OF CLINICAL MICROBIOLOGY, Apr. 1991, p. 726-730 0095-1137/91/040726-05$02.00/0
JOURNAL
Vol. 29, No. 4
Candida albicans Biotypes in Human Immunodeficiency Virus Type 1-Infected Patients with Oral Candidiasis before and after Antifungal Therapy MASSIMO BRUATTO, VALERIO VIDOTTO, GRAZIA MARINUZZI, RICCARDO RAITERI, AND ALESSANDRO SINICCO* Istituto di Malattie Infettive dell'Universitai di Torino, Ospedale Amedeo di Savoia, Corso Svizzera 164, 10149 Turin, Italy Received 9 July 1990/Accepted 24 January 1991
A prospective 19-month study of 26 human immunodeficiency virus type 1-infected patients with episodes of erythematous or pseudomembranous oral candidiasis was done to evaluate the significance of Candida albicans biotypes in patients treated with antifungal therapy. Changes in the biotype of C. albicans were frequently noted in recurrent oral candidiasis. However, no correlation was found between the various biotypes and the clinical features of oral candidiasis, the clinical stage of human immunodeficiency virus type 1 infection, or the number of CD4+ lymphocytes. On the contrary, a significant correlation appeared among clinical lesion features, CD4+ cell numbers, and time of clinical disappearance of the oral lesions. Changes in the biotype of C. albicans were observed at the end of the antifungal therapy in 17 of 26 patients who had a second appearance of oral candidiasis as well as in 10 of 14 subjects who experienced a third reappearance of oral candidiasis.
Oral candidiasis is a common feature associated with human immune deficiency virus type 1 (HIV-1) infections. Four major clinical types of oral candidiasis have been noted: pseudomembranous, erythematous (atrophic), and hyperplastic and angular cheilitis (5). These types frequently appear in HIV-1-infected subjects, and they are considered reliable markers of disease progression (7). Several different approaches have been used to differentiate biotypes of pathogenic fungi. For some fungal pathogens, epidemiologic data have been obtained from biotype studies (1, 3, 9-11, 14). Many reports have shown the presence of various biotypes of Candida albicans in different clinical presentations (1, 9, 12, 15), but few studies concerning biotyping in HIV-infected patients with oral candidiasis have been performed (6, 18). The purpose of this study was to evaluate the distribution of and the variation in biotypes of C. albicans from HIV-1infected patients with erythematous or pseudomembranous oral candidiasis during the course of their HIV-1 infection as well as after antifungal therapy.
longed to the IV-Cl subgroup, and 1 patient belonged to the IV-D subgroup (2). Anti-HIV-1 antibodies were tested by both ELAVIA Ab HIV-1 (Pasteur AC1; Istituto Pasteur Vaccins, Marnes la Coquette, France) and enzyme immunoassay [Anti-HIV (HTLV III/LAV) EIA "Roche"; Hoffmann-La Roche Ltd., Basel, Switzerland] kits and were confirmed by Western immunoblot analysis (DuPont De Nemours, DuPont Company, Wilmington, Del.). Lymphocyte subsets were determined and monitored by the cytofluorimeter Ortho-Spectrum III kit by using monoclonal antibodies against CD3+ (OKT3), CD4+ (OKT4), and CD8+ (OKT8) (Ortho Diagnostic Systems, High Wycombe, United Kingdom). The mouth of each patient with either erythematous or pseudomembranous candidiasis was rinsed with sterile saline; an oral swab was then used to culture the tongue, buccal, or oropharyngeal surfaces. After the oral swabbing, each patient was treated with oral ketoconazole (200 mg/day) until clinical disappearance of the oral lesion(s). Yeasts were isolated by streaking the oral swab on Sabouraud agar (Difco, Detroit, Mich.) plates supplemented with ampicillin and streptomycin. The plates were incubated at 25°C for 72 h. Yeasts which grew within that time were presumptively identified as C. albicans by the germ-tube test in calf serum (Biomerieux, Lyon, France) and by chlamydoconidial production on corn meal agar (Difco) in Dalmau plates. The identifications were confirmed with ATB 32 C auxanographic galleries (Biomerieux). All the strains isolated and identified as C. albicans were biotyped by using the killer system methods described by Polonelli et al. (14). Twenty-four killer strains obtained from L. Polonelli's collection were used (Table 1). Each isolate was assigned an 8 digit biotype as exemplified in Fig. 1. When the patients showed another episode of oral candidiasis, the isolation, identification, and biotyping of the colonizing strain were repeated as described above. Statistical analysis was performed by using the x2 test, Student's t test, and Spearman's correlation test (4).
