Molecular Identification, Dimorphism and Virulence of C. albicans

 

Mohsen A. Sayed, Gihad A. Sayed*, Eman Abdullah M. Ali

Botany and Microbiology Department, Faculty of Science, Cairo University, 12613, Giza, Egypt.

*Corresponding Author E-mail: gihadahmed311@gmail.com

 

ABSTRACT:

C. albicans causes human diseases, especially in immune-compromised patients. The current study aimed to identify Candida albicans using different techniques. Dimorphism and virulence behaviour were also studied. A Candida albicans strain was firstly identified by biochemical methods using VITEK 2 Compact automated technique and chromogenically using CHROMagar differential media that differentiate between Candida spp. Based on an enzymatic reaction. Molecular identification using ITS primers was also used to confirm Candida albicans identification. Accession number of the identified C. albicans was obtained as OK104215. The enhancement of dimorphism was studied using RPMI 1640 media (Roswell Park Memorial Institute Medium), while monitoring growth at different time intervals under microscope to investigate dimorphic changes. C. albicans showed its optimum dimorphism after 36-66 hours at 37C. HPLC analysis for the enzyme product S-adenosylmethionine (SAM) was carried out at different time intervals. By increasing time, SAM production increased until optimum production reached after 72h of incubation on RPMI 1640. After that the production of SAM began to decrease.

 

KEYWORDS: Candida albicans, Dimorphism, Virulence, Molecular identification, HPLC.

 

 

 

INTRODUCTION: 

C. albicans causes candidiasis or oral thrush in human, especially in immune-compromised patients1,2. It frequently colonizes skin and mucous membranes; it causes nosocomial bloodstream infections worldwide3. C. albicans is a dimorphic fungus has the ability to change its morphology from single yeast cell to filamentous form4. The morphogenic switch between hyphae and yeast form is associated with different factors, that is environmental (pH, temperature, oxygen and glucose concentration) or biochemical (enzyme activity and protein biosynthesis)5,6. Dimorphism is the key of virulence behavior in C. albicans7. Chromogenic Candida agar is used in identification of Candida8,9.

 

Parka et al.10 used biochemical kits such as API 20 C AUX and Vitek-2C in identification of Candida spp. in addition to molecular identification. Identification of Fungi on DNA bases is gaining more importance11. Many identification systems depend on sequencing of DNA to accurately identify organisms12.

 

However, global system for fungal identification is a need13. Barcoding of DNA is an important method applied for species identification depending on sequences of DNA14. Region of internal transcribed spacer (ITS) is the common sequenced region of DNA used in fungal identification15 and is recommended as a global fungal sequence of barcoding16. Morphogenesis of some eukaryotic organisms is affected by some enzymes which are specified for fungus, some enzymes can affect virulence and pathogenic capacity of the microorganisms such as phospholipases, lipases, proteinases, and dimorphic growth in some Candida       sp. 1,17. Genes that initiate C. albicans dimorphism are regulated by a network of key signaling pathway and transcriptional factors18. S-adenosylmethionine synthetase (SAMs), also named as methionine adenosyltranferase (MAT), controls methylation reactions, and speeds up methylation of L-methionine (non-polar amino acid) to form S-adenosylmethionine (SAM)19 which is a methyl donor that facilitates methylation of DNA and once DNA is methylated it switch the genes off; therefore SAM can be considered to control gene expression, and that’s why it play an important role in yeast dimorphism process18,20,21.

 

The main objectives of this study were to identify Candida albicans using chromogenic, biochemical and molecular techniques. Dimorphism and virulence behavior were also studied using HPLC analysis for SAM product.

 

MATERIALS AND METHODS:

MATERIALS:

C. albicans:

C. albicans was kindly provided by microbiology laboratory, Micro-Analytical Centre, Faculty of Science, Cairo University, Giza, Egypt.

 

Cultivation of Candida:

Candida was cultivated on the following media: Yeast malt extract agar (g/l): Glucose, 10; Yeast extract, 3; Malt extract, 3; agar, 20. (pH 7.4). Sabouraud’s Dextrose agar (g/l): Dextrose, 40; Peptone, 10; Agar, 15. (pH 7.4). Sabouraud’s glucose broth (g/l) Glucose 40; Peptone; (pH 7.4). Candida was incubated at 37C for 48 hours.

