Maria Mataftsi

Lemonia Skoura

Dimitra

D D S Lemonia

Dimitra Sakellari

HIV-infection and periodontal diseases: an overview of the post-HAART era

Keywords: Immunology and Microbiology, Public HIV, periodontitis, HAART

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Although oral candidiasis appears to be the infection more significantly decreased after the introduction of HAART, the current literature suggests that the prevalence and course of periodontal lesions have also been The development of highly active antiretroviral therapy (HAART) especially after 1995, has significantly modified the course of HIV disease, at least in the industrialized world, into a manageable chronic disease with longer survival and improved quality of life in HIV-infected subjects.

HAART generally consists of a dual nucleoside analogue reverse transcriptase inhibitor (NRTI) "backbone" and a third or "cornerstone" drug, such as a nonnucleoside inhibitor (NNRTI) or a protease inhibitor (PI), usually a "boosted" one.The use of a NNRTI as a third drug is less potent and therefore, in most settings not a preferred option, and it is recommended that baseline resistance testing should guide the specific regimen design.

HAART increases CD4+ cell count, decreases levels of HIV RNA, and extends AIDS-free survival, at least in the shorterm. Moreover, HIV suppression with antiretroviral therapy may decrease inflammation and immune activation thought to contribute to higher rates of cardiovascular and other co-morbidities reported in HIV-infected cohorts.

Eradication of HIV infection cannot be achieved with available antiretroviral regimens. This is mainly attributed to the fact that the pool of latently infected CD4+ T-cells is established during the earliest stages of acute HIV infection and persists with a long half-life, even with prolonged suppression of plasma viremia.

It is known that HAART is associated with significant problems, including toxic side effects, development of virological resistance and great financial expense. Up to half of patients on antiretroviral therapy may experience adverse effects of the medications [START_REF] Fellay | Prevalence of adverse events associated with potent antiretroviral treatment: Swiss HIV Cohort Study[END_REF] , 2008).

While most side-effects diminish over time, some can be life-threatening, underscoring the importance of careful patient monitoring (UNAIDS, 2008).

Due to the intensity of combined antiretroviral treatment and widespread use of HAART, the incidence of many AIDS-related opportunistic infections in patients with advanced HIV infection has significantly decreased but despite dramatic declines in the incidence of opportunistic infections in many resource-rich nations, opportunistic infections remain a leading cause of hospitalization and death for persons with HIV infection.

Among the HIV-associated infections, oral lesions have been recognized

as prominent features of HIV infection since the beginning of the epidemic and continue to be important.

Purpose of the present review is to overview the features, prevalence, bacteriology and host response characteristics of periodontal infections in HIV patients, especially as modified during the HAART era.

FEATURES OF PERIODONTAL LESIONS IN HIV INFECTED PATIENTS

HIV infection in adults is linked with the expression of various types of periodontal lesions, which include specific forms of gingivitis and necrotizing periodontal diseases, as well as with possible exacerbation of preexisting periodontal disease [START_REF] Winkler | Periodontal disease associated with HIV infection[END_REF][START_REF] Robinson | The significance and management of periodontal lesions in HIV infection[END_REF][START_REF] Ec-Clearinghouse | EC-Clearinghouse on Oral Problems Related to HIV Infection and WHO Collaborating Centre on Oral Manifestations of the Immunodeficiency Virus Classification and diagnostic criteria for oral lesions in HIV infection[END_REF]. Periodontal diseases strongly associated with HIV infection are classified as Linear Gingivitis Erythema (LGE),

Necrotizing Ulcerative Gingivitis (NUG), and Necrotizing Ulcerative Periodontitis (NUP) and are included among the seven cardinal oral lesions, which have been identified and recognized internationally, as follows: oral candidiasis, oral hairy leukoplakia, Kaposi sarcoma, LGE, NUG, NUP and non-Hodgkin lymphoma [START_REF] Armitage | Development of a classification system for periodontal diseases and conditions[END_REF][START_REF] Ec-Clearinghouse | EC-Clearinghouse on Oral Problems Related to HIV Infection and WHO Collaborating Centre on Oral Manifestations of the Immunodeficiency Virus Classification and diagnostic criteria for oral lesions in HIV infection[END_REF][START_REF] Coogan | Oral lesions in infection with human immunodeficiency virus[END_REF].

The criteria for diagnosis of HIV-related oral lesions are not well defined in children. Orofacial manifestations have been categorised into three

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Together with other oral infections, HIV-associated periodontal diseases are regarded as serious complications of HIV infection and have an important diagnostic and prognostic value [START_REF] Ec-Clearinghouse | EC-Clearinghouse on Oral Problems Related to HIV Infection and WHO Collaborating Centre on Oral Manifestations of the Immunodeficiency Virus Classification and diagnostic criteria for oral lesions in HIV infection[END_REF]Glick et al, 1994a;[START_REF] Shangase | Necrotising ulcerative gingivitis/periodontitis as indicators of HIV-infection[END_REF][START_REF] Coogan | Oral lesions in infection with human immunodeficiency virus[END_REF]. They belong among the earliest clinical features of the infection and could predict progression of HIV disease to AIDS. [START_REF] Robinson | The significance and management of periodontal lesions in HIV infection[END_REF][START_REF] Coogan | Oral lesions in infection with human immunodeficiency virus[END_REF]. It should also be mentioned that for patients on antiretroviral therapy, HIV-related oral lesions in general, may suggest possible treatment failure as will be further discussed in the present review. [START_REF] Margiotta | HIV infection: oral lesions, CD4+ cell count and viral load in an Italian study population[END_REF]Ramirez-Amador et al, 2007;Gaitan-Cepeda et al, 2005;[START_REF] Eyeson | Oral manifestations of an HIV positive cohort in the era of highly active anti-retroviral therapy (HAART) in South London[END_REF][START_REF] Flint | B3)Markers of immunodeficiency and mechanisms of HAART therapy on oral lesions[END_REF]. However, HIV-associated periodontal infections are less common than οral candidiasis and oral hairy leukoplakia and thus not included as criteria in the Centers for Disease Control (CDC) classification [START_REF] Cdc | 1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults[END_REF]. HIV-associated periodontal infections have characteristic clinical appearance which has been well described [START_REF] Winkler | Periodontal disease associated with HIV infection[END_REF][START_REF] Greenspan | Oral complications in HIV infection[END_REF][START_REF] Reznik | Oral manifestations of HIV disease[END_REF][START_REF] Murray | Periodontal diseases in patients infected by human immunodeficiency virus[END_REF].

Linear Gingival Erythema (LGE) is a form of gingivitis characterized by a distinct fiery red band along the margin of the gingiva (EC-Clearinghouse, 1994). It is usually associated with anterior teeth, commonly extended to the posterior teeth, accompanied in some cases by bleeding and discomfort [START_REF] Reznik | Oral manifestations of HIV disease[END_REF]. In other cases it presents as petechia-like patches on attached or free gingiva. Currently, Candida species have been implicated to the aetiopathology of LGE as well as other HIV-associated periodontal pathology. [START_REF] Murray | Periodontal diseases in patients infected by human immunodeficiency virus[END_REF][START_REF] Greenspan | Oral complications in HIV infection[END_REF][START_REF] Reznik | Oral manifestations of HIV disease[END_REF].

The rapid establishment and course of necrotizing forms of periodontal disease in patients with HIV/AIDS infection, contrary to the gradually progressing periodontal disease in adults in the general population has been outlined in many studies and had not been reported before AIDS epidemic. [START_REF] Murray | The microbiology of HIVassociated periodontal lesions[END_REF][START_REF] Murray | Periodontal diseases in patients infected by human immunodeficiency virus[END_REF][START_REF] Barr | Periodontal changes by HIV serostatus in a cohort of homosexual and bisexual men[END_REF]Yeung et al, 1993a) HAART appears to have profoundly influenced the prevalence, severity and course of periodontal lesions as will be further discussed in the next section of the present review [START_REF] Parveen | Effects of highly active antiretroviral therapy on HIV-1associated oral complications[END_REF].

Risk factors for periodontal disease in HIV-infected individuals besides the general factors of age, smoking, preexisting gingivitis, poor oral hygiene and poor diet, include counts of CD4+ cells (Glick et al, 1994b), viral load and specific species of microbiota.

