Specimen collection in this study was done following strict aseptic precautions from clinically suspected cases of candidiasis. Three types of clinical Specimens- High vaginal swabs (HVS), Urine, and Oral Swabs (OSs) were collected in this study using sterile swabs and bottles for appropriate specimens. The specimens were collected from inpatients and outpatients from the Departments of Gynecology, Pediatrics and Intensive Care unit (ICU), as well as Department of Medicine and Surgery of RSUTH. Comprehensive clinical history of all patients in this study was recorded but only age and sex were considered as demographic data in this study.

The following guidelines as described by 14 for the collection of clinical specimens were duly followed during the study.

1. Sterile collection devise and containers were used to collect the specimens

2. Specimens were collected under strict aseptic precautions

3. Sufficient specimens were collected

4. Specimens were collected from an active lesion containing viable organisms

5. The specimens were labeled appropriately

The methods employed in collecting these specimens from various sources were as follows:

1. High vaginal Swabs (HVSs): As speculum examination was done and the vaginal discharge was collected with the aid of sterile swabs from the posterior fornix.

2. Urine sample: Fresh mid-stream and clean catch urine of about 20-50ml was collected in sterile screw-capped containers.

3. Oral (Mouth) Swabs: With the aid of a tongue depressor, the lesions were visualized and the specimens were collected using a sterile swab. All specimens were transported immediately to the laboratory for analysis.

A total of 206 clinical samples with suspected cases within the period of July to September, 2023 made up the sample size for this investigation.

Candida species in this study were cultured using the streak method. All the clinical samples of HVS and oral swabs were streaked on the sterile surface of Saboraud Dextrose Agar (SDA) (Titen Biotech Ltd India) amended with Tetracycline. For the urine samples, a sterile swab stick was dipped into the urine before it was streaked onto the surface of the sterile SDA. They were labeled accordingly and incubated at room temperature for 2 – 5 days and examined daily for growth 1514. Following incubation, colonial examinations of each isolate with respect to the clinical sample were identified for colour, shape, size and texture 16.

Yeast cultures were isolated by streaking using a sterile wire loop on freshly prepared SDA and incubated for 3 days at room temperature so as to obtain pure isolates. Following incubation, the discrete single colony of each isolate that were grown along the line of streak was further grown on SDA using a sterile cotton wool. The pure isolate was then preserved in 15% sterile glycerol solution for further use.

The colonies of yeast isolates were identified by the colony characters (cultural characteristics) as describe by 16, Gram staining and sugar fermentation.

Smears of each colony were made on a clean grease free slide, then heat fixed after air drying by passing the reverse side of the slide over a flame of Bunsen burner. The smears were stained by Gram’s method using crystal violet (60 sec) Lugol’s iodine (60 seconds), and decolorized with 95% alcohol till no colour appeared to ooze out, then counterstained for 40 seconds using Safranin, while washing was done after each stain was used. The smears were blot dried and observed under oil immersion for shapes and cream reactions 17.

Three carbohydrate broths were used in this study for the sugar fermentation test, each containing 1% glucose, Lactose and sucrose separately with 1% peptone and 0.005% phenol red indicator. Inverted Durham’s tubes were immersed for gas detection. All the broths were sterilized by autoclaving at 121^oC for 15 minutes. On cooling the yeast colonies were inoculated into each, broth and incubated at 25oC for 2-3 days and examined at 24 hour intervals for acid (yellow colouration) and gas (space in Durham tubes) production.

