CANDIDIA AND HIV CO-INFECTIONS AMONG WOMEN ATTENDING HIV CLINIC

Candidiasis has been identified as a ubiquitous fungal infection commonly affecting people living with HIV/AIDS. This research ascertained the prevalence and risk factors for Oral and vaginal candidiasis among 180 HIV out-patients at Ebonyi State General Hospital, Ezzangbo. Oral and vaginal swabs were aseptically collected from the participants in duplicate. One of the swabs was streaked on Sabouraud's Dextrose Agar (SDA) and isolates were identified using macroscopic characteristics while the second swab was used for microscopic examination. Structured questionnaires were administered to participants to obtain risk factors associated with the infections. The antifungal susceptibility testing of the Candida isolates was performed using disc diffusion method. Data obtained was statistically analysed using Statistical Package for Social Scientist (SPSS). Results obtained from the study indicated 120(66.7%) were positive for Candidiasis; 31.7% was positive for only vaginal Candidiasis, 22.2% were positive for only oral Candidiasis while 12.8% had both. Candida albicans (CA) was dominant with 71% while none albicans Candida (NAC) was 29%. Isolates were most sensitive to Itraconazole and Griseofluvin and less sensitive to Ketoconazole, Nystatin and Fluconazole. The results of this study underlined essentials of good hygiene practices and importance of regularly assessing HIV patients for candidiasis.

Candidiasis, also known as thrush or moniliasis, is one of the most typical HIV-associated opportunistic infections caused primarily by Candida albicans (Monika et al., 2016).When cluster of differentiation 4 (CD4) levels are extremely low (below 200), candidiasis outbreaks can be quite dangerous.It has been reported that the prevalence of Candida infection during HIV infection is inversely connected to the patient's CD4 levels, which in turn rely on the usage of anti-retroviral therapy (Smith et al., 2018).One of the worst pandemics continues to be the human immunodeficiency virus (HIV) infection.Globally, 21.7 million individuals get antiretroviral medication, 1.8 million people contracted the virus for the first time, and 940,000 people died from HIVrelated illnesses in 2017 (Adam, 2018;CDC, 2018;UNAIDS, 2019).
Nigeria has one of the highest rates of new HIV infections in Sub-Saharan Africa and the second largest globally (UNAIDS, 2019).Currently, 33% of Nigerians aged 15 to 49 (or 1.9 million people, or 1.4% of the population) do not know their HIV status.In Nigeria, there are about 130,000 new instances of HIV infection, 53,000 deaths from AIDS, about 55% of adults and 35% of children are receiving antiretroviral therapy (ART), and 80% of HIV-positive patients receiving ART have viral suppression.The highest prevalence rate, 5.6%, is found in Akwa Ibom State, while the lowest prevalence rates, 0.3%, are found in Jigawa and Katsina States.Particularly in Ebonyi State the study area, the frequency of 0.8% has been reported (NACA, 2020).
To lower the mortality and morbidity rates of infections caused by Candida species, particularly in patients with HIV infection, early detection of candidiasis is essential.It has been suggested that the first person to notice oral lesions may be a general practitioner or dentist, who may encourage the patient to get tested for HIV and perhaps benefit from early management (Naga et al., 2019).The examination for oral and vaginal candidiasis in HIV-positive women using Antiretroviral treatment (ART) will aid in identifying the primary Candida species responsible for the illness through proper characterisation.Identification of the risk factors for oral and vaginal candidiasis in HIV-positive females as well as the sensitivity profile of the Candida species to the existing antifungal medications are very crucial.It is unknown how frequently oral and vaginal candidiasis affects HIV-positive out-patient women receiving Antiretroviral treatment (ART) at Ebonyi State General Hospital, Ezzamgbo.Therefore, this study is necessary to bridge the knowledge gap of Candidiasis-HIV coinfection in this area.

Exclusion Criteria:
The following people were left out of the study: HIVpositive females under medication who have had antifungal treatment in the past two weeks, and HIV-positive females under 18 years.

