Isolation and Evaluation of Candida Species and Associated Microbes causing Gastrointestinal Tract Infections Among Patients Accessing Care at a Tertiary Health Facility in Lagos, Southwest Nigeria

Introduction: Candida is a normal flora of the oral cavity, gastrointestinal tract (GIT) and the vagina but has been reported to become opportunistic pathogen (switch) in certain disease conditions. An investigation into this switch in the GIT will give a clear insight into their progression from commensal to opportunistic pathogens. Therefore, this study aimed to isolate and evaluate the presence of Candida species and associated microbes causing GIT infections among patients accessing care at a tertiary healthcare facility in Lagos, southwest Nigeria Methodology: A total of 150 consenting participants (M=82; F=68) age ranged 1-60 years were recruited into this study between July and December 2019 at the Lagos University Teaching Hospital, Idi-araba using standard methods. Demographic details were taken and characte¬ristics of the stool specimens were noted. Samples were examined macroscopically and microscopically, then cultured on Sabouraud dextrose agar with corn meal extract along with other bacteria culture media and incubated appropriately. CHROMagar TM for Candida was used for presumptive identification of isolates and was further confirmed with the API32C system for Candida. Results: An overall rate for Candidiasis in this study was


Introduction
Candida species reside in the human gastrointestinal tract as part of the body's microbiota. Due to changes in host environment as a consequence of disease conditions (such as immunosuppression, metabolic imbalances and dysbiosis), they can proliferate as opportunistic pathogens [1,2]. They are the predominant cause of opportunistic human mycoses, and are capable of causing superficial as well as invasive mycoses [3] Candida proliferation, especially with heavy growth, within the gut may result in and abdominal discomfort [4,5]. There are many Candida species harvesting the GIT and any of them can cause gastrointestinal candidiasis among disease patients; however, Candida albicans is the most common [6].
Studies reporting prevalence of gastrointestinal candidiasis at the global and continental levels are scarce, and studies in various countries have yielded varying results [2,7,8]. Previous studies carried out in different centres in African continent reported a non-uniform prevalence of gastrointestinal candidiasis with a varying rate of 12.4% but with a slightly higher prevalence in sub-Saharan Africa at 12.8% [9]. Both C. albicans and non-albicans Candida species revealed high rates of intrinsic and acquired forms of antifungal resistance [5,[10][11][12]. Other microscopic fungi that may become opportunistic pathogens apart from Candida species include Aspergillus, Mucor, and Cryptococcus [11]. When specimens from infected patients are cultured, Candida spp are considered as pathogens when isolated either in pure or mixed culture and counts ≤ 10 3 , 10 4 and ≥10 5 cfu/ml are considered to be significant [13].
Successful treatment of opportunistic mycoses depends on identification of the specific organism causing the infection.
Without effective therapy a systemic infection of this type can be fatal [14]. Furthermore, the yeast is also common commensal of the gastrointestinal tract (GIT). Most Candida infections are opportunistic, occurring in debilitated persons and also in persons with prolonged broad-spectrum antibiotics therapy. Roberth et al. [15], Edwards, [16] noted that even when the digestive flora remained balanced in otherwise healthy persons who lack beneficial bacteria in their gut (due to bad life style or bad feeding habits, probably on antibiotic, chemotherapy or on other strong chemicals); C. albicans was seen to over-proliferate and cause ill health.
Opportunistic fungal infections are increasing around the world with limited information on their prevalence in gastrointestinal (GIT) infections [3]. Different clinical forms of Candidiasis are known involving primarily the mucosal surfaces (thrush), GIT or urogenital tract, and deep-seated infections such as Candidaemia or Meningitis [17]. It is therefore pertinent to determine the level of pathogenicity of intestinal fungal infections in the study environment. In Nigeria the problem is compounded due to laboratory challenges and inadequate facilities in many healthcare centres, which hinders the timely diagnosis and treatment of such infections. This study therefore aimed to isolate and evaluate the presence of Candida species and associated microbes causing GIT infections among patients accessing care at a tertiary health facility in Lagos, southwest Nigeria

Methods Ethical Considerations
This study was approved by the Ethical consent and clearance from the Management of the Lagos University Teaching Hospital (LUTH), Idi-araba with Reference approval number No: NHRECO4/04/2008. All participants were provided with written informed consent before the enrolment, and for participants younger than age 18 years, written informed accent was provided by a parent or legal guardian.

