Clinical and Investigative Medicine

 

Asymptomatic oral carriage of Candida albicans in patients with HIV infection

Ignatius W. Fong, MB, BS
Manuel Laurel, MD
Aileen Burford-Mason, MD

Clin Invest Med 1997;20(2):85-93.

[résumé]

Drs. Fong and Laurel are with St. Michael's Hospital, and Dr. Burford-Mason is with the Toronto Hospital, University of Toronto, Toronto, Ont.

(Original manuscript submitted June 4, 1996; accepted Nov. 21, 1996; submitted in final form Jan. 17, 1997)

Reprint requests to: Dr. Ignatius W. Fong, St. Michael's Hospital, 30 Bond St., Toronto ON M5B 1W8; fax 416 864-5870

Contents
Abstract

Objective: To assess the relationship of asymptomatic carriage of Candida albicans and clinically apparent thrush in patients with HIV infection.

Design: Prospective, longitudinal, controlled study.

Setting: The HIV clinic at St. Michael's Hospital, University of Toronto.

Participants: One hundred and twenty-seven patients with HIV infection were divided into 3 groups according to the CD4+ lymphocyte count, and 37 healthy volunteers served as controls.

Interventions: Determination of blood type, baseline CD4+ lymphocyte count in patients with HIV infection, and immunophenotyping. Samples of saliva (2 mL) were obtained from each patient and control.

Main outcome measures: Carrier status, clinical presence of thrush, the association between carriage of C. albicans and blood type, secretor status and history of oral infection.

Results: In patients with HIV infection and C. albicans colonization no correlation was found with blood type or secretor status of blood group antigen in the saliva. The frequency of oral carriage of yeast was greater in patients infected with HIV than in controls, but the difference was not significant for asymptomatic subjects with a CD4+ lymphocyte count greater than 500/µL. Persistent carriage of yeast and development of clinical thrush were associated with lower CD4+ counts. Clinical thrush developed only in patients with persistent asymptomatic carriage of C. albicans and CD4+ counts less than 500/µL.

Conclusion: The greater risk of oral colonization with C. albicans in patients with HIV infection partly explains the high prevalence of thrush found in this group.

Résumé

Objectif : Évaluer le lien entre l'état de porteur asymptomatique du Candida albicans et la présence de muguet apparent sur le plan clinique chez des patients infectés au VIH.

Conception : Étude contrôlée longitudinale prospective.

Contexte : La clinique de VIH ŕ l'Hôpital St. Michael de l'Université de Toronto.

Participants : On a réparti 127 patients infectés au VIH dans 3 groupes selon la numération lymphocytaire CD4+ et 37 bénévoles en bonne santé ont servi de témoins.

Interventions : Détermination du type sanguin, numération lymphocytaire de base CD4+ chez les patients infectés au VIH et immunophénotypage. On a obtenu des spécimens de salive (2 mL) de chaque patient et de chaque témoin.

Principales mesures des résultats : État de porteur, présence clinique du muguet, lien entre l'état de porteur du C. albicans et le groupe sanguin, état sécréteur et antécédents d'infection buccale.

Résultats : Chez les patients infectés au VIH et porteurs de colonies de C. albicans, on n'a établi aucun lien entre le type sanguin ou l'état sécréteur de l'antigčne du groupe sanguin dans la salive. La fréquence de lévulose buccale était plus élevée chez les patients infectés au VIH que chez les témoins, mais la différence n'était pas significative dans le cas des sujets asymptomatiques chez lesquels la numération lymphocytaire CD4+ était supérieure ŕ 500/µL. On a établi un lien entre la persistance de la lévulose et l'apparition du muguet clinique et des numérations CD4+ moins élevées. Le muguet clinique n'a fait son apparition que chez les patients toujours porteurs asymptomatiques du C. albicans et dont la numération CD4+ était inférieure ŕ 500/µL.

