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 ~  Materials and Me...
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ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 3  |  Page : 364-368
 

Changing virulence factors among vaginal non-albicans Candida species


1 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
2 Department of Obstetrics and Gynaecology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Web Publication14-Nov-2018

Correspondence Address:
Dr. Rakesh Singh
Additional Professor, Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_94

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 ~ Abstract 

Background: Vulvovaginal candidiasis (VVC) is caused by overgrowth of Candida species in the female lower genital tract and most commonly caused by Candida albicans. The production of various virulence factors may attribute to their pathogenicity. Hence, this study was aimed to determine the production of various virulence factors of Candida spp. causing VVC. Materials and Methods: A total of 51 Candida spp. were isolated prospectively from 50 patients among 211 clinically suspected cases of VVC. The haemolytic activity, biofilm production, proteinase activity, phospholipase activity and esterase activity were detected by standard methods. Statistical analysis was performed using OpenEpi version 3.01. Results: Haemolytic activity was observed in 42 Candida isolates (82.4%), biofilm activity in 21 Candida isolates (41.2%), proteinase and esterase activity in 19 Candida isolates (37.3%) each and phospholipase activity in 15 Candida isolates (29.4%). Phospholipase activity was observed in all of the C. albicans strains, whereas all strains of Candida krusei were able to produce biofilm. All strains of Candida parapsilosis and 87% strains of Candida glabrata were haemolytic. Five of the eight C. glabrata strains were found to produce strong proteinase (Prz score ≤0.63). About 30.4% strains of C. glabrata and 20% strains of C. krusei were found to be positive for esterase activity. This is one of the few studies which revealed esterase activity among C. glabrata and C. krusei strains. Conclusions: This study highlighted that there is a change in the virulence factors among the non-albicans Candida species, especially C. glabrata strains which were haemolytic and produce strong proteinase activity and esterase activity. It may be one of the explanation of the most common causative agent of VVC in our study. Multicentric studies from this area might be required to get a more generalised conclusion.


Keywords: Candida glabrata, non-albicans Candida, virulence factors, vulvovaginal candidiasis


How to cite this article:
Kalaiarasan K, Singh R, Chaturvedula L. Changing virulence factors among vaginal non-albicans Candida species. Indian J Med Microbiol 2018;36:364-8

How to cite this URL:
Kalaiarasan K, Singh R, Chaturvedula L. Changing virulence factors among vaginal non-albicans Candida species. Indian J Med Microbiol [serial online] 2018 [cited 2019 Aug 18];36:364-8. Available from: http://www.ijmm.org/text.asp?2018/36/3/364/245400



 ~ Introduction Top


Vulvovaginal candidiasis (VVC) is caused by overgrowth of Candida species in the female lower genital tract, the vulva and the vagina.[1],[2] Candida albicans is the most common species isolated in 85%–90% of VVC.[3],[4] C. albicans is considered as the most virulent species and infections caused by this species represent an important public challenge worldwide. C. albicans adheres to vaginal epithelial cells in significantly higher numbers than do other Candida species. The second most common pathogen detected in VVC is Candida glabrata. It is followed by other non-albicans Candida (NAC) - Candida tropicalis, Candida parapsilosis, and Candida krusei.[3],[5] There is a recent shift in the fungal profile and rise in the NAC species infections. Many virulence factors of Candida spp. are responsible for its pathogenesis such as; ability to evade host defence, adherence to the surface epithelium, biofilm production, haemolytic activity and invasion by the production of extracellular enzymes such as phospholipase, proteinase and esterase.[6] Each species of Candida spp. exhibit different degree of virulence factors.[7] VVC by NAC are gradually increasing as a causative agent.[8] We observed C. glabrata (45.1%) as the most common causative agent of VVC in our hospital.[9] Very limited studies from India analysed the virulence factors from VVC isolates. This study was undertaken to know the virulence factors of Candida isolates obtained from VVC cases in our hospital.


 ~ Materials and Methods Top


A total of 211 clinically suspected cases of VVC were presented during 6 month period - July to December 2015 at the Department of Obstetrics and Gynaecology of Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India. High vaginal swabs were collected from the patients and inoculated on chromogenic culture medium (Hi-Media, Mumbai, India). The study was approved by the Institute Ethics Committee (Human studies). Fifty-one Candida spp. were isolated prospectively from 50 patients. Identification was made by standard conventional tests.[10],[11] C. glabrata (n = 23) was the most causative agent. It was followed by C. tropicalis (n = 12), C. albicans (n = 9), C. krusei (n = 5), and C. parapsilosis (n = 2). One mixed infection of C. glabrata and C. albicans was observed in one patient. It is published in J Clin Diagn Res.[9] Candida spp. were subcultured on Sabouraud's dextrose agar (SDA) slopes and stored in the refrigerator. Fresh subculture was made on SDA for testing of virulence factor characteristics. It was suspended in normal saline which was matched to a concentration of 2 McFarland standard. A volume of 10 μl of this yeast suspension (except biofilm production) was tested for the following virulence factors:

Haemolytic activity

The haemolytic activity was determined using glucose enriched SDA-based blood agar as described by Mann et al. with minor modification.[12],[13] A volume of 4 ml of fresh sheep blood was added to 100 ml of SDA which was supplemented with 3% glucose as a final concentration. The pH of the final medium was 5.6 ± 2. Plates were stored in the refrigerator. A volume of 10 μl of test strain was inoculated on a sugar-enriched sheep blood agar medium. The plates were incubated at 37°C in 5% CO2 for 48 h. The presence of positive haemolytic activity was determined as a distinct translucent halo around the inoculums site when viewed against transmitted light.

Biofilm production

Biofilm production was detected as described by Branchini et al.[14] after staining with safranin. A loopful of test strain from the SDA plate was inoculated into a tube containing 10 ml Sabouraud's dextrose broth supplemented (Hi-Media, Mumbai, India) with 8% of glucose. The tubes were incubated at 37°C for 24 h. After incubation, the broth was aspirated out, and the walls of the tubes were stained with 1% safranin. It was kept for 7 min, and then safranin was removed. Biofilm production was graded visually as negative, weak positive (1+), moderate positive (2+) and strongly positive (3+).

Proteinase activity

The Candida proteinase activity was detected as described by Staib with minor modification.[15],[16] The test was performed using 1% bovine serum albumin (BSA) medium supplemented with 2% dextrose, 0.1% KH2 PO4, 0.05% MgSO4 and 2% agar. All the reagents except BSA were mixed and autoclaved. About 1% of BSA solution was added to the autoclaved reagents at 50°C. BSA plates were stored in the refrigerator. A volume of 10 μl of test strain was inoculated onto the surface of BSA plate. After 5 days of incubation at 37°C, the plates were fixed with 20% trichloroacetic acid and stained with 1.25% amido black. It was decolourised by 15% acetic acid. The absence of amido black stain around the growth was considered the zone of proteolysis. The diameter was measured for proteinase production. The proteinase activity was calculated as the ratio of colony diameter to the proteolytic unstained zone diameter (Prz score). Prz score of 1 was considered negative, <1 was taken as positive, those with ≤0.63 was taken as strong positive which was described for phospholipase activity.[17]

Phospholipase activity

The estimation of phospholipase activity was detected as described by Price et al. with minor modification.[13],[17] Briefly, 200 ml of egg yolk agar medium was prepared with a final concentration of 13 g of SDA, 11.7 g NaCl, 0.111 g CaCl2 and 10% sterile egg yolk. All the reagents except egg yolk were mixed in 180 ml of distilled water and autoclaved. The 20 ml of supernatant of the egg yolk which was obtained after centrifugation (500 ×g for 10 min at room temperature) was added to the 180 ml of sterilised reagents. Egg yolk agar plates were stored in the refrigerator. A volume of 10 μl of test strain was inoculated onto the surface of the egg yolk agar medium. The plate was incubated at 37°C for 7 days. After the incubation, to determine the phospholipase activity, precipitation zone diameter around the yeast growth was measured. The ratio between colony and diameter of the zone and colony was calculated (Pz score). Pz score of 1 was considered negative, Pz score between 0.64 and 0.99 was taken as positive and the Pz score ≤0.63 was taken as strong positive.[17]

Esterase activity

The esterase activity was detected by tween 80 medium as described by Slifkin with minor modification.[18] It was prepared by taking 1000 ml of distilled water with peptone (10.0 g), NaCl (5.0 g), CaCl2(0.1 g) and agar (15 g). All the reagents were mixed well and autoclaved. After cooling the autoclaved reagents to 50°C, 5 ml of tween 80 was added. This 25 ml of the medium was poured into 90 mm petri-dish plate. The plates were stored in the refrigerator. 10 μl of test strain was inoculated on tween 80 medium by making an inoculum size of 10 mm in diameter. The inoculated plates were incubated at 37°C and observed regularly up to 10 days. The production of esterase by Candida isolate was determined by the presence of a halo around the inoculum when observed against the transmitted light. The diameter of the colony and diameter of the zone along with colony was measured. The ratio between colony and diameter of the zone and colony was calculated (Ez score). Ez score of 1 was considered negative, Ez score between 0.64 and 0.99 was taken as positive and the Ez score ≤0.63 was taken as strongly positive as for phospholipase activity.[17]

Statistical analysis

The categorical variables were expressed as frequency and percentages. The association of virulence factors and Candida spp. was compared using Chi-square test or Fisher's exact test using OpenEpi version 3.01 software. All statistical analysis was carried at 5% level of significance with a value of P < 0.05 was considered as statistically significant.


 ~ Results Top


Fifty-one Candida spp. obtained from clinically suspected cases of VVC were analysed for their virulence activity [Figure 1]. Haemolytic activity was observed in 42 Candida isolates (82.4%), biofilm activity in 21 Candida isolates (41.2%), proteinase and esterase activity in 19 Candida isolates (37.3%) each and phospholipase activity in 15 Candida isolates (29.4%). The species-wise distribution of virulence factors in vaginal Candida isolates is shown in [Table 1]. All strains of C. parapsilosis, 87% strains of C. glabrata, 83.3% strains of C. tropicalis, 77.8% strains of C. albicans and 60% strains of C. krusei were found to be haemolytic.
Figure 1: Virulence factors (a) haemolytic activity on glucose and blood enriched sabouraud's dextrose agar plate (b) biofilm production (c) proteinase activity on bovine serum albumin plate (d) phospholipase activity on egg yolk agar plate and (e) esterase activity on tween 80 opacity medium

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Table 1: Candida spp. wise distribution of virulence factors in vaginal Candida isolates

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All strains of C. krusei, 58.3% strains of C. tropicalis, 30.4% strains of C. glabrata, 22.2% strains of C. albicans and none of the strains of C. parapsilosis were found to be positive for biofilm production [Table 1]. Although low in numbers, significant high number of C. krusei strains were positive for biofilm production as compared with other Candida spp. (other than C. krusei isolated from VVC) strains (P = 0.02) as shown in [Table 2]. The biofilm production was found more with NAC (45.2%) as compared to C. albicans (22.2%). The highest grade (3+) of biofilm production was seen in all strains of C. krusei and 2 strains of C. tropicalis. Moderate grade (2+) of biofilm production was seen in 2 strains each of C. glabrata, C. albicans and C. tropicalis. Mild grade (1+) of biofilm production was seen in 5 strains of C. glabrata and three strains of C. tropicalis.
Table 2: Significant association of virulence factors and the Candida spp. among vulvovaginal candidiasis

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A total of 19 isolates (37.3%) were positive for proteinase activity. About 44.4% strains of C. albicans, 41.7% strains of C. tropicalis, 40% strains of C. krusei and 34.8% strains of C. glabrata were positive while none of the strain of C. parapsilosis was found to be positive for proteinase activity [Table 1]. Proteinase activity of C. albicans was compared with NAC and not observed as statistically significant (P = 0.89) as shown in [Table 2]. Prz score ≤0.63 was observed in 5 strains of C. glabrata, 2 strains each of C. albicans and C. krusei and a single strain of C. tropicalis. Prz score between >0.63 and <1 was observed in 4 strains of C. tropicalis, three strains of C. glabrata and two strains of C. albicans [Figure 2].
Figure 2: Species-wise distribution of vaginal Candida isolates with positive proteinase activity

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A total of 15 isolates (29.4%) showed phospholipase activity. All strains of C. albicans, 40% strains of C. krusei, 25% strains of C. tropicalis, 4.3% strains of C. glabrata were positive while none of the strains of C. parapsilosis were found to be positive for phospholipase activity [Table 1]. Significantly, high number of strains of C. albicans were positive for phospholipase activity as compared with NAC (P < 0.01) strains [Table 2]. All isolates of Candida spp. showed Pz score ≤0.63.

Nineteen (37.3%) vaginal Candida isolates showed positive for esterase activity. About 66.7% strains of C. tropicalis, 33.3% strains of C. albicans, 30.4% strains of C. glabrata, 20% strains of C. krusei were positive while none of the strains of C. parapsilosis were found to be positive for esterase activity [Table 1]. Significantly, high number of strains of C. tropicalis were positive [Table 2] for esterase activity as compared with other Candida spp. (other than C. tropicalis isolated from VVC) strains (P = 0.04). All of the yeast showed Ez score ≤0.63 of esterase activity except for single strain of C. albicans which was having Ez score >0.63.


 ~ Discussion Top


Candida species express several virulence factors to escape from the host defence mechanisms. Expression of virulence factors may vary depending on the infecting species, geographical origin, type of infection, site of infection and host reaction. Virulence factors play an important role to understand the pathogenesis of candidiasis. We analysed the detection of several virulence factors of Candida species such as haemolytic activity, biofilm production, proteinase activity, phospholipase activity and esterase activity. Very few studies from India analysed these virulence factors from VVC isolates.

Candida species produce haemolysin in glucose-enriched blood agar. This study was in contrast to the other study where all the strains of C. albicans and C. tropicalis were haemolytic.[7],[19] In correlation with our study, Udayalaxmi et al. observed that all strains of C. glabrata and C. krusei were haemolytic whereas 97.5% of the C. albicans and 94.7% of C. tropicalis strains were haemolytic.[20] The biofilm production was found more with NAC (45.2%) as compared to C. albicans (22.2%) and the similar results are illustrated by other authors.[21] Significantly, high number of strains of C. krusei was positive for biofilm production as compared with other Candida spp. (other than C. krusei isolated from VVC) and highest grade (3+) of biofilm production was seen in all strains of C. krusei. Hence, strong biofilm production was seen in strains of C. krusei than C. albicans. More research is required in this area to find out more cases of C. krusei from VVC producing biofilm.

Nineteen Candida isolates (37.3%) were positive for proteinase activity. Similar to our study Sachin et al.[16] reported that high proteinase production in C. albicans (82.1%) followed by C. tropicalis (80%); which were isolated from different clinical specimens. A study from Ozcan et al.[22] reported that proteinase activity was observed in 19 strains (90.5%) of C. albicans and in all C. tropicalis isolates. Surprisingly, five of the eight positive C. glabrata strains [Figure 2] were found to produce strong proteinase (Prz score ≤0.63). It may be an indication of a change in the virulence factors of C. glabrata strains in our geographical area. More study may be conducted in this field. Fifteen Candida isolates (29.4%) showed phospholipase activity and significantly high number of strains of C. albicans was positive for phospholipase activity as compared with NAC (P < 0.01) strains as shown in [Table 2]. This is in correlation with other studies which also observed more numbers of C. albicans strains with phospholipase activity.[16],[23] Our study is in correlation with Sachin et al. who also reported phospholipase activity among NAC isolates[16] and in contrast to the Fule et al. who did not observe phospholipase activity among vaginal NAC isolates.[23] Interestingly, all of the Candida spp. in this study showed strong phospholipase activity (Pz score ≤0.63).

In our study, we observed that a maximum number of strains having esterase activity was C. tropicalis and significantly high number of strains of C. tropicalis were positive for esterase activity [Table 2] as compared with other Candida spp. (other than C. tropicalis isolated from VVC) strains (P = 0.04). Slifkin[18] reported all strains of C. albicans and C. tropicalis were positive for esterase activity, but none of the C. glabrata and C. krusei showed esterase activity. Aktas et al.[24] also observed all strains of C. tropicalis and 98.3% strains of C. albicans have esterase activity while none of the strains of C. glabrata, C. parapsilosis, and C. krusei showed it. This is one of the few studies which is describing about esterase activity in C. glabrata and C. krusei isolates of VVC. This again reemphasis the change in virulence factors among C. glabrata strains which enable them to cause VVC in this geographical area. All of the yeast showed Ez score ≤0.63 of esterase activity except for single strain of C. albicans which was having Ez score >0.63.

The study was conducted on a low number of isolates and in one tertiary care hospital which is the limitation of the study. More study or multicentric study may be required in this field of virulence factors among strains of VVC.


 ~ Conclusions Top


Results of the present study depict that the high number of C. glabrata strains were haemolytic and produce strong proteinase activity. To the best of our knowledge, it is one of the few studies which describe esterase activity in C. glabrata strains. It indicates that there is a change in the virulence factors among the NAC species, especially C. glabrata strains which may also explain as it was observed as the most common causative agent of VVC. Hence, the multicentric study may be required to get a more generalised conclusion.

Financial support and sponsorship

We are thankful to Jawaharlal Institute of Postgraduate Medical Education and Research for providing financial support.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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