IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Available online at IJTID Website: https://e-journal.unair.ac.id/IJTID/ Vol. 11 No. 1 January–April 2023 Original Article Germ Tube Induction Test Comparing Total of Six Liquid and Three Solid Media in Candida albicans Rivaldi Ruby1 , Erlangga Saputra Arifin1 , Sandy Vitria Kurniawan2 , Sem Samuel Surja3* 1School of Medicine and Health Sciences, Universitas Katolik Indonesia Atma Jaya, Jakarta, Indonesia 2Department of Pharmacology and Pharmacy, School of Medicine and Health Sciences, Universitas Katolik Indonesia Atma Jaya, Jakarta, Indonesia 3Department of Parasitology, School of Medicine and Health Sciences, Universitas Katolik Indonesia Atma Jaya, Jakarta, Indonesia Received: March 3rd, 2022; Revised: November 6th, 2022; Accepted: February 27th, 2023 ABSTRACT Invasive candidiasis (IC) has a high mortality rate of 70%, thus diagnosis should be established without delay. Given its fast result, serological test such as β-d-glucan (BDG) test is one alternative diagnosis modalities. However, it lacks specificity. Candida albicans germ tube antibody (CAGTA) test is an alternative serological test which has a high sensitivity of 76.2% and specificity of 80.3%. Manufacturing CAGTA serological test requires provision of specific germ tube antigen. In this study, various culture media were tested to find the best media for germ tube induction. This study was an experimental in vitro study. The number and length of the germ tube were recorded in two- and three-hour incubation periods. A total of six samples containing one C. albicans ATCC 90028, four C. albicans wild type strains, and one C. krusei wild type strain were used. Nine media were tested to induce germ tube formation: human and sheep serum, fetal bovine serum, mueller hinton agar and broth, tryptic soy agar and broth, brain heart infusion agar and broth. At both incubation periods, the medium with the highest number of germ tube was human serum (p=0.001 and p=0). The longest germ tube was found in sheep serum at two-hour incubation period (p=0.005). Mueller hinton broth (MHB) showed comparable results with human and sheep serum (p>0.05). Human serum is a superior inducer of morphogenesis. However, the use of MHB is recommended in this study, since provision of fresh human and sheep serum on a regular basis is impractical. Keywords: Candida albicans; germ tube; human serum; mueller hinton broth; sheep serum Highlights: Several media could induce not only numbers of germ tube, but also its length. Therefore, they could benefit for easier diagnosis and also higher amounts of germ tube protein. How to Cite: Ruby, R., Arifin., E. S., Kurniawan, S. V., Surja, S. S. Germ Tube Induction Test Comparing Total of Six Liquid and Three Solid Media in Candida albicans. Indonesian Journal of Tropical and Infectious Disease. 11(1). 18–26. Apr. 2023. DOI: 10.20473/ijtid.v11i1.34097 * Corresponding Author: sem.samuel@atmajaya.ac.id https://e-journal.unair.ac.id/IJTID/ https://orcid.org/0000-0002-6609-8413 https://orcid.org/0000-0002-5094-9957 https://orcid.org/0000-0002-1707-0166 https://orcid.org/0000-0001-5981-0014 19 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Indonesian Journal of Tropical and Infectious Disease, Vol. 11 No. 1 January–April 2023: 8–26 INTRODUCTION Candidiasis is a disease with a high prevalence rate globally. This disease generally affects the skin and mucosal tissue, causing mild conditions such as oral and vulvovaginal candidiasis.1 At the systemic level, it has high mortality and morbidity, referred to as invasive candidiasis (IC). It is associated with prolonged intensive care unit (ICU) admission and immunocompromised conditions such as acquired immune deficiency syndrome (AIDS).2 Globally, candidiasis occupies the top three incidences of diseases caused by fungi in the year 2017—the first is oral candidiasis with an incidence rate of 2,000,000, followed by esophageal candidiasis with 1,300,000, and then IC with 750,000 incidences.1 Invasive candidiasis yields a high mortality rate of 70%.3 Invasive candidiasis is