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A cost-effective carbohydrate fermentation test for yeast using microtitre plate
Pertaining to substantial increase in number of patients with suppressed immunity as a
result of human immunodeficiency virus (HIV) infection, advances in cancer chemotherapy
and immunosuppressive therapy for organ transplantation, opportunistic yeast infections
are becoming increasingly common. Non‑albicans candida species have emerged as a
significant healthcare problem with the development of resistance to multiple antifungal
agents in recent years. Consequently, laboratory identification of yeasts has crucial role in
patient‑care as well as in research. Carbohydrate fermentation tests are essential for
speciation of bacteria and fungi and are particularly relevant in setups where automated
identification systems are unavailable. Miniaturization of biochemical tests have been found
to be beneficial in reducing the cost of reagents. Although the miniaturized fermentation
tests have been developed for bacteria, [1] it has not been standardized for fungi. In
this study, we describe a cost‑effective in‑house simple modification of carbohydrate
fermentation test for yeasts.
The objective of this study was to evaluate the performance of miniaturized carbohydrate
fermentation test for yeast identification in comparison to conventional
identification tests. In this study, a culture collection of 31 clinical yeast isolates, comprising
Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida
krusei and Cryptococcus neoformans were identified by microscopy, germ tube formation,
morphology on cornmeal agar and HiCrome Candida differential agar and tested for
carbohydrate fermentation by both microtitre plate method and conventional method.
C. albicans ATCC 90028, Candida tropicalis ATCC 750, Candida parapsilosis ATCC 22019
and Candida krusei ATCC 6258 were used for quality control. The miniaturized
fermentation test was carried out in a 96 well (8 × 12 wells) autoclavable polypropylene
microtitre plate. The wells of horizontal rows were utilized for testing fermentation reaction
of a particular carbohydrate viz. glucose, sucrose, lactose and maltose. 300 l of basal
media containing phenol red indicator and 2% test sugar was added to each well of a
horizontal row. The panel was sealed and stored at 4°C. Before use, it was brought to room
temperature and 12 vertical columns were labelled with 12 test isolate numbers. Yeast
suspension in sterile distilled water equivalent to No. 1 McFarland standard was prepared
and one drop of it was inoculated in each well of the vertical column [Figure 1]. The
microtitre plate was covered with the lid and incubated at 25–30°C for 7 days.
Development of yellow colour was considered as positive result. Same isolates were also
tested by conventional sugar fermentation method in 12 × 100 mm glass test tubes
containing 3 ml media. In this study, test sugars were prepared as 10% aqueous solutions
and were sterilized by free steaming at 100°C.[2] Twenty millilitre and 5 ml of this stock
solution were used for testing 24 isolates by conventional and modified sugar fermentation
test, respectively. Phenol red indicator was used in both conventional and modified
carbohydrate fermentation tests.
Results
Identification based on microscopy, germ tube formation, morphology on
cornmeal agar and HiCrome Candida differential agar had same results as
conventional sugar fermentation. Candida albicans was most common isolate
(n = 14), followed by Candida tropicalis (n = 12), Candida glabrata (n = 2),
Candida parapsilosis (n = 1), Candida krusei (n = 1) and Cryptococcus neoformans
(n = 1). Except two discordant results, we found no significant
difference in results of conventional and miniaturized sugar
fermentation method. Two C. albicans isolates which were sucrose negative
in conventional sugar fermentation (no acid and gas formed), displayed
positive result (acid produced) in microtitre plate method. There were
no discrepancies noted, when only acid production was compared to gas
production in interpretation of results of conventional fermentation
test. All tests which produced gas also had acid reaction. The
sensitivity and specificity of this modified technique was 85.7% and
100%, respectively, when compared with conventional sugar fermentation
test with observation of gas production.
Discussion
The principle of fermentation tests is based on detection of acid and
gas produced as a result of sugar fermentation. Usually a pH indicator
is incorporated in the media to reflect the change in pH in terms of
colour change. Unlike bacteria, its use for speciation of fungi has a
major limitation. Owing to the slow growth rate of fungi, prolonged
incubation up to 3 week is recommended. We found that on an average, the
time taken to produce positive reaction was 24 hours for microtitre
plate method, in comparison to 72 hours in case of conventional test.
This finding is in keeping with other studies where smaller volume of
media and a relatively heavy inoculum has been reported to yield a rapid
reaction.[3] Although the test in this study was performed with 300 l
of carbohydrate fermentation media, reducing this volume to 100 l may
avoid spillage or contamination during handling. However, this
miniaturization from 300 l to 100 l needs further evaluation.
Aliquoting the sugar solution in smaller volumes and storing these for
later use may be helpful in reducing risk of contamination of unutilized
sugar solution if stored in a single container. Miniaturized biochemical
tests were initially developed for bacterial pathogens. These are also
available commercially. Microtitre plate has been used for testing
fermentation reactions in Neisseria and anaerobic bacteria.[1,4] However,
this method has not been tested and standardized for yeasts.
Cost‑effective analysis was done and the results are shown in Table 1a
and b. As per our estimate, this modified technique will reduce the cost
of identification when compared with conventional method by 80% (Rs.
39.56 for conventional sugar testing and Rs. 8.17 for the modified
method). Although commercial tests (Candifast[5] from ElitechGroup,
Puteaux, France and GLABRATA RTT[3] from Fumouze Diagnostics, Levallois
Perret, France) with similar methodology are in use for prompt
identification of yeasts, the higher costs often limit their application
in most mycology laboratories. Hence, this simple in‑house modification
of carbohydrate fermentation test may be used as an adjunct to the
conventional identification tests for rapid identification.
In conclusion, miniaturized carbohydrate fermentation test using
microtitre plate may be employed as a Table 1 (a): Cost of preparation of sugar
fermentation media
Media and sugars Cost Phenol red base media
(M054, Himedia, Mumbai) 16 grams to be suspend in
1000 ml distilled
water 100 gm = Rs. 460
16 gm = 1000 ml = Rs. 73.6
100 ml = 1.6 gm = Rs. 7.36
Dextrose (RM016) 500G = Rs. 215
Sucrose (RM601) 500G = Rs. 333
Lactose (RM565) 500G = Rs. 648
Maltose (RM3050) 500G = Rs. 850
(All Hi‑media, Mumbai) Average cost of sugar = Rs. 511.5
For 2 gm on average cost = Rs. 2.046 for each sugar
For 0.5 gm on average
cost = Rs. 0.51
References
1. Kellogg DS Jr, Turner EM. Rapid fermentation confirmation of Neisseria
gonorrhoeae. Appl Microbiol 1973;25:550‑2.
2. Collee JG, Miles RS, Watt B. Tests for identification of bacteria. In: Collee JG,
Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical
Microbiology. 14th ed. Chap. 7. London: Churchill Livingstone; 2010. p. 132.
3. Freydiere AM, Robert R, Ploton C, Marot‑Leblond A, Monerau F, Vandenesch F. Rapid
identification of candida glabrata with a new commercial test, GLABRATA RTT. J Clin
Microbiol 2003;41:3861‑3.
4. Stargel D, Thompson FS, Phillips SE, Lombard GL, Dowell VR Jr. Modification of the
minitek miniaturized differentiation system for characterization of anaerobic
bacteria. J Clin Microbiol 1976;3:291‑301.
5. Gundes SG, Gulenc S, Bingol R. Comparative performance of fungichrom I, candifast
and API 20C Aux systems in the identification of clinically significant yeasts. J Med
Microbiol 2001;50:1105‑10.
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