ORIGINAL�ARTICLE ABSTRACT Objective: To evaluate the performance of CHROMagar and API 20C AUX for the documentation of different Candida species. Study Design: Descriptive cross-sectional study. Place and Duration of Study: The study was conducted in the Department of Microbiology at AFIP (Armed Forces Institute of Pathology), CMH Rawalpindi and Army Medical College Rawalpindi in collaboration with Departments of Pathology (Microbiology) at Pakistan Railways Teaching Hospital (PRH), Islamic International th th Medical College Rawalpindi from 01 April 2017 to 30 September 2017. Materials and Methods: Collectively 100 isolates of candida yielded from HVS clinical samples. Phenotypic tests including growth on CHROMagar Candida and API 20C AUX were used for reporting different Candida species. Clinical Candida isolates along with reference institutional control strains of Candida species were used in the study. Data was analyzed using simple descriptive statistics (frequencies, percentages) for each categorical variable. Results: Among 100 candida isolates 92 (92%) isolates of Candida were identified correctly to level of species by CHROMagar Candida, in comparison to 100% identification of candida species using API 20C AUX. Results of present study revealed that CHROMagar Candida can be used to report three species of Candida considering the morphology and colour of colonies of these particular species, and to distinguish them as C. albicans, C. tropicalis, and C. glabrata. Conclusion: Both phenotypic tests CHROMagar plates and API 20 C AUX are effective in the documentation of Candida species. However API 20 C Aux is found to be more accurate than CHROMagar because less commonly isolated Candida species cannot be documented using CHROMagar. Moreover being less costly; use of CHROMagar Candida is helpful in rapid identification and constructing suitable therapeutic plan for patient's management in laboratories with limited resources. Key Words: Candida Species, HVS, Identification Methods. 1980s, a steady rise in incidence as well as prevalence of these infections has been found contributing significantly to morbidity and mortality. Mycotic infections are frequently found in patients 1,2,3 with depressed immunity like cancer patients receiving chemotherapy, transplant patients and AIDS patients who are more prone to develop 4 infections caused by candida. Infections due to Candida albicans remains the most frequent etiology of human diseases due to genus candida, but the incidence of infections due to non albicans candida is 5,6 also increasing. There is a variety of methods for identifying Candida species from clinical samples, among them CHROMagar Candida differential medium is commonly used to isolate presumptive C. albicans, C. dubliniensis, C. tropicalis and C. krusei. Its sensitivity and specificity is considered satisfactory Introduction Fungal infections have worldwide spread. Since Comparative Evaluation of CHROMagar and API 20C AUX in Isolation and Identification of Candida Species 1 2 3 Uzma Mussarat Malik , Abdul Bari Khan , Muhammad Luqman Satti Correspondence: Dr. Uzma Mussarat Malik Department of Pathology Islamic International Dental College Riphah International University, Islamabad E-mail: uzma_arslan5@yahoo.com 1 Department of Pathology Islamic International Dental College Riphah International University, Islamabad 2 Department of Pathology Islamic International Medical College Riphah International University, Islamabad 3 Department of Microbiology Armed Forces Institute of Pathology, AFIP Rawalpindi Funding Source: NIL; Conflict of Interest: NIL Received: Feb 29, 2018 Revised: May 10, 2018 Accepted: May 15, 2018 Efficacy of CHROMagar and API 20 C AUXJIIMC 2018 Vol. 13, No.2 85 7,8 for these species. The biochemical characterization is done using the API® 20C AUX (BioMerieux, France), which relies on variations in the assimilation of 9 carbohydrates. However, it presents limitations related to cost and to distinguish between some 7 species. Presently the emergence of non albicans Candida species is a major problem to be addressed 10,11 in several institutions. In 70% to 90% vulvovaginal candidiasis cases, C. albicans is found to be main causative agent to be followed by C. glabrata causing 5,6,12 10% - 20% of vaginal candidiasis. Most species of Candida are involved in causing vulvovaginitis but C. krusei, C. parapsilosis ,and C. tropicalis are 13 infrequent causative agents. Conventional methods used for candida species identification like assimilation and fermentation reactions are described as clumsy and beyond the range of expertise in local laboratories. Evaluation of identification methods in resource-limited settings for candida species such as microscopy, colonial morphology and biochemical studies require unique research studies for effective management and 14 prompt diagnosis of fungal infections. Candida vaginitis is usually diagnosed without proper diagnostic procedures but there is possibility that women may be uninfected or may be suffering from another illness. Culture on Sabouraud's dextrose agar (SDA) for diagnosis of fungal infections is considered as gold standard, while isolation and identification using different phenotypic assays can 15,16 take up to 2 – 4 days. Rapid identification of mycotic infections is possible with use of different brands of chromogenic media. These chromogenic agars, reduces the time required for the identification of yeast by distinguishing common Candida species on the basis of specific color that generated because of reaction of substrate with enzymes secreted by microorganisms after 0 17,18 incubation for 48 hours at 37 C. Use of API 20C AUX kit for identification of