Oral opportunistic infections, mainly oral candidiasis (OC) and oral hairy leukoplakia, (OHL) have been associated with CD4+ count in both the pre-HAART and the HAART era in several studies. Based on these findings, low CD4+ counts are now considered as the main risk factor associated with the development of oral lesions and especially of oral candidiasis [START_REF] Margiotta | HIV infection: oral lesions, CD4+ cell count and viral load in an Italian study population[END_REF].

Regarding periodontal disease, there is little and unclear data, especially during the HAART era. In 1994, Glick and coworkers have reported an association between NUP and CD4+ count below 200 cells/mm 3 in HIVinfected patients and suggested that NUP may be a good marker of immune deterioration. The same authors reported in another 1994 study a positive predictive value (95.1%) for periodontal diseases, which was higher than the values reported for oral hairy leukoplakia (70.1%) and oral candidiasis (69.9%) (Glick et al, 1994a). High positive predictive values have also been reported for necrotizing ulcerative periodontitis (80%) and a moderate (54.5%) one for LGE. [START_REF] Begg | Oral lesions as markers of severe immunosuppression in HIVinfected homosexual men and injection drug users[END_REF][START_REF] Patton | Changing prevalence of oral manifestations of human immuno-deficiency virus in the era of protease inhibitor therapy[END_REF]. In agreement with the previous studies, Margiota et al (1999) reported that NUP and NUG were significantly associated with CD4+ [START_REF] Schuman | Oral lesions among women living with or at risk for HIV infection. HIV Epidemiology Research Study (HERS) Group[END_REF].

Contradictory results have also been reported in a 2000 study by Patton.

The author reported that the viral load was significantly related to the presence of strongly HIV-associated oral lesions [START_REF] Patton | Changing prevalence of oral manifestations of human immuno-deficiency virus in the era of protease inhibitor therapy[END_REF] but that among periodontal lesions, only LGE has a significant predictive value (70%) for immune suppression when measured by CD4 cell counts below 200 cells/mm 3 . In the same study, the predictive value for necrotizing ulcerative diseases was lower (47.4%) compared to the values reported previously, a finding which could be attributed to the improved antiretroviral management of HIV disease of the population under investigation. A significant correlation between necrotizing ulcerative diseases and CD4+ T cells number below 200/mm 3 was also reported in a study from South Africa, with a positive predictive value of 69.6% for HIV infection in otherwise asymptomatic subjects [START_REF] Shangase | Necrotising ulcerative gingivitis/periodontitis as indicators of HIV-infection[END_REF].

As HIV infection gradually becomes a chronic disease, the features and course of chronic periodontal disease in HIV infected patients require more extensive investigation. The "conventional" periodontal diseases in the HAART era have been mentioned in very few studies. [START_REF] Kroidl | Prevalence of oral lesions and periodontal diseases in HIV-infected patients on antiretroviral therapy[END_REF][START_REF] Alpagot | Risk factors for periodontitis in HIV patients[END_REF]. Conventional periodontitis progresses gradually, causing no or minimal pain or discomfort, being thus undiagnosed, until considerable tissue loss occurs. [START_REF] Alpagot | Risk factors for periodontitis in HIV patients[END_REF]. Generally, periodontal inflammation seems to be more severe in cases where CD4+ counts are low [START_REF] Kroidl | Prevalence of oral lesions and periodontal diseases in HIV-infected patients on antiretroviral therapy[END_REF] and research nowadays is focused on the accelerated rate with which chronic adult periodontitis presents in seropositive patients. [START_REF] Lamster | Epidemiology and diagnosis of HIV-associated periodontal diseases[END_REF] Overall, findings from the above mentioned studies suggest the value of the identification of periodontal disease, even in patients on HAART therapy, in screening the immune suppression, both in diagnosed and undiagnosed HIV infection in adults.

The relation between oral lesions in general and immune and virologic the prevalence of periodontal disease such as age, immune system competence, smoking habits, oral hygiene level, are not always taken into consideration [START_REF] Barr | Periodontal changes by HIV serostatus in a cohort of homosexual and bisexual men[END_REF][START_REF] Alpagot | Risk factors for periodontitis in HIV patients[END_REF]. The type of lesion is often not mentioned, while there is some confusion with the terminology.

Additionally, it is usually unclear whether diagnosis is made by trained examiners or if universally accepted criteria are used. [START_REF] Ec-Clearinghouse | EC-Clearinghouse on Oral Problems Related to HIV Infection and WHO Collaborating Centre on Oral Manifestations of the Immunodeficiency Virus Classification and diagnostic criteria for oral lesions in HIV infection[END_REF].

Introduction of antiretroviral therapies and mainly the HAART in 1995 has changed the epidemiology of opportunistic infections in HIV-infected patients [START_REF] Holtzer | Decline in the rate of specific opportunistic infections at San Francisco General Hospital, 1994-1997[END_REF][START_REF] Paul | Impact of antiretroviral therapy on decreasing hospitalization rates of HIV-infected patients in 2001[END_REF], and has decreased the mortality and morbidity of HIV infection (Pallela et al, 1998). A significant decrease of the overall prevalence of oral lesions from 47-85%, before the introduction of HAART, to 32-46%, post-HAART has been reported [START_REF] Schmidt-Westhausen | Decline in the rate of oral opportunistic infections following introduction of highly active antiretroviral therapy[END_REF][START_REF] Patton | Changing prevalence of oral manifestations of human immuno-deficiency virus in the era of protease inhibitor therapy[END_REF][START_REF] Gaitan Cepeda | Oral lesions and immune reconstitution syndrome in HIV+/AIDS patients receiving highly active antiretroviral therapy. Epidemiological evidence[END_REF]. Oral manifestations significantly decreased in patients on dual and triple therapy in comparison with patients on monotherapy and those on no antiretroviral therapy [START_REF] Tappuni | The effect of antiretroviral therapy on the prevalence of oral manifestations in HIV-infected patients: a UK study[END_REF]. Moreover, a lower prevalence (32%) of oral lesions was found in patients on HAART, including efavirenz, compared to patients on HAART including a PI (63%).

( [START_REF] Aquino-García | Short communication: oral lesions in HIV/AIDS patients undergoing HAART including efavirenz[END_REF]. Recently, in a retrospective epidemiological analysis performed in Brazil from 1988 to 2004, HAART was found to be associated with significantly lower prevalence of oral manifestations [START_REF] Ferreira | Prevalence of oral manifestations of HIV infection in Rio De Janeiro, Brazil from 1988 to 2004[END_REF] 2009). Possibly, pathogens such as P. gingivalis, that are commonly associated with periodontal disease, do not consist the principle pathogenic factor, while both atypical oral organisms and typical periodontopathogenic bacteria influenced the pathogenesis of periodontitis in HIV-infected patients.

More over the recognition of different microbial profiles in the subgingival area of these patients may be significant. Μore complex microbial profiles were demonstrated in diseased sites than in healthy periodontium in HIVinfected patients [START_REF] Paster | Bacterial diversity in necrotizing ulcerative periodontitis in HIVpositive subjects[END_REF], while certain combinations of microbes were detected exclusively in HIV-infected individuals. These specific "complexes" may be responsible for chronic periodontitis in this group of patients [START_REF] Patel | Periodontal pathogens in subgingival plaque of HIV-positive subjects with chronic periodontitis[END_REF] since it is known that changes in the humoral and cellular immunity can affect the establishment and growth of pathogens and the resultant combination of microbes in the subgingival pockets of HIV-positive subjects.

HIV -HOST INTERACTION IN THE PERIODONTAL ENVIRONMENT

Periodontal disease may result from a loss of regulation of immune responses to oral microbiota [START_REF] Jotwani | Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies[END_REF].

However, in HIV-infected patients, pathogenetic mechanisms involved in immune responses and in tolerance at the oral mucosa in health and [START_REF] Lamster | Host mediators in gingival crevicular fluid: implications for the pathogenesis of periodontal disease[END_REF][START_REF] Van Dyke | Resolution of inflammation: a new paradigm for the pathogenesis of periodontal diseases[END_REF]. In HIV-infected patients with periodontitis an increase of inflammatory mediators has also been detected. [START_REF] Alpagot | Longitudinal evaluation of GCF IFNgamma levels and periodontal status in HIV+ patients[END_REF] reported that the higher GCF levels of pro-inflammatory cytokine interferon-γ (IFN-γ) is associated with the periodontal disease progression in HIV-positive patients similarly to reports for non-HIV individuals with chronic periodonditis [START_REF] Dutzan | Levels of interferon-gamma and transcription factor T-bet in progressive periodontal lesions in patients with chronic periodontitis[END_REF]. High levels of significant mediators of inflammation involved in the pathogenesis of periodontal disease such as prostaglandin E 2 (PGE 2 ), [START_REF] Leibur | Prostaglandin E2 levels in blood plasma and in crevicular fluid of advanced periodontitis patients before and after surgical therapy[END_REF], transforming growth factor-beta (TGF-b1), matrix metalloproteinase -1 (MMP-1) were also found in gingival crevicular fluid (GCF) of periodontitis sites in HIV/AIDS patients and could serve as prognostic factors for the progression of tissue destruction in HIV-infected adults.

