RESEARCH

 

Rice vinegar removes Candida albicans from denture acrylic resins

 

 

T Garach^I; S Shangase^II; M Molaudzi^III; J Molepo^IV

^IBChD, MSc (Dent), Dentist/Lecturer, Department of Oral Medicine and Periodontology, School of Oral Health Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. ORCID Number: 0000-0002-5577-579X
^IIBDS, MDent, Head of Department, Department of Oral Medicine and Periodontology, School of Oral Health Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. ORCID Number: 0000-0003-0234-8089
^IIIBSc (Hons), MSc, Lecturer, Department of Oral Biological Sciences, School of Oral Health Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. ORCID Number: 0000-0002-9000-5715
^IVBSc, BSc (Hons), Head of Department, Department of Oral Biological Sciences, School of Oral Health Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. ORCID Number: 0000-0002-5547-097X

Correspondence

 

 

---

ABSTRACT

INTRODUCTION: Denture stomatitis, mainly caused by Candida albicans, often affect denture wearers. To manage these patients, denture hygiene is of utmost importance. There is a need for low cost, easily accessible denture disinfectants
AIMS: To investigate the efficacy of rice vinegar and other disinfecting solutions in removing C. albicans from acrylic resins
MATERIALS AND METHODS: Hundred and eighty acrylic resin plates were contaminated with C. albicans strains and divided into Ave groups. These were immersed in apple cider vinegar (ACV), white wine vinegar (WWV), rice vinegar (RV), chlorhexidine (CHX), and sterile distilled H2O (control). The plates were incubated at room temperature for 30 minutes, 1 hour and 8 hours. Candida removing ability of the disinfecting solutions was evaluated, and data was analyzed using two-way ANOVA with Tukey post-test. Significance level of p<0.05 was used.
RESULTS: RV, ACV, WWV and CHX showed the highest efficacy (100%) in removing both C. albicans strains at 8 hours (p>0.05). CHX was the most effective disinfectant in removing both C. albicans strains at 30 minutes, 1 hour, and 8 hours (99%-100%).
CONCLUSION: RV was as effective as ACV, WWV and CHX in removing C. albicans from acrylic plates at 8 hours.

Keywords: Rice vinegar. Denture acrylic resin. Candida albicans. Disinfection.

---

 

 

INTRODUCTION

According to the World Health Organization, globally approximately 30% of people aged between 65-74 years are edentulous,¹ with many replacing lost teeth with dentures. Denture hygiene remains the mainstay preventive measure in the management of denture-wearing patients.²

Denture stomatitis, a mucous membrane inflammation, occurs on the denture-bearing mucosa of denture wearers. It is a common finding amongst denture wearers (15-70%), with women commonly affected.^3,4 Several factors, including patient age, age of denture, oral hygiene, diet, smoking, denture trauma, continuous denture wearing, salivary flow, denture base material, cellular immunity, pH of the denture plaque, and oral microorganisms contribute to the onset of the disease.^4-8C. albicans is reported as the primary causative agent of the disease.^9,10

Denture base acrylic resin is a significant contributing factor to the oral colonization of the denture by C. albicans. Dentures have rough Atting surfaces, which are conducive for adherence of Candida species and formation of the biofilm.¹¹ In order to control this colonization and prevent Candida infections, the removal of these organisms from dentures is imperative. Antifungal agents are used for treatment of Candida associated denture stomatitis. However, if the dentures are not properly cleaned and disinfected, denture stomatitis recurs when treatment is ceased.¹² Whilst more people have access to dental care, thus retaining their natural teeth for longer, most elderly people continue to lose their teeth, rendering them prone to denture stomatitis.¹³

Chlorhexidine gluconate (2%) antiseptic mouthwash has been used as a soaking solution to suppress adhesion of Candida to acrylic dentures.¹⁴ However, the use of chlorhexidine leads to a brown discoloration of the denture, and has a bitter taste, which may reduce compliance.¹⁵

Although previous studies have tested the removal of C. albicans from a denture base using acetic acid/vinegar,¹⁶²⁰ no South African study has tested and compared the effect of different types of vinegars on the removal of C. albicans from acrylic resin bases over different time periods. In addition, there are no documented studies on the use of rice vinegar (RV) as a denture cleanser.