Study Design
This was a cross-sectional study carried out at the General Outpatient Department of the Lagos University Teaching Hospital. A total of 150 consenting participants (M=82; F=68) age ranged 1-60 years were recruited into this study between July and December 2019 at the Lagos University Teaching Hospital, Idi-araba along with the controls at random from both out-patient and in-patients clinics. The 80 control participants were the apparently healthy participants recruited from schools and offices at random within the ages of 1 and 60 years. Subsequently, informed consent of the participants was duly obtained in writing where they are adults and informed assent of guardians in the case of minor.

Inclusion Criteria
Only participants presenting with provisional diagnosis of GIT related diseases, clinical history of being immunocompromised, on chemotherapy, admission and between the ages of 1and 60 years were considered.

Exclusion Criteria
Participants with no GIT related illness or clinical history of being recently hospitalized or immunocompromised was not recruited for this study. Participants were randomly selected and informed consent was obtained. Eligible patients who picked cards with numbers that were multiples of three were enrolled in the study. The cards were reshuffled each time a card was picked and picked cards were not replaced. Where repeated numbers were generated, only one card with such numbers was left in the box.

Data and Sample Collection
Clinical data were obtained from the consenting participants' medical records for demographic characteristics such as age, sex and antifungal medication. Stool specimens and blood samples were collected from the recruited participants. Participants were advised to look out for blood stained or mucoid portions of their freshly voided early morning stool sample and aseptically put directly into sterile wide mouth universal transparent container.
The stool specimens were received and properly labeled with the participant's name, date and time of collection. Briefly, a sterile, wide-mouthed spoon fitted with a graduated stool container was labeled for each participant and participants were instructed to produce about 10 mL of stool. The specimens were immediately delivered to the laboratory for subsequent analysis. One 4-mL fasting blood specimen was collected from each participant into fluoride/oxalate tubes and the plasma separated from the cells within 30 min after collection.

Sample Laboratory Analyses
Stool samples were examined macroscopically for presence of blood, mucus, texture, colour, present or absence of adult worm.
Wet preparation of each stool sample including control was done on clean grease free slides using physiological saline, 40% dimethyl sulphide (DMSO) KOH and Lacto phenol cotton blue then allowed to stand for 10minutes. Slides were then viewed with X10 objective of the microscope for presence of yeast cells measuring 2 -4µ in diameter and fungi hyphae. Gram stained smears were also examined for yeast cells and fungi hyphae under the microscope using the X100 objective of the microscope. The yeasts may often be seen attached to pseudohyphae that stain Gram positive [18].
Culture Media: Sabouraud dextrose agar (pH 5.6) containing corn meal extract was prepared to give a final pH (at 25°C) 6.0 which will enhance growth and preserve the chlamydiospores according to the manufacturer's instructions. Bacteria stool culture media (Selenite F, MacConkey, DCA, SS agar) was prepared according to manufacturer's instruction and used to detect or exclude bacterial isolates [18]. As for inoculation, with the use of sterile wire loop and streaking method to obtain discreet colonies on culture plates, all stool samples including control were inoculated into two different sterile freshly prepared Sabouraud dextrose agar plates containing corn meal extracts and antibiotics (chloramphenicol, streptomycin, gentamycin at a concentration of 100mg/liter of medium respectively) because of the bacteria load in stool specimens and also cultured on the different routine bacteria stool culture media mentioned above to identify and then exclude gastroenteritis of bacteria origin. All inoculated media including the Selenite F broth was incubated at 35°C -37°C respectively. One of the Sabouraud agar plate were incubated at room temperature (18°C -25°C).
Plate Reading: Presumptive fungi colonies were read based on their colonial appearance: size, shape, elevation, consistency (mucoid, dry or semi-mucoid), colour or pigmentation, and distinctive yeast (bread) smell. The number of colonies on plates showing pure fungal growth was counted. Those with counts ranging from ≤10 3 , 10 4 and ≥10 5 cfu/g after 24 -72hrs incubation were regarded as significant [13]. Plates that showed no growth after 7days incubation on the Sabouraud dextrose corn meal extract agar plates were disregarded and discarded appropriately.
Bacterial isolates from the routine bacteriology culture plates were identified and their pathogenicity determined appropriately.

