Wood’s Light can help to distinguish tinea versicolor from dermatophytosis not caused by certain fluorescing species such as Microsporum spp. or T. schoenleinii and from erythrasma, which follows infection with Corynebacterium minutissimum (Cheesbrough, 1997). Ringworm lesions caused by other Trichophyton spp., Epidermophyton spp. or few geophilic Microsporum spp. generally do not fluoresce at all, and erythrasma would appear coral-red, whereas tinea versicolor lesions show a yellow-green fluorescence (Gupta, 2002; Sunenshine, 1998). There are various options to treat tinea versicolor, both topical or systemic variants may be effective. Examples of topical agents are selenium sulfide, propylene glycol 50% in water (Faergemann, 1994), ciclopiroxolamine or imidazoles (Gupta, 2002). Imidazoles are also effective if administered orally, whereas terbinafine and griseofulvine are not (Gupta, 2002). However, the treatment is associated with a relatively high rate of recurrence reaching 60 to 80%, according to Arenas et al. (Arenas et al., 1982). 22 Introduction 1.2 Dermatophyte infections (synonym: Ringworm infections)

Dermatophyte infections are limited to the superficial layers of the epidermis and its appendices such as the hair and nails (Shrum, 1994) and are able to invade these structures (Elewski, 1986). They are classified according to genera, namely Trichophyton, Epidermophyton and Microsporum (Canizares, 1983; Hay, 1996), according to ecology (antropophilic, geophilic or zoophilic dermatophytes) (Shrum, 1994) and according to anatomic locations of infection (Weitzman et al., 1995). Infections caused by geophilic or zoophilic fungi are generally more severe than those caused by anthropophilic fungi (Canizares, 1983). The prevalence of dermatophyte infections is higher in developing than in industrialized countries because of prevailing poverty, overcrowding, sharing of fomites, poor hygiene and new drug resistant pathogens (Ayaya et al., 2001; Canizares et al., 1993). Increasing use of immunosuppressive therapy, chemotherapy, antibiotics and the HIV/AIDS pandemic, which affects particularly the poorest areas of the world, further contribute to the emergence of these infections (Ayaya et al., 2001; Elmets, 1994; Elewski, 2000). Patients with AIDS may present with clinically atypical or more extensive lesions, but the incidence is not higher than among immunocompetent individuals (Hay, 1996).

1.2.1 Tinea capitis

In the developing world tinea capitis remains the predominant clinical type among children (Welsh et al., 2002; Al-Sogair, 1991; Porter, 1980; Shrum, 1994; Canizares, 1983). The spectrum of causative agents is variable depending on the setting of the studies. Prevalence at primary schools ranges between 11.3% in Ivory Coast (Hervé et al., 2002) and 33.3% in Kenya (Ayaya et al., 2001). Tinea capitis may present with different symptoms (Shrum, 1994). The clinical manifestations can be divided into three groups (Canizares et al., 1993): 1. One comprises scaly, dry, non-inflammatory lesions sometimes presenting grey patches or “black dots”, which appear when hairs break off close to the hair follicle. This type usually does not produce permanent alopecia. It is often caused by anthropophilic species and can be subdivided into a microsporic and a trichophytic variety. 2. Another presentation is the more acute, wet, and inflammatory type with possible suppuration or kerion formation (Shrum, 1994). Kerion celsi are characterized by erythematous nodules, swelling, and regional lymphadenitis (Hussain et al., 1999) and represents a hypersensitivity, often self-limiting, reaction to the invading pathogens (Hussain et al., 1999; Weitzman et al., 1995; Canizares, 1983). 23 Introduction 3. The third type is dry, crusted, occasionally inflammatory and suppurative, caused mainly by T. schoenleinii and other faviform fungi. This clinical picture is also referred to as “favus”. It is characterized by yellow crusts which are referred to as scutulae (Canizares et al., 1993; Canizares, 1983) (cup-shaped areas of densely interwoven mycelium, scales, and debris). They may become confluent and appear as mats of honeycomb-like areas (Shrum, 1994) leading to permanent alopecia (Canizares et al., 1993). Bacterial superinfection and cervical lymphadenopathy may be present, and the patient may develop an id reaction (Shrum, 1994; Canizares, 1983). Sometimes the infection may also be accompanied by fever and malaise (Weitzman et al., 1995). The differential diagnosis includes other disorders involving scaling and/or hair loss such as seborrhoeic dermatitis, scalp psoriasis, alopecia areata and trichotillomania (Shrum, 1994). Tinea capitis should be treated systemically as topical treatment is much less effective and in many cases useless (P. Schmid-Grendelmeier, personal communication). Usually, topical antifungal agents are recommended only as adjuncts to systemic treatment because they cannot penetrate into hair follicules sufficiently (Chan et al., 2004; Canizares et al., 1993; Weitzman et al., 1995; Schmeller, 1998). The systemic application of griseofulvin has revolutionised treatment, especially of endothrix infections. However, the dosage needed to cure tinea capitis has been slowly increasing over the years (Gupta et al., 2003). Due to rapid elimination, long duration of therapy is necessary, which may reduce patient compliance (Gupta et al., 2003). Newer antifungal drugs like terbinafine, itraconazole or fluconazole are comparable in efficacy (Chan et al., 2004) and have longer retention periods in the affected tissues (Gupta et al., 2003). In theory Terbafine is now available as an oral formulation, but not in the study setting that is described in the present study. The newer agents are generally more expensive than griseofulvin, which is still the only FDA-approved drug and for the treatment of tinea capitis (Gupta et al., 2003), but have much less side effects. Even though in temperate climates the drugs of choice are nowadays terbinafine or itraconazole, griseofulvin or ketoconazole remain the first choice in the tropics (Welsh et al., 2002; P. Schmid-Grendelmeier, personal communication). Whitfield’s Ointment (5% benzoic acid, 5% salicylic acid and basic cream), tolfnaftate, haloprogin and clotrimazole have been recommended for topical treatment. Besides, the application of topical or systemic antibiotics or prednisolone may become necessary (Canizares et al., 1993). Apart from drug treatment, general sanitation measures have to be taken to prevent the disease from spreading (Canizares et al., 1993; Weitzman et al., 1995). Taking these conditions into consideration, it can be concluded that a realistic approach to prevent and reduce ringworm infections appears to be a change in hygiene behaviour. This has been 24 Introduction achieved for example in Sri Lanka where the custom of frequent head baths with soap and water is likely to be responsible for the relatively low prevalence of tinea capitis in certain studied communities (Attapattu, 1989). Alebiousu et al. from Nigeria were able to cure more than half of their study population with either tinea capitis or corporis by applying a soap containing plant- derived ingredients for several weeks (Alebiosu et al., 2003).

1.2.2 Tinea corporis

This term usually describes all dermatophyte infections that do not involve the scalp, ear, face, beard, axillae, hands, feet and groin (Shrum, 1994). In the present study, facial lesions are described as tinea corporis lesions as well. Tinea corporis is common in children, but can also be found frequently among adolescents and adults (Soyinka, 1978; Weitzman et al., 1995). Prevalence rates in tropical regions such as Gambia have been reported to range between 3% in the wet season and 0.7% in the dry season (Porter, 1980); prevalence rates among primary schoolchildren were found to be 2.7% in rural Ethiopia (Figueroa et al., 1996) and 0.8% in Nigeria (Soyinka, 1978). Tinea corporis involves glabellar skin and presents with single or multiple oval, scaly patches with central clearing, sometimes delineated by versicular borders (Weitzman et al., 1995; Canizares, 1983). Especially in the tropics the presentation may be polymorphic (Canizares et al., 1993; Shrum, 1994). In case of invasion, the formation of “Majocchi’s Granuloma” may occur, usually after minor trauma (Shrum, 1994). A distinct clinical type is tinea imbricata showing a polycyclic pattern of the scaly lesions (Canizares et al., 1993; Shrum, 1994; Hay, 1996). As for tinea capitis, topical treatment may not be sufficient and must then be supplemented by griseofulvin or ketoconazole or other systemic agents (Shrum, 1994; Weitzman et al., 1995). However, in single lesions topical treatment is mostly effective (P. Schmid-Grendelmeier, personal communication).

1.2.1 and 1.2.2 Diagnosis

Diagnosis for tinea corporis and capitis is made by microscopic examination of skin scrapings and hair clippings (in case of tinea capitis) with KOH solution or by fungal cultures. Spores can be seen either in or outside the hair (endothrix or ectothrix infection), depending on the causative species. Besides, hyphae and mycelia may be found. The diagnostic test with best specifity is fungal culture on Sabouraud’s agar; additionally, Wood’s Lamp or biopsy followed by PAS or silver staining can be used (Shrum, 1994). Differential diagnosis of tinea corporis should include nummular eczema, psoriasis, drug eruptions, mycosis fungoides and pityriasis versicolor (Gupta, 2002; Shrum, 1994). 25 Introduction 1.2.3 Tinea pedis

This disease, also known as “athlete’s foot”, is a common diagnosis in white urban populations (Shrum, 1994; Canizares, 1983; Canizares et al., 1993). According to Shrum et al. it is a rare condition in African populations, as well as in populations of other tropical regions (Shrum, 1994). However, Soyinka found that the incidence of tinea pedis among secondary school children in Nigeria was higher than that of other dermatomycoses (Soyinka, 1978). Another school survey in Nigeria yielded a prevalence of 1.5% among the pupils (Enweani, 1996). Generally it is more common among the older age groups (Shrum, 1994; Canizares, 1983). Clinically, there are three varieties of tinea pedis (Canizares et al., 1993). 1. The first is the intertriginous type, with interdigital maceration, accompanied by whitish thickening of the web spaces of the feet. It may extend to both the toes and the soles (Masri- Fridling, 1996), pruritus and foul odour may be present. 2. The second type is the dishydrotic type with acute vesicular, bullous or vesiculo-bullous lesions with erythema and fissuring. This type is usually located on the mid-sole. 3. The third form is the hyperkeratotic moccasin type, which tends to be chronic and presents with scaling of the plantar surface (Shrum, 1994). Vesiculation is usually absent. Asymptomatic infection is common in tinea pedis (Masri-Fridling, 1996), but severe cases involving mixed infection by dermatophytes, candida and bacteria may also occur (Weitzman et al., 1995; Hay, 1996). Bacterial superinfection is mostly noted in the interdigital type (Masri-Fridling, 1996). This feature is then referred to as “dermatophytosis complex” (Canizares et al., 1993; Odom, 1993). Diagnosis is made using KOH preparations and culture, as described above for the other dermatophyte infections. Differential diagnosis includes psoriasis, contact dermatitis, dyshidrosis and pompholyx (Shrum, 1994; Canizares, 1983). Candidiasis, erythrasma, acrodermatitis contiua, pyodermas and secondary syphilis should also be considered (Masri-Fridling, 1996). Treatment with topical fungistatic or fungicidal agents may be successful. Antibacterial soaps are recommended in cases of mixed bacterial and fungal interdigital infections. Severe cases may require the addition of systemic agents such as itraconazole or terbinafine and oral antibiotics (Masri-Fridling, 1996).

1.2.4 Tinea cruris

This term describes dermatophyte infections of the groin, perianal and perineal areas and the upper thighs. The infection is most common in adult men (Weitzman et al., 1995). Lesions usually appear as erythematous or brown scaling patches. Involvement of the groins can be bilateral, and lesions may present as annular patches, with active margins showing vesiculation and crusting (Weitzman 26 Introduction et al., 1995; Canizares et al., 1996). It is more common in hot and humid climates, especially if the standard of hygiene is low (Anezia, 1981). Differential diagnosis between tinea cruris caused by dermatophytes, Erythrasma caused by Corynebacterium minutissimum, intertrigo, which may be of bacterial origin, and candidiasis may be difficult if no microscopic diagnosis or fungal culture are performed (Canizares, 1975). Wood’s light is a helpful device to identifiy C. minutissimum (also see 1.1.1). In this study, it was not in all cases possible to examine the inguinal or genital area. Furthermore, only few samples could be obtained as privacy or an investigator of the same sex as the examined child could not always be guaranteed. Since differential diagnosis could thus not be made, inguinal lesions are referred to as “intertrigo”, summarizing all the above mentioned differential diagnoses affecting the inguinal area.

1.2.5 Tinea manuum

Tinea manuum usually presents as unilateral diffuse hyperkeratotic lesions, localised in the interdigital spaces or the palms (Weitzman et al., 1995; Canizares et al., 1993). Simultaneous involvement of the feet is common (Canizares et al., 1993).

1.2.6 Tinea unguium

Dermatophytes may also invade nails. This infection is called tinea unguium. Infection of the nail by non-dermatophytic fungi is referred to as onychomycosis. The invasion can be subungual or superficial, which is also known as leukonychia trichophytica (Weitzman et al., 1995). T. rubrum and T. mentagrophytes are the most common dermatophytes causing this infection (Weitzman et al., 1995). The management of fungal nail infections has greatly improved with the introduction of systemic agents as well as topical nail polishes containing ciclopirox and amorolfine (Gupta et al., 2003).

1.2.7 Dermatomycoses caused by non-dermatophytic fungi

Primary infections of the skin and nails may also be caused by fungi other than dermatophytes or Malassezia and Candida yeasts. These are for example Exophiala werneckii, Piedra hortae, Trichosporon belgii, which cause tinea nigra palmaris, black piedra and white piedra respectively. Aspergillus, Acremonium spp. and Scopulariopsis brevicaulis often invade nails and thus produce onychomycosis (English et al., 1994; Elewski et al., 1989). Other species such as Hendersonula toruloidea and Scytalidium hyalinum are non-dermatophyte saprophytic moulds found in soil, water, air and on fomites (Masri-Fridling, 1996; Elewski et al., 1991). These facultative pathogens may cause dermatomycoses mimicking the clinical picture of 27 Introduction certain ringworm infections. Pathogenic moulds should be suspected if the infection is resistant to standard antifungal treatment, if standard cultures fail to grow and if KOH preparations are nevertheless positive (Masri-Fridling, 1996). Since species identification was not possible in all cases in the present study, dermatomycoses other than tinea versicolor will be referred to as “other superficial dermatomycotic infections”.

1.3 Scabies

Scabies remains a major public health problem in developing countries, especially because of the HIV/AIDS pandemic (Usha et al., 2000; Green, 1989). The prevalence of scabies has been found to be associated with similar factors as that of dermatophytoses, such as poverty, crowding in households and poor hygiene (Inanir et al., 2002). Several further factors such as population movements, wars, misdiagnosis, inadequate treatment, and changes in the immune status of the population have been suggested to affect the epidemiology of scabies (Green, 1989). In 1962 Schaller found that between 11 and 57% of schoolchildren in various Ethiopian provinces were infected (Schaller, 1969). Since 1990 prevalence rates ranging between 1.7% and 17% have been reported from Nigeria and Ethiopia (Enweani, 1996; Figueroa et al., 1996). Scabies is a contagious diseases, and its transmission is being favoured by warm closeness (Canizares et al., 1993), thus affecting most likely the whole familiy at the same time (Anezib, 1981; White, 1996). The infection is caused by the itch mite, Sarcoptes scabiei. The female parasites deposit their eggs and faeces into burrows in the stratum corneum of the skin and die 14 days after fertilization. The male parasites die immediately after fertilization. Larvae hatch after three to four days (Anezib, 1981; White, 1996). The six-legged larva passes through a nymphal state and finally turns into an eight-legged adult (Canizares, 1983; Canizares et al., 1993). Three features are diagnostic of scabies (Canizares, 1983). 1. Itching, the most characteristic symptom, starts between three days and six weeks after the first infection, due to an Type IV hypersensitivity reaction against the mites and their products (Burkhart et al., 2000). In subsequent infections pruritus appears within a few days. Typically, pruritus is most severe at night. 2. Observing scabies burrows between the fingers or at other typical sites is pathognomonic for the infestation (Burkhart et al., 2000). However, in children they may be difficult to find (White, 1996). When opening a burrow, the mite can be extracted with a sharp instrument and examined directly or microscopically (Canizares, 1983). Superficial scrapings from ulcers may contain mites, scybala or eggs (White, 1996). 28 Introduction 3. The distribution of the lesions is characteristic. Burrows are typically localized in the webs and on the sides of the fingers, wrists and ulnar border of the hands, elbows, anterior axillary folds, areola of the breasts, umbilicus, waist, external genitalia and perineum, natal cleft, thighs, knees, ankles and the palms and soles (Anezib, 1981). The distribution of the urticarial rash does not necessarily correspond with the infestated sites (White, 1996). In tropical environments scabies has to be distinguished from insect bites, papular urticaria, and pediculosis. In immunocompromised individuals, crusted scabies may be found, involving profuse crusting of the skin and the formation of hyperkeratotic plaques (White, 1996). Asymptomatic cases of scabies have been reported (Jiminez-Lucho et al., 1995); some may be heavily infestated with millions of mites but not show no crusted lesions (Estes et al., 1993). Scabies in children is often atypical in appearance making differential diagnosis difficult at times. Differential diagnosis includes eczema, pyoderma, and in the tropics also onchocerciasis (White, 1996). Although scabies may not represent a major disease per se, it can be associated with secondary complications such as bacterial superinfection, eczematisation and poststreptococcal glomerulonephritis (White, 1996). Systemical treatment of scabies with Ivermectin is effective (Terri et al., 1995), but topical treatment also yields good results. 5% permethrin cream (Canizares et al., 1993), 0.3% lindane cream, topical ivermectin, benzyl benzoate emulsion, crotaminon and sulfur are used as topical agents (Pruksachatkunakorn et al., 2002; Pönnighaus, 1995; Anezib, 1981; Victoria et al., 2001). At the dispensaries in and around Ifakara, scabies is usually treated with BBE (Benzyl benzoate emulsion (Dr. M. Mbata, personal communication).

2. Information about Triclosan

The broad-spectrum antimicrobial agent Triclosan has been used worldwide for skin care products during the last 30 years. It is a nonionic, off-white, odorless and tasteless powder. The chemical name is 2,4,4’-trichloro-2’-hydroxy-diphenyl-ether (C12H17Cl3O2) (Jones et al., 2000). Triclosan was originally developed by Ciba-Geigy Company, Basel, Switzerland, and has been used in the United States since the 1960s in under-arm deodorants and bar soaps. In 1972 it was for the first time introduced into health care as a surgical scrub (Jones et al., 2000). In many countries Triclosan can nowadays be found in a wide range of further products apart from deodorants and soaps, such as shower gels, health care personal hand washes, lotions, creams, toothpastes etc (© Ciba Specialty Chemicalsb, 1998). The chemical is currently distributed as IRGASAN® DP 300 for skin applications and IRGACARE® MP for oral applications (Jones et al., 2000). 29 Introduction 2.1 Properties

2.1.1 Safety

IRGASAN® DP 300 / IRGACARE® MP is not toxic in acute toxicity tests when used in formulations, not irritating to skin and eyes, non-sensitizing, non-carcinogenic, non-mutagenic, not toxic to reproduction, non-teratogenic, completely eliminated and does not accumulate in organs or tissues. It is well tolerated in humans (© Ciba Specialty Chemicalsb, 1999). Based on the broad safety package and the long-term experience using this antimicrobial, IRGASAN® DP 300 / IRGACARE® MP is considered to be safe for humans when used in the recommended concentrations (Bhargava, et al., 1996).

2.1.2 Anti-inflammatory effect and anti-irritative effects

Triclosan was proven to be an effective inhibitor of cyclo-oxygenase and lipo-oxygenase, key enzymes of the arachidonic acid metabolism, thus reducing the formation of pro-inflammatory metabolites such as prostaglandin E2 and leukotriene B4 (Nissen et al., 1998). Triclosan is therefore able to reduce inflammatory skin reactions and skin irritations (Kjaerheim et al., 1995; Skaare et al., 1997; Barkvoll et al., 1994). Barkvoll and Rölla showed that Triclosan is capable of reducing skin irritation caused by sodium lauryl sulphate (Barkvoll et al., 1994) as well as allergic reactions to nickel in nickel sensitized patients (Barkvoll et al., 1995). In another study, Nissen and Ochs demonstrated the concentration dependent anti-inflammatory efficacy on erythema caused by UV- radiation (Nissen et al., 1998).

2.1.3 Broad-spectrum antimicrobial activity

Triclosan shows broad-spectrum antimicrobial activity against most gram-positive, gram-negative bacteria, moulds and yeasts (Nissen et al., 1998). The antimicrobial spectrum and the speed of activity of Triclosan have been documented both as various formulations and as an active ingredient (Bhargava, et al., 1996; Larson, 1988; The Soap and Detergent Association and The Cosmetic, Toiletry, and Fragrance Association, 1995; Jampani et al., 1998). These documentations include in vivo as well as in vitro tests (The Soap and Detergent Association and The Cosmetic, Toiletry, and Fragrance Association, 1995). Data collected in several in vitro tests with respect to the antimicrobial efficacy against fungal pathogens are listed below (see Table 1).

30 Introduction Table 1: Minimum inhibitory concentrations (MIC) of IRGASAN®DP300 /IRGACARE®MP: Yeasts and Moulds (© Ciba Specialty Chemicalsa, 1998):

Microorganism Strain Medium* MIC-value Aspergillus fumigatus ATCC 9197 SMA 10 ppm Aspergillus niger ATCC 6275 M 30 ppm Aspergillus repens ATCC 44409 M 5 ppm Blastomyces dermatitidis ATCC 10225 SMA 3 ppm Candida albicans A NA 3 ppm Candida albicans A NA 33 ppm Candida albicans ATCC 10259 M 3 ppm Candida albicans ATCC 10259 BHI 4 ppm Candida albicans ATCC 10231 BHI 4 ppm Candida albicans ATCC 11651 10 ppm Candida albicans A SMA 10 ppm Candida albicans A 10 ppm Candida albicans A K 75 10 ppm Candida guillermondii A K 334 10 ppm Candida guillermondii A SMA 33 ppm Candida krusei DSM 70075 M-H 10 ppm Candida lipolytica DSM 1345 M-H 1.0 ppm Candida parakrusei A SMA 4 ppm Candida parapsilosis A SMA 33 ppm Candida parapsilosis A K 332 30 ppm Candida pulcherrima A SMA 33 ppm Candida stellatoidea A SMA 10 ppm Candida tropicalis A SMA 10 ppm Candida tropicalis A K 644 10 ppm Candida tropicalis DSM 1346 M-H 10 ppm Candida utilis A SMA 33 ppm Chaetomium globosum ATCC 6205 SMA 10 ppm Cryptococcus neoformans A SMA 10 ppm Epidermophyton floccosum ATCC 10227 SMA 10 ppm Epidermophyton floccosum CBS 55384 M-H 2.5 ppm Hansenula anomala A SMA 10 ppm Keratinomyces ajelloi A SMA 10 ppm Malassezia furfur ATCC 14521 SMA >1000 ppm Malassezia furfur DSM 6171 SMA 500 ppm Microsporum canis ATCC 10214 M 3 ppm Microsporum canis ATCC 10214 SMA 1-10 ppm Microsporum cookei A SMA 33 ppm Microsporum cookei A K 406 M 3 ppm Microsporum gypseum A SMA 10 ppm Microsporum nanum A SMA 10 ppm Neurospora sitophilia A SMA 0.1 ppm Paecilomyces varioti ATCC 18502 SMA 10 ppm Penicillium egyptiacum A SMA 33 ppm Penicillium expansum DSM 1282 SMA 31.2 ppm Penicillium funiculosum ATCC 9645 SMA 10 ppm Pichia farinosa A SMA 33 ppm Piedraia hortai A SMA 10 ppm Rhodotorula mucilaginosa A SMA 33 ppm Saccaromyces cervisiae ATCC 2601 BHI 4 ppm 31 Introduction Scopulariopsis brevicaulis ATCC 36840 M 15.6 ppm Schizosaccharomyces pombe A SMA 3 ppm Sporothrix schencki ATCC 10212 SMA 3-10 ppm Torulopsis dattila A SMA 10 ppm Torulopsis famata A SMA 10 ppm Trichoderma viride ATCC 18502 SMA 20 ppm Trichomonas foetus A 100 ppm Trichophyton concentricum A SMA 10 ppm Trichophyton cutaneum A SMA 10 ppm Trichophyton mentagrophytes ATCC 9533 M 3 ppm Trichophyton mentagrophytes ATCC 9533 SMA 1 ppm Trichophyton mentagrophytes ATCC 9533 M-H 1.25 ppm Trichophyton quinckeanum A SMA 10 ppm Trichophyton rubrum A K 351 M 3 ppm Trichophyton rubrum DSM 4167 M-H 0.63 ppm Trichophyton rubrum A SMA 10 ppm Trichophyton schoenleinii A SMA 3 ppm Trichophyton terrestre A SMA 10 ppm Trichophyton tonsurans A SMA 10 ppm

* Media: NA = Nutrient Agar M = Mycophil Agar M-H = Müller-Hinton Agar SMA = Sabouraud Maltose Agar BHI= Brain Heard Infusion Agar

Sources: ATCC = American Type Culture Collection NCTC = National Collection of Type Cultures (London) CITM = Centre Intern. de distribution de Souches (Lausanne) NCIB = National Collection of Industrial Bacteria (U.K.) C.-G. = Ciba Geigy A = Isolates from bacteriological or veterinary institutes

The formulation of the product may affect the antimicrobial efficacy due to differences in pH, detergent base, emollients and humectants (a substance that promotes retention of moisture), ionic nature of the formulation, and the type of surfactants used (Jones et al., 2000). In order to restore the antimicrobial activity in surfactant-based solutions, the interaction between Triclosan and the surfactant molecules forming micelles around the Triclosan molecules can be reduced by a technique called de-blocking (Jones et al., 2000). Triclosan has been used successfully to reduce the nosocomial outbreaks involving antibiotic- resistant bacteria (Jones et al., 2000). In vivo studies have revealed that Triclosan in surgical scrubs is effective against the transient bacterial flora (Jones et al., 2000; Larson, 1995). Triclosan’s effect on the skin flora is persistent and cumulative (Bhargava, et al., 1996). A long-lasting activity on the skin has been proven for products containing Irgasan DP 300. If applied as a rinse-off formulation, the ingredient displays a protection against the growth of pathogens even after multiple washes with a non-antimicrobial soap (© Ciba Specialty Chemicalsb (1998). 32 Introduction Triclosan applied as a 1% formulation offers excellent immediate, broad-spectrum and persistent antimicrobial activity combined with gentleness to the skin, even if applied frequently (Jones et al., 2000).

2.1.4 Mechanism of action against dermatophytes, moulds and yeasts

The in vitro efficacy against many dermatophytes, moulds and yeasts can be explained by interactions of the agent with the fungal cell membrane. Mainly unspecific effects such as disorganization of the cytoplasmatic membrane and leakage of low molecular weight cellular components as well as inhibition of nutrient uptake are considered to be important factors with regard to the mode of action of Triclosan. The extent to which the inhibition of the enzyme enoyl reductase, which is essential for synthesis of fatty acids and is an important target for Triclosan in bacteria, particularly at low concentrations of Triclosan, plays a role in the activity against fungi is not yet fully understood (Regös et al., 1974; D. Ochs, personal communication).

2.1.5 Concentrations currently in use

In cosmetic products Triclosan is usually available in concentrations ranging between 0.1 and 0.3%. These products include under arm deodorants, deodorant bar and liquid soaps, liquid hand washes, shower and bath products, lotions, creams, hair shampoos and shaving products. If applied as a medical soap, the concentration of Triclosan has to be higher and reaches 1%. As a rule, rinse-off formulations require higher concentrations than leave-on formulations.

2.2 Triclosan-containing products currently in use in Africa

There are several products currently available in East Africa, which contain Triclosan. Examples are Protex® Antibacterial Soap (Colgate, Nairobi; Bodycare, Dar es Salaam), Lifebuoy®/Asespo® Soap (Unilever Nairobi), Roberts® Antiseptic Soap (Cussons Nairobi), Familiy Medicated Soap® (G&N Soap, Dar es Salaam), and Tropcial Deo® (Buyline, Nairobi).

33 Methods Methods

1. Study Area

A placebo-controlled, double-blind clinical trial was carried out in Ifakara, Kilombero District, Morogoro Region, in southern Tanzania, to assess the efficacy of Triclosan in soap against selected dermatomycoses and scabies. The district is a rural area with an estimated population of approximately 322,000 (United Republic of Tanzania, 2002). The district capital, Ifakara, is situated in the river plain of Kilombero River, 270 m above sea level and 320 km southwest of Tanzania’s largest city, Dar es Salaam. The estimated annual rainfall is 1350 mm/year. There are two rainy seasons, from October to December and from February to May (Armstrong Schellenberg et al., 2003). Ambient temperatures are highest in December and lowest in July (The management and ecology of Tanzanian forests; 2001) (also see Table 1). Most inhabitants are subsistence farmers who grow mainly rice, maize, cassava and bananas (aTanner et al., 1987). Other common occupations are fishing and small-scale trading (Armstrong Schellenberg et al., 2002).

Figure 1: Tanzania (Source: SuperTravelNet.Com) Figure 2: Morogoro Region and Ifakara © Acosta et al., 2001

Table 1: Overview of climatic conditions in Ifakara during the time of the study (Source: IPS Meteostar®) Average High Average Low N° of days Month (°C) (°C) with rain April 30.6 25.0 11 Screening Exami- May 28.3 23.3 17 nation Follow-up 1 June 27.2 22.2 12 Follow-up 2 July 26.2 21.6 12

The health situation in Kilombero District is still generally poor, prevalence rates of malnutrition and communicable diseases are high, and availability of food, clean and safe water, and sound environmental sanitation are limited. Common water sources are communal boreholes, natural 34 Methods spring or river water, and hand-dug wells. The roads are unpaved and transport is difficult in the rainy season. The public health in system is organized through dispensaries, health centres and hospitals. Maternal and child health clinics are widely available (Armstrong Schellenberg et al., 2002). The dermatologic outpatient department at Ifakara’s St. Francis Designated District Hospital (SFDDH) with one dermatologist serves a population of over 600,000 of the whole diocese (Kilombero and Ulanga District combined) (Kibatala, 2002). According to the 1997 St. Francis Designated District Hospital’s (SFDDH) Annual Report, fungal, parasitic and bacterial skin disorders in the rural population, are still highly prevalent among the local population compared to the urban population (Kibatala, 1997).

2. Selection of Primary Schools

After the study protocol was approved by the ethical committees in Basel, Switzerland, (EKBB) and in Tanzania (IHRDC), two primary schools (3 - 8 km from Ifakara town centre) were selected for the study. This selection occurred in agreement between the local supervisor, Dr. B. Idindili, the representative in charge of the District Medical Office and the District Educational Officer (DEO). A written permission documenting this decision was obtained from the DEO. The field investigators then introduced themselves to the teachers of the schools in order to explain the concept and the procedures of the study and to present them the written permission. Both schools agreed to take part in the study. The screening examinations were started at Lihami Primary School and then continued at Michenga Primary School until enough children could be recruited for the soap trial.

