Evaluation of Antifungal Susceptibility Profile of Candida Species Isolated from Female Patients Attending Aminu Kano Teaching Hospital (AKTH)

The increasing incidence of Candidiasis genitourinary tracts as well as the introduction of new antifungal drugs has recently encouraged the need for performing fungal susceptibility tests. The study was aimed at evaluating the antifungal susceptibility profile of Candida species isolated from female patients attending Aminu Kano Teaching Hospital (AKTH), with suspected Candidal infections August, 2012 to June, 2013. Five hundred and one (521) clinical samples comprising 342 urine and 179 high vaginal swabs were cultured on Sabouraud dextrose agar. The Candida species isolated were identified to species level using Chromogenic agar and API 20 C AUX test kit. Antifungal susceptib tests were performed using commercially prepared single antifungal disc (Bioanalyse Turkey). Out of these 521 samples analyzed only 59 yielded species giving the overall prevalence of 11.3% with Candida albicans 22 (37.3%) as the common speci isolated followed by C. glabrata 19 (32.2%), tropicalis 5(8.5%), C. krusei 3 (5.1%), C. magnoliae (5.1%), C. lusitaniae 2 (3.4%), C. parapsilosis (3.4%), C. famata 2 (3.4%) and C. guilliermondii (1.7%). The antifungal susceptibility test shows 81.4% of the isolates were susceptible to ketoconazole and only 3.4% to nystatin. However, 33.9% were susceptible, 13.6% intermediate susceptible and 52.5% resistant to fluconazole. Similarly 28.8% were susceptible, 5.1% intermediate susceptible and resistant to voriconazole. Additionally 25.4% were susceptible, 11.9% intermediate susceptible and | Volume – 1 | Issue – 5 | July-Aug 2017 6470 | www.ijtsrd.com | Volume Journal of Trend in Scientific and Development (IJTSRD) International Open Access Journal Yakubu, G. 2 Panda, T. W. Dagona, A.G University, P.M.B.3011, KanoKano Teaching Hospital, Kano Faculty of Science, Federal University, Gashua


INTRODUCTION
Candida species are opportunistic yeast affecting the genitourinary tracts. Candida for disease control (CDC) as a cause of sexually transmitted disease (Prescott mycotic pathogen produces as diverse a spectrum of opportunistic disease in humans as does Candida species are important nosocomial pathogens and can be transmitted sexually (Tatfeng Candida species are the normal micro biota within the gastrointestinal tracts, respiratory tracts, vag and the mouth (Prescott et al., refers to a range of infection caused by species of fungal genus Candida. The infections can be acute or chronic, localized or systemic. Disseminated Candidiasis is frequently life threatening. T majority of these infections are caused by albicans (Greenwood et al., 1992). The incidence of genitourinary tract infection is much higher in females during adolescence and childbearing years (Sobel, 2004 , 1993, Tatfeng et al., 2004. In broader sense fungal diseases became recognized as being of clinical importance in the second half of the last century. This is mainly due to advancement in medical technologies (Rizvi et al., 2011). With the remarkable modern advances in medicine, there has been an increase in the number of immuno compromised individuals who need extensive care in the hospitals (Rizvi et al., 2011). This has resulted in a rise in the incidence of fungal infections, especially those due to Candida species (Rizvi et al., 2011). Among the species only nine are frequent pathogens for human (Andy et al., 2004). Candida albicans is the most common fungal causative agent in superficial and deep seated Candidiasis. However, non-albicans are also being implicated in recent years (Gullo, 2009). However, infection of the vulva and vagina with Candida albicans is termed vulvovaginal Candidiasis. This is characterized by intense vulval and vaginal irritation (itching) which often gives rise to inflammation, soreness, redness and white spots in and around the genital tract. Also, painful urination and intercourse are associated with the infection. In about 5% cases of Candidal vulvovaginitis, the disease has a chronic course showing frequent and refractory episodes (Ferrer, 2000). Additionally, various studies have shown that nearly 75% of all women will experience at least one attack of Candidal vulvovaginitis during their life time and suffer multiple episodes (Ferrer, 2000;Saporiti et al., 2001).The presence of Candida in urine is called candiduria. This is rarely encountered in otherwise healthy people with structurally normal urinary tract (Schonebeck and Ansehn, 1972;Kauffman, 2005 andBukhary, 2008). It is however of common occurrence in hospitalized patients. Candida species account for almost 10-15% of nosocomial urinary tract infections (UTIs) (Kauffman et al., 2000;Lundstrom andSobel, 2001 andKauffman, 2005).In the United States it is estimated that the incidence of candiduria was 25,000 cases per year. And approximately one third of hospitalized patients with urine cultures yielding Candida were in the Intensive care unit (ICU) where bladder catheter use was high (Shay and Miller, 2004). The aim of the present research was to carry out antifungal potency evaluation test against Candida species isolated from female patients attending Aminu Kano Teaching Hospital. These antifungal agents inhibit macromolecule synthesis (Flucytosine), impair membrane barrier function (polyenes), inhibit ergosterol synthesis (allylamines, thiocarbamates, azole derivatives, and morpholines) or interact with microtubules (Vanden et al., 1997).. Emergence of drug resistance among yeast isolates and consequent increase in serious fungal infections have been reported (DeMuri et al., 1995). The mechanism of resistance to these antifungal agents by yeast isolates are purely chromosomal as Candida species lack plasmid or other natural mechanism capable of transferring genetic materials between strains (Odds et al., 2003). Knowledge of mechanisms of antifungal resistance has been valuable in identifying resistant isolates and using them to validate in vitro measurement systems (Ghannoum,

