Fungal infections associated with HIV infection.

Oral candidiasis is perhaps the commonest infection seen in HIV disease. The aim of this workshop was to provide a sketch of the multifarious aspects of the disease from a global perspective. To this end the panellists addressed issues such as the virulence of Candida, emergence of antifungal resistance, management of candidiasis and other exotic, oral mycotic diseases. An all-pervasive theme was the dramatic differences in the management of fungal infections consequential to the availability (or the lack) of anti-HIV drugs in the developed and the developing world. Further, the social stigmata associated with the HIV disease in many developing regions in Africa and Asia appears to modify the therapeutic strategies. Additionally, the lesser-known regional variations in the disease manifestations and therapeutic approaches were stark. Further work is direly needed to address these issues.

Saliva and inhibition of HIV-1 infection: molecular mechanisms.

Oral fluids are rarely a vehicle for HIV-1 infection in vivo, unlike other mucosal secretions. This unique property raises questions regarding (1) the molecular mechanisms responsible for the lack of salivary transmission, (2) the extent to which oral immunological responses mirror responses at other mucosal sites, (3) the use of promising salivary markers of HIV-1 disease progression, (4) the relationship between oral and blood viral loads, (5) cofactors that influence oro-genital transmission, and (6) the feasibility of oral-based antibody testing for HIV-1 diagnosis in the home. This paper discusses these questions and provides background summaries, findings from new studies, consensus opinions, practical relevance to developing countries, and suggestions for future research agenda on each of the key topics.

Oral mucosal immunity and HIV infection: current status.

There is a paradox that profound HIV-induced immunodeficiency is present systemically, whereas the majority of infections associated with HIV disease are present or initiated at mucosal surfaces. There is therefore a need to understand both specific and non-specific mechanisms of mucosal protection against HIV and its copathogens. The majority of HIV infections occur as a result of the passage of virus across mucosal membranes. Resistance to HIV infection at mucosal surfaces may be related to HIV-specific CD8+ T cell responses in some individuals and may be the basis for protective vaccine design. However, T-cells, macrophages and dendritic cells in mucosa may be a portal of entry for HIV. Transcytosis of HIV can occur from the mucosal to the submucosal surface and vice versa, and may be inhibited by mucosal immunoglobulins and neutralizing IgA within epithelial cells. HIV-induced alterations to oral epithelial cells, together with impairment of mucosal CD4+ T-cells and consequent altered cytokine secretion, may contribute to secondary infections. It also appears that HIV infection is associated with decreased salivary IgA levels, although a dichotomy between IgA concentrations in saliva and serum has been reported. Mucosal antibody responses, however, seem to be maintained. Considerable attention has been given to the possibility of mucosal immunization against HIV and there is evidence that secretory IgA antibody is neutralizing to different HIV strains. In addition to specific immune factors, it is likely that innate nonspecific factors may be significant in protecting mucosal surfaces, including lactoferrin, secretory leukocyte protease inhibitor, mucins, proline rich proteins and cystatins. These may be useful candidate virucides in topical preparations. Thus humoral, cellular and innate immune mechanisms, as well as lymphocyte-epithelial interactions, may all be impaired at mucosal surfaces as a result of HIV infection and may contribute to the susceptibility of mucosa to infective processes.

Salivary calprotectin levels are raised in patients with oral candidiasis or Sjögren's syndrome but decreased by HIV infection.

Calprotectin levels were determined in whole saliva from patients predisposed to oral candidiasis due to HIV infection or Sjögren's syndrome and from patients with candidiasis associated with various oral disorders (e.g. lichen planus, oral ulceration). Mean calprotectin levels were higher in whole saliva (2 microgram/ml) than in parotid saliva (0.3 microgram/ml). Oral candidiasis was associated with raised whole saliva calprotectin levels in all groups studied. HIV infection was associated with lower levels of salivary calprotectin, in the presence of high or low salivary Candida counts, although CD4+ lymphocyte counts did not significantly correlate with calprotectin concentrations. Calprotectin levels were elevated in saliva from Sjögren's syndrome patients with oral candidiasis, consistent with mucosal transudation of calprotectin from inflamed mucosa and limited dilution due to decreased salivary flow rates. This study indicates that oral candidiasis is associated with raised calprotectin levels secondary to mucosal inflammation, but that diminution of this candidacidal factor due to HIV infection may be a predisposing factor in the aetiology of oral candidiasis.

