DIAGN MICROBIOL INFECT DIS 521 1989;12:521-523

NOTES

Rapid Identification of Candida albicans Using 4-Methylumbelliferyl N-acetyl- -galactosaminide

Maurice T. Dalton, David J.M. Haldane, and June MacDonald

Rapid identification of Candida albicans is performed mainly by the germ-tube test. However, recent reports have suggested that up to 5% of C. albicans species can give false negative results. We describe the use of 4-Methylumbelliferyl N-acetyl- ~-D-galactosaminid~ (4-MAG) conjugate as an alternative to the germ-tube test. Our results indicate that, in comparison to

the germ-tube test, the 4-MAG test has a sensitivity of 100% and a specificity of 92%. Candida tropicalis can give false- positive results, and that a further screening test is required to identify this species. Problems reading end-points were not encountered.

Candida albicans continues to be the most commonly recovered yeast in clinical laboratories. The more frequent use of immunosuppressing therapies and the prolonged survival time of immunocompro- mised hosts promotes opportunities for C. albicans as a major primary and opportunistic pathogen. Fur- thermore, the widespread use of broad spectrum antibiotics has also played a part in raising the clin- ical profile of candida infections (Muenier-Carpenter et al., 1981; Horant et al., 1988).

Rapid presumptive identification of C. albicans has been based on the demonstration of germ tubes fol- lowing incubation at 35°C for 4 hr in pooled human or animal serum (Mackenzie, 1962). The use of com- mercial pooled human serum is now considered by many to be hazardous, and, in addition, up to 5% of C. albicans isolates have been reported as germ- tube negative (Perry and Miller, 1988).

From the Departments of Microbiology, Victoria General Hos- pital and Dalhousie University, Halifax, Nova Scotia, Canada.

Address reprint requests to: Maurice T. Dalton, Department of Microbiology, Victoria General Hospital, Halifax, B3H 1V8, Nova Scotia, Canada.

Received March 27, 1989; revised and accepted August 8, 1989. © 1989 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/89/$03.50

Bobey and Ederer (1981) and Perry and Miller (1988) previously indicated the hydrolysis product of 4-Methylumbelliferyl N-acetyl-~-D-galactos- aminide (4-MAG) conjugate is 4-Methylumbell- iferone, which produces a pale blue fluorescence when excited by a 365 nmol UV light source. 4-MAG is hydrolysed by either preformed or recently formed ~-D-galactos-aminidases present in C. albicans. Perry and Miller (1988) recently suggested that this test may be a useful alternative to the germ-tube test. We report here a comparison of the germ-tube test and 4-MAG breakdown by 292 clinical isolates of yeasts in our laboratory.

Two hundred three consecutive yeasts recovered from clinical specimens and 92 clinical yeast isolates previously identified by the API 20C ® System (An- alytab Products, Plainsview, New York) were ex- amined by both the germ-tube test and the 4-MAG test.

Stock cultures and fresh isolates were first sub- cultured onto 5% sheep blood agar (B.B.L., Cock- eysville, Maryland) for purity testing. The germ-tube test was performed by making a dilute suspension of yeast with pooled human serum (Difco, Detroit, Michigan), in borosilicate tubes 13 mm x 100 mm (Johns Scientific, Toronto, Ontario), using a sterile Pasteur pipette. The suspension was incubated at

522 M.T. Dalton et al.

35°C for 4 hr in open-topped tubes. A drop was then transferred to a clean glass slide, using the same Pasteur pipette, for examination by light microscopy at x 450. A germ tube was defined as a short lateral hyphal element, nonseptate, with parallel sides and no constriction at the yeast cell end (Merowitz et al., 1977). One hundred consecutive cells were exam- ined in at least ten high-powered fields in each case.

The 4-MAG test was performed as described by Perry and Miller (1988) with the exception of the concentration of 4 MAG. We initially experienced difficulties in reading results with known C. albicans controls. Using A.T.T.C. control strains we varied the concentration of 4-MAG that we found to give most readable results at 1.5 mmol in 0.1 mol acetate buffer pH 5.1. The 4-MAG test tubes were examined at 30, 60, and 120 min and at 4 hr using a 365 mm UV light source (Ultra-Violet Products Inc., Los An- geles, California). An inoculum equivalent to 5.5- 6.5 x 10 s was used for the test throughout. The presence or absence of characteristic light blue flu- orescence was recorded as positive or negative.

Previously identified strains of yeasts were tested blindly and the identification code was broken only at the end of the study. Strains of fresh clinical iso- lates yielding discordant results between the the germ tube test and the 4-MAG test were subsequently subjected to our identification procedure using API 20C.

Candida albicans ATCC 44831, Candida kefyr ATCC 28838 (as a negative control), and Candida tropicalis ATCC 28707 were used throughout as controls. The C. tropicalis ATCC 28707 control gave positive results in the 4-MAG test.

Comparison of 4-MAG and germ-tube test results for 203 consecutive yeast isolates is shown in Table 1. One hundred fifty-three (76%) isolates were pos- itive by both the germ-tube and the 4-MAG tests, and 45 (22%) isolates were negative by both tests. Five isolates were positive by 4-MAG but negative by the germ-tube test. Further identification of these five isolates showed them to be Trichosporon beigelii (1), Candida parapsilosis (1), Candida tropicalis (2), and one unidentified yeast.

Analysis of results from 92 identified yeasts is

shown in Table 2. All 47 C. albicans strains were correctly identified by both methods. However, four strains of C. tropicalis gave false positive results with the 4-MAG test. None of 21 other isolates of five different species of yeast gave discrepant results. All 4-MAG results are reported after 4 hr incubation. Twenty-three strains of C. albicans gave negative re- sults, and one C. tropicalis gave a false positive result, after 2 hr incubation.

Candida spedes are of increasing concern in many health-care institutions both as primary pathogens and as opportunist pathogens, particularly among immunocompromised patient populations. Because of developments in effective antifungal therapies and the clinical significance attached to C. albicans it is becoming increasingly important to identify C. al- bicans as rapidly as possible. Rapid presumptive identification of C. albicans has been based since 1962 mainly on the demonstration of germ-tube produc- tion in pooled human or animal serum at 37°C (Hor- ant et al., 1988). C. stellatoidea, previously considered a separate species that also gave positive results in the germ-tube test, has been taxonomically included with C. albicans. (Martin 1979) noted C. tropicalis from the oral cavity could also produce germ tubes under the same conditions as C. albicans. This, in addition to the need for expertise in reading the germ tube test, has resulted in a search for new rapid and re- liable techniques for the presumptive identification of C. albicans. Among possible alternatives for pre- sumptive identification Perry and Miller (1988) have shown the 4-MAG test to have a similar sensitivity and specificity to the germ-tube test and to have the advantage of being readable at 2 hr rather than at 4 hr.

In our study we found that the germ-tube test correctly allocated all 92 known isolates of yeasts, whereas the 4-MAG test correctly allocated only 88 isolates (Table 2). The four misallocations were all C. tropicalis that, as expected, gave negative results in the germ-tube test. There were no false-negative or false-positive germ-tube tests recorded for any of the other seven species tested. With this group of organisms, the sensitivity and specificity of the 4- MAG test, compared to the germ-tube test, were

TABLE 1. Comparison of 4-Methylumbelliferyl N-Acetyl-f~-D- Galactosaminide Test (4-MAG) and Germ-Tube Test Results on 203 Consecutive Yeasts Recovered from Clinical Specimens

4-MAG

Germ-tube test Positive Negative

Positive 153 0 Negative 5 45

Notes 523

TABLE 2. Compar ison of 4-Methylumbelliferyl N-Acetyl-~-D- Galactosaminide Test and Germ-Tube Test Results and Identification of 92 Known Yeasts from Clinical Specimens

Total GT+/ GT+/ GT+! GT+! number 4-MAG + 4-MAG- 4-MAG + 4-MAG-

C. albicans 47 47 - - - - - - C. tropicalis 26 - - - - 4 22 T. glabrata 10 - - - - - - 10 C. parapsilosis 3 - - - - - - 3 C. lusi tanae 3 - - - - - - 3 C. krusei 2 - - - - - - 2

C. pseudotropicalis 1 - - - - - - 1

GT ÷, germ-tube test results positive; GT-, germ-tube test result negative; 4 MAG +, 4-methylum- belliferyl N-acetylq3-D-galactosaminide test results positive; 4 MAG-, 4-methylumbelliferyl N- acetyl-~-D-galactosaminide test results negative.

100% and 92%, respectively, and the efficiency of the 4-M.A.G. test was 96%.

Results for 203 consecutive clinical isolates, Table 1, show five (2.5%) isolates were positive for the 4- MAG test while giving negative results for the germ- tube test. Two of these five isolates were subse- quent ly identified as C. t ropica l i s . Under these con- ditions, the sensitivity and specificity of the 4-MAG test as compared to the germ-tube test were 100% and 90%, respectively, with an efficiency of 97.5%.

Results were not reliable before 4 hr incubation. Only 24 of 45 test results recorded positive for C. a lb icans and one false-positive result were positive at 2 hr. Prolonged incubation overnight resulted in six extra false-positive 4-MAG results. By using pos- itive and negative controls and the darkened inside of an incubator to read specimens, all results were easily readable and had clear-cut end points at 4 hr.

It is notable that positive results remained positive for up to 2 weeks despite cont inuous incubation in sealed tubes.

From this s tudy, the 4-MAG test appears to have similar sensitivity but is less specific than the germ- tube test. False-positive results were caused mainly by C. t rop ica l i s , which may be dist inguished from C. alb icans by cellobiose assimilation or by using a tech- nique similar to the 4-MAG. Bobey et al. (1981) de- scribed the use of a 4-Methylumbelliferyl conjugate to detect py rophospha te diesterase in nine out of nine strains of C. t ropica l i s . We also found that the 4-MAG test was not readable at less than 4 hr in- cubation. The main advantages of the test were ease of reading and less "hands -on" time for technolo- gists. Material costs of the 4-MAG test were similar to those of the germ-tube test.

R E F E R E N C E S

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Horant T, Culver D, Jarvis W, Emori G, Banerjee S, Mar- tone W, Thornsberry C (1988) Pathogens causing no- socomial infections: Preliminary data from the National Nosocomial Infection Surveillance System. Antimicrobic News le t t e r 5:65-67.

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