Several denture cleansers are readily available on the market; however, the high cost leads to less than 60% of denture wearers using commercially available cleansers.¹⁶ Therefore there is a need for low cost, easily accessible denture disinfectants. Vinegar is cheap and readily available in South Africa. The objective of the study was to investigate the efficacy of RV and other disinfecting solutions in the removal of C. albicans from denture acrylic resin.

 

MATERIALS AND METHODS

Denture acrylic resin plates and disinfecting solutions

One hundred and ninety-two denture acrylic resin plates measuring 10x10x3mm were prepared according to the manufacturer's instructions (Confi-Dent agencies, Johannesburg, South Africa). The plates were kept in a flask containing sterile normal saline, and sterilized in an autoclave at 121°C/15 min.

The vinegars tested were 6% white wine vinegar (WWV), 5.5% RV, and 5% apple cider vinegar (ACV), all obtained from Instant Trading Co. (Pty) Ltd, Durban, South Africa. Chlorhexidine (CHX) (0.12%; Sunstar Americas, Inc, Ontario, Canada) was included as a positive control and sterile distilled water as a negative control.

C. albicans biofilm formation on denture acrylic resin plates

This study was conducted in the Department of Oral Biological Sciences, University of the Witwatersrand. C. albicans strains used in the study were the ATCC 90028 (National Health Laboratory Services (Johannesburg, South Africa), and a clinical C. albicans isolate. This isolate was obtained from an HIV positive patient (hereafter referred as clinical C. albicans strain) from a previous study and was stored in a -80°C freezer in the Department of Oral Biological Sciences. Ethical clearance to conduct the study was obtained from the Human Research Ethics Committee (Certificate No: W-CJ-150205-3).

For biofilm formation, a method described by Jafari, et al.¹⁷ (2012) was followed, with slight modifications. The two Candida strains were grown on Sabouraud's Dextrose Agar (SDA) plates at 37°C for 48 hours. Candida suspensions were prepared by dissolving colonies in 20ml of sterile normal saline and turbidity adjusted to 0.5 Mc-Farland standard (Difco Laboratories, Detroit, MI, USA), equivalent to 1.5 x 108 Candida cells per milliliter. Ninety sterile denture acrylic resin plates were contaminated with the C. albicans ATCC 90028 strain, while 90 were contaminated with a clinical C. albicans strain. This was done by immersing the plates in 3 ml sterile Sabouraud's Broth in test tubes and adding 200 of Candida suspension to each tube. The tubes were incubated at 37°C in a shaking incubator (100 rpm) for 48 hours.

Disinfection of denture acrylic resin plates

The 90 denture acrylic resin plates contaminated with the Candida ATCC 90028 strain and 90 contaminated with a clinical C. albicans strain were randomly divided into Ave groups of 18 each. Group 1 plates were immersed in 20ml of 5% ACV, group 2 in 20ml of 6% WWV, group 3 in 20ml of 5.5% RV, group 4 in 20ml of 0.12% CHX and group 5 in 20ml of distilled water (H₂O; control).

The plates from each group were all incubated at room temperature and after 30 minutes, 6 plates were removed from each group and processed to determine viable Candida cells (colony forming units; CFUs). The same procedure was repeated after 1 hour and after 8 hours. Six acrylic plates contaminated with Candida ATCC 90028 strain and six contaminated with a clinical strain, but not exposed to any of the disinfecting solutions were included as controls.

Cell viability

To determine Candida cell viability, the denture acrylic resin plates were aseptically suspended in 20ml of sterile saline and vortexed for microbial cell detachment. Serial dilutions were prepared from this suspension by mixing 0.1 ml of each suspension with 0.9ml of sterile phosphate buffered saline (1:10), which was further diluted by adding 0.1ml of this suspension into another 0.9ml of PBS (1:100). One hundred microliters of these dilutions were spread in duplicate on SDA plates and incubated for 48 hours at 37°C.

These tests were repeated three times for each of the two C. albicans strains for reproducibility of results. The Candida removing ability of ACV, WWV, RV, CHX, and H2O was calculated by using the formula: (CFU_b - CFU_a) ÷ CFU_b x100, where CFU_b=Number of CFUs before dis-infection and CFU_a =Number of CFUs after disinfection.

