Sains Malaysiana 50(3)(2021): 735-742


Evaluation of Chitin as a Biomarker of Pathogenic Fungal Isolates

(Penilaian Kitin sebagai Biopenanda Pencilan Kulat Patogen)




1Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor Darul Takzim, Malaysia


2Center of Desert Studies, University of Anbar, Ramadi, Iraq


3Biomedical Science Programme, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Federal Territory, Malaysia


4Department of Medical Microbiology and Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latiff, Bandar Tun Razak Cheras, 56000 Kuala Lumpur, Federal Territory, Malaysia


5Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor Darul Takzim, Malaysia


Diserahkan: 28 Mei 2020/Diterima: 27 Ogos 2020



Chitin is a polysaccharide component of the inner cell wall of fungi that has been used to estimate fungal invasion in plant products. However, its detection in major pathogenic fungal species has not been investigated. The present study aimed to determine the chitin contents of pathogenic fungal species in order to evaluate its diagnostic potential as a biomarker for fungal infections. High performance liquid chromatography (HPLC) was used to measure chitin content. Pure chitin was acid hydrolyzed and the fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine produced were measured. The chitin contents of ten pathogenic fungal isolates were determined per mycelial dry weight. They varied from 18.61 (± 0.09) to 47.12 (± 0.50) µg/mg dry mycelial weight. Candida albicans and Cryptococcus neoformans exhibited the highest and lowest levels of chitin, respectively. Based upon relative amounts of chitin produced, three groups namely: high (Candida albicans, Cryptococcus gattii, Aspergillus niger and Penicillium at 47.12, 46.98, 46.05, and 44.15 µg/mg respectively), medium (Rhizopus, Aspergillus fumigatus, Fusarium solani, and Mucor at 36.61, 36.30, 35.03, and 34.84 µg/mg, respectively), and low (Candida tropicalis and Cryptococcus neoformans at 20.78 and 18.61 µg/mg, respectively), were identified. Chitin was not detectable in bacterial isolates used as controls. The chitin detection method offers a sensitive and specific tool for the quantification of chitin in pathogenic fungal isolates. The detection of chitin may be a useful assay for the diagnosis of fungal infections in clinical samples.

Keywords: Biomarker; chitin; diagnosis; HPLC; pathogenic fungi



Kitin adalah komponen polisakarida pada dinding sebelah dalam kulat yang telah digunakan untuk menentukan kehadiran kulat pada hasil tumbuhan. Walau bagaimanapun, pengesanan kitin dalam kulatpatogen yang utama belum lagi dikaji. Kajian ini bertujuan menentukan kandungan kitin dalam spesieskulat patogen untuk menilai potensi diagnostiknya sebagai biopenanda jangkitan kulat. Kaedah kromatografi cecair prestasi tinggi (HPLC) telah digunakan untuk mengukur kandungan kitin. Hidrolisasi asid dilakukan terhadap kitin tulen untuk menghasilkan terbitan glukosamin 9-fluorenilmetilkloroformat (FMOC-CI) yang berpendafluor, lalu diukur. Kandungan kitin bagi sepuluh pencilan kulat patogen ditentukan berdasarkan berat kering miselia. Hasil yang diperoleh adalah dalam julat 18.61 (± 0.09) ke 47.12 (± 0.50) µg/mg berat kering miselia. Kandungan tertinggi kitin terdapat pada Candida albicans manakala kandungan yang paling rendah pada Cryptococcus neoformans. Berdasarkan kandungan relatif kitin, tiga kumpulan dikenal pasti, iaitu tinggi (Candida albicans, Cryptococcus gattii, Aspergillus niger dan Penicillium dengan 47.12, 46.98, 46.05 dan 44.15 µg/mg masing-masing), sederhana (Rhizopus, Aspergillus fumigatus, Fusarium solani dan Mucor dengan 36.61, 36.30, 35.03 dan 34.84 µg/mg masing-masing) dan rendah (Candida tropicalis dan Cryptococcus neoformans dengan 20.78 dan 18.61 µg/mg masing-masing). Kitin tidak dapat dikesan pada pencilan bakteria yang diguna sebagai kawalan. Kaedah pengesanan kitin boleh digunakan sebagai suatu alat khusus dan sensitif untuk kuantifikasi kitin pada pencilan kulat patogen. Pengesanan kitin merupakan suatu asai yang mungkin berguna untuk diagnosis jangkitan kulat dalam sampel klinikal.