caused by Candida spp. with the most common etiology being Candida albicans.4 In humans, this fungus is a normal flora of the skin, oropharynx, digestive, and urogenital tract.5 Infection occurs when there is hyphal growth and biofilm formation in the tissue. These mechanisms also allow resistance of C. albicans to traditional antifungal agents.6 Timely diagnosis is required in order to reduce mortality. Currently, IC is diagnosed through the findings of hyphae on microscopic examination or through a time- consuming culture.1 Serological tests provide relatively faster and easier way to diagnose IC. β-d- glucan (BDG) test is one widely used Candida serological test. However, it lacks specificity due to cross reaction with other fungi.7 A serological test detecting antibody against germ tube could be used as an alternative to diagnose IC, namely C. albicans germ tube antibody (CAGTA) test.8 Germ tubes are formed by C. albicans in a number of conditions such as starvation, presence of serum or N-acetylglucosamine, physiological temperature, and CO2. 9 It has high sensitivity of 76.2% and specificity of 80.3% since morphological transition from yeast to germ tube and hyphae is important for pathogenicity of C. albicans.8,10 Combination of BDG and CAGTA serological tests is recommended for IC early diagnosis.11 The first step in manufacturing CAGTA serological test is isolation of the germ tube antigen. It is important to seek the best medium for C. albicans since this antigen is obtained by inducing its growth in a suitable environment. Various media can be used for induction of germ tube, each with unique compositions and function. Human serum is the most used medium for germ tube test.12 Its main limitation is the requirement of fresh human serum on a regular basis. For this reason, this study aims to find the best media in inducing germ tube formation of C. albicans. While previous studies mainly assessed the sensitivity of each medium for germ tube test, this study also measured the number and length of germ tube formed after certain incubation period. MATERIALS AND METHODS Study Design This experimental in vitro study was conducted in the Parasitology Laboratory, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia from August 2020 to October 2020. Ethical clearance was obtained from the Atma Jaya ethical committee with the number 01/06/KEP-FKUAJ/2020. Fungi Strains Candida albicans wild type, C. albicans ATCC 90028, and C. krusei wild type were used in this study. All C. albicans wild type were obtained from patient’s sputum in Microbiology Laboratory, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia, while C. albicans ATCC 90028 and C. krusei wild type were obtained from the collection of Department Parasitology, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia. Each strain 20 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Rivaldi Ruby, et al. Germ Tube Induction Test was identified and confirmed through macroscopic and microscopic examination. Macroscopic identification was made using CHROMAgar (Oxoid, United Kingdom) to analyze the color and characteristics of the fungal colonies. Candida albicans was characterized by the formation of green colonies, in contrast with C. krusei which appeared as pink colonies.13 Microscopic identification was made through lactophenol cotton blue (LPCB) staining and germ tube test. Light microscope (Olympus CX21) is used to identify the morphologies. Ovoid and spherical yeast cell shapes are a characterization of C. albicans, distinguished from C. krusei that commonly appear as a more elongated (long grain rice) shape. A positive germ tube test is also only found in C. albicans.14,15 A total of six isolates containing one C. albicans ATCC 90028, four C. albicans wild type, and one C. krusei wild type were used. Candida albicans ATCC 90028 and C. krusei were used as the positive and negative control, respectively. Medium The media used for induction of germ tube were serum, broth, and agar. Sera used were human serum, sheep serum, and fetal bovine serum (FBS, Biowest, France). The