3. Screening Examinations

The screening examinations were performed at two primary schools (Lihami and Michenga Primary Schools), 3 and 8 km from Ifakara town centre. All the physical examinations in the course of this randomized, double-blind, prospective, placebo-controlled study were performed in Swahili by the dermatologist of the local district hospital (SFDDH), Dr. M. Mbata (MM), a clinical officer, C. Maswi (CM), and the medical students from the University Hospital Freiburg, Germany, Julia Ferié (JF) and Almuth Dinkela (AD) (principal investigator of the soap trial). At Lihami Primary School the interview and examination were carried out in two small rooms (each approximately 2.5 m2 x 2 m2) with two windows serving as light source. In Michenga, a larger room with a curtain generating privacy was available. The first screening of each child consisted of four parts: (i) the examination of the skin, (ii) written documentation of the history and clinical presentation of skin diseases currently present, following standardized definitions and (iii) supported by digital photography, (iv) 35 Methods sample-taking (skin scrapings and/or hair clippings) at the active borders of the lesions. These steps were carried out as follows: (i): The entire body surface, hairs and nails were examined. The examination of the genital region was not possible in every child because privacy could not always be guaranteed depending on the room in which the examinations were carried out. The largest diameter of the biggest lesion was recorded. (ii): Several questions concerning the hygiene behaviour and living conditions were asked. These questions were evaluated in another study (For details please see Ferié, 2005). If during the physical examination a skin disease was discovered, the history-taking and description of the skin disease was documented using a standardized questionnaire in Kiswahili. In case that several diagnoses were present, the “principal diagnosis” was described in detail and characteristic signs and symptoms of the disease were assessed. The findings related to the other diagnoses were also documented. In the case of dermatomycoses and scabies the characteristic parameters were summarized in a clinical score to assess the degree of infection (see “Patients, Approaches and Methods: 6. Degree of infection”). In case of the presence of several diagnoses of interest for soap trial, the “principal diagnosis” was selected according to the priorities of this study: - if several superficial fungal infections were present, the order was as follows: 1. Tinea capitis 2. Tinea corporis 3. Tinea versicolor 4. Tinea pedis - scabies was always the main disease (iii): For each skin disease identified the main skin lesion was documented by digital photography. In order to identify the child in the picture a piece of paper displaying the seven digit identification number number of the child was placed beside the skin lesion. The attempt was to take pictures of the same lesion during each of the three examinations. All uncertain and various representative cases, were cross-checked by the expert dermatologist, PD Dr. P. Schmid-Grendelmeier in Zürich, a fully trained dermatologist, skilled with experience in tropical skin diseases. (iv): If tinea corporis, tinea capitis or tinea versicolor infection was suspected, a sample was taken at the active borders of the lesions as described by Weitzman et al., and Gupta et al. and Stein (Weitzman et al., 1996; Gupta, 2002; Gupta et al., 2001; Stein, 1983). In the case of tinea capitis, apart from the skin scrapings, hair clippings or scrapings from hair stumps were taken if possible. The samples were taken by using sterile surgical blades (n° 15). The scales were placed on pieces of white paper, which were then folded, closed with stapler pins and stored in plastic envelopes, which 36 Methods are normally used by the local pharmacies to store the pills to be sold. A new plastic envelope was used for each child. If several samples were taken of one child, the different body sites were documented on the piece of paper on which the scales were placed. In the case of clinically diagnosed scabies, samples were taken from suspicious lesions with a sterile surgical blade (n° 15) using immersion oil. They were directly placed on a microscopic slide, covered with a cover slip and then preserved with clear polish and stored in a plastic envelope. All samples were brought to the laboratory at IHRDC for microscopic examination on the same day or after a maximum of two days. The microscopic examination was performed by AD and JF. The fungal scales were put on a microscopic slide on which 20% potassium hydroxide solution had been applied, and covered with a cover slip. After 20 minutes the samples were examined using 10 and 40 fold magnification. The result was regarded as positive if hyphae and/or mycelia and/or large amounts of spores were seen. No sample was taken from tinea pedis lesions. The scabies samples were examined with 10 fold magnification. The result was regarded as positive if mites, scybala or eggs could be discovered. Quality control was ensured by controls performed by H. Urassa, microbiologist and research scientist at IHRDC (Ifakara Health Research and Development Centre). In all cases of suspected fungal infections, particularly if differential diagnosis between tinea corporis and tinea versicolor infections was difficult, additional skin samples were taken after one, two and approximately three months after the baseline examination, stored in the plastic envelopes in the usual manner and then transported to Zürich via air courier services to the laboratory of mycology at the University Hospital Zürich. Here three drops of Congo Red solution were used instead of KOH solution for microscopic diagnosis, six to seven pieces of scales were added and covered with a coverslip. Examination of the samples was performed using a 20 to 40 fold magnification. The remaining scales were placed on three different agars for culture (see Table 2).

Table 2: Composition of different media used for microscopic examination and culture Composition of Congo Red solution: Composition of Dixon Agar: SDS (sodium dodecyl sulphate) 5 g Bacto Malt Extract 5 mL Congo Red 0.5% in Ethanol 50% 5 mL Bacto oxygal 20 g Distilled water 1000 mL Bacto glycerol 2.5 mL Tween 40 10 mL Distilled water 1000 mL Bacto Agar 15 g Chloramphenicol 50 mg Gentamycin 5 mg Cycloheximide + Aceton 0.4 g Composition of Sabouraud Glucose Agar: Composition of Mycosel Agar (containing cycloheximide): Glucose 40 g Mycosel Agar 54 g Neopeptone 10 g Distilled water 1500 ml Agar granulated 16 g Choramphenicol 50 mg Distilled water 1000 mL Gentamycin 5 mg Chloramphenicol 50 mg Gentamycin 5 mg 37 Methods Dixon agar was used for cultivating Malassezia spp. Sabouraud Glucose Agar allows the growth of dermatophytes, molds and yeasts apart from Malassezia spp. and contains chloramphenicol and gentamycin to inhibit bacterial growth. Mycosel Agar is similar to the latter, but additionally contains cyclohexmide to prevent the growth of molds. Cultures were left at about 20° C for 3 to 4 weeks. If Malassezia spp. cultivation on Dixon Agar was successful, a specimen of the culture was stained using gram technique and observed under light microscope with a 100 fold magnification. If dermatophytes were identified by colony morphology, two subcultures were prepared using Phenol Red Agar and Potato Dextrose Agar. Specimens of cultures positive for dermatophytes were stained with cotton blue and observed under a 40 fold magnification. A further identification technique was that of the “hanging drop”.

4. Admission to the Study

4.1 Inclusion Criteria

The children were included in the study if they were diagnosed clinically at the screening examination to suffer from tinea versicolor, tinea capitis, tinea corporis, tinea pedis or scabies. The inclusion of cases of tinea cruris was not possible for two reasons. First, privacy and an investigator of the same sex as the examined child could not be always guaranteed. Second, no Wood Lamp was available, which would have facilitated the differential diagnosis between erythrasma and tinea cruris. At both schools meetings were held during which the parents or caretakers were informed about the procedures of the soap trial and were given the opportunity to ask questions (see Appendix D 2.1 and 2.2). They were provided with information material regarding the skin disorder their child was suffering from (see Appendix D 3.1.1 to 3.2.2). Besides, they received a form sheet guaranteeing the access to free treatment for the skin disease which had been the reason for admission to the study, if the study participant would not be cured after two months of soap use or if he or she would suffer from any side-effects related to the soap use in the course of the study (see Appendix D 4.1). At the end of these meetings the parents or caretakers were asked to give their oral informed consent to let the child participate in the soap trial (see Appendix D 1.1 and 1.2).

4.2 Excludsion Criteria

On admission children were excluded if the degree of their skin disorder was so severe they required immediate treatment with either antifungal or antiscabietic drugs, which were then administered directly after the screening examination. If the child explained in the course of the 38 Methods history-taking that he or she had already been given any kind of standard treatment at other health facilities participation in the present study was not possible. During follow-up, study participants were excluded if they had decided they did not wish to participate any longer; if they had received any of the above mentioned treatments other than the soap since the first screening examination; if no more lesions were seen at the first soap distribution; if they had received their soap less than four times or if they were not present at the second follow-up examination. No child developed side-effects thus none had to be excluded for this reason. If the local dermatologist (Dr. M. Mbata) decided during the period of soap use that the infection required urgent treatment, the children were excluded from the study and received a prescription for free standard treatment (see 8. Follow-up Examinations) for their skin disorders from the dermatologist.

5. Case Definitions

Tinea versicolor: The following criteria characterize the clinical picture of tinea versicolor. Shape and appearance of the lesions (Canizares et al., 1993; Sunenshine, 1998; Piamphongsant, 1983; Gupta, 2002): • Multiform macules or patches • Lesions may coalesce and/or be perifollicular • Hypo- or hyperpigmented • Fine, adherent scales (may be absent after the use of Vaseline) • Pruritus may be present. Typical localizations (Canizares et al., 1993): Areas with the greatest density of sebaceous glands such as the upper trunk, neck and face, sometimes on upper arms and abdomen. Rarely on other body parts. Other superficial dermatomycoses (tinea capitis, corporis and pedis): Species identification could not be performed in all cases. Apart from dermatophytes, yeasts or moulds may have been among the etiologic fungi. Therefore cases of tinea capitis, corporis and pedis will be referred to as “other superficial dermatomycoses”. Tinea capitis: One of the three clinical pictures has to be present for diagnosis. Shape and appearance of the lesions (Canizares et al., 1993; Shrum, 1994; Weitzman et al., 1996): 39 Methods • Scaly, dry, non-inflammatory lesions sometimes presenting grey patches or “black dots” • Acute, wet, and inflammatory lesions with possible suppuration or kerion formation • Dry, crusted, occasionally inflammatory and suppurative lesions, yellow crusts (also called “favus”). Folliculitis, bacterial superinfection, alopecia and scarring may occur. Pruritus may be present. Positive microscopy confirms diagnosis but is not regarded as mandatory in this study. Tinea corporis: The following criteria characterize the clinical picture of tinea corporis. Shape and appearance of the lesions (Canizares et al., 1993): • Sharply demarcated round or oval scaly patch(es) • Active vesicular border and central clearing may be present. Occasionally pustular lesions Pruritus may be present. Typical localizations (Canizares et al., 1993): • Glabrous (=hairless) skin including non-hairy parts of face, neck, trunk, limbs (not axillae, groins, interdigital spaces of hands or feet, palms or soles) The aspect of the lesions may in some cases resemble those of tinea versicolor, especially if multiple lesions are present. In these cases, the final diagnosis was based on the culture performed in Zürich. Tinea pedis: One of the three clinical pictures has to be present for diagnosis. Shape and appearance of the lesions (Canizares et al., 1993; Masri-Fridling, 1996; Shrum, 1994): • Interdigital type with maceration. It may extend to both the toes and the soles • Dishydrotic type with acute versicular, bullous or vesiculo-bullous lesions with erythema, fissuring and sometimes bacterial superinfection. Usually located on the mid-sole • Hyperkeratotic moccasin type; tends to be chronic and presents with scaling of the plantar surface. No vesiculation. Pruritus may be present. Scabies: Four features are diagnostic of scabies (Canizares et al., 1993; Canizares, 19983; Van Hees, 2001; Anezib, 1981; Haas , 2001; Burkhart, et al., 2000): • Itching (most severe at night) • Typical rash (small blisters and papules) 40 Methods • Scabies burrows • Common localization: sides of the fingers and hands, flexor sides of the wrists, anterior parts of elbows, on the genitals and buttocks etc. A history of contact with infestated persons supports the diagnosis.

6. Degree of Infection

Based on several other studies the degree of infection was assessed by using a separate symptom score for tinea versicolor, tinea capitis, corporis and pedis as well as for scabies infection (Hira et al., 1986; Lange et al., 1998; Hickman, 1996; Hussain et al., 1999; Martinez-Roig et al., 1988; Gupta et al., 1998) (see Tables 3a and 3b). This score reflects the degree of certain clinical signs and symptoms typical of each of the four types of fungal infections. In order to calculate this score the appropriate parameters were chosen, and score values were given by the four examiners on examination for each parameter (see Tables 3a and 3b; also see Appendix C Part 1.3). These score values were then added to yield the total score.

Tables 3a and 3b: For each of these diseases the appropriate parameters were chosen, and separate score values were added to a total score (based on Hira et al., 1986; Lange et al., 1998; Hickman, 1996; Hussain et al., 1999; Martinez- Roig et al., 1988; Gupta et al., 1998). The score reflects the degree of certain clinical signs and symptoms typical of each of the four types of fungal infections targeted for the study. Table 3a: Score parameters and score values Size of Degree of Number of Aspect of Additional largest pruritus lesions lesions clinical signs Diagnosis lesion Total Possible range of partial score according to parameter score T. 0 to 3 0 to 8 0 to 14 N.A. N.A. 0 to 25 versicolor T. capitis 0 to 3 0 to 8 0 to 18 0 to 6 0 to 6 0 to 41 T. 0 to 3 0 to 8 0 to 12 0 to 6 N.A. 0 to 29 corporis T. pedis 0 to 3 0 to 8 0 to 4 0 to 6 N.A. 0 to 21 Scabies 0 to 3 0 to 6* 0 to 16 N.A. N.A. 0 to 27 * number of common localizations (Pinkerton et al., 2003; Anezia, 1981)

41 Methods Table 3b: Degree of infection according to possible total score values and classification of degree of infection Degree of infection: Degree of infection: Degree of infection: Important/ Mild Moderate Diagnosis Severe Possible total score values and classification of degree of infection T. versicolor 3 to 6 7 to 9 10 to 25 T. capitis 6 to 13 14 to 20 21 to 41 T. corporis 7 to 13 14 to 19 20 to 29 T. pedis 4 to 7 8 to 13 14 to 21 Scabies 4 6 to 11 12 to 27

7. Randomisation

Randomization of the study population was carried out before the onset of the clinical trial for 400 possible study units by attributing the letters A to U randomly to the serial numbers of 1 to 400 using EpiInfo. During the screening examination all the study participants living in one household were identified and formed one unit. A unit could consist of one or more children. At both schools the serial numbers assigned to the randomised letters were distributed in the order of the names on the list of the study participants. The list had been created by the field investigator in the order in which the children had been included in the study. The participants attending Lihami Primary School were assigned the first serial numbers. They were given soap for the first time before the screening in Michenga was completed, in order to allow a reasonable timing and coordination of the soap distributions and follow-up examinations at both schools. In order to achieve an equal distribution of the multi-HHM-units (household member = HHM) between the placebo and Triclosan group, the one-HHM-units were attributed the first serial numbers, followed by the two- HHM-units, then the three-HHM-units etc. The same procedure was employed to attribute the soap letters at Michenga Primary School, beginning with the next higher serial number after the last child at Lihami School. If children were identified to live in one household after the randomisation had been completed but before receiving the soap for the first time, they were all assigned the serial number of the HHM appearing in the list first. If children were identified to live in one household after the randomisation had been completed and after having received the first piece of soap, they remained with the same serial number and soap letter until the end of the study, but additional instructions were given in order to emphasize the importance of never exchanging the soap with any household member taking part in the study.

42 Methods Table 4: Composition of the placebo and active soaps

Placebo Soap: Active Soap: 0.2% Titanium Dioxide 0.2% Titanium Dioxide 0.023% Tetrasodium EDTA 0.023% Tetrasodium EDTA 3.0% Stearic Acid 3.0% Stearic Acid 0-5% Glycerine / Water 1:1 0-5% Glycerine / Water 1:1 q.s. Dye /Perfume q.s. Dye /Perfume ad 100.0% Soap Noodles* ad 100.0% Soap Noodles** 1.0% IRGASAN DP300 **Mettler Basic Soap: 80.0% Sodium tallowate 20.0% Sodium cocoate

Through the attribution of the letter each child was assigned to receive his or her soap bar pieces from one kind of box (labelled alphabetically, A to U) only. Each box contained 30 pieces of soap (100g per piece). The letter printed on the boxes indicated if the soap contained Triclosan or not. Until data entry and data cleaning was finished, all four field investigators were blind with regard to who received Triclosan or the placebo (for composition of soap see Table 4). Considering the possibility that the whole family would use the child’s soap, it was provided generously. The prinicpal investigator (AD), assisted by MM, CM and JF, visited both schools weekly to distribute a new soap bar per child.

8. Follow-up Examinations

Two follow-up examinations were performed, after the first and second month of soap use. Apart from history-taking and questions concerning hygiene behaviour and living conditions, these follow-up examinations involved the same steps as the first screening examination (also see Figure 3): (i) examination of the skin, (ii) documentation of the skin lesions by digital photography, (iii) only at the second follow-up examination: sample-taking if a superficial fungal infection (not in candidiasis, tinea pedis or cruris infections) was present. The sample-taking was not repeated in the case of scabies infections. Three additional questions were introduced: (i) whether the child had experienced any improvement of the skin disorder due to the soap consumption; (ii) if specifically the pruritus had improved; (iii) and in case of tinea capitis infection if new hair growth was visible. When possible, pictures were taken of the same lesion during each of the three examinations for evaluation of improvement or resolution of symptoms during the follow-up examinations. On comparison special attention was paid to the aspect of the lesions at the first and second follow-up. The following parameters were assessed and summarised by classifying the result of the comparison into “improvement”, “same” or “deterioration”: 43 Methods • size of the lesion • number of lesions • presence of “active” lesions (see 5. Case definitions) • presence of only residual hypopigmentation (Weitzman et al., 1996; Sunenshine, 1998) or postinflammatory hyperpigmentation • presence of superinfection (in case of dermatophyte or scabies infection) • new hair growth visible (in case of tinea capitis infection)

If a child was cured at the time of the first follow-up, no picture was taken. Samples were taken during the second follow-up at the site of the former lesions, even if no one were present at this stage. All children who were not cured by the end of the soap trial were given a presription from the dermatologist Dr. M. Mbata to receive free treatment at the SFDDH dispensary. Drugs prescribed were oral ketoconazole for tinea versicolor, tinea capitis, corporis or pedis; griseofulvine for tinea capitis, corporis and pedis; clotrimazole cream or Whitfield’s Ointment for minor cases of tinea versicolor, tinea capitis, corporis, or pedis and BBE to treat scabies. Terbinafine was provided to the children who were severely affected by tinea capitis at the end of the soap trial.

9. Evaluation of Effectiveness - Dermatomycoses

9.1 General

In order to assess the effectiveness of the soap containing Triclosan, several endpoints (clinical signs and symptoms typical of the disease) were evaluated during all examinations. The relative risk of having these symptoms if receiving placebo versus active soap was calculated for the screening and follow-up 2.

9.2 Pruritus

In order to improve the reliability of the answers of the children, the examiners asked twice during the history-taking if the child had pruritus. Not all the children gave coherent answers. Therefore the results may be sometimes contradictory, e.g. when compared with the degrees of pruritus. The calculation of the relative risk combines answers provided at the two different stages of the history- taking. If one of the two questions was answered positively, it was considered that pruritus had been present. 44 Methods 9.3 Clinical and Microscopic Resolution

In order to decide whether the study participants were cured at time of the second follow-up seven (five in case of tinea pedis) evaluation categories were defined, which combined clinical signs and symptoms and the result of the microscopic examination (only clinical signs in the case of tinea pedis). The Chi2 test or the Fisher’s exact test were used to find out if the evaluation categories were equally distributed between active and placebo groups.

9.4 Definition of Evaluation Categories at Second Follow-up

9.4.1 Tinea versicolor

CATEGORY: DEFINITION: Clinical and mycological resolution (based on Lange et al., 1998; Hickman, 1996): 1: clinical and microscopic resolution: score = 0, child cured 2: microscopic resolution: microscopy negative and few clinical symptoms: Score < 4 (only if score had been > 4 at screening) No resolution: 3: improvement: negative microscopic evidence with significant clinical improvement: reduction of score to 5 or 6 & score at screening > 6 or reduction by 4 points & score at screening > 6 4: negative microscopic evidence with no significant clinical improvement reduction of score to > 7 or reduction by < 4 points or score at screening and at second follow-up = 4 5: positive microscopic evidence with significant clinical improvement: reduction of score to 5 or 6 & score at screening > 6 or reduction by 4 points & score at screening > 6 6: lack of improvement: positive microscopy with a lack of significant clinical improvement: score > 7 or reduction by < 4 points 7: deterioration: increased score

9.4.2 Tinea capitis

CATEGORY: DEFINITION: Clinical and mycological resolution (based on Hussain et al., 1999; Gupta et al., 1998): 1: clinical and microscopic resolution: score = 0, child cured 45 Methods 2: microscopic resolution: microscopy negative and few clinical symptoms: Score < 6 No resolution: 3: improvement: negative microscopic evidence with significant clinical improvement: reduction of score to < 14 or reduction by > 6 points 4: negative microscopic evidence with no significant clinical improvement: reduction of score to > 14 or reduction by < 6 points 5: positive microscopic evidence and clinical resolution with significant clinical improvement: reduction of score to < 14 or reduction by > 6 points 6: lack of improvement: positive microscopy with a lack of significant clinical improvement: reduction of score to > 14 or reduction by < 6 points 7: deterioration: increased score

9.4.3 Tinea corporis

CATEGORY: DEFINITION: Clinical and mycological resolution: (based on Gooskens et al., 1994; Montero-Gei et al., 1992) 1: clinical and microscopic resolution: score = 0, child cured 2: microscopic resolution: microscopy negative and few clinical symptoms: Score < 7 No resolution: 3: improvement: negative microscopic evidence with significant clinical improvement: reduction of score to < 14 or reduction by > 6 points 4: negative microscopic evidence with no significant clinical improvement reduction of score to > 14 or reduction by < 6 points 5: positive microscopic evidence and clinical resolution with significant clinical improvement: reduction of score to < 14 or reduction by > 6 points 6: lack of improvement: positive microscopy with a lack of significant clinical improvement: reduction of score to > 14 or reduction by < 6 points 7: deterioration: increased score

9.4.4 Tinea pedis

CATEGORY: DEFINITION: Clinical resolution (abbreviations: N.A.= non-applicable) 1: clinical resolution: score = 0, child cured no resolution: 46 Methods 2: improvement : score was reduced 3: lack of improvement: score remained the same 4: no resolution: score was N.A. at screening, but child was not cured at second follow-up 5: deterioration: increased score

10. Evaluation of Effectiveness - Scabies

In order to assess the effectiveness of the soap containing Triclosan, several endpoints (certain signs and symptoms typical of the disease) were evaluated during all the examinations. No significance tests could be performed because the sample sizes were too small (only two children in the active group). Apart from the parameters used for the symptom score, the time of itching, involvement of other family members, the localization of the lesions, and the result of the microscopic examination were recorded. Burrows were hardly found during the baseline examination, which impaired the sample- taking to a great extent. This procedure was therefore not repeated at the second follow-up.

11. Assessment of Soap Use (Compliance)

One piece of soap per week and child was distributed at the two schools. The availability of soap was ensured for every study participant, by personal delivery through the field investigator, assisted by MM, CM and JF at the two primary schools. The following additional tools for checking the use of soaps were used:

1) Upon receipt of a new bar of soap, the remains of the former piece had to be returned. 2) If the child was not present in the school at the time of soap distribution, a student who knew where the child lived was identified in order to deliver the soap to the child’s home, or a teacher was given clearly labelled envelopes containing the soaps, which were then to be delivered through him/her. 3) At each soap distribution a record was kept whether the child collected the soap him/herself or who was selected as a courier instead. 4) If the child informed the investigator in advance that he/she would not be present for a certain number of weeks, he or she received the appropriate number of soaps at the time when the child was present. 5) During the second follow-up each child was asked if he/she received the soap the week before and by whom. 47 Methods 6) After the last soap distribution, control visits lasting nine days in Lihami and seven days in Michenga, were conducted by another field worker (professional nurse) at the homes of 10% of the study participants who had been randomly selected. Before randomisation, two clusters were independently formed at each school. One included children who had collected the soap themselves at the last soap distribution while the other included children who had been sent the soap by another person. The remaining pieces of soap had to be shown to the field worker during her visit. This was done in order to check the presence of the study soap pieces at the children’s homes. The field worker also questioned the child or the interviewed household member about who had given the latest piece of soap to the child. 7) The field worker also asked how many pieces of soap the child had received in total and how many people in the household had used the soap. In the case of the 10% randomly selected study participants, this information was evaluated for coherence. In case children had been delivered the soap at home, we calculated which proportion of them could either show a piece of soap to the field worker or confirmed when asked in the course of the second follow-up that they had received their piece of soap by someone else. The tools employed to check the use of soaps proved that the measures taken to deliver the soaps can be considered as reliable. Unlike originally planned, the children were given a new piece of soap even if the remains of the former piece could not be returned, since most children had used the whole piece or could not show it for other reasons. Their future soap consumption was regarded as more likely if they were provided with a new piece of soap rather than being sent back home to collect the old piece before obtaining the new one. 48 Results

12. Determination of Minimal Inhibitory Concentrations (MIC’s) of Placebo Soap and of Triclosan

The field study in Ifakara were followed by an in vitro assessments of the MIC’s of placebo soap and of pure Triclosan against M. sympodialis cultured from six different patients at the University Hospital, Zürich. To assess the MIC of the soap, the following steps were performed:

Placebo soap: 1) Preparation of stock solution with placebo soap: 1 g of pre-diluted soap (2% and 0.2%) were given into 10 mL of sterile water, stirring at 35-40°C; yielding a concentration of 100 mg/mL (10%) 2) Preparation of dilutions (each twice): 1 mL of stock solution were given in N° 1; in N° 2-11: 0.5 mL of the dilution successively, 0.5 mL of N° 1 in N°2, 0.5 mL of N° 2 in N° 3 etc.; N° 12 control without soap, only sterile water; yielding the following dilution series (Table 4a) (Conc.=concentration):

Table 4a: Dilution series of stock solution (Placebo soap) Concen- tration 100000 50000 25000 12500 6250 3125 1562.5 781.25 390.63 195.31 97.66 0 (μg/mL) N° 1 2 3 4 5 6 7 8 9 10 11 12 3) Preparation of final concentrations (each twice): 0.3 mL of each dilution was added into 15 mL of agar yielding the following dilution series on Dixon Agar (Table 4b) (Conc.=concentration):

Table 4b: Dilution series of stock solution on agar (Placebo soap) Concen- tration 2000 1000 500 250 125 62.5 31.25 15.63 17.81 3.91 1.95 0 (μg/mL) N° 1 2 3 4 5 6 7 8 9 10 11 12

4) Drying overnight at approximately 22 °C 5) Preparation of Malassezia Suspension: 5 mL NaCl (0.85%; pH 7.2) and 0.01% Triton X-100 were added onto 6 plates with 7 to 14 days old agar, on which Malassezia spp. (from six patients) had been cultivated. Air mycelia and spores were removed, and the suspension was filtered through sterile glass wool. The glass wool was rinsed again with another 5 mL NaCl (0.85%; pH 7.2) and 0.01% Triton X-100. The filtered spores 49 Results were diluted again with 5 mL NaCl (0.85%; pH 7.2) and 0.01% Triton X-100. The plates were inoculated with 0.01 mL of this solution (10 μl) using a micropipette. Triclosan Powder: 1) Preparation of stock solution with placebo soap: 500 mg are given into 10 mL absolute Ethanol; yielding a concentration of 50000 μg/mL (5%) 2) Preparation of dilutions: 1 mL of stock solution were given in N° 1; in N° 2-11: 0.75 mL of the dilution successively, 0.75 mL of N° 1 in N°2, 0.75 mL of N° 2 in N° 3 etc.; N° 12 control without Triclosan, only sterile water; yielding the following dilution series (Table 4c) (Conc.=concentration):

Table 4c: Dilution series of stock solution (Triclosan powder) Concen- tration 50000 37500 28125 21093.75 15820.31 11865.23 8898.93 6674.19 5005.65 3754.2 2815.7 0 (μg/mL) N° 1 2 3 4 5 6 7 8 9 10 11 12

3) Preparation of final concentrations: 0.3 mL of each dilution was added into 15 mL of agar yielding the following dilution series on Dixon Agar (Table 4d) (Conc.=concentration): Table 4d: Dilution series of stock solution on agar (Triclosan powder) Concen- tration 1000 750 562.5 421.8 316.4 237.3 177.98 133.49 100.11 75.09 56.31 0 (μg/mL) N° 1 2 3 4 5 6 7 8 9 10 11 12

4) Drying overnight at approximately 22 °C 5) Preparation of Malassezia Suspension: 5 mL NaCl (0.85%; pH 7.2) and 0.01% Triton X-100 were added onto 6 plates with 7 to 14 days old agar, on which Malassezia spp. (from six patients) had been cultivated. Air mycelia and spores were removed, and the suspension is filtered through sterile glass wool. The glass wool was rinsed again with another 5 mL NaCl (0.85%; pH 7.2) and 0.01% Triton X-100. The filtered spores were diluted again with 5 mL NaCl (0.85%; pH 7.2) and 0.01% Triton X-100. The plates were inoculated with 0.01 mL of this solution (10 μl) using a micropipette.

13. Data Entry and Analysis

The data were entered in FoxPro 2.6, and the evaluation was performed with STATA 8.2. Chi Square and Fisher's exact test were used to calculate p-values for contingency tables. In order to compare the diameter of the largest lesion in tinea capitis and corporis infections at the different examinations, Wilcoxon's rank sum test was used. Symmetry tests were applied to compare 50 Results separately the degree of pruritus, number of lesions and degree of infection at the three different examinations for tinea versicolor and dermatophyte infections. The symmetry of treatment benefits or deterioration was compared between the active and placebo group by using contingency tables and testing these for difference of proportions using 95% confidence intervals. For all three examinations, the relative risk of the placebo versus the active group was calculated for different parameters. In order to show the whole spectrum of clinical evolution of all the cases of tinea versicolor, tinea capitis and corporis, a scatter plot presentation together with cubic splines was chosen. This presentation displays the clinical score at screening (y-axis) and follow-up 2 (x-axis). The cubic spline fit incorporates the score values of all subjects with the specific disease. Its position relative to the reference line is discussed. The graphical presentations were made separately for active and placebo groups, and, in the case of tinea versicolor and tinea capitis infections, according to the positive or negative microscopic examination results during the second follow-up.

14. Additional Information

The findings which are not presented in the chapter “Results” can be found in the Appendix A. 51 Results Results 1. General information In the course of this study a total number of 820 primary school children were examined. The distribution of sexes was balanced at both schools, with a total female versus male proportion of 48.2 (394/818) versus 51.8% (424/818). An almost even proportion of children belonged to the age groups 6 to 10 years and 11 to 14 years, whereas the age group 15 to 19 years included only 16.1% (131/812) of the children. The mean age was 11.6 years. In the oldest age group there were more boys, whereas among the youngest age group the proportion of girls was higher (see Table 1). These differences in proportions were significant (Chi2 test p-value = 0.049).

