Prevalence and Antifungal Susceptibility Pattern of Candida Isolates from Women with Recurrent Vulvovaginal Candidiasis in Ghana - A Review of Laboratory Reports.

Vulvovaginal candidiasis (VVC) is a common infection among women of childbearing age, and few of these women experience recurrent vulvovaginal candidiasis (RVVC). The study was aimed at determining the virulent factors, and antifungal susceptibility of the Candida species isolated from women with RVVC attending the Nkawie Government Hospital, Ashanti-Region, Ghana. Over a 6–month period (October 2016 to March 2017), a total of 288 women with RVVC were evaluated. Isolation of the yeast was performed after the inoculation of the vaginal specimens onto Sabouraud Dextrose Agar (SDA), and incubated for 24-48 hours at 37 o C. The isolates were identied by standardized conventional methods. The enzymatic activities of esterase, phospholipase, haemolysis and biolm production were evaluated for the identication of the yeast isolates. Susceptibility to antifungal agents was determined by using the Kirby-Bauer disk diffusion method. Azole resistant isolates were further tested for ERG11 gene which encodes the enzyme (cytochrome P450 lanosterol 14-α-demethylase) by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Vaginal swabs cultures of 200 women (64.4) from 288 samples yielded Candida species. Candida albicans was the commonest isolated specie (33.0%), followed by Candida glabrata (29.5 %), Candida tropicalis (23.0%), and Candida krusei (15.5%). Hemolysin production, phospholipase enzyme activity, and biolms formation were found in 84.5%, 83%, 77.5%.of the isolates respectively. Most phospholipase producing Candida isolates also formed biolms. All Candida spp isolated were susceptible to itraconazole while majority of them were resistant to voriconazole. ERG11 genes were detected in 11.1% of Azole resistant Candida species. There is a signicant increase in the rate of antifungal resistance among the Candida isolates to uconazole and voriconazole. There is need for continuous surveillance as well as antifungal susceptibility testing on the Candida spp to guide therapy. A larger epidemiological study is also advocated to determining the degree of spread of ERG11 genes. ERG11-genes encode the enzyme (cytochrome P450 lanosterol


Introduction
Vulvovaginal candidiasis (VVC) is a common infection caused by Candida species among women of childbearing age, and 5-8% of these women experience recurrent vulvovaginal candidiasis (RVVC). Symptoms include vaginal itching or soreness, dyspareunia, discomfort when urinating and in some cases abnormal vaginal discharge (Melo et al., 2019). Even though most cases of VVC are mild, some women can develop severe infections that lead to redness, swelling and cracks in the vaginal wall (Melo et al., 2019). It has been estimated RVVC affects about 138 million women annually with a global prevalence of 3871 per 100, 000 women while 372 million women are affected over life time (Denning et al., 2018). [25][26][27][28][29][30][31][32][33][34] year group has the highest prevalence (9%) according to Denning et al. (2018). The risk of acquiring RVVC depends on many host-related and behavioral factors like age, pregnancy, diabetes mellitus, therapeutic immune suppression, locality and social economic status ( Candida species are commensals in various ecological niches of the human body (Rajilić-Stojanović and de Vos, 2014), by co-existing with the normal bacterial ora in human hosts. The growth of Candida species is continually being restricted by the innate immune system and complex bacterial microbiome dynamics.(Ilkit and Guzel, 2011) Some of these bacteria produce molecules which inhibit the growth of the yeast cells (Maheronnaghsh et al., 2019). Candida can become a pathogen when the normal bacterial ora are disturbed leading to opportunistic infections in the immunocompromised host when the organism overgrows, and causes infection (Viegas et al., 2019). Pathogenicity is enhanced as a result of the activation of the virulent factors in the organism (Liken and Kaufman, 2018), which the fungus uses to establish itself, and interact directly with the host cells (Chen and Huang, 2018). Candida species use nutrient acquisition, stress adaptation and immune evasion mechanisms as well as polymorphism, bio lm formation, and extracellular hydrolytic enzymes to overcome normal bacteria ora. Others include the host's immune system and subsequent invasion of the host tissues (Desai, 2018).
The azole drugs which include miconazole, clotrimazole, uconazole, and itraconazole are the commonest antifungal agents that are usually prescribed for treating VVC (Choukri et al., 2014). These imidazole and triazole compounds inhibit the production of the enzymes needed for the demethylation step in the synthesis of ergosterol, a sterol found in the plasma membrane of fungi, and it is known to maintain the integrity of the fungal cell wall (Whaley et al., 2017). Azole drugs are cheap and safe to use; however, the frequent use of these drugs can lead to the emergence of resistant strains (Wiederhold, 2017). The mechanism of resistance is the failure of the drugs to cross the yeast cell membrane due to over-expression and alteration of the target enzyme gene ERG11 that is involved in ergosterol production (Morschhäuser, 2016). Also, it has been found that the up-regulation of drug e ux pump genes such as CDR1 and CDR2 in some species of Candida also can results in the development of resistance to azole drugs. Missense mutations in the ERG11 gene can also lead to the alteration of the drug target site and result in drug resistant in Candida species (Whaley et al., 2017). Studies have documented the aetiology, pathogenesis, immunity and development of vaccines for Candida species that have been causing RVVC in women in different parts of the world (Sobel, 2002;Cassone, 2015