The antifungal agents used were 200mg Fluconazole capsule BP (Mancare Pharmaceuticals PVT LTD, India), 200mg Ketoral (Ketoconazole) Tablet USP (Laborate Pharmaceuticals LTD, India) Microzol-Plus containing 200mg Micronazole Nitrate and 750mg Metronidazole Tablet (Drugfield Pharmaceuticals LTD, Nigeria); 500000IU Nystatin Tablet (Mekophar Chemical Pharmaceutical Joint- Stock Company, Vietnam) and 250mg Itracap (Itraconozole)(Genix Pharma PVT, LTD, Pakistan). A twofold dilution of each stock solution of antifungal agent was prepared by diluting from the stock to the least concentration. Concentrations of 100µg/ml, 50µg/ml, 25µg/ml, 12.5µg/ml, 6.25µg/ml, 3.124µg/ml and 1.56µg/ml were prepared and were used to impregnate the sterile perforated WhatmanNo. 1 filter paper discs

The inoculums of yeast isolates were grown in Tryptic Soy broth (TSB) medium for 18-24hrs, and the turbidity of the medium was adjusted by adding sterile normal saline until the turbidity matches that of 0.5 McFarland standard 18.

With a sterile swab was dipped into the already prepared inoculums and rotated firmly against the upper inside wall of the test tube for many times in order to remove excess fluid, the test yeast isolates were inoculated onto the dried sterile surface of SDA amended with tetracycline by streaking until the entire surface was covered. The plates were left for about 5 minutes in order for the surface moisture to be absorbed before applying the drug impregnated discs.

In compliance with Clinical and Laboratory Standards Institute 19 guidelines the agar disc diffusion techniques were employed in the antifungal susceptibility testing. The drug impregnated discs were taken out form the containers using a pair of sterile forceps, and they were then placed onto of the yeast infected Saboraud Dextrose Agar. The discs were tightly pressed down to make contact with the SDA’s surface using the sterile forceps. All plates were incubated at 37⁰C for 48-72hrs 14.

After the incubation period, the plates were evaluated. Using a meter rule, the diameter of the zone of complete inhibition was measured and the Mean values were recorded in millimeters, rounding to the nearest whole number. The susceptibility (sensitivity), Intermediate or Susceptible-Dose Dependent (S-DD), and Resistance to the antifungal drugs to the test isolates were measured and compared to the standard zone interpretive breakpoints provided by the CLSI M44-A2 recommendations ^{14; 15}.

Patients’ information was collected through oral questionnaires. The risk factors of infection type and the data obtained in this study were in this study were statistically analyzed using IBM SPSS statistic for windows version 26/IBM Corp Armonk. NY. USA. Descriptive statistics such as Mean, Standard Deviation (SD), Tables and Proportional Annova were used to describe the data. Difference between propositions was analyzed using X² tests, if the sample sizes were small or unbalanced. A two tailed p-value< 0.05 was considered statistically significant Table 1.

Results

An uneven distribution of patient ages was seen in this study as shown in Table 2, with a large number of patients (82.0%) falling into the 11-40 years age group, with a mean age of 1.86+ 0.344. The range of ages among the patients was 4 months old to 73 years. The age range of 11 to 30 has the greatest frequency.

The distribution of patients in this investigation based on gender is presented in Table 3. The results showed a higher percentage (86.4%) for female patients as compared to the male patients (13.6%) and this implies that the females outnumbered the males in this study. At p = 0.000, the patients’ gender distribution showed a statistically significant difference of 36.59, p<0.01.

The total of 44 samples out of the 206 specimens collected, tested positive in this study for Candida species giving a prevalence of 66.1% (p<0.513), as shown in Table 4. Out of these 44 positive cases from the various specimens, 25(23.8%) were positive for HVS, 14(17.3%) from urine and 5(25.0%) from oral swabs. There was no statistically significance difference at p<0.513 among the specimens.

The results of the culture positive cases of Candida species according to age and gender in this study is shown in Table 5. Out of the 44 isolates obtained from various clinical specimens, 20 (48.7%) were isolated from the female patients within the age group of 31-40 years and this was the highest prevalence of Candida species observed in this study. Following that, 14 (34.1%) positive cases were isolated from female patients in the age group of 21-30 years while 5 (12.2%) were isolated from those of the age 11-20 years of age as the least 1 (2.4%) were found within the age group of 41 years and above for the females. For the male patients, 1 (33.3%) positive case was observed for those within the age group of 0-10 years, 11-20 years and 41-50 years. The prevalence percentage of positive cases in this investigation showed that females (93.2%) were greater than males (6.8%) with statically significant difference of 25.112 at p=0.000 Figure 1.

