Inadequate laboratory support in the sub-Saharan African has hampered the laboratory diagnosis involving culture, isolation and identification of various Candida species. This present study attempted to identify the most prevalent Candida species causing RVVC. Previous study conducted in Ghana reported C. albicans followed by C. glabrata as the most common isolated species (Adjapong et al., 2014). Good Laboratory Practices could improve the data on the aetiology of Candida infection among women in Africa.
VVC can be managed with either topical or oral antifungal agents. It has been reported that a single oral dose of fluconazole can lead to total cure of VVC (Pappas et al., 2004). In Ghana over-the-counter acquisition of drugs and other pharmaceuticals renders many drugs potential for abuse. Most abused drugs are the topical clotrimazole creams and vaginal tablets. They are easy to administer and are safe and effective, so are usually the first options for most women when they get VVC. In majority of the cases fluconazole is the antifungal agent used after failed attempts with clotrimazole. Nevertheless fluconazole is the drug used for first-line treatment of VVC in most parts of the world (Fan et al., 2008; Matheson and Mazza, 2017).
Due to the overuse without proper laboratory diagnosis and medical prescription of both orthodox, herbal medicines and other natural products in the treatment of different forms of vaginal infections in Ghana, there has been increasing number of Candida species causing infections. Just as the Candida species are varied in the aetiology of their infections, so also drug is resistance among Candida species vary to commonly prescribed antifungals in Africa (Africa and dos Santos Abrantes, 2016). Majority of the studies have attributed the resistance of the yeast, to yeast varieties, immunosuppression and age of the host (Africa and dos Santos Abrantes, 2016) in addition to intrinsic resistance among Candida strains. None of Candida isolates in our present study was resistant to itraconazole, a situation which may be attributable to less availability of itraconazole on the Ghanaian market and also hardly prescribed. Resistance to itraconazole has been previously reported in the United Kingdom (UK) among C. dubliniensis and C. albicans isolates (Venkateswarlu et al., 1996; Whaley et al., 2017; Tsitsopoulou et al., 2018). Fluconazole resistance was demonstrated in 18% of C. albicans, and 93% among C. krusei isolates from this present study. This high proportion of C. krusei isolates resistance to fluconazole is similar to values obtained elsewhere (Massa et al., 2018) and the results were attributed to over dependence on fluconazole for treatment of Candida vaginitis infections (Revie et al., 2018). In this study, it was discovered that 64–83% of the Candida species isolated from the women with RVVC in the Nkawie Government Hospital, Ashanti Region, Ghana were resistant to amphotericin B. This pattern of Candida resistance to amphotericin B is unexpected because the drug is usually reserved for treating serious systemic fungal infections. Amphotericin B resistance has also been observed in 10–15% of C. krusei fungaemia (Dudiuk et al., 2019). Reports of emergence of resistance in some of the strains of Candida could be worrisome especially in resource limited settings like Africa. The highest and widest range of resistance (10–83%) of the Candida isolates to the antifungal agents investigated in the study was observed for voriconazole. Overexpression of multidrug resistance genes is responsible for the reduced susceptible proportions of Candida strains to voriconazole. These have been identified in fluconazole and voriconazole-resistant strains of C. parapsilosis too (Balkan et al., 2019; Hu et al., 2019).
There are different resistance mechanisms but resistance to azole antifungal drugs is mostly due to modifications in the target enzyme as a result of mutations (Balkan et al., 2019; Brilhante et al., 2019). Low access of azole drugs to the target site is also due to the over-expression of the ERG11 gene. Resistance to azole antifungal agents is of much concern as these drugs are mostly dependent upon for the treatment of candidiasis. This is because of their safety and effectiveness against most Candida strains isolated from vulvovaginal infections (Felix et al., 2019). The detection of ERG11 genes as observed in the present study is similar to previous findings in other parts of the world (Hou et al., 2019; Sardari et al., 2019). Apart from the over-expression and mutations in the ERG11 gene, another major mechanism mostly exhibited by C. glabrata which confers resistance to azole antifungals is the up-regulation of multidrug efflux transporter genes such as CDR (Navarro-Rodríguez et al., 2019). Most C. glabrata strains therefore express CDR1 and CDR2 genes over ERG11 genes and this may have accounted for the non-detection of ERG11 in azole-resistant C. glabrata in this study (Whaley et al., 2017).
Virulent factors facilitate the ability of Candida species to cause infections (El-Houssaini et al., 2019). These aid the yeast to protect itself against antibodies and phagocytic activity of the host immune system (Oliver et al., 2019). About 95.5% of C. albicans produced the most biofilms followed by 78% C. glabrata produced biofilms, and then C. krusei (67.4%) and C. tropicalis (51.7%) as presented in Table 3. Biofilm formation increases the ability of Candida species to withstand host defenses and helps in establishing a reservoir for continuing and recurrent infections (Ishchuk et al., 2019). Infections from biofilm forming Candida species are therefore associated with higher morbidity, recurrence and then mortality rates increase in systemic infections.
Hemolysins are putative virulence factors contributing to the pathogenicity of Candida species by facilitating hyphal invasion (El-Houssaini et al., 2019). Hemolytic activity is therefore a factor used to initiate an infection. It is used to break down cells and enables the yeast to proliferate to cause the irritation and oedema of the vulva which is often associated with Candida infections. The high hemolytic activities were observed for all the Candida species investigated (Table 6). Candida species produce hydrolases which play important roles in adherence, penetration, invasion and destruction of host tissues (El-Houssaini et al., 2019). Candida species are capable of producing exo-enzymes but the potency varies among species and depends on the site of infection (Naglik et al., 2019). Phospholipase is a hydrolytic enzyme which can act by damaging the host cell membrane and can facilitates the invasion by the yeast. This is made possible as the enzyme hydrolyzes phospholipids into fatty acids therefore exposing receptors for adhesion (Maheronnaghsh et al., 2019; Melo et al., 2019). Majority of C. albicans (90.9%) produced phospholipases, and so did the other non-albicans Candida species isolated (Table 6). All the biofilm forming Candida isolates found in this study also had phospholipase activity. Studies have shown that enzyme activity, hydrophobicity and the ability for biofilm formation are important factors that are responsible for the pathogenicity of various Candida species (Sobel, 2016; Oliver et al., 2019). These virulent factors together with resistance mechanisms may act synergistically to be responsible for the recurrent infections in women from Nkawie Government Hospital, Ashanti Region, Ghana. This study had established that antifungal resistance was widespread among the isolates, though no isolate was resistant to itraconazole. Resistance proportion to voriconazole was high and was associated with C. glabrata. The most common Candida specie noted for women with recurrent vaginal discharge was C. glabrata. It is recommended that culture and antifungal sensitivity testing should be performed routinely on Candida isolates as it is done on many bacteria isolates from clinical specimens. This may help in the selection of appropriate antifungal agent(s) to stem recurrence among the women. A larger study to determine degree of spread of ERG11 genes in Ghana is suggested.