 

Identification of Candida:

The yeast was firstly identified biochemically by VITEK 2 Compact technique22,23,24, then the identification was confirmed chromogenically by CHROMagar media25,26, 27,28. The medium consists of (g/l): Glucose, 20; Peptone, 10; Chloramphenicol, 0.5; Agar, 15; Chromogenic mixture, 2. The plates were incubated at 37C for 48 hours.

 

Molecular identification of C. albicans:

DNA extraction from the fungus was performed using SDS extraction method described by Hidalgo et al.,29. Amplification of the ITS gene was performed using the primers ITS1- F (5´- TCC GTA GGT GAA CCT TGC GG-3´) and ITS4- R (5´-TCC TCC GCT TAT TGA TAT GC-3´)30,31,32. PCR amplifications were carried out in 25 µl reaction mixture containing 2µl of the primers 31. The amplified products were purified and sequenced using automated DNA sequencer. Fungal strain were identified by submitting the ITS sequences in the National Centre for Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) search program34,35,36. Accession number of the identified C. albicans was obtained.

 

Enhancement of Dimorphism:

Roswell Park Memorial Institute Medium (RPMI         1640) 37 that enhances virulence, and dimorphism phenomenon 38, under aseptic conditions was inoculated from Sabouraud’s agar slants, then morphologically examined under microscope every 1, 6, 12, 18, 24, 30, 36, 42, 48, 54, 66, 72, 78 and 84 hours. The time taken for best dimorphic phenomena to happen was observed. RPMI-1640 media contain the following (mg/L): Ca (NO3)2·H2O, 100; KCl, 400; MgSO4, 48.8; NaCl, 6000; Na2HPO4, 800; NaHCO3, 2000; L-arginine, 200; L-asparagine; H2O, 56.8; L-aspartic acid, 20; L-cystine. 2HCl, 65.2;  L-glutamic acid, 20; L-glutamine, 300; glycine, 10; L-histidine, 15; hydroxy-L-proline, 20; L-isoleucine, 50; L-leucine, 50; L-lysine. HCl, 40; L-methionine, 15; L-phenylalanine, 15; L-proline, 20; L-serine, 30; L-threonine, 20; L-tryptophan, 5; L-tyrosine.2Na.2H2O, 28.83; Lvaline, 20; biotin, 0.20; D-calcium pantothenate, 0.25; choline chloride, 3; folic acid, 1; i-Inositol, 35; nicotinamide, 1; p-aminobenzoic acid, 1; pyridoxine. HCl, 1; riboflavin, 0.20; thiamine. HCl, 1; vitamin B12, 0.05; D-glucose, 2000; glutathione, 1 and phenol red, 5. The final pH of the media was 7.4.

 

Assay of SAM synthetase activity using high performance liquid chromatography (HPLC):

Cultivated cells exposed to sonicator for cell disruption and intercellular enzyme extraction39. Standard reaction mixture (mM): tris HCl pH 8.0 (100), KCl (200), MgCl2 (20), dithiotheritol (1), ATP (5), L-Methionine (5) (pH 7.8). Standard SAM was purchased from Sigma Company for HPLC analysis (4.6mm x 250mm, 5µm) to identify samples concentration at 254nm. SAM was diluted by an equal volume of (NH4)2SO4 cold saturated solution. The solution was stirred for 20min in ice bath and then the precipitate was removed by centrifugation at -4C, 1500rpm for 10 min. 50g of (NH4)2SO4 per 100 ml of original volume was added to the supernatant over 45min. The precipitate was collected by centrifugation at -4C (1500rpm x 10 min), and then the precipitate was dissolved in 20ml of 2% Na2CO3 in 0.1 N NaOH40,41.

 

RESULT:

Identification of Candida:

C. albicans was firstly identified biochemically using VITEK 2 Compact technique (Table1). The identification of Candida was carried out after 48 hours of incubation at 37°C. The data showed 99% probability C. albicans with excellent identification confidence. Also, C. albicans was identified chromogenically using CHROMagar differential media showing green colonies after 48 hours incubation at 37(Figure1). The identification of C. albicans was finally confirmed using molecular techniques. Amplification of the ITS region was carried out during PCR reaction using ITS1 and ITS4 primers. Accession number of the identified C. albicans was obtained as OK104215. Its relation with the other related fungal species was displayed by phylogenetic tree (Figure 2).