After the introduction of HAART, HIV-infection is considered as a chronic infection characterized by persistency of the virus in the infected host, and, despite the undetectable plasma levels of the HIV, cessation of therapy results in viral reappearance in circulation. [START_REF] Chun | Re-emergence of HIV after stopping therapy[END_REF].

Persistency of the virus is possibly due to a very low level of replication and continuous secretion of virus by long-lived infected cells, undetectable by conventional assays, or HIV latency and silencing (reviewed in [START_REF] Mok | Waking up the sleepers: HIV latency and reactivation[END_REF][START_REF] Williams | Regulation of HIV-1 latency by T-cell activation[END_REF][START_REF] Dahl | HIV reservoirs, latency, and reactivation: Prospects for eradication[END_REF][START_REF] Colin | Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies[END_REF].The oral cavity seems to be an important reservoir of HIV-1 as the virus is found in saliva, GCF, and oral epithelial cells. To date, HIV-1 [START_REF] Schrager | Cellular and anatomical reservoirs of HIV-1 in patients receiving potent antiretroviral combination therapy[END_REF] Therefore, the role of oral immune factors and periodontal disease in the persistency of HIV infection, the possibility of oral transmission and the re-emerging of HIV infection, should be investigated.

The oral cavity has rarely been reported as a site of HIV transmission. [START_REF] Klein | Low occupational risk of human immunodeficiency virus infection among dental professionals[END_REF][START_REF] Cohen | Limits on oral transmission of HIV-1[END_REF][START_REF] Jotwani | Increase in HIV receptors/coreceptors/alpha-defensins in inflamed human gingiva[END_REF][START_REF] Cutler | Oral mucosal expression of HIV-1 receptors, co-receptors, and alpha-defensins: tableau of resistance or susceptibility to HIV infection?[END_REF]. In saliva, HIV is present at very low levels [START_REF] Spear | Relationship of HIV RNA and cytokines in saliva from HIV-infected individuals[END_REF] possibly due to low levels of macrophages and lymphocytes and to inhibitory factors in the saliva of HIV-infected patients. A number of host defense factors are present in the saliva including, the hypotonic nature of saliva [START_REF] Baron | Why is HIV rarely transmitted by oral secretions? Saliva can disrupt orally shed, infected leukocytes[END_REF], endogenous inhibitors of HIV, particularly secretory leukocyte protease inhibitor (SLPI) that blocks HIV infection in several cell-culture systems [START_REF] Shugars | Endogenous salivary inhibitors of human immunodeficiency virus[END_REF], salivary mucins MUC5B and MUC7 which trap and aggregate the virus and can inhibit it by 100% [START_REF] Habte | The role of crude human saliva and purified salivary MUC5B and MUC7 mucins in the inhibition of Human Immunodeficiency Virus type 1 in an inhibition assay[END_REF], sIgA antibodies which neutralize HIV, antimicrobial peptides such as a-and b-defensins [START_REF] Nakashima | Defensins inhibit HIV replication in vitro[END_REF][START_REF] Jotwani | Increase in HIV receptors/coreceptors/alpha-defensins in inflamed human gingiva[END_REF][START_REF] Zhang | Contribution of human alpha[END_REF][START_REF] Mackewicz | alpha-Defensins can have anti-HIV activity but are not CD8 cell anti-HIV factors[END_REF]Quinones-Mateu et al, 2003), histatins [START_REF] Groot | Histatin 5-derived peptide with improved fungicidal properties enhances human immunodeficiency virus type 1 replication by promoting viral entry[END_REF] and lactoferrin. It seems that the inhibitory factors may act synergistically [START_REF] Bolscher | Inhibition of HIV-1 IIIB and clinical isolates by human parotid, submandibular, sublingual and palatine saliva[END_REF].

Recently, HIV-specific antibody dependent cell-mediated cytotoxicity (ADCC) activity, an important part of cell mediated immunity, was demonstrated in saliva. [START_REF] Kim | Saliva can mediate HIV-1-specific antibody-dependent cellmediated cytotoxicity[END_REF]. More over, studying the possible effect of microbial components on HIV, inhibition of virus entry by a binding domain (HGP44) of P. gingivalis was demonstrated [START_REF] Xie | Inhibition of human immunodeficiency virus type 1 entry by a binding domain of Porphyromonas gingivalis gingipain[END_REF]).

HAART appears not to adversely affect inherent salivary oral host defense in HIV-patients with mild to moderate immune dysfunction. [START_REF] Lin | Salivary gland function in HIV-infected patients treated with highly active antiretroviral therapy (HAART)[END_REF] In many studies RNA [START_REF] Spear | Relationship of HIV RNA and cytokines in saliva from HIV-infected individuals[END_REF][START_REF] Shugars | Hyper-excretion of human immunodeficiency virus type 1 RNA in saliva[END_REF] and DNA of HIV have been detected in saliva [START_REF] Goto | Detection of proviral sequences in saliva of patients infected with human immunodeficiency virus type 1[END_REF]Yeung et al, 1993 b;[START_REF] Levy | HIV in saliva[END_REF]. Possible sources of infectious virions and proviral HIV-1 DNA in saliva include serum and HIV-containing macrophages and lymphocytes from GCF, which is increased during periodontal infection. In most studies, HIV is present in patients' saliva at that 5 out of 67 HIV-positive subjects expressed higher levels in saliva than blood and also had more advanced HIV-associated periodontal disease, suggesting that HIV can be produced locally in the oral cavity and may be influenced by oral tissue inflammation.

Although, relatively little and contradictory information on HIV excretion patterns in GCF is available in the literature, however the presence of periodontitis may be a contributing factor. Proviral HIV-1 DNA, viral RNA and p24 antigen has been detected in up to 50% of GCF samples from HIV-infected subjects with periodontitis [START_REF] Sanz | Detection of HIV virus in saliva and gingival crevicular fluid. Its correlation with periodontal statusand AIDS symptomatology (abstract)[END_REF]Chebbi et al,1997;[START_REF] Maticic | Proviral HIV-1 DNA in gingival crevicular fluid of HIV-1-infected patients in various stages of HIV disease[END_REF] while in some reports the virus or the p24

antigen have not been detected in GCF samples [START_REF] O'shea | HIV excretion patterns and specific antibody responses in body fluids[END_REF][START_REF] Chebbi | Search for infectious HIV in gingival crevicular fluid and saliva of advanced AIDS patients with severe periodontitis[END_REF]. These results suggest that infected mononuclear cells present in GCF could be a potential source of HIV-1.

More over it has been demonstrated that HIV-1 infects and replicates in vitro in keratinocytes isolated from normal oral mucosa [START_REF] Moore | Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro[END_REF] However, recent studies showed that, during health, in lamina propria cells usually express the DC-SIGN receptors and mannose receptors, but very few of the cells present the CCR5 on their surface and none present the CXCR4 HIV co-receptors. [START_REF] Jotwani | Increase in HIV receptors/coreceptors/alpha-defensins in inflamed human gingiva[END_REF]). In the epithelium, cells do not express CD4 but instead glycospingolipid-galactosylceramide (GalCer) and Langerin receptors (Jotvani et al, 2004;[START_REF] Challacombe | The effects of HIV infection on oral mucosal immunity[END_REF]. HIV co-receptors CCR5 and/or CXCR4 were found closer to the basal layer far from the surface-associated layers [START_REF] Jotwani | Increase in HIV receptors/coreceptors/alpha-defensins in inflamed human gingiva[END_REF]. So, in health, low expression of CCR5, and restricted expression of CXCR4 in oral mucosa suggest an unfavourable environment for the virus and this may play a significant role in the resistance of gingiva to infection with HIV-1. [START_REF] Jotwani | Increase in HIV receptors/coreceptors/alpha-defensins in inflamed human gingiva[END_REF][START_REF] Jameson | Expression of DC-SIGN by dendritic cells of intestinal and genital mucosae in humans and rhesus macaques[END_REF].