FBS used was not diluted with 100% concentration. Human serum was prepared by centrifugating blood from a healthy donor.16 The broth and agar media used were mueller hinton agar (MHA, Oxoid, United Kingdom), mueller hinton broth (MHB, Conda, Spain), tryptic soy agar (TSA, Oxoid, United Kingdom), tryptic soy broth (TSB, Merck, Germany), brain heart infusion agar (BHIA, Oxoid, United Kingdom), and brain heart infusion broth (BHIB, Oxoid, United Kingdom). All media were obtained from the Department of Microbiology, Parasitology, and Pharmacology, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia. Broth and agar media were prepared by combining 1 L of aquadest with 38, 21, 45, 30, 53, and 37 g of MHA, MHB, TSA, TSB, BHIA, and BHIB, respectively. These suspensions were heated until completely dissolved, followed by sterilization using an autoclave at 121°C with a pressure of 15 Psi for 15 minutes. Broth medium was poured into the 1.5 ml test tube, while agar medium was poured into a petri dish. For all media, pH was adjusted at 7.4 which is confirmed by pH meter. Gem Tube Induction Candida albicans and C. krusei were inoculated in sabouraud dextrose agar (SDA, Oxoid, United Kingdom) for 48 hours at room temperature (25°C). Two hundred μL of 3 McFarland fungi suspension was added into 800 μL of each serum and broth medium. The mixture was incubated for 24 hours at 37°C.12 The number and length of the germ tube were recorded in two-, three-, and 24-hour incubation periods. Ten μL of the mixture was dripped into an improved Neubauer counting chamber and the germ tube was observed under the microscope (Figure 1a).17 All processes were performed in duplicate. Germ tube induction in agar media was conducted by dripping 10 μL of 0.5 McFarland fungi suspension into 1 x 1 cm2 agar.18 Cover slip was placed to facilitate easier examination. The agar was then incubated for three hours at 37°C. The number and length of germ tube induction were recorded in two- and three-hour incubation periods using the microscope (Figure 1b). All processes were performed in duplicate. Germ Tube Calculation This study measured the number and length of germ tubes formed. The number of germ tube was calculated in five small squares of the counting chamber using standardized formula (Figure 2).19 Germ tube length measurement was conducted by comparing the length of the germ tube and the counting chamber small squares. The longest germ tube in the five small squares was 21 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Indonesian Journal of Tropical and Infectious Disease, Vol. 11 No. 1 January–April 2023: 8–26 recorded. Germ tube length measurement was done only in serum and broth media. There are no difficulties in performing the measurement methods. Figure 1. Germ tube appearance under the microscope in different preparations. (a) Improved Neubauer Counting Chamber – Germ tube (red circle); (b) Slide culture 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑒𝑟𝑚 𝑡𝑢𝑏𝑒 / 𝜇𝐿 = 𝑛 𝑉 𝑉 = 𝑖𝑚𝑝𝑟𝑜𝑣𝑒𝑑 𝑁𝑒𝑢𝑏𝑎𝑢𝑒𝑟 𝑐𝑜𝑢𝑛𝑡𝑖𝑛𝑔 𝑐ℎ𝑎𝑚𝑏𝑒𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 = 1 50 𝑚𝑚3 𝑛 = 𝑡𝑜𝑡𝑎𝑙 𝑜𝑓 𝑔𝑒𝑟𝑚 𝑡𝑢𝑏𝑒 𝑖𝑛 5 𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 Figure 2. Improved Neubauer Counting Chamber Calculation of the number of germ tube on agar media was carried out by taking three representative images using the high-power field (HPF) microscope. Then the number of germ tubes was grouped into several categories (Table 1). Table 1. Germ Tubes Counts Categories Using Agar Media Inductions Categories Germ Tube Count/HPF 1+ 1-10 2+ 11-20 3+ 21-30 4+ 31-40 5+ 41-50 6+ >50 HPF – high-power field Data Analysis Data analysis was done using Statistical Product and Service Solution (SPSS) version 22. Data on serum and broth medium was analyzed using a One-Way ANOVA statistical test or Kruskal-Wallis test, depending on data normality. It was then followed by a Bonferroni post hoc test if significant results were found. In agar medium, Fisher-Exact or Chi-Square test were used depending on the terms and criteria of the statistical test. A significant value was