Statistical analysis

The data was analyzed using GraphPad Prism (Graph Pad Software Inc., La Jolla, CA). Two-way-ANOVA with Tuk-ey's post-test was used for comparison of the Candida removing ability of ACV, RV, WWV, CHX, and H₂O at different times. A significance level of less than 0.05 (p<0.05) was used.

 

RESULTS

The removing ability of different disinfecting solutions at different time intervals are illustrated in Table 1 (ATCC strain) and Table 2 (Clinical strain). At 8 hours, ACV, WWV, RV, and CHX showed the highest efficacy (100%) in removing ATCC 90028 and clinical C. albicans strains from acrylic resins. CHX was the most effective disinfectant in removing both strains of C. albicans at 30 minutes, 1 hour, and 8 hours (99% - 100%).

For ATCC at 30 minutes and 1 hr, WWV was the next most effective after CHX (92 and 97% respectively). For clinical strain, at 30 minutes and 1 hr, WWV was the next most effective after CHX (82 and 73% respectively).

Sterile water could not completely remove either of the C. albicans strains at 30 minutes, 1 hour and 8 hours.

For the ATCC 90028 strain, there was no statistically significant difference between ACV, WWC, RV and CHX at 30 minutes, 1 hour and 8 hours (p>0.1) in removing this strain from denture acrylic resin plates. There was statistically significant difference between ACV, WWV, RV, CHX when compared to water at 8 hours (p=0.0002).

There was no statistically significant difference betweenACV, WWC, RV and CHX at 30 minutes, 1 hour and 8 hours in removing the clinical strain from denture acrylic resin plates (p>0.3). However, there was statistically significant difference between ACV, WWC, RV, CHX, and water at 8 hours (p<0.05).

 

DISCUSSION

Denture stomatitis is caused mainly by the adherence and colonization of C. albicans on the fitting surface of the denture. In order to control this infection, it is important to remove this organism by disinfecting dentures, and there is a need for low cost and easily accessible denture disinfectants.

Vinegar is cheap, has low toxicity²¹ and is readily available in South Africa. It is a sour liquid comprised mainly of acetic acid, which is prepared by the fermentation of alcoholic beverages, mainly white and red wines.²² C. albicans is the main causative agent of denture stomatitis, and thus using this solution in the disinfection of dentures might prove beneficial both therapeutically and as a preventive measure.²³

This study aimed to investigate the efficacy of RV and other disinfecting solutions in the removal of C. albicans from denture acrylic resin. In this study, RV was found to be as effective as CHX, ACV and WWV in removing C. albicans from acrylic denture resin at 8 hours.

Previous studies have tested the removal of C. albicans from a denture base using acetic acid/vinegar.^{16,17120,23,24} However, there are no documented studies on the use of RV as a denture cleanser. In this study, RV (5.5%), WWV (6%) and ACV (5%) removed 100% of C. albicans ATCC 90028 and clinical C. albicans strains from denture acrylic resin plates at 8 hours but did not completely remove these strains at 30 minutes and 1 hour. This shows that longer soaking periods are required to remove C. albicans from acrylic dentures using these vinegars.

There are no documented studies where the efficacy of RV against C. albicans on denture acrylic resin plates was tested. To the best of our knowledge, this is the first documented study where RV was tested and found to be effective as a denture cleanser. However, Choi, et al.²⁵ (2015) reported that dark RV has strong antioxidant and antimicrobial activity, and was more effective than carbe-nicillin (50 pg/ml) and tetracycline (50 pg/ml) in inhibiting growth of bacterial species.

Mota, et al.²⁰ (2014) reported 4% ACV to be fungicidal between 30-180 minutes, in contrast to the current study. They also found that ACV prevented C. albicans from adhering to the acrylic resin, and suggested that ACV could be used as an alternative therapeutic agent in denture stomatitis patients.