In the presence of inflammation, there is evidence of up-regulation of various receptors, including HIV receptors, on the surface of oral epithelium, and the epithelium may become more permeable. [START_REF] Challacombe | The effects of HIV infection on oral mucosal immunity[END_REF]. More over, in patients with chronic periodontitis there is a significant increase in the number of dermal dendritic cells (DDCs) expressing DC-SIGN receptors and a trend for increased mannose receptors identified in the inflamed gingival lamina propria (Jotvani et al, 2004). It is suggested that HIV uses both the above C-type lectin receptors to attach to different dentritic cells subsets [START_REF] Turville | Bitter-sweet symphony: defining the role of dendritic cell gp120 receptors in HIV infection[END_REF]. It has been shown that dentritic cells, DDCs and LCs, form immune conjugates with CD4+ T cells in the lamina propria (Jotvani & Cutler, 2003) and under these conditions it is possible for dendritic cells to transfer HIV in the T-lymphocytes in the inflamed gingival lamina propria.

It has also been reported that in the presence of oral lesions and periodontal disease there is a continuous shedding of HIV-infected blood into the oral cavity from mucosal and gingival lesions in HIV-infected patients, resulting in the detectable presence of the HIV at a high frequency in the oral cavity, with an increased possibility for HIV transmission. [START_REF] Bolscher | Inhibition of HIV-1 IIIB and clinical isolates by human parotid, submandibular, sublingual and palatine saliva[END_REF].

According to the above mentioned findings, inflammation is considered as a risk factor for HIV infection although defensive mechanisms However, during chronic periodontitis there is a ten fold increase in α-defensin-1 [START_REF] Jotwani | Increase in HIV receptors/coreceptors/alpha-defensins in inflamed human gingiva[END_REF], known to have potent anti-HIV activity, while HBD2 and HBD3 are also up-regulated during inflammation. [START_REF] Dale | Periodontal epithelium: a newly recognized role in health and disease[END_REF][START_REF] Quiñones-Mateu | Human epithelial beta-defensins 2 and 3 inhibit HIV-1 replication[END_REF].

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Notably, co-infection with the endogenous pathogen P.gingivalis in vitro, revealed an upregulation of CCR5 receptors of oral keratinocytes, which are not usually expressed in health, through LPS stimulating the Toll-like receptors and gingipains. The R5-type HIV-1 co-receptors CCR5, is the target of R5-type HIV-1 associated with most primary systemic infections.

Thus infection with P. gingivalis could increase transmission of HIV infection through the oral cavity. [START_REF] Giacaman | Porphyromonas gingivalis selectively up-regulates the HIV-1 coreceptor CCR5 in oral keratinocytes[END_REF].

In addition, periodontal diseases and other oral opportunistic infections in HIV-infected patients could influence HIV reactivation. They represent chronic infections and associated inflammation, with a possibility of latently infected host cell stimulation. Transcription of the HIV-provirus is dependent on the interaction between cellular and viral transcription factors (reviewed in [START_REF] Mok | Waking up the sleepers: HIV latency and reactivation[END_REF][START_REF] Williams | Regulation of HIV-1 latency by T-cell activation[END_REF].The mechanisms involved in the reactivation of latency remain to be elucidated, however a number of factors such as different cellular environments and LTR (Long Terminal Repeat) variations in different HIV-1 isolates have been proposed that may play a role [START_REF] Rohr | Regulation of HIV-1 gene transcription: from lymphocytes to microglial cells[END_REF].

In vitro exposure of latently infected resting CD4+ cells to a number of conditions paradoxically worsen with a substantial increase in inflammation during the initial months of host immune reconstitution, as a result of HAART [START_REF] Feller | Herpes zoster infection as an immune reconstitution inflammatory syndrome in HIV-seropositive subjects: a review[END_REF][START_REF] Murdoch | Immune reconstitution inflammatory syndrome (IRIS): review of common infectious manifestations and treatment options[END_REF].

Opportunistic oral infections have not yet been characterized as IRIS, but

Nicolatou-Galitis et al (2004) and [START_REF] Greenspan | Incidence of oral lesions in HIV-1infected women: reduction with HAART[END_REF]

  a global health problem of unprecedented dimensions, although the development of highly active antiretroviral therapy (HAART) has significantly modified the course of HIV disease into a manageable chronic disease with longer survival and improved quality of life in HIV-infected subjects. Among the HIV-associated infections, oral lesions have been recognized as prominent features since the beginning of the epidemic and continue to be important. Periodontal diseases strongly associated with HIV infection are classified as Linear Gingival Erythema (LGE), Necrotizing Ulcerative Gingivitis (NUG), and Necrotizing Ulcerative Periodontitis (NUP), and are included among the cardinal oral lesions.

  modified. Higher prevalence of opportunistic microorganisms has been frequently detected in the subgingival flora of HIV-infected individuals, probably due to the immune status of those patients, as colonization research is required regarding biological issues such as the role of oral immune factors and periodontal disease in the persistency of HIV infection, the possibility of oral transmission and the re-emerging of HIV infection. INTRODUCTION HIV infection remains a global health problem of unprecedented dimensions. Unknown 27 years ago, HIV has already caused an estimated 25 million deaths worldwide and has generated profound demographic changes in the most heavily affected countries. While the percentage of people living with HIV has stabilized since 2000, the overall number of people living with HIV has steadily increased, as new infections occur each year, HIV treatments extend life, and in addition, new infections still outnumber AIDS deaths.

  status is still not well established in children. No association was found between the prevalence of oral lesions and immunological status or viral load in children, while there are no data for periodontal diseases(Gaitan- Cepeda et al, 2002). periodontal diseases in HIV-infected individuals remains a controversial issue. Data from relevant studies vary widely due to several factors. Many studies refer to HIV-infected individuals, without mentioning the stage of AIDS or the use and the type of antiretroviral therapy, the use of protease inhibitors or not, as well as the use of adjuctive antimicrobials (antibiotics, antifungals). Factors which influence

  as well as in vivo in oral mucosal epithelial cells[START_REF] Rodríguez-Iñigo | Detection of human immunodeficiency virus type 1 RNA by in situ hybridization in oral mucosa epithelial cells from anti-HIV-1 positive patients[END_REF], which could represent a reservoir for the virus, although this is not a universal finding(Quinones-Mateu et al, 2003).Regarding gingival tissues, studies have shown that dendritic cells (DCs) and macrophages in gingiva express C-type lectin receptors DC-SIGN (Dendritic-cell-specific ICAM-3-grabbing non-integrins, CD209), MR mannose receptors, CD206), and Langerin (CD 207), which are targets for HIV and other microbes[START_REF] Van Kooyk | Pathogens use carbohydrates to escape immunity induced by dendritic cells[END_REF]. Using these receptors HIV could advance by down-regulating intracellular signalling and effective immune response and cause chronic infections that persist for life.

  cytokines, bacterial antigens, mitogens or monoclonal antibodies directed to T-cell receptors CD3 can induce viral replication, but these findings have not been reproduced in vivo. It is also suggested that in the progress of an opportunistic infection microorganisms or their components, such as LPS, stimulate and activate Toll-like receptors (TLRs), and subsequently NFκB and other transcription factors. In addition, transcription factors can be activated indirectly by the large amounts of pro-inflammatory cytokines and chemokines which are produced during infection (reviewed in[START_REF] González | Oral infectious diseases: a potential risk factor for HIV virus recrudescence?[END_REF]. Regarding periodontal pathogens, it has recently been shown that P. gingivalis produces high concentrations of butyric acids causing histone acetylation which is involved in repressing HIV transcription and results in virus persistency[START_REF] Imai | Reactivation of latent HIV-1 infection by the periodontopathic bacterium Porphyromonas gingivalis involves histone modification[END_REF] The results of the study and the above mentioned possible mechanism of reactivation of HIV, suggest that periodontal disease could act as a risk factor for HIV reactivation in infected individuals and might contribute to the systemic dissemination of the virus. This hypothesis could be the biological basis linking a chronic infection such as periodontitis to the "immune reconstitution inflammatory syndrome" (IRIS) (Gaitan-Cepeda et al, 2008) a situation in which, preexisting asymptomatic or mildly symptomatic infections or inflammatory