Study Area
The study was conducted in the microbiology laboratory of Aminu Kano Teaching Hospital (AKTH) Kano. The hospital is located in the metropolitan city of Kano in Tarauni LGA. It serves as a referral centre for diseases that could not be manage by other hospitals within the Kano environs and some neighbouring states.

Study Population
The study targeted only female patients that were sent to microbiology laboratory by clinicians with a request form and are suspected having Candidiasis. Those that did not meet the above criteria were not included in this study.

Sample Size
A total of 521 clinical samples including 342 urine and 179 high vaginal swabs were collected and analyzed from female patients attending Aminu Kano Teaching Hospital with suspected cases of Candidiasis between august, 2013 to June, 2014.

Ethical Clearance
This was obtained from the hospital ethical committee prior to the commencement of the study. In addition, an informed consent was sought from patients before International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 @ IJTSRD | Available Online @ www.ijtsrd.com | Volume -1 | Issue -5 | July-Aug 2017 Page: 978 their enrolment. Those that refused to be recruited after much explanation were not considered in the research.

SAMPLE COLLECTION
Urine Collection and Processing Fresh morning midstream urine samples were aseptically collected in clean, dry culture bottles. Patients have been told to first allow urine to flow out and collect the midstream in a sterile screw cap container and then replace the lid firmly and bring the samples to the microbiology laboratory. For each specimen collected, Age, Sex, Laboratory number, and Name of patients were clearly labelled. At laboratory, the urine was first transferred into a sterile test tube and centrifuged at 3000 rpm for 5 minutes. After centrifugation, the supernatant was decanted and the sediment was streaked on to SDA (Himedia) with the aid of a standard wire loop calibrated to hold (0.01) ml urine. Specimens were incubated at 37 0C for 24-48 hrs. Wet film was then prepared and the remaining portion of the sediment was placed on a dried grease free slide and stained using Gram`s method. Microscopy was followed to observe the presence of yeast cells. High Vaginal Swab Collection and Processing .High vaginal swab samples were collected aseptically in sterile cotton tipped swab stick by a trained female nurse officer. The specimens were properly labelled and then transported to the laboratory for analysis. Upon receipt at the laboratory samples were inoculated on SDA (Himedia) directly using the cotton tipped swab stick and then spread by streaking with standard sterile wire loop. Specimens were incubated at 37 OC for 24 -48 hrs. The swab was then replaced back to its container and 2-3 drops of normal saline were added. This was shaken vigorously and dispensed in to a sterile test tube. The specimen was centrifuged at 3000 rpm for 5 minutes, supernatant was decanted and a wet film prepared to observe the presence of yeast cells. Part of the sediment was used for Gram staining. The stained slides were examined under microscope for budding yeast cells. Identification and Characterization Yeast isolates were identified to specie level using Chromogenic Agar and API 20C Aux kit Biomeriuxµ company of France. Inoculation on Chromogenic Agar Yeast isolates were first sub cultured on SDA (Himedia) to obtained pure/ fresh colonies of the organism to be tested. Chromogenic agar was then prepared according to manufacturer guidelines. The yeast isolates were sub culture on to the media and incubated for 24 -48 hrs. API 20 AUX C Kit, Yeast isolates were first sub culture on SDA in order to obtain fresh colonies of the organism. Colonies were picked by successive touches and introduced in to a sterile test tube containing 2ml (0.85%) normal saline. The turbidity was measured visually to match with 2 Mc Farland standard as directed by the manufacturer. And then 