Table 1: Frequency distribution and relative frequency of age groups among all the children examined Age groups: Number of children (percentage) Sex *row percentage Total** 6 to 10 years 11 to 14 years 15 to 19 years Female 177 (52.5) 162 (47.1) 51 (38.9) 390 (48.0) Male 160 (47.5) 182 (52.9) 80 (61.1) 424 (52.0) Total 337 (41.5*) 344 (42.4*) 131 (16.1*) 812 (100.0) Chi2 test p-value = 0.049 ** The information concerning sex and/or age is missing for eight children

2. Epidemiology of superficial dermatomycoses

2.1 Prevalence rates

Children suffering from one of the skin infections listed below (Table 2) were enrolled according to the inclusion and exclusion criteria on admission (see “Patients, Materials and Methods: 4. Admission to the Study”). Unless otherwise specified, cases of tinea capitis, corporis and pedis will be referred to as “other superficial dermatomycoses” since culture could not be performed in all cases, and yeasts or moulds may thus not be excluded as the etiologic fungi. Tinea versicolor was the most common skin disorder, being prevalent in 26.2% (215/820) children. 5.5% (45/820) of the children had tinea capitis, and 2.6% (21/820) were suffering from tinea corporis. Slightly more children had tinea pedis (3.2%; 26/820). Tinea versicolor infections accounted for 70.0% of all fungal infections. Tinea capitis ranked second representing 14.7% of all dermatomycoses. Tinea pedis was more common than tinea corporis, accounting for 8.5% versus 6.8% of all fungal infections found among these primary schoolchildren. There was one case of clinically diagnosed candidiasis of the hand. It was not included in the study (Table 2).

52 Results Table 2: Prevalence rates of superficial fungal infections and scabies according to age groups and sex Number of observations (Prevalence rates) Age groups (1 to 3) and sex (f or m): number of cases 3 (15 to 19 Total*: 2 (11 to 14 Diagnosis 1 (6 to 10 years): n= years): years): 337 812 344 131 f:177 m: 160 f: 162 m: 182 f: 51 m: 80 f: 390 m: 422 Tinea 24 19 59 56 21 35 104 110 versicolor 13.6 11.9 36.4 30.8 41.2 43.8 26.6 26.1 Total 43 (12.8) 115 (33.4) 56 (42.7) 214 (26.4) Tinea 12 14 711 01 19 26 capitis 6.7 8.6 4.3 6.0 0.0 1.3 4.8 6.2 Total 26 (7.7) 18 (5.2) 1 (0.7) 45 (5.5) Tinea 54 4710 10 11 corporis 2.8 2.5 2.5 3.8 1.9 0.0 2.6 2.6 Total 9 (2.7) 11 (3.2) 1 (0.7) 21 (2.6) Tinea 71 6354 18 8 pedis 3.9 0.6 3.7 1.6 9.8 5.0 4.6 1.9 Total 8 (2.1) 9 (2.6) 9 (6.9) 26 (3.2) Total number of fungal infections 306 35 1201 4 8 Scabies 1.7 3.1 0.6 1.0. 0.0 1.3 1.0 1.9 Total 8 (2.4) 3 (0.9) 1 (0.8) 12 (1.5) ** The information concerning sex and/or age is missing for eight children.

2.2 Prevalence rates and degree of infection according to sex

2.2.1 Tinea versicolor

The prevalence was found to be slightly higher among girls: 26.7% (105/394) versus 25.9% (110/424) (Chi2 test p-value = 0.819). The degree of infection among the study participants with tinea versicolor (n=174) was assessed by using a symptom score. Values at the screening examination ranged from 3 to 18. The score was normally distributed with a mean score of 9.96. In 10 children the score was not calculated as the essential information could not be gathered from the examination sheet. More than half of the study participants had an important infection. Mild and moderate infections were found to be almost equally frequent (see Table 3). The degree of the infection among the study participants was not found to differ significantly between the two sexes (Chi2 test p-value = 0.074). The proportion of important infections was only slightly higher among boys (61.9% (m) versus 57.5% (f)). Two thirds of the children with mild infections were boys, and two thirds of the children with moderate infections were girls. Important infections were almost equally distributed between the sexes (see Appendix 2.2). 53 Results

Table 3: Tinea versicolor: Degree of infection: Number of observations and percentage Range of score and resulting degree of infection 3 to 6: 7 to 9: 10 to 25: N.A. Mild Moderate Important Total

Number of 32 (18.4) 34 (19.5) 98 (56.3) 10 (5.8) 174 (100.0) observations (Percentage)

2.2.2 Other superficial dermatomycotic infections

Among the fungal and parasitic skin infections of interest to this study, only tinea pedis prevalence rates differed significantly between the two sexes. The prevalence rate was 1.9% (8/424) among boys and 4.6% (18/394) among girls. The difference in prevalence rates was significant (Fisher’s exact test p-value = 0.044). The prevalence rate of tinea capitis was slightly higher among boys (6.1%; 26/424) than girls (4.8%; 19/394). This difference was not statistically significant (Fisher’s exact test p-value = 0.412). The prevalence rate of tinea corporis was nearly equal for girls (2.5%; n=7) and boys (2.6%; n=6) (see Appendix 2.2). The degree of infection among the 76 study participants with tinea capitis, corporis or pedis was assessed by using a symptom score (see Table 4).

Table 4: Degree of infection: Relative frequencies (number of observations) and frequency distribution (percentage) Number of observations (Percentage): Diagnosis Degree of infection* Mild Moderate Severe NA Total 7 (17.5) 25 (62.5) 5 (12.5) 3 (7.5) 40 (100.0) Tinea capitis Tinea corporis 9 (60.0) 3 (20.0) 1 (6.7) 2 (13.3) 15 (100.0) Tinea pedis 0 (0.0) 2 (9.5) 5 (23.8) 14 (66.7) 21 (100.0) Total 16 (21.0) 30 (39.5) 11 (14.5) 19 (25.0) 76 (100.0) *Score range Mild 6 to 13 (Tinea capitis) 7 to 13 (Tinea corporis) 4 to 7 (Tinea pedis) Moderate 14 to 20 (Tinea capitis) 14 to 19 (Tinea corporis) 8 to 13 (Tinea pedis) Severe 21 to 41 (Tinea capitis) 20 to 29 (Tinea corporis) 14 to 21 (Tinea pedis) At the first screening nearly two-thirds (25/40) of the cases of tinea capitis were moderate infections. Mild and severe infection were almost equally frequent. Cases of tinea corporis were found to be less frequently moderate (20.0%; 3/15) or severe (6.7%; 1/15), but mild infections 54 Results accounted for 60% (9/15) of cases. The degree of the tinea capitis and corporis infection did not differ significantly between the two sexes (Fisher’s exact test p-values > 0.05). No statement can be made with regard to the whether the degree of tinea pedis infections depended on sex, since it the score could only be calculated for one boy. For 14 out of 21 cases the score could not be calculated (see Table 4). Only the interdigital, macerated type (see Background: 1.1.2.3 Tinea pedis) was discovered among the examined primary school children.

2.3 Prevalence rates and degree of infection according to age groups

There were significant differences between prevalence rates among the three age groups for tinea versicolor, tinea capitis and tinea pedis infections (Fisher’s exact test p-values = 0.000, 0.04 and 0.033 (Chi2 test p-value), respectively). The prevalence rates of tinea versicolor and tinea pedis were found to increase with age, whereas the prevalence rates of tinea capitis and tinea corporis (Fisher's exact test p-value = 0.295) were found to decrease with age.

2.3.1 Tinea versicolor

The degree of infection among the study participants was found to be significantly different for the three age groups. (Fisher’s exact test p-value = 0.000). The proportion of children with severe infection (81.4% of infections in this age group) was found to be highest in age group 3 (15 to 19 years; n=43) and lowest (48.8%) in age group 1 (6 to 10 years; n=29). Moderate infection was found to be most frequent (29.4%) among the 11 to 14 year-old children (n=92), whereas mild infections were most frequent (41.4%) among the 6 to 10 year-old children (see Appendix 2.3).

2.3.2 Other superficial dermatomycotic infections

The degree of tinea capitis and corporis infections was assessed according to two age groups (6 to 10 years; n=32, and >10 years; n=24). The degree of infection was not significantly different for the two age groups (Fisher’s exact test p-values > 0.05). Severe infection was almost equally frequent in both groups (see Appendix 2.3). The degree of the infection did not depend on the age group (Fisher’s exact test p-value = 0.429). However, this statement is not reliable since the degree of infection could only be determined for one third of the children. 55 Results 3. Assessment of the effect of soap

3.1 Assessment of the efficacy of Triclosan in soap

Study participants and dropouts: 248 children were enrolled in the study based on the inclusion and exclusion admission criteria. 20 had to be excluded after admission during the follow-ups. Before and after excluding these, the number of children belonging to the placebo and active group was balanced (see Figure 1 and Table 5a). Among the 228 study participants 48.3% were female and 51.7% male. The age distribution was different from that of the originally examined children. 28.1% (64/228) were 6 to 10 years old, 51.3% (117/228) to the 11 to 14 years old, and 20.6% (47/228) were adolescents aged 15 to 19 years. Some study participants had two or more skin infections at the time of the screening examination. They were regarded as separate cases of infection when dealing with the individual diseases. Table 5a: Relative frequencies and frequency distribution of study participants (cases of infection) receiving placebo versus active soap according to individual diagnoses – all dropouts excluded Number of cases (Percentage) Placebo Active Total Diagnosis Tinea versicolor 82 (47.1) 92 (52.9) 174 Tinea capitis 21 (52.5) 19 (47.5) 40 Tinea corporis 7 (46.7) 8 (53.3) 15 Tinea pedis 11 (52.4) 10 (47.6) 21 Scabies 4 (66.7) 2 (33.3) 6 Total 125 (48.8) 131 (51.2) 256

Among the study participants 11 children had simultaneous tinea versicolor infection and tinea capitis or and 10 had simultaneous tinea versicolor and tinea pedis infection (see Appendix 3.1 for Table 5b). Evaluation of follow-ups: The data collected at the first follow-up were analyzed, but are listed only in the Appendix. The emphasis placed on these results shall be small, since only 163 out of the 228 study participants were present. 56 Results

820 children examined at baseline examination: • clinical examination • clinical diagnosis of tinea versicolor, other superficial fungal infections and scabies • digital photography • microscopic examination of samples

248 children enrolled in soap trial: randomization for placebo and active group

Placebo group: Active group: 120 children 128 children

First treatment cycle: placebo or active soap for one month

First Follow-up: • clinical re-examination • sample-taking for microscopy and culture in Zürich for quality control

Second treatment cycle: placebo or active soap for one month

Second Follow-up: • clinical re-examination • digital photography • microscopic examination of samples l • sample-taking for microscopy and culture in Zürich for quality control Exclusion of dropouts: 8 from placebo group, 14 from active group

For evaluation: For evaluation: Placebo group: Active group 112 children 116 children

Figure 1: Study participants of soap trial

57 Results Table 6a:Characteristic Endpoints: Proportions at Screening and Follow-up 2 Placebo (P) versus Active soap (A) Tinea versicolor Tinea capitis Tinea corporis Endpoints Number of Number of Number of observations (clinical Exami- n observations n observations n (percentage of n) signs and nation (percentage of n) (percentage of n) symptoms) P A P A P A P A P A P A Screening 6 (7.9) 12 (14.0) 7 (100.0) 7 (100.0) 3 (25.0) 1 (20.0) Scaling 76 86 7 7 7 6 Follow-up 2 3 (3.9) 3 (3.5) 5 (71.4) 4 (57.1) 0 (0.0) 0 (0.0) Screening 34 (55.7) 41 (56.2) 9 (81.8) 7 (77.8) 4 (80.0) 3 (60.0) Pruritus 61 73 11 9 5 5 Follow-up 2 25 (40.1) 28 (38.4) 4 (44.4) 4 (44.4) 1 (20.0) 1 (20.0) Pruritus Follow-up 2 82 90 12 (85.4) 23 (74.4) 21 18 19 (90.5) 16 (88.9) 8 6 7 (83.3) 6 (100.0) improved Subjective impression: Follow-up 2 53 59 49 (92.6) 57 (89.9) 20 17 18 (90.0) 17 (100.0) 6 8 6 (100.0) 8 (100.0) improved Hypo- Screening 73 (96.1) 80 (93.0) Tinea capitis: pigmen- 76 86 The proportion of children with Follow-up 2 54 (74.1) 65 (75.6) tation occipital adenopathy increased in the Screening 4 (5.2) 9 (10.5) active group, whereas it decreased in Hyper- the placebo group. Besides, the child pigmen- 77 86 Follow-up 2 4 (5.2) 8 (9.3) with kerion lesions was not cured tation through the use of the active soap. Screening 10 (61.1) 11 (62.5) Tinea versicolor, tinea capitis and corporis: Alopecia 18 16 More than three quarters of all children Follow-up 2 4 (22.2) 5 (31.3) in each group stated that pruritus had Occ./post. Screening Tinea versicolor: 6 (33.3) 2 (12.5) improved and that their infection in cervical The proportion of children 18 16 Follow-up 2 3 (16.7) 4 (25.0) general had improved in the course of adenopathy presenting the individual the soap trial. Grey Screening endpoints was reduced from the 1 (5.9) 3 (17.7) 17 17 patches Follow-up 2 screening examination to the 0 (0.0) 0 (0.0) Black Screening second follow-up apart from the 3 (16.7) 5 (31.3) 18 16 dots Follow-up 2 proportion of children in the 0 (0.0) 0 (0.0) Screening placebo group with 0 (0.0) 1 (6.3) Kerion 18 16 Follow-up 2 hyperpigmented lesions. 0 (0.0) 1 (6.3) Super- Screening 1 (5.9) 3 (17.7) 17 17 infection Follow-up 2 0 (0.0) 0 (0.0) Table 6b: Clinical parameters: Number of observations according to diagnosis from Screening (SCR) to Follow-up 2 (Fu2): 58 Results Placebo (P) versus Active soap (A) The degree of pruritus and infection and the number of lesions were reduced in tinea versicolor, tinea capitis and corporis infections. More than 50% of the children in all groups showed improvement of the lesions in the photographs from Clinical parameters screening to the second follow-up. Tinea versicolor Tinea capitis Tinea corporis P A P A P A P A P A P A Degree re- re- 77 78 reduceda reduceda 18 17 reducedc reducedc 7 5 of pruritus ducedc ducedc Degree of re- re- 77 84 reduceda reduceda 19 17 reduceda reduceda 6 3 infection ducedc ducedc Number re- re- 76 85 reduceda reduceda 18 17 reducedc reducedc 7 6 of lesions ducedc ducedc 58 68 23 16 10 3 19.0 17.7 57.1 22.2 28.6 16.7 Microscopy (SCR) (SCR) (SCR) (SCR) (SCR) (SCR) negativeb 77 76 13 18 5 9 54.5 48.7 52.9 64.3 85.7 42.9 (Fu2) (Fu2) (Fu2) (Fu2) (Fu2) (Fu2) Photos: % with 48 57 54.1% 64.2% 14 10 71.4 60.0 7 6 85.7 66.7 improvement a = significant reduction (Symmetry test p-value < 0.05); b = different total number of observations at screening and follow-up 2 (assessment of frequency distribution and relative frequencies); c=reduction not significant (Symmetry test p-value > 0.05)

Table 6c: Tinea pedis: Clinical parameters: Tendencies from Screening to Follow-up 2 Placebo (P) versus Active soap (A) Due to the small number of cases of tinea pedis infections only the degree of pruritus and infection Clinical parameters and the size of the largest lesions could be evaluated. Total number of Placebo Active observations Degree of reduced reduced pruritus (different total number of Degree of observations at reduced infection screening and Size of the follow-up 2) reduced reduced largest lesion 59 Results

3.1.1 Tinea versicolor infections

3.1.1.1 Signs and symptoms/effectiveness

In order to assess the effectiveness of the soap containing Triclosan, several endpoints (clinical signs and symptoms typical of the disorder) were evaluated during all three examinations (see Table 6a to 6c). The proportions having these signs and symptoms were calculated for both examinations. There was no significant difference found between placebo and active group for any parameter. The relative risk of having these symptoms if receiving placebo versus active soap was calculated during these three examinations. It was not found to be significantly different from 1 at any stage if using placebo soap (see Appendix 3.1.1.1 for Table 6e). For various endpoints the symmetry test showed a significant improvement for active and placebo soap. However, there was no significant difference in the symmetry regarding these parameters (see Appendix 3.1 for Table 6d).

3.1.1.2 Degree of pruritus

It was found that the overall proportion of children without pruritus increased. The proportions of children having mild and moderate pruritus decreased significantly from the screening to the second follow-up if taking placebo and active groups (Symmetry test p-value<0.05) (Table 7a), and if taking them separately (see Appendix 3.1.1.2 for Figure 2b and Symmetry tables 7b and 7c).

Degree of pruritus: Both groups

80,00 70,00 60,00 none 50,00 mild 40,00 30,00 moderate 20,00 severe 10,00

Percentage of children of Percentage 0,00 Screening Follow-up 2 Examination

Figure 2a: Degree of pruritus (n=155) Both groups (Screening and follow-up 2)

Symmetry table: Number of children and degree of pruritus at screening and second follow-up: Table 7a: BOTH GROUPS Degree of Degree of pruritus at follow-up 2 pruritus at Total None Mild Moderate screening None 62 10 0 72 Mild 40 24 2 66 Moderate 8 6 3 17 Total 11 40 5 155 Symmetry test p-value = 0.000 60 Results 3.1.1.3 Number of lesions

When summarizing the two groups, a reduction in the proportion of children with 1 or 2, 3 to 5 and more than 5 lesions can be observed from the screening examination to the second follow-up (see Figure 3a, and Appendix 3.1.1.3 for Figure 3b). There is only a slight decrease in the proportion of children with disseminated lesions.

Number of lesions: Both groups

100%

80% disseminated > 5 but countable 60% 3 to 5 40% 1 or 2 20% none

Percentage children of 0% Screening Follow-up 2 Examination

Figure 3a: Number of lesions (n=161) Both groups (Screening and follow-up 2)

The symmetry test reveals that the reduction in the number of lesions from screening to the second follow-up was statistically significant if taking the children of both groups together (Symmetry test p-value<0.05) and if taking them separately (see Table 8a and Appendix 3.1.1.3 for Tables 8b and 8c). Symmetry table: Number of children and number of lesions at screening and second follow-up: Table 8a: BOTH GROUPS Number of lesions at Follow-up 2 Number of Disse- Total lesions at screening None 1 or 2 3 to 5 >5 minated None 0 0 0 0 0 0 1 or 2 9 9 4 4 2 28 3 to 5 6 1 1 6 3 17 >5 12 3 4 9 13 41 Disseminated 9 4 5 5 52 75 Total 36 17 14 24 70 161 Symmetry test p-value = 0.0000

3.1.1.4 Photographs

Pictures of the same lesion were taken during both examinations. The morphology visible in the picture was compared. It was assessed whether improvement (“better”) had taken place, if the morphology of the lesion remained the same (“same”) or if there was deterioration (“worse”). 61 Results Approximately 60 % of the children presented with visibly improved lesions and only 11% with deteriorated lesions (see Figure 5). Example for improved lesions (Figure 4): There are multiple, slightly scaling confluent macules on the chin at the first screening examination. At the second follow-up the child is cured, and only ´Figure 4: Pictures taken during: few residual, non- Screening Follow-up 2 scaling macules remain, which are not regarded as active lesions. (Please find further examples examples in Appendix B).

Evaluation of Photographs (SCR to Fu2): Placebo versus active group

100%

80%

60% worse same 40% better 20%

Percentage ofPercentage children 0% Placebo: n=61 Active: n=67

Figure 5: Evaluation of photographs: Comparing pictures taken at screening (SCR) and the second follow-up examination (Fu2)

3.1.1.5 Microscopic examination of skin scrapings

The relative risk of positive microscopic evidence if receiving placebo versus active soap was calculated at screening and the second follow-up. It was not significantly higher at any of the two examinations if using placebo soap (Chi2 test p-values>0.05). The proportion of positive results decreased strongly in the placebo group from 81.0% (47/58) to 45.5% (35/77) and in the active group from 82.3% (55/68) to 51.3% (39/76).

62 Results Table 9a: Results of the microscopic examination at screening and second follow-up: Number of observation and Relative risk of placebo versus active group Number of observations (Percentage) Relative Chi2 test Examination Placebo Active risk p-value Positive Negative Positive Negative Screening 47 (81.0) 11 (19.0) 55 (82.3) 13 (17.7) 1.00 0.849 Total 102/126 (81.0%) positive

Total 24/126 (19.0%) negative Follow-up 2 35 (45.5) 42 (54.5) 39 (51.3) 37 (48.7) 0.86 0.897 Total 74/153 (48.4%) positive

Total 79/153 (51.6%) negative The samples of 45 children taken during the screening examination could not be evaluated. At the second follow-up examination the samples of 8 children could not be evaluated (see Table 9a and Appendix 3.1.1.5).

3.1.1.6 Quality control of microscopy; cultures

Direct microscopy and cultures were conducted at the “Mycological laboratory of the Department of Dermatology, University Hospital of Zürich” in all cases of uncertain diagnoses one, two and three months after the screening. No cultures could be made of the samples taken after three months. In most cases (88.2% (30/34) and 65.0% (26/40) respectively) the causative agent identified by direct microscopy belonged to Malassezia spp. The frequency distribution of the thus confirmed cases of tinea versicolor did not differ significantly between the placebo and the active group (Fisher’s exact test p-values>0.05). 5.8% (follow-up 1) and 9.3% (follow-up 2) of the samples were negative, the remaining proportions revealed either spores or filaments characteristic of dermatophytes. On culture, only 35.3% (12/34) and 13.8% (4/29) of the samples were positive for Malassezia spp. during the first and the second follow-up respectively. Two cases of dermatophyte infection caused by T. tonsurans (identified by culture) coincided with the identification of Malassezia spp. by direct microscopy indicating a potential co-infection (see Tables 9b, Figures 6a to 6c, Appendix 3.1.1.6 and Apendix B: Child 14). 63 Results Direct microscopy of Pityriasis versicolor (stained with Congo Red, original magnification: x40) Pictures provided by the Mycological laboratory of the Department of Dermatology, University Hospital of Zürich

Figure 6a: Abundant large, round thick-walled spores and mycelia, “spaghetti and meatballs pattern”

Figure 6b: Hyphae forming groups of mycelia

Figure 6c: Few spores and short septate hyphae penetrating epidermal cells

64 Results Table 9b: Culture results of cases with uncertain diagnosis, performed at the Mycological laboratory of the Department of Dermatology, University Hospital of Zürich (note: at the second follow-up no culture was could be performed in 11 cases) Number of observations (Percentage among all Pathogen samples taken) 1 month 2 months Malassezia spec. 12 (35.3) 4 (13.8) Mould 16 (47.1) 16 (55.2) S. brevicaulis 1 (2.9) 1 (3.5) T. tonsurans 2 (5.9) 1 (3.5) T. violaceum 1 (2.9) 0 (0.0) T. mentagrophytes 0 (0.0) 1 (3.5) Malassezia spec. + T. tonsurans 0 (0.0) 1 (3.5) Phoma spec. 0 (0.0) 1 (3.5) Ulocladium spec. 0 (0.0) 1 (3.5) negative 2 (5.9) 3 (10.3) Total 34 (100.0) 29 (100.0)

3.1.1.7 Subjective Impression (See Table 6a)

3.1.1.8 Degree of infection

If summarizing the two groups, a slight decrease in the proportion of children with important (60.3% to 47.8%) and moderate degree of infection (20.5% to 14.3%) could be observed from the first screening examination to the second follow-up. (see Figure 7a, and Appendix 3.1.1.8 for Figure 7b).

Degree of infection: Both groups (n=161)

80 60 none mild 40 moderate 20 severe 0

Percentage children of Screening Follow-up 2 Examination

Figure 7a: Degree of infection (n=161): Both groups (Screening follow-up 2)

The symmetry test confirms that the reduction in the degree of infection from screening to follow- up 2 wass statistically significant if taking active and placebo group together and if assessing the symmetry separately for the two groups (Symmetry test p-value<0.05) (see Table 10a and Appendix 3.1.1.8 for tables 10b and 10c).

65 Results Symmetry table: Number of children according to degrees of infection at screening (SCR) and follow-up 2(Fu2): Table 10a: BOTH GROUPS Degree of Degree of infection at Follow-up 2 infection at Total None Mild Moderate Important Screening None 0 0 0 0 0 Mild 10 14 3 4 31 Moderate 11 3 10 9 33 Important 14 9 10 64 97 Total 35 26 23 77 161 Symmetry test p-value = 0.000

3.1.1.9 Clinical improvement

20 S S 20 c c o o r 15 r e e 15

S S 10 c c 10 r r e e e 5 e 5 n n i i n n 0 g 0 g 0 5 10 15 20 Score Follow-up 2 0 5 10 15 20 Score Follow-up 2 Cubic spline fit Score Reference line Cubic spline fit Score Reference line Figure 8a Placebo: Microscopy positive Figure 8c Placebo: Microscopy negative

S 20 S 20 c c o o r 15 r 15 e e

S 10 S c c 10 r r e e e 5 e 5 n n i i 0 n 0 n g g 0 5 10 15 20 0 5 10 15 20 Score Follow-up 2 Score Follow-up 2

Cubic spline fit Score Cubic spline fit Score Reference line Reference line

Figure 8b: Active: Microscopy positive Figure 8d: Active: Microscopy negative not evaluated for cubic spline curve Figures 8a to 8d: Tinea versicolor: Score at screening (SCR) and follow-up 2 (Fu2), according to microscopic result at follow-up 2

66 Results The overall improvement and the lack of superiority of active soap, as described in the paragraphs 3.1.1.1 to 3.1.1.8 above, can be confirmed when presenting the clinical score of each individual stratified according to active and placebo group by using a scatter plot presentation. Figures 8a to 8d display the score at screening (y-axis) and follow-up 2 (x-axis). It becomes evident that there was a marked improvement until follow-up 2, since the estimated cubic spline, lies mostly above the reference line. The decrease in the score was slightly more pronounced in the cases showing negative microscopic results during the second follow-up. In the placebo group with positive microscopic results, the end of the fitted cubic spline curve, which was an ascending slope, lies below the reference line. This indicates that cases which were more severe at screening rather had a tendency to deteriorate. In the cases of negative microscopic results, the cases of deterioration had been mild infections during the screening examination.

3.1.1.10 Clinical and microscopic resolution

Evaluation Categories at Follow-up2: Placebo versus active group

30 25 20 Placebo 15 10 Active children 5 Percentage of of Percentage 0 1234567 Evaluation Categories

* 1= resolution: child cured 2= microscopic resolution and only few clinical symptoms 3= improvement: negative microscopic evidence with significant clinical improvement 4= no significant clinical improvement but negative mycologic evidence 5= improvement: microscopy positive with significant clinical improvement 6= lack of improvement: positive mycologic evidence with no significant clinical improvement 7= deterioration Figure 9: Proportions of children (placebo versus active soap) belonging to different evaluation categories (1 to 7) at the second follow-up examination (Fu2) after 2 months of soap use

There was no significant difference in the frequency distribution of the 7 evaluation categories comprising the clinical signs and symptoms and the microscopic diagnosis between the placebo and active group after 2 months. (Fisher’s exact test p-value= 0.194) (see Table 11 (Appendix 3.1.1.10) and Figure 9). The proportion of children showing resolution (category 1 plus category 2) is nearly identical for the placebo versus active group: 28.0% (23/82) versus 28.9% (24/83) (Chi2 test p-value=1.000). The overall cure rate was of 28.4% (47/165) (see Table 12). The proportion of children belonging to category 2 (microscopic resolution but no complete clinical resolution) was greater in the active 67 Results group. A larger proportion of children belonging to the active group had a positive microscopic result. Lack of significant clinical improvement (category 4 plus 6) and deterioration (category 7) was more common in the placebo group.

Table 12: Relative frequencies and frequency distribution of children with and without resolution (placebo versus active) Clinical and Number of observations (Percentage) microscopic Total Placebo Active resolution Yes 23 (28.0) 24 (28.9) 47 (28.4) No 59 (72.0) 59 (71.1) 118 (71.5) Total 82 (100.0) 83 (100.0) 165 (100.0) Fisher's exact test p-value = 1.000

3.1.2 Other superficial dermatomycotic infections: Tinea capitis, corporis and pedis infections

3.1.2.1 Signs and symptoms/effectiveness

In order to assess the effectiveness of the soap containing Triclosan, several endpoints (clinical signs and symptoms typical of the disease) were evaluated during all three examinations. The proportions having these signs and symptoms were calculated for both examinations. There was no significant difference found between placebo and active group for any parameter. The relative risk of having these symptoms if receiving placebo versus active soap was calculated. It was not found to be significantly different from 1 at any stage if using placebo soap (see Appendix 3.1.2.1). For various endpoints the symmetry test showed a significant improvement for active and placebo soap. However, there was no significant difference in the symmetry regarding these parameters (see Appendix 3.1 for Table 6d).