Page 6 of 34 Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy

	that LGE and NUG were not related to HIV serostatus or CD4+ lymphocyte
	count
	O r a
	l
	D i
	s e
	a s
	e
	s
	-
	M a
	n u s
	c r
	i p
	t
	C
	counts lower than 200 cells/mm 3 in a cohort of Italian subjects infected o p y
	with HIV. In contrast to these reports, Schuman et al, in a study
	conducted in a US population, after the introduction of HAART, reported
	7

  As shown in Table3, bacteria that are not usually linked with periodontal disease, such as Enterococcus faecalis, Acinetobacter baumanii, Pseudomonas aeruginosa, Campylobacter pylori, were frequently detected in HIV-infected patients, in most of the studies[START_REF] Lde | Association of T CD4 lymphocyte levels and subgingival microbiota of chronic periodontitis in HIV-infected Brazilians under HAART[END_REF][START_REF] Lde | Clinical and microbiological profiles of human immunodeficiency virus (HIV)-seropositive Brazilians undergoing highly active antiretroviral therapy and HIV-seronegative Brazilians with chronic periodontitis[END_REF][START_REF] Aas | Subgingival plaque microbiota in HIV positive patients[END_REF][START_REF] De Souza | Detection of Helicobacter pylori, Enterococcus faecalis, and Pseudomonas aeruginosa in the subgingival biofilm of HIV-infected subjects undergoing HAART with chronic periodontitis[END_REF]. Putative periodontopathogenic bacteria, such as T. forsythia, P. gingivalis, P. intermedia, were associated with periodontitis[START_REF] Alpagot | Risk factors for periodontitis in HIV patients[END_REF] Goncalves et al, 2004) in HIV-positive patients and were considered as risk factors[START_REF] Lde | Association of T CD4 lymphocyte levels and subgingival microbiota of chronic periodontitis in HIV-infected Brazilians under HAART[END_REF], whereas in many studies the prevalence of these classical periodontopathogenic bacteria was found smaller in HIV-positive than in HIV-negative subjects[START_REF] Paster | Bacterial diversity in necrotizing ulcerative periodontitis in HIVpositive subjects[END_REF][START_REF] Patel | Periodontal pathogens in subgingival plaque of HIV-positive subjects with chronic periodontitis[END_REF][START_REF] Lde | Clinical and microbiological profiles of human immunodeficiency virus (HIV)-seropositive Brazilians undergoing highly active antiretroviral therapy and HIV-seronegative Brazilians with chronic periodontitis[END_REF][START_REF] Aas | Subgingival plaque microbiota in HIV positive patients[END_REF] Gonçalves et al,

	Page 9 of 34	Oral Diseases -Manuscript Copy
		Regarding the prevalence of HIV-associated periodontal diseases in the as Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum,
		pre-HAART era, data vary widely both in developed and developing Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and
	O r a countries. Indicatively, reported rates of prevalence for LGE range between 9 and 50%, for NUG between 11 and 25% and for NUP between 1 and 18% (Laskaris et al, 1992; Masouredis et al, 1992; Tukutuku et al, 1990; Glick et al, 1994b). O r a Treponema denticola, in HIV-positive patients, in comparison to HIV-negative patients, (Murray et al, 1989; Cross & Smith, 1995; Scully et al, 1999; Alpagot et al, 2004), while there are studies that present the exact opposite, i.e. that putative pathogens are less prevalent in HIV-O r a
		l After the introduction of HAART, findings from relevant studies also vary l positive patients. (Tenebaum et al, 1997; Paster et al, 2002; Patel et al, l
		D i s e and cannot be compared, partly because of the different types of therapy received by participating patients. Data from representative studies in developed and developing countries concerning adult and pediatric populations are shown in Table1. The effect of HAART on prevalence of D i s e 2003; Gonçalves et al, 2007; Botero et al, 2007) D i Several authors agree that certain microbial species such as Candida spp s e
		a s a s a s
		e e e
		s s s
		---
		M a M a M a
		n u s n u s n u s
		c r c r c r
		i p i p i p
		t t t
		C o p y C Data from studies in the HAART era, which apply culture-independent molecular techniques are displayed in Table 3. These techniques as well as C Results from studies on the subgingival microbiota in HIV-infected individuals are quite diverse. Some studies have shown that the microbiota is similar in HIV-positive and HIV-negative patients with o p y other approaches such as proteomics and the study of biofilms will allow an extensive investigation of the microbiota in HIV-infected individuals and the pathogenetic role of "unusual" species. o p y
		Oral Diseases -Manuscript Copy	9 10 11

. Among oral manifestations, oral candidiasis appears to be the lesion most significantly decreased after the introduction of HAART as shown by several studies. HIV-associated periodontal disease is shown in Table

2

. It appears that HAART is associated with a lower prevalence of HIV-associated periodontal disease in adults. The difference between pre-and post-HAART in most of the studies was found to be statistically significant.

On the contrary, HAART does not appear to significantly affect the prevalence of periodontal disease in children

[START_REF] Flanagan | Prevalence of oral soft tissue lesions in HIV-infected minority children treated with highly active antiretroviral therapies[END_REF][START_REF] Khongkunthian | Oral manifestations in 45 HIV-positive children from Northern Thailand[END_REF][START_REF] Parveen | Effects of highly active antiretroviral therapy on HIV-1associated oral complications[END_REF]

BACTERIA ASSOCIATED WITH PERIODONTAL DISEASE IN HIV -INFECTED

PATIENTS

The development of periodontal disease is generally accepted to depend on the interaction between the host response and the resident oral microbiota, which constitutes a complex dynamic biofilm of multiple microbial communities. Considering that it is a microbial community disease, a distinct microbial profile in these patients, if identified, could assist our understanding of the aetiopathological mechanisms (Kuboniva et al, 2009). periodontitis.

[START_REF] Zambon | Studies of the subgingival microflora in patients with acquired immunodeficiency syndrome[END_REF][START_REF] Brady | Oral diseases, mycology and periodontal microbiology of HIV-1infected women[END_REF][START_REF] Nakou | Periodontal microflora of HIV infected patients with periodontitis[END_REF][START_REF] Tsang | Predominant cultivable subgingival microbiota of healthy and HIV-infected ethnic Chinese[END_REF][START_REF] Teanpaisan | Isolation and genotyping of black-pigmented anaerobes from periodontal sites of HIVpositive and non-infected subjects in Thailand[END_REF]

. Other studies have shown a higher prevalence of putative periodontal pathogens such (Jabra-Rizk et al, 2001), Enterobacter faecalis

[START_REF] Lde | Association of T CD4 lymphocyte levels and subgingival microbiota of chronic periodontitis in HIV-infected Brazilians under HAART[END_REF][START_REF] Zambon | Studies of the subgingival microflora in patients with acquired immunodeficiency syndrome[END_REF][START_REF] Nakou | Periodontal microflora of HIV infected patients with periodontitis[END_REF][START_REF] Lde | Clinical and microbiological profiles of human immunodeficiency virus (HIV)-seropositive Brazilians undergoing highly active antiretroviral therapy and HIV-seronegative Brazilians with chronic periodontitis[END_REF]

, Clostridium clostridiiforme

[START_REF] Zambon | Studies of the subgingival microflora in patients with acquired immunodeficiency syndrome[END_REF]

Clostridium difficile

[START_REF] Zambon | Studies of the subgingival microflora in patients with acquired immunodeficiency syndrome[END_REF][START_REF] Nakou | Periodontal microflora of HIV infected patients with periodontitis[END_REF][START_REF] Lde | Clinical and microbiological profiles of human immunodeficiency virus (HIV)-seropositive Brazilians undergoing highly active antiretroviral therapy and HIV-seronegative Brazilians with chronic periodontitis[END_REF]

, Klebsiella pneumoniae

[START_REF] Zambon | Studies of the subgingival microflora in patients with acquired immunodeficiency syndrome[END_REF][START_REF] Nakou | Periodontal microflora of HIV infected patients with periodontitis[END_REF][START_REF] Lde | Clinical and microbiological profiles of human immunodeficiency virus (HIV)-seropositive Brazilians undergoing highly active antiretroviral therapy and HIV-seronegative Brazilians with chronic periodontitis[END_REF][START_REF] Botero | Occurrence of periodontopathic and superinfecting bacteria in chronic and aggressive periodontitis subjects in a Colombian population[END_REF]