yielded if p<0.05. RESULTS AND DISCUSSION Fungi Identification Macroscopic identification of all samples was done using CHROMagar. It is depicted in Figure 3. Moreover, the morphologies found in microscopic identification using LPCB confirmed the samples’ species (Figure 4). Figure 3. Macroscopic characterizations on CHROMagar. The green colonies are C. albicans and the pink colony is C. krusei (a) (b) Figure 4. Microscopic characterizations of Candida species grown in SDA. (a) Ovoid appearance of C. albicans (black circle) (b) Elongated appearance of C. krusei (red circle) 22 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Rivaldi Ruby, et al. Germ Tube Induction Test Germ Tube Induction in Serum and Broth All Candida samples were used in germ tube induction. Candida albicans ATCC 90028 and C. krusei function serve as a positive and negative control, respectively. In all media, C. albicans ATCC 90028 showed positive germ tube results, while C. krusei had negative results. Candida albicans wild type was used for the evaluation of each media’s ability to induce germ tube formation. Several media could facilitate germ tube formation. Results were variable between media at two- and three-hour incubation period. At two-hour incubation period, it was found that the medium with the highest number of germ tube was human serum. Sheep serum also showed comparable results in germ tube induction. Roughly, the order of media that could facilitate germ tube induction was as follows: human serum, sheep serum, FBS, MHB, TSB, and BHIB; while at three-hour incubation period, the order was as follows: human serum, MHB, sheep serum, FBS, TSB, and BHIB as shown in Table 2. Table 2. Number of Germ Tubes Formed on Serum and Broth Media (number/μL) Time Fungi Media p-value Human Serum Sheep Serum FBS MHB TSB BHIB 2 hours CA 1 38125 40625 26875 11250 5625 5000 0.001 CA 2 23125 20625 10000 22500 11250 3750 CA 3 25000 23125 8750 15625 13750 0 CA 4 21250 15000 18125 11250 0 625 3 hours CA 1 42500 16250 13750 15625 3750 0 0 CA 2 26250 15000 5000 37500 15625 6875 CA 3 32500 20625 7500 25000 16250 0 CA 4 31875 13125 18750 15000 3750 0 *Post hoc (2 hours) Human Serum vs. BHIB p=0.003; Human Serum vs. TSB p=0.024 **Post hoc (2 hours) Sheep Serum vs. BHIB p=0.006 ***Post hoc (3 hours) MHB vs. BHIB p=0.004 ****Post hoc (3 hours) Human Serum vs. BHIB p=0; Human Serum vs. Sheep Serum p=0.03; Human Serum vs. TSB p=0.001; Human Serum vs. FBS p=0.003 No difference was shown between MHB and human serum in post hoc analysis (p>0.05). The number of germ tubes between broth media were highest in MHB, compared to TSB and BHIB (p=0.015 and p=0.009 at two- and three-hour incubation periods, respectively) as shown in Table 3. At the 24- hour incubation period, the fungi experienced rapid growth into hyphae. Therefore, further analysis was not performed in this incubation period. Table 3. Number of Germ Tubes Formed on Broth Media (number/μL) Time Fungi Media p-value MHB TSB BHIB 2 hours CA 1 11250 5625 5000 0.115 CA 2 22500 11250 3750 CA 3 15625 13750 0 CA 4 11250 0 625 3 hours CA 1 15625 3750 0 0.009 CA 2 37500 15625 6875 CA 3 25000 16250 0 CA 4 15000 3750 0 *Post hoc (2 hours) MHB vs. BHIB p=0.015 **Post hoc (3 hours) MHB vs. BHIB p=0.009 23 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Indonesian Journal of Tropical and Infectious Disease, Vol. 11 No. 1 January–April 2023: 8–26 Table 2. Number of Germ Tubes Formed on Serum and Broth Media (number/μL) Time Fungi Media p-value Human Serum Sheep Serum FBS MHB TSB BHIB 2 hours CA 1 14 28.89 22.22 17.775 24.445 2.22 0.005 CA 2 26 38 37.78 22.22 26 17.78 CA 3 20 33.335 26.665 23.33 31.115 0 CA 4 30 55.555 38 33.335 0 10 3 hours CA 1 26.665 53.33 26 31.115 14 0 0.155 CA 2 57.78 50 42 77.775 95.555 48.89 CA 3 28.89 36 34 37.78 57.78 0 CA 4 77.78 44.665 22 73.335 26.665 0 *Post hoc (2 hours) BHIB vs. Sheep Serum p=0.003 *Post hoc (2 hours) BHIB vs. FBS p=0.039 In the measurement of length, the longest germ tube was found in sheep serum at two- hour incubation period, followed by FBS, MHB, TSB, human serum, and BHIB. No difference