Colonization of Human Immunodeficiency Virus-Infected Outpatients in Taiwan with Candida Species

    Division of Infectious Diseases, Department of Internal Medicine, National Taiwan University Hospital, and Department of Parasitology, National Taiwan University College of Medicine, Taipei
    Department of Biological Science and Technology, National Chiao Tung University, Hsinchu
    Division of Clinical Research
    Division of Biostatistics and Bioinformatics, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan, Republic of China

    ABSTRACT

    To understand the Candida colonization of human immunodeficiency virus (HIV)-infected outpatients in Taiwan, we have conducted a prospective cohort study of Candida colonization and its risk factors at the National Taiwan University Hospital from 1999 to 2002. More than 50% of the patients were colonized with Candida species, and 12% developed symptomatic candidiasis. Patients colonized with fluconazole-resistant strains of Candida species had a higher prevalence of candidiasis than those colonized with susceptible strains. Our analysis found that antibiotic treatment and lower CD4+ counts (<200 cells/mm3) increased the rate of oropharyngeal candidiasis in HIV-infected patients, while antiretroviral therapy protected patients from the development of candidiasis.

    INTRODUCTION

    Mucosal candidiasis, including oropharyngeal, esophageal, and vaginal candidiasis, is common among human immunodeficiency virus (HIV)-infected patients (4, 11). In particular, oropharyngeal candidiasis occurs in up to 90% of patients during the course of HIV infection (17). Progressive cell-mediated immunodeficiency, with CD4+ lymphocyte counts less than 200 cells/mm3, is a risk factor for colonization with Candida species and the development of candidiasis (3). The widespread use of azole antifungal agents for the treatment of mucosal candidiasis results in colonization with less susceptible organisms and the development of resistance (4, 15). Thus, oropharyngeal candidiasis due to drug-resistant fungi is an emerging problem for patients infected with HIV (18).

    The overall prevalence of known HIV infection in Taiwan remains relatively low (0.01%) (9). As in most other industrialized countries, the majority of HIV-infected patients in Taiwan receive care in the outpatient setting. Therefore, to better understand the epidemiology of Candida species carriage among HIV-infected outpatients in Taiwan, we undertook a study to determine the prevalence of oropharyngeal colonization. Our objectives were to assess the colonization status and the risk factors for colonization and the development of candidiasis in HIV-infected outpatients in Taiwan. The susceptibilities of those Candida isolates to antifungal drugs were also determined.

    MATERIALS AND METHODS

    Study population and data collection. HIV-infected patients were monitored regularly in the outpatient infectious diseases clinic of National Taiwan University Hospital, a major referral hospital for the management of HIV-related complications. The patients were enrolled after they provided informed verbal consent. This was a prospective study performed by the use of three surveys, conducted from May to June 1999, May to September 2001, and January to April 2002. A standardized data collection form was used to retrieve demographic information, the most recent CD4+ lymphocyte count, and the highly active antiretroviral therapy (HAART) prescribed. In addition, clinical information for the previous 3 months was obtained and included information on whether the patient had a history of oral or esophageal candidiasis or hospitalization and the antibacterial and antifungal drugs received.

    Sampling and microbiologic processing. Oropharyngeal swab specimens for culture were obtained from all patients by using a dry sponge swab (EZ Culturette; Becton Dickinson, Sparks, Md.). All swabs were maintained at room temperature and were transported to the laboratory within 24 h. They were then plated on solid medium within 12 h of arrival. The swabs collected in 1999 were plated on Sabouraud dextrose with chloramphenicol and gentamicin (BBL), and those collected in 2001 and 2002 were plated on Chromagar Candida (BBL). All plates were incubated at 30°C. Three independent colonies were selected from each positive culture. Additional colonies were selected from cultures with more than one morphotype. All isolates were first subjected to the germ tube assay. For germ tube assay-positive isolates, a temperature sensitivity assay was performed to differentiate Candida albicans from Candida dubliniensis (growth defect at 42°C). The VITEK yeast biochemical card (YBC; bioMerieux, St. Louis, Mo.) was used to identify those isolates that failed to form germ tubes and isolates that formed germ tubes but that failed to grow at 42°C. We used the API 32C system (bioMerieux) to assess our results when the VITEK YBC yielded results of less than 90% certainty.

    Antifungal susceptibility testing. The MICs of antifungal drugs were determined by in vitro antifungal susceptibility testing according to the M27-A guidelines published in 1997 by the National Committee of Clinical Laboratory Standards (NCCLS) (10). RPMI 1640 medium (31800-022; Gibco BRL) was used for dilution and growth of the yeast culture. The growth end point of each isolate was determined with a spectrophotometer (Spectra MAX Plus; Molecular Devices Corp., Sunnyvale, Calif.). The MICs were also interpreted according to NCCLS guidelines. The amphotericin B MIC was determined, as the MIC of amphotericin B is needed to completely inhibit the growth of isolates after 48 h of incubation at 35°C. Isolates for which the amphotericin B MIC was 2 μg/ml were considered amphotericin B resistant.

    The fluconazole MIC was defined as the concentration of fluconazole needed to reduce the turbidity to 50% after 48 h of incubation at 35°C. Isolates for which fluconazole MICs were 64 μg/ml, 16 to 32 μg/ml, and 8 μg/ml were defined as resistant, susceptible-dose dependent, and susceptible to fluconazole, respectively.

    Statistical analysis. All clinical laboratory data were entered into a relational database designed in Access 97 software (Microsoft, Redland, Wash.). The chi-square test was used to study the association of factors with incident or persistent oral yeast species colonization. Risk factors for patients with oropharyngeal colonization were identified by multiple logistic regression.

    RESULTS

    Patients. A total of 122, 243, and 276 patients were enrolled in this study in 1999, 2001, and 2002, respectively (Table 1). The majority (91%) of the patients were men. The CD4+ counts were available for 599 patients, and the average CD4+ count was 279.5 cells/mm3. Only 15.3% of the patients had CD4+ counts greater than 500 cells/mm3, while 43.9% of the patients had CD4+ counts less than 200 cells/mm3 and 13% of the patients had CD4+ counts less than 50 cells/mm3. Consequently, 84.4% of the patients were receiving HAART. A total of 35.3% of patients also received antibiotics as treatment or primary or secondary prophylaxis for opportunistic infections.

    A total of 127 patients were enrolled in two of the three surveys. Of these, 32 were negative for yeasts by culture, 36 were positive for yeasts in both surveys, and 59 were positive for yeasts by culture only once. Of 51 patients who were enrolled in all three surveys, 11 patients were negative for yeasts by culture and 11 patients were positive for yeasts by culture in all three surveys. Of the remaining 29 patients, 13 patients were positive for yeasts by culture once and 16 patients were positive for yeasts by culture twice.

    Distribution of yeasts. Yeast culture positivity rates in 1999, 2001, and 2002 were 56.6% (69 of 122 patients), 53.5% (130 of 243 patients), and 49.6% (137 of 276 patients), respectively. One isolate of each Candida species from each patient in each survey was analyzed (Table 2). C. albicans was the most common species and accounted for 91, 86, and 85.2% of the isolates in 1999, 2001, and 2002, respectively. A total of 338 isolates were recovered; and these consisted of 293 (86.7%) C. albicans isolates, 10 (3%) Candida glabrata isolates, 10 (3%) Candida parapsilosis isolates, 7 (2.1%) Candida tropicalis isolates, 5 (1.5%) Candida lusitaniae isolates, and 13 (3.7%) other isolates. Four, nine, and nine different Candida species were isolated from patients in the 1999, 2001, and 2002 surveys, respectively.

    Antifungal susceptibilities of yeasts. The susceptibilities to antifungal agents of one Candida species isolate among multiple isolates collected from each patient were analyzed (Table 2). Of 262 isolates, the amphotericin B MICs for 12 (4.6%), 188 (71.8%), 61 (23.3%), and 1 (0.4%) isolates were 0.25, 0.5, 1, and 2 μg/ml, respectively. The only resistant isolate was a Candida famata isolate. A total of 244 (93.1%), 12 (4.6%), and 6 (2.3%) isolates were susceptible, susceptible-dose dependent, and resistant to fluconazole, respectively (Table 3). Of six fluconazole-resistant isolates, five were C. albicans. The prevalence of candidiasis was higher among patients colonized with fluconazole-resistant Candida species than among those colonized with fluconazole-susceptible isolates (P < 0.05).

    On the basis of multivariate analysis, antibiotic treatment and lower CD4+ counts (<200 cells/mm3) were independent risk factors for oropharyngeal colonization among the patients. In contrast, treatment with HAART and antifungal drugs decreased the odds of oropharyngeal colonization. There were 83 episodes of candidiasis among 641 patients within the 3 months prior to the oropharyngeal swab specimen culture, and 12.3% of the patients had received antifungal therapy during that period. On the basis of the multivariate analysis, antibiotic treatment and lower CD4+ counts (<200 cells/mm3) were also independent risk factors for the development of oropharyngeal candidiasis among the patients (Table 4). In contrast, the odds of oropharyngeal candidiasis decreased 5.2-fold for HIV-infected patients receiving antiretroviral therapy.

    DISCUSSION

    In this study 52.4% of HIV-infected outpatients were colonized with yeasts. This rate is slightly lower than those found in previous surveys (i.e., 60 to 63%) (1, 11). Symptomatic oral candidiasis has been reported to occur in 7 to 48% of HIV-infected patients and in 43 to 93% of patients with progressive immunodeficiency (8, 16). In the present study, 12.9% of HIV-infected patients had developed candidiasis within the 3 months before the surveys.

    Fluconazole is widely used for the treatment of mucosal candidiasis, resulting in colonization with less susceptible organisms and the development of resistance among usually susceptible species, such as C. albicans (4, 15). Candida krusei was isolated once in the present study. Surprisingly, the fluconazole MIC for this species was 0.25 μg/ml, even though C. krusei is considered less susceptible to fluconazole than other species (12). Another interesting result was the finding that all five C. lusitaniae isolates were susceptible to amphotericin B, even though C. lusitaniae has been reported to be relatively resistant to amphotericin B (6). The overall resistance rates to amphotericin B and fluconazole were 0.4 and 2.3%, respectively, which are lower than those indicated in previous reports from Taiwan (7, 19).

    Oral colonization with yeasts is known to be significantly higher among HIV-infected patients than healthy individuals (11). In addition to a reduction in the HIV load and restoration of the immune system, antiretroviral therapy may have a more intrinsic role in the elimination of Candida species in HIV-infected patients (2). Thus, the frequency of oropharyngeal candidiasis decreased with antiretroviral therapy in the present study, as well as in previous studies (2, 5). Our findings of a significantly increased risk of oropharyngeal colonization and candidiasis in HIV-infected patients with progressive immunodeficiency (CD4+ count less than 200 cells/mm3) is consistent with the findings described in previous reports (3, 11). In a previous report by Ohmit et al. (11), antibiotic treatment was associated with incident or persistent oral Candida species colonization but not oropharyngeal candidiasis. In contrast, we found that antibiotic treatment was associated not only with oropharyngeal colonization but also with candidiasis. The difference between these two studies may lie in the populations studied.

    Systemic candidemia is recognized as an important, albeit uncommon, cause of mortality (14). Up to 47% of HIV-infected patients with candidemia may succumb to their infection (13). In contrast, mucosal candidiasis does not contribute to morbidity or a significant reduction in quality of life but, rather, contributes to increased medical costs for the treatment of HIV infection. We therefore recommend that HIV-infected patients who are receiving antibiotics and who have CD4+ counts below 200 cells/mm3 be carefully monitored for candidiasis.

    ACKNOWLEDGMENTS

    We thank Bristol-Myers Squibb and Pfizer for supplying the pure powders of amphotericin B and fluconazole, respectively.

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