The prevalence of Candida species according to Clinical samples as shown in Table 6 showed a total of 44 isolates with 25 (56.8%) from HVS, 14 (31.8%) from urine and 5 (11.4%) from oral swabs. Candida albicans in this study was the most prevalent species accounting 22 (50%) whereas the non-albicans Candida (NAC) made up the other 50% of the total isolates. The prevalence rate of Candida albicans were 13 (59.1%), 7 (31.8%) and 2 (9.1%) for HVS, urine and oral swabs respectively, C. glabrata was 2 (50%), 1 (25%) and 1 (25%), C. kruseiwas 4 (50%), 3 (37.5%) and 1 (12.5%); C. parapsilosis4 (80%) and 1 (20%) from HVS and urine respectively; C. pelliculosa 1 (100%) from HVS only whereas C. tropicalis was 1 (25%), 2 (50%) and 1 (25%) from HVS, urine and oral swabs, respectively Figure 2.

The results of the Antifungal Susceptibility Pattern of Candida species in this study is presented in Table 7. In the present investigation, out of the 44 Candida isolates tested, Itraconazole demonstrated resistance in 34.1% cases studied. This was observed to be the most resistant among the antifungal agents. Miconazole and Nystatin showed resistance of 18.2%. The susceptible –Dose dependent was high in Itraconazole (36.4%), followed by Nystatin (29.3%) and Miconazole (22.7%). The highest sensitivity was noted in this study for Fluconazole (79.5%) and Ketoconazole (77.3%), 18.2% showed resistance and 2.3% and 4.5% showed Susceptible-Dose Dependent to Fluconazole and Ketoconazole respectively. Among the Candida species examined, C. albicans was most sensitive to all the antifungal agents followed by C. krusei, C. glabrata and C. parapsilo

The current investigation revealed that among the patients who visited Rivers State University Teaching Hospital, Port Harcourt, both sexes and all ages can develop candidiasis as the study identified the particular types of Candida that results infections. The oldest participant in our study was 73 years old, and the youngest was a four-month – old baby giving a mean age of MeanAge 1.86+ 0.344 years. The age range of 11-40 years old accounted for bulk of the patients in this study, which may be explained by the fact that this is the age group with the highest rates of pregnancy, sexual activity and hormonal volatility. This results is however, lower when compared to the study of 20 who in their study reported the mean age group of 43.4 years. The result of this study is consistent with other studies as many investigations noted high rate in the age group of 20-49 years ^{14; 21}. Patients’ age has a major influence on how susceptible they are to candidiasis especially if they are older than 65 years. Candida infections are more likely to cause death in the elderly especially in those over 65 years. Individuals in the reproductive age group are particularly susceptible to immune system and hormone variations. The distribution of Candida infections is influenced by these variables as well as physiological changes associated with ageing 22

The study revealed that female patients 178 (86.4%) were higher when compared to male patients 28 (13.6%), which indicated that the females surpassed in number the males. This high distribution of females as noticed in this study has proven that they are mostly infected than the males. This finding is in conformity with the report of 14. Similarly, the study also agrees with the report of 23, and 24, who had stated high distribution of females than males in the study but disagrees with the study of 25, who stated that males are commonly infected than females with an incidence of 94 (62.6%) and 56 (37.3%), respectively. Gender has a major role in the clinical research of candidiasis in different age groups, male and female exhibit different infection rates and yeast growth intensities 26. This emphasizes how gender differences in the epidemiology and clinical symptoms of candidiasis must be taken into account.

