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Journal of Clinical Microbiology, April 2007, p. 1315-1318, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.01688-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Systematic Survey of Nonspecific Agglutination by Candida spp. in Latex Assays{triangledown}

Karsten Becker,1,2* Ahmed S. Almasri,1 Christof von Eiff,1,2 Georg Peters,1,2 Christine Heilmann,1,2 and Wolfgang Fegeler1

Institute of Medical Microbiology,1 Interdisciplinary Centre of Clinical Research (IZKF), Münster, University Hospital of Münster, 48149 Münster, Germany2

Received 16 August 2006/ Returned for modification 3 January 2007/ Accepted 21 January 2007


   ABSTRACT
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In order to demonstrate that cells of Candida spp. may show considerable nonspecific agglutination in latex agglutination tests, 150 clinical and reference isolates of 12 yeast species were systematically studied by applying various test parameters. In fact, 40 (26.7%) of these isolates revealed nonspecific results, significantly associated with the time allowed for agglutination.


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Immunoassays based on latex technology have become widely applied as both rapid and easy-to-use diagnostic approaches and research tools. Latex agglutination tests (LATs) are based on the linkage of antigen or antibody molecules to the surfaces of a large variety of latex polymer particles, leading to the formation of immunocomplexes large enough to allow for optical detection. As other immunoassays, LATs are subject to interference that leads to false-positive or false-negative results (10). However, the special characteristics of the latex particles such as the surface charge, hydrophilic or hydrophobic nature, and adsorption and agglutination conditions may cause additional failures (15). Thus, in addition to nonspecific reactions due to the antigen-antibody reactions (e.g., those involving heterophilic antibodies and rheumatoid factors), factors affecting the specificity through the direct interaction of cross-reacting substances or (infectious) particles with the latex surface have to be considered.

For routine microbiological diagnostics, LATs are increasingly popular as confirmatory tests for, e.g., the identification of Staphylococcus aureus and its methicillin resistance (14). Whereas in general the sensitivities and specificities of these tests have improved in the past years, problems due to false-positive and false-negative reactions still remain (16, 20, 22). Among these, nonspecific results caused by Candida albicans are reported in the notes on the Pastorex Staph-Plus from the manufacturer (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France). Since C. albicans is known to express mannan adhesins known to bind to the surfaces of latex microbeads and microspheres (12, 13) and no data are available regarding the influence of the nonspecific binding of yeast cells on routine microbiological agglutination procedures, there is an obvious need to study systematically (i) the prevalence of C. albicans isolates agglutinating nonspecifically in LATs and (ii) the respective behaviors of non-C. albicans yeasts.

We tested a total of 150 clinical and reference strains comprising anamorphs of "C. africana" (n = 1), C. albicans (n = 50, including ATCC 90028, DSM 11943, DSM 11944, DSM 11946, and DSM 11948), C. dubliniensis (n = 10), C. (Torulopsis) glabrata (n = 10, including ATCC 90030 and DSM 11950), C. guilliermondii (n = 10, including DSM 11947), C. kefyr (n = 9, including DSM 11954), C. krusei (n = 10), C. lusitaniae (n = 10), C. parapsilosis (n = 10, including ATCC 22019, DSM 11224, and DSM 11955), C. tropicalis (n = 10, including DSM 11953), Cryptococcus neoformans var. neoformans (n = 10, including DSM 11959, DSM 11960, and DSM 11961), and Saccharomyces cerevisiae (n = 10, including ATCC 9763). The clinical isolates were recovered from German, Indian, and Syrian patients (3, 5). Only one isolate per patient was included. The isolates were identified by the API 32C identification system for yeasts (bioMérieux, Marcy-l'Etoile, France), and the identifications were confirmed by standard taxonomic procedures (11). In the case of ambiguous or equivocal results, specific PCR procedures and/or sequencing of fungal rRNA genes was performed as previously described (1, 23). The yeast isolates were maintained in parallel on Columbia sheep blood agar (CBA) and Kimmig agar (Merck, Darmstadt, Germany) for application of the agglutination test.

To investigate nonspecific agglutination by yeast cells, first the Pastorex Staph-Plus LAT (Bio-Rad, Marnes-la-Coquette, France) for the specific detection of Staphylococcus aureus was used. According to the manufacturer's recommendations, one to three colonies were taken after 24 and 48 h of cultivation on CBA and Kimmig agar, respectively. After gentle rotation of the card, tests were read after 30 s (manufacturer's recommendation) and 120 s of agglutination time. The formation of aggregates visible to the naked eye under normal lighting within the test periods was assessed as a positive reaction. All reactions were performed in duplicate. Furthermore, the MRSA-Screen test (Denka Seiken, Tokyo, Japan) representing a further LAT designed for the rapid detection of staphylococcal penicillin binding protein 2a expressed by methicillin-resistant Staphylococcus aureus strains was applied for selected isolates testing positive in the Pastorex Staph-Plus LAT. To apply the same study conditions, the MRSA-Screen test was read at the same times used for the Pastorex Staph-Plus test, thereby underrunning the recommended agglutination time of 3 min. All other test conditions, i.e., the media used and the incubation times, were also identical.

Differences between the test parameters were assessed using the chi-square test.

Overall, 40 (26.7%) of the 150 yeast isolates revealed positive agglutination results in the Pastorex Staph-Plus LAT. Of the 12 yeast species included, C. albicans (15/50; 30.0%), C. dubliniensis (6/10; 60.0%), C. glabrata (2/10; 20.0%), C. guilliermondii (4/10; 40.0%), C. lusitaniae (4/10; 40.0%), C. parapsilosis (5/10; 50.0%), and C. tropicalis (4/10; 40.0%) were characterized by having at least one isolate test positive (Table 1). Thus, by analyzing only these species, 36.4% (40/110) of the isolates showed the potential for a nonspecific LAT reaction. No nonspecific reactions were found by testing "C. africana," C. kefyr, C. krusei, Cryptococcus neoformans, and Saccharomyces cerevisiae.


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  		TABLE 1. Nonspecific agglutination of yeast isolates

 
Overall, 1,200 agglutination reactions with sensitized latex particles of the Pastorex Staph-Plus test were read after 24 and 48 h of cultivation, with a total of 600 reactions for each parameter studied, i.e., the different solid media and times allowed for agglutination (Table 1). No significant differences in nonspecific reactions were found by comparing the agglutination reactions of colonies taken from different solid media (CBA, 104 nonspecific reactions out of 600; Kimmig agar, 107 nonspecific reactions out of 600; P = 0.82). Also, the ages of the colonies (24 h, 102 nonspecific reactions out of 600; 48 h, 109 nonspecific reactions out of 600) showed no significant influence on the number of false-positive agglutination reactions with the sensitized latex particles (P = 0.82) (Table 1). In contrast, the time allowed for the agglutination process showed a significant effect on the number of nonspecific agglutination reactions (P < 0.00001). Whereas 57 false-positive reactions out of 600 were found by applying the 30-s agglutination time, significantly more nonspecific reactions (n = 154) were found by allowing 120 s before reading the LAT.

Noteworthily, the simultaneously performed negative controls consisting of latex particles sensitized by bovine albumin solution showed only positive test results when colonies were incubated for 48 h (not when colonies were incubated for <48 h).

Selected isolates (n = 13) of each Candida species exhibiting nonspecific agglutination results in the Pastorex Staph-Plus LAT were subsequently retested using a further LAT (MRSA-Screen). Of these, 10 (76.9%) isolates showed at least one nonspecific agglutination reaction, with 6 and 10 isolates testing nonspecifically positive by applying agglutination times of 30 and 120 s, respectively. The three retested isolates with negative results included isolates of C. albicans, C. dubliniensis, and C. tropicalis.

Due to the increasing number of automated systems and easy-to-use kits applied in routine microbiological laboratories, an escalating proportion of untrained staff is unfortunately employed for procedures reserved in the past only for highly skilled personnel. Thus, only cursory examination and nonobservance of major identification criteria for staphylococci and yeasts may also result in failures through the erroneous application of yeast colonies in LATs restricted for the identification of Staphylococcus aureus and methicillin resistance in staphylococci.

In addition to van der Waals forces and hydrophobic and electrostatic interactions, protein adhesion accelerates the initial adhesion and attachment onto polymer surfaces such as latex particles. While fewer adhesins from fungal pathogens than from staphylococci and other bacterial microorganisms have been identified, some Candida spp. adhesins and ligands involved in interactions with epithelial and endothelial surfaces and fibrin-platelet matrices as well as in biofilm formation on polymer surfaces have been described previously (7, 18, 19). In particular, for infections associated with medical devices such as central venous catheters, dialysis accesses, central nervous system and cardiovascular devices, and joint prostheses, the impact of Candida biofilms has been reported (8, 21).

