INTRODUCTION:

Vulvovaginal candidiasis (VVC) is a multifactorial infectious disease that causes pathologic inflammation in the lower female reproductive system. While VVC is a condition that affects immunocompetent and otherwise healthy women, it is most prevalent during their child-bearing years. Despite the fact that VVC is not fatal, its huge global incidence and severe detrimental impact on quality of life demands additional research¹.

A third of non-pregnant or single women are at risk of contracting it, VVC is significantly higher among women with polygamous husband². Despite breakthroughs in treatment, candidiasis is still a common fungal illness caused primarily by Candida albicans. It can manifest as vulvovaginal candidiasis or thrush, a mucocutaneous candidiasis.

Candida infections are common in infants, immune-compromised persons such as AIDS patients, and people using broad-spectrum medications. It is mostly caused by Candida albicans, with other species such as Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida krusei becoming increasingly isolated³. Candida infection can manifest themselves in a variety of ways. This dimorphic yeast colonizes the cutaneous, gastrointestinal, and reproductive tracts as a commensal. Species of Candida albicans are emerging pathogens that can infect human mucocutaneous surfaces⁴. Opportunistic infections, such as Candida spp., are common in a variety of clinical conditions that produce immunodeficiency. Candida spp., for the most part, acted as an opportunistic infection, attacking human body parts and colonization. Candida albicans infects the mucosal membrane and causes candidiasis, especially in immunocompromised people⁵. Candidiasis can emerge when the equilibrium between human immunity and this opportunistic pathogen is disrupted⁶.

Metal oxide nanoparticles' antimicrobial activities are expressed through the production of reactive oxygen species. In the absence and presence of irradiation, the decline of Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus with Ag was investigated^7-12. The antibacterial effect of ZnO NPs was related to the formation of free radicals on the surface, as well as free radical damage of lipids in the bacterial cell membrane, and cell membrane breakdown. ZnO NPs exhibit antibacterial and antifungal properties, especially against Candida albicans. When zinc oxide nanoparticles are introduced into materials like paints, fabrics, and polymers, they act as antibacterial agents^13-16.

The copper–silver–chitosan nanocomposite, which has had a substantial influence on the growth of C. albicans in the laboratory setting when compared to other nanocomposite analyzed, has had the most antifungal effect of the nanocomposite studied. nanocomposite. Graphic abstract¹⁷. Because of its potential antibacterial agents, silver nanocomposite has sparked a lot of interest, and they're being used in a lot of biological and medical disciplines. Because of its biocompatibility and biodegradability, in addition to its antibacterial and antifungal activity against a wide variety of microbes, Ag-based nanocomposites, such as chitosan-silver nanocomposite, represent an emerging group of bio-nanostructured materials with a mix of properties¹⁸. Silver nanocomposites are implanted Ag NP/polymer nanocomposites are excellent antibacterial. They've been used in food packaging to beat out competitors to enhance the time frame depending on the actual use of the food, as well as the control of toxigenic fungus and plant pathogens infections¹⁹

MATERIALS AND METHODS:

Collection of Samples:

50 samples were collected from patients with infection of vaginitis women were obtained from Al-Hakim and Al-Furat Hospitals in Al-Najaf province. All of the samples were transferred to the Faculty of Science's Laboratory of Mycology for pathogen isolation and diagnosis of yeasts.

Swap Samples:

Samples were taken by sterilizing cotton swabs from inside of the vagina and the cervix, cotton swabs were aseptically dipped in sterile culture tubes containing sterile medium and delivered to the laboratory as soon as feasible. They were then streaked in sabouraud dextrose agar and incubated at 37°C for 24-48 hours.

Yeasts diagnosis:

In sabouraud dextrose agar, morphological aspects of growingin the chrom agar test was used to aid in the diagnosis of candida species based on color colony reverse and pigments, texture, margin, were formed²⁰. To determine the frequency, the following approach was used to calculate isolation, frequency, and visibility:

Number of genus or species fungal isolates

Frequency (%) = --------------------------------------------------- ×100…(1)

Totally of all fungal isolates

Number of samples appeared genus or species

Visibility (%) = --------------------------------------------------- ×100…..(2)

Totally of all fungal isolates

Isolated Yeasts Maintenance:

After purification, the isolates were cultured in vials. S.D.A media were incubated in 37°C for 2 days before being stored in the refrigerator at 4°C for future studies.