was found at the three-hour incubation period as shown in Table 4. No broth media was found superior to the other in terms of the germ tubes length as shown in Table 5. Table 5. Length of the Germ Tube Formed on Broth Media (μm) Time Fungi Media p-value MHB TSB BHIB 2 hours CA 1 17.775 24.445 2.22 0.998 CA 2 22.22 26 17.78 CA 3 23.33 31.115 0 CA 4 33.335 0 10 3 hours CA 1 31.115 14 0 0.132 CA 2 77.775 95.555 48.89 CA 3 37.78 57.78 0 CA 4 73.335 26.665 0 Germ Tube Induction in Agar The number of germ tubes formed was highest in MHA. However, results were not significant both in two- and three-hour incubation periods as shown in Table 6. Table 6. Length of the Germ Tube Formed on Agar Media (μm) Time Fungi Media p-value MHA TSA BHIA 2 hours CA 1 +3 +1 0 0.408 CA 2 +6 +1 +2 CA 3 +4 +1 +1 CA 4 +1 0 0 3 hours CA 1 +1 0 0 1 CA 2 +6 +1 +1 CA 3 +2 +1 +1 CA 4 0 0 0 Hyphal morphogenesis is one of the most investigated virulence attributes of C. albicans. The ability of C. albicans to undergo reversible morphological transition could be triggered by variety of environmental condition.20–22 Serum, especially human serum, contains several important components that promote germ tube formation; therefore, it accounts as strong inducer for yeast-to-hyphae formation. Burch et al. (2018) stated that human serum fraction revealed signs of bacterial peptidoglycan (PGN)-like molecules which highly active for hyphae induction.23 Glucose could also act as morphogen, which in the certain amount could stimulate morphogenesis of C. albicans.24,25 Human serum, sheep serum, and FBS have approximately 1 mM to 10 mM of glucose.26– 28 This amount of glucose is optimal for germ tube formation according to previous study.24 Moreover, combined with exposure of 37°C and neutral pH environment, serum could inhibit NRG1 transcription, a potent inhibitor for hyphal formation (Su et al. 2018).29 This exposures to 37°C and neutral to alkaline pH, could induce hyphal growth through the Cek1 mitogen-activated protein kinase pathway (MAPK pathway) and the Rim101-pH sensing pathway, respectively.21 Studies by Hilmioglu et al. (2007) found that human serum was superior with the highest number of positive germ tube.30 In concordance to previous data, present study found that human and sheep serum were capable of inducing the highest number and longest germ tube, respectively, compared to other 24 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Rivaldi Ruby, et al. Germ Tube Induction Test media. This study also found that extended incubation period on serum led to an increasing number of hyphae. Long incubation period causes deprivation of nutrient and energy which leads to more effective hyphal growth.31,32 Utilization of human serum for germ tube induction has few drawbacks despite its superiority to other media. Serum has to be fresh otherwise stored serum could decrease germ tube production.12 Some serum could have biological inhibitor present in it. Wich et al. (2021), found that human serum antibodies have the capability to inhibit adherence of C. albicans to epithelial cells.33 Moreover, Ding et al. (2014) stated that germ tube formation in RPMI 1640 medium was delayed in the initial stage of the culture (within 90 min) in the presence of serum, although the number of hyphae was gradually increased to normal after extension of incubation period (from 2h to 3h).31 Presence of these biological inhibitor could cause inconsistent result in different batches of serum. Lastly, serum preparation poses possible risk of biohazard.12 In this study, several standardized media were studied to find comparable alternative of serum. Application of commercially available media also facilitates further culture and production of germ tube antigen for serological test. Broth medium also have essential substances for the growth and morphogenesis of C. albicans. MHB, TSB, and BHIB were tested in their ability to promote germ tube formation. As stated above, elevated temperature and neutral pH were the one main inducer for morphogenesis.29 Moreover, all media contains nutritive compounds that