candida species including C.albicans and non albicans candida is easier and has greatly reduced the laboratory time involved in the speciation of Candida 19, 20 isolates. Owing to limited knowledge and practice of these phenotypic methods, this study was aimed to compare and evaluate the efficacy of CHROMagar and API 20C AUX in identification of Candida species. Materials and Methods A descriptive cross-sectional study was conducted st th from 01 April 2017 to 30 September 2017 after approval from Research and Ethical Review Committee. Non-probability convenient sampling was done and non-parametric data were collected. The data of Candida species were analyzed by using frequencies distribution test on SPSS (version 20) software. A total of 100 candida isolates were collected from Microbiology labs of Armed Forces Institute of Pathology (AFIP), Army Medical College (AMC) and Islamic International Medical College (IIMC) in collaboration with Combined Military Hospital (CMH), Military Hospital (MH) and Railway Hospital Rawalpindi respectively. Study included HVS s p e c i m e n s o f p re g n a nt , n o n p re g n a nt a n d postmenopausal women that revealed growth of , candida species on Sabouraud s culture plate. Direct Gram-stained smear examination was done for all collected candida isolates after culture on Sabouraud's dextrose agar (SDA) (Oxoid, UK) incubating at 37 °C for 24 – 48 hours. Institutional control strains were used to compare the results of present study. Conventional methods, such as germ tube test, macroscopic appearance and structural description of colonies i.e. colour, size and texture on Sabouraud's dextrose agar (SDA), and CHROMagar Candida were used to confirm the growth of control strains. CHROMagar Candida medium in each liter contained peptone (10 g), glucose (20 g), agar (15 g), and chloramphenicol (0.5 g) and Chromogenic mixture. (2 g), while pH of the medium was maintained at 6.1 according to instructions of manufacturer. Candida colonies of study samples from SDA agar were inoculated onto CHROMagar. Specimens were streaked for isolation onto the surface of the medium. The plates were kept for incubation at 30°C for 48-72 hrs in an inverted position. Forty two hours incubation time is obligatory for complete color development of Candida colonies. The diverse species of Candida species revealed dissimilar colours of colonies i.e. C. albicans colony appeared light to medium green, C. tropicalis colonies gave dark blue to metallic-blue colour and C.glabrata colonies looked light mauve to mauve. Moreover, these colonies were flat with a whitish border. Other candida species like C. krusei can give rise to light to Efficacy of CHROMagar and API 20 C AUXJIIMC 2018 Vol. 13, No.2 86 dark mauve colour. Rest of non albicans candida species produced light creamy to light pink colour that were later on identified with API kit. API 20 C AUX (Biomerieux, France) was used to p e r fo r m C a r b o hyd rate fe r m e ntat i o n te st s . Dehydrated substrates were added in 20 different cupules which allowed the performance of 19 assimilation tests. Semi solid minimal medium was used to inoculate the cupules and the growth of yeast was seen when utilized the added substrate as the soul carbon source. The reactions were read by linking them to growth controls and documentation was done by referring to the Analytical Profile Index. colonies and were termed as C. tropicalis, while 68 (68%) samples showed green coloured colonies of candida albicans. Among rest of 8 samples, 4 samples showed light pink colonies and 4 samples revealed blue to mauve coloured colonies which after API testing identified as C.famata 4(4%), C. guilliermondii 2(2%), Saccharomyces cerevisiae 1(1%) and C. lusitaniae 1(1%). Moreover, Candida albicans was found to be dominant over the non albicans species. The comparative identification results are shown in Table I. Fig 1: API 20C AUX Results of API were finally compared with the culture results and speciation of Candida was also done. Results Results of germ tube test revealed 68 out of 100 isolates were positive for germ tube and remaining 32 were negative as shown in Fig 2. Fig 2:F requency of Germ tube posi�ve isolates Among 100 samples grown on CHROMagar plates, 16 (16%) showed pinkish purple growth and were labeled as C. glabrata, 8 (8%) revealed blue coloured Table I: Iden�fica�on of Samples using CHROMagar and API 20C Aux The API 20 C AUX tests recognized the Candida species on the basis of fermentation and utilization of different sugars. The confirmatory test by API yielded seven species of Candida in total i.e. C. albicans, C. glabrata, C. tropicalis, C. guilliermondii, C. famata, Saccharomyces cerevisiae and C. lusitaniae. API test results were matched with CHROMagar results, germ tube test results and microscopy of these selected samples. Overall CHROMagar identified strains correctly with more than 90% discrimination while comparing with API 20 C AUX results. The results of API are tabulated in Table 1. The API 20 C AUX system correctly identified about 100% of the isolates compared to 92% by CHROMagar culture technique. The CHROMagar culture plates correctly identified all organisms except 8 isolates. These 8 isolates revealed pink to Efficacy of CHROMagar and API 20 C AUXJIIMC 2018 Vol. 13, No.2 87 purple coloured colonies that were identified differently using API 20C AUX. The reason for this deviation is because CHROMagar has been known to identify frequently found species of Candida i.e. C. albicans, C. glabrata, C. tropicalis. Discussion In developing countries especially where resources are limited, deficiency of training skills and non- availability of proper reagents that are contributory factors in making final diagnosis of mycotic infections, identification of fungal infections to species level become quite difficult. Moreover to m i n i m i z e t h e m o n e t a r y b u r d e n o n t h e underprivileged patients, laboratories only perform germ tube test and limit their report only to 21 identification of C. albicans. In present study, all 100 candida isolates gave distinct colours on CHROMagar thus helped in the recognition of candida species causing vulvovaginitis in study population. This data conforms to finding of 22 a study conducted by Horvath et al. There is nonconformance of our data with a study conducted by Grace L et al who reported 78% identification of 23 candida species using CHROMagar. This finding might be due to direct subculture of specimen on CHROMagar plates. LYnn L et al reported that CHROMagar readily identify C. albicans, C.glabrata, 22 C.krusei, and C. tropicalis. Diagnosis of vaginal candidiasis is usually based on clinical symptoms and direct microscopic examination as stated by Nyirjesy 24,25 et al and Faraji et al ; Although microscopic examination of clinical samples is quick, easy method and may recognize the probable causative agent, but CDC recommend that vaginal culture is mandatory 26 to confirm the diagnosis. In this study, different laboratory methods were used for prompt diagnosis of candida species, among these Gram staining and germ tube test were found easy and trustworthy techniques for the documentation of Candida spp. CHROMagar is found to be a novel medium enabling isolation and identification of different Candida species. This media correctly identified 92% of Candida strains which is in accordance with the prior 27 study done by Ozcan et al.in 2010. Based on different colors and morphology of colonies “CHROMagar Candida”, provided a fast and convincing recognition of frequently found yeasts species, which would ordinarily be missed during conventional plating on solid medium. According to Nejad et al the major advantage of “CHROMagar Candida” was its ability to detect the presence of 28 mixed species, and results of this study prove that use of CHROMagar was helpful in correct identification of C.albicans, C.glabrata and C.tropicalis depending on the colour and morphology of colonies. Chromogenic culture media 29 are very helpful in identification of C. albicans but its main limitation is less power of discrimination 30 among non albicans Candida species. In present study, 92 % of Candida isolates were correctly identified after growth on CHROMagar , in contrast to the study conducted by Dalia et al they stated that this medium correctly identified C. albicans with excellent sensitivity and specificity, but revealed 31 lower sensitivity for non albicans candida. The data suggested that species which were identified by CHROMagar were almost the same as were confirmed by API 20C Aux. It means that in the presence of CHROMagar, germ tube test to confirm C.albicans can be excluded. On completion of study it is acknowledged that CHROMagar medium is appropriate and affordable diagnostics medium in a resource-limit setting because approximate cost per culture for complete identification of Candida using SDA, Corn meal agar, and API 20C Aux in Pakistan is around Rs. 1,200 while CHROMagar Candida is around Rs. 250 per specimen culture. Additionally, 4 - 5 samples can be inoculated on one CHROMagar plate without compromising its effectiveness that makes it more cost efficient. Present study supports the statement that the most suitable and popular procedure for candida species identification is the use of commercially available kits for carbohydrate assimilation and / or enzyme 31 detection. In the present work, all Candida isolates were correctly identified to the species level by API 20C AUX. Results of present study about effectiveness of API 20C are in accordance with other 32,33,34,35 studies. API tests are evaluated as best methods for the final documentation of all Candida species but owing to the fact that in our present hospital settings where limited resources are available this technique is difficult to practice on routine basis. Comparison of CHROMagar and API 20C AUX (Biomerieux, France) reveals that use of CHROMagar is less costly while API identification Efficacy of CHROMagar and API 20 C AUXJIIMC 2018 Vol. 13, No.2 88 method is costly and laborious but API 20 C has an advantage that it can identify more species of non albicans Candida as compared to CHROMagar, which can identify only three to four commonly involved Candida species. Conclusion This study reveals CHROMagar Candida and API 20C AUX having good potential to rapidly identify candida species. In resource limit settings, CHROMagar can be used as useful adjunctive medium in the clinical laboratory but for identification of yeasts at species level, the use of API 20C Aux with a wider database is preferable. API 20C is better for the diagnosis of candidiasis especially due to less frequently found candida species and should be adopted as routine diagnostic procedure in the clinical microbiological laboratories. This study has involved evaluation of two phenotypic methods in identification of candida species in small number of cases with vaginal candidiasis because of cost and availability issues. Future studies including large sample size and comparison of these two methods with other diagnostic methods used for identification of candida species are needed. Moreover comparison and evaluation of these two methods in identifying candida species in cases other t h a n v u l v o v a g i n a l c a n d i d i a s i s a r e a l s o recommended. REFERENCES 1. 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