, Mycoplasma salivarium

[START_REF] Moore | Periodontal microflora of HIV positive subjects with gingivitis or adult periodontitis[END_REF][START_REF] Zambon | Studies of the subgingival microflora in patients with acquired immunodeficiency syndrome[END_REF][START_REF] Nakou | Periodontal microflora of HIV infected patients with periodontitis[END_REF][START_REF] Lde | Clinical and microbiological profiles of human immunodeficiency virus (HIV)-seropositive Brazilians undergoing highly active antiretroviral therapy and HIV-seronegative Brazilians with chronic periodontitis[END_REF]

, Pseudomonas aeruginosa

[START_REF] Nakou | Periodontal microflora of HIV infected patients with periodontitis[END_REF][START_REF] Lde | Clinical and microbiological profiles of human immunodeficiency virus (HIV)-seropositive Brazilians undergoing highly active antiretroviral therapy and HIV-seronegative Brazilians with chronic periodontitis[END_REF][START_REF] Botero | Occurrence of periodontopathic and superinfecting bacteria in chronic and aggressive periodontitis subjects in a Colombian population[END_REF]

, Acinetobacter baumanii

[START_REF] Nakou | Periodontal microflora of HIV infected patients with periodontitis[END_REF][START_REF] Lde | Clinical and microbiological profiles of human immunodeficiency virus (HIV)-seropositive Brazilians undergoing highly active antiretroviral therapy and HIV-seronegative Brazilians with chronic periodontitis[END_REF]

, Enterobacter cloacae

[START_REF] Botero | Occurrence of periodontopathic and superinfecting bacteria in chronic and aggressive periodontitis subjects in a Colombian population[END_REF][START_REF] Nakou | Periodontal microflora of HIV infected patients with periodontitis[END_REF]

, which are frequently found in the periodontal environment of HIV-positive patients, are uncommon in other individuals. The role of these "uncommon" species in the pathogenesis of periodontal disease in HIV-infected individuals is not yet fully understood, while it is suggested that the higher prevalence of such opportunistic microorganisms is due to the immune status of those patients as colonization and overgrowth of atypical pathogenic species is facilitated by severe immunosuppression.

[START_REF] Lde | Association of T CD4 lymphocyte levels and subgingival microbiota of chronic periodontitis in HIV-infected Brazilians under HAART[END_REF]

.

  12inflammation remain unclear and studies are required in order to define the interaction between the immuno-compromised host and microbes. In general, it is poorly understood how HIV or HIV-infected cells affect oral mucosal epithelium and influence innate and acquired immunity, and how the altered local or systemic immune response of these patients contributes to the pathogenesis of periodontal disease(Alpagot et al,

	Page 11 of 34	Oral Diseases -Manuscript Copy
	O r a
	l 2004; Challacombe & Naglik, 2006). Subgingival biofilm microorganisms
	have polymononuclears (PMN), lymphocytes and macrophages, which produce the capacity to activate inflammatory cells including D i
	inflammatory mediators, and subsequently induce MMPs and their s e inhibitors production. It is known that, in periodontal disease, most of the
	s a tissue damage is caused by host response
		e
		s
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		C
		o p y
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Table 1

 1 Prevalence of HIV-associated and conventional periodontal disease in the HAART era

	have reported a

Page 31 of 34 Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy

  Τable 2 Effect of HAART on prevalence of HIV-associated periodontal disease in HIV-infected adults HAART=Highly active antiretroviral therapy ,HAART/ PI= Highly active antiretroviral therapy with protease inhibitor as the third drug, ART= Any type of antiretroviral therapy, HIV-accociated periodontal disease:LGE=linear gingival erythema, NUG=necrotizing ulcerative gingivitis, NUP= necrotizing ulcerative periodontitis

			Oral Diseases -Manuscript Copy	Page 32 of 34
	O r a l Authors Country Aguirre et al (1999) Spain O r a l	Subject sample 72 HIV+ patients CD4+ <499 on HAART		HIV-associated periodontal disease LGE NUG NUP 48.6% 31.9% LGE are down, Effect of therapy NUP has remained steady comparison to two earlier studies in from the Spain
	Schmidt et al, (2000) Patton et al (2000) Ceballos et al (2000) Tappuni and Fleming (2001) Ramirez et al	D i s e a s e s Germany 103 HIV+ patients one month on HAART USA Pre-HAART, 271 HIV+ 8% on HAART Post HAART, 299 HIV+ 42% on HAART). Spain 154 HIV/AIDS on HAART /PI for at least 6 months UK 195 HIV+ not on ART 89 HIV+ on ART Brazil Study of 12 years D i s e a s e s	1.9%. 0.6% Periodontal disease 2.9% 4.8% 1.7% 0.6% 6% 2%	After 6 months of therapy, from 61 re-examined patients only one had NUP. Significant decrease of NUP (p=0.03) More than 30% decrease of HIV associated periodontal disease in comparison with historical controls Decrease of NUG Significant decrease of periodontal
	(2003)		before HAART (1989-1995)	-4.1% (1989-1991) 1.7% (1992-1995) -	disease between before and after HAART periods
	Ferreira et al (2007) Nicolatou et al (2004)	Brazil Greece	after HAART (1996-2001) 1230 HIV+ (1988-2004) on HAART HIV+ not on ART HIV+ on double ART and HAART/PI	M a n u s c r 0.4% (1996-1998) 0.7% (1999-2001) (p=0.002) 2.5% 1.6% 1.3% HAART associated with a significant lower prevalence of LGE (p<0.001) 8.1% 0% M a n Decrease of NUG u s c r
					i p t i p t
					C o p C o p
					y y
			Oral Diseases -Manuscript Copy

Table 3 .

 3 Studies of subgingival plaque microbiota in HIV infected patients in HAART era using culture-independent methods

	Page 33 of 34		Oral Diseases -Manuscript Copy
	Authors	Subject	methodology	Principal findings
		sample
	Paster et al	8 HIV+/NUP	Checkerboard DNA	108 species identified (65 uncultivable)
	2002		hybridization assay	Most frequent: Bulleidia extructa, Dialister,
		HAART: data	Over 200 probes	Fusobacterium, Selenomonas, Phylum TM7
		not available		Peptostreptococcus, Veillonella,
				Classical periodontal pathogens not detected
				Different and more complex microbial profiles
				in periodontitis than in healthy periodontium
	Patel et al	20 HIV+/CP	PCR for	T.denticola and P.gingivalis less prevalent in HIV+
	2003		P nigrescens, C.rectus,	subjects
		HAART: data	P.intermedia,P gingivalis,	Three microbial profiles exclusively in HIV+
		not available	T.denticola, E.corrodens,	P. nigrescens / C.rectus
			A.actinomycetemcomitans	P. nigrescens / P. gigivalis
				P. nigrescens / T.denticola
	Alpagot et al	152 HIV+/CP	Fluorescent assay for	F.nucleatum, P. intermedia, A.
	2004		selective Gram-negative	actinomycetemcomitans , P. gingivalis:
		63% HAART	species	risk factors for periodontitis in HIV+
	Goncalves et	64 HIV+/CP	Checkerboard DNA	Several classical pathogens more prevalent in
	al 2004		hybridization assay	HIV+/CP than in HIV+/healthy periodontium:
		100% HAART	Probes for 22 species	E. faecalis, F. nucleatum more prevalent
				in patients with lower T CD4+cells
	Goncalves et	37 HIV+/CP	Checkerboard DNA	Bacterial species and classical periodontal
	al 2007	35 HIV+/HP	hybridization assay	pathogens less frequent in HIV+/CP, than in
			Probes for 33 species	HIV-/CP (T. forsythia, S. gordonii. P.gingivalis,
		100% HAART		S. intermedius,)
				Unusual for CP species more commonly in
				HIV+ (A.baumannii, E. faecalis)
	Aas et al	14 HIV+/CP,	16S and 18S rRNA-cloning	109 species (42% uncultivable) were identified
	2007	gingivitis, LGE	and sequensing	Gemella, Dialister, Streptococcus, Veilonella
				were predominant
		HAART: data		Classical periodontal pathogens not detected
		not available		(T.denticola, P.gingivalis, T. forsythia)
				Unsual for CP microbes (Pseudomonas,
				Neisseria) more commonly in HIV+ and severe
				immunosupression
	Goncalves et	13 HIV+/CP	PCR for	Unusual for CP microbes more frequent in
	al 2009	10 HIV+/HP	H. pylori, E.faecalis,	CP than in healthy periodontium (E.faecalis,
		100% HAART	P.aeruginosa	E.pylori, P.aeruginosa)
				E.pylori most prevalent in CP in HIV+
	CP= chronic periodontitis	HP=healthy periodontium
	NUP= necrotizing ulcerative periodontitis	HAART=Highly active antiretroviral therapy	LGE= linear gingival erythema
			Oral Diseases -Manuscript Copy

Page 34 of 34 Oral Diseases -Manuscript Copy Oral Diseases -Manuscript Copy

Oral Diseases -Manuscript CopyOral Diseases -Manuscript Copy

[START_REF] Patton | Changing prevalence of oral manifestations of human immuno-deficiency virus in the era of protease inhibitor therapy[END_REF]. Sensitivity, specificity, and positive predictive value of oral opportunistic infections in adults with HIV/AIDS as markers of immune suppression and viral burden. Oral Surg Oral Med Oral Pathol Oral


Dr Ian A Jenkins
email: ian.jenkins@uhbristol.nhs.uk

Edin Frca

Tolerance and Addiction; the patient, the parent or the clinician?