Study Period:
The duration of this study was from January to April 2022.A record of the patients who attended the weekly Anti-retroviral therapy (ART) clinic was kept throughout the study term.The patients' compliance with the therapy regimen was observed, and the findings were included in the socio-demographic data.
Study Population: A total of 180 HIVpositive adult female patients attending Ebonyi State General Hospital, Ezzangbo HIV clinic were the study population for this cross-sectional institutional-based research.

Sample size determination:
The sample size of 180 for this research was calculated using the method described by Charan and Biswas (2013)

Collection of Survey Data:
Prior to collecting samples, individuals were asked to complete a well-structured questionnaire to provide demographic and clinical data.There was a unique participant identification number (PIN) for each study participant.The biodata of the patients, such as name, age, educational level and marital status, was included in the first section of the questionnaire.The second section includes the history of candidiasis (such as poor breath, a white tongue, dry mouth, trouble swallowing, peculiar taste, vaginal discharge, vaginal itching, etc.), risk factors (if any), personal cleanliness, and medical care seeking behavior.Age, marital status, and ART use was used to stratify the research population.Data was gathered on the epidemiology and demographic trends of oral and vaginal candidiasis in HIV infection via responses to the structured questionnaire.All information gathered from participants were kept private and confidential then, using standardised formats the survey and clinic data was collated for analysis.

Collection, Transportation, and Storage of the Clinical Swab Specimens:
With the aid of trained nurses each Okoh et al., 2023 research participant provided high vaginal swabs (HVS) and mouth swabs.Samples were aseptically collected in duplicate using swab sticks.Each participant's specimens were collected, and the specimen container was tagged with their Identification Number.All samples were brought as quickly as possible and without delay to the laboratory, where they were processed the same day, they were collected.The samples were stored in the refrigerator at 37 o C in cases where a delay was anticipated.

Laboratory Analyses of the Clinical Swab Specimens
Direct microscopy: Each patient had one of their duplicate swab sticks (mouth and vaginal swabs) stirred in 1ml of normal saline.Each sample was suspended in a drop on a distinct greasefree glass slide, gently covered with a cover slip to keep air bubbles out and examined under a microscope with 10x and 40x lenses.On a clean, grease-free slide, a drop of the suspension was applied before a drop of potassium hydroxide (KOH) was added.Using 10x and 40x objectives, the mixture was examined for Candida species after being combined and covered with a cover slip.

Culture of the Clinical Swab Specimens:
Each patient's second duplicate oral and vaginal swabs were streaked on Sabouraud dextrose agar (SDA), which also included 50.0mg of chloramphenicol to prevent bacterial growth.The culture plates were incubated at 37 0 C for 24 hours and checked for growth.

Identification of Candida isolates:
The macroscopic characteristics of the fungal isolates from the oral and vaginal swabs, such as elevation, surface, colour, edge, and opacity, were used to identify the isolates.Microscopic analysis was performed with the methods described by Ochei and Kolhatkar (2006) for Wet mount, Permanent direct mount, Gram staining, and Germ tube test.

Symptomatic and Asymptomatic
Candidiasis: In this study symptomatic candidiasis was the presence of one or more symptoms and indications that are consistent with oral and vaginal candidiasis together with the identification of Candida species in the oral and vaginal swab cultures of the study participants.While asymptomatic candidiasis was the finding of Candida species in the oral/vaginal swab cultures of the research participants in the absence of one or more symptoms and signs typical of oral/vaginal candidiasis Antifungal Resistance testing of the Candida Isolates: Bauer et al. (1966) and Cheesbrough (2006)'s described modified Kirby-Bauer disc diffusion technique was used to assess the antifungal sensitivity pattern of the Candida isolates.The zones sizes of each antibiotic were analysed using the Interpretative Chart, and the isolates were reported as Resistant or Sensitive.
Data analysis: Microsoft Excel was used to enter the data gathered from the test procedures and the questionnaire.The statistical application SPSS-20.0 (Statistical Package for Social Scientist version 20.0) was used to conduct the statistical analysis.Tukey-kramer Multiple Comparisons and Chi-square, the prevalence rates of oral and vaginal candidiasis among adult females with HIV was compared to see if there were any significant difference.In order to establish the connection between the occurrence of candidiasis and related risk variables, data was also subjected to Spearman correlation and regression analysis to identify significant risk variables for candidiasis.The P-Value was set at 0.05.

