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

doi:10.21203/rs.3.rs-33278/v1

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

https://doi.org/10.21203/rs.3.rs-33278/v1

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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^oC. The isolates were identified by standardized conventional methods. The enzymatic activities of esterase, phospholipase, haemolysis and biofilm production were evaluated for the identification 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 biofilms formation were found in 84.5%, 83%, 77.5%.of the isolates respectively. Most phospholipase producing Candida isolates also formed biofilms. 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 significant increase in the rate of antifungal resistance among the Candida isolates to fluconazole 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.

Infectious Diseases

Vulvovaginitis

Candida species

antifungal resistance

fluconazole

voriconazole

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). The 25–34 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 (Donders and Sobel 2017). In a retrospective analysis of pertinent data compiled by the Microbiology and Infection Control Unit of the Latifa Hospital, in Dubai, the incidence of vaginal candidiasis significantly increased from 10.76% in 2005 to 17.61% in 2011, with average prevalence of 13.88% (Hamad et al., 2014). Different strains of Candida have been isolated from 21% of women who reported abnormal vaginal discharge at the Gynecological Department of the Komfo Anokye Teaching Hospital (KATH) in Kumasi, Ghana (Feglo and Narkwa 2012). Similar results were confirmed by Konadu et al. (2019), who reported prevalence of 36.5% of VVC among pregnant women attending antenatal clinic in the middle belt of Ghana.

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 flora 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 flora 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, biofilm formation, and extracellular hydrolytic enzymes to overcome normal bacteria flora. Others include the host’s immune system and subsequent invasion of the host tissues (Desai, 2018).

The azole drugs which include miconazole, clotrimazole, fluconazole, 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 efflux 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). However, in Ghana there are paucity of studies on the aetiology of RVVC especially infections caused by Candida species (Apea-Kubi et al., 2005; Abuquah, 2012; Essel et al., 2014). Even in those studies the virulent factors associated with the Candida species were not reported. This study was aimed to determining the prevalence of Candida species, their virulence factors, antifungal susceptibility pattern, and molecular characteristics of the isolates from women with RVVC attending the Nkawie Government Hospital, Ashanti-Region, Ghana.

Study design and sample collection

It was a cross sectional study carried out from October 2016 to March 2017 among women diagnosed with RVVC at the Out-Patient’s Department (OPD), Ante-Natal Clinic (ANC) and the Anti-Retroviral Therapy (ART) Unit of the Nkawie-Toase Government Hospital, Ashanti Region, Ghana. High vaginal samples (HVS) were collected from women who presented with history of RVVC by a trained midwife after duly signed informed consent. The samples were placed into the tubes containing Amies transport medium and transported immediately to the laboratory for analysis.

Wet film 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 field (> 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 modification of the germ tube test as described by Matare et al. (2017). Briefly, triplicates sets of test tubes containing 0.5-1.0 ml of pooled human serum were inoculated with 2–3 colonies of each isolate. The tubes were incubated at 37^oC for 3 hours, after which a drop of each suspension was placed on labeled microscopically for examination. The presence of germ tubes was used as presumptive identification of C. albicans.

Isolation of and identification of Candida species

The vaginal samples were cultured on Sabouraud Dextrose Agar (SDA), and incubated for 24–48 hours at 37^oC to isolate the Candida strains. Isolated strains on the SDA were inoculated on CHROMAgar™ Candida (HiMedia, Laboratories, India) and incubated at 37^oC for 48 hours for presumptive differentiations and identification of Candida albicans from non-albicans Candida. Growths with various colours were employed in the identification of Candida species (Fig. 3).

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^oC with 5% carbon dioxide (CO₂) 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 fluid to dry. The plates were then covered and incubated at 37^oC for 3 days. Formations of zones of precipitation around the colonies represented phospholipase enzyme production by the isolate as presented in Fig. 5.

Biofilm 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^oC for 24 hours without agitation. This was followed by aspirating gently the top fluid 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 filled with 2% safranin stain, and incubated in the dark for 15 min, after which the excess stain was washed off with PBS. Visible film adherent on the wall of the polystyrene tube was taken as indicative for biofilm formation by the yeast isolate (Fig. 6).