3.1.2.2 Degree of pruritus

Tinea capitis, corporis and pedis infections It was found that the proportion of children without pruritus increased, whereas the proportion of children having mild, moderate or severe pruritus decreased significantly from the screening to the second follow-up examination. This is if taking the two groups together (see Figure 10a) and if taking placebo and active groups separately (see Appendix 3.1.2.2 for Figure 10b). If looking at the three diagnoses separately, the degree of pruritus decreased significantly only for tinea capitis infections (Symmetry test p-value = 0.0098) if both groups are summarized (see Appendix 3.1.2.2). With regard to the other diagnoses the decrease was not significant. 68 Results

Degree of pruritus: Both groups (n=53)

80,00

60,00 none mild 40,00 moderate 20,00 severe

0,00 Percentage of children Percentage of Screening Follow-up 2 Examination

Figure 10a: Other superficial dermatomycoses: Degree of pruritus (n=53) Both groups (Screening and follow-up 2)

Other superficial dermatomycoses: Symmetry table: Number of children according to degrees of pruritus at screening and second follow-up: Table 13a: BOTH GROUPS Degree of Degree of pruritus at Follow-up 2

pruritus at None Mild Moderate Severe Total Screening None 12 1 0 0 13 Mild 18 7 0 1 26 Moderate 5 5 2 0 12 Severe 2 0 0 0 2 Total 37 13 2 1 53 Symmetry test p-value = 0.000

2.1.2.3 Size of the largest lesion and number of lesions

Size of the largest lesion - Tinea capitis

The average diameter of the largest lesion decreased to a similar extent in both groups from the screening to the second follow-up (Wilcoxon rank sum test p-value =0.896) (see Table 14a). Table 14a: Tinea capitis: Number of observations and mean diameter of the largest lesion at screening (SCR) and second follow-up (Fu2) Number of observations Mean diameter (cm) Examination Placebo Active Placebo Active Screening 15 13 2.6 2.9 Follow-up 2 6 8 1.9 2.4

Size of the largest lesion - Tinea corporis

The average diameter of the largest lesion decreased in the active group, whereas it increased in the placebo group from the screening to the second follow-up. However, this was not significant (Wilcoxon rank sum test p-value=0.339) (see Table 14b). 69 Results Table 14b: Tinea corporis: Number of observations and mean diameter of the largest lesion at screening (SCR) and second follow-up (Fu2) Number of observations Mean diameter (cm) Examination Placebo Active Placebo Active Screening 7 5 2.1 2.2 Follow-up 2 4 7 3.1 1.0

Number of lesions - Tinea capitis

The symmetry test showed that there was a significant reduction in the number of lesions from screening the second follow-up if taking the children of both groups together (Symmetry test p- value = 0.0038), but not if taking the two groups separately (see Table 15a and Appendix 3.1.2.3 for Tables 15b and 15c). Table 15a: BOTH GROUPS Number of lesions at Follow-up 2 Number of Disse- Total lesions at Screening None 1 or 2 3 to 5 >5 minated None 1 0 0 0 0 1 11 or 2 4 2 1 0 1 8 3 to 5 7 2 2 1 2 14 >5 6 0 0 0 2 8 Disseminated 2 1 0 0 1 4 Total 20 5 3 1 6 35 Symmetry test p-value = 0.0038

Number of lesions - Tinea corporis

The symmetry test reveals that there was a significant reduction in the number of lesions from screening the second follow-up if taking the children of both groups together (Symmetry test p- value 0.0460) but not if taking the two groups separately (see Table 15d and 15e and 15f (Appendix)). Table 15d: BOTH GROUPS Number of lesions Follow-up 2 Number of Disse- Total lesions at Screening None 1 or 2 3 to 5 >5 minated None 0 0 0 0 0 0 1 or 2 5 3 0 0 0 8 3 to 5 0 1 0 0 0 1 >5 0 0 2 0 0 2 Disseminated 0 0 0 0 2 2 Total 5 4 2 0 2 13 Symmetry test p-value = 0.0460 70 Results 3.1.2.4 Photographs

Tinea capitis and Tinea corporis The largest proportion of children showed improvement (tinea capitis: 66.7% (16/24) and tinea corporis 76.9% (10/13)) of the lesions as compared to deterioration or unchanged lesions. This is true for both groups in case of tinea capitis and corporis infections (see Figures 11,12 and 13).

Evaluation of photographs

100% 80% worse 60% same 40% better 20% 0%

Pecentage of children T. capitis T. capitis T. T. Placebo: Active: corporis corporis n=18 n=41 Placebo: Active: n=7 n=6

Figure 11: Evaluation of photographs: Comparing pictures taken at screening (SCR) and the second follow-up examination (Fu2) (n=number of individuals)

Examples for improved lesions (Figures 12 and 13):

Figure 12: Pictures taken during Screening Follow-up 2

Figure 12: The child was cured at the second follow-up: grey patches, alopecia and hair stumps are no longer visible. There is new hair growth at all formerly affected sites. The whitish reflection of the flash light is mimicking a grey patch and must not be mistaken for an active lesion. There is no permanent alopecia, scarring or skin atrophy. 71 Results

Figure 13: Pictures taken during Screening Follow-up 1

Figure 13: At the first screening the child presents with a typical ringworm-like lesion, characterised by a scaly patch with a sharply demarcated and slightly raised concentric margin. There is some beginning central clearing and postinflammatory hyperpigmentation in the centre of the lesion. At the first follow-up only very minor scaling at the upper border of the formerly active lesion is present. Residual postinflammatory hyperpigmentation remains and minor skin atrophy are still present at the follow-up.

3.1.2.5 Microscopic examination of skin scrapings

Tinea capitis

Hyphae, mycelia and/or spores were found in 59.0% (21/39) of all children with tinea capitis at the screening examination, whereas at the second follow-up this proportion had decreased to only 41.9% (13/31) (see Table 16a). During the screening examination, the relative risk for the placebo group to yield a positive microscopic diagnosis was 0.551 times that of the active group because the proportions of positive and negative results had not been distributed equally during the screening examination despite randomization. This was significant (Fisher's exact test p-value = 0.0492). However, this finding has to be regarded to be due to chance because no intervention had taken place at that stage. On the second follow-up the proportions the relative risk of yielding a positive result was not significantly different from 1. However, the proportion of negative results increased only in the active group. During the screening examination the samples of one child could not be evaluated. During the second follow-up examination the samples of 11 children could not be evaluated.

Tinea corporis

Hyphae, mycelia and/or spores were found in 76.9% (10/13) of all children with tinea corporis at the screening examination, whereas at the second follow-up this proportion had decreased to only 72 Results 35.7% (5/14) (see Table 16b). The relative risk of positive microscopic evidence if receiving placebo versus active soap was not significantly different from 1 during the second follow-up examination if using placebo soap. During the screening examination the samples of 2 children could not be evaluated. During the second follow-up examination the samples of 5 children could not be evaluated. Direct microscopy and cultures were conducted in at “Mycological laboratory of the Department of Dermatology, University Hospital of Zürich” in all cases of uncertain diagnoses at the first and/or the second follow-up examinations. The dermatophyte species identified were T. tonsurans, T. violaceum and T. mentagrophytes (see 3.1.1.6).

Table16a: Tinea capitis: Results of the microscopic examination at screening and second follow-up: Number of observation and Relative risk of placebo versus active group Number of observations (#) (Percentage) Fisher’s Relative exact Examination Placebo Active risk test Positive Negative Positive Negative p-value Screening 9 (42.9) 12 (57.1) 14 (77.8) 4 (22.2) 0.551 0.0492 Total 21/33 (59.0) positive Total 18/33 (31.0) negative Follow-up 2 8 (47.1) 9 (52.9) 5 (35.7) 9 (64.3) 1.318 0.3942 Total 13/31 (41.9) positive Total 18/31 (58.1) negative

Table 16b: Tinea corporis: Results of the microscopic examination at screening (SCR), second follow-up (Fu2) (n=number of samples evaluated; (#) difference in numbers because data could not be obtained or evaluated in some cases) Number of observations (#) (Percentage) Fisher’s Relative exact Examination Placebo Active risk test Positive Negative Positive Negative p-value Screening 5 (71.4) 2 (28.6) 5 (83.3) 1 (16.7) 0.857 1.000 Total 10/13 (76.9) positive Total 3/13 (23.1) negative Follow-up 2 1 (14.3) 6 (85.7) 4 (57.1) 3 (42.9) 0.25 0.133 Total 5/14 (35.7) positive Total 9/14 (64.3) negative

73 Results 3.1.2.6 Subjective impression (See Table 6b)

3.1.2.7 Degree of infection (See Table 6b and Appendix 3.1.2.7 for Tables 17a to 17c)

3.1.2.8 Clinical improvement

The marked overall improvement after two months of soap use and the lack of superiority of active soap can be confirmed when presenting the clinical score of each individual stratified according to active and placebo group by using a scatter plot presentation. Figures 14a to 14d (tinea capitis) and 15a to 15b (tinea corporis; see Appendix 3.1.2.8) display the score during screening (y-axis) and follow-up 2 (x-axis). The improvement becomes evident, since the estimated cubic spline lies mostly above the reference line.

25 S S 20 c c o 20 o r r 15 e e 15 S S10 c 10 c r r e e 5 e 5 e n n i i 0 n 0 n 0 5 10 15 20 g 0 5 10 15 20 g Score Follow-up 2 Score Follow-up 2 Cubic spline fit Score Cubic spline fit Score Reference line Reference line

Figure 14 a Placebo: Microscopy positive Figure 14c Placebo: Microscopy negative

S 15 S 20 c c o o r r 15 e 10 e S S 10 c c r 5 r e e 5 e e n n i i 0 n 0 n g 0 5 10 15 20 g 0 5 10 15 20 Score Follow-up 2 Score Follow-up 2 Cubic spline fit Score Cubic spline fit Score Reference line Reference line

Figure 14b Active: Microscopy positive Figure 14d Active: Microscopy negative

Figures 14a to 14d: Clinical improvement of tinea capitis: Score at screening and follow-up 2, according to microscopic result (M+ or M-) at follow-up 2

74 Results Tinea capitis

In both groups of positive microscopic cases, the end of the cubic spline curve lies below the reference line. Only cases of moderate infection at screening had the tendency to deteriorate. In the cases of negative microscopic results at screening there was no deterioration. In the cases of negative microscopic results at the second follow-up the decrease of the score was more pronounced. No difference can be observed between the placebo and active group: the fitted cubic spline has a similar shape for both groups, even if the subjects with negative or positive microscopic results at the second follow-up are taken separately (see Figure 14a to 14d):

Tinea corporis (See Appendix 3.1.2.8 for Figure 15e, 15f and Table 18).

3.1.2.9 Clinical and microscopic resolution

Tinea capitis

The proportion of children showing resolution (category 1 plus category 2) was higher for the placebo (n=20) versus active group (n=16): 65.0% versus 56.2% (Fisher’s exact test p-value = 0.734), leading to an overall resolution rate of 61.1% (see Table 19a (Appendix 3.1.2.9), Table 19b and Figure 16). A positive microscopic result despite significant clinical improvement (category 5) as well as deterioration (category 7) was found more frequently in the placebo group (Appendix 3.1.2.9 for Table 19a).

Evaluation categories at Follow-up 2: Placebo versus active group

80,00 60,00 Placebo 40,00 Active

children 20,00

Percentage of of Percentage 0,00 1234567 Evaluation categories*

* 1= resolution: child cured 2= microscopic resolution and only few clinical symptoms 3= improvement: negative mycologic evidence with significant clinical improvement 4= negative mycologic evidence with no significant clinical improvement 5= improvement: microscopy positive with significant clinical improvement 6= lack of improvement: positive mycologic evidence with no significant clinical improvement 7= deterioration Figure 16: Tinea capitis: Proportions of children (placebo versus active soap) belonging to different evaluation categories (1 to 7) at the second follow-up examination (Fu2) after 2 months of soap use

75 Results Table 19b: Tinea capitis: Relative frequencies (number of observations) and frequency distribution (percentage) of children having received placebo versus active soap: resolution versus no resolution Clinical and Number of observations (Percentage) microscopic resolution Placebo Active Total Yes 13 (65.0) 9 (56.2) 22 (61.1) No 7 (35.0) 7 (43.8) 14 (38.9) Total 20 (100.0) 16 (100.0) 36 (100.0)

Fisher's exact test p-value = 0.734

Tinea corporis

The proportion of children showing resolution (category 1 plus category 2) was nearly equal in the placebo (n=9) versus active group (n=7): 55.6% versus 57.1% (Fisher’s exact test p-value = 1.000), leading to an overall resolution rate of 56.3% (see Table 19d and Figure 17). Deterioration only occurred in one case in the placebo group. A positive microscopic result despite significant clinical improvement (category 5) was not found (see Appendix 3.1.2.9 for Table 19c).

Evaluation categories at Follow-up 2: Placebo versus active group

50,00 40,00 30,00 Placebo 20,00 Active

children 10,00

Percentage of 0,00 1234567 Evaluation categories

* 1= resolution: child cured 2= microscopic resolution and only few clinical symptoms 3= improvement: negative mycologic evidence with significant clinical improvement 4= negative mycologic evidence with no significant clinical improvement 5= improvement: microscopy positive with significant clinical improvement 6= lack of improvement: positive mycologic evidence with no significant clinical improvement 7= deterioration Figure 17: Tinea corporis: Proportions of children (placebo versus active soap) belonging to different evaluation categories (1 to 7) at the second follow-up examination (Fu2) after 2 months of soap use

Table 19d: Tinea corporis: Relative frequencies (number of observations) and frequency distribution (percentage) of children having received placebo versus active soap: resolution versus no resolution Clinical and Number of observations (Percentage) microscopic resolution Placebo Active Total Yes 5 (55.6) 4 (57.1) 9 (56.3) No 4 (44.4) 3 (42.9) 7 (43.7) Total 9 (100.0) 7 (100.0) 16 (100.0) Fisher's exact test p-value = 1.000

76 Results Tinea pedis

In order to decide whether the study participants were cured during the second follow-up examination five categories were defined, which combined clinical signs and symptoms. There was no significant difference in the frequencies of the categories between the two groups (see Table 19e (Appendix) and Figure 18) (Fisher’s exact test p-value = 0.183). The proportion of children showing complete resolution of symptoms was greater in the active group (n=7) than in the placebo group (n=9) (85.7% versus 55.6%). However, this difference in proportions was not significant (Fisher’s exact test p-value = 0.308) (see Table 19f).

Evaluation categories: Placebo versus active group

100 80 60 Placebo 40 Active children 20 Percentage of of Percentage 0 12345 Evaluation Categories*

* 1= clinical resolution 2= improvement: score was reduced 3= lack of improvement: score remained the same 4= no resolution: score was non-applicable at screening, but child is not cured 5= deterioration: score increased Figure 18: Tinea pedis: Proportions of children (placebo versus active soap) belonging to different evaluation categories (1 to 5) at the second follow-up examination after 2 months of soap use

Table 19f: Tinea pedis infections: Relative frequencies (number of observations) and frequency distribution (percentage) of children having received placebo versus active soap: resolution versus no resolution: Number of observations (Percentage) Clinical resolution Placebo Active Total Yes 5 (55.6) 6 (85.7) 11 (68.7) No 4 (44.4) 1 (14.3) 5 (32.3) Total 9 (100.0) 7 (100.0) 16 (100.0) Fisher's exact test p-value = 0.308

3.2 Determination of minimal inhibitory concentrations (MIC’s) of placebo soap and of Triclosan

There was abundant growth of the fungus of certain some isolates (e.g. N° 4 and 5), in others only few or no colonies were found to grow after inoculation (e.g. N°2 and N°3). This is true for both soap dilutions (0.2% and 2%) and for the whole dilution series. The control showed the same isolate-dependent pattern of growth. For some sets of the same dilution, the results were contradictory, i.e. abundant growth in one case, and no colony growth in the other. No concentration-dependent inhibition of growth was observed in any of the Malassezia spp. isolates 77 Results from the Department of Mycology, University of Zürich (see Table 20a and Appendix 3.2). Therefore no MIC could be determined for placebo soap. For Triclosan an inhibition of growth was only observed in the significant concentrations of 1000 to 562.5 μg/mL. In the successive dilutions, no concentration dependent inhibition of growth could be found. As in placebo soap, the growth of the fungus varied according to the isolate (see Table 20b).

Table20a: Growth of Malassezia spp. colonies on Dixon Agar inoculated with different concentrations of placebo soap. Concentration of soap solution: 0.2%; for 2% soap solution see Appendix 3.2 Number of colony forming units (CFU) (### > 50 CFU) according to Malassezia spp. isolate (N°1 to 6) Concentration of soap Isolate Isolate Isolate Isolate Isolate Isolate solution 1 2 3 4 5 6 (0.2%) on agar (μg/mL) 1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd series series series series series series series series series series series series

400 ### 3 ### 0 0 0 ### ### ### ### ### 1 200 2 1 0 0 ### 0 ### ### ### ### ### ### 100 5 1 2 0 0 0 ### ### ### ### 0 0 50 10 2 0 0 0 0 ### ### ### ### ### 4 25 5 2 0 0 0 0 ### ### ### ### 9 2 12.5 5 3 1 0 ### 0 ### ### ### ### 0 1 6.25 2 2 0 0 0 0 ### ### ### ### ### ### 3.13 1 0 0 5 0 0 ### ### ### ### 2 ### 1.56 2 ### 0 3 0 0 ### ### ### ### ### ### 0.78 1 4 0 1 1 0 ### ### ### ### ### ### 0.39 1 ### 0 0 2 1 ### ### ### ### ### 1 0 2 4 0 2 1 0 ### ### ### ### ### 0

Table 20b: Growth of Malassezia spp. colonies on Dixon Agar inoculated with different concentrations Triclosan Dilution series of Triclosan: Number of colonies according to Malassezia spp. isolate (1 to 6) Concentrations of Triclosan Isolate Isolate Isolate Isolate Isolate Isolate on agar (μg/mL) 1 2 3 4 5 6 1000 0 0 0 0 0 0 750 0 0 0 0 0 0 562.5 0 0 0 0 3 0 421.8 6 0 0 5 40 9 316.4 5 1 0 2 50 8 237.3 1 1 0 5 40 23 177.98 1 1 2 2 42 21 133.49 2 1 2 11 39 19 100.11 2 4 2 2 43 23 75.09 2 1 2 3 49 21 56.31 1 1 2 3 42 14 0 (only ethanol on agar) 4 2 2 2 40 19 Only agar 2 1 4 2 39 15 81 Results 4. Scabies

4.1 Epidemiology and Study population

Scabies infestation was a comparatively rare condition, with a prevalence of only 1.5% (12/820). The prevalence was found to decrease with age, and boys were more often affected than girls. Two of the 12 affected children were given Ivermectin because their infection was too severe to take part in this study. Another two children were also not included because they had healing scabies. One of them had already received BBE treatment. The eight remaining study participants were pupils at Lihami Primary School. Two of them were girls, six were boys. Seven children were between 6 and 10 years of age, one child was 13 years old. The mean age was 9.25 years. After randomisation four children received placebo and four children active soap.

4.2 Assessment of the effect of soap and the efficacy of Triclosan

4.2.1.2 Case descriptions Active Group

Screening

Diagnosis was made clinically. In one case microscopic examination of a skin sample was successful. All four boys, who were aged between 8,8,9 and 10 years, had moderate infections (the score ranged between 6 and 11). Their skin eruptions presented as typical papular pruritic rashes or the characteristic primary lesions (burrows) on their forearms, hands and the anterior axillary folds. Other localisations found were the umbilical and pubic region, the gluteal region and the thighs. Three children complained about moderate pruritus, the child with the most severe infection in this group about severe pruritus. In all four cases the itching was more severe at night. Other family members were affected in 2 out of these four children.

Follow-up 2:

The two study participants aged 8 years had to be excluded from the study. One was lost to follow- up, and the other one only came once to pick up his soap during the whole study. Nevertheless, this child was cured. Only post-inflammatory excoriated lesions could be observed. The remaining two boys showed a contrary evolution of their scabies infection. In one of them the infection got significantly worse. The score had increased from 6 to 14. Some lesions had become superinfected. The skin was dry and in some areas atrophic. Both entire arms including wrists and hands were affected, and the infestation had spread to both anterior and posterior lower legs and the genital region. Eight common localisations were affected. In the other boy, the signs of infection had 82 Results improved. The score was reduced to 4 points. Lesions were found at common localisation only. There was no more superinfection. Both boys reported that pruritus had improved.

4.2.1.3 Case descriptions Placebo Group

Screening

This group consisted of two girls, who were sisters and living together and of two boys, who were brothers and also staying together in the same house. They were between 8 and 13 years of age. Diagnosis was made clinically. No mites could be detected on microscopic examination of the skin scrapings. Both girls had severe infection with score values of 13 and 19 respectively, whereas the degree of infection of the two boys was moderate with score values of 6 and 9. Typical papular pruritic rash on forearms, hands, knees and the umbilical region was observed. Besides, macules, burrows, crusts and generalized lichenification could also be observed. In her sister some lesions were excoriated, and most lesions were pink in colour. There was bacterial superinfection, which was treated with a five day course of cloxacillin. The number of common localisations affected varied between 1 and 5 in all four children. Bacterial superinfection was present in three of the four children. The two boys complained about moderate pruritus. The child with the severest infection in this group described her pruritus as only mild. Her sister had severe pruritus. In all four cases the itching was more severe at night.

Follow-up 2

Both girls were completely cured, and the infestation of the boys had improved. The younger boy was nearly cured, with only some residual signs and mild degree of infection (score=3). Most of the lesions were now healing; active lesions were found in the genital region. Residual postinflammatory hyperpigmentation was still found on the right anterior and posterior upper arm, chest, abdomen and the genital region. Pruritus was no longer present. His brother had only improved slightly. However, the score was reduced markedly to 11 because there was no more bacterial superinfection or thick crusts. His skin showed generalized lichenification and was in some areas atrophic. The lesions were not sharply demarcated and disseminated over nine common localisations. Pruritus was now mild. In three children the improvement of the skin disease could be confirmed by the photographs taken at the first screening examination and the second follow-up. Deterioration of the lesions was seen in case of the 13 year-old boy from screening to the first follow-up. 83 Results 4.2.1.4 Evaluation of effectiveness

There were no evident differences between the placebo and the active group. No significance tests could be performed because the sample sizes were too small (only two children in the active group) (see Appendix 3.1.3.4.1 to 4 and Table 21). However, in both groups an overall tendency towards a reduction in the proportion of children having these symptoms could be observed.

4.2.1.5 Degree of pruritus

The difference in pruritus between the two groups cannot be assessed because the sample size was too small. However, it can be stated that if summarizing the two groups the proportion of children having mild pruritus increased from the screening to the second follow-up examination. No more children had moderate or severe pruritus at the second follow-up, but two thirds had no pruritus (see Figure 18 and for Appendix 3.1.3.5).

Degree of pruritus

0,8 0,6 none 0,4 mild

scabies 0,2 moderate children with Proportion of Proportion 0 SCR: n=8 Fu1: n=4 Fu2: n=6 severe Examination

Figure 18: Degree of pruritus: Proportions of children at the three examinations (SCR, Fu1, Fu2)

4.2.1.6 Degree of infection

Table 21: Degree of infection: Number of observations: Degree of infection Number of observations Examination Total (score) Placebo Active Mild (4) 0 0 0 Screening Moderate (5 to 11) 2 4 6 Severe (12 to 27) 2 0 2 Total 4 4 8 cured 2 0 2 Mild (4) 1 1 2 Follow-up 2 Moderate (5 to 11) 1 1 2 Severe (12 to 27) 0 0 0 Total 4 2 6 The degree of infection improved in both groups: Two out of six children still had moderate infection at the second follow-up. Two children (both placebo group) were cured, and the remaining two children had only residual mild infection. No difference can be discovered between the two groups because the sample sizes were too small (see Table 21).

85 Discussion Discussion:

1. Epidemiology of superficial dermatomycoses

1.1 Tinea versicolor

In the present study the prevalence of tinea versicolor was found to be 26.2% and accounted for 70% of all dermatomycoses. A proportion of tinea versicolor infections among primary schoolchildren as high as in the present study, and therefore relatively low proportion of tinea capitis infections (14.7%), has to this author’s knowledge not been reported so far from Tanzania or another sub-Saharan African country (Satimia et al., 1998; Schmeller, 1998; Schmeller et al., 2001). This pattern cannot be easily explained, and only few assumptions shall be presented here. Tinea versicolor is not regarded as a contagious disease, and Malassezia yeasts were also isolated from healthy individuals (Gupta, 2002; Faergemann, 1994; Schmidt, 1997; Leeming et al., 1989). The growth conditions present among the individuals in Ifakara must therefore have been highly favourable for Malassezia yeasts thus leading to a successive overgrowth and shift to the pathogenic filamentous form. This might at the same time have inhibited the growth of dermatophytes competitively. Certain predisposing factors for tinea versicolor infections might play an important role, e.g. endogenous factors such as malnutrition, hyperhidrosis, a positive family history and immunodeficiency possibly due to HIV infection. There is no information available with regard to the actual rate of HIV positive children in Kilombero District, but in Ifakara 13.6% of the general population are believed to be infected (Source: IHRDC, 2003). However, the proportion of HIV positive children attending primary school may be relatively low. This is is due to the fact that those infected during pregnancy, delivery or through breastfeeding usually die before reaching the school age because of their increased susceptibility towards the effects of malnutrition and infectious diseases like PCP or tuberculosis (Dr. B. Idindili, personal communication). The presence of any of the above mentioned endogenous factors was not assessed in the course of this study. Exogenous factors include heat, moisture and occlusion through clothes or vaseline, palm oil or cocoa butter. Occlusion may increase CO2 production resulting in an altered microbiologic flora and a change in the pH of the skin (Pontash et al., 1989; Dave et al., 1987; aTerragni et al., 2001; bTerragni et al., 2001; Bouassida et al., 1998). In the region where the present study was performed, the application of vaseline – called “mafuta” is a very common procedure as shiny greasy skin is looked at as being very attractive (P. Schmid-Grendelmeier, personal communication). During history-taking and physical examination, this feature was not assessed in a systematic way, but our observations confirmed the statement. Increased susceptibility of African in comparison to 86 Discussion Caucasian children has also been discussed to play a part (bTerragni et al., 2001; Borelli, 1985). The influence of the age distribution among the study population should not be neglected. 58.5% of the children were older than 11 years. The group of adolescents was slightly over-represented thus increasing the overall prevalence rate.

1.2 Other superficial dermatomycotic infections

In the Study Proposal of the present study, the prevalence rate of tinea capitis and/or tinea corporis had been assumed to be approximately 15%. This assumption was based on studies conducted in various comparable settings. In Ivory Coast 11.3% of the examined pupils (Hervé et al., 2002) had tinea capitis infections whereas 16.9% (Melake et al., 1990) were in infected in north-west Ethiopia, 29% in south-west Ethiopia (Figueroa et al., 1997) and in Zimbabwe (Robertson, 1990) and another study in even 33.3% (Ayaya et al., 2001) in Kenya. Figueroa et al. identified 24% of school children as asymptomatic dermatophyte carriers (Figueroa et al., 1997). It is noteworthy that in the present study samples were only taken from symptomatic children. The possibility that many children were not identified to be infected with tinea capitis or corporis should be considered. In the present study, 5.5% of the children showed clinical signs of tinea capitis infection, 3.2% of tinea pedis and 2.6% of tinea corporis infections. The prevalence rate of 2.6% of tinea corporis was similar to those described by Enweani et al. (1996) and Figueroa (1996) who discovered rates of 2.7% for Nigerian and Ethiopian schoolchildren respectively. It also coincides with the prevalence described for children aged 6 to 14 years in various Tanzanian districts (approx. 1 to 4%) (KCMC, unpublished data). Tinea pedis infections were slightly more frequent in the pupils from Ifakara (3.2%) than in pupils from Nigeria (1.5%) (Enweani, 1996) and from Turkey (0.5%) (Inanir et al., 2002). The prevalence rate compares more closely to the average infection rate of 3.7% among British pupils as described by English et al. (English et al. , 1959). It remains uncertain whether hygiene conditions, health education and access to treatment were better as compared to the other studies thus explaining the relatively low prevalence rate of tinea capitis. Furthermore, it is possible though not very likely that the children attending the two primary schools in Ifakara have less contact with animals, dust, contagious fomites or infected individuals. The fact that all children attending standard 1 and 2 at any of the Ifakara Primary Schools had to have short hair may protect them from tinea capitis infections. 87 Discussion 2. Assessment of the effect of soap and the efficacy of Triclosan in soap

Large proportions of cases of dermatomycoses were cured during two months of regular soap use. Cure rates of tinea versicolor, tinea capitis, corporis and pedis were 28.4%, 61.1%, 56.3% and 36.8% respectively (overall cure rate for dermatomycoses: 36.0%).

2.1 Superficial dermatomycoses

2.1.1 Tinea versicolor infections

No significant differences were found between the active and placebo soap for any of the observed parameters in any of the selected dermatoses. Some parameters were supportive of a slightly superior effectiveness of Triclosan soap. It has to be concluded that Triclosan is not clinically effective against Malassezia spp. However, for both placebo and active soap significant tendencies towards improvement and resolution could be identified (see Tables 6a and 6b). The fact that the proportion of children presenting the following parameters: scaling, pruritus, and hypopigmentation; decreased from the first examination to the second follow-up supports this statement. Severe extensive cases showed less improvement than milder ones. Triclosan as an inhibitor of cyclo-oxygenase and lipo-oxygenase has an anti-inflammatory and anti- irritative properties (Nissen et al., 1998). However, since there was no difference in pruritus between the Triclosan and the placebo group, it has to be concluded that Triclosan alone was probably not responsible for the overall decrease of the degree of pruritus over two months. The lacking of the additional effect by Triclosan might be explained by the fact that in spite of the long- lasting antimicrobial efficacy of all products containing Triclosan, the anti-inflammatory or anti- irritative effect is not as strong in rinse-off-formulations as in leave-on-formulations (M. Schnyder, personal communication). The leave-on time might therefore have been too short for a more pronounced anti-irritative effect of Triclosan to become evident. The decrease in the intensity of pruritus could be explained by the procedure of intense and regular washing which was promoted in the course of this study. Controlled soap use per se thus seems to have had a beneficial effect on the degree of pruritus. The soap consumption during the study period has probably been higher than normally, leading to a reduction of dust and other irritative particles on the skin surface. The decrease in the intensity of pruritus is also associated with the decrease in the overall degree of infection. Climatic conditions may have influenced the degree of pruritus indirectly, since, according to Faergemann (1993) it seems to depend on the degree of sweating. 88 Discussion The overall proportion with disseminated lesions remained nearly the same. The soap might thus be able to reduce the extension of the lesions, if lesions have not been disseminated before. In these cases complete resolution may take longer to occur or may not at all be achieved by regular soap use. Regular soap use has strongly reduced the overall proportion of positive microscopic results from 81.0% during the screening to 48.4% during the second follow-up examination (see Results for Table 10a). There was no significant difference between active and placebo group. Early changes in the aspects of the filaments that may have taken place due to the use of the soap may not have even been visible by light microscopy. McDaniel et al. have shown that already shortly after the onset of treatment, the fungal cells may appear wrinkled and disorganized. These changes might have been visible using electronmicroscopic techniques thus underlining even more the effectiveness of the soap (McDaniel et al., 1984; Gupta, 2002). On the other hand it has to be considered that a very thorough and frequent mode of washing of the skin may obscure a microscopic diagnosis, thus producing some false negative results on microscopic examination (Faergemann, et al., 1982). This may be the case in the present study, but would affect both groups to a similar extent. In the present in vivo study Triclosan was not found to be mycologically more effective against Malassezia spp. than the placebo. This coincides with in vitro experiments, which have shown that Triclosan is not effective against M. furfur (MIC>1000 ppm) (© Ciba Specialty Chemicalsa, 1998). Even though no efficacy of Triclosan could be proven for M. furfur, it is important that this species may represent only 1 to 10% of all the species isolated. According to Gupta et al. the epidemiology of Malassezia yeasts depends on the geographic region (Gupta et al., 2003). The results of the microscopic examination and cultivation of fungi performed for quality control at the “Mycological laboratory of the Department of Dermatology, University Hospital of Zürich” revealed that, in nearly 90% of uncertain differential diagnoses between tinea versicolor and tinea corporis, the lesions were caused by Malassezia spp. This implies that the clinical picture of tinea versicolor shows a great variety of presentations. An over-diagnosis of tinea corporis may be the consequence and should be considered as a possible source of bias. In the placebo and active group the majority had experienced improvement at both follow-up examinations. In conclusion, the subjective assessment shows that on a subjective level the soap use was well-accepted and regarded as effective in reducing pruritus or the cosmetic disturbance, which are the most relevant complaints of the children. A significant improvement of the degree of infection could be achieved through the application of both soaps (see Results for Figure 7a and 7b). There are several possible explanations for this. First, the amount of oily or sebaceous substances on the skin surface might be reduced due to the regular use of soap thus making the growth of the lipophilic yeasts more difficult. 89 Discussion

Second, less occlusion of skin through oily substances might provoke a decrease of CO2 concentration and an increase of the pH value on the skin surface. Third, the change of season between the screening and the follow-up examinations, bringing less heat and humidity than before (see Table 1, Patients, Approaches and Methods), also has to be considered. These climatic factors are believed to contribute largely to the high prevalence in tropical regions (Gupta, 2002; Taplin et al., 1965) by promoting the shift to the pathogenic mycelial form. Miskeen et al. have described in India a marked increase in the number of cases during the hot and humid summer months and a sudden decrease during the cooler and drier winter months (Miskeen et al., 1984). Kamalam et al. found that the incidence of tinea versicolor in Madras was highest in May (month of highest maximum temperatures) and October (month of most extensive rainfalls) and lowest in January (month of lowest minimum temperatures) (Kamalam, et al., 1981). In Venezuela a significantly higher prevalence rate of tinea versicolor was found in the semi-arid in comparison to the sub-humid zone (Perez Blanco et al., 1990). Porter et al. also reported a markedly higher prevalence during the rainy season from a Gambian village (Porter, 1979). According to him, this decrease can be attributed to the higher hydration level of the stratum corneum associated with the greater dispersal of sebum during the hot and humid season. It is possible that in the present study vigorous scrubbing with soap and water, as recommended by Canizares (1982) as an adjunct to further topical treatment, contributed to the overall improvement seen. Spontaneous resolution is not believed to occur frequently (Canizares et al., 1993). However, it is not possible to prove whether the improvement in the degree of infection in the present study, representing a tendency towards resolution of the disorder over time, was due to the soap use or the climatic conditions, as no control group could be included in the study for ethical reasons. The scatter plot presentation (see Results for Figures 8a to 8d) allows the reader to visualize that a large proportion of children showed improvement of their lesions. Important cases with positive microscopy during the second follow-up rather had the tendency to deteriorate than cases that were mild during the screening examination. This finding underlines that regular soap use was not effective in more important cases particularly if spores and filaments could not be eliminated. These cases would thus require other forms of topical or even systemic treatment.