are necessary for fungal growth. Amino acids are one potent inducer present in the medium. It promotes yeast-to- hyphal transition through the cAMP-PKA pathway.21 In this study, MHB, TSB, and BHIB contain amino acids which further facilitate hyphal formation.21,22 Furthermore, in MHB, C. albicans showed the highest number of germ tube formation. One possible explanation is that it contained starch components with protective colloid roles against toxic compounds in the medium.34 BHIB is the most nutritious medium with combination of brain and beef infusion (a total of 17.5 g/l), protease peptone (10 g/l), and dextrose (2 g/l) which provide carbon, nitrogen, amino acids, and other nutrients.34 However, C. albicans showed lowest number of germ tube in BHIB. An exact explanation of this phenomenon is unknown. It seems that high nutrient provision is not an inducer of morphogenesis. According to Mba et al. (2020), C. albicans exhibits metabolic flexibility and filamentous growth in the condition of nutrient starvation.34 It is difficult to identify which media facilitate better morphogenesis based on previous studies, since results were mostly conflicting. Hilmioglu et al. (2007) showed that BHIB surpassed TSB as morphogenesis inducer, while Yakasiri et al. (2020) concluded that TSB exceeds MHB and BHIB.30,35 Different results could be attributed to different strain, media quality, and research conditions. Interestingly, Atalay et al. (2017) found that MHA was the best media for germ tube induction compared to human serum.18 Distinct factor affects the yeast-to-hyphal transition in agar media. Villa et al. (2020) stated that C. albicans hyphal growth in agar is influenced by the embedded surroundings or conditions. This is achievable through the upregulation of CZF1 transcription factor when the fungi are inoculated within the agar matrix.9 In present study, higher number of germ tube was found in MHA compared to other agar media although the result was not significant. STRENGTH AND LIMITATION To our knowledge, this is the first study that compares several media by measuring the number and length of the germ tubes. However, this study has several limitations. Lack of fungal strain prevents generalization of the result to other strain of C. albicans. 25 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Indonesian Journal of Tropical and Infectious Disease, Vol. 11 No. 1 January–April 2023: 8–26 Wider variety of culture medium could be used, since RPMI 1640 and YEPD broth also showed potential in previous studies.12,31 CONCLUSIONS This study showed that certain media in a specific environmental condition could facilitate hyphal growth that initially appears as germ tube formation. Human serum is a strong inducer of morphogenesis. Incubation of C. albicans in standardized medium such as MHB and TSB, coupled with 37°C environmental temperature and neutral pH, is also adequate to facilitate such phenomenon. Those media are preferred to human serum because it is readily available, routinely used in daily microbiology laboratories, and it provides stable result between batches. ACKNOWLEDGEMENT We would like to thank Linda, Yasir, and Riska, laboratory workers in the Department of Parasitology and Department of Microbiology, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia for assisting all laboratory procedures in this study. ETCHICAL CLEARANCE Ethical clearance was obtained from the Atma Jaya ethical committee with the number 01/06/KEP-FKUAJ/2020. FUNDING This research was funded by the School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia. CONFLICT OF INTEREST The authors declare that they have no conflict of interest. AUTHOR CONTRIBUTION Designed the study, collected and analyzed the data, and also prepared the manuscript: RR and EAS. A scientific adviser in the field of mycology: SS and SVK. All authors read and approved the final manuscript. REFERENCES 1. Bongomin F, Gago S, Oladele RO, Denning DW. Global and multi-national prevalence of fungal diseases—estimate precision. 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