Impaired secretory immunity in dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa is a congenital disorder characterized by blistering of the skin and oral mucosa. This study investigated the hypothesis that children with dystrophic epidermolysis bullosa have impaired oral secretory immunity. Immunoglobulin A (IgA), secretory IgA and IgG concentrations, and IgA and secretory IgA antibody levels to Candida albicans, Lactobacillus casei and Streptococcus mutans were measured in whole saliva from 22 children with dystrophic epidermolysis bullosa and 22 matched controls. Salivary total IgA and total IgG concentrations were significantly raised in dystrophic epidermolysis bullosa due to serum leakage from oral blistering, but the converse was seen with secretory IgA. This suggestion of a mucosal immune defect was supported by decreased secretory IgA antibody responses to all three microorganisms tested. This apparent defect in secretory immunity in dystrophic epidermolysis bullosa may be due to mucosal involvement and damage resulting in impaired antigen sampling in mucosal associated lymphoid tissue or to impaired transport of secretory IgA across the salivary gland mucosa.

In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis.

Secreted aspartyl proteinases are putative virulence factors in Candida infections. Candida albicans possesses at least nine members of a SAP gene family, all of which have been sequenced. Although the expression of the SAP genes has been extensively characterized under laboratory growth conditions, no studies have analyzed in detail the in vivo expression of these proteinases in human oral colonization and infection. We have developed a reliable and sensitive procedure to detect C. albicans mRNA from whole saliva of patients with oral C. albicans infection and those with asymptomatic Candida carriage. The reverse transcription-PCR protocol was used to determine which of the SAP1 to SAP7 genes are expressed by C. albicans during colonization and infection of the oral cavity. SAP2 and the SAP4 to SAP6 subfamily were the predominant proteinase genes expressed in the oral cavities of both Candida carriers and patients with oral candidiasis; SAP4, SAP5, or SAP6 mRNA was detected in all subjects. SAP1 and SAP3 transcripts were observed only in patients with oral candidiasis. SAP7 mRNA expression, which has never been demonstrated under laboratory conditions, was detected in several of the patient samples. All seven SAP genes were simultaneously expressed in some patients with oral candidiasis. This is the first detailed study showing that the SAP gene family is expressed by C. albicans during colonization and infection in humans and that C. albicans infection is associated with the differential expression of individual SAP genes which may be involved in the pathogenesis of oral candidiasis.

Adherence of Candida albicans to denture-base materials with different surface finishes.

OBJECTIVES: To assess the in vitro adherence of Candida albicans to heat-cured hard and soft denture-base materials with varying surface roughness, and to observe the effect of a mixed salivary pellicle on candidal adhesion to these surfaces. METHODS: In vitro adhesion assays on heat-cured acrylic resin (Trevalon), Molloplast B and Novus using the type strain of C. albicans (NCPF 3153A). Surfaces for the assays were prepared using clinically appropriate rotary instruments. Unstimulated, pooled and clarified whole saliva was used to assess its effect on adhesion. RESULTS: Significantly greater adhesion of C. albicans to rough rather than smooth surfaces was found (P < 0.001), as well as increased adhesion to the machined soft lining materials compared with acrylic. Pre-coating denture-base materials with saliva reduced candidal adhesion on all materials. CONCLUSIONS: Rough surfaces on denture-base materials promote the adhesion of C. albicans in vitro. However, saliva reduces adhesion of C. albicans and thus diminishes the effect of surface roughness and free surface energy differences between materials.

Salivary and mucosal immune responses to HIV and its co-pathogens.