Determination of minimal inhibitory concentrations (MIC’s) of placebo soap and of Triclosan

In order to further understand the possible mycological effectiveness of placebo soap against tinea versicolor after all the analyses had been performed, it was attempted to assess the MIC’s of placebo soap and of pure Triclosan against Malassezia species isolated in Zürich. For the Triclosan dilution series, only the significant concentrations were able to inhibit growth. For relevant concentrations (<10 (μg/mL) and for both dilution series of placebo soap, the growth of the fungus 90 Discussion depended only on the properties of the isolate and was very irregular. The results provided no explanation for the improvement found in vivo, since they do not allow any quantitative conclusions. However, they support the hypothesis that the improvement seen is rather associated with unspecific aspects related to regular soap use, and not a specific effectiveness of either the soap or Triclosan against Malassezia spp. It has to be concluded that neither placebo soap nor Triclosan soap inhibit the growth of Malassezia spp. at relevant concentrations.

2.1.2 Other superficial dermatophytic infections

There was an overall tendency towards improvement and resolution of the other dermatomycotic infections during the two months of controlled soap use (see Results for Table 6a and 6b). The proportion of children presenting the characteristic parameters decreased in both groups from the screening to the second follow-up. It can be stated that Triclosan did not prove to be clinically effective against tinea capitis, corporis or pedis infections, since both soaps seem to have had the same overall curative effect (see Appendix 3.1.2.1). The degree of pruritus decreased significantly in both groups. In the case of tinea pedis infections the decrease was very striking. Only mild pruritus was still present in only one fifth of the children. Controlled soap use per se seemed to have a beneficial effect on the degree of pruritus. Possible explanations are analogous to those discussed with regard to the degree of pruritus in case of tinea versicolor infections. Calculation of the average diameter of the largest lesion can be regarded as an important tool to assess the clinical effectiveness of the active and placebo soaps. For tinea capitis there was a similar decrease in both groups: in the active group from 2.9 cm to 2.4 cm, and in the placebo group from 2.6 cm to 1.6 cm (see Results for Table 14a). For tinea corporis in the active group the mean diameter of the largest lesion decreased from the screening to the second follow-up from 2.2 cm to 1.0 cm, whereas it increased in the placebo group (see Results for Table 16b). This finding is one of the few parameters in favour of the active soap even though the difference was not significant. Nevertheless this finding may rather be due to unspecific variation than a better effectiveness of active soap. The importance of the number of lesions when assessing the degree of the skin disorder should not be overemphasized, because these two features do not necessarily coincide, since in severe and extensive cases multiple lesions may coalesce to form one large patch. On interpretation of the series of photographs, visible deterioration of the morphology of the lesions only occurred in the placebo group (see Results for Figure 10). Nevertheless, improvement of tinea capitis as well as tinea corporis infections was also found more frequently in the placebo group than in the active group. Triclosan soap might thus prevent deterioration better than placebo soap. 91 Discussion However, these differences were not significant, and one has to keep in mind, that the number of children for whom the pictures could be evaluated was unbalanced: altogether 18 children in the active and 41 in the placebo group. The mycological effectiveness of Triclosan against some dermatophyte pathogens has been proven in vitro (© Ciba Specialty Chemicalsa, 1998) (see Background: Information about Triclosan), but this finding could not be reliably confirmed in the present clinical trial, since the relative risk did not differ significantly. Both soaps seemed to have a certain mycological effectiveness against dermatophytes, which may be due to the alkaline pH of the soap (pH=10), which itself displays an antimicrobial effect (D. Ochs, personal communication). The effectiveness of Triclosan may have become more evident if the treatment period had been longer. It should also be considered that the filaments that were seen during the second follow-up might have been dead hyphae in some cases (Gooskens et al., 1994). In tinea capitis this decrease of positive results occurred only in the active group, which may be considered as supportive of the findings made in vitro. In the scatter plot presentation for tinea capitis the overall improvement is even more striking than in the cases of tinea versicolor infections. Increases of score values occurred in seven cases and only resulted in an increase in the degree of in infection in one case. In all cases with negative microscopic results the clinical score was reduced. This confirms that the score used in this study is useful in reflecting the clinical course of the infections. Overall improvement of tinea capitis and corporis: There are different possible explanantions for the overall improvement of the cases of tinea capitis and corporis infections included in the study. First, this might be due to the elimination of the pathogens from the lesion sites simply through the washing process, supported by the antimicrobial properties of the soap. Figueroa et al. have suggested that the burden of the dermatophytes may determine the clinical stages of tinea capitis infections among Ethiopian schoolchildren (Figueroa et al., 1997). In the present study, elimination of the pathogens from the scalp and glabrous skin have also have reduced the chance of spreading from asymptomatic sites, which may simply be contaminated or sites of asymptomatic infection, as well as the chance of re-infection. A protective as well a curative effect of soap use have been described by other authors. Attapattu has reported from Sri Lanka, that the custom of daily head baths of young children with water and soap may account for the low prevalence of tinea capitis in the community he observed in spite of poverty and overcrowding (Attapattu, 1989). More than half of the study participants with fungal infections were cured by using a soap containing certain plant derivates during a study conducted by Alebiosu et al. in Nigeria (Alebiosu et al., 2003). Second, many children might have shown spontaneous resolution (Schmeller, 1998; Weitzman et al., 1996). Resolution of the infection occurs more rapidly due to cell-mediated immunity if a 92 Discussion severe primary infection has taken place before, which is very likely for most children in this study (Canizares, 1982). Neither the incidence nor the prevalence among untreated children was assessed at the time of the second follow-up and no control group was included. Therefore it is not possible to determine whether resolution occurred spontaneously. Third, the change of season from the screening (hot, rainy season) to the second follow-up examination (cooler and drier; see also Table 1, Patients, Approaches and Methods) might have influenced the overall improvement of the degree of infection (Canizares, 1982). Porter et al. reported an increase in the prevalence rates of tinea coporis from the wet to the dry season in a Gambian village, whereas the prevalence of tinea capitis decreased significantly (Porter, 1979). Abu-Elteen et al. found that in a hot, semi-arid region at the north-eastern part of the Jordanian desert, tinea capitis and corporis infections occurred predominantly in the spring and winter months (Abu-Elteen et al., 1999). However, other authors believe that the growth of dermatophytes is generally favoured by heat and humidity (Abu-Elteen et al., 1999; Hay, 1996; Kamalam et al., 1981). Investigations by Kamalam in Madras confirmed that tinea capitis and corporis infections were correlated with minimum and maximum temperatures as well as rainfall (Kamalam et al., 1981). It is not possible to draw conclusions as to whether the prevalence rates in Ifakara also changed due to the change of the season, since no control group without any treatment was examined and because the numbers of cases were very small in the case of tinea corporis infections. The rates of clinical and microscopic resolution are comparable to the clinical success rates reported by Alebiosu et al. from Nigeria (Alebiosu et al., 2003), who treated 446 individuals with fungal infections and 64 subjects with scabies with either “Toto Soap”, “Toto Ointment”, soap and ointment or sulphur ointment for five to twelve weeks. “Toto Soap” is comparable to the placebo soap used in the present study. It contains palm oil (Butyrosporum paradoxum oils and Elaeis guineesis) as a plant derivate that has acclaimed medical value and a chlorinated phenol, which might also have acted as the active substance. Out of 107 patients with fungal infections treated with Toto soap alone, 51.4% of the patients with dermatomycoses could be cured after a median period of five weeks. All five patients with scabies receiving Toto soap were cured. The authors emphasize that even better effectiveness could be achieved by combining the soap with the ointment. Both studies strongly suggest that regular soap use may have an important effectiveness against these dermatoses, which are common in tropical Africa. Effectiveness of Triclosan: Triclosan is known to show in vitro effectiveness against a wide range of dermatophyte species (© Ciba Specialty Chemicalsa, 1998). Among the pathogens that could be identified through cultures in Zürich, Triclosan has been proven to be effective against T. mentagrophytes and T. tonsurans, with minium inhibitory concentrations ranging between 1 and 10 ppm. Because of these in vitro findings, 93 Discussion one might have expected a more pronounced mycological effectiveness of the active soap against these dermatophytes. It is possible that in the present study pathogens which were not identified through culture in Zürich are more resistant to topical treatment with Triclosan. This might explain the lack of superior efficacy in most cases. The assumption that the leave-on time of the soap might have been too short to produce any effect at all, cannot explain the findings. Triclosan displays a long-lasting antimicrobial efficacy, even if applied as a rinse-off formulation (Ciba Specialty Chemicalsa, 1999). Active dermal concentrations can be detected after 24 hours, and the substance accumulates on the skin after repeated use. However, the mycological effectiveness of Triclosan against the dermatophyte species might have been influenced by certain interactions with vaseline applied onto the skin. It was observed during the trial that many children used vaseline regularly. Excessive application of this substance after the soap use might have reduced the amount of Triclosan that could accumulate on the skin, since Triclosan is a lipophilic molecule, which would therefore be deposited in vaseline to a great extent. If vaseline was applied before washing, an emulsion might be formed during the washing process, which would be easliy rinsed off, thus reducing the long-term efficacy (Dr. W. Baschong, M. Schnyder, personal communication). It should also be mentioned that in the tropics a large proportion of dermatomycoses may be caused by yeasts such as Candida spp. or by moulds, against which Triclosan is not highly effective (e.g. MIC of C. albicans is 33 ppm). These fungi might have been among the causative species particularly in the cases in which the samples could be evaluated. It is important that apart from one case of tinea corporis infection caused by T. mentagrophytes, all pathogens cultivated in Zürich either from tinea corporis or capitis lesions were T. tonsurans or T. violaceum, which cause endothrix infections (see Results for Tables 9b and 9c). It can be assumed that they were the most common causative species among the schoolchildren in Ifakara. Topical treatment of tinea capitis probably is probably not capable of eliminating hyphae in the deeper parts of the follicle or penetrate hair shafts (Schmeller, 1998; Hussain et al., 1999; Chan et al., 2004). Therefore many authors doubt whether topical treatment e.g. with Whitfield’s Ointment is effective, especially in cases of endothrix infections (Ayaya et al., 2001; Gooskens et al., 1994; Canizares et al., 1993). Weitzman et al. recommend that topical agents may be used as adjuncts to eliminate viable material from the lesions and thus prevent further spreading (Weitzman et al., 1996). In the future, an effective antifungal soap available for the majority of the population might fulfill this purpose better than any other sort of topical treatment, which would only be used by patients who are aware of their disease. With this kind of soap, asymptomic carriers (Weitzman et al., 1996; Figueroa et al., 1997) as well as non-infected individuals could be “treated” effectively at the same time. 94 Discussion Tinea pedis: The proportion of children showing complete resolution of symptoms was greater in the active than in the placebo group (85.7% versus 55.6%) (see Results for Tables 19e and 19f). For all remaining uncured children in the active group there is no information concerning the degree of infection at the screening examination. Active soap might therefore have been clinically more effective, but this assumption cannot be reliably proven by significance tests. The possibly superior efficacy of Triclosan may be explained by the pathomechanism particularly common in interdigital tinea pedis infections, which was the only type discovered in the present study population: After destruction of the barrier role of the stratum corneum by the dermatophyte, bacterial overgrowth at the expense of the dermatophyte may occur, especially under favourable conditions such as occlusion and humidity. Bacterial organisms commonly isolated from these “dermatophytosis complex” lesions include gram-positive species such as S. aureus or C. minutissimum, and gram-negative bacteria such as Pseudomonas and Proteus spp. (Masri-Fridling, 1996), against all of which Triclosan is effective, apart from Pseudomonas spp. (Ciba Specialty Chemicalsa, 1998). In the present study the total number of observations was very small, and for 13 children the clinical score could not be evaluated at the second follow-up. An overall proportion of 36.8% showed clinical resolution at the second follow-up. The possibilities that this has occurred spontaneously or was due to the change of season cannot be ruled out. Reports which investigate the influence of seasonal changes on the prevalence of tinea pedis infections are slightly contradictory in their findings. A decrease of the incidence of tinea pedis among Korean children in the cooler season of the year has been described by Jang et al. (Jang et al., 2000). However, Kamalam found the highest incidence rates in the months of medium temperatures and lowest rainfalls. The incidence rate was lowest in November, when humidity is highest and rainfalls are abundant (Kamalam et al., 1981). Abu-Elteen et al. described the highest incidence of tinea pedis for the summer months (Abu-Elteen 1999).

3. Scabies

3.1 Epidemiology of scabies

In the Study Proposal of the present study, the prevalence rate of ectoparasitoses (scabies and/or pediculosis) had been assumed to be 10%. This assumption was based on the reports by bTanner et al. (1987), Henderson (1992), Schmeller (1998), Figueroa et al. (1996) and Heukelbach et al. (2003). Surveys in various districts of Tanzania revealed overall prevalence rates between 4 and 30.4% among the general population (KCMC, unpublished data). According to Tanner et al., 95 Discussion between 1982 and 1984 scabies was the third most common reason to seek treatment at any level of the health care delivery system in Kilombero District (bTanner et al., 1987). Considering a drop-out-rate of 40%, the minimum sample size calculated in the Proposal was 127 children in order to observe significant differences between active and placebo group. This aim could not be achieved since none of the 820 examined children were suffering from pediculosis and only 1.5% had scabies. Therefore only a descriptive analysis of the eight cases was possible.

3.2 Assessment of the effect of soap

3.2.1 Assessment of the efficacy of Triclosan in soap

The difference in pruritus between the two groups could not be assessed because the sample size was too small. As in the case of fungal infections, the soap may be able to relieve pruritus since it reduces the amount of dust and irritative substances on the skin, and also has antimicrobial properties. This effect does not appear to be due to Triclosan, but the sample is too small to verify this assumption (see Results for Figure 18). Both soaps have displayed a curative effect on the signs and symptoms of scabies (see Results for Table 21). With regard to complete resolution of scabies, it is surprising that it only occurred in the placebo group. Since the number of cases was very small, possible explanations are speculative. The children with severe infestations, even though otherwhise instructed by the principal investigator, may have sought further medical help at local health facilities. It is also possible that other affected family members had been provided with the necessary medication and had been instructed to include the whole family in the treatment. However, the possibility that the soap has been effective should not be neglected. Due to the antimicrobial properties of the soap base, particularly bacterial superinfection may have been reduced. Alebiosu et al. were able to cure all five patients with scabies whom they treated with “Toto Soap" (Alebiosu et al., 2003 ). It is not clear whether the active ingredients (Butyrospermum paradoxum oils and Elaesis guineesis) are responsible for the resolution of scabies or whether the soap base itself produced this effect. Lesions may have improved or even resolved spontaneously. Seasonal variation of the incidence has been discussed. Most authors indicate a higher incidence in fall and winter (Christophersen, 1978; Mimouni et al., 2003). However, Porter et al. found no difference in prevalence rates between the wet and dry season in Gambia (Porter, 1979). Any possible association between climatic conditions and point prevalence rates does not seem to be an appropriate explanation for the decrease in the degree of infection in the present study, since the second follow-up was performed during the drier and cooler period of the year. 96 Discussion Due to the small number of cases, it is difficult to make any statements regarding the possible superior efficacy of the active soap. However, Triclosan did not prove to be strongly effective against scabies infestations, and regardless of possible minor benefits such as anti-inflammtory effects and antibacterial effectiveness (Ciba Specialty Chemicalsa, 1998 ) it should not be used as the only therapy of scabies, but possibly as an adjunct to standard treatments. In a study conducted in central Tanzania, Henderson et al. have shown that the addition of soap and scabicidal ointment to standard BBE treatment schemes led to a significant rise in cure rates (Henderson et al., 1992). The soap may be beneficial in supporting the prevention of re-infections from untreated relatives and improve the hygiene conditions in the children’s homes.

4. Strength and Limitations

4.1 Physical examinations and photographic images

Poor illumination therefore affected to a certain extent the physical examinations in the classrooms. At Lihami Primary School the illumination of the examination room was better during the two follow-ups, as it had been possible to shift to a larger classroom. Since the conditions were not exactly the same at the different examinations, it is possible that minor lesions had not been visible in their full extension during the first screening. Some pictures taken at the first screening could not be evaluated. The illumination was better at Michenga Primary School since a brighter room with a curtain generating privacy could be used during the whole trial. A strength of the study of the study is the fact that all examinations were performed by the same four investigators. Since the clinical experience of the two medical students (Almuth Dinkela and Julia Ferié) was still limited at the beginning of the study, each finding was briefly presented to Dr. M. Mbata, who is a fully trained local dermatologist (Assistant Medical Officer). At a later stage all unclear findings were still discussed with him. Quality control of the diagnosis was furthermore ensured by the assessment of the digital photographs by the expert dermatologist in Zürich.

4.2 Design of questionnaires

The documentation system used in the questionnaires only allowed the examiners to describe lesions due to the defined principal diagnosis in full detail. Further skin disorders could only briefly be recorded. Open questions might have yielded more reliable information with regard to the subjective description of the symptoms by the study participants. However, an unstructured interview would have exceeded the language skills of the European investigators (AD and JF). 97 Discussion 4.3 Sample-taking and microscopic examination

The paper, scalpel or envelope may have become contaminated in some cases, since there was no possibilty to store them in clean places during the whole examination period. Many samples could not be evaluated because not enough material had been taken. It was not possible to cultivate the fungi at the laboratory facilities available at IHRDC in Ifakara itself. The identification of all the pathogens by culture or other techniques might have contributed to answering some of the questions which this study has raised. Maybe Triclosan would have proven to be effective against certain species. Furthermore, mere microscopic examination did not allow us to distinguish between dead and viable material, which might have been important since the study period lasted only two months and effects might not have been so evident yet. Tinea pedis infections were not confirmed by microscopic diagnosis since they were not one of the diseases that were to be evaluated with priority in the soap trial. Only clinical improvement was assessed. However, one should bear in mind that, apart from dermatophytes, the infections might be caused by many different species such as Candida spp., S. brevicaulis, H. toruloidea and Fusarium spp. (Masri-Fridling, 1996). The number of cases was very small, and reliable statements can only be made to a very little extent. Only in one case a mite was detected on microscopic examination and thus allowed the confirmation of clinically diagnosed scabies. Eggs were not seen in any case. Detection of scybala was complicated by dirt. The sample-collection may have failed because burrows were hardly found due to scratch effects and superinfection. Sample-taking was therefore not performed at the follow- up examinations and the diagnosis was thus based on clinical findings.

4.4 Quality control of microscopy and additional cultivation of fungi in Zürich

Quality control of the microscopic examinations performed in Ifakara was not possible in all cases. In case of dermatophyte infections, species identification in all cases would have been desirable to explain the lack of efficacy of Triclosan. Isolation of Malassezia yeasts from human skin does not necessarily indicate tinea versicolor infections there is also skin colonization by this fungus without resulting skin pathology (Gupta, 2002; Roberts, 1969). Gupta et al. therefore postulate that the characteristic micromorphology of fungal elements in the skin is identified to confirm the diagnosis, which could not be done with the means available in Ifakara (Gupta et al., 2001). However, as the skin samples were taken from affected skin and no other fungal elements were cultured, Malassezia spp. seem to be responsible for the observed skin lesions in our patients. 98 Discussion 4.5 Results during the first follow-up

The first follow-up fell into the period of school holidays when many children moved with their family into the far away “shamba (field) house”. Only 136 children came to this examination and few to the soap distributions, even though attendance and compliance were promoted with the help of the local Balozi leaders. Even though an overall improvement could be observed for all diagnoses (Tinea verisocolor, tinea capitis, corporis and pedis, and scabies), some results at the first follow-up (see Appendix) do not reflect this tendency. Maybe these observations are due to unspecific variance. Irritation or exacerbation under treatment would be another possible explanation. Furthermore, the assessment may not have been as exact as at the second follow-up.

4.6 Characteristics of the study population

As Lihami Primary School could not provide the examiners with complete and up – to – date class lists, it was difficult to check which children had already been seen. It is possible that some children were not examined at all and that other children who otherwise would not have attended school preferred to come to school to be examined. Information obtained from the younger children with regard to the subjective impression of the skin disorder and the degree of pruritus might not have been reliable, since they might have felt pressured to provide the answer they believe to be expected to give. This has maybe hampered the assessment of any possibly existing difference between the two groups. One may assume that the compliance during these two months of the soap trial was good. Nevertheless, a longer study period and maybe even greater amounts of soap distributed per week would have been required to reduce the chance that the soap was used for other purposes or exchanged, possibly even between the study participants.

4.7 Spontaneous resolution and climatic influences

It is not possible to determine whether resolution of the dermatoses occurred spontaneously. Neither the incidence nor the prevalence among untreated children was assessed during the second follow- up. Besides, a control group would have allowed us to draw conclusions as to whether the prevalence rates in Ifakara also changed due to the change of the season.

99 Discussion 5. Conclusions

• Among 820 children aged 6 to 19 years, 277 children (33.9%) presented with fungal infections and only 12 (1.5%) with scabies. • Among the 306 cases of dermatomycoses, 214 (70.0%) were due to tinea versicolor, proven by microscopy in 81.0%, 45 (14.7%) were due to tinea capitis, 21 (6.8%) to tinea corporis and 26 (8.5%) to tinea pedis. • In tinea versicolor, controlled soap use has resulted in significant improvement of the degree of infection as well as an increase of in the proportion with negative microscopic results from 19.0% to 51.6%. 28.4% of all individuals were cured; however there was no additional effect if Triclosan was added to the soap. This is consistent with the lack of efficacy in vitro of Triclosan against Malassezia spp. reported in the literature (© Ciba Specialty Chemicalsa, 1998) and found at the “Mycological laboratory of the Department of Dermatology, University Hospital of Zürich”. • In tinea capitis, corporis and pedis, a significant improvement was also observed after controlled soap use, with or without Triclosan. Cure rates ranged from 36.8% to 61.1%. There was no significant difference between placebo and active group. However, a decrease of the average lesion size in tinea corporis only occurred in the active group, whereas it increased in the placebo group. In tinea capitis the proportion of negative microscopic findings decreased only if Triclosan soap was used. • The mild beneficial effect of Triclosan can be explained by its known in vitro activity against dermatophytes such as T. rubrum or mentagrophytes. The lack of a stronger efficacy of Triclosan could be explained by the used rinse-off application instead of leave-on and the additional use of petrolatum (vaseline). • The group of patients with scabies was too small to allow any statistical analysis. The use of soap seemed to be beneficial regarding resolution and pruritus, but there was no difference when the soap contained Triclosan. • In summary, the controlled use of soap over two months showed a beneficial effect against tinea versicolor, other superficial dermatomycotic infections and scabies. While there is no additional effectiveness of Triclosan against tinea versicolor, Triclosan may be useful against superficial dermatomycotic infections if other application modes are used. Our results argue for the use of soap, but are not sufficient to support the addition of Triclosan to treat superficial skin mycoses and scabies. However, the fact that Triclosan is highly effective against bacterial infections - also playing an important role in a tropical setting -, and its putative beneficial effects do not argue against the addition of this agent. 100 Discussion

© Photo: Cosmos Popperfoto © PHOTO COSMOS 6. Implications

In low-income countries such as Tanzania, treatment options for benign but very common skin disorders such as fungal and parasitic skin infections are poor, since numerous life-threatening conditions have higher priority. Nevertheless, the provision of safe, effective and inexpensive treatment of common skin diseases is essential for successful tropical primary health care, since those diseases may lead to severe cosmetic disfigurement, permanent alopecia or secondary bacterial superinfection including complications such as poststreptococcal glomerulonephritis. The prevention and treatment of the most relevant skin disorders should include the training of paramedical personnel, care-takers, teachers and the children themselves, and also the availability of cheap prevention measures and effective treatment options such as an effective topical agent applied as bar soap. More information is needed on the efficacy of Triclosan and improved hygiene compared to established topical antifungals and antiscabietic drugs on a long-term basis. Control groups should be included to assess the likelihood of spontaneous resolution or improvement due to seasonal changes. However, it can be concluded from our findings that just the regular use of soap has lead to a substantial improvement of superficial fungal infections of the skin, namely tinea versicolor, tinea capitis, corporis and pedis among the study group in the investigated area of Tanzania. 101 Bibliography Bibliography Abu-Elteen KH, Malek MA (1999) Prevalence of dermatophytoses in the Zarqua district of Jordan. Mycopathologia 145: 137-142

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(Excerpt from the Study Proposal)

“We assume the prevalence rate of Tinea capitis to be 15% among the children, and of ectoparasitoses to be 10%. The confidence interval is 95%, the power 80%. In order to proof the effectiveness the prevalence of Tinea capitis within the group treated with the Triclosan-containing soap has to be reduced from 100% to at least 40%, making a difference of at least 60% significant. In the placebo group we expect the ordinary soap to reduce the prevalence from 100% to about 70%. When comparing the two groups after two months the superior efficacy of the Triclosan- containing soap is proven if the prevalence rate is at least 30% smaller than in the group treated with the placebo soap. Both groups affected by Tinea capitis have to consist of 48 children each. Considering a drop-out- rate of 40% during the study this sample size has to be at least 160 children (2*48+40%=160). When comparing placebo and the verum group after two months the superior efficacy of the Triclosan-containing soap on ectoparasitoses is significant if the prevalence rate is at least 30% smaller than in the group treated with the placebo soap. The placebo and the verum group have to consist of 38 children each. Considering the drop-out-rate of 40% this sample size has to consist of at least 127 children (2*38+40%=126.6). Adding the two sample sizes, approx. 287 children will be included in the study population.” The prevalence rates assumed in the Study Proposal were 15% and 10% for dermatophytoses and ectoparasitoses respectively. 114 Tables, symmetry tables and figures Appendix A: Tables (*Number of observations, row percentages and column percentages), Symmetry Tables and Figures

2. Tinea versicolor and other superficial dermatomycotic infections

2.2. Prevalences and degree of infection according to sex

2.2.1 Tinea versicolor: Degree of infection according to sex*:

Degree of infection: Number of observations: female male Total mild 12 20 32 37.50 62.50 100.00 15.00 23.81 19.51 moderate 22 12 34 64.71 35.29 100.00 27.50 14.29 20.73 important 46 52 98 46.94 53.06 100.00 57.50 61.90 59.76

Total 80 84 164 48.78 51.22 100.00 100.00 100.00 100.00 Chi2 test p-value = 0.074

2.2.2 Tinea capitis: Degree of infection according to sex*:

Degree of infection: Number of observations: female male Total mild 2 4 6 33.33 66.67 100.00 22.22 44.44 33.33 moderate 3 3 6 50.00 50.00 100.00 33.33 33.33 33.33 severe 4 2 6 66.67 33.33 100.00 44.44 22.22 33.33

Total 9 9 18 50.00 50.00 100.00 100.00 100.00 100.00 Fisher's exact test p-values = 0.835

115 Tables, symmetry tables and figures Tinea corporis: Degree of infection according to sex*: Degree of infection: Number of observations: female male Total mild 5 4 9 55.56 44.44 100.00 71.43 66.67 69.23 moderate 1 2 3 33.33 66.67 100.00 14.29 33.33 23.08 severe 1 0 1 100.00 0.00 100.00 14.29 0.00 7.69

Total 7 6 13 53.85 46.15 100.00 100.00 100.00 100.00 Fisher's exact test p-values = 1.000

2.3 Prevalence rates and degree of infection according to age groups (in years=yrs)

2.3.1 Tinea versicolor: Degree of infection according to age group*:

Degree of Number of observations: infection: 6 to 10 yrs 11 to 14 yrs 15 5o 19 yrs Total mild 12 16 4 32 37.50 50.00 12.50 100.00 41.38 17.39 9.30 19.51 moderate 3 27 4 34 8.82 79.41 11.76 100.00 10.34 29.35 9.30 20.73 severe 14 49 35 98 14.29 50.00 35.71 100.00 48.28 53.26 81.40 59.76

Total 29 92 43 164 17.68 56.10 26.22 100.00 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.000

2.3.2 Tinea capitis: Degree of infection according to age group*: Degree of Number of observations: infection: 6 to 10 years >10 years Total mild 1 6 7 14.29 85.71 100.00 8.33 24.00 18.92 moderate 10 15 25 40.00 60.00 100.00 116 Tables, symmetry tables and figures 83.33 60.00 67.57 severe 1 4 5 20.00 80.00 100.00 8.33 16.00 13.51

Total 12 25 37 32.43 67.57 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.491