DISCUSSION
The study underlined the importance of checking HIV-Positive patients for presence of other infections particularly candidiasis, which is often common with HIV patients who are not well balanced with hygiene.It has been reported that co-infections are cause of complications on people living with HIV/AIDS due to weak immune system of HIV patients.The results obtained in this study agreed with other related research in Nigeria and elsewhere on occurrence of candidiasis.Esebelahie et al. (2013) reported a 52.5% overall prevalence rate of candidiasis in research conducted among HIV patients in Benin City, Southern Nigeria but our study's 66.7% overall prevalence rate was higher.Similarly, it was higher than the 30.1% recorded by Njunda et al., (2011) in a study conducted in Cameroon, a Okoh et al., 2023 neighboring African nation that shares an eastward border with Nigeria.Different research conducted in China and Taiwan by Li et al. (2013) and Lin et al. (2013) revealed 49.5% and 51.4% respectively.Additionally, it was observed to be comparable to the findings of Paula et al. (2015) and Goulart et al. (2018) 's findings, who reported 50.4% and 51.3%, respectively from research conducted in Brazil.
The 22.2% rate of oral candidiasis found in this study was higher than the 9.68% rate reported among HIV-positive people in Jos, Nigeria by Lar et al., (2012).It was also higher than the 12.5% recorded by Okonkwo et al. (2013) in research conducted in Abakaliki, Nigeria, and 10.8% reported by Enitan et al. (2019) from Ogun State South-west Nigeria.The work of Vijeta et al. (2013), who reported a HIV prevalence rate of 11% among patients in Northern India was also shown to be extremely lower than this one.Other studies conducted outside of Africa observed much higher prevalence rates and enjoys more close comparison to this particular work.These studies include those by Schuman et al. (1998) who reported 22% in USA, Tsang and Samaranayake (2000) reported 54.8% in Hong Kong, Campisi et al. (2002) reported 61.9% in America, Gugnani et al. (2003) reported 65.3% in research conducted in India and Pongsiriwet et al. (2004) who reported prevalence rate of70% in Northern Thailand.
In this study, vaginal candidiasis had a single occurrence rate of 31.7%, which is significantly lower than the 88.8% reported by Umeh and Umeakanne (2010) in Benue State, Nigeria.Njunda et al. (2011) obtained prevalence rates of 36.3% in Cameroon while Schuman et al. (1998) reported similar prevalence rates of 37% in Atlanta, Georgia, USA.These are slightly higher than the finding of this research.Differences in methodology, geographic location, sample size, educational, socioeconomic, and cultural status, level of personal hygiene, adherence to HAART, and prophylaxis for opportunistic infection by the study participants, among other factors, may account for variations in prevalence rates reported from different parts of the world.
In this study, a prevalence of 12.8% for the co-occurrence of oral and vaginal candidiasis was found which is obviously below the 17.5% reported by Enitan et al. (2019) in a study conducted on Candidiasis in HIV female adults in Ogun State southwest Nigeria.The majority of earlier studies on candidiasis in HIV-infected individuals either took oral and vaginal candidiasis as a separate occurrence or in conjunction with candidaemia and/or candidiuria.When oral and vaginal candidiasis occur at the same time in one person, it may be a sign of concurrently poor oral and vaginal hygiene while using HAART medicine.This necessitates an overall improvement in their personal hygiene standard.
Candida albicans (71% of the Candida species isolated in this study) was the most prevalent, while Non-albicans Candida (NAC) made up the remaining 29%.This demonstrates that Candida albicans (CA) is the primary and commonest cause of candidiasis, outpacing Non-albicans Candida (NAC).Other works done in Africa and elsewhere concur with this.For instance, in a study conducted in Tanzania, Hamza et al., (2018) reported 84.5% (CA) and 15.5% (NAC), In Abakaliki, Nigeria, Okonkwo et al., (2013) recorded 80% (CA) and 20% (NAC), and in Accra, Ghana, Kwamin et al. (2013) reported 68.5% (CA) and 31.5% (NAC).However, in Northern India, Vijeta et al. (2013) observed 90.5% (CA) and 9.5% (NAC).Spalanzani et al., (2018) reported 66.7% (CA) and 33.3% (NAC) in research done in Brazil, whereas Goulart et al. (2018) reported 80% (CA) and 20% (NAC).Okoh et al., 