Nevertheless, systematic studies on the interaction of Candida cells or their adhesion factors with latex particles are rare. Since latex particles are aqueous suspensions of polymer particles (mostly polystyrene microspheres and also other hydrocarbons and derivatives), manifold interactions with respective Candida adhesins and ligands as well as interactions due to cell surface hydrophobicity may be expected. Here, Candida isolates were shown to bind on negative controls (latex particles nonspecifically sensitized by bovine albumin) as well as on specifically sensitized latex particles. In particular, colonies incubated for ≥48 h were found to cause nonspecific positive results with bovine albumin-sensitized latex particles, whereas 24-h-old cultures remained negative. Colling et al. attributed some of the binding activity of yeast cells characterized by surface hydrophobicity to styrene microspheres to mannose-containing surface components (4). Furthermore, these authors showed that surface-modifying substances are able to block the binding of styrene microspheres to yeast, warranting more studies to determine those compounds and chemical entities capable of diminishing the surface hydrophobicity of yeast cells. Hawser and Islam examined the binding of C. albicans synchronized yeast-phase cells to bovine albumin and showed that this yeast binds efficiently and specifically to bovine albumin immobilized on tissue culture plastic (6). Consequently, cultures incubated longer than 24 h should not be used in LATs.

In the LATs investigated, sensitized latex particles also showed nonspecific results with colonies after 24 h of incubation. Thus, in addition to the nonspecific binding to bovine albumin or the pure latex particles, interactions with the purposely coated latex particles should not be disregarded. The Pastorex Staph-Plus latex particles are sensitized by fibrinogen, immunoglobulin G, and monoclonal antibodies directed against capsular polysaccharides of Staphylococcus aureus. The MRSA-Screen uses latex particles sensitized by monoclonal antibodies (against penicillin binding protein 2a). Interestingly, Rodier et al. observed that the incubation of C. albicans with immunoglobulin G, specific or not, causes a decrease in the capacity for adherence to polystyrene and to some extracellular matrix components (17).

Besides C. albicans, three of the four most frequent non-C. albicans species, C. parapsilosis, C. tropicalis, and C. glabrata, as well as the emerging yeasts C. dubliniensis, C. guilliermondii, and C. lusitaniae, were found to agglutinate nonspecifically with the latex particles to a substantial extent. In particular, C. parapsilosis isolates are related to foreign body insertions, as known otherwise for C. albicans. While the non-albicans Candida species have been reported to be less virulent in vitro and also in animal models, they have the ability to cause severe infections in humans (9). Surprisingly, another of the most common non-C. albicans species, C. krusei, showed no nonspecific agglutination although only a limited number of isolates were included. The actual pathogenicity of this yeast is still a matter of debate; however, based on experimental data, it was postulated that C. krusei has a lower level of virulence than C. albicans (24). Also, the Cryptococcus neoformans isolates included showed no nonspecific agglutination reactions. While this species is known to cause severe invasive infections in apparently immunocompetent patients, but preferentially in the immunodeficient host, many gaps in knowledge about the effectiveness of cryptococcal virulence factors beyond the antiphagocytic properties of the capsule still remain (2). Furthermore, C. kefyr and Saccharomyces cerevisiae, both only rarely described as being involved in severe infections, exhibited no agglutination.

Whereas the type of solid medium used for the cultivation of the yeasts as well as the cultivation time was shown to have no influence on the rate of nonspecific agglutination results, the LAT results were significantly influenced by the agglutination time allowed. For both tests used, it was shown that an excess of agglutination time led to an increase in false-positive reactions due to nonspecific agglutination by respective Candida isolates. This finding held true not only when the limit specified by the manufacturer was exceeded, as it was in using 120 s of agglutination time in the Pastorex Staph-Plus LAT, but also when the agglutination time was less than the recommended time, as in the case of the MRSA-Screen LAT. Thus, the recommended time allowed for agglutination should not be exceeded; however, adherence to the manufacturer's recommendations will not safeguard fully against nonspecific reactions.

In this first systematic study of nonspecific agglutination by yeast isolates in LATs designed for the identification of Staphylococcus aureus and the determination of methicillin resistance, a surprisingly high percentage of yeast isolates comprising different species were found to cause nonspecific agglutination of the latex particles. More efforts are necessary to solve this nontrivial problem of the erroneous misuse of easy-to-perform approaches in the microbiological laboratory.


   ACKNOWLEDGMENTS
 
We are grateful to M. Schulte, A. Hassing, and E. Kruse for excellent technical assistance. We thank H. C. Gugnani, Delhi, for providing the Indian Candida isolates.

This work was supported in part by a grant of the Interdisciplinary Center for Clinical Research (IZKF), Münster (Hei2/042/04).


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology, University of Münster, D-48149 Münster, Germany. Phone: (49) 251 83-55375. Fax: (49) 251 83-55350. E-mail: kbecker{at}uni-muenster.de Back

{triangledown} Published ahead of print on 31 January 2007. Back


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Journal of Clinical Microbiology, April 2007, p. 1315-1318, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.01688-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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