 

 

Figure 1: Chromogenic identification of C. albicans using CHROMagar differential medium

 

 

Figure 2:  Phylogenetic tree of C. albicans (OK104215).

 

Table 1: Biochemical identification of C. albicans through VITEK 2 Compact automated technique.

Biochemical Details

LysA

-

lMLTa

+

LeuA

+

ARG

+

ERYa

-

GLYLa

+

TyrA

-

BNAG

-

ARBa

-

AMYa

-

dGALa

+

GENa

-

dGLUa

+

LACa

-

MAdGa

+

dCELa

-

GGT

-

dMALa

+

dRAFa

-

NAGA1

+

dMNEa

+

dMELa

-

dMLZa

-

lSBEa

-

IRHAa

-

XLTa

+

dSORa

+

SACa

+

URE

-

AGLU

+

dTURa

+

dTREa

+

NO3a

-

lARAa

-

dGATa

(-)

ESC

-

lGLTa

+

dXYLa

+

LATa

+

ACEa

+

CITa

+

GRTas

+

lPROa

+

2KGa

+

NAGa

+

dGNTa

+

 

 

 

 

 

Figure 3: Microscopic examination of C. albicans growth after different time intervals (Light microscope 40 X).

 

 

 

Dimorphism enhancement:

Dimorphism phenomenon enhanced by sub-culturing C. albicans on RPMI 1640 media. Growth was monitored every 1, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, and 84 h to investigate dimorphic changes. Growth after one h on RPMI 1640 growth media, showed initiation of germ tubes and mycelial formation. After 6 h, a transition phase between yeast form and mycelial (filamentous) form was observed. After 30 h, the filamentation of Candida was developed. After 36 h, the filamentation and development of mycelia was completed. After 66 h, degeneration of filaments began to develop. After 72 h, degeneration of filaments was completed (Figure 3).

 

HPLC analysis for SAM (enzyme product):

HPLC analysis for SAM (enzyme product) after 6, 24, 48, 72, and 96 h was carried out. It was found that SAM increased with increase in time, and the optimum production of SAM was attained after 72 h of incubation on the dimorphism enhancing media (RPMI 1640). After that the production of SAM began to decrease (Figure 4).

 

Description: Oval:  fDescription: Oval: e

 

 

 

 

 

Figure 4: HPLC analysis for SAM (enzyme product) after different time intervals.

 

 

 

DISCUSSION:

Over one million patients die every year suffering from invasive fungal infections17. Candidiasis is a very serious problem due to its highly developed resistance against many drugs42. Targeting enzymes involved in this phenomenon may weaken fungal growth in the host 7. Dimorphism of C. albicans is a serious phenomenon, and is considers the key of its virulence22,23,51,52,53.

 

In the present work, C. albicans was firstly identified biochemically using VITEK 2 Compact technique, and then the identification was made using chromogenic identification. The conventional methods of identification of Candida species are time consuming and difficult to perform. VITEK method is a rapid and accurate method for identification of Candida species43. All chromogenic media tested appeared to be useful in presumptive identification of Candida spp44.

 

The identification of C. albicans was confirmed using molecular identification technique for accurate and precise identification, as the fungal DNA is its biological stamp as mentioned by Neppelenbroek et al.45,46; Gharanfoli et al.47 and Sankari et al.26. PCR-based detection of internal transcribed spacer regions of the rRNA genes was evaluated as a means of fungal identification by using internal transcribed spacers48.

 

Dimorphic fungi are those fungi which exist in two forms of growth, morphologically and biochemically different (parasitic form and commensally form).Yeast form unicellular and multicellular form4. This transference process from one form to another occurs under certain conditions with the catalysis of enzymatic activity of SAMs which plays an important role in dimorphism process5.

 

Regarding HPLC analysis for SAM after different time intervals, it was found that SAM concentration increases with increase in filamentation of C. albicans and as the culturing media aged, it loses its nutrients so filamentation declined as well as SAM level        declined5, 49. SAM plays an important role in biological methylation, and it also linked to polyamine               metabolism 50.