There are very few documented studies on the effectiveness of ACV in removing C. albicans from denture materials. However, using gel diffusion methods, ACV was effective against C. albicans after 24 hours' exposure.^{26, 28}

A study by Jafari, et al.¹⁷ (2012) showed that 5% and 10% white vinegar removed 99% and 100% of the C. albi-cans attached to acrylic plates respectively after 8 hours, confirming the efficacy of white vinegar, in agreement with the current study. Another study found that using a higher concentration (125ml/200ml) of vinegar was effective in removing C. albicans from acrylic plates in a shorter time (1 hour), while a lower concentration (75ml/ 200ml) took longer to remove C. albicans (8 hours).²⁹ Their results suggest that increasing vinegar concentration would shorten the efficacy time.

ln contrast to our results, Pinto, et al.¹⁶ (2008) reported that 10% vinegar could not eliminate C. albicans from dentures and saliva of denture wearers after 45 days of overnight exposure, but reduced the amount (cfu/mL) of Candida species in the saliva.

Vinegar was ineffective in reducing C. albicans colonization on denture materials after 10 minutes' exposure,^30,31 whilst 50% vinegar diluted in water was less effective than alkaline peroxide in removing C. albicans biofilms after 30 minutes.¹⁸ Kumar, et al.³² (2012) reported 4% and 50% vinegar to be less effective in removing C. albicans from acrylic resin after 8 hours exposure compared to commercial solutions, which is also in contrast to our study findings. Higher concentrations of vinegar (100%) with shorter exposure times were effective in removing C. albicans from dentures.^23,24,33,34

The results of the current study show that 5.5% RV, 6% WWV, and 5% ACV are effective (100% efficacy) and can be used as denture cleansers, allowing denture wearers to soak their dentures overnight, ready for use the next day. This will be beneficial for denture wearers, as all these vinegars are cheap and are available in every household around the world.

In the current study, 0.12% CHX was the most effective disinfectant in removing both tested C. albicans strains (99 - 100%) at 30 minutes, 1 hour, and 8 hours. This is not surprising as CHX is considered the gold standard agent for controlling dental plaque effectively.³⁵

Water could not completely remove either C. albicans strains at 30 minutes, 1 hour, and 8 hours. These results are in agreement with previous studies, where water was less effective in removing C. albicans from dentures^36-38 confirming that water alone is not suitable for disinfection of dentures. The ability of water to reduce C. albicans from acrylic resin can be explained by that water is hypotonic as compared to the cellular contents of microorganisms, and this lead to osmotic pressure, causing the flow of water into the cells and disrupting the micro-organisms.³⁹

The limitation of this study is that it was an in vitro study. Further studies are needed to confirm the efficacy of RV in vivo. ln addition, the long term effects of RV on the denture acrylic requires further study to assess its effect on the mechanical strength of denture acrylic over time, amongst others. Further studies are needed to determine the duration of time or the number of times the same RV solution could be used, before it loses its effectiveness. Such a study will further confirm the cost-effectiveness of vinegar as a disinfectant.

 

CONCLUSION

Within the confines of this study, 5.5% RV was as effective as 5% ACV, 6% WWV and 0.12% CHX in removing C. albicans from dentures after overnight soaking. This is the first study where RV was tested and found effective as a denture cleanser.

 

Acknowledgements

We express our appreciation to the University of the Wit-watersrand for funding this research (grant number: 001 40184511035121105 RM ORMP015).

 

References

1. World Health Organization. Fact sheet No 318. April 2012. http://www.WHO.int [Accessed 4 Sept. 2020].         [ Links ]

2. Douglas L. Candida biofilms and their role in infection. Trends Microbiol. 2003; 11: 30-6.         [ Links ]

3. Arendorf TM, Walker DM. Denture stomatitis: a review. J Oral Rehab. 1987; 14: 217-27.         [ Links ]

4. Gendreau L, Loewy Z. Epidemiology and Etiology of Denture Stomatitis. J Prosthodont. 2011; 20: 251-60.         [ Links ]

5. Shulman J, Rivera-Hidalgo F, Beach M. Risk factors associated with denture stomatitis in the United States. J Oral Path Med. 2005; 34: 340-6.         [ Links ]

6. Coco B, Bagg J, Cross L, Jose A, Cross J, Ramage G. Mixed Candida albicans and Candida glabrata populations associated with the pathogenesis of denture stomatitis. Oral Microbiol lmmun. 2008; 23: 377-83.         [ Links ]

7. Gasparoto T, Vieira N, Porto V, Campanelli A, Lara V. Ageing exacerbates damage of systemic and salivary neutrophils from patients presenting Candida-related denture stomatitis. lmmun Ageing. 2009; 6: 3.         [ Links ]