Tinea corporis: Degree of infection according to age group*: Degree of Number of observations: infection: 6 to 10 years >10 years Total mild 5 4 9 55.56 44.44 100.00 71.43 66.67 69.23 moderate 1 2 3 33.33 66.67 100.00 14.29 33.33 23.08 severe 1 0 1 100.00 0.00 100.00 14.29 0.00 7.69

Total 7 6 13 53.85 46.15 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 1.000

Tinea pedis: Degree of infection according to age groups*: Degree of Number of observations: infection: 6 to 10 years 11 to 14 years 15 5o 19 years Total moderate 1 3 1 5 20.00 60.00 20.00 100.00 50.00 100.00 50.00 71.43 severe 1 0 1 2 50.00 0.00 50.00 100.00 50.00 0.00 50.00 28.57

Total 2 3 2 7 28.57 42.86 28.57 100.00 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.429

117 Tables, symmetry tables and figures 3. Assessment of the effect of soap

3.1 Assessment of the efficacy of Triclosan in soap

Table 5b: Number of observations and percentage of study participants with more than one diagnosis Number of Diagnoses observations (out Percent of 228) Tinea versicolor and Tinea capitis 11 4.8 Tinea versicolor and Tinea pedis 10 4.4 Tinea capitis and Tinea pedis 4 1.7 Tinea versicolor and Tinea corporis 2 0.9 Tinea versicolor, Tinea capitis and Tinea pedis 1 0.4 Scabies and Tinea pedis 1 0.4 Scabies and Tinea capitis 1 0.4 Tinea capitis and Tinea corporis 0 0.0 Tinea versicolor, Tinea capitis and Tinea corporis 0 0.0

Table 6d: Comparison of the symmetry between the symmetry of placebo and active soap: Confidence Intervals Diagnosis Clin. parameters CI (95%) Tinea versicolor Degree of [-0.5312, 0.1522]. pruritus Number of lesions [-0.2671, 0.1637] Degree of [- 0.3851, 0.2417] infection Tinea capitis, Degree of [-0.8016, 0.6592] corporis, pedis pruritus Tinea capitis Number of lesions [-0.6999, 0.2304] 3.1.1 Tinea versicolor infections 3.1.1.1 Signs and symptoms/effectiveness Table 6e: Relative risk of showing certain signs or symptoms when comparing placebo to active group at second follow-up Chi2 test p- Parameter value Total number 95% Confidence (clinical signs and Relative risk (* Fisher’s of observations Interval symptoms) exact test p- value) Scaling 162 1.1 1.000 [0.2353552; 5.44059] Pruritus 134 1.1 0.859 [0.7030138; 0.624001] Pruritus not improved 172 0.6 0.089 [0.3048115; 1.075803] Hypopigmentation 162 0.9 0.594 [0.77963; 1.133553] Hyperpigmentation 163 0.6 0.379 [0.1750343; 1.781691] Subjective impression: 170 0.7 0.597 [0.2726817; 1.967757] not improved

118 Tables, symmetry tables and figures 3.1.1.2 Degree of pruritus Tables for Figure 2a:

Tinea versicolor: Degree of pruritus at screening* Degree of pruritus: Number of observations (and percentages): Placebo Active Total none 35 37 72 48.61 51.39 100.00 45.45 47.44 46.45 mild 37 29 66 56.06 43.94 100.00 48.05 37.18 42.58 moderate 5 12 17 29.41 70.59 100.00 6.49 15.38 10.97

Total 77 78 155 49.68 50.32 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.140

Tinea versicolor: Degree of pruritus at follow-up 2*: Degree of pruritus: Number of observations (and percentages): Placebo Active Total none 52 58 110 47.27 52.73 100.00 67.53 74.36 70.97 mild 22 18 40 55.00 45.00 100.00 28.57 23.08 25.81 moderate 3 2 5 60.00 40.00 100.00 3.90 2.56 3.23

Total 77 78 155 49.68 50.32 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.626

119 Tables, symmetry tables and figures Tinea versicolor: Symmetry table: Number of children according to degrees of pruritus at screening and second follow-up: Table 7b: PLACEBO GROUP Degree of Degree of pruritus at follow-up 2 pruritus at Total None Mild Moderate screening None 29 6 0 35 Mild 21 15 1 37 Moderate 2 1 2 5 Total 52 22 3 77 Symmetry test p-value = 0.0159

Table 7c: ACTIVE GROUP Degree of Degree of pruritus at follow-up 2 pruritus at Total None Mild Moderate screening None 33 4 0 37 Mild 19 9 1 29 Moderate 6 5 1 12 Total 58 18 2 78 Symmetry test p-value = 0.0004

3.1.1.3 Number of lesions and aspect of the lesions:

Tables for Figure 3a: Tinea versicolor: Number of lesions at screening* Number of Lesions: Number of observations (and percentages): Placebo Active Total 1 or2 13 15 28 46.43 53.57 100.00 17.11 17.65 17.39

3 to 5 12 5 17 70.59 29.41 100.00 15.79 5.88 10.56

>5 17 24 41 41.46 58.54 100.00 22.37 28.24 25.47 disseminated 34 41 75 45.33 54.67 100.00 44.74 48.24 46.58

Total 76 85 161 47.20 52.80 100.00 100.00 100.00 100.00 Chi2 test p-value = 0.223

120 Tables, symmetry tables and figures Tinea versicolor: Number of lesions at follow-up 2*: Number of Lesions: Placebo Active Total none 20 16 36 55.56 44.44 100.00 26.32 18.82 22.36

1 or 2 5 12 17 29.41 70.59 100.00 6.58 14.12 10.56

3 to 5 4 10 14 28.57 71.43 100.00 5.26 11.76 8.70

>5 11 13 24 45.83 54.17 100.00 14.47 15.29 14.91 disseminated 36 34 70 51.43 48.57 100.00 47.37 40.00 43.48

Total 76 85 161 47.20 52.80 100.00 100.00 100.00 100.00 Chi2 test p-value = 0.228

Tinea versicolor: Symmetry table: Number of children according to number of lesions at screening and first follow-up: Table 8b: PLACEBO GROUP Number of lesions at Follow-up 2 Number of Disse- Total lesions at screening None 1 or 2 3 to 5 >5 minated None 0 0 0 0 0 0 1 or 2 5 2 2 3 1 13 3 to 5 6 1 1 2 2 12 >5 6 1 0 4 6 17 Disseminated 3 1 1 2 27 34 Total 20 5 4 11 36 76 Symmetry test p-value = 0.0042 Table 8c: ACTIVE GROUP Number of lesions at Follow-up 2 Number of Disse- Total lesions at screening None 1 or 2 3 to 5 >5 minated None 0 0 0 0 0 0 1 or 2 4 7 2 1 1 15 3 to 5 0 0 0 4 1 5 >5 6 2 4 5 7 24 Disseminated 6 3 4 3 25 41 Total 16 12 10 13 34 85 Symmetry test p-value = 0.0068

121 Tables, symmetry tables and figures Tinea versicolor: Morphology of lesions at screening*: a: 1= sharply deliniated (possibly with central healing) 2= grey patches 3= both Aspect of Lesionsa: Number of observations (and percentages): Placebo Active Total None/ N.A. 62 57 119 52.10 47.90 100.00 67.39 69.51 68.39

1 17 15 32 53.13 46.88 100.00 18.48 18.29 18.39

2 10 8 18 55.56 44.44 100.00 10.87 9.76 10.34

3 3 2 5 60.00 40.00 100.00 3.26 2.44 2.87

Total 92 82 174 52.87 47.13 100.00 100.00 100.00 100.00 Fisher's exact test p-value =0.990

Tinea versicolor: Aspect of lesions at follow-up 2*: a: 1= sharply deliniated (possibly with central healing) 2= grey patches 3= both Aspect of lesionsa: Placebo Active Total None/ N.A. 77 75 152 50.66 49.34 100.00 82.80 88.24 85.39

1 9 3 12 75.00 25.00 100.00 9.68 3.53 6.74

2 2 3 5 40.00 60.00 100.00 2.15 3.53 2.81

3 5 4 9 55.56 44.44 100.00 5.38 4.71 5.06

Total 93 85 178 52.25 47.75 100.00 100.00 100.00 100.00 Fisher's exact test p-value =0.377 122 Tables, symmetry tables and figures 3.1.1.4 Photographs: Tinea versicolor: Evaluation of photographs: Comparing pictures taken at screening and follow-up 1*: Comparison: Number of observations: Placebo Active Total better 15 12 27 55.56 44.44 100.00 42.86 26.09 33.33 same 14 31 45 31.11 68.89 100.00 40.00 67.39 55.56 worse 6 3 9 66.67 33.33 100.00 17.14 6.52 11.11

Total 35 46 81 43.21 56.79 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.036

Tinea versicolor: Evaluation of photographs: Comparing pictures taken at follow-up 1 and follow-up 2*: Comparison: Number of observations: Placebo Active Total better 14 15 29 48.28 51.72 100.00 36.84 35.71 36.25 same 17 24 41 41.46 58.54 100.00 44.74 57.14 51.25 worse 7 3 10 70.00 30.00 100.00 18.42 7.14 12.50

Total 38 42 80 47.50 52.50 100.00 100.00 100.00 100.00 Fisher's exact test p-value =0.279

Table for Figure 5:Tinea versicolor: Evaluation of photographs: Comparing pictures taken at screening and follow-up 2* Comparison: Number of observations: Placebo Active Total better 33 43 76 43.42 56.58 100.00 54.10 64.18 59.38 same 18 20 38 123 Tables, symmetry tables and figures 47.37 52.63 100.00 29.51 29.85 29.69 worse 10 4 14 71.43 28.57 100.00 16.39 5.97 10.94

Total 61 67 128 47.66 52.34 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.158

3.1.1.6 Microscopic results (Quality control)

Table 9c: Examinations performed at the Mycological laboratory of the Department of Dermatology, University Hospital of Zürich Number of observations (Percentage among all samples Pathogen taken) 1 month 2 and 3 months Malassezia spp. 30 (88.2) 26 (65.0) Spores 2 (5.9) 8 (20.0) Mycelia 0 (0.0) 1 (2.5) Not enough scales 0 (0.0) 2 (5.0) Negative 2 (5.9) 3 (7.5) Total 34 (100.0) 40 (100.0)

3.1.1.8 Degree of infection:

Degree of infection: Placebo versus active group

n 100% 80% important moderate 60% mild 40% none 20% 0% Percentage of childre Placebo - Active - SCR Placebo - Fu2 Active - Fu2 SCR Soap - Examination

Figure 7b: Degree of infection (n (Placebo) =77 ; n (Active) = 84) Placebo versus active group (Screening (SCR) and follow-up 2 (Fu2))

Tables for Figure 7a and 7b: Tinea versicolor: Degree of infection at screening* Degree of infection: Number of observations: Placebo Active Total mild 15 16 31 48.39 51.61 100.00 19.48 19.05 19.25 moderate 18 15 33 54.55 45.45 100.00 23.38 17.86 20.50

124 Tables, symmetry tables and figures severe 44 53 97 45.36 54.64 100.00 57.14 63.10 60.25 Total 77 84 161 47.83 52.17 100.00 100.00 100.00 100.00 Chi2 test p-value = 0.658

Tinea versicolor: Degree of infection at follow-up 2* Degree of infection: Number of observations: Placebo Active Total resolution 20 15 35 57.14 42.86 100.00 25.97 17.86 21.74 mild 6 20 26 23.08 76.92 100.00 7.79 23.81 16.15 moderate 12 11 23 52.17 47.83 100.00 15.58 13.10 14.29 important 39 38 77 50.65 49.35 100.00 50.65 45.24 47.83

Total 77 84 161 47.83 52.17 100.00 100.00 100.00 100.00 Chi2 test p-value = 0.046

Tinea versicolor: Symmetry table: Number of children according to degree of infection at screening (SCR) and second follow-up (Fu2): Table 10b: PLACEBO GROUP Degree of Degree of infection at Follow-up 2 infection at Total None Mild Moderate Important Screening None 0 0 0 0 0 Mild 7 4 2 2 15 Moderate 6 1 5 6 18 Important 7 1 5 31 44 Total 20 6 12 39 77 Symmetry test p-value = 0.0020

Table 10c: ACTIVE GROUP Degree of Degree of infection at Follow-up 2 infection at Total None Mild Moderate Important Screening None 0 0 0 0 0 Mild 3 10 1 2 16 Moderate 5 2 5 3 15 125 Tables, symmetry tables and figures Important 7 8 5 33 53 Total 25 20 11 38 84 Symmetry test p-value = 0.0035 3.1.1.10 Clinical and microscopic resolution Table 11: Relative frequencies and frequency distribution of children having received placebo versus active soap, stratified according to evaluation categories Placebo Active Total *Evaluation number of Clinical and Number of Number of categories Percentage Percentage observations microscopic observations observations (percentage) resolution 1 21 25.6 18 21.7 39 (23.6) 2 2 2.4 6 7.2 8 (4.8) Subtotal 23 28.0 24 28.9 47 (28.4) No 3 6 7.3 8 9.6 14 (8.5) resolution 4 13 15.9 10 12.1 23 (14.0) 5 3 3.6 10 13.3 13 (7.9) 6 17 20.7 19 22.9 36 (21.8) 7 20 24.4 12 14.5 32 (19.4) Total 82 100.0 83 100.0 165 (100.0) * 1= resolution: child cured 2= microscopic resolution and only few clinical symptoms 3= improvement: negative microscopic evidence with significant clinical improvement 4= no significant clinical improvement but negative mycologic evidence 5= improvement: microscopy positive with significant clinical improvement 6= lack of improvement: positive mycologic evidence with no significant clinical improvement 7= deterioration Fisher's exact test p-value = 0.194

3.1.2 Other superficial dermatomycotic infections

3.1.2.1 Signs and symptoms/effectiveness Relative risk of showing certain signs or symptoms: comparing placebo to active group at the second follow-up (Fu2): Tinea capitis Parameter Number of Fisher’s exact 95% Confidence (clinical signs and observations Relative risk test p-value Interval symptoms) at follow-up 2 (2-sided) Scaling 14 1.0 NA [0.5648063; 2.766435] Alopecia 34 0.7 0.703 [0.2298904; 2.19965] Occipital/posterior 34 0.7 0.682 [0.1751029; 2.53819] cervical adenopathy Grey patches 34 No cases NA NA Black dots 34 No cases NA NA Kerion 34 0.0 0.471 NA Superinfection 34 NA NA NA No new hair growth 39 1.3 1.000 [0.2408787; 6.86262] Pruritus 20 0.8 1.000 [0.2806726; 2.38506] Pruritus not improved 39 0.9 1.000 [0.1339547; 5.48464] Subjective impression: No cases in 37 0.490 NA not improved active group

Tinea corporis: Relative risk of showing certain signs or symptoms: comparing placebo to active group at second follow-up Parameter Fisher’s Number of (clinical signs and exact test p- 95% Confidence observations at Relative risk symptoms) value (2- Interval follow-up 2 sided) 126 Tables, symmetry tables and figures Scaling 9 NA NA NA Concentric lesions 13 0.9 1.000 [0.1681699; 4.368761] Satellite lesions 13 0.85 1.000 [0.0670402; 10.959] Pruritus 10 1.0 1.000 [0.0838119; 11.93148] Pruritus not No cases in 12 1.000 NA improved active group Subjective impression: not 13 No cases NA NA improved T inea pedis: Relative risk of showing certain signs or symptoms: comparing placebo to active group at second follow-up Parameter Fisher’s exact Number of 95% Confidence (clinical signs and Relative risk test p-value (2- observations Interval symptoms) sided) Macerated, whitish 7 1.6 1.000 [0.3739831; 6.845228] lesions Pruritus No cases in 6 1.000 NA active group Pruritus not 19 No cases NA NA improved Subjective impression: not 19 0.5 0.582 [0.048623; 4.164618] improved

3.1.2.2 Degree of pruritus:

Tinea capitis, corporis and pedis: Degree of pruritus at screening*: Degree of pruritus: Number of observations: Placebo Active Total none 6 7 13 46.15 53.85 100.00 20.00 30.43 24.53 mild 13 13 26 50.00 50.00 100.00 43.33 56.52 49.06 moderate 9 3 12 75.00 25.00 100.00 30.00 13.04 22.64 severe 2 0 2 100.00 0.00 100.00 6.67 0.00 3.77

Total 30 23 53 56.60 43.40 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.264

Tinea capitis, corporis and pedis: Degree of pruritus at follow-up 2*: 127 Tables, symmetry tables and figures Degree of pruritus: Number of observations: Placebo Active Total none 19 18 37 51.35 48.65 100.00 63.33 78.26 69.81 mild 9 4 13 69.23 30.77 100.00 30.00 17.39 24.53 moderate 2 0 2 100.00 0.00 100.00 6.67 0.00 3.77 severe 0 1 1 0.00 100.00 100.00 0.00 4.35 1.89

Total 30 23 53 56.60 43.40 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.251

Tinea capitis, corporis and pedis: Symmetry table: Number of children according to degrees of pruritus at screening and second follow-up: Table 13b: PLACEBO GROUP Degree of Degree of pruritus at Follow-up 2 pruritus at Total None Mild Moderate Severe Screening None 5 1 0 0 6 Mild 8 5 0 0 13 Moderate 4 3 2 0 9 Severe 2 0 0 0 2 Total 19 9 2 0 30 Symmetry test p-value = 0.006

Table 13c: ACTIVE GROUP Degree of Degree of pruritus at Follow-up 2 pruritus at Total None Mild Moderate Severe Screening None 7 0 0 0 7 Mild 10 2 0 1 13 Moderate 1 2 0 0 3 Severe 0 0 0 0 0 Total 18 4 0 1 23 Symmetry test p-value = 0.0073

128 Tables, symmetry tables and figures

Degree of Pruritus: Both Groups

100% 80% severe 60% moderate 40% mild 20% none 0% Percentage of children Placebo - Active - Placebo - Active - Fu2 SCR SCR Fu2

Figure 10b: Other superficial dermatomycoses (n (Placebo) = 30; n (Active) = 23) Placebo versus active group (Screening (SCR) and follow-up 2 (Fu2))

Tinea capitis: Symmetry table: Number of children according to degrees of pruritus at screening and second follow-up: BOTH GROUPS: Degree of Degree of pruritus at Follow-up 2

pruritus at None Mild Moderate Severe Total Screening None 10 1 0 0 11 Mild 11 4 1 0 16 Moderate 3 3 1 0 7 Severe 1 0 0 0 1 Total 25 8 2 0 35 Symmetry test p-value = 0.0098

PLACEBO GROUP: Degree of Degree of pruritus at Follow-up 2 pruritus at Total None Mild Moderate Severe Screening None 4 1 0 0 5 Mild 5 3 0 0 8 Moderate 2 1 1 0 4 Severe 1 0 0 0 1 Total 12 5 1 0 18 Symmetry test p-value = 0.1546

ACTIVE GROUP: Degree of Degree of pruritus at Follow-up 2 pruritus at Total None Mild Moderate Severe Screening None 6 0 0 0 6 Mild 6 1 1 0 8 Moderate 1 2 0 0 3 Severe 0 0 0 0 0 Total 13 3 1 0 17 Symmetry test p-value = 0.0620

129 Tables, symmetry tables and figures Tinea corporis: Symmetry table: Number of children according to degrees of pruritus at screening and second follow-up: BOTH GROUPS: Degree of Degree of pruritus at Follow-up 2

pruritus at None Mild Moderate Severe Total Screening None 2 0 0 0 2 Mild 5 1 0 1 7 Moderate 1 1 1 0 3 Severe 0 0 0 0 0 Total 8 2 1 1 12 Symmetry test p-value = 0.0916

PLACEBO GROUP: Degree of Degree of pruritus at Follow-up 2 pruritus at Total None Mild Moderate Severe Screening None 1 0 0 0 1 Mild 3 0 0 0 3 Moderate 1 1 1 0 3 Severe 0 0 0 0 0 Total 5 1 1 0 7 Symmetry test p-value = 0.1718

ACTIVE GROUP: Degree of Degree of pruritus at Follow-up 2 pruritus at Total None Mild Moderate Severe Screening None 1 0 0 0 1 Mild 2 1 0 1 4 Moderate 0 0 0 0 0 Severe 0 0 0 0 0 Total 3 1 0 1 5 Symmetry test p-value = 0.2231

Tinea pedis: Symmetry table: Number of children according to degrees of pruritus at screening and second follow-up: BOTH GROUPS: Degree of Follow-up 2 pruritus at* Total *Screening None Mild Moderate Severe None 0 0 0 0 0 Mild 1 2 0 0 3 Moderate 1 1 0 0 2 Severe 1 0 0 0 1 Total 3 3 0 0 6 Symmetry test p-value = 0.406

130 Tables, symmetry tables and figures 3.1.2.3 Size of the largest lesion and number of lesions:

Tinea pedis: Size of the largest lesion at screening*: Size of the largest Number of observations: lesion: Placebo Active Total N.A. 0 1 1 0.00 100.00 100.00 0.00 50.00 14.29

<1 cm 1 1 2 50.00 50.00 100.00 20.00 50.00 28.57

1 to 3 cm 4 0 4 100.00 0.00 100.00 80.00 0.00 57.14

Total 5 2 7 71.43 28.57 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.143

Tinea pedis: Size of the largest lesion at follow-up 1*: Size of the largest Number of observations: lesion: Placebo Active Total none 2 2 4 50.00 50.00 100.00 40.00 50.00 44.44

<1cm 1 0 1 100.00 0.00 100.00 20.00 0.00 11.11

1 to 3cm 2 2 4 50.00 50.00 100.00 40.00 50.00 44.44

Total 5 4 9 55.56 44.44 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 1.000

Tinea pedis: Size of the largest lesion at follow-up 2*: Size of the largest Number of observations: lesion: Placebo Active Total none 5 8 13 38.46 61.54 100.00 50.00 88.89 68.42

<1cm 1 0 1 100.00 0.00 100.00 10.00 0.00 5.26 131 Tables, symmetry tables and figures

1 to 3 cm 4 1 5 80.00 20.00 100.00 40.00 11.11 26.32

Total 10 9 19 52.63 47.37 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.211

Tinea capitis: Number of lesions at screening*: Number of Lesions: Number of observations (and percentages): Placebo Active Total N.A. 1 0 1 100.00 0.00 100.00 5.26 0.00 2.78

1 or 2 4 4 8 50.00 50.00 100.00 21.05 23.53 22.22

3 to 5 7 7 14 50.00 50.00 100.00 36.84 41.18 38.89

>5 6 3 9 66.67 33.33 100.00 31.58 17.65 25.00 disseminated 1 3 4 25.00 75.00 100.00 5.26 17.65 11.11

Total 19 17 36 52.78 47.22 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.606

Tinea capitis: Number of lesions at follow-up 2*: Number of Lesions: Placebo Active Total None/N.A. 12 9 21 57.14 42.86 100.00 63.16 52.94 58.33

1 or 2 1 4 5 20.00 80.00 100.00 5.26 23.53 13.89

3 to 5 2 1 3 66.67 33.33 100.00 10.53 5.88 8.33

132 Tables, symmetry tables and figures >5 1 0 1 100.00 0.00 100.00 5.26 0.00 2.78 disseminated 3 3 6 50.00 50.00 100.00 15.79 17.65 16.67

Total 19 17 36 52.78 47.22 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.551

Tinea capitis: Symmetry table: Number of children according to number of lesions at screening and first follow-up: Table 15b: PLACEBO GROUP Number of lesions Follow-up 2 Number of Disse- Total lesions at Screening None 1 or 2 3 to 5 >5 minated None 1 0 0 0 0 1 1 or 2 2 0 1 0 1 4 3 to 5 4 0 1 1 1 7 >5 4 0 0 0 1 5 Disseminated 0 1 0 0 0 1 Total 11 1 2 1 3 18 Symmetry test p-value = 0.0818

Table 15c: ACTIVE GROUP Follow-up 2 Number of Disse- Total lesions at Screening None 1 or 2 3 to 5 >5 minated None 0 0 0 0 0 0 1 or 2 2 2 0 0 0 4 3 to 5 3 2 1 0 1 7 >5 2 0 0 0 1 3 Disseminated 2 0 0 0 1 3 Total 9 4 1 0 3 17 Symmetry test p-value = 0.0721

Tinea corporis: Number of lesions at screening*: Number of Lesions: Number of observations (and percentages): Placebo Active Total 1 or 2 5 3 8 62.50 37.50 100.00 71.43 50.00 61.54

3 to 5 0 1 1 0.00 100.00 100.00 0.00 16.67 7.69

>5 1 1 2 50.00 50.00 100.00 133 Tables, symmetry tables and figures 14.29 16.67 15.38 disseminated 1 1 2 50.00 50.00 100.00 14.29 16.67 15.38

Total 7 6 13 53.85 46.15 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.837

Tinea corporis:Number of lesions at follow-up 2*: Number of Lesions: Placebo Active Total none 4 1 5 80.00 20.00 100.00 57.14 16.67 38.46

1 or 2 1 3 4 25.00 75.00 100.00 14.29 50.00 30.77

3 to 5 1 1 2 50.00 50.00 100.00 14.29 16.67 15.38 disseminated 1 1 2 50.00 50.00 100.00 14.29 16.67 15.38

Total 7 6 13 53.85 46.15 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.627

Tinea capitis: Symmetry table: Number of children according to number of lesions at screening (SCR) and first follow-up (Fu2): Table 15e: PLACEBO GROUP Number of lesions Follow-up 2 Number of Disse- Total lesions at Screening None 1 or 2 3 to 5 >5 minated None 0 0 0 0 0 0 1 or 2 4 1 0 0 0 5 3 to 5 0 0 0 0 0 0 >5 0 0 1 0 0 1 Disseminated 0 0 0 0 1 1 Total 4 1 1 0 1 7 Symmetry test p-value = 0.0821

Table 15f: ACTIVE GROUP Number of lesions Follow-up 2 Number of Disse- Total lesions at Screening None 1 or 2 3 to 5 >5 minated 134 Tables, symmetry tables and figures None 0 0 0 0 0 0 1 or 2 1 2 0 0 0 3 3 to 5 0 1 0 0 0 1 >5 0 0 1 0 0 1 Disseminated 0 0 0 0 1 1 Total 1 3 1 0 1 6 Symmetry test p-value = 0.3916

3.1.2.4 Photographs:

Tinea capitis: Evaluation of photographs : Comparing pictures taken at screening and follow- up 2*: Comparison: Number of observations: Placebo Active Total better 10 6 16 62.50 37.50 100.00 71.43 60.00 66.67 same 2 4 6 33.33 66.67 100.00 14.29 40.00 25.00 worse 2 0 2 100.00 0.00 100.00 14.29 0.00 8.33

Total 14 10 24 58.33 41.67 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.301

Tine corporis: Evaluation of photographs : Comparing pictures taken at screening and follow-up 2*: Comparison: Number of observations: Placebo Active Total better 6 4 10 60.00 40.00 100.00 85.71 66.67 76.92 same 0 2 2 0.00 100.00 100.00 0.00 33.33 15.38 worse 1 0 1 100.00 0.00 100.00 14.29 0.00 7.69

Total 7 6 13 53.85 46.15 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.315

135 Tables, symmetry tables and figures 3.1.2.7 Degree of infection: If summarizing the two groups, a decrease in the proportion of children with severe (13.9% to 0.0%), moderate (67.6% to 33.3%) and mild degree of infection (19.4% to 8.3%) could be observed from the first screening examination to the second follow-up. The proportion of clinically cured children reached 61.5% at the second follow-up (see Appendix 3.1.2.7 and Figures 11a and 11b).

Degree of infection: Both groups (n=36)

80 60 none mild 40 moderate 20 severe 0

Percentage of children Percentage of Screening Follow-up 2 Examination

Figure 12a: Tinea capitis: Degree of infection (n=36) Both groups (Screening and follow-up 2) The symmetry test reveals that the reduction in the degree of infection from screening to follow-up 2 is statistically significant if summarizing active and placebo group and if assessing the symmetry separately for the two groups (Symmetry test p-value <0.05) (see Table 17a to 17c).

Tinea capitis: Symmetry table:Number of children according to degrees of infection at screening and follow-up 2 Table 17a: BOTH GROUPS Degree of Degree of infection at Follow-up 2

infection at None Mild Moderate Severe Total Screening None 0 0 0 0 0 Mild 7 0 0 0 7 Moderate 12 3 9 0 24 Severe 2 0 3 0 5 Total 21 3 12 0 36 Symmetry test p-value = 0.0001

An improvement of the degree of infection could be achieved through the application of both soaps. It occurred in 73.7% (placebo group) versus 76.5% (active group).

Table 17b: PLACEBO GROUP Degree of Degree of infection at Follow-up 2 pruritus at Total None Mild Moderate Severe Screening None 0 0 0 0 0 Mild 6 0 0 0 6 Moderate 5 0 5 0 10 Severe 1 0 2 0 3 Total 12 0 7 0 19 Symmetry test p-value = 0.0073

Table 17c: ACTIVE GROUP Degree of Degree of infection at Follow-up 2 pruritus at Total None Mild Moderate Severe Screening None 0 0 0 0 0 136 Tables, symmetry tables and figures Mild 1 0 0 0 1 Moderate 7 3 4 0 14 Severe 1 0 1 0 2 Total 9 3 5 0 17 Symmetry test p-value = 0.0234

Tinea capitis: Degree of infection at screening* Degree of infection: Number of observations: Placebo Active Total mild 6 1 7 85.71 14.29 100.00 31.58 5.88 19.44 moderate 10 14 24 41.67 58.33 100.00 52.63 82.35 66.67 severe 3 2 5 60.00 40.00 100.00 15.79 11.76 13.89

Total 19 17 36 52.78 47.22 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.122

Tinea capitis: Degree of infection at follow-up 2* Degree of infection: Number of observations: Placebo Active Total none 12 9 21 57.14 42.86 100.00 63.16 52.94 58.33 mild 0 3 3 0.00 100.00 100.00 0.00 17.65 8.33 moderate 7 5 12 58.33 41.67 100.00 36.84 29.41 33.33

Total 19 17 36 52.78 47.22 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.242

Tinea corporis: Degree of infection at screening* Degree of infection: Number of observations: Placebo Active Total mild 3 2 5 60.00 40.00 100.00 50.00 66.67 55.56 137 Tables, symmetry tables and figures moderate 2 1 3 66.67 33.33 100.00 33.33 33.33 33.33 severe 1 0 1 100.00 0.00 100.00 16.67 0.00 11.11

Total 6 3 9 66.67 33.33 100.00 100.00 100.00 100.00

Fisher's exact test p-value = 1.000

Tinea corporis: Degree of infection at follow-up 2* Degree of infection: Number of observations: Placebo Active Total none 4 1 5 80.00 20.00 100.00 66.67 33.33 55.56 mild 0 1 1 0.00 100.00 100.00 0.00 33.33 11.11 moderate 1 1 2 50.00 50.00 100.00 16.67 33.33 22.22 severe 1 0 1 100.00 0.00 100.00 16.67 0.00 11.11

Total 6 3 9 66.67 33.33 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.762

Tinea corporis: Symmetry table: Number of children according to degrees of infection at screening and second follow-up: BOTH GROUPS: Degree of Degree of infection at Follow-up 2

infection at None Mild Moderate Severe Total Screening None 0 0 0 0 0 Mild 4 1 0 0 5 Moderate 1 0 1 1 3 Severe 0 0 1 0 1 Total 5 1 2 1 9 Symmetry test p-value = 0.1718

138 Tables, symmetry tables and figures PLACEBO GROUP: Degree of Degree of infection at Follow-up 2 infection at Total None Mild Moderate Severe Screening None 0 0 0 0 0 Mild 3 0 0 0 3 Moderate 1 0 0 1 2 Severe 0 0 1 0 1 Total 3 0 1 1 6 Symmetry test p-value = 0.2615

ACTIVE GROUP: Degree of Degree of infection at Follow-up 2 infection at Total None Mild Moderate Severe Screening None 0 0 0 0 0 Mild 1 1 0 0 2 Moderate 0 0 1 0 1 Severe 0 0 0 0 0 Total 1 1 1 0 3 Symmetry test p-value = 0.3173

Tinea pedis: Degree of infection at screening*: Degree of infection: Number of observations: Placebo Active Total moderate 1 1 2 50.00 50.00 100.00 16.67 100.00 28.57 severe 5 0 5 100.00 0.00 100.00 83.33 0.00 71.43

Total 6 1 7 85.71 14.29 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 0.286

Tinea pedis: Degree of infection at follow-up 2*: Degree of infection: Number of observations: Placebo Active Total none 2 1 3 66.67 33.33 100.00 33.33 100.00 42.86 moderate 3 0 3 100.00 0.00 100.00 50.00 0.00 42.86 severe 1 0 1 100.00 0.00 100.00 16.67 0.00 14.29 139 Tables, symmetry tables and figures

Total 6 1 7 85.71 14.29 100.00 100.00 100.00 100.00 Fisher's exact test p-value = 1.000

3.1.2.8 Clinical Improvement Tinea corporis: Score at screening (SCR) and follow-up 2 (Fu2)

25 S 25 S c c o o 20 r 20 r e e 15 S 15 S C C R R 10 10

5 5

0 0

0 5 10 15 20 25 0 5 10 15 20 25 Score Fu2 Score Fu2 Cubic spline fit Score: M- Cubic spline fit Score: M- Reference line Score: M+ Reference line Score: M+

Figure 13e: Placebo: Microscopy positive (M+)/ Figure 13f: Active: Microscopy positive (M+)/ Microscopy negative (M-) Microscopy negative (M-)

Table 18: Clinical improvement of tinea corporis: Change of score (placebo versus active group) from screening to follow-up 2, according to positive (M. pos) or negative (M. neg.) microscopic result at the second follow-up. Score N.A.: No score value available for follow-up 2, but child not cured. Number of children Reduc- In- Cured: Score Score at follow-up 2 tion of creased Total N.A. = 0 score score M. pos. 1 0 0 2 3 Placebo M. neg. 2 1 1 1 5 Subtotal Placebo 3 1 1 3 8 M. pos. 0 1 0 1 2 Active M. neg. 0 3 0 0 3 Subtotal Active 0 4 0 1 5 Grant Total 3 5 1 4 13

3.1.2.9 Clinical and microscopic resolution

Table 19a: Tinea capitis: Relative frequencies (number of observations) and frequency distribution (percentage) of children having received placebo versus active soap, stratifies according to evaluation Placebo Active Total Clinical *Evaluation Number of Number of number of and categories observations Percentage observations Percentage observations microscopic (percentage) resolution 1 13 65.0 9 56.3 22 (61.1) 2 0 0.0 0 0.0 0 (0.0) Subtotal 14 65.0 10 56.3 22 (61.1) No 3 1 5.0 4 25.0 5 (13.9) resolution 4 0 0.0 0 0.0 0 (0.0) 140 Tables, symmetry tables and figures 5 3 15.0 1 6.3 4 (11.1) 6 0 0.0 0 0.0 0 (0.0) 7 3 15.0 2 12.4 5 (13.9) Total 20 100.0 16 100.0 36 (100.0) * 1= resolution: child cured 2= microscopic resolution and only few clinical symptoms 3= improvement: negative mycologic evidence with significant clinical improvement 4= negative mycologic evidence with no significant clinical improvement 5= improvement: microscopy positive with significant clinical improvement 6= lack of improvement: positive mycologic evidence with no significant clinical improvement 7= deterioration Fisher's exact test p-value = 0.420

Table 19c: Tinea corporis: Relative frequencies (number of observations) and frequency distribution (percentage) of children having received placebo versus active soap, stratifies according to evaluation categories Placebo Active Total

*Evaluation Number of Number of number of Clinical categories observations Percentage observations Percentage observations and (percentage) microscopic 1 4 44.8 2 28.6 6 (37.5) resolution 2 1 11.1 2 28.6 3 (18.8) Subtotal 5 55.9 4 57.2 9 (56.3) 3 1 11.1 1 14.3 2 (12.5 4 0 0.0 0 0.0 0 (0.0) No 5 2 22.2 2 28.6 4 (25.0) resolution 6 0 0.0 0 0.0 0 (0.0) 7 1 11.1 0 0.0 1 (6.25) Total 9 100.0 7 100.0 16 (100.0)

Fisher's exact test p-value = 0.937

Table 19e: Tinea pedis: Relative frequencies (number of observations) and frequency distribution (percentage) of children having received placebo versus active soap, stratifies according to evaluation categories Placebo Active Total Number of Number of number of Clinical *Evaluation observations Percentage observations Percentage observations resolution categories (percentage) 1 5 55.6 6 85.7 11 (68.8) 2 2 22.2 0 0.0 2 (12.5) No 3 2 22.2 0 0.0 2 (12.5) resolution 4 0 0.0 1 14.3 1 (6.2) 5 0 0.0 0 0.0 0 (0.0) Total 9 100.0 7 100.0 16 (100.0) * 1= clinical resolution 2= improvement: score was reduced 3= lack of improvement: score remained the same 4= no resolution: score was non-applicable at screening, but child is not cured 5= deterioration: score increased

141 Tables, symmetry tables and figures 3.2 Determination of minimal inhibitory concentrations (MIC’s) of placebo soap and of Triclosan Table 1: Growth of Malassezia spp. colonies on Dixon Agar inoculated with different concentrations of placebo soap. Concentration of soap solution: 2.0% Number of colony forming units (CFU) (### > 50 CFU) according to Malassezia spp. isolate (N°1 to 6) Concentration of soap Isolate Isolate Isolate Isolate Isolate Isolate solution 1 2 3 4 5 6 (0.2%) on agar (μg/mL) 1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd series series series series series series series series series series series series

400 11 ### 4 0 0 0 ### ### ### ### 5 ### 200 7 ### 0 0 0 0 ### ### ### ### 2 ### 100 0 3 0 0 0 0 ### ### ### ### 1 2 50 1 4 0 0 0 0 ### ### ### ### 6 ### 25 1 1 1 0 0 0 ### ### ### ### 0 0 12.5 1 0 1 0 0 0 ### 1 ### ### 0 0 6.25 0 2 0 3 0 0 ### ### ### ### 9 0 3.13 5 3 0 ### 0 0 ### ### ### ### 8 ### 1.56 4 1 2 0 0 0 ### ### ### ### 0 ### 0.78 2 2 2 1 2 0 ### ### ### ### ### ### 0.39 2 6 4 1 0 0 0 ### ### ### ### 0 0 0 9 0 0 0 0 ### ### ### ### ### 0

Table 2: Growth of Malassezia spp. colonies on Dixon Agar inoculated with different concentrations of Triclosan (* colony growth visible, but number of colonies was not counted), after 7 and 14 days Concentration Concentration Number of colony forming units (CFU) according to Malassezia spp. isolate (5 to 8) of Triclosan in of Triclosan in 7 days 14 days 7 days 14 days 7 days 14 days 7 days 14 days stocking Dixon Agar solution Isolate Isolate Isolate Isolate (μg/mL) (μg/mL) 5 6 7 8 1 50 000 1000 0 0 0 0 0 0 0 0 2 25 000 500 0 7 1 2 7 11 1 34 3 12 500 250 30 29 9 6 24 34 50 55 4 6 250 125 23 34 7 8 24 40 60 38 5 3 125 62.5 19 25 5 9 28 40 56 44 6 1562.5 31.25 24 23 4 4 21 44 53 53 7 781.24 15.63 12 16 2 4 19 39 46 52 8 390.63 7.81 10 23 1 1 9 33 50 45 9 195.31 3.91 10 20 3 4 8 36 51 47 10 97.66 1.95 13 20 0 2 10 30 51 47 11 48.83 0.98 9 19 1 6 11 28 45 51 12 24.41 0.49 6 20 1 3 10 41 51 54 13 12.21 0.24 13 17 1 4 15 32 54 47 14 6.10 0.12 9 15 2 3 5 23 45 42 15 3.05 0.06 9 23 1 1 9 30 45 34 16 1.53 0.03 11 16 1 2 7 27 35 40 17 0.08 0.015 6 15 1 1 2 24 40 35 18 0.04 0.008 2 9 2 3 1 19 42 41 19 0.02 0.004 6 11 3 4 3 41 43 43 Control 20 * * * * * * * * (only ethanol) 142 Tables, symmetry tables and figures 4. Scabies

4.2 Evaluation of effectiveness

Scabies: Number of observations : Characteristic clinical parameters at screening (SCR: Placebo n=4, Active n=4), first (Fu1: Placebo n=4, Active n=0) and second (Fu2: Placebo n=4, Active n=2) follow-up : Parameter Exami- Number of observations nation Placebo Active Total (out of total number of observations made) pruritus SCR 4 4 8/8 Fu1 4 - 4/4 Fu2 1 1 2/6 more severe at night SCR 4 4 8/8 Fu1 3 - 3/4 Fu2 1 1 2/6 other family members affected SCR 4 2 6/8 Fu1 4 - 4/4 Fu2 1 2 3/6 rash SCR 3 3 6/8 Fu1 2 - 2/4 Fu2 0 1 1/6

Scabies: Number of observations : Site of lesions at screening (SCR: Placebo n=4, Active n=4), first (Fu1: Placebo n=4, Active n=0) and second (Fu2: Placebo n=4, Active n=2) follow-up: Common loca- Exami- Number of observations lisation nation Placebo Active Total ( out of total number of observations made) Hands: sides of the fingers SCR 4 3 7/8 Fu1 3 - 3/4 Fu2 1 2 3/6 wrists SCR 4 3 7/8 Fu1 4 - 4/4 Fu2 1 1 2/6 ulnar borders SCR 3 4 7/8 Fu1 3 - 3/4 Fu2 1 1 2/6 palms SCR 1 1 2/8 Fu1 1 - 1/4 Fu2 0 0 0/6 143 Tables, symmetry tables and figures

Other parts: anterior parts of elbows SCR 3 4 7/8 Fu1 2 - 2/4 Fu2 1 0 1/6 anterior axillary folds SCR 0 2 2/8 Fu1 2 - 2/4 Fu2 1 1 2/6 umbilical region SCR 1 2 3/8 Fu1 2 - 2/4 Fu2 2 1 3/6 external genitali, peri- neum, glans penis SCR 0 2 2/8 Fu1 1 - 1/4 Fu2 2 1 3/6 areloae SCR 0 0 0/8 Fu1 1 - 1/4 Fu2 0 1 1/6 gluteal region SCR 0 1 1/8 Fu1 1 - 1/4 Fu2 1 0 1/6 waist SCR 0 1 1/8 Fu1 1 - 1/4 Fu2 1 0 1/6 thighs SCR 0 1 1/8 Fu1 1 - 1/4 Fu2 0 0 0/6 knees SCR 1 0 1/8 Fu1 1 - 1/4 Fu2 0 1 1/6 ankles SCR 0 0 0/8 Fu1 3 - 3/4 Fu2 1 1 2/6 soles SCR 0 0 0/8 Fu1 3 - 3/4 Fu2 0 0 0/6 abdominal region SCR 1 1 2/8 Fu1 1 - 1/4 Fu2 0 1 1/6 144 Tables, symmetry tables and figures

others: SCR 4 4 8/8 . Fu1 1 - 1/4 Fu2 1 1 2/6

Scabies: Number of observations : Number of lesions at screening (SCR: Placebo n=4, Active n=4), first (Fu1: Placebo n=4, Active n=0) and second (Fu2: Placebo n=4, Active n=2) follow- up: Number of loca- Exami- Number of observations lisation nation Placebo Active Total ( out of total number of observations made) none SCR N.A. N.A. N.A. Fu1 1 - 1/4 Fu2 2 0 2/6

1 to 3 SCR 2 1 2/8 Fu1 2 - 2/4 Fu2 1 1 2/6

4 to 6 SCR 1 1 2/8 Fu1 1 - 1/4 Fu2 1 0 1/6

>6 SCR 1 0 5/8 Fu1 2 - 2/4 Fu2 1 1 2/6

Scabies: Number of observations : Aspect of lesions at screening (SCR: Placebo n=4, Active n=4), first (Fu1: Placebo n=4, Active n=0) and second (Fu2: Placebo n=4, Active n=2) follow- up: Aspect of lesions Exami- Number of observations nation Placebo Active Total ( out of total number of observations made) none SCR N.A. N.A. N.A. Fu1 0 - 0/4 Fu2 1 0 1/6 papules, macules, burrows SCR 3 4 7/8 Fu1 4 - 4/4 Fu2 1 2 3/6 crusts, generalized lichenification SCR 1 0 1/8 Fu1 4 - 4/4 Fu2 1 1 2/6

145 Tables, symmetry tables and figures superinfection SCR 2 0 2/8 Fu1 1 - 1/4 Fu2 0 1 1/6 thick crusts SCR 0 0 0/8 Fu1 1 - 1/4 Fu2 0 0 0/6 4.2.1.5 Degree of pruritus: Scabies: Number of observations : Degree of pruritus at screening (SCR: Placebo n=4, Active n=4), first (Fu1: Placebo n=4, Active n=0) and second (Fu2: Placebo n=4, Active n=2) follow- up: Degree of Exami- Number of observations pruritus nation Placebo Active Total ( out of total number of observations made) none SCR 0 0 0/8 Fu1 0 - 0/4 Fu2 3 1 4/6 mild SCR 1 0 1/8 Fu1 1 - 1/4 Fu2 1 1 2/6 moderate SCR 1 3 4/8 Fu1 2 - 2/4 Fu2 0 0 0/6 severe SCR 2 1 3/8 Fu1 1 - 1/4 Fu2 0 0 0/6 pruritus improved Fu1 2 - 2/4 Fu2 0 0 0/6

146 Appendix B: Photographic Imags: Placebo//Active Soap Appendix B: Photographic Images: 1. Tinea versicolor: 1.1 Improvement: Child 1: Screening: Child 1: Continuous improvement can be seen from screening to both follow-ups and between the two follow-ups. At the first follow-up the improvement manifests as a reduction in the number of lesions and the size of the lesions. Furthermore, the lesions seem to be less scaly. No statement can be made about the difference in pigmentary changes due to the different illumination conditions. At the second follow-up no more lesions can be identified.

Child 1: Follow-up 1:

147 Appendix B: Photographic Imags: Placebo//Active Soap Child 1: Follow-up 2:

Child 2: Screening: Child 2: Even though at all three examinations multiple macules can be seen, the aspect of the lesions changes over time. At the follow-up examinations they appear to be less scaly, less hypopigmented and less sharply demarcated from non-lesional skin.

148 Appendix B: Photographic Imags: Placebo//Active Soap Child 2: Follow-up 1:

Child 2: Follow-up 2:

149 Appendix B: Photographic Imags: Placebo//Active Soap 1.2 No change: Child 3: Screening: Child 3: The aspect of the lesions changes from that of dark scaly, fine and wrinkled macules and patches with clearly demarcated borders, to that of hyperpigmented follicular bound macules. However, the degree of infection does not appear to become more severe, since the distribution and pattern of lesions at the observed site (right infraclavicular region) remains fairly similar.

Child 3: Follow-up 1:

150 Appendix B: Photographic Imags: Placebo//Active Soap Child 3: Follow-up 2:

Child 4: Screening: Child 4: The number, size and shape of the hypopigmented macules remain the same over the period of three months. At all three examinations the lesions seem to be slightly scaly.

151 Appendix B: Photographic Imags: Placebo//Active Soap Child 4: Follow-up 1:

Child 4: Follow-up 2:

152 Appendix B: Photographic Imags: Placebo//Active Soap Child 5: Screening: Child 5: The number, size and shape of the hypopigmented macules remain the same from the screening examination to the second follow-up. At the first examination the macules may be slightly less scaly than at the other examinatons. Nevertheless improvement is not obvious.

Child 5: Follow-up 1:

153 Appendix B: Photographic Imags: Placebo//Active Soap

Child 5: Follow-up 2:

1.3 Deterioration: Child 6: Screening: Child 6: The confluent macules/ patches show a marked increase in size. Apart from this the aspect of the lesions does not change. Hypopigmentation and scaling remain until the second follow-up.

154 Appendix B: Photographic Imags: Placebo//Active Soap Child 6: Follow-up 1:

Child 6: Follow-up 2:

155 Appendix B: Photographic Imags: Placebo//Active Soap Child 7: Screening: Child 7: The hyperpigmented follicular bound macules and papules show a marked increase in size. Hyperpigmentation if more pronounced the second follow-up.

Child 7: Follow-up 2:

156 Appendix B: Photographic Imags: Placebo//Active Soap Child 8: Screening: Child 8: The confluent macules/patches show a marked increase in size, but the aspect of the lesions does not change. Hypopigmentation and scaling appear to be more pronounced at the first follow-up.

Child 8: Follow-up 1:

157 Appendix B: Photographic Imags: Placebo//Active Soap 2. Tinea capitis: 2.2 No change: Child 9: Screening: Child 11: Several large slightly greyish patches with “black dot pattern” are well visible at the first screening and the first follow-up. The aspect of the lesion remains the same. The non-affected sites between the patches are shaved, so alopecia resulting from the dermatophyte infection cannot be exactly compared between the two examinations.

Child 9: Follow-up 1:

2.3 Deterioration: Child 10: Screening: 158 Appendix B: Photographic Imags: Placebo//Active Soap Child 12: The large scaly grey patch increases markedly in diameter from the screening examination to the first follow- up. Alopecia also becomes more extensive. Besides, crusting is more pronounced at the first follow-up. The margins of the lesions are no longer clearly demarcated. The type of lesions at the first follow-up can be characterised as “kerion- like”.

Child 10: Follow-up 1:

3. Tinea corporis: 3.2 No change: Child 11: Screening: 159 Appendix B: Photographic Imags: Placebo//Active Soap At the screening and the second follow-up, the lesions were disseminated and slightly scaly. No improvement took place. Interestingly, no Malassezia yeasts, but only spores, were identified on microscopic examination of the skin samples of this child. Therefore this case was considered as a dermatophyte infection caused by T. tonsurans (identified by culture) despite the fact that the clinical picture is very unusual for tinea faciei. It strongly resembles tinea versicolor infections. Child 11: Follow-up 2: A potential co-infection cannot be ruled out, due to false negative results for Malesszia spp. According to Kane et al., the dermatophyte T. tonsurans may persist for a long time in the environment or on fomites such as combs or headgear (Kane et al., 1988). Some people may develop subclinical scalp infections and shed the pathogen for many years (Hay, 1996). This might be the reason why in the present study for two further children clinically diagnosed to suffer from tinea versicolor, T. tonsurans could be isolated from the same site without currently producing any relevant skin disease.

160 Appendix B: Photographic Imags: Placebo//Active Soap 3.3 Deterioration: Child 12: Screening: Child 15: The slightly scaly patch is not entirely typical of a ringworm lesion, since there is no central healing. However, direct microscopy was positive at the first screening. At the first follow-up the patch has markedly increased in size.

Child 12: Follow-up 1:

161 Appendix C: Questionnaires and Data Entry Sheets Appendix C: 1. Questionnaire (English/Kiswahili) 1.1 PART A : History and hygiene evaluation (only Screening):

ID Number of the child: □. □. □. □□□□ Date:______# Screening - # School - # Form – Serial number child Name of the child: Form: Sex: Age: 1 = YES 2= NO NA = Not applicable DNK = child does not know

1. Do you have any skin problem at present? yes □ no □ Una tatizo la ngozi yako?

2. If so, which of the following describes your problem? (only refers to question number one) Tatizo lako ni nini? 1. itching kuwashwa yes □ no □ 2. depigmetation kuwa na mabaka (patch) yes □ no □ 3. change in colour: black inabadirika rangi: nyeusi yes □ no □ 4. change in colour: red inabadirika rangi: nyekuindu yes □ no □ 5. change in colour: white inabadirika rangi: nyeupe yes □ no □ 6. rash vipele yes □ no □ 7. sores vidonda (NO if it does not refer to the MAIN skin problem!) yes □ no □ 8. does it hurt je inauma yes □ no □ 9. is it numb? Unasikia ganzi yes □ no □

3. Do you have any other skin problem (if the child presents with several significant skin diseases, please comment in detail at the end of the questionnaire) Je una tatizo lingine la ngozi? yes □ no □

4. if yes kama ndiyo: Mention eleza: CATEGORIZE! 1. tinea versicolor Matangatanga 2. Ringworm Mapunje 3. NA 4. DNK 5. wounds vidonda 6. rash vipele 7. itching uwasho 8. Pustules which rupture leaving ulcers 9. Eczema

5. When did this skin problem start? (only refers to question number one) Tatizo lako hili la ngozi lilianza lini? 1. < two weeks ago . chini ya mwiki mbili 2. 2-4 weeks zaidi ya wiki mbili na chini ya wiki nne If 5.1/ 5.2 YES – please enter 9.5/6/7 = 3 (NA) 162 Appendix C: Questionnaires and Data Entry Sheets 3. 2-6 months ago zaidi ya miwezi mibili na chini ya miwezi sita 4. <1 year ago mwaka 1 5. 2 years ago miaka 2 6. 3 years ago miaka 3 7. > years ago zaidi ya miaka mitatu

6. Where did this skin problem start? (only refers to question number one) Tatizo hili limeanzia wapi? 1. scalp ngozi ya kichwa yes □ no □ 2. face uso yes □ no □ 3. neck shingo yes □ no □ 4. chest kifua yes □ no □ 5. abdomen tumbo yes □ no □ 6. back mgongo yes □ no □ 7. axilla makwapa yes □ no □ 8. arms mkono yes □ no □ 9. hands viganjwa yes □ no □ 10. finger webs uwazi wa vidole yes □ no □ 11. genital region sehemu za siri yes □ no □ 12. thighs mapaja yes □ no □ 13. knees magoti yes □ no □ 14. lower leg miguu yes □ no □ 15. feet nyaao yes □ no □ 16. between the toes baina ya kidole cha miguu yes □ no □

7. Size Ukubwa: 1. Does it remain the same? Inabakia vilevile? 2. Is it increasing in size? Inaongezeka ukubwa? 3. Is it decreasing in size? Inapungua? 4. Is it varying in size? Inarudiarudia?

8. Does this skin disease itch (lesions described in question no. 1)? Je ugonjwa wako huu unawasha? yes □ no □

9. When does it itch? Ni wakati gani unawasha zaidi? 1. in the hot sun jua kali yes □ no □ 2. in bed/sleeping kitandani/ unapolala yes □ no □ 3. after bathing with hot water baada ya kuoga na maji joto yes □ no □ 4. after bathing with cold water maji baridi yes □ no □ IF Question No. 5.1/ 5.2 = YES: Please fill in 3 (NA) in Question No. 9.5/ 9.6/ 9.7 = 5. rainy season masika yes □ no □ 6. hot/dry season kiangazi yes □ no □ 7. cold season wakati wa baridi yes □ no □

13. Are there any others at home who have the same skin disease or the same signs of the skin disease as you? How many Taja wangapi: IF NO, PLEASE ENTER “0” □ 15. Apart from the skin problem(s) do you have another problem such as the following? Una tatizo lolote kati ya haya yafuatayo: 1. fever homa yes □ no □ 163 Appendix C: Questionnaires and Data Entry Sheets 2. fatigue kuchoka yes □ no □ 3. headache unaumwa kichwa yes □ no □ 4. nausea kichefuchefu yes □ no □ 5. vomiting kutapika yes □ no □ 6. joint pain maumivu ya viungo yes □ no □ 7. edema uvimbe yes □ no □ 8. diarrhoea kuharisha yes □ no □ 9. abdominal pain maumivu ya tumbo yes □ no □ 10. generalized pruritus uwashwa mwili mzina yes □ no □ 11. itchy scalp Kitchwa chako kina uwashwa? yes □ no □ 12. others: vitu vingine: CATEGORIZE!

1. coughing kuhoa 2. NO other complains 3. NA 4. DNK 5. wounds kidonda 6. abdominal problems tumbo 7. back hurts 8. pain in different body parts after having played football/ shamba work etc. 9. eye problem 10. ear problem 11. itching 12. running nose

16. Have you received any treatment for your main skin problem? Je umetumia dawa yoyote kutibu tatizo hili? yes □ no □

17. If yes: which kind of treatment? Kama ndiyo: ni dawa gani? 1. crème krimu yes □ no □ 2. tablets vidonge yes □ no □ 3. injection sindano yes □ no □ 4. leaves majani yes □ no □ 5. roots mizizi yes □ no □ 6. charm kombe yes □ no □ 7. necklace hirizi yes □ no □ 8. Name of the drug: Jina la dawa: CATEGORIZE!

1. traditional medicine 2. NO name given 3. NA 4. DNK 5. Panadol 6. Aspirin 7. breaking fluid 8. mafuta (Vaseline/ Oil) 9. Gentian Violet 10. ointment

164 Appendix C: Questionnaires and Data Entry Sheets 19. How long have you been taking this drug? 18. Umetumia dawa hii kwa muda gani? 1.< 1 week kidogo zaidi wiki moja 2.< 2 weeks kidogo zaidi wiki mbili 3.< 4 weeks kidogo zaidi wiki nne 4. 2-6 months miezi miwili hadi miezi sita 5.< 1 year kidogo zaidi mwaka mmoja 6.> 1 year zaidi ya mwaka mmoja

20. Has this drug helped? Je imekusaidia? yes □ no □ 19. 21. If several drugs have been used: which one has helped? Mention. CATEGORIZE! Kama umetumia madawa mabali – dawa gani imesaidia? 20. 0. NA 1. tablets 2. leaves from traditional healer 3. NA 4. DNK 5.

22. Have you ever been admitted to hospital? Je, umewahi kulazwa hospitalini? yes □ no □ 21. 23. if yes kama ndiyo: 22.

How many times? Mara ngapi? □ Please enter number of admissions, if no admissions, write 0, if question 22: DNK, write 0 24. What were you suffering from? Ulikuwa unamwa nini? CATEGORIZE! 23. 1. malaria 2. abdominal problems tumbo 3. NA 4. DNK 5. abscess tambasi 6. headache/problems kichwa 7. fever homa 8. meningitis 9. fracture 10. seizures 11. asthma 12. wounds 13. pneumonia

25. Does any family member at home or any relative have asthma? Je nyumbani kuna mtu au ndugu mwenye pumu? yes □ no □ 24. 26. Does any family member at home or any relative have a skin disease different from the one 25. you have? Je nyumbani kuna mtu au ndugu mwenye ugonjwa wa ngozi mbali ya ugonjwa huu ? yes □ no □ 165 Appendix C: Questionnaires and Data Entry Sheets

27. If yes kama ndiyo, 26. mention taja: CATEGORIZE! 1. tinea versicolor Matangatanga 2. ringworm Mapunje 3. NA 4. DNK 5. rash vipele 6. wounds vidonda

28. Apart from the drug for your (main) skin disease, do you take any other drugs? 27. Je unatumia dawa nyingine yoyote (mbali na dawa ya ugonjwa wa ngozi)?yes □ no □

29. Which drugs? Dawa gani? CATEGORIZE! 28. 1. traditional medicine 2. NO other drug used 3. NA 4. DNK 4. Panadol 5. Aspirin 6. breaking fluid 7. mafuta (Vaseline/ Oil) 8. antiseptic solution 9. Panadol 10. leaves

32. What is your religion? 31. Dini yako ni ipi? 1. Christian Mkristo 2. Muslim Mwislamu 3. NA 4. DNK 5. No religion hamna

1.2 PART B : Physical Examination: (all children at Screening; Study participants at follow-up examinations) Lesions suspicious of skin diseases found on: 58. Scalp: □ 59. Face: □ 60. Gums □ 61. Tongue □

Neck: 62. anterior □ 63. posterior □

64. Chest: □ 65. Abdomen: □ 66. Back: □

166 Appendix C: Questionnaires and Data Entry Sheets Axillar region: 67. right □ 68. left □

Upper arm: 69. Right ant. □ 70. post. □ 71. Left ant. □ 72. post. □

Forearm: 73.Right ant. □ 74.post. □ 75.Left ant. □ 76.post. □

Wrist: 77.Right dorsal □ 78. volar □ 79. Left dorsal □ 80. volar □

Hands: 81. Right dorsal □ 82.palm □ 83. Left dorsal □ 84.palm □

Nails: 85. right hand □ 86. left hand □ 87. right foot □ 88. left foot □

Gluteal region: 89.Right □ 90. Left □ 91. Perianal region: □ 92. Inguinal region: □ 93. Pubic region: □

Thighs: 94.Right ant. □ 95.post. □ 96.Left ant. □ 97.post. □

Knee: 98. Right ant. □ 99. post. □ 100. Left ant. □ 101. post. □

Lower leg: 102.Right ant. □ 103.post. □ 104.Left ant. □ 105.post. □

Ankles: 106.Right med.□ 107. lat. □ 108.Left med. □ 109. lat. □

Feet: 110.Right dorsal □ 111. Left dorsal □ 112. Right sole □ 113. Left sole □

IF SPACES ARE NOT SUFFICIENT, PLEASE USE FOLLOWING LOCALIZATIONS: 114: Right arm/wrist/ hand 115: Left arm/ wrist/ hand 116: Gluteal region 117: Right leg/ ankle/ foot 118: Left leg/ ankle/ foot

Main Lesion:

114. Type of efflorescence: PLEASE MARK WITH “YES” OBSERVED EFFLORESCENCE!

Primary lesions: Macule: non-elevated, less than 1 cm Patch: non-elevated, more than 1 cm Papule: elevated lesion less than 1 cm 167 Appendix C: Questionnaires and Data Entry Sheets Nodule: deeply-seated, indurated, larger than 1 cm Vesicle: with fluid less than 1 cm Bulla: with fluid, more than 1 cm Pustule: circumscribed with pus Vesiculo-pustules Wheal: edematous elevation of the skin of varying size and shape, pale pink in colour Tumour: circumscribed swelling larger than 2.5 cm in diameter

Secondary lesions: scales, crust, plaque, fissure, excoriations, erosion, ulcer, atrophy, sclerosis

Other: burrow, comedo, teleangiectasia, lichenification, cyst SKIN DRY/ WET/ SCALING?