Antifungal susceptibility testing of the yeast isolates

Antibiotic susceptibility pattern of the yeast isolates and the reference controls (ATCC strains) were determined using the Kirby-Bauer disk diffusion method. Turbidity standard of 0.5 MacFarland of each of the yeast suspension was seeded on the surface of a sterile RPMI-1640 agar (HiMedia Laboratories, India). The following six different antifungal paper disks (Axiom Laboratories, India) were tested: amphotericin B (20 µg), clotrimazole (10 µg), fluconazole (25 µg), miconazole (50 µg), itraconazole (10 µg) and voriconazole (30 µg). Controls were set up using C. albicans (ATCC 10231) and C. albicans (ATCC 60193). All the plates were incubated aerobically at 37 °C for 24–48 hours. Diameters of zones of inhibition around the antifungal disks (Fig. 7) were measured and compared with the Clinical and Laboratory Standard Institute (CLSI, 2009) breakpoints.

Genotypic identification of the Candida species

Genotypic identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for the species identification. C. albicans (ATCC 10231) was used as a positive control in the PCR-RFLP analysis.

DNA extraction

DNA extraction was performed using the modifications of the methods of Elfeky et al. (2016). Briefly, 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 amplified by the PCR method using primers with the following sequences: ERG11-F: (5’-CAAGAAGATCATAACTCAAT-3’ and ERG11-R: (5’-AGAACACTGAATCGAAAG-3’)’

The amplification was carried out using a gradient thermal cycler (Techne TC-512, Bibby Scientific Limited, USA) as previously described by Xiang et al. (2013). Each reaction contained 10 µl of 2X Dream Tag Green Master mix, 1.5 µl of genomic DNA (template), 2 µl each of primer (10 µmol/l), in addition to change the annealing temperature (53^oC). Amplified PCR products were run on 2% agarose gel electrophoresis and visualized by UV transilluminator (BiometraT 3, Analytikjena, USA). RFLP analysis was performed as described by Elfeky et al. (2016).

Data analysis

Data obtained were entered into Microsoft Excel (2007) and presented in summary tables. Chi-square (χ²) test was used for statistical analysis with SPSS version 19.0 (SPSS Inc., Chicago, IL, USA).P-value < _0.05was assigned for statistical significance.

Two hundred and eighty-eight women were enrolled for the study. Their mean age was 28 ± 5.2 years (range 17–55 years). Major risk factors identified to be associated with RVVC were pregnancy and human immunodeficiency virus (HIV) infection (Table 1). Pregnancy was found not to be a major predisposing risk factor associated with Candida vaginal infections. Majority of the women however had HIV infection as an underlying illness that predisposes them to Candida infections and the predisposing risk factor responsible for acquisition of Candida glabrata (N = 33; 55.9%) Candida tropicalis (N = 36; 78.3%) and Candida krusei (N = 18; 62.1%) was HIV infection (Table 2). HIV infection and pregnancy were not predisposing risk factors to infections by Candida albicans. Comparative analysis of risk factors associated with C. albicans and non-albicans Candida depicted significant difference (P = 0.002) for HIV infection (Table 2)

Table 1

Risk factors associated with *Candida* infections
Risk factor	C. albicans (n = 66) %	C. glabrata (n = 59) %	C. tropicalis (n = 46) %	C. krusei (n = 29) %
Pregnancy
Yes	25 (37.9)	21 (35.6)	11 (23.9)	6 (20.7)
No	41 (62.1)	38 (64.4)	35 (76.1)	23 (79.3)
HIV
Yes	27 (40.9)	33 (55.9)	36 (78.3)	18 (62.1)
No	39 (59.1)	26 (44.1)	10 (21.7)	11 (37.9)
Total number of isolates (n = 200); (%), Percentage

Table 2

Comparative analysis of risk factors associated with *Candida albicans* and non-*albicans Candida*
Risk factor	C. albicans (n = 66) %	Non-albicans Candida (n = 134) %	p-value
Pregnancy
Yes	25 (37.9)	39 (28.4)	0.197
HIV
Yes	27 (40.9)	87 (64.9)	0.002
Total number of isolates (n = 200); (%), Percentage

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 significant differences (P = 0.0005 and P = 0.043) for itching and burning sensation respectively (Table 3).

Table 3

Comparative analysis of symptoms associated with *Candida albicans* and non-*albicans Candida*
Symptoms	C. albicans (n = 66) %	Non-albicans Candida (n = 134) %	p-value
Itching
Yes	49 (74.2)	64 (47.8)	0.0005
Burning sensation
Yes	59 (89.4)	130 (97.0)	0.043
Odour
Yes	61 (92.4)	129 (96.3)	0.303
Total number of isolates (n = 200); (%), Percentage

There were 200 (69.4%) vagina swabs that produced culture positive of Candida spp out of the 288 tested. The most common Candida species isolated was C. albicans with 33% (66/200) followed by non-albicans Candida species comprising of Candida glabrata 29.5% (59/200), Candida tropicalis 23% (46/200) and Candida krusei 15.5% (Table 4). None of the vagina swab samples from the women with RVVC at the Nkawie Government Hospital, Ashanti Region, Ghana yielded Candida dubliniensis and Candida parapsilosis. Antifungal susceptibility test results indicated that all the isolates (n = 200) 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 fluconazole 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.