100 µl of the suspension was pipetted with micro titre pipette and dispensed into API media. Further transfer of suspension was done from API media to API strip that is made up of 20 cupules. The cupules were filled with the API media already containing the test organism and then incubated at 29 0C for 48 -72 hrs as directed by the manufacturer. The cupules are already pre-coated with sugars except the first cupule that serve as control. After incubation, growth in each cupule was compared with the control. A cupule that is more turbid than the control was recorded in the result sheet as positive. On the result sheet the tests are separated into seven groups with each group bearing three digits numbers of 1, 2 and 4. Therefore, by adding the numbers corresponding to positive reactions within each, a 7 digits number is obtained which constitute the numerical profile. The final identification was done by checking the seven digits number in the API booklet, where yeast species are provided with codes that correspond to Excellent, Very Good, Good and Acceptable Identifications. Antifungal Susceptibility Test Disk diffusion method using commercially prepared antifungal single disk (Bioanalyse) Turkey was employed as recommended by (National committee for clinical laboratory standards (NCCLS-M44A, 2004).
Procedure: Fresh culture of each isolate of not more than 18-24 hrs old as recommended by the company were suspended in a sterile test tube containing 5ml (0.85%) normal saline. The turbidity was measured visually to match with 0.5 Mc Farland standard. Sterile cotton tipped swab stick was then dipped into the suspension containing the organism to be tested. The cotton tipped swab stick was then raised above the suspension and pressed along the inner wall of the test tube to remove excess moisture. This was inoculated on dried plate of Muller Hinton agar supplemented with 2% Glucose and 0. In this case only fluconazole and voriconazole interpretive break points were provided. However, the response to other antifungal agents for which no interpretive break points provided by CLSI, these were interpreted according to the manufacturer's instructions (Table  1). Also, two quality control strains of Candida namely: C. albicans ATCC 90028 and Candida tropicalis ATCC 750 were used.

RESULTS
In this research a total of 521 samples including 342 and 179 Urine and High vaginal swabs respectively were processed and analyzed, out of which only 59 (11.3%) were Candida species (Table 1). However, the prevalence of Candida species in urine was 8 (2.3%). The species isolated were C. albicans of about 3 (37.5%), C. glabrata, C. tropicalis, C. famata, C. parapsilosis, and C. magnoliae each with 1(12.5%) respectively. Higher prevalence of 51(28.5%) was recorded in high vaginal swab samples which includes C. albicans 19 (37.3%) followed by C. glabrata 18(35.3%), C. tropicalis 4(7.8%), C. krusei 3(5.9%), C. magnoliae and C. lusitaniae 2(3.9%) each, C. parapsilosis, C. famata, and C. guilliermondii 1(1.96%) respectively. The predominant specie isolated in both the clinical samples was C. albicans (Table 2). With the respect to antifungal susceptibility profile of urine isolates, the result shows that 75% of the isolates were susceptible to Ketoconazole. While 62.5% of the isolates were susceptible to Fluconazole, only 50% were susceptible to Voriconazole and Flucytosine respectively. Interestingly, C. famata was resistant to all the agents and in addition none of the isolates was susceptible to Nystatin. Similarly C. tropicalis was only susceptible to ketoconazole while C. albicans was susceptible to all the agents except Nystatin (Table 3) In addition, C. famata and C. guilliermondii were resistant to all the agents except that C. famata was susceptible to only Ketoconazole.    N FLU  VOR  NYS  5FC  KTC  S  I R  S  I  R  S  I  R S  I R  S  I  R  1 C. albicans  19 5 1 13  5 0  14  1 -18 1  0 18  16  -3  2 C. glabrata  18 8 4  6  5  3 10  1 -17 9  5  4  17 -