Kata kunci: Biopenanda; diagnosis; HPLC; kitin; kulat patogen



Appuhn, A. & Joergensen, R.G. 2006. Microbial colonisation of roots as a function of plant species. Soil Biology and Biochemistry 38: 1040-1051.

Arvanitis, M., Anagnostou, T., Fuchs, B.B., Caliendo, A.M. & Mylonakis, E. 2014. Molecular and nonmolecular diagnostic methods for invasive fungal infections. Clinical Microbiology Reviews 27(3): 490-526.

Brakhage, A.A. 2005. Systemic fungal infections caused by Aspergillus species: Epidemiology, infection process and virulence determinants. Current Drug Targets 6: 875-886.

Donald, W.W. & Mirocha, C.J. 1977. Chitin as a measure of fungal growth in stored corn and soybean seed. Cereal Chemistry 54: 466-474.

Ekblad, A. & Nasholm, T. 1996. Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine. Plant Soil 178: 29-35.

Ekblad, A., Wallander, H. & Nasholm, T. 1998. Chitin and ergosterol combined to measure total and living fungal biomass in ectomycorrhizas. New Phytologist 138: 143-149.

Feofilova, E.P., Nemtsev, D.V., Tereshina, V.M. & Memorskaya, A.S. 2006. Developmental change of the composition and content of the chitin-glucan complex in the fungus Aspergillus niger. Applied Biochemistry and Microbiology 42(6): 545-549.

Free, S.J. 2013. Fungal cell wall organization and biosynthesis. Advances in Genetics 81: 33-82.

Garey, K.W., Rege, M., Pai, M.P., Mingo, D.E., Suda, K.J., Turpin, R.S. & Bearden, D.T. 2006. Time to initiation of fluconazole therapy impacts mortality in patients with candidemia: A multi-institutional study. Clinical Infectious Diseases 43(1): 25-31.

Gay, L. 1991. Chitin content and chitin synthase activity as indicators of the growth of three different anaerobic rumen fungi. Federation of European Microbiological Societies Microbiology Letters 80(1): 99-102.

Joergensen, R.G. & Wichern, F. 2008. Quantitative assessment of the fungal contribution to microbial tissue in soil. Soil Biology and Biochemistry 40(12): 2977-2991.

Kedzierska, A., Kochan, P., Pietrzyk, A. & Kedzierska, J. 2007. Current status of fungal cell wall components in the immunodiagnostics of invasive fungal infections in humans: Galactomannan, mannan and (1A3)-b-D-glucan antigens. European Journal of Clinical Microbiology & Infectious Diseases 26(11): 755-766.

Kim, S.K. 2013. Chitin and Chitosan Derivatives: Advances in Drug Discovery and Developments. Florida: CRC Press, Taylor & Francis Group.

Latge, J.P. 2007. The cell wall: A carbohydrate armour for the fungal cell. Molecular Microbiology 66: 279-290.

Lin, H.H. & Cousin, M.A. 1985. Detection of mold in processed foods by high performance liquid chromatography. Journal of Food Protection 48(8): 671-678.

Maertens, J., Theunissen, K., Verbeken, E., Lagrou, K., Verhaegen, J., Boogaerts, M. & Eldere, J.V. 2004. Prospective clinical evaluation of lower cut-offs for galactomannan detection in adult neutropenic cancer patients and haematological stem cell transplant recipients. British Journal of Haematology 126(6): 852-860.

Maertens, J., Van Eldere, J., Verhaegen, J., Verbeken, E., Verschakelen, J. & Boogaerts, M. 2002. Use of circulating galactomannan screening for early diagnosis of invasive aspergillosis in allogeneic stem cell transplant recipients. The Journal of Infectious Diseases 186(9): 1297-1306.

Munro, C.A. & Gow, N.A.R. 2001. Chitin synthesis in human pathogenic fungi. Medical Mycology 39(Suppl 1): 41-53.

Muzzarelli, R.A.A., Boudrant, J., Meyer, D., Manno, N., DeMarchis, M. & Paoletti, M.G 2012. Current views on fungal chitin/chitosan, human chitinases, food preservation, glucans, pectins and inulin: A tribute to Henri Braconnot, precursor of the carbohydrate polymers science, on the chitin bicentennial. Carbohydrate Polymers 87(2): 995-1012.

Nandi, B. 1978. Glucosamine analysis of fungus-infected wheat as a method to determine the effect of antifungal compounds in grain preservation. Cereal Chemistry 55: 121-126.