 

CONCLUSION:

C. albicans can easily be identified by various ways, biochemically using VITEK 2 Compact technique, chromogenically using CHROMagar media, and molecularly using ITS technique. The responsible factor of C. albicans virulence is the dimorphism, which shows its optimum behavior after 36-66 hours incubation on RPMI-1640 liquid medium at 37C under aseptic conditions. Maximum SAM production was attained after 72 h.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

REFERENCES:

1.      Badiee P. Hashemizadeh Z. Opportunistic invasive fungal infections: diagnosis & clinical management. Indian J Med Res 2014; 139(2):195-204.

2.      Vila T. Sultan AS. Montelongo-Jauregui D. Jabra-Rizk MA. Oral Candidiasis: A Disease of Opportunity. J Fungi (Basel) 2020; 6(1):15.

3.      Brissaud O. Guichoux J. Harambat J. Tandonnet O. Zaoutis T. Invasive fungal disease in PICU: epidemiology and risk factors. Ann Intensive Care 2012; 2(1):6.

4.      Arkowitz RA.  Bassilana M. Recent advances in understanding Candida albicans hyphal growth. F 1000 research 2019; 8:700.

5.      Mysyakina S. Funtikova NS. The role of sterols in morphogenetic processes and dimorphism in fungi. Microbiology 2007; 76(1):1-13.

6.      Montelongo-Jauregui D. Lopez-Ribot JL. Candida Interactions with the Oral Bacterial Microbiota. J Fungi (Basel) 2019; 4(4):122.

7.      Kim J. Lee J. Rapid method for chromatin immunoprecipitation (ChIP) assay in a dimorphic fungus, Candida albicans. J Microbiol 2020; 58(1):11-16.

8.      Nawrot U. Wlodarczyk K. Skala J. Prondo-Morarska APM. Evaluation of Candiselect 4. a chromogenic medium for yeast differentiation. Clin Microbiol Infect 2005; 11(suppl 2):715-716.

9.      Ghelardi E. Pichierri G. Castagna B. Barnini S. Tavanti A. Campa M. Efficacy of Chromogenic Candida Agar for isolation and presumptive identification of pathogenic yeast species. Clin Microbiol Infect 2008; 14:141–147.

10.    Parka J. Oha J.  Sanga H. Shresthaa B. Leeb H , Koob J. Chob S . Choib JS.  Min Leeb M. Kimb J and Gi-Ho Sunga G. Identification and Antifungal Susceptibility Profiles of Cyberlindnera fabianii in Korea. Mycobiol 2019; 47(4):449-456.

11.    Hoang MTV. Irinyi L. Chen SCA. Sorrell TC. Meyer W. Dual DNA barcoding for the molecular identification of the agents of invasive fungal infections. Front Microbiol 2019; 10: 1647.

12.    Hering D. Borja A. Jones JI. Pont D. Boets P. et al. Implementation options for DNA-based identification into ecological status assessment under the European Water Framework Directive. Water Res. 2018; 138:182-205.

13.    Wu B. Hussain M. Zhang W. Stadler M. Liu X. Xiang M. Current insights into fungal species diversity and perspective on naming the environmental DNA sequences of fungi. Mycol 2019; 10(3):127-40.

14.    Letchuman S. Short Introduction of DNA Barcoding. Int J Res 2018; 5(4):673-86.

15.    Fajarningsih ND. Internal Transcribed Spacer (ITS) as DNA Barcoding to Identify Fungal Species: a Review. Squalene Bull  Mar Fish Post Harvest Biotech 2016; 11(2):37-44.

16.    Cheng T. Xu C. Lei L. Li C. Zhang Y. Zhou S. Barcoding the kingdom Plantae: New PCR primers for ITS regions of plants with improved universality and specificity. Mol Ecol Resour 2016; 16: 138-49.

17.    Fernandes CM. Goldman GH.  Poeta MD. Biological Roles Played by Sphingolipids in Dimorphic and Filamentous Fungi. mBio 2018; 9(3):e00642-18.

18.    Sharma J. Rosiana S. Razzaq I. Shapiro RS. Linking Cellular Morphogenesis with Antifungal Treatment and Susceptibility in Candida Pathogens. J Fungi (Basel) 2019; 5(1):17.

19.    Yoon S. Lee W.  M Kim. Kim T D. Ryu Y. Structural and functional characterization of S-adenosylmethionine (SAM) synthetase from Pichia ciferrii. Bioprocess Biosyst Eng 2012; 35:173–181.