DISCUSSIONS
Vaginal discharge is one of most frequent gynaecological problems encountered in females, especially during their reproductive stage (Akingbade et al., 2013). This study has identified the different Candida species responsible for various degrees of infections among female patients attending Aminu Kano Teaching Hospital. The study also determined the antifungal susceptibility profile of all the C. species isolated. The results for the study revealed that, the overall prevalence of Candida isolates in the clinical samples was (11.3%) with C. albicans (37.3%) as the leading specie isolated. This is in line with (12.7%) detected by (Feglo et al., 2009) and (Mohanty et al., 2007) who also reported as much as (18.5%) prevalence in India, among sexually active married women attending rural primary health care. And in both the studies C. albicans was the commonest specie isolated. The prevalence of candiduria in this study was found to be (2.3%) with C. albicans (37.5%) as the highest specie isolated. This is lower than the (5.1%) reported by (Ayeh-Kumi et al., 2007) and (11%) observed by (Feglo et al., 2009). However, isolating C. albicans (37.5%) as the major C. specie in this study has completely agreed with the findings of (Gizachew et al. In the same vein, susceptibility to flucytosine was detected as (25.4%). This is absolutely lower than the (96.6%) documented by (Fundik and Tuncer 2002) and (83.6%) by (Feglo et al., 2009). The possible reasons to this could be suggestive to the fact that, sometimes results of in vitro antifungal susceptibility test do not always faithfully reflect events in vivo (Sobel et al., 2003 andFan et al., 2007).This is due to variations among individuals, characteristics of the drugs, and variable behaviour of the microorganisms in each individual (Srinivasan and Kenneth, 2006). It is important to note that the CLSI document (M44-A) refers to disk International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 @ IJTSRD | Available Online @ www.ijtsrd.com | Volume -1 | Issue -5 | July-Aug 2017 Page: 983 diffusion testing of standard conditions only for fluconazole and voriconazole. Other antifungal agents need further research to ensure the standardization of this method (Srinivasan and Kenneth, 2006).

CONCLUSION
In conclusion, C. albicans has been incriminated as the leading agent responsible for candidiasis among female patients attending Aminu Kano Teaching hospital followed by C. glabrata. Also, the various degree of resistance observed in this research has clearly signalised the need to closely monitor candidal infections by ensuring that antifungal susceptibility test is routinely carried out for better management of patients' conditions and avoidance of emergence of resistance Candida strains.

RECOMMENDATION
The need to introduce antifungal sensitivity test in Aminu Kano Teaching Hospital is of paramount importance. Development of interpreted break point for all antifungal agents using Agar diffusion method should be strongly considered by relevant bodies. Identification of Candida isolates to species level should be done routinely to enable brighter understanding of their distributions. Mobilization, sensitization as well as health education messages should be made public on the danger attached to selfmedications and indiscriminate use of drugs. All relevant agencies such as NAFDAC, Standard Organization and other regulatory agencies saddled with the responsibilities of checkmating the quality of drugs and food should double their effort in fighting sub-standard and fake drugs. With the present attention on yeast infections, more resources should be mobilized in order to strengthen research in the area.