Keywords: Addiction, Withdrawal, Neonatal abstinence, anesthesia < Brain, Intensive Care, Tolerance Tolerance, addiction, withdrawal, neonatal abstinence, anesthesiology, intensive care

Tolerance and Addiction; the patient, the parent or the clinician?

Introduction

The drugs most commonly associated with tolerance in anesthesia and critical care are opioids and benzodiazepines. Opioids are the mainstay of intraoperative analgesia for procedures that do not lend themselves to regional neural blockade and both classes of drugs are the most prevalent agents in use for sedation and analgesia in pediatric critical care [START_REF] Jenkins | Current United Kingdom sedation practice in pediatric intensive care[END_REF]. Whereas withdrawal phenomena on cessation of these agents in critical care have been recognised for some considerable time [START_REF] Arnold | Tolerance to isoflurane during prolonged administration[END_REF][START_REF] Fonsmark | Occurrence of withdrawal in critically ill sedated children[END_REF], their role in acute tolerance within the time-frame of pediatric surgery is becoming increasingly appreciated [START_REF] Borgland | Acute opioid receptor desensitization and tolerance: is there a link?[END_REF][START_REF] Crain | Antagonists of excitatory opioid receptor functions enhance morphine's analgesic potency and attenuate opioid tolerance/dependence liability[END_REF][START_REF] Crawford | Development of acute opioid tolerance during infusion of remifentanil for pediatric scoliosis surgery[END_REF][START_REF] Guignard | Acute opioid tolerance: intraoperative remifentanil increases postoperative pain and morphine requirement[END_REF][START_REF] Mcquay | Acute opiate tolerance in man[END_REF][START_REF] Vinik | Rapid development of tolerance to analgesia during remifentanil infusion in humans[END_REF].

Equally, tolerance and withdrawal phenomena have been reported with many different agents such as isoflurane [START_REF] Arnold | Tolerance to isoflurane during prolonged administration[END_REF], propofol [START_REF] Trotter | Neurological sequelae in children after prolonged propofol infusion[END_REF][START_REF] Ypsilantis | Tolerance to propofol's sedative effect in mechanically ventilated Rabbits[END_REF], barbiturates [START_REF] Tobias | Tolerance, withdrawal, and physical dependency after long-term sedation and analgesia of children in the pediatric intensive care unit[END_REF] and more recently dexmedetomidine [START_REF] Darnell | Withdrawal from multiple sedative agent therapy in an infant: is dexmedetomidine the cause or the cure?[END_REF] and clonidine [START_REF] Greenberg | Clonidine withdrawal in a 3 month old premature male infant[END_REF].

What causes tolerance?

This is best described with opioid biology where there is a complex interplay of local intracellular mechanisms and neural pathways that interact with the transmission of pain impulses affecting the response at cellular level. Opioid receptors act in the modulation of pain sensation in sites such as cerebral cortex layers III and IV, thalamic nuclei, peri-aqueductal grey matter, corpus striatum and dorsal horn laminae I and II. These neuronal cells have bi-modal opioid receptors, capable of releasing stimulatory messenger protein G s (hyperalgesia) or inhibitory messenger proteins, G 0 or G i (analgesia).

At adequate dosage opioids bind with receptors to release inhibitory G protein transmitters that decrease intracellular cyclic AMP (cAMP) and neuronal nitric oxide synthetase so resulting in analgesia. However, opioids can also produce hyperalgesia and this depends on the effect of the molecule at the receptor and whether this may release stimulatory G s proteins [START_REF] Anand | Tolerance and withdrawal from prolonged opioid use in critically ill children[END_REF].

Tolerance develops as a result of adaptation to the presence of opioids binding to receptors and the resultant biochemical changes in the cell. Receptors have been observed to internalize into the cell wall (desensitization) or decrease in number (down-regulation). Up-regulation of intracellular adenylyl cyclase production of cAMP can occur, as can increased release of dynorphins from descending spinal pathways that produce hyperalgesia by negative feedback [START_REF] Borgland | Acute opioid receptor desensitization and tolerance: is there a link?[END_REF][START_REF] Anand | Tolerance and withdrawal from prolonged opioid use in critically ill children[END_REF]. Opioids vary in their structure and their specific actions on opioid receptors and subsequent release of intracellular transmitters. In part this explains their variable potential for producing analgesia and also tolerance.

Many cells with opioid receptors also contain N-methyl-D-aspartate (NMDA) receptors, which if activated, cause increased intracellular calcium and hyperalgesia. Opioids may activate antinocioceptive but also pronocioceptive systems, such as these NMDA receptors, and these seem to have longer duration of action [START_REF] Anand | Tolerance and withdrawal from prolonged opioid use in critically ill children[END_REF][START_REF] Celerier | Evidence for opiate-activated NMDA processes masking opiate analgesia in rats[END_REF][START_REF] Laulin | The role of ketamine in preventing fentanyl-induced hyperalgesia and subsequent acute morphine tolerance[END_REF]. Theoretically therefore, NMDA receptor antagonists such as ketamine may be expected to inhibit this pronocioceptive system, and this has been observed to be the case with production of analgesia at concentrations of ketamine below that which would produce analgesia by independent action. This has been exploited clinically as indicated below [START_REF] Laulin | The role of ketamine in preventing fentanyl-induced hyperalgesia and subsequent acute morphine tolerance[END_REF].

Acute Tolerance with Opioids

The development of acute tolerance (or tachyphylaxis) to opioids is well recognised [START_REF] Mcquay | Acute opiate tolerance in man[END_REF]. Acute tolerance to the analgesic effects of infusions of remifentanil developed within 90-120 minutes in human adult volunteers [START_REF] Vinik | Rapid development of tolerance to analgesia during remifentanil infusion in humans[END_REF]. However, sedative tachyphylaxis was not similarly noted thus raising the possibility that administration of larger doses to achieve analgesic effects may lead to increased sedation and respiratory depression. There is also evidence that increasing the dose of remifentanil leads to increasing tolerance. In adult abdominal surgery, where the remifentanil dose was kept constant and desflurane titrated against patient autonomic response, the morphine needed postoperatively was significantly less than in the comparator group where the desflurane was held at 0.5 MAC and the remifentanil titrated against patient response [START_REF] Guignard | Acute opioid tolerance: intraoperative remifentanil increases postoperative pain and morphine requirement[END_REF]. This acute tolerance has also been seen with fentanyl [START_REF] Mcquay | Acute opiate tolerance in man[END_REF][START_REF] Laulin | The role of ketamine in preventing fentanyl-induced hyperalgesia and subsequent acute morphine tolerance[END_REF].

As stated above tolerance may be expected to differ according to the specific actions of differing opioid molecules that bind to receptors. For example, methadone acts not only at the µ-opioid receptor but also the δ-opioid receptor, thus activating supplemental spinal antinocioceptive pathways, and the NMDA receptor where it inhibits pronocioceptive activity [START_REF] Davis | d-Methadone blocks morphine tolerance and N-methyl-D-aspartate-induced hyperalgesia[END_REF]. The use of NMDA receptor antagonists used as adjuncts to opioid anesthesia to avoid acute tolerance has proved effective in rats [START_REF] Laulin | The role of ketamine in preventing fentanyl-induced hyperalgesia and subsequent acute morphine tolerance[END_REF] but ineffective in children [START_REF] Engelhardt | Intraoperative low-dose ketamine does not prevent a remifentanil-induced increase in morphine requirement after pediatric scoliosis surgery[END_REF]. It this latter study it was given only per-operatively but some authors claim that pre-emptive NMDA co-administration should include the surgery and the postoperative phase to be effective [START_REF] Laulin | The role of ketamine in preventing fentanyl-induced hyperalgesia and subsequent acute morphine tolerance[END_REF][START_REF] Kissin | Preemptive analgesia[END_REF]. Further studies are needed in this regard.