20.    Hayashi T. Teruya T. Chaleckis R. Morigasaki S. Yanagida M. S-Adenosylmethionine Synthetase Is Required for Cell Growth, Maintenance of G0 Phase, and Termination of Quiescence in Fission Yeast. iScience 2018; 5:38-51.

21.    Liu W. Tang D. Shi R. Lian J. Huang L. Cai1 J. Xu Z. Efficient production of S-adenosyl-L-methionine from DL‐methionine in metabolic engineered Saccharomyces cerevisiae. WILEY 2019; 10:1-12.

22.    Al-Tekreeti ARA. Al-Halbosiy MMF. Dheeb BI. Hashim AJ. Al-Zuhairi AFH. Mohammad FI. Molecular identification of clinical Candida isolates by simple and randomly amplified polymorphic DNA-PCR. Arabian Journal for Science and Engineering 2018; 43:163-170.

23.    Ambaraghassi G. Dufresne PJ. Dufresne SF. Vallières É. Muñoz JF. Cuomo CA. Berkow EL. Lockhart SR. Luong ML. Identification of Candida auris by Use of the Updated Vitek 2 Yeast Identification System, Version 8.01: a Multilaboratory Evaluation Study. J Clin Microbiol 2019; 23; 57(11).

24.    Yi Q. Xiao M. Fan X. Zhang G. Yang Y. et al. Evaluation of Autof MS 1000 and Vitek MS MALDI-TOF MS System in Identification of Closely-Related Yeasts Causing Invasive Fungal Diseases. Front Cell Infect Microbiol 2021; 18;11:628828.

25.    Bellanger A. Gbaguidi-Haore H. Liapis E. Scherer E. Millon L. Rapid identification of Candida sp. by MALDI-TOF mass spectrometry subsequent to short-term incubation on a solid medium. APMIS 2019; 127(4):217-221.

26.    Sankari SL. Mahalakshmi K. Kumar VN. Chromogenic medium versus PCR-RFLP in the speciation of Candida: a comparative study. BMC Res Notes 2019; 12(1):681.

27.    Patil A. Boparai NK. Shankargouda SB. Doddamani MH. Vora A. Dave  T. Candida dubliniensis: The New Culprit on the Block Causing Denture Stomatitis? An In Vivo Study. J Contemp Dent Pract 2021; 1;22(5):517-521.

28.    Tha TP. Gopinath P. Differentiation of Candida dubliniensis on CHROM agar and Pal’s agar. Research J. Pharm. and Tech 2016; 9(12):2150-2154.

29.    Hidalgo A. Melo A. Romero F. Hidalgo V. Villanueva J. Fonseca-Salamanca F. DNA extraction in Echinococcus granulosus and Taenia spp. eggs in dogs stool samples applying thermal shock. Exp Parasitol; 2018. 186:10-16.

30.    Korabeena M. The variability in the fungal ribosomal DNA (ITS1, ITS2, and 5.8 r RNA gene): Its biological meaning and application in medical mycology. In: Mendez-Vilas A, editor. Communicating Current Research and Educational Topics and Trends in Appplied Microbiology; 2007. p. 783-787.

31.    Divya D.  Rishad KS.  Arjunan S.  Gopinath LR and Merlin Christy P. ITS - PCR Based Molecular Identification of Fungi Associated With Piper nigrum And Its Growth Sensitivity Against Pseudomonas fluorescens. Int J of interdisci res and reves 2013; 1(3):26-33.

32.    Nawan.  Septi. Handayani. Molecular identification of Streptomyces sp. isolated from peat land of Palangka Raya, Kalimantan Tengah using 16S rRNA gene sequences analysis. Research Journal of Pharmacy and Technology. 2021; 14(12):6639-4.

33.    Kadry AA. El-Ganiny AM , El-Baz AM. Comparison of methods used in identification of Candida albicans. Research J. Pharm. and Tech. 2018; 11(3): 1164-1168.

34.    Altschul SF. Madden TL. Schäffer AA.  Zhang J. Zhang Z.  Miller W. Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997; 1; 25(17):3389-402.

35.    Dhanjal DS. Chopra C. Anand A.  Chopra RS. Accessing the Microbial Diversity of Sugarcane Fields from Gujjarwal Village, Ludhiana and their Molecular Identification. Research J. Pharm. and Tech 2017; 10(10):3439-3442

36.    Shree JK. Krishnaveni C. Isolation, Identification and Molecular Characterization of Endophytic Fungi from the leaves of Coelogyne species, and their role as an Antimicrobial agent. Research Journal of Pharmacy and Technology. 2021; 14(11):5613-7.