Wet lm preparation and Gram staining
The swabs were processed immediately upon arrival in the laboratory. The tube was shaken to dislodge materials from the swab into the saline to form a suspension. A drop of the saline suspension was placed on a clean grease free microscopic glass slide with cover slip, and then examined under the light microscope for budding yeast cells (Fig. 1). Cell counts of greater than 5 per high per eld (> 5 /HPF) and hyphae or pseudohyphae were taken as indicative of an active infection. The swabs were also subjected to Gram staining for the presence of budding yeast cells and pseudohyphae or hyphae (Fig. 2).

Germ-tube test
Colonies of the Candida isolates were speciated using modi cation of the germ tube test as described by

Determination of hemolytic activity
Sheep blood Sabouraud dextrose agar (SB-SDA) plates (HiMedia, Laboratories, India) were inoculated by placing 10 µl of Candida suspensions in saline on the plates using the spot inoculation method. The inoculated plates were incubated at 37 o C with 5% carbon dioxide (CO 2 ) jar for 48 hours. Hemolytic activity was indicated by the presence of a distinct translucent halo around the colony (Fig. 4).

Determination of phospholipase enzyme
Phospholipase enzyme production in the yeast isolates was determined by inoculating 10 µl drop of the yeast isolates on egg yolk agar. The agar plates were left opened on the laboratory bench for 5 min for the uid to dry. The plates were then covered and incubated at 37 o C for 3 days. Formations of zones of precipitation around the colonies represented phospholipase enzyme production by the isolate as presented in Fig. 5.

Bio lm production
After resuscitating the yeast by incubating in saline for 4 hours, 10 µl of the broth were added to Sabouraud dextrose hicynth broth (HiMedia Laboratories, India) contained in polystyrene tube and then incubated at 37 o C for 24 hours without agitation. This was followed by aspirating gently the top uid from the tube with a Pasteur pipette. Then the tube without the broth was washed three times with phosphate buffered saline (PBS) at a pH of 7.2 (HiMedia Laboratories, India). The tube was then half lled with 2% safranin stain, and incubated in the dark for 15 min, after which the excess stain was washed off with PBS. Visible lm adherent on the wall of the polystyrene tube was taken as indicative for bio lm formation by the yeast isolate ( Fig. 6).
Antifungal susceptibility testing of the yeast isolates fresh pure colonies of the Candida cells were dissolved in 500 µl, sterile distilled water in a sterile 1.5 ml/L micro-centrifuge tube. The Candida cells were disrupted using tissue homogenizer (Qiagen) for 3 min followed by centrifugation at 13,000 rpm for 3 min. The sediment was then subjected to DNA extraction using Q1Aamp DNA Mini kit according to the manufacturer's instructions.