The prevalence of Candida species with respect to the clinical specimens showed high distribution of 66.1%. The prevalence rate observed in this study is said to be higher compared to those reported by 27, who reported 26%. 28 and 29 had reported the prevalence rate of 30.7% and 30% in Jamaica and Nigeria respectively. This finding is however higher than the above results. The prevalence of Candida isolates in this study fluctuates with respect to the clinical specimens, and these results is in agreement with the report of 24, who documented in Ghana that high prevalence of Candida infection was isolated from HVS compared to urine. The high frequency of HVS and Oral Swabs observed in this research could be attributed to the vaginal and oral environment which could be influenced by hormonal changes, pH level, and the presence of glycogen.

Age and sex are two main factors influencing candidiasis, with older people more prone to infections and invasive candidiasis is more common in women. These pre-disposition are further influenced by variation in the strength of yeast growth in males and females 30. Comprehending these variables is imperative for proficient handling and avoidance of candidiasis. In our study, the prevalence of positive cases of Candida species with respect to gender and their age showed an inequitable distribution. The high prevalent of Candida species among the age group of 21-40years conforms with the study earlier reported by 25, that the majority of patients were in the age group of 21-60 years. 31, had similarly reported high incidence of Candida species in women in the age range of 17-44 years, while 14, reported high incidence of Candida species in the age group of 20-49 years.

Our investigation revealed that both sexes and all ages can get candidiasis. High infection rate in the age group as observed in this study for the females may be probably due to the indiscriminate drug usage, especially contraceptives among females; decreases levels of protection cervical antibodies in the reproductive tract, practice poor hygiene, history of antibiotics or drug misuse, hormonal factors, anatomical differences and diabetes.

In the research we conducted, substantial gender disparities were seen when the number of yeast colonies in the entire group were analyzed with respect to age. The number of yeast colonies in female group varies with age and this agrees with the study of 30. Rural women had an elevated rate of infections owing mostly to conditions of poor medical care, lack of health education, scarce economic resources and difficulty in timely medical treatment as documented by 32 and this also may contribute to the high prevalence of Candida species in this study.

In our study we identified 44 species of Candida, with Candida albicans 22 (50%) being the most prevalent. Among the non-albicans Candida species it was observed in this study that C. krusei 8 (18.2%) demonstrated increased prevalence proceeded by C. parapsilosis 5 (11.4%), C. glabrataand C. tropicalis 4 (9.1%), respectively and the least was C. pelliculosa1 (2.2%) which aligns with the publicly available reports from various global location ^{14; 1; 16. 15; and 33}. The high prevalence of Candida species from HVS in this study is concordant with the study of 34. Candida species has been stated to be the fifth most frequent nosocomial infections in hospitals with C. albicans accounting for 76-89% in VVC and 25% in Urine, followed by non-albicans Candida and fourth most frequently isolated pathogens from blood stream [35; 25; 4. and 14). C. albicans being the most prevailing Candida species in this study has been reported for several decades to be the primary cause of invasive infections that can be fatal. Globally, it is the most frequent cause of mucosal and systemic infections that accounts for over 70% fungal infection. C. albicans is the primary cause of candidemia 36.

The in vitro susceptibility of Candida species recovered from different clinical samples was determined in this study. Fluconazole, Ketoconazole, Miconazole, Nystatin and Itraconazole are the antifungal medications that were examined. Based on their widespread availability in the local market and their status as hospital prescription medications, the antifungal agents utilized in this investigation were selected. The results of our study indicated that 34.1% of all the Candida species were resistant to Itraconazole, whereas 18.2% were resistant to Fluconazole, Ketoconazole, Miconazole and Nystatin respectively. This is in conformity with the result of 25, and 15. Among the species that infect humans most frequently is Candida albicans. The non-albicans Candida in this study corresponds with Candida albicans and their identification is vital since non-albicans Candida are more resistant to azoles than C.albicans. Despite their similarities, Candida albicans and non-albicans Candida (NAC) species differ in terms of their virulence traits, susceptibility to antifungal agents and incidence studies 14.