37.    Leney-Greene MA. Boddapati AK. Su HC. Cantor JR. Lenardo MJ. Human Plasma-like Medium Improves T Lymphocyte Activation. iScience 2020; 23(1):100759.

38.    Uribe B. González O.  Ourliac-Garnier I. Le Pape P. Ba BB.  Alonso RM. Gaudin K. Determination of antifungal caspofungin in RPMI-1640 cell culture medium by column-switching HPLC-FLD. J Pharm Biomed Anal 2020; 5;188:113366.

39.    Borthwick KAJ. Coakley WT. McDonnell MB. Nowotny H.  Benes E, Gröschl M. Development of a novel compact sonicator for cell disruption. J Microbiol Methods 2005; 60(2):207-16.

40.    Murugan S. Janardhan CHU. Babu MN. RP-HPLC Method for Simultaneous Estimation of Albendazole and Niclosamide in Oral Suspension for Veterinary Use. Research J. Pharm. and Tech. 9(1): Jan., 2016; Page 27-32.

41.    Agrahari MV. Bajpai. Nanda S. Essential Concepts of Mobile Phase Selection for Reversed Phase HPLC. Research J. Pharm. and Tech. 6(5): May 2013; Page 459-464

42.    Kollu NV. Lajeunesse DR. Cell Rupture and Morphogenesis Control of the Dimorphic Yeast Candida albicans by Nanostructured Surfaces. ACS Omega 2021; 4;6(2):1361-1369.

43.    Sundaram M. Navaneethakrishnan RM. Evaluation of Vitek 2 system for clinical identification of Candida species and their antifungal susceptibility test. J Evolution Med Dent Sci 2016; 5(47):2948-2951.

44.    Cooke VM. Miles RJ. Price RG. Midgley G. Khamri W. Richardson AC. New Chromogenic Agar Medium for the Identification of Candida spp. App Environ Microbiol 2002; 3622-3627.

45.    Neppelenbroek KH. Seó RS. Urban VM. Silva S. Dovigo LN. Jorge JH. Campanha NH. Identification of Candida species in the clinical laboratory: a review of conventional, commercial, and molecular techniques. Oral Dis 2014; 20(4):329-44

46.    Dey A. Ragavan ML. Mandal SK. Das N . Isolation, Identification and In vitro Characterisation of Probiotic Yeast Strains. Research J. Pharm. and Tech. 2017; 10(3): 726-732.

47.    Gharanfoli A. Mahmoudi E. Torabizadeh R. Katiraee F. Faraji S. Isolation, characterization, and molecular identification of Candida species from urinary tract infections. Curr Med Mycol 2019; 5(2):33-36.

48.    Ali HH.  Al-Obaidi RM. Fattah CH. Molecular identification of Candida species isolated from ears of dogs infected with Otitis externa by detecting internal transcript spacer (ITS1 and ITS4) in Sulaimania. Iraq Adv Anim Vet Sci 2015; 3(9):491-499.

49.    Berger BJ. Knodel MH. Characterization of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberclosis, BMC Microbiol 2003; 3:12.

50.    Rato C. Amirova SR.  Bates DG.  Stansfield I.  Wallace HM. Translational recoding as a feedback controller: systems approaches reveal polyamine-specific effects on the antizyme ribosomal frameshift. Nucleic Acids Res 2011; 39(11):4587-4597.

51.    Tamizh Paavai Tha. Gopinath P. Differentiation of Candida dubliniensis on CHROM agar and Pal’s agar. Research J. Pharm. and Tech 2016; 9(12):2150-2154.

52.    Zagazig Egypt. Comparison of methods used in identification of Candida albicans. Research J. Pharm. and Tech. 2018; 11(3): 1164-1168.

53.    Rybalkin M. Diadiun T. Khokhlenkova N. Azarenko Y. Stepanenko S. Determination of Candida albicans fungus proteins concentration by Elisa method at Intramuscular Introduction in Candidiasis Therapy. Research Journal of Pharmacy and Technology. 2021; 14(6):3249-2

 

 

 

 

Received on 07.05.2022             Modified on 03.07.2022

Accepted on 10.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1007-1011.

DOI: 10.52711/0974-360X.2023.00168