Identification Test Methods
These are test procedures that were used for the identification  [18].
The inoculated plates were incubated aerobically at 37°C for 24 hours. After incubation, the colours of the isolates were noted and identified on the colour chat.
Sugar Fermentation: Tests API 32C System: With the use of API 32C system, sugar fermentation test was carried out. Isolates were first purified by sub culturing on Sabouraud dextrose agar at least 2 to 3 times. Intermittent staining was done with Lacto phenol cotton blue and viewed under the X100 objective lens to be sure the isolate was yeast. A suspension from the culture medium was made in Sabouraud dextrose broth with a turbidity equivalent to 2 McFarland. From each of the suspension, 250µl was then transferred into an ampoule of the API 32C System medium. A volume of 135µl of prepared yeast basal medium suspension was then pipetted into wells containing 32 different freeze-dried sugars and incubated at 30°C for 24 to 48 hours. After incubation, growth was seen as turbidity in the well containing the sugars (substrate) in each cupule and was read using the ATB TM Expression TM or mini API instrument, or visually. Identification was obtained using the identification software into which the positive substrate code combinations are imputed and printed out [19]. Software identification was done at albicans [20].
Chlamydiospore Formation: Suspected culture isolates were inoculated onto Sabouraud dextrose agar plates using the Dalmau technique i.e., on a microscopic glass slide and a light-tomoderate inoculum of the greisen tube test was transferred by loop and streaked lightly over an area slightly greater than that to be covered by the subsequently placed, sterile, 22mm cover glass which was then incubated at 37°C for up to 3hours. Periodically, this begins between 1-1.5hours. Cultures were observed through the cover glass for germ tube production. The low-power objective was used to scan the inoculated area; the high-dry objective was used to confirm the presence of germ tubes. After germ tube detection of after 3hours at 37°C, the plates were incubated at room temperature (18°C -25°C) for chlamydiospore formation, a drop of Lacto phenol cotton blue was added to improve clarity and staining of chlamydiospore [21].

Statistical Analysis
Descriptive statistics was used to describe the study characteristics of the participants. Continuous variables were      (Table 1). Eighty-two (82) participants were males but only ten (10) Table 1).

Distribution of Isolates by Disease Conditions
Twenty-six of the participants were clinically diagnosed to have  (Table 2).

Age and Sex Distribution of Pure Candida Albicans Isolates Among Participants
Out of the 150 participants tested, 50 were positive for fungi culture but only 25 participants stool culture yielded pure Candida albicans isolates. The age group 51 -60 years with 24 (11 females and 13 males) participants had pathogenic C. albicans infection in 25% of the participants in the group as the highest. As for C. albicans, the female had higher rate (10%) than their male (6.7%) counterparts. However, by gender, this was not statistically significant (p=0.012).  [29] in this study with 4 participants being infected and a frequency of 3% of the total population (Table 3).
Chi-square= 72.  Table 4). The total number of control participants recruited for this study was eighty, thirty-six (45%) of them were females and forty-four (55%) were male participants.
All the stool samples collected from the participants in the control group yielded growth of normal flora (bacteria and fungi) of the gastrointestinal tract (GIT). Their stool microscopy revealed very scanty or no yeast cells (Table 5).