8. Skupien JA, Valentini F, Boscato N, Pereira-Cenci T. Prevention and treatment of Candida colonization on denture liners: a systematic review. J Prosthet Dent. 2013; 110: 356-62.         [ Links ]

9. Ramage G, Tomsett K, Wickes BL, Lopez-Ribot JL, Redding SW. Denture stomatitis: a role for Candida biofilms. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004; 98: 53-9.         [ Links ]

10. Jackson S, Coulthwaite L, Loewy Z, Scallan A, Verran J. Biofilm development by blastospores and hyphae of Candida albicans on abraded denture acrylic resin surfaces. J Prosthet Dent. 2014; 112: 988-93.         [ Links ]

11. Wady A, Machado A, Zucolotto V, Zamperini C, Berni E, Vergani C. Evaluation of Candida albicans adhesion and bio-film formation on a denture base acrylic resin containing silver nanoparticles. J Appl Microbiol. 2012; 112: 1163-72.         [ Links ]

12. Glass RT, Bullard JW, Conrad RS, Blewett EW. Evaluation of the sanitization effectiveness of a denture-cleaning product on dentures contaminated with known microbial flora. An in vitro study. Quintessence Int. 2004; 35: 194-9.         [ Links ]

13. Petersen P, Yamamoto T. Improving the oral health of older people: the approach of the WHO Global Oral Health Programme. Comm Dent Oral Epidemiol. 2005; 33: 81-92.         [ Links ]

14. McCourtie J, MacFarlane T, Samaranayake L. A comparison of the effects of chlorhexidine gluconate, amphotericin B and nystatin on the adherence of Candida species to denture acrylic. J Antimicrob Chemother. 1986; 17: 575-83.         [ Links ]

15. Flötra L, Gjermo P, Rölla G, Waerhaug J. Side effects of chlor-hexidine mouth washes. Eur J Oral Sci. 1971; 79: 119-25.         [ Links ]

16. Pinto T, Neves A, Leão M, Jorge A. Vinegar as an antimicrobial agent for control of Candidaspp.in complete denture wearers. J Appl Oral Sci 2008; 16: 385-90.         [ Links ]

17. Jafari A, Falah-Tafti A, Lotfi-Kamran M, Zahraeii A, Kazemi A. Vinegar as a removing agent of Candida albicans from acrylic resin plates. Jundishapur J Microbiol. 2012; 5: 388-92.         [ Links ]

18. Yadav V, Garg S, Garg R, Mittal S, Yadav R. Effectiveness of different denture cleansing methods on removal of biofilms formed in vivo. J Craniomaxillofac Surg. 2013; 2: 22.         [ Links ]

19. Gouveia CL, Freire ICM, Figueiredo RDA, Almeida LDFDD, Cavalcanti YW, Padilha WWN. Antifungal activity of components used for decontamination of dental prostheses on the growth of Candida albicans. Rev Odontol UNESP. 2014; 43: 137-42.         [ Links ]

20. Mota A, de Castro R, de Araújo Oliveira J, de Oliveira Lima E. Antifungal activity of apple cider vinegar on candida species involved in denture stomatitis. J Prosthodont. 2014; 24: 296-302.         [ Links ]

21. Makino SI, Cheun H-I, Tabuchi H, Shirahata T. Antibacterial activity of chaff vinegar and its practical application. J Vet Med Sci. 2000; 62: 893-5.         [ Links ]

22. Lastauskiene E, Zinkeviciene A, Girkontaite I, Kaunietis A, Kvedariene V. Formic Acid and Acetic Acid Induce a Programmed Cell Death in Pathogenic Candida Species. Curr Microbiol. 2014; 69: 303-10.         [ Links ]

23. da Silva FC, Kimpara ET, Mancini MN, Balducci I, Jorge AO, Koga-Ito CY. Effectiveness of six different disinfectants on removing Ave microbial species and effects on the topographic characteristics of acrylic resin. J Prosthodont. 2008; 17: 627-33.         [ Links ]

24. Lavanya Kumar C. Study to Assess the Disinfection of Dental Prostheses to Clear Locally Prevalent Microbial Strains. Journal of Evidence Based Medicine and Healthcare 2015; 2: 8304-7.         [ Links ]

