Istituto di Malattie Infettive e Medicina Pubblica, Università degli Studi di Ancona, Ospedale Umberto I°, Via Conca, 60121, Torrette di Ancona, Ancona, Italy
Received 18 April 2002; returned 6 August 2002; revised 29 August 2002; accepted 6 September 2002
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Abstract |
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Introduction |
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Following the introduction of highly active antiretroviral therapy (HAART) in clinical practice, the prevalence of most opportunistic infections, including OPC, is declining.6,7 In addition, the control of azole-resistant OPC has been reported.810 However, the beneficial effects of HAART on the occurrence of OPC had not been entirely elucidated. Although the recovery of immune function that follows the use of HAART is believed to be the most important factor responsible for the declining rates of OPC, other mechanisms seem to play a fundamental role as well. It has been suggested that, independent of CD4 cell count, low plasma HIV RNA levels significantly correlate with low oropharyngeal carriage of Candida spp., thereby reducing the risk of developing symptomatic infections.11 In addition, recent experimental data showed that some antiretroviral protease inhibitors (PIs) can interfere with Candida infection by inhibiting the fungal secretory aspartyl proteinases (SAPs) that play a pathogenic role in mucosal invasion.12,13 Whether these findings have clinical implications is still unknown. Another unanswered question is whether, and to what extent, HIV-infected patients undergoing HAART can harbour fluconazole-resistant yeasts in their oral cavities.
In an attempt to answer these questions, we conducted a prospective study of 102 HIV-infected patients undergoing HAART. All patients were asymptomatic for OPC and had been undergoing the same HAART regimen for at least 3 months before entry into the study. Point prevalence, microbiology and fluconazole susceptibility patterns of yeast isolates colonizing the oral cavities of these patients were evaluated.
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Materials and methods |
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This prospective study was conducted at the outpatient AIDS Clinic of the Institute of Infectious Diseases and Public Health of the University of Ancona from June to December 2001. Patients were eligible for the study if they were HIV-positive without clinical evidence of OPC; if they had no topical or systemic antifungal use in the 6 months before study entry; if they were undergoing a stable HAART regimen (combination of the same three drugs in the 3 months before study entry); and if their compliance with the antiretroviral regimen was >95%. The following exclusion criteria were used: oral prosthetic devices and diabetes. Data on patient demographics, history of prior fungal infections, antifungal treatments and medications at the time of enrolment were collected. All patients were subjected to clinical evaluation, HIV RNA and CD4 cell measurements on the day of enrolment. Informed consent was obtained from all patients.
Mycological assessment and identification of yeasts
The study subjects were asked to swish 20 mL of 0.9% sterile saline in their mouths for 15 s and to collect the contents into a sterile container. Subsequently, 1 mL was plated on to Sabouraud dextrose agar (SDA) plates (diameter 14 cm) supplemented with chloramphenicol (0.5 g/L). Plates were incubated at 35°C for 48 h. Growth on the plates was quantified and the Candida load was expressed as cfu/mL. If a dense growth of yeasts was noted after 24 h of incubation, the culture was diluted 10-fold and re-plated. Two to three individual colonies with the same morphology from each specimen culture were individually streaked on to SDA plates and stored for standard identification. Yeasts were identified to the species level by standard methods (which included germ-tube formation, corn meal agar for blastoconidia, pseudohyphae and true hyphae, as well as chlamydospore production) and by the API 20 C system for sugar assimilation. In addition, for presumptive identification of Candida dubliniensis, all isolates identified as C. albicans by standard methods were subcultured at 45°C.14 Yeasts were then stored in 10% glycerol at 70°C until use.
Fluconazole susceptibility testing
Fluconazole susceptibility was tested by a broth microdilution method according to the guidelines of the NCCLS.15 Briefly, testing was performed in RPMI-1640 buffered to pH 7.0 with 0.165 M MOPS. MICs were determined visually and defined as a prominent decrease in turbidity compared with the growth control. In accordance with the scheme in the NCCLS document, isolates were defined as flucon azole susceptible if MICs were 8.0 mg/L, susceptible dose dependent if MICs ranged from 16 to 32 mg/L and resistant if MICs were
64 mg/L.15 C. albicans ATCC 90029 was used as quality control strain in each run of the experiments.
Statistical analysis
Differences between continuous variables were assessed by means of two-tailed Students t-test for differences in means. Differences between categorical variables were evaluated by 2 test. A P value of <0.05 was considered statistically significant.
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Results |
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Discussion |
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Recent in vitro and in vivo experimental data have shown that some antiretroviral molecules belonging to the class of PIs can interfere with Candida infection by inhibiting the fungal SAPs.12,13 Some of these enzymes are similar to HIV proteases and had been shown to play a pathogenic role in mucosal invasion.16 However, in this study, we did not find any correlation between the status of yeast carrier and the type of antiretroviral regimen. These apparently controversial results might be due to at least two different reasons. First, since all isolates considered in this study were commensal and not infecting strains, one can speculate that SAPs were produced at constitutively low levels, thereby reducing the potential target for PIs. This hypothesis is strengthened by the observation that strains of C. albicans isolated from the oral cavities of patients with symptomatic thrush are significantly more proteolytic than those isolated from asymptomatic yeast carriers.17 Secondly, PI concentrations achievable in human tissues are somewhat lower than those utilized in these experimental studies.12,13
The appearance of fluconazole-resisistant isolates of C. albicans in these patients has been repeatedly documented.4,5,8 Before the widespread use of HAART, one study found that the point prevalence carriage of Candida spp. resistant to fluconazole (MIC 64 mg/L) was 21% in HIV-infected patients with OPC and 14% in asymptomatically colonized patients. When the same cohort of patients was re-evaluated after the introduction of HAART, the authors found a declining rate of both OPC and oral carriage of Candida spp. as well as a trend toward less frequent in vitro resistance to fluconazole.8,18 In the present study, we found that 93% of the isolates were susceptible in vitro to the triazole (MIC
8.0 mg/L) and the remaining 7% were susceptible dose dependent.
In conclusion, our data reveal important characteristics of carriage of yeasts in the oropharynx of HIV-infected patients undergoing HAART. We have shown here that the status of yeast carrier is not associated with the number of CD4 cells or the viral load. Similarly, the type of antiretroviral regimen does not influence the carriage of Candida spp., the only predictor of which is a previous history of OPC. The majority of the patients harbour strains that are in vitro susceptible to fluconazole.
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Acknowledgements |
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Footnotes |
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References |
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