Conclusion : Le risque plus élevé d'apparition de colonies buccales de C. albicans chez les patients infectés au VIH explique en partie la prévalence élevée de muguet constatée dans ce groupe.

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Introduction

Candida albicans is the most frequently diagnosed opportunistic infecting agent of the oral cavity, pharynx and esophagus in immunocompromised patients.[1­3] Clinically, the infection is a marker of moderate or severe immunosuppression,[4] and patients with unexplained oral candidiasis have a high risk of developing AIDS.[3] The presence of thrush in newly diagnosed HIV-positive patients has prognostic significance.[3,5,6] Whereas symptom-free HIV-positive patients with CD4+ lymphocyte counts below 400/µL have a 50% risk of progression to full-blown AIDS within 3 years, such patients who also have thrush have a 90% risk of progression in the same period.[6]

Although oral C. albicans infection may occur at any stage of HIV infection, it is most commonly associated with low CD4+ lymphocyte counts,[4,7] and increased isolation of C. albicans from the oropharynx has been correlated with low T-lymphocyte:helper-T-suppressor ratios[7] and T-cell dysfunction.[8] AIDS has also been associated with various monocyte functional abnormalities including defective chemotaxis[9] and phagocytosis.[10] Since monocytes have fungicidal activity,[11,12] their dysfunction, combined with defects and depletion of T lymphocytes, could explain the predisposition of these patients to oral and esophageal candidiasis.

Asymptomatic oral C. albicans carriage has been demonstrated in HIV-positive patients,[13] and an increased incidence of asymptomatic oral C. albicans carriage in HIV-positive patients compared with other at-risk groups, such as denture wearers, has also been noted.[14] A higher prevalence of oral C. albicans colonization in patients with AIDS may therefore be a predisposing factor for the subsequent development of clinical thrush. Although the predisposing factors for increased oral carriage of C. albicans in HIV-positive patients are not known, studies in normal subjects and patients with peptic ulcer have shown that both blood group O and nonsecretion of blood group antigens in saliva are separate and cumulative risk factors for asymptomatic oral C. albicans carriage.[15,16]

A prospective study of C. albicans carriage in subjects with HIV infection and in healthy control subjects was therefore carried out. Results were correlated with ABO blood type, ABO secretor status, and CD4+ lymphocyte count. Longitudinal studies and the use of a quantitative mouth-rinse technique to assess oral C. albicans carriage[15­17] enabled us to study the temporal association in HIV-positive patients between carrier status, oral yeast load and the development of clinical thrush over a 6-month period.

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Subjects and methods

We conducted a prospective, 6-month longitudinal study involving patients attending the HIV Clinic at St. Michael's Hospital, Toronto, in 1994. Approval was obtained from the institution's research ethics board, and consent was obtained from all patients and volunteers.

Population

One hundred and twenty-seven patients with HIV infection were enrolled in the study and stratified into 3 groups according to CD4+ lymphocyte count: group 1 patients had CD4+ counts of greater than 500/µL; group 2 patients had CD4+ counts of 200 to 500/µL; and group 3 patients had CD4+ counts less than 200/µL. Data were obtained on previous history of thrush, use of antimicrobial and antifungal agents, the wearing of dentures and smoking. Thirty-seven healthy volunteers (with no risk for HIV infection) were selected as controls.

Methods

On admission to the study, blood type was determined by standard laboratory techniques. A baseline CD4+ count was determined for all subjects with HIV infection. Immunophenotyping was performed by the whole-blood lysis method, employing monoclonal antibodies labelled with either fluorescein isothiocyanate or phycoerythrin (Becton­Dickinson, San Jose, Calif.). A total of 2000 gated events in the lymphocyte gate were acquired with use of a FACScan flow cytometer equipped with a 15 mW argon ion laser and analysed with simulet software (Becton­Dickinson).