2023 Regarding the symptoms of oral and vaginal candidiasis (oral sores, white tongue, difficulty swallowing, bad breath, and mouth dryness) in relation to the presence of candida species, it appears that these symptoms were more pronounced among HIV-positive patients who were infected with Candida albicans (CA) as opposed to Non-albicans Candida (NAC).This raises the possibility that CA is a more aggressive and virulent opportunistic pathogenic fungus than the NAC, which would explain why CA-infected research participants had greater signs of oral and vaginal candidiasis.In all types of candidiasis taken into account, the former was shown to be more common than the latter among research participants who had symptomatic and asymptomatic candidiasis.This demonstrates that asymptomatic instances are quite common.Additionally, it has been established that ongoing, asymptomatic Candida species carrying may be a risk factor for a later infection (Goulart et al., 2018).
In this study, we define symptomatic candidiasis as the presence of one or more symptoms and indications that are consistent with oral/vaginal candidiasis together with the identification of Candida species in the oral/vaginal swab culture of the study participants.On the other hand, asymptomatic candidiasis was defined as the finding of Candida species in the oral/vaginal swab cultures of the research participants in the absence of one or more symptoms and signs typical of oral/vaginal candidiasis.The community's most important infection reservoirs are asymptomatic people.They may not show any visible signs or symptoms of the illness, but the virus is present in their saliva or vaginal discharge, and they can spread it to others through kissing or sexual contact.The cycle of infection within the community must be stopped by identifying and treating asymptomatic people in light of the aforementioned information.Additionally, having oral sores alone does not always indicate that a person has oral candidiasis, at the first instance, the reason is straightforward: oral infections have been linked to pathogens other than Candida, such as some viruses (such as Herpes Simplex Virus-1), parasites (such as Entamoeba gingivalis, Trichomonas tenax), and many bacteria, such as Streptococcus mutans, Staphylococcus aureus, and Klebsiella pneumoniae.Similar to this, having vaginal itching alone does not always indicate that a person has vaginal candidiasis.The same cause as mentioned above; other organisms such Chlamydia trichomatis, Neisseria gonorrheae, and Trichomonas vaginalis may also be the cause.For this reason, differential diagnosis is essential to identify the infection's true cause when it manifests with identical symptoms.Incorrect reporting and treatment might result from incorrect diagnosis.In addition, self-diagnosis based on personal experience or in another way may result in the erroneous diagnosis and treatment.Antimicrobial resistance is largely caused by the purchase of drugs over the counter without the proper laboratory test results.Clinicians are presently fighting such practice.

CONCLUSION
The findings of this research revealed that a majority of the HIV patients screened were infected with Candida.The most prevalent species of Candida that affected the sampled population was the Candida albicans this suggest that Candida albicans are the commonest and more pathogenic strain of the Candida species.The antifungal susceptibility profile of the Candida isolates revealed that Candida albicans isolates were more sensitive to the five drugs tested against them than the non-albicans Candida isolates.To avoid infection with Candida Okoh et al., 2023 species proper hygiene practices should be enhanced.

Figure 1 :Figure 2 :
Figure 1: Prevalence of symptomatic and asymptomatic candidiasis among participants ,242,500 for the Okoh et al., 2023 year 2022.Nigerians from other States also reside in different parts of the State.

Table 1 :
prevalence of oral candidiasis in relation to the sociodemographic details Okoh et al., 2023 Frequency of oral candidiasis in relation to the demographics of participants

Table 2 :
Frequency of vaginal candidiasis in relation to the demographics of participants

Table 3 :
Frequency of Oral and Vaginal candidiasis in relation to the demographics of participants

Table 4 :
Risk factors for oral and vaginal candidiasis among the study participants *P-value >0.05 is not significant.P-value < 0.05 is significant.Okoh et al., 2023