Chitosan/Silver Nanocomposites Preparation:

To make the nanocomposites, Ag NPs were added in two different weight percentages (0.5wt%, 1wt%, 1.5wt%) and mixed for 10 minutes using a sonicator. The weight of the NPs that must be added to the Chitosan solution to generate nanocomposites was calculated using the Eq.(3) below.²¹

Where: Wm is the matrix weight, Wp is the Weight of particles , ρ_m is the stands for matrix density, ρ_p is the particle density and Vf is Volume fraction

The nanoparticles were mixed with chitosan to create a nanocomposite solution.

0.5weight % = 50mg nano silver

1 weight % = 100mg nano silver

1.5 weight % = 150mg nano silver

Concentration silver/chitosan nanocomposites= ………………..(4)

The ability of Chitosan-silver nanocomposite to inhibit or reduce yeast growth:

The sabouraud dextrose agar medium was made in 12 flasks divided into four groups, each with three flasks.After sterilizing the flasks in an autoclave and allowing them to cool, the antibiotic Amoxicillin at a concentration of 100/L was added. At 40^oC. Chitosan-silver nanocomposite suspension in concentrations of (50,100,150) ppm were added to the culture media in a 1:00 ratio for each concentration independently.

1ml of different concentrations of Chitosan-silver nanocomposite were added to the Petri dishes, then SDA was poured over it and mixed well with the substance and the dishes were allowed to harden. Then, one by one, the cultivating disk of each yeast culture. At 37°C, plates were incubated for 24-48 hours²². The percentages of inhibition were then computed using the Eq. (5) below.

Percentage inhibition (%) = ……………..(5)

R₁ is the Candida colony's greatest radial growth control treatment.

R₂ is the colony's maximal radial growth plates containing Chitosan-silver nanocomposite for each Candida.

RESULT AND DISCUSSION:

Samples of vagina swabs:

Table (1) shows the appearance of two types of Candida were isolated from the samples of vagina with frequency (44,11) % respectively. Two types of Candida appeared are C.albicans, C. krusei²³.

Table (1) Percentage of visibility and frequency of fungi from vaginitis women.

S. No	Fungi	Visibility (%)	Frequency (%)
1	C.albicans	70	44
2	C. krusei	26	11

Diagnosis of isolated Candida:

Candida spp. had a smooth, shiny, or dry colony texture and matured in 24 to 48 hours. Figure 1 illustrates how to diagnose Candida using chromagar, which is a differential agar for the colonies. C.albicans has light green colonies with smooth edges, but C.krusei has pink colonies with white borders or fuzzy edges ²⁴ who identified the identical features of Candida spp. colonies that formed on chromagar. Chromogenic media are an efficient and fast test for diagnosing candida at the species level.²⁵

Figures (1) A, B Candida spp. after 2 days at 37 Co, colonies on chromagar a-C.albicans , b- C.krusei.

The ability of a Chitosan-silver nanocomposite to stop or slow yeast growth:

The influence of Chitosan-silver nanocomposite on growth inhibition is depicted in (table 2; fig 2,3 ) demonstrating that Chitosan-silver nanocomposite has a high efficiency in inhibiting yeast growth. Candida albicans, by percentage of (54)% in the concentration of 50ppm and 92% in the concentration 100ppm ,While 150ppm of Chitosan-silver nanocomposite causes complete inhibition growth of C.albicans .The inhibition of C.krusei in concentration of 50ppm; with percentage (40)%, Chitosan/silver Nanocomposites cause a The creation of pits in the cell walls and pores in the plasma membrane characterizes damage to the fungal cell walls and cell membrane. This outcome is completed with²⁶.

Table (2) The effects of Chitosan-silver nanocomposite concentrations in average of inhibition percentage of Candida spp. after 2 days.

Concentrations (ppm)	Average of inhibition percentage
Concentrations (ppm)	*Candida Albicans*	*Candida* *kruse*
0	0	0
50	54	40
100	92	80
150	100	100

Figures (2) The effect of Chitosan-silver nanocomposite inhibition proportion of concentrations of C.albicans by combining with the media 37 C⁰ after two day. A = 50 ppm B= control treatment.