PCR-RFLP analysis
The ERG11 genes which encode the enzyme (cytochrome P450 lanosterol 14-α-demethylase) from 27 azole resistant isolates and controls were ampli ed by the PCR method using primers with the following sequences: ERG11-F: (5'-CAAGAAGATCATAACTCAAT-3' and ERG11-R: (5'-AGAACACTGAATCGAAAG-3')' The ampli cation was carried out using a gradient thermal cycler (Techne TC-512, Bibby Scienti c Limited, USA) as previously described by Xiang    For the symptoms associated with C. albicans and non-albicans Candida, all the women who had RVVC (N = 200) had discharges. All the women had malodourous vagina discharges accompanied by vulva itching and burning sensation. Comparative analysis of the remaining three (3) symptoms; itching, burning sensation and odor associated with C. albicans and non-albicans Candida showed signi cant differences (P = 0.0005 and P = 0.043) for itching and burning sensation respectively (Table 3). were susceptible to itraconazole. The highest resistance proportion was against voriconazole where C. tropicalis recorded 79% followed by C. albicans (71%), C. glabrata (71%) and C. krusei (10%). High resistance proportions were recorded against uconazole with C. krusei registering 93% and C. glabrata (78%), C. tropicalis (20%) and C. albicans (18%). Details of the resistance proportions are presented in Table 5.  Vor-voriconazole ERG11 genes were ampli ed and detected in only two C. albicans and one C. tropicalis isolates investigated (Fig. 8). Hemolytic activity was the virulence factor found to be mostly expressed by all the Candida species recovered from the women with RVVC. About 90.9% of C. albicans were found to produce phospholipase whilst 95.5% formed bio lms and 97% produced hemolysin. With C. glabrata, 89.8% of the isolates produced phospholipase, 78% formed bio lms and 93.2% produced hemolysin. There was high production of hemolysins among the isolates of C. krusei (93.5%) and C. tropicalis (93.1%). Almost all Candida isolates which produced phospholipase were found to form bio lms ( Table 6). VVC can be managed with either topical or oral antifungal agents. It has been reported that a single oral dose of uconazole 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 rst options for most women when they get VVC. In majority of the cases uconazole is the antifungal agent used after failed attempts with clotrimazole. Nevertheless uconazole is the drug used for rst-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 uconazole is similar to values obtained elsewhere (Massa et al., 2018) and the results were attributed to over dependence on uconazole 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 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 ndings 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 upregulation of multidrug e ux 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 bio lms followed by 78% C. glabrata produced bio lms, and then C. krusei (67.4%) and C. tropicalis (51.7%) as presented in Table 3. Bio lm 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 bio lm 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 bio lm forming Candida isolates found in this study also had phospholipase activity. Studies have shown that enzyme activity, hydrophobicity and the ability for bio lm 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.

Conclusions
From the present study, the most prominent isolated species in RVVC infections in Ghana were C. albicans, C. glabrata, C. tropicalis and C. krusei. Non-albicans Candida species were noted to have almost all the virulent factors as C. albicans for the establishment of an infection. However, non-albicans Candida species may not be as virulent as C. albicans. C. glabrata species do not have all virulent factors as compared to C. albicans. This is because the C. glabrata species were unable to form hyphae and pseudohyphae. Also all the C. glabrata species had relatively small yeast cell sizes compared to C. albicans. The antifungal susceptibility testing showed that there was a signi cant increase in the rate of resistance among the Candida isolates to uconazole and voriconazole. It is therefore suggested that there is need for continuous surveillance as well as antifungal susceptibility testing on Candida spp and other microbial infections to guide therapy in Ghana. A larger epidemiological study is also advocated to determining the degree of spread of ERG11 genes.

Declarations
Ethics approval and consent to participate: The study was approved by the Joint Committee on Human Ethics, Research and Publication, School of Medicine and Dentistry, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana as well as from the Management of the Nkawic Government Hospital, Ghana. The objectives of the study were also explained to the study participants and those that were willing to take part were asked to sign or thumbprint a consent form before the samples were collected from them.
Adherence to national and international regulations: