R. Bharathi

Department of Microbiology, Sri Devaraja Urs Medical College, Kolar – 563 101, Karnataka, India.

Abstract

In recent days, non albicans candida species are emerging, many of these species are inherently resistant to routinely used antifungals. Hence, the need for speciation of candida is important in the treatment point of view. Speciation of candida can be done by conventional methods, using chromogenic media, serological, molecular methods. Most of the laboratories use Hicrome for identification of yeast species considering its ease, rapidity. To determine the usefulness of Hicrome agar in the identification of different species of candida in comparison with corn meal agar. The candida  isolated from cutaneous candidiasis were included. Species identification was done by colony color on Hicrome, and confirmed by morphology on corn meal agar for 80 candida isolates. The 10 different species of candida were identified. Emerging species of candida – Candida kefyr, C.zeylanoides, C.lusitaniae C.lipolytica were identified by morphology on corn meal agar which were misidentified as C.glabrata by Hicrome. Identification of C.albicans, C.tropicalis, C.krusei by colony colour on Hicrome correlated with the morphology on corn meal agar. So, the  incorporation of corn mealagar in the routine yeast identification is more judicious than Hicrome as it increase the accuracy in the identification of candida species within in the same time span as that of Hicrome.

Keywords: Hicrome, corn meal agar, Candida.

Introduction

Candida albicans remains the most  common causative agent of both superficial and deep fungal  infections1 .  But recent reports suggest that a shift has occurred in the distribution of infections, with NAC being increasingly detected2.

Due to the epidemiological alteration in the distribution of Candida species as well as significant increasing trend of either intrinsic or acquired  resistance in  some of these fungi, the precise identification of Candida species is necessary for effective antifungal therapy and also for prevention of nosocomial infections3. The  strains of  C. lusitaniae  may  show resistance to amphotericin B  so, for example, the automatic prescription of amphotericin B in a patient with a septicemia due to C. lusitaniae 4.

A large variety of methods have been developed with the aim of facilitating rapid, accurate yeast identification4. Many tests with different techniques from conventional to molecular methods are available for yeast identification.

Clinical microbiology laboratories face an important challenge to select a system for yeast identification that is accurate, cost-effective, easily interpreted and reasonably rapid 4.

Now a days, chromogenic media  are frequently used  in  the direct and rapid identification of yeasts because different Candida species produce unique colors on these media.5

Our study is to evaluate the usefulness of  Hicrome  agar for  speciation of candida  in comparison with  the  corn meal agar.

MATERIALS  AND  METHODS

The samples from cutaneous candidiasis cases (nail clippings from  onychomycosis, swabs from  intertrigo / napkin rash ) were included in the study .  A total  number of  80  candida were isolated following culture on the SDA slants. Candida  isolates  were speciated using Hicrome . Hicrome  Candida  agar  product code  number  M-677 was obtained commercially. Plates were  prepared  as  per  manufacturer’s  instructions. 5-8 colonies from 24-48 hr old SDA slants were inoculated on Hicrome agar and incubated at 37°C for 48 hrs. Colony morphology and colour  was  noted and compared with  HiMedia instructions6. As  per  HiMedia  instructions,  depending on the colour of  colonies  species were identified as follows

On Hicrome agar  medium C.albicans appear as light green coloured smooth colonies, C.tropicalis appear as blue to purple coloured raised colonies. C.glabrata colonies appear as cream to white smooth colonies, while C.krusei appear as purple fuzzy colonies. C.parapsilosis  appears as cream coloured colonies with mauve tinge6. Then the colony were inoculated on 1cm x 1 cm block of  corn meal agar (CMA)  block. The agar  block was covered with sterile cover slip and placed in a  sterile  petridish  moistened  with  filter paper and incubated at room temperature for 48 hours7,8.

After 48 hrs, the slide was placed on the microscopic stage and the edge of the cover slip was observed using 10x and 40x objectives for chlamydospores, pseudohyphae, hyphae, blastospores,  blastoconidia etc and the Candida were speciated7,8

RESULTS

Table 1  Showing the  identification of various candida species by Hicrome and  cornmeal agar. In our study 10 different candida species were identified by each method. But Candida kefyr, C.zeylanoides,  C.lusitaniae,  C.lipolytica were not identified by Hicrome.

Table 1. Showing the identification of various candida species by Hicrome and cornmeal   agar 

Isolates Detected on CMA Detected on CA
Candida albicans 24 24
Candida tropicalis 18 18
Candida parapsilosis 17 12
Candida glabrata 04 16
Candida guilliermondii 05 0
Candida krusei 06 06
Candida kefyr 03 0
Candida  zeylanoides 01 0
Candida lusitaniae 01 0
Candida lipolytica 01  0
Total 80 80

Table 2  Showing sensitivity and specificity of Hicrome agar against  CMA 100% sensitivity of Hicrome agar was observed in identification of C.albicans, C.tropicalis, C. krusei,  C.glabrata. Moderate sensitivity of 70.58% was observed in C.parapsilosis. 100% specificity was observed in C.parapsilosis, C. krusei. 96.77% & 96.42% specificity was observed in C. tropicalis , C.abicans  respectively. 84.21% specificity was observed in C.glabrata.

Table 2. Showing sensitivity and specificity of Hicrome agar   against   the cornmeal agar     

Species Sensitivity  % Specificity  %
Candida albicans 100% 96.42%
Candida tropicalis 100% 96.77%
Candida parapsilosis 70.58% 100%
Candida glabrata 100% 84.21%
Candida krusei 100% 100%

Table 3  Showing  sensitivity, specificity of  Hicrome as  observed  in other similar  studies. The  sensitivity, specificity of  C.albicans , C.tropicalis , C. krusei  are in agreement with  the similar studies done by VP Baradkar 5, Manisha 9.

Table 3. Showing sensitivity, specificity of   candida  of   Hicrome agar in comparison with other  similar studies

Species  Our study VP Baradkar5 Manisha9
C albicans 100%, 96.42% 96.55, 96.42 100%,100%
C  tropicalis 100%,96.77% 100%,100% 92.9,100%
C  parapsilosis 70.58%,100% 80%, 98.03% —–
C  glabrata 100%,84.21% 90.9%, 88.23% 100%, 96.42%
C  krusei 100%, 100% ————— 100%, 98.42 %

Table  4  Appearance  of  different candida isolates on Hicrome,  Cornmeal agar Hicrome agar  for candida  falsely identified C.parapsilosis as C.glabrata based on colony colour. Candida kefyr,  C.zeylanoides, C.lusitaniae  C.lipolytica  were  identified by morphology on corn meal agar which were misidentified as C.glabrata by Hicrome. The appearance of candida isolates on Hicrome agar, microscopic appearance on  Cornmeal agar are given in  Fig. 1.

Fig. 1. Microscopic appearance on corn meal agar ( left; magnification ,x400) and colony colors on Hicrome agar for candida after 48 hours of incubation. (A) C albicans (B) C. tropicalis (C) C. parapsilosis (D) C. glabrata (E) C. krusei

Table 4. Appearance of different candida isolates on  Hicrome , cornmeal agar

Species Colony colour on Hicrome Morphology on Corn meal agar(CMA) Identification with
Hicrome     
Identification with
Hicrome   vs  CMA  
C.albicans (24) Light green colored smooth colonies Pseudohyphae with terminal chlamydospores; clusters of blastoconidia at septa. Identified all strains as C.albicans Accurate identification all 24
C.tropicalis (18) Blue to purple coloured raised colonies Blastoconidia anywhere along pseudohyphae Identified all strains as  C.tropicalis Accurate identification all 18
C.parapsilosis (17) Cream colored colonies with mauve tinge Blastoconidia along curved pseudohyphae; giant mycelial  cells  Only 12 identified , other 5 strains misidentified as  C.glabrata Identified   all 17
C. glabrata(4) Cream to white smooth colonies No pseudohyphae; cells small; terminal budding 16 strains were identified , morphology essential   4 strains correlated by  both methods
C.krusei  (6)  Purple fuzzy colonies Pseudohyphae with cross – match sticks or treelike blastoconidia Identified all strains as C.krusei Accurate identification all 6
C.guillerimondii(5)

 