115. Number of lesions: if lesions can be counted, please enter numbers if disseminated, please enter DISS, if numerous, enter NUM if extensive, enter EXT if none-aplicable, leave blank

116. Distribution: 1. localized 2. generalized on one part of the body 3. generalized on more than 1 part of the body

117. Colour: 0. NA 1. hypopigmented 2. hyperpigmented 3. NA 4. normopigmented 5. yellowish 6. pink 7. bluish 8. greyish 9. black 10. reddish 11. whitish

118. Shape: 0. NA 1. circular 2. polygonal = irregular 3. NA 4. cylindrical 5. linear 6. No special shape

119. Demarcation: 0. NA 1. sharply 2. not sharply 3. NA

120. Diameter/length: IN CM, ONE DECIMAL! If NA: leave blank 168 Appendix C: Questionnaires and Data Entry Sheets

121. Configuration: 0. NA 1. single lesion/s 2. one group 3. NA 4. in groups 5. herpetiform 6. serpinginous 7. continuous 8. rosettes 9. disseminated 10. confluent macules 11. generalized 12. irregular

DIAGNOSES:______122. Do you think you are ill at the moment? yes □ no □ Unafikiri una umwa sasa?

123. If yes: What are you suffering from?______Kama ndiyo, una umwa na nini? 124. Diagnosis correct: Ugonjwa sahihi yes □ no □

Patient examined by 1. MM □ 2. CM □ 3. AD □ 4. JF □

Number of photos taken ___

169 Appendix C: Questionnaires and Data Entry Sheets 1.3 PART C: Description of lesions of study participants (study participants at screening and follow-up examinations) All questions answered as (yes □ no □) (and score value) * to be asked at first and second follow-up

Fungal infections:

Tinea versicolor: Score range: 0 – 25:

126. Endpoints/Clinical parameters: 1. concentric lesions yes □ no □ 2. scaling in periphery yes □ no □ 3. scaling in concentric rings yes □ no □ 4. satellite lesions yes □ no □ 5. pruritus yes □ no □ 6. hypopigmentation yes □ no □

130. Description of pruritus: 1. no pruritus hamna kuwasha 0 2. it sometimes itches a little bit wakati wenngine inawasha kidogo 1 3. it is a real nuisance kweri inasumbua 2 4. patient cannot sleep because of pruritus 3 mgonjwa hawezi kulala kwa sababu ya kuwashwa/muwasho *5. Does it itch less than before? (subjective impression) yes □ no □

131. Number of lesions: 1. none 0 2. one or two 2 3. three to five 4 4. > five 6 5. disseminated 8

133. Aspect of lesions (several options at the same time possible): 1. none 0 2. hypopigmented/hyperpigmented lesions 2 3. scaling 2 4. grey patches 4 5. signs of bacterial superinfection 6

Tinea capitis: Score range: 0 – 41:

125. Endpoints/Clinical parameters: 1. scalp pruritus yes □ no □ 2. scaling yes □ no □ 3. diffuse or circumscribed alopecia yes □ no □ 4. occipital adenopathy yes □ no □ 5. black dots in the remaing hair follicle after loss of hair yes □ no □ 6. kerion (highly inflammatory lesions) yes □ no □ *7. New hair growth visible yes □ no □

170 Appendix C: Questionnaires and Data Entry Sheets 130. Description of pruritus: 1. no pruritus hamna kuwasha 0 2. it sometimes itches a little bit wakati wengine inawasha kidogo 1 3. it is a real nuisance kweri inasumbua 2 4. patient cannot sleep because of pruritus 3 mgonjwa hawezi kulala kwa sababu ya kuwashwa/muwasho *5. Does it itch less than before? (Pruritus improved) yes □ no □ 131. Number of lesions: 1. none 0 2. one or two 2 3. three to five 4 4. > five but countable 6 5. disseminated 8

132. Size of largest lesions (diameter): 1. none 0 2. <1 cm 2 3. 1 to 3 4 4. > 3 cm 6

133. Aspect of lesions (several options at the same time possible): 1. none 0 2. sharply deliniated with central healing 2 3. grey patches 4 4. yellowish-white adhearent masses (scutulae) 6 5. signs of bacterial superinfection 6

134. Hair stumps, Baldness, Skin atrophy: 1. none 0 2. one 1 3. two 2 4. all three 3

135. Additional clinical signs (occipital adenopathy, scaling, kerion, black dots) - none 0 - one 1 - two 2 - three or four 3

Tinea corporis: Score range: 0 – 29:

126. Endpoints/Clinical parameters: 1. concentric lesions yes □ no □ 2. scaling in periphery yes □ no □ 3. scaling in concentric rings yes □ no □ 4. satellite lesions yes □ no □ 5. pruritus yes □ no □ 6. hypopigmentation yes □ no □

130. Description of pruritus: 1. no pruritus hamna kuwasha 0 171 Appendix C: Questionnaires and Data Entry Sheets 2. it sometimes itches a little bit wakati wengine inawasha kidogo 1 3. it is a real nuisance kweri inasumbua 2 4. patient cannot sleep because of pruritus 3 mgonjwa hawezi kulala kwa sababu ya kuwashwa/muwasho *5. Does it itch less than before? (Pruritus improved) yes □ no □

131. Number of lesions: 1. none 0 2. one or two 2 3. three to five 4 4. > five but countable 6 5. disseminated 8

132. Size of largest lesions (diameter): 1. none 0 2. <1 cm 2 3. 1 to 3 4 4. > 3 cm 6

133. Aspect of lesions (several options at the same time possible): 1. none 0 2. sharply deliniated with central healing 2 3. scaling 4 4. signs of bacterial superinfection 6

Tinea pedis: Score range: 0 – 21:

130. Description of pruritus: 1. no pruritus hamna kuwasha 0 2. it sometimes itches a little bit wakati wengine inawasha kidogo 1 3. it is a real nuisance kweri inasumbua 2 4. patient cannot sleep because of pruritus 3 mgonjwa hawezi kulala kwa sababu ya kuwashwa/muwasho *5. Does it itch less than before? (Pruritus improved) yes □ no □

131. Number of lesions: 1. none 0 2. one or two 2 3. three to five 4 4. > five but countable 6 5. disseminated 8

132. Size of largest lesions (diameter): 1. none 0 2. <1 cm 2 3. 1 to 3 4 4. > 3 cm 6

127. Aspect of lesions: 1. none 0 4. erythematous, macerated whitish scaling, athlete’s foot (interdigital) 4 172 Appendix C: Questionnaires and Data Entry Sheets

Scabies: Clinical criteria: 136. itching yes □ no □ 137. itching more at night yes □ no □ 138. other family members also effected yes □ no □

139. Characteristic lesions in the common sites: 1.a sides of the fingers yes □ no □ 1.b wrists yes □ no □ 1.c ulnar border of the hands yes □ no □ 1.d palms yes □ no □ 2. anterior parts of elbows yes □ no □ 3. (anterior) axillary folds yes □ no □ 4. umbilicus yes □ no □ 5. external genitalia and perineum, glans penis yes □ no □ 6. areola of the breasts yes □ no □ 7. buttocks yes □ no □ 8. waist yes □ no □ 9. thighs yes □ no □ 10. knees yes □ no □ 11. ankles yes □ no □ 12. soles yes □ no □ 13. abdomen yes □ no □ 14. other:______yes □ no □

140. raised papules yes □ no □ 141. rash yes □ no □

Microscopic examination: 143. sample taken yes □ no □ 144. mites found on microscopic examination of scraping yes □ no □

All questions answered as (yes □ no □) (and score value) * to be asked at first and second follow-up

Score range: 0-47 145. Description of pruritus: 1. no pruritus hamna kuwasha 0 2. it sometimes itches a little bit wakati mwingine inawasha kidogo 1 3. it is a real nuisance kweri inasumbua 2 4. patient cannot sleep because of pruritus mgonjwa hawezi kulala kwa sababu ya kuwashwa/muwasho 3

147. Type of lesions (several answers possible): 1. none 0 2. only papules, macules, burrow 2 3. crusts, generalized lichenification 4 4. superinfection 6 5. thick crusts 6 *Do you think the soap helped you? Unafikiri umepungua baada ya kutumia sabuni yes □ no □ 173 Appendix C: Questionnaires and Data Entry Sheets 2. Data Entry Sheet: Screening Spectrum of Skin Diseases among Primary School Children

ID Number of the child: □. □. □. □□□□ Date:______# Screening - # School - # Form – Serial number child Name of the child: Form: Sex: Age: 1 = YES 2= NO NA = Not applicable DNK = child does not know

1 2 9.1 15.1 8 26 4 41.1 5 3 2.1 3 2 2 18.1 27 5 2 47 4 2 4 3 3 2 28 6 3 48 5 3 5 4 4 3 29 36 4 49 6 4 6 5 5 4 30 37 42 50 7 5 7 6 6 5 31 38 43 51.1 8 6 8 7 7 6 32.1 39.1 44.1 2 54.1 7 9 10.1 8 7 2 2 2 3 2 8 10 2 9 19.1 3 3 3 4 3 9 11 3 10 2 4 4 4 5 4 3 12 4 11 3 33.1 5 5 6 5 4 13 11.1 12 4 2 6 6 7 6 5.1 14 2 16 5 3 7 7 8 7 2 15 3 17.1 6 4 8 45.1 52.1 8 3 16 4 2 20 5 40.1 2 2 9 4 7.1 12 3 21 6 2 3 3 55.1 5 2 13 4 22 34 3 46.1 4 2 6 3 14.1 5 23 35.1 4 2 5 56.1 7 4 2 6 24 2 5 3 53.1 2 6.1 8 3 7 25 3 6 4 2 3 4.______33.6______15.12______17.8.______34.______21.______35.6.______24.______36.______39.8.______27.______44.7.______29.______46.5.______31.______174 Appendix C: Questionnaires and Data Entry Sheets ______4 63 87 111 4 3 5 2 4 6 57.1 64 88 112 5 4 6 3 147.1 161 2 65 89 113 6 133.1 7 4 2 162 3 66 90 114 127.1 2 8 5 3 163 4 67 91 115 2 3 9 PED 4 164 5 68 92 116 3 4 10 148.1 5 165.1 6 69 93 117 4 5 11 2 154.1 2 7 70 94 118 128.1 6 12 3 2 3 8 71 95 119 2 134.1 13 149 3 4 9 72 96 120 3 2 14 150.1 CLM DIAG 10 73 97 121 129.1 3 15 2 155 166 11 74 98 122 2 4 16 3 156.1 167 12 75 99 123 3 135.1 140 4 2 168 13 76 100 124 130.1 2 141 5 3 169 14 77 101 TIN 2 3 142 151.1 4 170 15 78 102 125.1 3 4 143 2 157 171 16 79 103 2 4 SCA 144 3 158 172 17 80 104 3 131.1 136 145.1 4 TUN 173 18 81 105 4 2 137 2 5 159 174 58 82 106 5 3 138 3 152.1 160.1 175 59 83 107 6 4 139.1 4 2 2 176 60 84 108 126.1 5 2 146.1 3 3 177 61 85 109 2 132.1 3 2 4 4 178 62 86 110 3 2 4 3 153.1 5 179.1

114-121 123.______2 ______3 ______4 139.16.______5 ______178 ______179 ______180 ______Comments :______Diagnosis : ______56.4.______57.18.______

175 Appendix C: Questionnaires and Data Entry Sheets

FOLLOW-UP Number ONE/TWO:

ID Number of the child: □. □. □. □□□□ Date:______# Screening - # School - # Form – Serial number child Name of the child: Form: Sex: Age: 1 = YES 2= NO NA = Not applicable DNK = child does not know

Tinea D-2-J D-13-E 3 4 141 151.1 D-1-A D-2-K D-13-F 4 135.1 142 2 D-1-B D-2-L D-13-G 130.1 2 145.1 3 D-1-C D-2-M D-13-H 2 3 2 4 D-1-D D-2-O D-13-I 3 4 3 5 D-1-E Scab D-13-J 4 136 S 4 152.1 D-1-F D-12-A D-13-K 5 137 5 2 D-1-G D-12-B D-13-L 131.1 3 138 146.1 3 D-1-H D-12-C D-13-M 2 139.1 2 4 D-1-I D-12-D D-13-O 3 2 3 5 D-1-J D-12-E 125.1 T 4 3 4 153.1 D-1-K D-12-F 2 5 4 147.1 2 D-1-L D-12-G 3 132.1 3 5 2 3 D-1-M D-12-H 4 2 6 3 4 D-1-O D-12-I 5 3 7 4 5 T. vers D-12-J 6 4 8 5 154.1 D-2-A D-12-K 7 133.1 9 148.1 P 2 D-2-B D-12-L 126.1 2 10 2 3 D-2-C D-12-M 2 3 11 3 179 D-2-D D-12-O 3 4 12 149 180 D-2-E Ped 4 5 13 150.1 D-2-F D-13-A 5 6 14 2 D-2-G D-13-B 6 134.1 15 3 D-2-H D-13-C 127.1 2 16 4 D-2-I D-13-D 2 3 140 5

114 - 121: 181 182 183 184 185 Subjec. Impres Comments: ______

176 Appendix D: Information for the study participants and parents Appendix D: 1. Informed Consent

1.1 English version: Invitation for parents, addressed to Local Leaders (“Balozi”)

Dear Balozi Leaders,

My name is Almuth Dinkela, I am a medical student sent to Ifakara by the Swiss Tropical Institute in Basel (Switzerland). I am working for IHRDC here in Ifakara in order to do my Medical Doctor research project in Kilombero District. I am interested in two skin diseases that are a very common problem in this area: Ringworm and scabies. Together with the specialist of dermatology at St. Francis Designated District Hospital, SFDDH, Dr. M. Mbata, Dr. Charles Maswi and Julia Ferié, I examined all the children attending Lihami Primary School to find out which skin diseases they are suffering from. We have identified those who have fungal infections and scabies to take part in my study.

On Monday, May 12th 2003, a meeting will we held at Lihami Primary School (Michenga Primary School). We would like to invite you and all the parents of these children to attend the meeting at - ______. The parents will have the chance to ask questions about the study and will be asked for their permission to let their children participate. We would be very happy if you could inform the parents about this meeting.

Thank you very much for your co-operation!

Almuth Dinkela

177 Appendix D: Information for the study participants and parents Kiswahili versicon: Invitation for parents, addressed to Local Leaders (“Balozi”):

Ndugu balozi,

Mimi naitwa Almuth Dinkela. Mimi ni daktari mwanafunzi, nimekuja Ifakara kwa niaba ya kituo cha utafiti wa magonjwa katika nchi za joto cha uswisi, kilichopo Basel uswisi. Kwa hapa Ifakara nafanya kazi kituo cha utafiti wa magonjwa ya binadamu (IHRDC) ikiwa ni utafiti katika kufanikisha shahada yangu ya udaktari. Ninaangalia magonjwa mawili ya ngozi ambayo yameenea sana katika eneo hili, nayo ni mapunye/matangatanga na upele. Kwa kushirikiana na mtaalamu wa magonjwa ya ngozi toka hospitali ya mtakatifu Francis, Dr M. Mbata, na Charles Maswi na Julia Ferie, tumewapima watoto wote wanaosoma shule ya msingi Lihami ili kuona ni ugonjwa gani wa ngozi wanooumwa. Tumewatambua wale wenye mapunye/matangatanga na upele ambao ndio watakaoshiriki kwenye utafiti wangu.

Jumanne tarehe 13/05/2003 kutakuwa na mkutano Lihami shuleni. Tunapenda kuwaalika wazazi wa watoto hawa kuhudhuria mkutanoni saa ______. Wazazi watapewa fursa yakuuliza maswali yanayohusiana na utafiti huu, na tatawataka ruhusa ili watoto wao waweze kushiriki. Itakuwa ni furaha yetu kubwa kama utawataarifu iliwaweze kuhudhuria mkutanoni.

Tunashukuru sana kwa ushirikiano wako.

Almuth Dinkela

178 Appendix D: Information for the study participants and parents 2. Information for the Parents – provided at school meeting:

2.1 English version:

Dear Parents, My name is Almuth Dinkela, I am a medical student sent to Ifakara by the Swiss Tropical Institute in Basel (Switzerland). I am working for IHRDC here in Ifakara in order to do my Medical Doctor research project in Kilombero District. I am interested in two skin diseases that are a very common problem in this area: Ringworm and scabies. Together with the specialist of dermatology at St. Francis Designated District Hospital, SFDDH, Dr. M. Mbata, Dr. Charles Maswi and Julia Ferié, I examined all the children attending Lihami Primary School. We found out that your child______is suffering from Ringworm/Scabies. Therefore he/she has been identified to take part in my study. What I am trying to find out: I would like to find out if regular body washing with soap can cure and prevent Fungal infetions/Scabies. There are two kinds of soap that I will be using. I would like to find out which of the two soap its more effective. One is a normal soap for washing the body, the other one contains an additional substance, called Triclosan. This drug has been used with great success in industrialised countries for more than 25 years to prevent and treat many skin diseases. The soap will contain it in the same concentration (1%) as soaps that are sold in the United States of America and Europe. What will happen during the study: The study will last for 2 months. During these 2 months your child will receive its soap for free. This soap has been produced in Switzerland for this study. Until the end of the 2 months you and I will not know which type of soap he/she has received because the two types of soap look the same. I will visit the school every week and distribute new soap bars. Your child can only receive a new bar, if he/she brings the remaining piece of the previous one back to school. If your child does not use the soap at least once per day, he/she will not receive any more bars. I will examine your child again after one month and after two months. If the skin disease of your child could not be cured by the soap, your child will receive free treatment of the skin disease at SFDDH. How your child has to use the soap: Your child has to use the soap every day. It must be used for washing the body only. All the body parts should be covered with foam, especially the parts where the skin is affected by the skin disease. The foam should be rinsed off with clear water afterwards. Eye contact with the soap or the foam should be avoided to prevent irritation. What is important for the child’s well-being: • Triclosan has never had any severe or toxic side effects • If for some reason your child does not tolerate the soap, he/she does not have to use it any longer and will be referred to SFDDH for treatment .Agreeing to participate in this study: It is entirely your choice whether you let the child participate in this study or not. A decision not to let him/her participate will not affect your rights to treatment at SFDDH. We will respect your decision. If you choose to let your child participate in the study, all the information collected in this study will remain confidential and will be used for research purposes only. Whenever you have questions about the study please contact any of the persons mentioned above or Dr. Boniphace Idindili from IHRDC.´ Thank you for your time and co-operation! 179 Appendix D: Information for the study participants and parents 2.2 Kiswahili version:

Taarifa kwa wazazi

Wazazi wapendwa, Mimi naitwa Almuth Dinkela. Mimi ni daktari mwanafunzi, nimekuja Ifakara kwa niaba ya kituo cha utafiti wa magonjwa katika nchi za joto cha uswisi, kilichopo Basel uswisi.Kwa hapa Ifakara nafanya kazi kituo cha utafiti wa magonjwa ya binadamu (IHRDC) ikiwa ni utafiti katika kufanikisha shahada yangu ya udaktari. Ninaangalia magonjwa mawili ya ngoziambayo yameenea sana ktika eneo hili, nayo ni mapunye/matangatanga na upele. Kwa kushirikiana na mtaalamu wa magonjwa ya ngozi toka hospitali ya mtakatifu Francis, Dr M. Mbata, Charles Maswi na Julia Ferie , tumewapima watoto wote wanaosoma shule ya msingi Lihami. Tumegundua kuwa watoto ______wanaumwa upele. Kwa hiyo atakuwa mshiriki katika mradi huu. Ninachokiangalia: Ninajaribu kuangalia kama kuoga mwili kwa sabuni wakati wote kunaweza kutibu na kukinga upele. Kuna aina mbili za sabuni nitakazotumia. Ninataka kuangalia ni aina ipi ya sabuni kati ya hizo inasaidia zaidi. Ya kwamza ni sabuni ya kawaida ya kuogea, na nyingine ni sabuni iliyoongezwa dawa maalum inayoitwa Triclosan. Dawa hii imekuwa ikitumiwa kwa mafanikio makubwa katika nchi zilizoendelea kwa zaidi ya miaka 25 kutibu na kukinga magonjwa ya ngozi. Sabuni hii ina kiwango sawa cha 1%, kama sabuni zinazotumika marekani na ulaya. Kitakachofanyika katika utafiti huu: Utafiti huu utadumu kwa miezi miwili. Katika miezi hiyo watoto watapewa sabuni bure. Sabuni hizi zimetoka Uswiwi kwa ajili ya utafiti huu. Mwishoni mwa miezi hii miwili wewe na mimi hatutakuwa tukijua mtoto amepata aina ipi ya sabuni kwa maana sabuni zote mbili zinafanana. Nitatembelea shuleni kila wiki na kugawa sabuni mpya. Mtoto atapata sabuni mpya pale atakapoleta kipande cha sabuni iliyoisha shuleni. Kama mtoto atakuwa hatumii sabuni angalau mara moja kila siku , hatapewa sabuni nyingine. Nitampima tena mtoto baada ya mwezi mmoja na miwili. Kama uganjwa wake wa ngozi haujapona kwa sabuni, atatibiwa ugonjwa huo hospitalini St Francis bure. Namna mtoto atakavyotumia sabuni: Mtoto antakiwa kutumia sabuni yake kila siku. Itatumika kwa kuogea tu. Mwili mzima unatakiwa kupakwa sabuni na zaidi sehemu yenye ugonjwa wa ngozi. Povu la sabuni lapashwa kuondolewa kwa kuoga maji safi. Inabidi kujihadhari povu la sabuni lisiingie machoni ili kuepuka kuwashwa.

Mambo muhimu katika afya ya mtoto. • Triclosan hijawahi kuonyesha madhara yoyote. • Kama kwa sababu yoyote ile mtoto akiwa hapatani na sabuni hiyo anatakiwa kuiacha na atapelekwa hospitalini kwa matibabu.

Kukubali kushiriki kwenye utafiti: Una hiari ya kumruhusu mtoto kushiriki katika utafiti huu au la. Uamuzi wa kutomruhusu kushiriki kwenye utafiti huu hautamnyima haki yake ya kutibiwa hospitalini. Kama utamruhusu mtoto kushiriki kwenye utafiti huu, habari zote zitakazopatikana kwenye utafiti huu zitabaki kuwa siri na zitatumika kwa ajili ya utafiti tu. Wakati wowote ukiwa na maswaliyahusuyo utafiti huu wasiliana na Dr. Boniface Idindili kutoka IHRDC.

Tunashukuru kwa ushirikiano wako! Almuth Dinkela 180 Appendix D: Information for the study participants and parents 3. Information for the Parents – about the skin diseases: Handouts for parents 3.1.1 Ringworm: English version:

Your child has been diagnosed to suffer from:

Ringworm:

This skin disease appears as small rings of raised skin. Ringworm itches. It is most often: • on the head • between the toes • in the groin

CAUSES: Ringworm is caused by a kind of germ called a fungus. Ringworm is not caused by a worm.

The disease spreads by touching or other contacts with the infected skin. Ringworm can spread if a healthy person • uses the brush, comb or used clothing of an infected person, or sleeps in a bed with an infected person. Ringworm • on the head makes patches of hair come out and the skin can itch • between the toes can cause much itching

TREATMENT: Washing helps stop the disease: • wash itchy places with soap and water and dry the places very well • wash the head with soap especially thoroughly and dry well after washing • wash between the toes with soap and dry well after washing • wash in the groins with soap and dry well after washing

But most important is cleanliness: Wear clean clothes – dirty socks and shoes help the disease to spread!

PREVENTION: Ringworm and other fungus infection spread quickly: • do not let a person with fungus infection sleep with others • do not use a comb or clothing of a person who has fungus infection unless the comb or clothing are washed very well

181 Appendix D: Information for the study participants and parents 3.1.2 Kiswahili version:

Habari kuhusu magonjwa ya ngozi kwa washiriki wa mafunzo:

Mtoto wako amebainika anaumwa na:

Mapunye na Matangatanga:

Mabadiliko haya wa ngozi yanatokeza kama kiduara kwenye ngozi ambayo imevimba. Mapunye inawasha sana na inatokeza sana:

• kichwani • katikati ya vidole vya miguu • kinenani

SABABU: Mapunye husababishwa na aina ya vijidudu ambavyo vinaitwa kuvu/fungus. Mapunye hayasababishwi na minyoo.

Ugonjwa husambaa kwa kugusana au kwa mashirikiano mengine na ngozi ambayo imeambukizwa. Mapunye yanaweza kusambaa ikiwa mtu mwenye afya • anatumia kitana au anatumia nguo za mtu ambaye ameambukizwa, au analala kitandani na mtu ambaye ameambukizwa Mapunye • kichwani hufanya mabaka ya nywele ambazo zimetoka na ngozi inaweza kuwasha • katikati ya vidole vya miguu huweza kusababisha kuwasha kwingi

MATIBABU: Kuosha kunasaidia kuzuia ugonjwa: • osha sehemu zenye kuwasha kwa sabuni na maji. Halafu safisha sehemu ziwe kavu sana • osha kichwa kizima kwa sabuni, halafu safisha kiwe kikavu • osha baina ya vidole vya miguu kwa sabuni, halafu safisha viwe vikavu • osha katika kinena kwa sabuni, halafu safisha kiwe kavu

Lakini muhimu zaidi ni usafi: Vaa nguo safi – soksi chafu na viatu husaidia ugonjwa kusambaa!

KUJIKINGA: Mapunye na unyono husambaa haraka: • Usimruhusu mtu mwenye maambukizo ya mapunye kulala na wengine • Usitumie kitana au nguo za mtu mwenye maambukizo ya mapunye mpaka kitana na nguo kioshwe vizuri 182 Appendix D: Information for the study participants and parents 3.2.1 Scabies: English version: Your child has been diagnosed to suffer from:

Scabies:

This itchy skin disease appears as very small irritation or marks. It can be all over the body, but appears most often: • between the fingers • on the wrists • around the waist • on the genitals • on the head Children often get this skin disease.

CAUSES: Scabies is caused by little insects under the skin, as these little germs (insects) are making their path under the skin, this causes itching on the skin and the person to scratch. It spreads by touching the diseased skin of a person with scabies, or by wearing clothes to sleeping in bedding used a person with scabies.

TREATMENT: Hard washing (scrubbing) with soap and water will cure this skin problem. • rub the skin hard with soap where there are marks and lumps • use a brush or a maize comb • do this every morning and evening • cut the child’s finger nails short • wash the clothes and bedding in hot water and hang it on a clothes line into the sun • wear every day clean clothing

NOTE: if the sores have a thick white yellow liquid in them, they are infected. Scabies, infected very badly, needs to be treated in the hospital.

PREVENTION: • keep clean • wash the whole body with soap and water every day • wear clean clothing • wash the bed sheets many times and and hang them on a clothes line into the sun Scabies can be prevented by cleanliness! 183 Appendix D: Information for the study participants and parents 3.2.2 Kiswahili version:

Habari kuhusu magonjwa ya ngozi kwa washiriki wa mafunzo:

Mtoto wako amebainika anaumwa na:

Upele:

Mabadiliko haya ya vipele vya ngozi yanatokeza kama kiwasho au alama ndogo. Vinaweza kuwa mwili mzima, lakini hutokeza mara nyingi: • Katikati ya vidole (between the fingers) • Kwenye viwiko (on the wrists) • Karibu na kiumo (around the waist) • Katika sehemu za siri (on the genitals) • Kichwani (on the head) Watoto mara nyingi hupata ugonjwa huu wa ngozi.

SABABU: Vipele husababishwa na vijidudu vidogo chini ya ngozi. Ilivyokuwa vijidudu hivi hufanya njia chini ya ngozi, vinasababisha kuwasha na mtu hujikuna. Husambaa kwa kugusa ngozi ya mgonjwa wa vipele au kwa kuvaa nguo na kulalia sehemu ambazo zilitumiwa na mgonjwa wa vipele.

MATIBABU: Kusugua kwa sabuni na maji kutibu matatizo ya ngozi. • Sugua ngozi sana kwa sabuni sehemu zenye alama au • Tumia brashi au kibunzi cha muhindi • Fanya hivi kila asubuhi na jioni • Kata kucha ya ziwe fupi • Fua nguo na mashuka kwa maji ya moto halafu anika kwenye kamba juani • Vaa nguo safi kila siku! Angalia: Ikiwa vidonda vina ute wa majimaji yenye rangi ya manjano laini, kwa hivyo vidonda hivyo vimeambukizwa. Vipele ambayo vimeambukizwa sana, vinahitaji matibabu hospitalini. KUJIKINGA: • Kuwa msafi • Oga mwili mzima kwa sabuni na maji kila siku • Vaa nguo safi • Fua mashuka mara nyingi na anika kwenye kamba juani Upele unaweza kuzuwiwa kwa kuwa msafi! 184 Appendix D: Information for the study participants and parents 4. Treatment Cards:

4.1 Distributed to children at first soap distribution:

IHRDC BOX 53 Ifakara

This is to certify that the holder of this document

NAME: ______

ID-Number: ______has the right to receive free medical treatment at St. Francis Designated District Hospital for the skin disease mentioned below

• Tinea capitis • Tinea corporis • Tinea pedis • Tinea versicolor • Scabies

1) if the holder of this document has not been cured of these skin diseases after the Triclosan- Soap study has officially been declared to be finished or if he/she cannot take part in the study any longer because of severe side effects

2) if the holder of this document shows any signs of intolerance towards the soap and requires immediate treatment of these side effects

All diagnoses have to be confirmed by Dr. M. Mbata before the treatment can be provided.

This sheet has to be presented to prove the right to access free treatment for the conditions mentioned above.

Dr. M. Mbata

Charles Maswi

Almuth Dinkela

Julia Ferié

185 Appendix D: Information for the study participants and parents 4.2 Distributed to children after the trial to receive free treatment at SFDDH pharmacy: St. Francis Hospital

KADI YA DAWA: PROJECT: TRICLOSAN SOAP STUDY

Issued on:

Valid until:

Name of the patient: ______

ID-Number:

Age: ____ Sex: ____

Diagnoses: 1)______

______

______

Prescription: 1)______

______

______

Signature:

Please collect and store this card in the blue folder labelled “TRICLOSAN SOAP STUDY” at the SFDDH Dispensary after handing out the drug.

THANK YOU!

186 Appendix D: Information for the study participants and parents 5. ID card: Distributed to children at first soap distribution:

Name Date Present Soap returned

ID Age Sex Age Soap letter Serial N° Comments: