Publicação
Molecular epidemiology and evaluation of Candida spp. persistence in the vaginal mucosa, in chronic cases of vulvovaginal candidosis
| Resumo: | Vulvovaginal candidosis is an infection that affects most women of childbearing age; however epidemiological data on its incidence and risk of chronicity still need clarification, knowing that it varies from region to region. This study aimed to evaluate, in 93 vaginal isolates from 36 women of childbearing age with episodic (16) and/or chronic (20) vulvovaginal candidosis, virulence factors, genotypic characteristics and their relationship with chronicization of vulvovaginal candidosis, and to compare the characteristics of fungi from the two clinical groups in order to obtain differences that allow us to understand the causes of chronicity. We identified the strains phenotypically using the chromogenic medium CHROMagar and analysis of biochemical profiles. We evaluated the minimum inhibitory concentration of azole compounds (fluconazole and clotrimazole) using the in vitro microdilution method according to the CLSI standard at two different pHs (pH4.5 and pH7) for fluconazole, the ability to form biofilms using the crystal violet method according to Sherry et al, 2017, the ability to: a) form germ tube in Candida albicans strains according to the methodology described by Ellepola and Samaranayake, 2008, b) produce phospholipase using the adapted method Price et al, 1982, c) produce proteinase described by Vinodhini et al, 2016, d) cell wall hydrophobicity and e) ability to resist high pH. Adhesion to HeLa cells (human cervical cancer epithelial cell line) and resistance to phagocytosis by RAW 264.7 cells (murine macrophages) were tested in vitro, as well as evaluating their genetic similarity through the analysis of DNA profiles (molecular typing by RAPD). The antifungal activity of NaHCO3 against C. albicans ATCC 10231 and 12 clinical strains VVC was also determined using the microdilution method, biofilm formation and germ tube (hypha) formation in the presence of NaHCO3. For analysis and comparison of data from the two clinical groups, we used the graphpad prism 7 program for statistical analysis (χ2 test). Results: C. albicans was the most frequent species. When we compared the results of the determination of the minimum inhibitory concentration of the isolates from the two clinical groups, we did not find significant differences between them. We found that there were no significant differences between groups regarding the distribution of the number of biofilm-forming strains in sporadic cases of vulvovaginal candidosis or chronic vulvovaginal candidosis. In sporadic vulvovaginal candidosis (considering only strains with moderate germ tube capacity) there was a greater (p < 0.05) capacity to produce germ tubes in relation to chronic vulvovaginal candidosis. Regarding the proportion of isolates strong in producing phospholipase, no significant differences were found between sporadic and chronic isolates. The evaluation of the DNA profiles of consecutive isolates of chronic vulvovaginal candidosis revealed differences. Yeast isolates involved in sporadic infection adhered in greater proportion to HeLa cells (related to their ability to produce biofilm) (p < 0.05), while the ability to evade phagocytosis (related to high protein production) (p < 0.05) was more evident in the chronic isolates; the resistance of sporadic cases to phagocytosis related to a greater hydrophobicity of the cell wall (p < 0.05). We found that C. albicans had the ability to proliferate in broths over a wide range of pH values (7.40 to 9.77) and that the minimum inhibitory concentration of NaHCO3 against C. albicans was 12.5 mg/ml (pH 8.97). It was not possible to determine the minimum lethal concentration, which suggests that it is a fungistatic mechanism of action. C. albicans cells exposed to NaHCO3 (at concentrations at least twice the minimum inhibitory) showed a 1.5-fold reduction in the normal growth rate and a 93% reduction in hyphae-producing cells, when compared to non-exposed cells to NaHCO3. Furthermore, there was a 50% reduction in biofilm mass when C. albicans cells were exposed to four times the minimum inhibitory concentration. Conclusions: Our results show that, interestingly, one of the strategies for Candida spp persists in the host and express few virulence factors, thus triggering a lower defense reaction, and that isolates from episodic cases are strong producers of biofilms and germ tubes, which helps to adhere to Hela cells (and therefore to the vaginal epithelium), probably related in some way to the hydrophobicity of the wall. Furthermore, when in contact with macrophages these isolates persist, do not form as much biofilm or adhere as well to the epithelium, but are better resistant to phagocytosis because they produce more proteases and are more resistant to pH variations. The strains originating from chronic cases also have the ability to model virulence factors over time, which allows them to better persist in the host. We conclude that in the presence of NaHCO3 the growth of clinical and collection isolates (ATCC 10231) had, regardless of pH, reduced growth, attenuation of the formation of biofilms and hyphae, so this compound is profiled as an adjuvant for therapy with a view to controlling the chronicization of vulvovaginal candidosis. |
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| Autores principais: | Gonçalves, Paula Cristina Gonçalves Faria |
| Assunto: | Fatores de virulência Candidose vulvovaginal Cronicização Recorrência Persistência Resistência aos antifúngicos |
| Ano: | 2022 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade da Beira Interior |
| Idioma: | inglês |
| Origem: | uBibliorum |
| Resumo: | Vulvovaginal candidosis is an infection that affects most women of childbearing age; however epidemiological data on its incidence and risk of chronicity still need clarification, knowing that it varies from region to region. This study aimed to evaluate, in 93 vaginal isolates from 36 women of childbearing age with episodic (16) and/or chronic (20) vulvovaginal candidosis, virulence factors, genotypic characteristics and their relationship with chronicization of vulvovaginal candidosis, and to compare the characteristics of fungi from the two clinical groups in order to obtain differences that allow us to understand the causes of chronicity. We identified the strains phenotypically using the chromogenic medium CHROMagar and analysis of biochemical profiles. We evaluated the minimum inhibitory concentration of azole compounds (fluconazole and clotrimazole) using the in vitro microdilution method according to the CLSI standard at two different pHs (pH4.5 and pH7) for fluconazole, the ability to form biofilms using the crystal violet method according to Sherry et al, 2017, the ability to: a) form germ tube in Candida albicans strains according to the methodology described by Ellepola and Samaranayake, 2008, b) produce phospholipase using the adapted method Price et al, 1982, c) produce proteinase described by Vinodhini et al, 2016, d) cell wall hydrophobicity and e) ability to resist high pH. Adhesion to HeLa cells (human cervical cancer epithelial cell line) and resistance to phagocytosis by RAW 264.7 cells (murine macrophages) were tested in vitro, as well as evaluating their genetic similarity through the analysis of DNA profiles (molecular typing by RAPD). The antifungal activity of NaHCO3 against C. albicans ATCC 10231 and 12 clinical strains VVC was also determined using the microdilution method, biofilm formation and germ tube (hypha) formation in the presence of NaHCO3. For analysis and comparison of data from the two clinical groups, we used the graphpad prism 7 program for statistical analysis (χ2 test). Results: C. albicans was the most frequent species. When we compared the results of the determination of the minimum inhibitory concentration of the isolates from the two clinical groups, we did not find significant differences between them. We found that there were no significant differences between groups regarding the distribution of the number of biofilm-forming strains in sporadic cases of vulvovaginal candidosis or chronic vulvovaginal candidosis. In sporadic vulvovaginal candidosis (considering only strains with moderate germ tube capacity) there was a greater (p < 0.05) capacity to produce germ tubes in relation to chronic vulvovaginal candidosis. Regarding the proportion of isolates strong in producing phospholipase, no significant differences were found between sporadic and chronic isolates. The evaluation of the DNA profiles of consecutive isolates of chronic vulvovaginal candidosis revealed differences. Yeast isolates involved in sporadic infection adhered in greater proportion to HeLa cells (related to their ability to produce biofilm) (p < 0.05), while the ability to evade phagocytosis (related to high protein production) (p < 0.05) was more evident in the chronic isolates; the resistance of sporadic cases to phagocytosis related to a greater hydrophobicity of the cell wall (p < 0.05). We found that C. albicans had the ability to proliferate in broths over a wide range of pH values (7.40 to 9.77) and that the minimum inhibitory concentration of NaHCO3 against C. albicans was 12.5 mg/ml (pH 8.97). It was not possible to determine the minimum lethal concentration, which suggests that it is a fungistatic mechanism of action. C. albicans cells exposed to NaHCO3 (at concentrations at least twice the minimum inhibitory) showed a 1.5-fold reduction in the normal growth rate and a 93% reduction in hyphae-producing cells, when compared to non-exposed cells to NaHCO3. Furthermore, there was a 50% reduction in biofilm mass when C. albicans cells were exposed to four times the minimum inhibitory concentration. Conclusions: Our results show that, interestingly, one of the strategies for Candida spp persists in the host and express few virulence factors, thus triggering a lower defense reaction, and that isolates from episodic cases are strong producers of biofilms and germ tubes, which helps to adhere to Hela cells (and therefore to the vaginal epithelium), probably related in some way to the hydrophobicity of the wall. Furthermore, when in contact with macrophages these isolates persist, do not form as much biofilm or adhere as well to the epithelium, but are better resistant to phagocytosis because they produce more proteases and are more resistant to pH variations. The strains originating from chronic cases also have the ability to model virulence factors over time, which allows them to better persist in the host. We conclude that in the presence of NaHCO3 the growth of clinical and collection isolates (ATCC 10231) had, regardless of pH, reduced growth, attenuation of the formation of biofilms and hyphae, so this compound is profiled as an adjuvant for therapy with a view to controlling the chronicization of vulvovaginal candidosis. |
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