Figures (3) TEM (Transmission electron microscopy) of chitosan-silver nanocomposite on C.albicans. A- Yeast without treatment, B- Yeast after 24-hour Chitosan-silver nanocomposite

The morphological features of untreated cells were well preserved, with a distinct cell wall and cytoplasmic membrane. The breadth of the cell wall expands after treatment with Chitosan-silver nanocomposite, this is connected to the discovery that silver/Chitosan nanocomposites damage, fungal cell walls and cell membrane structure, causing pits in cell walls and a tiny hole in plasma membrane.

CONCLUSION:

Vaginitis women are appeared growth of yeast in 40 swab samples from the vagina. Two species of Candida spp. were isolated which of C.albicans and C.Kuseie. Two yeasts are isolated from the patients of vaginitis women, C.albicans more frequent than c.krusi, Chitosan-silver nanocomposite have shown a high efficiency to decrease growth of C.andida albicans and C. krusei.

ACKNOWLEDGMENTS:

I would like to thank Biology Department in Faculty of Science in University of Kufa.

REFERENCES:

1. Willems H. Ahmed S. Liu J. Xu Z. Peters B. Vulvovaginal Candidiasis: A Current Understanding and Burning Questions. Journal of Fungi. 2020; 6(1):27. DOI:10.3390/jof6010027.

2. Abdul-Aziz M. Mahdy M. Abdul-Ghani R. Alhilali N et al . Bacterial vaginosis, vulvovaginal candidiasis and trichomonal vaginitis among reproductive-aged women seeking primary healthcare in Sana’a city, Yemen . Tropical Medicin and Health. BMC Infect Dis. 2019; 19: 879 . doi: 10.1186/s12879-019-4549-3

3. Umme H. Hosakote S. Rudra V. Riyaz O. Atul S. Candidiasis a fungal infection-current challenges and progress in prevention and treatment. Infectious Disorders – Drug Targets, 2015; 15”42-52 . DOI:10.2174/1871526515666150320162036

4. Dabas P. An approach to Etiology, diagnosis and management of different types of candidiasis. Academic Journals. 2013; 4(6):63-74. DOI:10.5897/JYFR2013.0113

5. Brown G. Denning D. Levitz S. Tackling human fungal infections. Science 2012; 336-647. DOI: 10.1126/science.1222236

6. Gonçalves B. Ferreira C. et al. Vulvovaginal candidiasis: Epidemiology, microbiology and risk factors Critical Reviews in Microbiology. 2016=; (42): 27-50. doi.org/10.3109/1040841X.2015.1091805

7. Nair S. Jadhav V. Biosynthesis of Silver nanoparticles and comparing its Antifungal property with Ethanolic extract of Ixora coccinea plant. Asian J. Research Chem. 2020; 13(3): 198-202. DOI: 10.5958/0974-4150.2020.00038.3

8. Ravi L. Kannabiran K. Antifungal potential of green synthesized silver nanoparticles (AgNPS) from the stem bark extract of Kigelia pinnata. Research Journal of Pharmacy and Technology. 2021; 14(4):1842-6. DOI: 10.52711/0974-360X.2021.00326

9. Fathima T. Rajeshkumar S. Nagalingam M. Green Synthesis of Silver Nanoparticles using Symplocos racemosa and its Antifungal Activity against Candida albicans. Research J. Pharm. and Tech. 2021; 14(2):775-778. DOI: 10.5958/0974-360X.2021.00135.9

10. Kumararaja G. Saneesha N, Satheesh S., Shahana V. Abdur Rahim S. Shamna V. Shamna A. Sundaraganapathy R. Gayathri R. Development and Characterization of Silver nanoparticles (AgNPs) using Aqueous leaves broth of Artemisia vulgaris L., and its Anti-fungal activity. Research J. Pharm. and Tech. 2019; 12(10):4822-4826. DOI: 10.5958/0974-360X.2019.00834.5

11. Taha Z. Howar S. Sulaiman G. Isolation and Identification of Penicillium italicum from Iraqi Citrus Lemon Fruits and its Ability Manufacture of Silver Nanoparticles and their Antibacterial and Antifungal activity. Research J. Pharm. and Tech. 2019; 12(3): 1320-1326. DOI: 10.5958/0974-360X.2019.00221.X

12. Naveen Prasad S. Padmesh T. Common Duckweed (Lemna minor) Assisted Green Synthesis of Silver Nanoparticles as Potent Anti-Fungal Nanomaterial. Research J. Pharm. and Tech. 2014; 7(9): 955-958.