Identification of secretors

To identify secretor status, 2 mL of unstimulated saliva was obtained from each patient and volunteer. Each sample was boiled for 20 minutes and centrifuged at 4000 * g for 10 minutes; the supernatant was stored at -20°C until tested. The presence of ABO blood group antigens in saliva was determined by a dot enzyme-linked immunosorbent assay (dot-ELISA) according to the method of Zhou and associates,[18] with minor modifications. Aliquots of saliva (5 to 10 µL) diluted 1:100 in phosphate-buffered saline (PBS) were dotted onto 3 separate sheets of PVDF blotting membrane (Bio-Rad, Mississauga, Ont.) and the membranes incubated for 5 minutes at room temperature. The membranes were washed 3 times in PBS for 5 minutes with gentle agitation, and then nonspecific binding sites were blocked by incubating in 2.5% w/v skim milk powder in PBS at room temperature for 15 minutes. Membranes were probed with either anti-A (BioClone, Ortho Diagnostics, Markham, Ont.; 1:10), anti-B (BioClone, Ortho Diagnostics; 1:100) or anti-O(H) antibody (Dako 1:100, Dimension Labatories, Mississauga, Ont.) for 1 hour at room temperature, or overnight at 4°C, washed 3 times in PBS, and hybridized with peroxidase-conjugated affinity purified F(ab) 2 goat anti-mouse IgG (1:100, Jackson ImmunoResearch Laboratories Inc., West Grove, Penn.) for 1 hour at room temperature. Membranes were then exposed to 3´3-diaminobenzidine hydrochloride (DAB), and the reaction product was intensified by the addition of cobalt chloride and nickel chloride.

C. albicans carrier status

Patients and volunteers were tested for oral carriage of C. albicans species at the time of enrolment and at monthly intervals thereafter for 6 months. The subjects swilled 10 mL of sterile water vigorously around their mouth for 30 seconds and return the mouth rinse to a sterile container. Then 0.1 mL of the mouth rinse was plated onto Sabouraud's dextrose agar plates, and colonies of C. albicans were counted after 48 hours of incubation at 30°C. Yeasts were identified as to species by germ tube determination in calf serum, by chlamydiospore production on oxgall media and by Vitex-YBC card (Bio-Mérieux Inc., Hazelwood, Mo.) readings. Subjects' carrier status was defined as "persistent" when all cultures were positive for yeasts, "intermittent" when some of the cultures were positive for yeasts and "noncarrier" when all cultures were negative. Clinical assessment for the presence of oral thrush was noted at each clinic visit.

Clinical diagnosis of oral candidiasis

The diagnosis of thrush was made if there were typical white pseudomembranes or if there were erythematous lesions and the wet smear of the scrapings showed yeast pseudohyphae.

Statistical analysis

The data were analysed statistically by the SAS computer program. The groups were compared by Fisher's exact test and multiple pair-wise comparisons made. Adjusted p values for multiple comparisons were obtained using the Bonferroni's step-down method.[19] The Cochran­Mantel­Haenszel test was used to analyse correlations between quantitative carriage of yeasts and CD4+ lymphocyte counts.

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Results

General

Twenty-two patients and 2 controls were excluded from the analysis because follow-up cultures were not completed. Thus, 105 HIV-positive patients and 35 healthy control subjects were included in the analysis. The demographic features of patients and controls are shown in Table 1. There was no significant difference between patients and controls with respect to age, sex, smoking or the wearing of dentures. Of the 105 HIV-positive patients, 24 had CD4+ lymphocyte counts greater than 500/µL, 42 had counts between 200 and 500/µL and 39 had counts less than 200/µL. The number of patients taking antimicrobial drugs (primarily cotrimoxazole prophylaxis) and antifungal agents was higher in patients with CD4+ counts less than 200/µL, but the difference in drug use between controls and HIV-positive patients was significant only for antimicrobial usage (p = 0.01).

In the HIV-positive patients, 388 strains of yeast were isolated during the study. Of these, 361 (93%) were C. albicans and 27 (7%) were non-albicans species. Candida glabrata (Torulopsis glabrata) and Candida parapisilosis were the most common non-albicans species. In the control subjects, 46 strains of yeast were isolated: C. albicans in 41 (89%) and non-albicans species in 5 (11%). In both patients and control subjects, the non-albicans yeasts were carried only transiently and not isolated from persistent carriers.

Oral carriage of Candida species

The number of subjects with persistent oral carriage of yeasts was higher among the HIV-positive patients than among healthy subjects (Table 2), the difference being significant (p < 0.02) for HIV-positive subjects with CD4+ counts of 200 to 500/µL and of less than 200/µL but not for those with counts greater than 500/µL. Conversely, there were more noncarriers among healthy control subjects than HIV-positive patients, and this was significant (p < 0.04) for patients with CD4+ counts of 200 to 500/µL and less than 200/µL but not for those with counts greater than 500/µL. Combined persistent and intermittent carriers of yeasts constituted 82% of patients with CD4+ counts less than 200/µL, 86% of patients with counts of 200 to 500/µL, 67% of patients with counts greater than 500/µL and 51% of healthy control subjects.

Carriage of C. albicans, blood type and secretor status

There was no correlation between carriage of yeast and blood type and the secretor status of blood group antigen in the saliva of patients with HIV infection or of controls (Table 3). In HIV-positive patients, persistent carriage of yeast was greater in those with blood group O than in those with other blood types (46% v. 31%), but the difference was not significant.

Influence of concomitant antifungals and antimicrobials

The use of antifungal agents did not appear to suppress the carriage of yeast, since 9 of the 10 patients receiving treatment carried yeasts either intermittently or persistently. Antimicrobial agents for Pneumocystis carinii pneumonia prophylaxis also had little influence on the rate of yeast carriage: the rate of noncarriage of yeast in patients taking antimicrobials was 19%, whereas it was 21% among those not taking antimicrobials.

Relation between carrier state, history of infection and the development of thrush

Table 4 shows CD4+ lymphocyte counts in relation to the history of oral C. albicans infection and the development of clinical manifestations of thrush in HIV-positive patients during the study. Thrush was not seen in any of the healthy subjects nor was a history compatible with a diagnosis of previous C. albicans infection reported. Thrush developed in 6 HIV-positive patients during the study: in 2 patients with CD4+ counts of 200 to 500/µL and 4 patients with counts less than 200/µL. All infections occurred in HIV-positive patients who were persistent carriers, and 2 of the patients had a history of previous infection.

A history of oral thrush was reported by 20 patients, of whom 12 were persistent carriers, 5 were intermittent carriers and 3 were noncarriers. No patients with CD4+ counts greater than 500/µL had a history of oral C. albicans infection. Four patients with CD4+ counts between 200 and 500/µL had a history of oral C. albicans (2 were persistent carriers and 2 noncarriers). Sixteen patients with CD4+ counts less than 200/µL gave a history of oral C. albicans (10 were persistent carriers, 5 intermittent carriers and 1 a noncarrier).

Quantitative assessment of oral yeast carriage

The concentrations of yeast in the oral rinse were inversely correlated with the CD4+ lymphocyte count, with the highest concentrations being found in patients with CD4+ counts less than 200/µL (Table 5). The mean concentration in patients with CD4+ counts greater than 500/µL was similar to that seen in control subjects (Table 5). The concentration of yeast in the rinse was not significantly higher for patients with clinical thrush compared with those without thrush, for patients with similar CD4+ counts. Table 6 shows quantitative C. albicans carriage over the study period in the 6 patients who had clinical thrush. There was no temporal relation between the number of yeasts isolated and the diagnosis of clinical disease.

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Discussion

Asymptomatic oral carriage of C. albicans is a common finding in HIV-infected patients,[13,14] and it has been suggested that oral candidiasis results from yeast overgrowth in subjects with a history of colonization.[13] The results of this study support that hypothesis. We found asymptomatic carriage of yeasts more frequently in HIV-positive patients as a group, with more persistent carriers and fewer noncarriers among patients than among controls, and it was only among patients identified as persistent carriers that infections developed. However, the development of clinical infection was clearly related to clinical staging of the disease. The frequency of C. albicans carriage, the concentration of yeasts isolated and the development of thrush were all related to CD4+ counts: persistent and intermittent carriage occurred most frequently in patients with CD4+ counts less than 200/µL and least frequently in those with CD4+ counts greater than 500/µL; the concentration of yeast isolated from the mouth rinse of HIV-infected patients correlated inversely with CD4+ cell counts, the highest number of yeasts being isolated from patients with CD4+ counts less than 200/µL; of the 6 patients in whom thrush developed, 4 had CD4+ counts less 200/µL and 2 had counts between 200 and 500/µL. No case of thrush occurred in patients with CD4+ counts greater than 500/µL. Interestingly, HIV-positive patients with CD4+ counts greater than 500/mL did not differ significantly from controls in either the frequency of persistent or intermittent carriage, or the quantity of yeasts isolated. These findings support the view that the pathogenesis of HIV-associated oral candidiasis is related to underlying immune defects.[4,7­12]

However, since all 6 clinical infections diagnosed during the study occurred in patients who had previously been identified as persistent carriers, the development of clinical C. albicans infection may not depend solely on CD4+ lymphocyte count but also on asymptomatic C. albicans carriage. This premise is supported by the finding that asymptomatic carriage was a more reliable predictor of the development of clinical infection than was a history of C. albicans infection. In addition, 18% of patients with CD4+ counts less than 200/µL were noncarriers. C. albicans is a ubiquitious organism, and viable organisms can be recovered from a wide range of commonly consumed foods such as cereals, salads, fruit and vegetable juices, and from a variety of dried, canned and pasteurized items.[20] Despite the likelihood of oral exposure to yeasts, and although they were severely immunocompromised, these patients had no C. albicans infection or asymptomatic carriage throughout the study period. Further studies over a longer observation period would determine whether patients with CD4+ counts less than 200/µL who were noncarriers would eventually have become carriers or acquired infections as their disease progressed, or if some unknown protective mechanism exists in these patients. Moreover, there was no temporal relation between the quantity of yeasts isolated and the development of clinical thrush. Thus, quantitative cultures are not clinically useful.

Although previous studies have shown that blood group O and nonsecretion of blood group antigens in saliva are separate and cumulative risk factors for oral C. albicans carriage in normal subjects[15] and patients with peptic ulcer,[16] in this study there was no association between asymptomatic oral carriage of yeast and ABO blood group or secretor status, either in HIV-positive patients or in control subjects. Although in both groups more persistent and intermittent carriers had group O blood, the differences were not statistically significant. The healthy subjects in this study were chosen to reflect the age and sex distribution of the HIV-positive patients, who were predominantly young men, so the control group was not comparable to those of previous studies in healthy individuals. Those studies showed no association between oral carriage and age or sex, but they did not examine these factors in relation to blood group. An alternative explanation of our failure to confirm a relation between carriage and blood group or secretor status, or both, in healthy controls is the smaller number of healthy subjects included in this study. However, of the 6 clinical infections that occurred in the study, 5 were in patients with group O blood. All 6 were secretors of blood group antigens in saliva.

The presence of ABO blood group antigens in the mucus of secretors is thought to contribute nonspecifically to innate host defences against infections by binding to pathogens by way of carbohydrate moieties, thus preventing adherence to similar attachment sites on mucosal cell surfaces.[21] The inability to secrete the water soluble glycoprotein form of the ABO blood group antigen in bodily secretions has been shown to be associated with increased vulnerability to a number of infectious diseases, including superficial fungal infection.[22] In view of the susceptibility of HIV-positive patients to opportunistic infections, it would not have been surprising to find an excess of nonsecretors in the patient population. However, results suggest that nonsecretion of blood group antigens does not contribute to the development of opportunistic infections in patients with AIDS.

The reported results of studies on the rate of oral carriage of yeast in HIV-positive subjects have not been consistent. This is likely the result of different approaches to patient selection and varying methodologies for identifying carrier status. C. albicans is not uniformly distributed in the mouth,[23] and, because of this, studies using swabs to detect oral colonization may be less reliable than those using the mouth-rinse technique, which is reproducible and quantifiable.[17] In addition, few studies stratify the patients according to their CD4+ counts. In one study examining 28 HIV-positive patients with CD4+ counts greater than 400/µL and using the mouth-rinse technique, the carriage rate was 75% in the patients and 68% in the controls (not significant).[24]

In that study, the carriage rate in the group of HIV-positive subjects was similar to that in our patients with a CD4+ count greater 500/µL, but the carriage rate in the controls was higher than previously reported. The authors attributed this to the high percentage (40%) of women in their control group.

In contrast, Torssander and associates[25] found that the carriage rate of C. albicans in HIV-seropositive homosexual men was significantly higher than rates in HIV-seronegative homosexual and heterosexual men, but they noted no correlation with CD4+ counts. Felix and Wray[14] also noted higher oral C. albicans carriage in HIV-positive subjects (93.1%) compared with control subjects (57.4%) but did not examined the effect of CD4+ lymphocytopenia. Most of their patients probably had advanced HIV infection, because 52% had clinical candidiasis. Another study using oral swabs found C. albicans in only 63 (24%) of 261 HIV-positive patients,[13] and the colonization rate was related to Centers for Disease Control and Prevention staging, lymphocytopenia, CD4+ cell depletion and elevated ß2-microglobulin. The low rate of C. albicans colonization in this study may be related to the type of specimen cultured. However, their observation of the correlation of yeast colonization with CD4+ lymphocyte count and staging of the disease is consistent with our findings. Similarly, De Bernardis and associates[26] noted that increased oral colonization correlated with clinical disease and low CD4+ counts but found that the rate of colonization was similar for asymptomatic HIV-positive patients and controls (around 30%). In contrast, a study of subclinical colonization of the tongue detected from cytologic smears found that colonization did not correlate with the Walter Reed staging or T-lymphocyte depletion below 400/µL.[27] Such discrepancies between studies suggest that the adoption of more reliable methods for identifying carrier status and stratification of patients by disease stage is essential if results from different centres are to be compared.

To our knowledge, no previous study has examined the significance of asymptomic carriage of C. albicans as a risk factor for subsequent oral infection. The infrequency with which oral thrush is encountered in nonimmunocompromised subjects makes such studies difficult in healthy subjects. However, in patients with AIDS, oral thrush is common, making them an ideal population in which to undertake such an investigation. The findings of this study may have wider implications for other immunocompromised subjects such as patients who have undergone organ transplantation, in whom oral thrush may also be a problem.[28]

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Conclusion

Our study has demonstrated that the incidence of asymptomatic oral carriage of yeast is greater in HIV-positive subjects than in healthy subjects and that persistent asymptomatic carriage is a risk factor for subsequent oral C. albicans infection. Asymptomatic oral carriage was correlated with CD4+ lymphocyte counts but did not depend solely on this factor. There was no correlation between C. albicans colonization and ABO blood group or secretor status of blood group antigen in the saliva of HIV-infected patients. The increased rate of oral colonization with C. albicans species may partly explain the high prevalence of thrush in HIV-infected patients.

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Acknowledgements

We thank Mr. Jianli Li for his assistance in statistical analysis, and Ms. Dayle Noda and Mr. Srimohan Ragagopalan for their technical assistance.

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