Nilsson, K. & Bjurman, J. 1998. Chitin as an indicator of the biomass of two wood-decay fungi in relation to temperature, incubation time, and media composition. Canadian Journal of Microbiology 44(6): 575-581.

Penman, D., Britton, G., Hardwick, K., Collin, H.A. & Isaac, S. 2000. Chitin as a measure of biomass of Crinipellis perniciosa, causal agent of witches’ broom disease of Theobroma cacao. Mycological Research 104(6): 671-675.

Pfaller, M.A. & Diekema, D.J. 2004. Rare and emerging opportunistic fungal pathogens: Concern for resistance beyond Candida albicans and Aspergillus fumigatus. Journal of Clinical Microbiology 42(10): 4419-4431.

Phillips, M.W. & Gordon, G.L. 1989. Growth characteristics on cellobiose of three different anaerobic fungi isolated from the ovine rumen. Journal of Antimicrobial Chemotherapy 55(7): 1695-1702.

Pisa, D., Alonso, R., Rábano, A., Horst, M.N. & Carrasco, L. 2016. Fungal enolase, β-tubulin, and chitin are detected in brain tissue from Alzheimer’s disease patients. Frontiers in Microbiology 7: 1772.

Plaine, A., Walker, L., Da Costa, G., Mora-Montes, H.M., McKinnon, A., Gow, N.A.R., Gaillardin, C., Munro, C.A. & Richard, M.L. 2008. Functional analysis of Candida albicans GPI-anchored proteins: Roles in cell wall integrity and caspofungin sensitivity. Fungal Genetics and Biology 45(10): 1404-1414. 

Racil, Z., Kocmanova, I., Lengerova, M., Weinbergeroca, B., Buresova, L., Toskova, M., Winterova, J., Timilsina, S., Rodriguez, I. & Mayer, J. 2010. Difficulties in using 1,3-b-D-glucan as the screening test for the early diagnosis of invasive fungal infections in patients with haematological malignancies - high frequency of false-positive results and their analysis. Journal of Medical Microbiology 59(Pt9): 1016-1022.

Richardson, M.D. 2005. Changing patterns and trends in systemic fungal infections. Journal of Antimicrobial Chemotherapy 56(Suppl 1): i5-i11.

Ride, J.P. & Drysdale, R.B. 1972. A rapid method for the chemical estimation of filamentous fungi in plant tissue. Physiological Plant Pathology 2(1): 7-15.

Sendid, B., Poirot, J.L., Tabouret, M., Bonnin, A., Caillot, D., Camus, D. & Poulain, D. 2002. Combined detection of mannanaemia and antimannan antibodies as a strategy for the diagnosis of systemic infection caused by pathogenic Candida species. Journal of Medical Microbiology 51(5): 433-442.

Taylor, M.J., Ponikau, J.U., Sherris, D.A., Kern, E.B., Gaffey, T.A., Kephart, G. & Kita, H. 2002. Detection of fungal organisms in eosinophilic mucin using a fluorescein-labeled chitin-specific binding protein. Otolaryngology-Head and Neck Surgery 127(5): 377-383.

Tharanathan, R.N. & Kittur, F.S. 2003. Chitin - the undisputed biomolecule of great potential. Critical Reviews in Food Science and Nutrition 43(1): 61-87.

Vallabhaneni, S., Mody, R.K., Walker, T. & Chiller, T. 2016. The global burden of fungal diseases. Infectious Disease Clinics of North America 30(1): 1-11.

Walker, L.A., Munro, C.A., de Bruijn, I., Lenardon, M.D., McKinnon, A. & Gow, N.A.R. 2008. Stimulation of chitin synthesis rescues Candida albicans from echinocandins. PLoS Pathogens 4(4): e1000040. 

Wallander, H., Ekblad, A., Godbold, D.L., Johnson, D., Bahr, A., Baldrian, P., Bjork, R.G., Kieliszewska-Rokicka, B., Kjoller, R., Kraigher, H., Plassard, C. & Rudawska, M. 2013. Evaluation of methods to estimate production, biomass and turnover of ectomycorrhizal mycelium in forests soils - A review. Soil Biology and Biochemistry 57: 1034-1047.

Whipps, J.M. & Lewis, D.H. 1980. Methodology of a chitin assay. Transactions of the British Mycological Society 74(2): 416-418.


*Pengarang untuk surat-menyurat; email: