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Clade-related amphotericin B resistance among South African Candida albicans isolates
Diagnostic Microbiology and Infectious Disease 53 (2005) 29 – 31 www.elsevier.com/locate/diagmicrobio
Mycology
Clade-related amphotericin B resistance among South African Candida albicans isolates Elaine Blignauta, Julitha Molepoa, Claude Pujolb, David R. Sollb, Michael A. Pfallerc,T a
Department of Stomatological Studies, Medical University of Southern Africa, MEDUNSA 0204, Pretoria, Gauteng, South Africa b Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA c Department of Pathology, University of Iowa, Iowa City, IA 52242, USA Received 24 February 2005; accepted 29 March 2005
Abstract Molecular epidemiology revealed 5 distinct clades among clinical isolates of Candida albicans, using DNA fingerprinting with the complex Ca3 probe. Certain clades were found to be highly enriched in particular geographical areas (e.g., clade E in Europe and clade SA in South Africa, whereas clade II is completely absent in the southwest United States). From fingerprinting data, it is concluded that little interclade recombination takes place, and therefore, it would not be unusual to expect clade-specific phenotypic characteristics. The first clade-related phenotypic difference was found with 5-flucytosine resistance being almost exclusively restricted to clade I. When in vitro antifungal susceptibility testing revealed 8.4% of South African oral yeast isolates to be naturally resistant to amphotericin B, it was decided to investigate a possible clade relationship for this relatively high resistance. Thirty-eight amphotericin B–resistant C. albicans isolates were fingerprinted, and a mixed dendrogram was constructed, including previously fingerprinted isolates of known clade affiliation. With the exception of clade III, resistant isolates occurred in all clades (clade I = 3; clade II = 3; clade NG = 3; and clade SA = 29), except clade III. However, the higher number of resistant isolates that clustered in clade SA was statistically significant ( P V 0.02, m2 = 11.32). D 2005 Elsevier Inc. All rights reserved. Keywords: Antifungal resistance; Amphotericin B; Candida albicans; Clade relationship; South Africa; Oral yeasts
1. Introduction In developed countries, HIV/AIDS-related secondary complications have all but disappeared because all patients have access to highly active antiretroviral therapy (Sanglard and Odds, 2002; Ramirez-Amador et al., 2003). However, in South Africa, in the absence of highly active antiretroviral therapy, these complications persist, with candidiasis as the most common oral infection in HIV/AIDS patients (Arendorf et al., 1998; Blignaut et al., 1999). The main causative species is Candida albicans. Recently, 5 distinct clades of C. albicans (clades I, II, III, E, and SA) have been identified (Blignaut et al., 2002b; Pujol et al., 2002), using fingerprinting with the complex Ca3 probe (Anderson et al., 1993; Lockhart et al., 1995; Pujol et al., 1999). These clades occur in specific geographic areas, with clade E occurring primarily in Europe (Pujol et al., 2002) and clade SA in
T Corresponding author. Tel.: +1-319-384-9566; fax: +1-319-356-4916. E-mail address: [email protected] (M.A. Pfaller). 0732-8893/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2005.03.013
South Africa (Blignaut et al., 2002b; Soll and Pujol, 2003). Clade SA was significantly more prevalent among South African black HIV/AIDS patients and healthy black individuals (Blignaut et al., 2002b). South Africa currently has an estimated 6.5 million patients infected with HIV (Blignaut et al., 2002a), the majority being black, heterosexual, and dependent on government health services. Before any fluconazole or antiretroviral therapy was made available to these patients, antifungal susceptibility testing was performed first on more than 500 oral yeast isolates from South African HIV/AIDS patients and healthy individuals (Blignaut et al., 2002a). It is not surprising that no fluconazole resistance was found among the C. albicans isolates. However, 8.4% of the C. albicans isolates revealed a natural resistance (MIC z 2 Ag/mL) to amphotericin B. This prevalence is higher than the 0–0.4% found among oral yeast isolates by others (Waltimo et al., 2000; Pizzo et al., 2002; Dar-Odeh and Shehabi, 2003; Hajjeh et al., 2004). Amphotericin B is a polyene antifungal agent that acts by binding to ergosterol in the yeast cell membrane or on the ergosterol biosynthesis pathway and compromises the
30
E. Blignaut et al. / Diagnostic Microbiology and Infectious Disease 53 (2005) 29–31
osmotic integrity of the cell membrane, resulting in a higher permeability. In the South African government health sector, amphotericin B is reserved for patients with serious systemic fungal infections, in particular, cryptococcal meningitis. The first ever C. albicans clade-related phenotypic characteristic was recently described by Pujol et al. (2004), namely, a clade I–related 5-flucytosine resistance. This discovery prompted the investigation into the high prevalence of amphotericin B resistance among the South African C. albicans isolates. 2. Materials and methods Thirty-eight amphotericin B–resistant (MIC z 2 Ag/mL) C. albicans isolates were selected from a bank of isolates that were both fingerprinted (Blignaut et al., 2002b) and tested for antifungal susceptibility (Blignaut et al., 2002a). Antifungal susceptibility testing was performed according to the National Committee for Clinical Laboratory Standards M27-A2 document (2002). The isolates were fingerprinted using the complex Ca3 probe (Anderson et al., 1993; Lockhart et al., 1995; Pujol et al., 1999; Soll, 2000) for C. albicans. DNA was extracted according to the method of Scherer and Stevens (1987). The extracted DNA was digested with EcoRI and electrophoresed on a 0.8% agarose gel at 60 V over a distance of 16 cm, with DNA from the reference strain, 3153A, in the outer lanes. The DNA was transferred through capillary blotting to a BioBond Plus membrane (Sigma, St. Louis, MO). The Ca3 probe, labeled with 32P, was hybridized to the membrane and autoradiographed. The autoradiogram was scanned to the DENDRON software (SollTech, Iowa City, IA), and similarities between the fingerprinting patterns determined in a pairwise comparison and similarity coefficients (S AB) were calculated. A value of 0.0 indicated total unrelatedness; 1.0 indicates 100% similarity. A threshold of 0.7 was selected to delineate groups or clades. A mixed dendrogram was constructed, using the newly fingerprinted isolates and including previously analyzed isolates belonging to all 5 clades, to identify the groups or clades. Statistical significance of the amphotericin B isolates belonging to the different clades were calculated, considering the total number of previously fingerprinted C. albicans isolates in each clade (Blignaut et al., 2002b). 3. Results 3.1. Clade distribution A total of 346 C. albicans isolates were both fingerprinted and tested for antifungal susceptibility. The clade distribution of this collection, as well as the newly grouped amphotericin B–resistant isolates, is given in Table 1. A total of 61 isolates were assigned in group I, of which 3
Table 1 Clade distribution of amphotericin in B-resistant and B-susceptible C. albicans oral yeast isolates Clade I Amphotericin B–resistanta Amphotericin B–susceptible Total
Clade II
Clade III
Clade SA
Clade NG
3
3
0
29
3
58
53
23
150
24
61
56
23
179
27
m2 = 11.32, P V 0.02. a MIC z 2 Ag/mL.
were resistant to amphotericin B, 56 in group II (3 resistant isolates), 23 in group III (no resistant isolates), 179 in group SA (29 resistant isolates), and 27 in group NG (3 resistant isolates). Group SA had significantly more isolates that were amphotericin B–resistant when compared with the other groups (m2 = 11.93, P V 0.02). All the isolates were obtained from confirmed HIVpositive patients attending 2 AIDS clinics. There was no previous exposure to amphotericin B in any of the patients from whom these resistant isolates were obtained. However, 1 patient was previously treated with clotrimazole, 3 with miconazole, and 8 with nystatin. 4. Discussion The higher than previously reported amphotericin B resistance among South African C. albicans isolates can be explained in relation to the clade affiliation. The SA clade is highly enriched in South Africa (53%), but only 2% of North American isolates belong to this clade and 11% of isolates from Europe are clade SA (Blignaut et al., 2003). Antifungal treatment can lead to replacement of less susceptible strains or species. Alternatively, the colonizing or pathogenic strain can develop resistance (Sobel et al., 2000). However, none of the patients from whom the resistant isolates were obtained had previous exposure to amphotericin B. Nystatin oral solution is widely used as prophylactic agent in patients with a CD4 count below 200 cells/mm3 but has previously been found to be ineffective in preventing candidiasis in this particular cohort (Blignaut et al., 1999). The possible role that previous exposure to suboptimal levels of nystatin plays in the development of amphotericin B is uncertain. Unlike the clade-related 5-flucytosine resistance described by Pujol et al. (2004), in which resistant isolates were almost exclusively confined to clade I, all the clades, except clade III, contained isolates that appeared to be resistant to amphotericin B. However, significantly more resistant isolates belonged to the locally predominant clade, clade SA. This can hold serious implications for patients who harbor such resistant strains when this strain spreads systemically or if there should be a cross-resistance with other antifungal agents (Rex et al., 1995). In the South African government health sector, amphotericin B is
E. Blignaut et al. / Diagnostic Microbiology and Infectious Disease 53 (2005) 29–31
reserved for systemic fungal infections. The SA clade occurs predominantly among black HIV/AIDS patient and black healthy individuals, and it is these individuals who are dependent on government clinics and hospital for health services. These findings stress the importance of identifying pathogens that potentially can afflict the predicted 6.5 million HIV-infected individuals to subspecies level (Pfaller, 2000). Continued antifungal surveillance is equally important to predict the evolution of resistance in a particular population and to take timely measures. Acknowledgments This work was supported by an MRC grant to E.B. Previous fingerprinting and antifungal susceptibility testing was performed while E.B. was a Fogarty International Fellow (TWO5473) in the Departments of Pathology (MAP) and Biological Sciences (DRS), University of Iowa, Iowa City, IA, USA. References Anderson J, Srikantha T, Morrow B, Miyasaki SH, White TC, Agabian N, Schmid J, Soll DR (1993) Characterization and partial nucleotide sequence of the DNA fingerprinting probe Ca3 of Candida albicans. J Clin Microbiol 31:1472 – 1480. Arendorf TM, Bredekamp B, Cloete CA, Sauer G (1998) Oral manifestations of HIV infection in 600 South African patients. J Oral Pathol Med 27:176 – 179. Blignaut E, Botes M, et al (1999) The treatment of oral candidiasis in a cohort of South African HIV/AIDS patients. SADJ 54:605 – 608. Blignaut E, Messer S, Hollis RJ, Pfaller MA (2002a) Antifungal susceptibility of South African oral yeast isolates from HIV/AIDS patients and healthy individuals. Diagn Microbiol Infect Dis 44: 169 – 174. Blignaut E, Pujol C, Lockhart S, Joly S, Soll DR (2002b) Ca3 fingerprinting of Candida albicans isolates from human immunodeficiency viruspositive and healthy individuals reveals a new clade in South Africa. J Clin Microbiol 40:826 – 836. Blignaut E, Pujol C, Joly S, Soll DR (2003) Racial distribution of Candida dubliniensis colonization among South Africans. J Clin Microbiol 41:1838 – 1842. Dar-Odeh NS, Shehabi AA (2003) Oral candidosis in patients with removable dentures. Mycoses 46:187 – 191. Hajjeh RA, Sofair AN, Harrison LH, Lyon GM, Arthington-Skaggs BA, Mirza SA, Phelan M, Morgan J, Lee-Yang W, Ciblak MA, Benjamin LE, Sanza LT, Huie S, Yeo SF, Brandt ME, Warnock DW (2004) Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a
31
population-based active surveillance program. J Clin Microbiol 42:1519 – 1527. Lockhart SR, Fritch JJ, Meier AS, Schroppel K, Srikantha T, Galask R, Soll DR (1995) Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing. J Clin Microbiol 33:1501 – 1509. National Committee for Clinical Laboratory Standards (2002) Reference method for broth dilution antifungal susceptibility testing for yeasts (Document M27-A2). Wayne, PA7 NCCLS. Pfaller MA (2000) Epidemiology of nosocomial candidiasis: the importance of molecular typing. Braz J Infect Dis 4:161 – 167. Pizzo G, Barchiesi F, Falconi Di Francesco L, Giuliana G, Arzeni D, Milici ME, D’Angelo M, Scalise G (2002) Genotyping and antifungal susceptibility of human subgingival Candida albicans isolates. Arch Oral Biol 47:189 – 196. Pujol C, Joly S, Nolan B, Srikantha T, Soll DR (1999) Microevolutionary changes in Candida albicans identified by the complex Ca3 fingerprinting probe involve insertions and deletions of the full-length repetitive sequence RPS at specific genomic sites. Microbiology 145:2635 – 2646. Pujol C, Pfaller M, Soll DR (2002) Ca3 fingerprinting of Candida albicans bloodstream isolates from the United States, Canada, South America, and Europe reveals a European clade. J Clin Microbiol 40:2729 – 2740. Pujol C, Pfaller MA, Soll DR (2004) Flucytosine resistance is restricted to a single genetic clade of Candida albicans. Antimicrob Agents Chemother 48:262 – 266. Ramirez-Amador V, Esquivel-Pedraza L, Sierra-Madero J, Anaya-Saavedra G, Gonzalez-Ramirez I, Ponce-de-Leon S (2003) The changing clinical spectrum of human immunodeficiency virus (HIV)-related oral lesions in 1,000 consecutive patients: a 12-year study in a referral center in Mexico. Medicine 82:39 – 50. Rex JH, Cooper Jr CR, Merz WG, Galgiani JN, Anaissie EJ (1995) Detection of amphotericin B–resistant Candida isolates in a broth-based system. Antimicrob Agents Chemother 39:906 – 909. Sanglard D, Odds FC (2002) Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2:73 – 85. Scherer S, Stevens DA (1987) Application of DNA fingerprinting methods to epidemiology and taxonomy of Candida species. J Clin Microbiol 25:675 – 679. Sobel JD, Ohmit SE, Schuman P, Vazquez JA, Klein RS, Mayer K, Duerr A, Rompalo A (2000) The evolution of Candida species and fluconazole susceptibility among oral and vaginal isolates recovered from human immunodeficiency virus (HIV)–seropositive and at-risk HIV-seronegative women. J Infect Dis 183:286 – 293. Soll DR (2000) The ins and outs of DNA fingerprinting the infectious fungi. Clin Microbiol Rev 13:332 – 370. Soll DR, Pujol C (2003) Candida albicans clades. FEMS Immunol Med Microbiol 39:1 – 7. Waltimo TM, Orstavik D, Meurman JH, Samaranayake LP, Haapasalo MP (2000) In vitro susceptibility of Candida albicans isolates from apical and marginal periodontitis to common antifungal agents. Oral Microbiol Immunol 15:245 – 248.
Mycology
Clade-related amphotericin B resistance among South African Candida albicans isolates Elaine Blignauta, Julitha Molepoa, Claude Pujolb, David R. Sollb, Michael A. Pfallerc,T a
Department of Stomatological Studies, Medical University of Southern Africa, MEDUNSA 0204, Pretoria, Gauteng, South Africa b Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA c Department of Pathology, University of Iowa, Iowa City, IA 52242, USA Received 24 February 2005; accepted 29 March 2005
Abstract Molecular epidemiology revealed 5 distinct clades among clinical isolates of Candida albicans, using DNA fingerprinting with the complex Ca3 probe. Certain clades were found to be highly enriched in particular geographical areas (e.g., clade E in Europe and clade SA in South Africa, whereas clade II is completely absent in the southwest United States). From fingerprinting data, it is concluded that little interclade recombination takes place, and therefore, it would not be unusual to expect clade-specific phenotypic characteristics. The first clade-related phenotypic difference was found with 5-flucytosine resistance being almost exclusively restricted to clade I. When in vitro antifungal susceptibility testing revealed 8.4% of South African oral yeast isolates to be naturally resistant to amphotericin B, it was decided to investigate a possible clade relationship for this relatively high resistance. Thirty-eight amphotericin B–resistant C. albicans isolates were fingerprinted, and a mixed dendrogram was constructed, including previously fingerprinted isolates of known clade affiliation. With the exception of clade III, resistant isolates occurred in all clades (clade I = 3; clade II = 3; clade NG = 3; and clade SA = 29), except clade III. However, the higher number of resistant isolates that clustered in clade SA was statistically significant ( P V 0.02, m2 = 11.32). D 2005 Elsevier Inc. All rights reserved. Keywords: Antifungal resistance; Amphotericin B; Candida albicans; Clade relationship; South Africa; Oral yeasts
1. Introduction In developed countries, HIV/AIDS-related secondary complications have all but disappeared because all patients have access to highly active antiretroviral therapy (Sanglard and Odds, 2002; Ramirez-Amador et al., 2003). However, in South Africa, in the absence of highly active antiretroviral therapy, these complications persist, with candidiasis as the most common oral infection in HIV/AIDS patients (Arendorf et al., 1998; Blignaut et al., 1999). The main causative species is Candida albicans. Recently, 5 distinct clades of C. albicans (clades I, II, III, E, and SA) have been identified (Blignaut et al., 2002b; Pujol et al., 2002), using fingerprinting with the complex Ca3 probe (Anderson et al., 1993; Lockhart et al., 1995; Pujol et al., 1999). These clades occur in specific geographic areas, with clade E occurring primarily in Europe (Pujol et al., 2002) and clade SA in
T Corresponding author. Tel.: +1-319-384-9566; fax: +1-319-356-4916. E-mail address: [email protected] (M.A. Pfaller). 0732-8893/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2005.03.013
South Africa (Blignaut et al., 2002b; Soll and Pujol, 2003). Clade SA was significantly more prevalent among South African black HIV/AIDS patients and healthy black individuals (Blignaut et al., 2002b). South Africa currently has an estimated 6.5 million patients infected with HIV (Blignaut et al., 2002a), the majority being black, heterosexual, and dependent on government health services. Before any fluconazole or antiretroviral therapy was made available to these patients, antifungal susceptibility testing was performed first on more than 500 oral yeast isolates from South African HIV/AIDS patients and healthy individuals (Blignaut et al., 2002a). It is not surprising that no fluconazole resistance was found among the C. albicans isolates. However, 8.4% of the C. albicans isolates revealed a natural resistance (MIC z 2 Ag/mL) to amphotericin B. This prevalence is higher than the 0–0.4% found among oral yeast isolates by others (Waltimo et al., 2000; Pizzo et al., 2002; Dar-Odeh and Shehabi, 2003; Hajjeh et al., 2004). Amphotericin B is a polyene antifungal agent that acts by binding to ergosterol in the yeast cell membrane or on the ergosterol biosynthesis pathway and compromises the
30
E. Blignaut et al. / Diagnostic Microbiology and Infectious Disease 53 (2005) 29–31
osmotic integrity of the cell membrane, resulting in a higher permeability. In the South African government health sector, amphotericin B is reserved for patients with serious systemic fungal infections, in particular, cryptococcal meningitis. The first ever C. albicans clade-related phenotypic characteristic was recently described by Pujol et al. (2004), namely, a clade I–related 5-flucytosine resistance. This discovery prompted the investigation into the high prevalence of amphotericin B resistance among the South African C. albicans isolates. 2. Materials and methods Thirty-eight amphotericin B–resistant (MIC z 2 Ag/mL) C. albicans isolates were selected from a bank of isolates that were both fingerprinted (Blignaut et al., 2002b) and tested for antifungal susceptibility (Blignaut et al., 2002a). Antifungal susceptibility testing was performed according to the National Committee for Clinical Laboratory Standards M27-A2 document (2002). The isolates were fingerprinted using the complex Ca3 probe (Anderson et al., 1993; Lockhart et al., 1995; Pujol et al., 1999; Soll, 2000) for C. albicans. DNA was extracted according to the method of Scherer and Stevens (1987). The extracted DNA was digested with EcoRI and electrophoresed on a 0.8% agarose gel at 60 V over a distance of 16 cm, with DNA from the reference strain, 3153A, in the outer lanes. The DNA was transferred through capillary blotting to a BioBond Plus membrane (Sigma, St. Louis, MO). The Ca3 probe, labeled with 32P, was hybridized to the membrane and autoradiographed. The autoradiogram was scanned to the DENDRON software (SollTech, Iowa City, IA), and similarities between the fingerprinting patterns determined in a pairwise comparison and similarity coefficients (S AB) were calculated. A value of 0.0 indicated total unrelatedness; 1.0 indicates 100% similarity. A threshold of 0.7 was selected to delineate groups or clades. A mixed dendrogram was constructed, using the newly fingerprinted isolates and including previously analyzed isolates belonging to all 5 clades, to identify the groups or clades. Statistical significance of the amphotericin B isolates belonging to the different clades were calculated, considering the total number of previously fingerprinted C. albicans isolates in each clade (Blignaut et al., 2002b). 3. Results 3.1. Clade distribution A total of 346 C. albicans isolates were both fingerprinted and tested for antifungal susceptibility. The clade distribution of this collection, as well as the newly grouped amphotericin B–resistant isolates, is given in Table 1. A total of 61 isolates were assigned in group I, of which 3
Table 1 Clade distribution of amphotericin in B-resistant and B-susceptible C. albicans oral yeast isolates Clade I Amphotericin B–resistanta Amphotericin B–susceptible Total
Clade II
Clade III
Clade SA
Clade NG
3
3
0
29
3
58
53
23
150
24
61
56
23
179
27
m2 = 11.32, P V 0.02. a MIC z 2 Ag/mL.
were resistant to amphotericin B, 56 in group II (3 resistant isolates), 23 in group III (no resistant isolates), 179 in group SA (29 resistant isolates), and 27 in group NG (3 resistant isolates). Group SA had significantly more isolates that were amphotericin B–resistant when compared with the other groups (m2 = 11.93, P V 0.02). All the isolates were obtained from confirmed HIVpositive patients attending 2 AIDS clinics. There was no previous exposure to amphotericin B in any of the patients from whom these resistant isolates were obtained. However, 1 patient was previously treated with clotrimazole, 3 with miconazole, and 8 with nystatin. 4. Discussion The higher than previously reported amphotericin B resistance among South African C. albicans isolates can be explained in relation to the clade affiliation. The SA clade is highly enriched in South Africa (53%), but only 2% of North American isolates belong to this clade and 11% of isolates from Europe are clade SA (Blignaut et al., 2003). Antifungal treatment can lead to replacement of less susceptible strains or species. Alternatively, the colonizing or pathogenic strain can develop resistance (Sobel et al., 2000). However, none of the patients from whom the resistant isolates were obtained had previous exposure to amphotericin B. Nystatin oral solution is widely used as prophylactic agent in patients with a CD4 count below 200 cells/mm3 but has previously been found to be ineffective in preventing candidiasis in this particular cohort (Blignaut et al., 1999). The possible role that previous exposure to suboptimal levels of nystatin plays in the development of amphotericin B is uncertain. Unlike the clade-related 5-flucytosine resistance described by Pujol et al. (2004), in which resistant isolates were almost exclusively confined to clade I, all the clades, except clade III, contained isolates that appeared to be resistant to amphotericin B. However, significantly more resistant isolates belonged to the locally predominant clade, clade SA. This can hold serious implications for patients who harbor such resistant strains when this strain spreads systemically or if there should be a cross-resistance with other antifungal agents (Rex et al., 1995). In the South African government health sector, amphotericin B is
E. Blignaut et al. / Diagnostic Microbiology and Infectious Disease 53 (2005) 29–31
reserved for systemic fungal infections. The SA clade occurs predominantly among black HIV/AIDS patient and black healthy individuals, and it is these individuals who are dependent on government clinics and hospital for health services. These findings stress the importance of identifying pathogens that potentially can afflict the predicted 6.5 million HIV-infected individuals to subspecies level (Pfaller, 2000). Continued antifungal surveillance is equally important to predict the evolution of resistance in a particular population and to take timely measures. Acknowledgments This work was supported by an MRC grant to E.B. Previous fingerprinting and antifungal susceptibility testing was performed while E.B. was a Fogarty International Fellow (TWO5473) in the Departments of Pathology (MAP) and Biological Sciences (DRS), University of Iowa, Iowa City, IA, USA. References Anderson J, Srikantha T, Morrow B, Miyasaki SH, White TC, Agabian N, Schmid J, Soll DR (1993) Characterization and partial nucleotide sequence of the DNA fingerprinting probe Ca3 of Candida albicans. J Clin Microbiol 31:1472 – 1480. Arendorf TM, Bredekamp B, Cloete CA, Sauer G (1998) Oral manifestations of HIV infection in 600 South African patients. J Oral Pathol Med 27:176 – 179. Blignaut E, Botes M, et al (1999) The treatment of oral candidiasis in a cohort of South African HIV/AIDS patients. SADJ 54:605 – 608. Blignaut E, Messer S, Hollis