25. Choi H, Gwak G, Choi D, Park J, Cheong H. Antimicrobial Efficacy of Fermented Dark Vinegar from Unpolished Rice. Microbiol Biotech Lett. 2015; 43: 97-104.         [ Links ]

26. Hayder BJ, Fatin NA, Rana MK. In vitro assessment of anti-fungal potential of apple cider vinegar and acetic acid versus fluconazole in clinical isolates of otomycosis. Thi-Qar Med J. 2011; 5: 126-33.         [ Links ]

27. Al-Salihi SS, Jumaah IA. Activity of Some Disinfectants, Detergents and Essential Oils on Growth of the yeast Candida albicans. Al-Mustansiriyah J Sci. 2017; 28: 25-34.         [ Links ]

28. Yagnik D, Serafin V, Shah AJ. Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression. Sci Rep. 2018; 8: 1732.         [ Links ]

29. Ali AA, Al Harbi FA. The minimum inhibitory concentration of different candidal disinfecting agents. Saudi J Med Med Sci. 2015; 3: 26.         [ Links ]

30. Buergers R, Rosentritt M, Schneider-Brachert W, Behr M, Handel G, Hahnel S. Efficacy of denture disinfection methods in controlling Candida albicans colonization in vitro. Acta Odontol Scand. 2008; 66: 174-80.         [ Links ]

31. Sousa FACGD, Paradella TC, Koga-Ito CY, Jorge AOC. Effect of sodium bicarbonate on Candida albicans adherence to thermally activated acrylic resin. Braz Oral Res. 2009; 23: 381-5.         [ Links ]

32. Kumar MN, Thippeswamy HM, Swamy KR, Gujjari AK. Efficacy of commercial and household denture cleansers against Candida albicans adherent to acrylic denture base resin: An in vitro study. Indian J Dent Res. 2012; 23: 39.         [ Links ]

33. Yildirim-Bicer AZ, Peker I, Akca G, Celik I. In vitro antifungal evaluation of seven different disinfectants on acrylic resins. BioMed Res Int. 2014; ID 519098. http://dx.doi.org/10.1155/2014/519098 2014.         [ Links ]

34. Hashizume L, Hoscharuk M, Moreira M. Effect of affordable disinfectant solutions on Candida albicans adhered to acrylic resin for dental prosthesis. RGO - Revista Gaúcha de Odontologia. 2015; 63; 309-14.         [ Links ]

35. Jones C. Chlorhexidine: is it still the gold standard?. Peri-odontology 2000. 1987; 15: 55-62.         [ Links ]

36. Stafford GD, Arendorf T, Huggett R. The effect of overnight drying and water immersion on candidal colonization and properties of complete dentures. J Dent. 1986; 14: 52-6.         [ Links ]

37. Duyck J, Vandamme K, Krausch-Hofmann S, Boon L, De Keersmaecker K, Jalon E, et al. Impact of denture cleaning method and overnight storage condition on denture biofilm mass and composition: A cross-over randomized clinical trial. PLoS One. 2016; 11: e0145837.         [ Links ]

38. Lee H, Li C, Chang H, Yang Y, Wu J. Effects of different den-ure cleaning methods to remove Candida albicans from acrylic resin denture based material. J Dent Sci. 2011; 6: 216-20.         [ Links ]

39. Pelczar MJ Jr, Chan EC, Krieg NR eds. Microbiology: Concepts and Applications. Cultivation and growth of microorganisms, 2^nd ed. New York: McGraw-Hill. 1993; 175-98.         [ Links ]

 

 

Correspondence:
Julitha Molepo
Email: julitha.molepo@wits.ac.za

 

 

Author contributions:
1 . Tarana Garach: Study conception and design, acquisition of data, analysis and interpretation of data, drafting of manuscript - 35%
2 . Sindisiwe Shangase: Study conception and design, drafting of manuscript, critical revision - 20%
3 . Mulalo Molaudzi: Study conception and design, acquisition of data, analysis and interpretation of data, drafting of manuscript, critical revision - 25%
4 . Julitha Molepo: Study conception and design, analysis and interpretation of data, drafting of manuscript, critical revision - 20%