The profound effects that HIV induces in systemic immunity have been well characterised, but the situation with regard to mucosal immune responses is less clear. Oral cavity fluids have been used as a marker of the mucosal immune system. Whole and parotid saliva IgA, IgA1 and IgA2 concentrations have been found to be lower in both HIV infection and AIDS subjects, whereas serum IgA and IgA subclasses are markedly raised, suggesting a dichotomy between systemic and secretory immunity. Salivary antibodies to HIV can be readily detected and secretory IgA antibody can be neutralising to some strains of HIV. HIV vaccines can also induce antibody responses in saliva, but vaccination routes other than parenteral immunisation are needed. Antibody responses to oral microbes have also been studied and it has been shown that IgA, IgA1 and IgA2 subclass antibody titres to Candida albicans and to Streptococcus mutans are increased in whole or parotid saliva from HIV patients, but reduced in AIDS patients, suggesting a compensatory response which is overcome with progressive immunodeficiency. The avidity of salivary IgA antibodies to Candida in HIV seems unimpaired, whereas relative avidities of serum antibodies in HIV patients with candidiasis are lowered. Non-specific factors which may inhibit Candida and other opportunist pathogens are also found in saliva. The candidacidal, myelomonocytic protein calprotectin is present in saliva at levels which are biologically active, although levels are lowered in HIV infection. Overall, HIV infection appears to be associated with disregulation of a number of immune factors at the mucosal surface, but the ability of patients with HIV infection to mount specific antibody secretory responses seems to be relatively intact until late in infection.

IgA subclasses in HIV disease: dichotomy between raised levels in serum and decreased secretion rates in saliva.

This study sought to determine IgA, IgA1 and IgA2 concentrations and secretion rates in unstimulated whole saliva and stimulated parotid saliva and IgA, IgA1 and IgA2 concentrations in serum from asymptomatic human immunodeficiency virus (HIV)-infected, acquired immune deficiency syndrome (AIDS) and control subjects. In whole and parotid saliva the mean IgA, IgA1 and IgA2 concentrations in the HIV and AIDS groups were lower than the control group (P < 0.05). Unstimulated whole and stimulated parotid saliva flow rates were lower in the HIV and AIDS groups compared with the control group, and reached statistical significance with respect to the mean parotid saliva flow rate in the AIDS group (P < 0.05). Mean IgA, IgA1 and IgA2 secretion rates in both the HIV and AIDS groups were significantly less than the controls (P < 0.05). In contrast, serum IgA, IgA1 and IgA2 concentrations were markedly raised in the HIV and AIDS groups compared with the control group (P < 0.001). There was no correlation between saliva and serum IgA concentrations within individuals. This study suggests that, in spite of the raised, polyclonally activated serum IgA concentrations associated with HIV infection, salivary IgA concentrations and secretion rates are reduced, emphasizing the dichotomy between systemic and secretory immunity.

Candida albicans isolates from HIV-infected and AIDS patients exhibit enhanced adherence to epithelial cells.

The increased prevalence of oral candidosis associated with HIV infection must be intrinsically related to immunological changes in the host, but might also involve alterations to the infecting strains of yeast. This study aimed to determine if strains of Candida albicans isolated from asymptomatic HIV-infected individuals or AIDS patients possessed altered adherence properties in an in-vitro buccal epithelial cell (BEC) adherence assay. C. albicans isolates from 49 patients with HIV infection or AIDS adhered to BEC in significantly higher numbers than isolates from 49 control subjects (p < 0.001). No significant differences in adherence were detected between strains isolated from HIV-infected or AIDS subjects, or between strains isolated from C. Albicans carriers (low salivary C. albicans counts) or subjects with oral candidosis. The presence of whole saliva significantly inhibited the binding of candida to BEC (p < 0.001), but the significant difference in adherence between the HIV/AIDS and control isolates was maintained. The effect of saliva was independent of salivary candida antibodies and was abolished by treatment with protease or neuraminidase, suggesting the involvement of salivary mucins. The results of this study suggest that HIV infection is associated with the selection of strains of C. albicans with and increased ability to adhere to oral mucosa.

Immunoglobulin A (IgA), IgA1, and IgA2 antibodies to Candida albicans in whole and parotid saliva in human immunodeficiency virus infection and AIDS.

Human immunodeficiency virus (HIV)-infected individuals are predisposed to recurrent oral candidiasis, and, although it has been assumed that this is because of deficient mucosal immune responses, this has not been properly established. The present study aimed to compare the concentrations and secretion rates of immunoglobulin A (IgA) and IgA subclass antibodies to Candida albicans in whole and parotid saliva samples from HIV-infected patients, AIDS patients, and control subjects. Levels of IgA antibody to Candida species in whole saliva were higher in the HIV group than in the controls and were highest in the AIDS group (P < 0.05). In parotid saliva, the mean antibody levels were significantly greater in HIV-positive patients than in controls (P < 0.05) but fell to lower levels in the AIDS group. The secretion rates of Candida antibodies in parotid saliva were reduced in AIDS patients compared with HIV patients. The specific activities of the IgA antibodies and both subclasses were significantly higher in the HIV and AIDS patients than in the controls in both whole and parotid saliva (P < 0.05). Antibody levels were significantly correlated with the numbers of Candida organisms isolated from saliva (P < 0.05). These results suggest clear differences in salivary antibody profiles among HIV-infected. AIDS, and control subjects and are indicative of a response to antigenic challenge by infecting Candida species. No obvious defect in the mucosal immune response in the HIV or AIDS groups that might account for the increased prevalence of candidiasis was apparent.

Effect of iron concentration on siderophore synthesis and pigment production by Candida albicans.

Twelve strains of Candida albicans were grown in defined medium which had been deferrated by ion-exchange chromatography and then supplemented with FeCl3 to give iron concentrations ranging from 0.026 microM (growth-limiting) to 0.8 microM (excess). All of the strains secreted hydroxamate-type siderophores; phenolate siderophores were not detected. Isolates of C. lusitaniae, C. glabrata and C. parapsilosis also secreted hydroxamate but not phenolate-type iron chelators. Siderophore synthesis by C. albicans was maximal during growth in 0.026-0.2 microM iron. These low concentrations of iron also induced the synthesis of a green pigment, with maximal production at 0.026 microM. The pigment could be partially separated from hydroxamate siderophore activity on a column of Sephadex G-10 indicating that it probably does not function as an iron chelator.

Effect of iron deprivation on surface composition and virulence determinants of Candida albicans.

Six strains of Candida albicans were grown in defined medium which had been deferrated by ion-exchange chromatography and then supplemented with FeCl3 to give iron concentrations ranging from 0.026 microM to 0.8 microM. Growth in 0.026 microM-iron (measured as increase in biomass) was reduced by 26-59% as compared with that in excess (0.8 microM) iron. With five of the strains, adhesion to buccal epithelial cells was maximal after growth in 0.2-0.4 microM-iron, but strain GDH 2023 adhered best when grown in 0.026 microM-iron. Differences in yeast cell-wall composition were revealed by Zymolyase treatment of whole cells and by 125I-labelling of surface proteins. SDS-PAGE of iodinated proteins, followed by autoradiography, showed quantitative but no qualitative differences in protein profiles of iron-deficient and iron-replete organisms. The ability of all strains to form germ tubes in serum was near-maximal after growth in 0.2-0.4 microM-iron but was inhibited by up to 93% following growth in lower concentrations. These results indicate that expression of important virulence attributes by C. albicans is highly dependent on available iron and that expression in vivo may therefore be significantly different from that observed under conventional laboratory conditions.

An in vitro method to study the adherence of bacteria to saliva-treated tooth enamel sections.

An in vitro bacterial adherence assay which employed human tooth enamel sections precoated with saliva and an epifluorescent staining technique with acridine orange was developed. The assay was used to study the adherence properties of fresh and type strains of the following oral bacterial species: Bacteroides gingivalis, Bacteroides intermedius, Capnocytophaga species, Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Peptostreptococcus species, Veillonella species, Actinomyces israelii, Streptococcus salivarius and Streptococcus sanguis. Approximately half of the bacteria tested adhered well to enamel, including the fresh isolates of B. gingivalis, B. intermedius, Veillonella species and S. sanguis. Adherence did not correlate in all cases with the known distribution of these species in vivo. Three-quarters of the fresh strains adhered better than the type strains of the same species. The assay method is an alternative to the widely used hydroxyapatite bead assay.

An in vitro method to study the adherence of oral bacteria to HeLa cells.

Exfoliated buccal epithelial cells have been widely used in microbial adherence studies, but present a number of problems due to their variable nature and to contamination with indigenous bacteria. An adherence assay was developed using HeLa cell monolayers which were washed with buffer, or treated with saliva or serum to mimic buccal or crevicular epithelial cells, respectively. A total of eighteen strains of oral bacteria tested showed a low affinity for untreated HeLa cells, but most strains adhered in high numbers to saliva treated HeLa cells. A few strains, usually present in the gingival crevice, demonstrated a high affinity for serum treated HeLa cells. Thus, salivary and crevicular fluid components appear to be specifically implicated in the selective adherence and colonization of bacteria on oral surfaces.