RJ, Pfaller MA (2002a) Antifungal susceptibility of South African oral yeast isolates from HIV/AIDS patients and healthy individuals. Diagn Microbiol Infect Dis 44: 169 – 174. Blignaut E, Pujol C, Lockhart S, Joly S, Soll DR (2002b) Ca3 fingerprinting of Candida albicans isolates from human immunodeficiency viruspositive and healthy individuals reveals a new clade in South Africa. J Clin Microbiol 40:826 – 836. Blignaut E, Pujol C, Joly S, Soll DR (2003) Racial distribution of Candida dubliniensis colonization among South Africans. J Clin Microbiol 41:1838 – 1842. Dar-Odeh NS, Shehabi AA (2003) Oral candidosis in patients with removable dentures. Mycoses 46:187 – 191. Hajjeh RA, Sofair AN, Harrison LH, Lyon GM, Arthington-Skaggs BA, Mirza SA, Phelan M, Morgan J, Lee-Yang W, Ciblak MA, Benjamin LE, Sanza LT, Huie S, Yeo SF, Brandt ME, Warnock DW (2004) Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a
31
population-based active surveillance program. J Clin Microbiol 42:1519 – 1527. Lockhart SR, Fritch JJ, Meier AS, Schroppel K, Srikantha T, Galask R, Soll DR (1995) Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing. J Clin Microbiol 33:1501 – 1509. National Committee for Clinical Laboratory Standards (2002) Reference method for broth dilution antifungal susceptibility testing for yeasts (Document M27-A2). Wayne, PA7 NCCLS. Pfaller MA (2000) Epidemiology of nosocomial candidiasis: the importance of molecular typing. Braz J Infect Dis 4:161 – 167. Pizzo G, Barchiesi F, Falconi Di Francesco L, Giuliana G, Arzeni D, Milici ME, D’Angelo M, Scalise G (2002) Genotyping and antifungal susceptibility of human subgingival Candida albicans isolates. Arch Oral Biol 47:189 – 196. Pujol C, Joly S, Nolan B, Srikantha T, Soll DR (1999) Microevolutionary changes in Candida albicans identified by the complex Ca3 fingerprinting probe involve insertions and deletions of the full-length repetitive sequence RPS at specific genomic sites. Microbiology 145:2635 – 2646. Pujol C, Pfaller M, Soll DR (2002) Ca3 fingerprinting of Candida albicans bloodstream isolates from the United States, Canada, South America, and Europe reveals a European clade. J Clin Microbiol 40:2729 – 2740. Pujol C, Pfaller MA, Soll DR (2004) Flucytosine resistance is restricted to a single genetic clade of Candida albicans. Antimicrob Agents Chemother 48:262 – 266. Ramirez-Amador V, Esquivel-Pedraza L, Sierra-Madero J, Anaya-Saavedra G, Gonzalez-Ramirez I, Ponce-de-Leon S (2003) The changing clinical spectrum of human immunodeficiency virus (HIV)-related oral lesions in 1,000 consecutive patients: a 12-year study in a referral center in Mexico. Medicine 82:39 – 50. Rex JH, Cooper Jr CR, Merz WG, Galgiani JN, Anaissie EJ (1995) Detection of amphotericin B–resistant Candida isolates in a broth-based system. Antimicrob Agents Chemother 39:906 – 909. Sanglard D, Odds FC (2002) Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2:73 – 85. Scherer S, Stevens DA (1987) Application of DNA fingerprinting methods to epidemiology and taxonomy of Candida species. J Clin Microbiol 25:675 – 679. Sobel JD, Ohmit SE, Schuman P, Vazquez JA, Klein RS, Mayer K, Duerr A, Rompalo A (2000) The evolution of Candida species and fluconazole susceptibility among oral and vaginal isolates recovered from human immunodeficiency virus (HIV)–seropositive and at-risk HIV-seronegative women. J Infect Dis 183:286 – 293. Soll DR (2000) The ins and outs of DNA fingerprinting the infectious fungi. Clin Microbiol Rev 13:332 – 370. Soll DR, Pujol C (2003) Candida albicans clades. FEMS Immunol Med Microbiol 39:1 – 7. Waltimo TM, Orstavik D, Meurman JH, Samaranayake LP, Haapasalo MP (2000) In vitro susceptibility of Candida albicans isolates from apical and marginal periodontitis to common antifungal agents. Oral Microbiol Immunol 15:245 – 248.