Cream colonies Fairly short, fine pseudohyphae, clusters of blastoconidia at septa misidentified as  C.glabrata  5 strains correlated by  both methods
C.kefyr(3) Cream colonies Elongated blastoconidia resembling logs in a stream along pseudohyphae misidentified as  C.glabrata 3 strains correlated by  both methods
C.zeylanoides(1) Cream colonies Pseudohyphae give feather- like appearance at low power misidentified as C.glabrata 1 strains correlated by  both methods
C.luistaniae(1) Cream colonies Short chains of elongate blastoconidia along curved pseudohyphae misidentified as  C.glabrata 1 strains correlated by  both methods
C.lipolytica(`1) Cream colonies Elongated blastoconidia in short chains along pseudohyphae misidentified as  C.glabrata 1 strains correlated by  both methods

DISCUSSION

Approximately five Candida species were considered pathogenic in the 1960s, recent reviews listed at least 17 Candida species as being pathogenic 4. When  studies are limited to this genus, the most frequently isolated species were C.albicans, C.tropicalis, C.glabrata , C.parapsilosis 5 ,9.

Isolation of other Candida species, such as C. krusei, C. guilliermondii, C.lipolytica, C. kefyr, along with other unspecified species were also increased10. These results are reflected by  increase in the case reports concerning new and emerging yeasts11.

Many tests with different techniques from conventional to molecular methods are available for yeast identification. But  selection of the method by a lab depends on its affordability (sample size etc.),  reliability of the test  result,  and also the time factor.

Many  studies states that the potential advantage of chromogenic media is the straight forward identification of mixed yeast infections12. Now a day’s Hicrome agar is most commonly employed for the yeast identification in clinical microbiology laboratories.

In our study 10 different species of candida were identified by morphological study  on  corn meal agar. On Hicrome only 5 species of candida were identified  ie, C.albicans, C.tropicalis, C.glabrata, C.krusei , C. parapsilosis. (Table 1)

Hicrome agar was useful in identification of C.albicans, C.tropicalis, C.glabrata, C.krusei with 100 % sensitivity, moderate  sensitivity  of  70.58 % was observed in C.parapsilosis. 100 %  specificity  was observed in  C.parapsilosis, C.krusei. Moderate specificity was observed in  C.glabrata  84.21%. (Table 2)

The sensitivity, specificity of  C.albicans  C.tropicalis , C. krusei C. glabrata, C.parapsilosis  are in agreement with  the similar studies done by VP Baradkar 5, Manisha9 (Table 3). As observed in the previous studies done on speciation of candida using Hicrome 5,9 only the above mentioned species were identified, new emerging candida species identification were not stated.

In our study we  noted that while identifying candida species based on the color description by manufacturer 8, 21 isolates which  appeared as white colonies were interpreted as C. glabrata. When morphological identification by corn meal agar was done on these isolates, C. parapsilosis(5) , Candida kefyr(3) , C.zeylanoides(1), C.lusitaniae(1), C.lipolytica(1), C. guilliermondii (5) were identified. (Table 4)

Hicrome agar falsely identified C.parapsilosis as  C.glabrata was stated in the study done by Sagar et al13 which is similar to our study.

The advantage of Hicrome agar in identification of candida  species i e., ease of the test method compared to conventional methods and rapidity of identification cannot be ignored in  the era of emergence of NAC. Identification on Hicrome agar poses a problem as it is based on colour, features like fuzzy, hue etc and variations in intensity of color with passage of time. The interpretation becomes subjective, besides the media only recommends identification of C.albicans, C.tropicalis, C. glabrata, C.krusei, C.parapsilosis.

The study by Hazen 10 states the variable efficacy for fluconazole is evident with C.glabrata,  C.parapsilosis , C.rugosa,. C.tropicalis , S. cerevisiae, and T.beigelii which adds to the importance of accurate identification.

As observed in our study many new species identification were missed when identification on  only Hicrome considering its, ease and rapidity.

The turnaround  time  taken for identification by morphology on corn meal agar is 48hr 7  is similar  to that on Hicrome as per the manufacturer’s instructions 6.

But  identification by morphological study on corn meal agar demands the skill from the lab personnel, which can be mastered.

Koehler14 et al opines that careful observation of  the  yeast morphology on corn meal agar, adds confidence in the identification of candida species, which will also alert the microbiologist about the presence of unusual isolates.

CONCLUSION

In view of accurate identification, the limitations of Hicrome in yeast identification  not to be ignored by a clinical microbiology laboratory. The  incorporation  of corn meal agar in .routine yeast identification prevents  misidentification, adds confidence, improves the mycology skills among the lab personnel without compromising cost ,or time factor.

References

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