13. Baskar G. Chandhuru J. Sheraz K. Praveen A. Mycological Synthesis, Characterization and Antifungal Activity of Zinc Oxide Nanoparticles. Asian J. Pharm. Tech. 2013; 3(4): 142-146.

14. Sahib R. Effects of Zinc Oxide Nanoparticles on the Growth Inhibition of Fungi Isolated From Cream Cosmetics. Research J. Pharm. and Tech 2019; 12(1): 123-128. DOI: 10.5958/0974-360X.2019.00024.6

15. Ananthalakshmi R. Rajarathinam S. Mohamed A. Antioxidant activity of ZnO Nanoparticles synthesized using Luffa acutangula peel extract. Research J. Pharm. and Tech. 2019; 12(4):1569-1572 DOI: 10.5958/0974-360X.2019.00260.9.

16. Upadhyay P. Jain V. Sharma K. Sharma R. Synthesis and Applications of ZnO Nanoparticles in Biomedicine. Research J. Pharm. and Tech. 2020; 13(4):1636-1644. DOI: 10.5958/0974-360X.2020.00297.8

17. Wang L. Wang C. Yang C. Hsieh C. Chen S. Shen C. Wang J. Huang K. Synthesis and anti-fungal effect of silver nanoparticles-Chitosan composite particles. International Journal of Nanomedicine. 2015; 26 (10): 85-96. doi: 10.2147/IJN.S77410

18. Sunita R. Silver Nanoparticles and Its Polymer Nanocomposites-Synthesis, Optimization, Biomedical Usage, and Its Various Applications. Book: Polymer Nanocomposites in Biomedical Engineering. 2019; pp: 331-373.

19. Jung J. Kasi G. Seo J. Development of functional antimicrobial papers using Chitosan/starch-silver nanoparticles. International Journal of Biological Macromolecules. 2018;(112): 530–536. DOI: 10.1016/j.ijbiomac.2018.01.155

20. Moris D. Melhem M. Martins M. Mendes R. Oral Candida spp. colonization in human Immunodefecinciy Virus-Infected individuals. Journal of Venomous Animals and Toxins. 2008; 14(2): p. 231. https://doi.org/10.1590/S1678-91992008000200004

21. Rahimi M. Ahmadi R. Kafil H. Shafiei-Irannejad V. A novel bioactive quaternized Chitosan and its silver-containing nanocomposites as a potent antimicrobial wound dressing Structural and biological properties. Materials Science and Engineering. 2019; 101: 360–369. https://doi.org/10.1016/j.msec.2019.03.092

22. Bhavan P. Rajkumar R. Radhakrishnan S. Seenivasan C. Kannan S. Culture and identification of Candida albicans from vaginal ulcer and separation of enolase on SDS-PAGE. International Journal of Biology. 2010; 2 (1): 84-93. DOI:10.5539/ijb.v2n1p84

23. Gualco L. Debbia E. Bandettini R. Pescetto L. Cavallero A. Ossi M. Schito A. Marchese A. Antifungal resistance in Candida spp. Isolated in Italy between 2002 and 2005 for children and adults. International Journal of Antimicrobial Agents. 2007; 29( 2): 1979-1984.

24. Iyampillia T. Micheal J. Mathai E. Mathews M. Use of Chromagar medium in the differentiation of Candida species is it cost effective in developing countries. Tropical Medicine and Parasitology .2004; 98 :279-282.

25. Baradkar VP, Mathur M, Kumar S. Hichrom Candida agar for identification of Candida species. Indian Journal of Pathology and Microbiology. 2010;53(1):93–94.

26. Tammana M. Development of Chitosan/Graphene oxide/nano-silver Hydrogel for Photocatalytic Dye Degradation and Antibacterial Action. Dissertation at Department of Biotechnology/Thapar University /Patiala 2017.

Received on 29.09.2021 Modified on 05.02.2022

Research J. Pharm. and Tech. 2022; 15(8):3743-3746.

DOI: 10.52711/0974-360X.2022.00627