Table 4

Distribution of Candida species in women with recurrent vulvovaginal candidiasis
Isolate	Number of Isolates N (%)	Recurrent Infections N (%2)	Non-Recurrent Infections N (%₃)	P-value
Candida albicans	66 (33.0)	17 (25.8)	49 (74.2%)	< 0.01
Candida glabrata	59 (29.5)	41 (69.5)	18 (30.5%)	< 0.01
Candida tropicalis	46 (23.0)	12 (26.1)	34 (73.9%)	< 0.01
Candida krusei	29 (15.5)	15 (51.7)	14 (48.3%)	0.67
Total	200 (100)	85 (42.5)	115 (57.5%)
N: Number of Isolates; (%₁): Percentage of Isolate from the total number of Candida species;
(%₂): Percentage of Isolate found in recurrent infection; (%₃): Percentage of Isolate found in non-recurrent infection.

Table 5

The resistance patterns of the *Candida* species to the antifungal agents tested in the study.
Yeast species	Amp B (10 µg)	Cot (10 µg)	Flu (25 µg)	Mic (50 µg)	Itr (10 µg)	Vor (30 µg)
Candida albicans	42 (64%)	21 (32%)	12 (18%)	25 (38%)	0 (0)	47 (71%)
Candida glabrata	49 (83%)	38 (63%)	46 (78%)	32 (54%)	0 (0)	49 (83%)
Candida krusei	20 (69%)	14 (48%)	27 (93%)	6 (21%)	0 (0)	3 (10%)
Candida tropicalis	37 (80%)	21 (46%)	9 (20%)	13 (28%)	0 (0)	18 (79%)
Amp B-amphotericin B; Cot-cotrimoxazole; Flu-fluconazole; Mic-miconazole; Itr-itraconazole;
Vor-voriconazole

ERG11 genes were amplified 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 biofilms and 97% produced hemolysin. With C. glabrata, 89.8% of the isolates produced phospholipase, 78% formed biofilms 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 biofilms (Table 6).

Table 6

Virulence factors associated with the *Candida* isolates.
Yeast species	Phospholipase	Biofilm	Hemolysin
Candida albicans	60 (90.9%)	63 (95.5%)	64 (97.0%)
Candida glabrata	53 (89.8%)	46 (78.0%)	55 (93.2%)
Candida krusei	34 (73.9%)	31 (67.4%)	43 (93.5%)
Candida tropicalis	19 (65.5%)	15 (51.7%)	7 (93.1%)

TAXANOMY: Not applicable

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.

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 significant increase in the rate of resistance among the Candida isolates to fluconazole 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.

VVC-vulvovaginal candidiasis; RVVC-recurrent vulvovaginal candidiasis; CDR-multidrug efflux transporter genes; ERG11-genes which encode the enzyme (cytochrome P450 lanosterol 14-α-demethylase).

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:

Not applicable

Consent for publication:

Not applicable

Availability of data and materials:

The data used to support the findings of this study are available from the corresponding author upon request.

Competing interests:

None

Funding:

None

Author’s contributions:

Conceptualization, Methodology & Formal Analysis, P.K.F, G.A.P. and R.K.B.; Writing – Original Draft Preparation, G.A.P., P.K.F. and S.A.F Writing – Review & Editing, G.A.P., P.K.F. and S.A.F. All authors read and approved the final manuscript.

Acknowledgements:

We would like to appreciate the staff of the Nkawie Government Hospital, Ashanti Region for their support during the recruitment of the participants. We also thank staff at the Kumasi Centre for Collaborative Research (KCCR), Kumasi, Ghana for their support in molecular procedures and analysis at the Centre. We are very grateful to Prof. David W. Denning of Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, the University of Manchester, Manchester, M13 9PL, UK for his assistance during the writing and reviewing of this manuscript.

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

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Abstract

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Introduction

Materials And Methods

Study design and sample collection

Wet film preparation and Gram staining

Germ-tube test

Isolation of and identification of Candida species

Determination of hemolytic activity

Determination of phospholipase enzyme

Biofilm production

Antifungal susceptibility testing of the yeast isolates

Genotypic identification of the Candida species

DNA extraction

PCR-RFLP analysis

Data analysis

Results

Discussion

Conclusions

List Of Abbreviations

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