Study design and setting
This was a cross-sectional study in which 35 oropharyngeal Candida isolates obtained from twenty-nine (29) PLHIV with OPC on ART at TASO Mulago National Referral Hospital and TASO Mbarara Regional Referral Hospital in Uganda were retrieved from the sample repository of our previous study titled “Distribution and antifungal susceptibility profile of oropharyngeal Candida species isolated from People Living with HIV in the Era of Universal Test and Treatment Policy in Uganda’’.
Ethical approval
This study was approved by the Makerere University School of Biomedical Sciences Research Ethics Committee (Reference Number; SBS-2022-254). In addition, administrative clearance to conduct the study was obtained from The AIDS Support Organization (TASO) Uganda Limited headquarters (Reference; TASO/ADMCOO3/2023-UG-REC-009). Written informed consent was obtained from the study participants before being enrolled in the study
Retrieved oropharyngeal Candida isolates
A total of 35 Candida isolates were retrieved from sample repository, 20 were C. albicans, while 15 were NAC. The NAC species comprised of Candida tropicalis (n = 4), C. glabrata (n = 4), C. parapsilosis (n = 2), C. dubliniensis (n = 2), C. krusei (n = 2), and C. lusitaniae (n = 1) previously identified using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) BioTyper 4.1 system (Bruker Daltonics) from the Mycology/Microbiology (College of American Pathologists accredited) laboratory at the Department of Medical Microbiology, College of Health Sciences, Makerere University, Uganda.
Preparation of retrieved oropharyngeal Candida isolates for virulence studies
Candida isolates that were preserved in brain heart infusion (BHI) broth and 10% glycerol at -80oC glycerol and brain heart infusion (BHI) were retrieved from sample repository freezer. The isolates were thawed by gently warming them to room temperature for 1 hour. A loopful of the culture was streaked onto sabouraud dextrose agar (Oxoid, Basingstoke, UK) supplemented with 50µg/1 ml gentamicin and then incubated at 37°C for 24–48 hours to isolate Candida species and the strains were suspended in sterile phosphate-buffered saline (PBS) and matched to 0.5 Mc Farland to carry out the virulence assays. For each virulence factor each isolate was tested in duplicate on two different occasions, and the mean of the 2 values was calculated.
Determination of phospholipase activity
The phospholipase activity was analyzed using the egg yolk agar method [16], [17]. Briefly, the egg yolk medium comprised Sabouraud dextrose agar (45.5g), sodium chloride (20.43g), calcium chloride (0.193 g), 70 ml of 10% v/v egg yolk emulsion and 630 ml of distilled water as previously described by Tsang et al.[18]. Approximately 10 µl of a standardized yeast suspension (108 CFU/ml) was pipetted, spotted onto fresh egg yolk agar plates and left to dry in a biosafety cabinet. The culture was then incubated at 37°C aerobically for 48 hours, after which the diameter of the precipitation zone around the colony was determined. Phospholipase activity (Pz) was measured by dividing the diameter of the colony (Cd) by the total diameter of the colony plus the precipitation zone (Pd). C. albicans ATCC 10231 was chosen as a positive control, while C. kefyr 2512 was used as a negative control [19].
Pz value = \(\frac{\text{C}\text{d}}{\text{C}\text{d}+\text{P}\text{d}}\)
Pz activity was scored as negative when Pz = 1, weak when Pz = 0.64–0.99, and strong when Pz ≤ 0.63, as previously described by Price et al., [17], indicating that the lower the Pz is, the greater the phospholipase activity.
Determination of proteinase activity
Proteinase activity was determined using the bovine serum albumin agar (BSA) method [20]. The BSA consisted of 0.1% KH2PO4, 0.05% MgSO4, 2% dextrose, 2% agar and 1% bovine serum albumin as previously described by Tsang et al. [18], and its final pH was adjusted to 4.5 using 1 M HCl and 1 M NaOH. Using a 24-hour-old culture, a yeast suspension of approximately 1x108 CFU/ml was prepared using 1 ml of 0.85% normal saline and a turbidometer. Ten microliters of the standardized yeast suspension was pipetted and spotted onto sterile BSA agar plates. The inoculated plates were then incubated at 37°C for 5 days under aerobic conditions. After incubation, the plates were fixed with 20% trichloroacetic acid and stained with 0.25% w/v Coomassie blue. Decolorization was performed by flooding the culture plates with 15% acetic acid. Proteinase activity was detected by the presence of a clear halo around the yeast colonies. The diameter of the halo clearance (Hd) zone relative to the diameter of the colonies (Cd) was used to assess the degree of proteinase activity (Prz) as previously described by Price et al. [17]. C. albicans ATCC 10231 was used as a positive control, and C. kefyr 2512 was used as a negative control for this experiment. Prz value =\(\frac{\text{C}\text{d}}{\text{C}\text{d}+\text{H}\text{d}}\)
Prz activity was scored as negative when the Prz value = 1, weak when Prz = 0.64–0.99, and strong when Prz ≤ 0.63, meaning that a low Prz value indicated strong production of the proteinase enzyme [17].
Determination of hemolysin activity
The hemolytic activity of Candida species was determined by the blood agar plate method [21]. Briefly, SDA (Oxoid) containing 7% sheep blood and 3% w/v glucose with the final pH adjusted to 5.6 ± 0.2 was used. Ten (10) microliters of standardized yeast suspension (108) was inoculated onto blood agar plates, which were then incubated at 37°C in 5% carbon dioxide for 48 hours. After incubation, a transparent/semitransparent zone around the inoculation site was considered to indicate positive hemolytic activity [21]. Beta (β), alpha (α) and gamma (γ) hemolysis was indicated by clear, greenish and no hemolysis, respectively. The ratio of the diameter of the colony (Cd) to that of the translucent zone of hemolysis (Hd) (mm) was used as the hemolytic index (Hz value). C. albicans ATCC 90028 was used as a positive control, and C. parapsilosis ATCC 2201 was used as a negative control.
Hz value = \(\frac{Cd}{Hd}\)
Determination of esterase activity
The esterase activity of Candida species was detected using the Tween 80 opacity test medium method [22]. The test medium was adjusted to a pH of 6.8 and consisted of 1% peptone, 0.5% NaCl, 0.01% CaCl2 and 1.5% agar. After the medium cooled (to 50°C), 0.5% Tween-80 was added. Ten microliters of the previously prepared suspension was carefully spotted on Tween 80 opacity test medium and left to dry. This mixture was then incubated at 35°C for 2–5 days under aerobic conditions. The presence of a halo around an inoculated site on tween 80 opacity test medium indicated positive esterase activity [23]. Esterase activity (Ez) was determined as the ratio of the diameter of the colonies (Cd) to the diameter of the clear halo with calcium precipitates around the colony (Hd) as previously described by Price et al. [17]. C. albicans ATCC10231 served as a positive control for this experiment.
Ez value= \(\frac{Cd}{Hd}\)
Ez activity is negative when Ez = 1, weak when Ez = 0.64–0.99 and strong when Ez ≤ 0.63. Thus, a low Ez value indicated strong esterase production.
Determination of Coagulase Activity
Coagulase activity was determined by the classical tube coagulase method [24]. Briefly, Candida isolates were first standardized to match 0.5 McFarland’s turbidity standard, and 0.5 ml of this standardized cell suspension was added to 0.5 ml of 10% rabbit plasma in a tube. The inoculated tubes were then incubated at 37°C and observed for clot formation after 4 hours. The presence of a visible clot that could not be resuspended by gentle shaking was considered coagulase positive. Test tubes without clots were re-incubated at 37°C and re-examined after 24 hours. Staphylococcus aureus ATCC 25923 was used as a positive control, while Staphylococcus epidermidis ATCC 14990 served as a negative control.
Determination of biofilm formation
The ability of Candida isolates to form biofilms was analyzed using the microtiter plate assay (Mtp) method[25]. Fresh 24-hour-old broth from Candida cultures was used for this assay. The cultures were grown in yeast peptone dextrose (YPD) broth, and the cell suspension was then adjusted to McFarland’s standard using fresh YPD broth. This suspension was further diluted 20-fold to a concentration of approximately 5x106 CFU/ml. Then, 180 µl of sterile YPD broth was aseptically transferred to sterile 96-well polystyrene microtiter plates. For each isolate, 3 wells in a column were used to produce triplicate results. Then, 20 µl of the standardized yeast suspensions were added to each well containing 180 µl of YPD for a final concentration of approximately 5x105 CFU/ml. The microtiter plates were then incubated at 37°C for 48 hrs. At the end of the incubation period, excess planktonic cells and broth were removed, and the plates were washed with phosphate-buffered saline 3 times to remove unattached cells and media components. The plates were blotted with blotting paper, inverted, and left to dry. The sessile cells from which biofilms formed were then fixed by adding 150 µl of methanol to the wells for 20 minutes. The methanol was then removed by inverting the plates, and the liquid was removed. The plates were then washed with phosphate-buffered saline and dried off with blotting paper. Two hundred microliters of 1% crystal violet was added, and the mixture was left to stand for 15 min at 37°C. At the end of the incubation period, crystal violet was added, and the plates were washed 3 times with Dulbecco’s phosphate-buffered saline and left to air dry. After air drying, the stained biofilms were resolubilized using 150 µl of 33% glacial acetic acid, and the plates were carefully agitated using a rotatory shaker. The optical density of the microtiter plates was measured spectrophotometrically at 620 nm with a spectrophotometer at 600 nm. Candida albicans ATCC 10231 was used as a positive control, while uninoculated wells that contained sterile YPD were used as negative controls and treated as blanks. The blank absorbance values (ODc) were used to determine whether biofilms were formed by the isolates. The wells containing isolates whose optical density (OD) values were greater than that of the blank well were considered biofilm producers.
The OD values were used to calculate the cutoff values (ODc) of the isolates for biofilm formation categorization and interpretation as previously described by Kırmusaoglu et al.[25] as follows: OD ≤ ODc = no biofilm production, ODc < OD ≤ 2ODc = weak biofilm production, 2ODc < OD ≤ 4ODc = moderate biofilm production, 4ODc < OD = strong biofilm producer
Data analysis plan
Statistical analyses were performed using Stata version 17.0 software. Descriptive statistics, proportions and means were used to summarize the distributions of different virulence attributes. The chi-square test was used to test the association between biofilm formation, coagulase activity between C. albicans and NAC. Student’s t test was used to determine the mean difference in phospholipase, proteinase, hemolysin and esterase activities between NAC and C. albicans, considering a P value < 0.05 to indicate statistical significance.