All the isolates of Candida species in this study displayed susceptibility to Fluconazole, Ketoconazole, Miconazole and Nystatin at varying degrees except C. pelliculosa which showed 100% resistant to all agents. The percentage susceptibility of antifungal agents to the isolates showed 79.5% for Fluconazole, 77.3% for Ketoconazole which were the highest susceptible antifungal agents. Following this agent were Miconazole (59.1%) and Nystatin (52.3%) and this agrees with the document of 14 and 31 but was in contrast to the findings of 415. In this study, Candida albicans showed 86.4% and 81.2% to Fluconazole and Ketoconazole respectively and this agreed with the report of 3137, but in contrast to the study of 38 and 25 .Candida albicans in this study showed 22.7% resistance to Itraconazole and this conform with the study of 14.

Increase in the resistance to fluconazole had been documented to rise from 2.4% to 55.4% within 2006- 2012, but the rate dropped to 8.9% in 2013. In the same vein, there has been considerable increase in the resistance of Miconazole and Itraconazole from 2.4% and 7.1% respectively, from 2006-2023 39. Despite the similarities in the disease spectrums between Candida species, their susceptibilities to agents and degrees of severity vary, which helps to explain their epidemiology and manner of transmission 40.

Many potential reasons have been attributed to the high resistance rates observed in some Candida species in this study. Amongst these reasons are:

Adaptability of Genes: As documented by 41, one of the major reasons for high resistance rates in some Candida species is the adaptability of their genes. The genetic adaptability of Candida yeasts enables them to swiftly adapt to new surroundings due to their great chromosomal fluidity. By causing genetic modifications, this adaption may cause resistance populations to arise.

Secondly, antifungal use is another potential reason for high resistance rates observed in some Candida speciesin this study and this agrees with the study of 42. The accumulation of resistance in some Candida species has been facilitated by the overuse and neglect of antifungal medications, such as azoles. This happens as a result of the medications’ ongoing exposure, which favours mutant alleles that provides higher resistance.

Again, heterogeneity loss is another reason observed in this study that is responsible for high resistance rates observed in some Candida species. This has also been reported by 41. Alleles with point mutations in diploid fungi that have undergone genetic variability can become homozygotes. This may results in resistance building and infectious persistence.

The production of biofilm as seen in many of the Candida species in this study also contributed to their high resistance rates. Candida yeasts have the ability to produce biofilms, which let them to thrive when contacted by antifungal medications. This conforms to the study of 43. The development of resistance may be triggered by the capacity of Candida to build biofilms.

Furthermore, the techniques of Charles Darwin’s theory termed “Evolutionary Mechanism” in some Candida species also promote high resistance rates in some yeast in this study. Unlike bacteria, where resistance is frequently results from the transmission of DNA migratory components, the resistance in Candida often emerges through genetic changes within an evolutionary branch. This is in agreement with the study of 44, in their study of Evolutionary Emergence of Drug Resistance in Candida Opportunistic Pathogens

Moreso, variations in physiology has been reported by 44, as another key reason for the high resistance rates in some Candida species as seen in this study. When compared to other species of Candida, several species show clear variations in glycerolipids, sphingolipids, cell wall content, and sterol composition. The innate resistance in these Candida species isolated in this study may be influenced by these distinctions.

Species-Unique susceptibility also contributes to the high resistance in some of the studied Candida species and this agrees with the study of 44. When it come to antifungal medications, Candidapelliculosa, for example, shows different susceptibility profiles than other Candida species. This implies that various species may employ various strategies to avoid being resistant to antifungal drugs.

Demographic variables are other contributory factors of high resistance rates of some Candida species in this study and this agrees with the report of 45. The durability and dissimilation of resistance isolates may be influenced by the region of distribution, severity and circulation characteristics of the isolates.