MATERIALS AND METHODS From April 1988 to October 1989 at the Infectious Disease Institute of the University of Turin, 26 HIV-1-infected subjects presenting with erythematous or pseudomembranous candidiasis at the initial clinical examinations of their oral cavities were periodically followed. Among these patients, 15 were intravenous drug users, 9 were homosexual males, 1 was heterosexual, and 1 was a human blood recipient. They ranged in age from 22 to 42 years (mean + standard deviation, 31.0 + 5.1) and their sex ratio (male to female) was 5.5:1. According to the Centers for Disease Control criteria, at the commencement of the study, 22 subjects belonged to the IV-C2 subgroup, 3 patients be*
Corresponding author. 726
VOL. 29, 1991
CANDIDA ALBICANS BIOTYPES IN HIV-1 INFECTION
TABLE 1. Killer yeasts used in this studya Code
Species
Kl K2 K3 K4 K5 K6 K7 K8 K9 K10 K12 K16 K18 K27 K37 K41 K44 K48 K49 K50 K51 K113 K114 M83
Pichia sp. Pichia sp. Pichia anomala Pichia anomala Pichia anomala Pichia californica Pichia canadensis Pichia dimennae Pichia mrakii Pichia kluyverii Pichia bimundalis Saccharomyces cerevisiae Pichia fabianii Pichia farinosa Pichia mrakii Saccharomyces cerevisiae Kluyveromyces lactis Kluyveromyces lactis Kluyveromyces lactis Kluyveromyces fragilis Kluyveromyces fragilis Kluyveromyces lactis Kluyveromyces lactis Candida albicans
Collection'
1034 1035
UM CBS D. G. Ahearn D. G. Aheam D. G. Ahearn D. G. Aheam D. G. Ahearn C. Stumm D. G. Ahearn CDC D. G. Ahearn CBS UCSC J. Kandel N. Gunge CBS CBS
3 5739 Um866
ATCC N. Gunge N. Gunge
domembranous candidiasis, two with a prior pseudomembranous candidiasis developed an erythematous lesion, one patient who had pseudomembranous developed hyperplastic candidiasis, and one patient with a prior erythematous lesion developed a hyperplastic one. During this period, two patients developed AIDS. Among the 26 isolates, 17 were later replaced by a different biotype. Of 14 subjects who experienced a third appearance of oral candidiasis, 6 showed an alteration in the type of lesion. In particular, in three patients who at the moment of the first reappearance of oral candidiasis had an erythematous lesion, the pseudomembranous type changed to a hyperplastic one. Of the isolated strains, 10 were of a new biotype. Two subjects developed AIDS. All in all, biotype variation of the oral strains was observed in 73.1% of the patients. We differentiated 21 biotypes among 66 strains isolated from 26 patients who had been followed periodically. No significant cluster of C. albicans biotypes has been noticed. The most frequently observed biotypes were the following: 37774777 (10.1%), 37774377 (9.1%), 36774771 (9.1%), 37774357 (7.6%), and 37774371 (6.1%). A significant correlation appeared between pseudomembranous candidiasis and a CD4+ lymphocyte count of
JOURNAL
Vol. 29, No. 4
Candida albicans Biotypes in Human Immunodeficiency Virus Type 1-Infected Patients with Oral Candidiasis before and after Antifungal Therapy MASSIMO BRUATTO, VALERIO VIDOTTO, GRAZIA MARINUZZI, RICCARDO RAITERI, AND ALESSANDRO SINICCO* Istituto di Malattie Infettive dell'Universitai di Torino, Ospedale Amedeo di Savoia, Corso Svizzera 164, 10149 Turin, Italy Received 9 July 1990/Accepted 24 January 1991
A prospective 19-month study of 26 human immunodeficiency virus type 1-infected patients with episodes of erythematous or pseudomembranous oral candidiasis was done to evaluate the significance of Candida albicans biotypes in patients treated with antifungal therapy. Changes in the biotype of C. albicans were frequently noted in recurrent oral candidiasis. However, no correlation was found between the various biotypes and the clinical features of oral candidiasis, the clinical stage of human immunodeficiency virus type 1 infection, or the number of CD4+ lymphocytes. On the contrary, a significant correlation appeared among clinical lesion features, CD4+ cell numbers, and time of clinical disappearance of the oral lesions. Changes in the biotype of C. albicans were observed at the end of the antifungal therapy in 17 of 26 patients who had a second appearance of oral candidiasis as well as in 10 of 14 subjects who experienced a third reappearance of oral candidiasis.
Oral candidiasis is a common feature associated with human immune deficiency virus type 1 (HIV-1) infections. Four major clinical types of oral candidiasis have been noted: pseudomembranous, erythematous (atrophic), and hyperplastic and angular cheilitis (5). These types frequently appear in HIV-1-infected subjects, and they are considered reliable markers of disease progression (7). Several different approaches have been used to differentiate biotypes of pathogenic fungi. For some fungal pathogens, epidemiologic data have been obtained from biotype studies (1, 3, 9-11, 14). Many reports have shown the presence of various biotypes of Candida albicans in different clinical presentations (1, 9, 12, 15), but few studies concerning biotyping in HIV-infected patients with oral candidiasis have been performed (6, 18). The purpose of this study was to evaluate the distribution of and the variation in biotypes of C. albicans from HIV-1infected patients with erythematous or pseudomembranous oral candidiasis during the course of their HIV-1 infection as well as after antifungal therapy.
longed to the IV-Cl subgroup, and 1 patient belonged to the IV-D subgroup (2). Anti-HIV-1 antibodies were tested by both ELAVIA Ab HIV-1 (Pasteur AC1; Istituto Pasteur Vaccins, Marnes la Coquette, France) and enzyme immunoassay [Anti-HIV (HTLV III/LAV) EIA "Roche"; Hoffmann-La Roche Ltd., Basel, Switzerland] kits and were confirmed by Western immunoblot analysis (DuPont De Nemours, DuPont Company, Wilmington, Del.). Lymphocyte subsets were determined and monitored by the cytofluorimeter Ortho-Spectrum III kit by using monoclonal antibodies against CD3+ (OKT3), CD4+ (OKT4), and CD8+ (OKT8) (Ortho Diagnostic Systems, High Wycombe, United Kingdom). The mouth of each patient with either erythematous or pseudomembranous candidiasis was rinsed with sterile saline; an oral swab was then used to culture the tongue, buccal, or oropharyngeal surfaces. After the oral swabbing, each patient was treated with oral ketoconazole (200 mg/day) until clinical disappearance of the oral lesion(s). Yeasts were isolated by streaking the oral swab on Sabouraud agar (Difco, Detroit, Mich.) plates supplemented with ampicillin and streptomycin. The plates were incubated at 25°C for 72 h. Yeasts which grew within that time were presumptively identified as C. albicans by the germ-tube test in calf serum (Biomerieux, Lyon, France) and by chlamydoconidial production on corn meal agar (Difco) in Dalmau plates. The identifications were confirmed with ATB 32 C auxanographic galleries (Biomerieux). All the strains isolated and identified as C. albicans were biotyped by using the killer system methods described by Polonelli et al. (14). Twenty-four killer strains obtained from L. Polonelli's collection were used (Table 1). Each isolate was assigned an 8 digit biotype as exemplified in Fig. 1. When the patients showed another episode of oral candidiasis, the isolation, identification, and biotyping of the colonizing strain were repeated as described above. Statistical analysis was performed by using the x2 test, Student's t test, and Spearman's correlation test (4).
MATERIALS AND METHODS From April 1988 to October 1989 at the Infectious Disease Institute of the University of Turin, 26 HIV-1-infected subjects presenting with erythematous or pseudomembranous candidiasis at the initial clinical examinations of their oral cavities were periodically followed. Among these patients, 15 were intravenous drug users, 9 were homosexual males, 1 was heterosexual, and 1 was a human blood recipient. They ranged in age from 22 to 42 years (mean + standard deviation, 31.0 + 5.1) and their sex ratio (male to female) was 5.5:1. According to the Centers for Disease Control criteria, at the commencement of the study, 22 subjects belonged to the IV-C2 subgroup, 3 patients be*
Corresponding author. 726
VOL. 29, 1991
CANDIDA ALBICANS BIOTYPES IN HIV-1 INFECTION
TABLE 1. Killer yeasts used in this studya Code
Species
Kl K2 K3 K4 K5 K6 K7 K8 K9 K10 K12 K16 K18 K27 K37 K41 K44 K48 K49 K50 K51 K113 K114 M83
Pichia sp. Pichia sp. Pichia anomala Pichia anomala Pichia anomala Pichia californica Pichia canadensis Pichia dimennae Pichia mrakii Pichia kluyverii Pichia bimundalis Saccharomyces cerevisiae Pichia fabianii Pichia farinosa Pichia mrakii Saccharomyces cerevisiae Kluyveromyces lactis Kluyveromyces lactis Kluyveromyces lactis Kluyveromyces fragilis Kluyveromyces fragilis Kluyveromyces lactis Kluyveromyces lactis Candida albicans
Collection'
1034 1035
UM CBS D. G. Ahearn D. G. Aheam D. G. Ahearn D. G. Aheam D. G. Ahearn C. Stumm D. G. Ahearn CDC D. G. Ahearn CBS UCSC J. Kandel N. Gunge CBS CBS
3 5739 Um866
ATCC N. Gunge N. Gunge
domembranous candidiasis, two with a prior pseudomembranous candidiasis developed an erythematous lesion, one patient who had pseudomembranous developed hyperplastic candidiasis, and one patient with a prior erythematous lesion developed a hyperplastic one. During this period, two patients developed AIDS. Among the 26 isolates, 17 were later replaced by a different biotype. Of 14 subjects who experienced a third appearance of oral candidiasis, 6 showed an alteration in the type of lesion. In particular, in three patients who at the moment of the first reappearance of oral candidiasis had an erythematous lesion, the pseudomembranous type changed to a hyperplastic one. Of the isolated strains, 10 were of a new biotype. Two subjects developed AIDS. All in all, biotype variation of the oral strains was observed in 73.1% of the patients. We differentiated 21 biotypes among 66 strains isolated from 26 patients who had been followed periodically. No significant cluster of C. albicans biotypes has been noticed. The most frequently observed biotypes were the following: 37774777 (10.1%), 37774377 (9.1%), 36774771 (9.1%), 37774357 (7.6%), and 37774371 (6.1%). A significant correlation appeared between pseudomembranous candidiasis and a CD4+ lymphocyte count of