Discussion
In this study, the intestinal stool culture yielded pure fungi isolates of Candida species with an overall prevalence of 26.7% (40 isolates found among 150 patients). This is considerably higher than other findings such as 21.5% in Cameroon [22] but lower than 41.1% in Mexico (2) and 75.47% in Poland in 2015 [23] The prevalence in this study was also higher than the overall prevalence in sub-Saharan Africa, which was estimated to be 23.4% [7]. [8] in India, however, reported a lower prevalence (9.7%). Previous authors have noted a wide variation in the prevalence of Candidiasis depending on region, population surveyed and even the research methods used [24]. However, most studies used similar culturebased methods, the quantification threshold for colony-forming units of the culture colonies have diverse threshold. We adopted a threshold range of 105 colony forming unit (CFU) considered significant for growth, whereas in Poland study according to [23] a wider range (103 CFU -106 CFU) was considered. This may have resulted for the significantly higher prevalence in their study.
In the current study, female patients had a higher prevalence rate compared to male patients (18.7% vs 14.7%; p = 0.0439).
A study conducted in Vienna, Austria, on burn patients found a female predisposition in systemic and related candidiasis [25].
The predilection of gastrointestinal Candidiasis in female patients is poorly understood and there is no proper explanation for the predisposition [26]. In species distribution of Candida isolates, C. albicans accounted for the majority (16.7%) of isolates in this study. This is consistent with other studies that identified it as the most common isolate from clinical materials, [27,28] and could be attributed to the fact that C. albicans is highly adapted to the human mucosal surfaces and possesses virulence factors such as protease production and biofilm formation [24,29]. These enhance its chances of survival in the gastrointestinal tract. Omrani et al. [30] in their review of African studies also found a predominance of C. albicans.
Furthermore, the remaining 25 (16.7%) were reported as mixed culture of Candida spp. C. albicans and C. dubliniensis were found as a pair in seven stool samples from participants with cases of peptic ulcer disease 1(14.3%), gastrointestinal bleeding 4(57.1)), immune suppression 2(28.6%); others occurred in combinations of threes and fours of different species. This is consistent with the studies in Poland [23] and India [31]. Non-albicans species in general accounted for a larger number of isolates in studies by Banerjee et al. [31] in India, as well as in Brazil and Chile [32]. Generally, the immune system in HIV cases are suppressed hence the chances of C. albicans attaining pathogenic state were high [29,32].
In other disease conditions, the pervalence of C. albicans were significant, ranging from 25% in diarrhoea disease to 100% in the only participant with cancer and on chemotherapy included in this study. Co-existing infections is comoon when immunity is reduced and as a result, the prevalence obtained in the subgroups (HIV and Immune Supression) in this study were 85% and 43% respectively which were obtained for pure Candida albicans isolates. This was in line with the study obtained in Saudi Arabia with the overall prevalence of intestinal fungal infections of 58.7% in transplant recipients [32].
The most prevalent fungus in that study was Candida spp., which was seen in 22% of the study patients and 24.4% of the control group. Co-existing infection with two or more fungi was seen in 14.8% and 3.4% in the case and control groups. Interestingly, there was no significant difference (p-value= 0.05) in the prevalence of infection by a single organism between the two groups [33,34].
However, co-existing infection with two or more species was more prevalent in transplant recipients. There was a significant difference in the prevalence between the case and control groups in this study and this was seen in all the disease conditions especially in cases of GIT bleeding and immune suppression. Stool samples from all the participants used as control, yielded growth of gastrointestinal normal flora in this study. This may be as a result of other host factors like irritable bowel syndrome etc. that must be present for Candida to exist as a pathogen [34,35].
The pair of C. albicans and C. dubliniensis was isolated in seven stool samples from participants with cases of Peptic Ulcer Disease (1), Gastrointestinal Bleeding (4) and Immune Suppression (2) in this study; this may be attributed to the closely related genetic materials shared by these species which may have helped to increase their level of invasiveness in strain related virulence factors [36][37][38] though further study may be required to establish this fact. Other stool samples also yielded pairs (5) but in combination of various species. Ten (10) stool samples occurred as mixed cultures of three different species and only three of the participant's stool reported a combination of four different species (Table 4). Generally, fungal infection (gastroenteritis) was reported more in this study than bacteria (gastroenteritis) which indicates a shift from the normal cases. The reason for this shift may be attributed to Candida overgrowth which was supported by the study of Roberth et al. [15] and is in line with results from this work.