A curious and apparently paradoxical finding was that administration of 'ultra-low' doses of opioid antagonists such as naloxone appeared to enhance the analgesic effects of opioids and to attenuate opioid tolerance and liability to dependence. This is thought to be due to switching off release of antalgesic G s transmitter proteins which appears to occur at lower dosages that required to switch off release of the proanalgesic transmitters G 0 and G i [START_REF] Crain | Antagonists of excitatory opioid receptor functions enhance morphine's analgesic potency and attenuate opioid tolerance/dependence liability[END_REF].

Neonatal Neural Development, Tolerance and Addiction (including 'Abstinence')

The role of the parent directly affects neonatal addiction where this concerns substances ingested by mothers during pregnancy. Additionally, neonates present a different range of considerations due to the evolving architecture of their neural architecture and physiology, particularly as fetuses and preterm infants. Much has been written recently on the apparent neurotoxic effects of opioids, benzodiazepines, ketamine and nitrous oxide on neural integrity [START_REF] Durrmeyer | Use of analgesic and sedative drugs in the NICU: integrating clinical trials and laboratory data[END_REF]. Neuronal apoptosis has been observed in animal models in response to both infusions and single doses of benzodiazepines. Neural development, such as reductions in brain volume, neuronal density and dendritic growth are affected by intrauterine exposure to morphine [START_REF] Durrmeyer | Use of analgesic and sedative drugs in the NICU: integrating clinical trials and laboratory data[END_REF]. A multitude of agents have been implicated in the appearance of postpartum withdrawal phenomena in neonates exposed to substances in utero, including chronic ingestion of opioids [START_REF] Allegaert | Analgesics during pregnancy or breast-feeding: Effects on fetus and breast-fed infant[END_REF][START_REF] Baxter | Characterization of babies discharged from Cabell Huntington Hospital during the calendar year 2005 with the diagnoses of neonatal abstinence syndrome[END_REF][START_REF] Ebner | Management of neonatal abstinence syndrome in neonates born to opioid maintained women[END_REF][START_REF] Kraft | Sublingual buprenorphine for treatment of neonatal abstinence syndrome: A randomized trial[END_REF][START_REF] Lim | High-dose methadone in pregnant women and its effect on duration of neonatal abstinence syndrome[END_REF], benzodiazepines, selective serotonin reuptake inhibitors (SSRIs) [START_REF] Levinson-Castiel | Neonatal abstinence syndrome after in utero exposure to selective serotonin reuptake inhibitors in term infants[END_REF] and volatile substance abuse [START_REF] Tenenbein | Neonatal withdrawal from maternal volatile substance abuse[END_REF]. This 'neonatal abstinence syndrome' is associated with post partum behavioural differences in babies [START_REF] Finnegan | Neonatal abstinence syndrome: assessment and management[END_REF] and, with some agents, intrauterine growth retardation. Long lasting physical stigmata are seen in the 'fetal alcohol syndrome'.

However it must not be assumed that such animal model data extrapolates to human neonates. A very recent comprehensive review called into question the applicability of animal models in this regard [START_REF] Durrmeyer | Use of analgesic and sedative drugs in the NICU: integrating clinical trials and laboratory data[END_REF].

The lack of certainty concerning the gestational stage of rodent brain development that reliably 6 matches human brain development was noted. Also, doses of drugs and plasma levels needed to produce anesthesia in rodent can be 10-12 times higher than in humans. They also found data that document apoptotic activity of ketamine and midazolam in early gestational stages of the rodent fetus. However, if these same agents were administered shortly before term, brain development paradoxically appears to be enhanced. In the clinical context, a long term multi-centre study from France that included a 5-year neurological follow up of very preterm infants showed no difference between those receiving sedation and those not [START_REF] Roze | Prolonged sedation and/or analgesia and 5-year neurodevelopment outcome in very preterm infants: results from the EPIPAGE cohort[END_REF]. Whether short-term anaesthesia is harmful to the human neonates brain is still conjectural and is addressed elsewhere in this issue.

Neonatal abstinence may be made worse by concurrent brain hypoxia which is also associated with increased NMDA pathway activation [START_REF] Bachiocco | Severe withdrawal syndrome in three newborns subjected to continuous opioid infusion and seizure activity dependent on brain hypoxia -Ischemia. A possible link[END_REF]. With such infants presenting for surgery, it is important to know to what extent abstinence may affect the anesthetic technique. Unfortunately this does not appear to have been studied and reported. It might logically be expected that if the infant is withdrawing from opioids, more than usual of this class of drug may have to be employed perioperatively. However, data from neonatalogy suggests that other classes of agents such as chlorpromazine when directly compared to morphine as treatment to withdrawing neonates may be as effective in masking opioid withdrawal and even shorten the period of treatment of abstinence symptoms [START_REF] Mazurier | Comparison of chlorpromazine versus morphine hydrochloride for treatment of neonatal abstinence syndrome[END_REF].

Other strategies have been tried with the use of partial opioid agonists in the treatment of neonatal abstinence. Buprenorphine has been used as a substitute for opium using the Finnegan withdrawal scale as monitoring. There were no adverse effects and there seemed to be a tendency to both shorter treatment and length of stay but these did not reach statistical significance [START_REF] Kraft | Sublingual buprenorphine for treatment of neonatal abstinence syndrome: A randomized trial[END_REF].

Of relevance to this perhaps, when buprenorphine was compared to methadone in opioid abusing mothers, there was more illicit use of opioids detected in the buprenorphine group, suggesting that partial agonists were less effective in reducing opioid demand in these mothers [START_REF] Fischer | Methadone versus buprenorphine in pregnant addicts: a double-blind, double-dummy comparison study[END_REF].

Tolerance associated with Intensive Care

Tolerance occurs commonly during sedation in ventilated patients. In a UK-wide study this was seen in 13% of patients [START_REF] Jenkins | Current United Kingdom sedation practice in pediatric intensive care[END_REF] and this has been mirrored in other reports [START_REF] Anand | Tolerance and withdrawal from prolonged opioid use in critically ill children[END_REF][START_REF] Arnold | Tolerance and dependence in neonates sedated with fentanyl during extracorporeal membrane oxygenation[END_REF]. The use of midazolam is particularly associated with the occurrence of withdrawal (1) and a rate of 35% in a PICU population has been reported (3). Overall, withdrawal is associated with duration of sedation and the total doses of agents used [START_REF] Jenkins | Current United Kingdom sedation practice in pediatric intensive care[END_REF][START_REF] Fonsmark | Occurrence of withdrawal in critically ill sedated children[END_REF][START_REF] Arnold | Tolerance and dependence in neonates sedated with fentanyl during extracorporeal membrane oxygenation[END_REF]. In this respect the role of clinicians, nurses and intensivists, must not be underestimated. Where sedation is more tightly controlled by using objective scoring scales [START_REF] Carnevale | An item analysis of the COMFORT scale in a pediatric intensive care unit[END_REF] it may be possible to minimise excessive use of sedation agents which has been shown to be associated with the subsequent appearance of withdrawal symptoms (1-3, 12, 15, 34). The adoption of such scoring systems has been recommended by a consensus exercise [START_REF] Playfor | Consensus guidelines on sedation and analgesia in critically ill children[END_REF]. However, there is also emerging evidence of tolerance seen within 72 hours of sedation starting and consequently can occur with relatively short stays on the ICU [START_REF] Jenkins | Current United Kingdom sedation practice in pediatric intensive care[END_REF][START_REF] Anand | Tolerance and withdrawal from prolonged opioid use in critically ill children[END_REF]. Withdrawal symptoms in PICU patients may be seen after cessation of sedation within 6 hours in a third of children and in 98% by 24 hours (1).

Assessment of withdrawal symptomatology and severity has, for some years, followed that described in neonates by Finnegan. However, recent studies in PICU have described two purpose-built and validated scoring systems for children; the Withdrawal Assessment Tool -1 (WAT-1) (37) and the Sophia Observation Withdrawal Symptoms Scale (SOS) [START_REF] Ista | Construction of the Sophia Observation withdrawal Symptomsscale (SOS) for critically ill children[END_REF]. Both these scoring systems now provide an opportunity to record withdrawal phenomena both in terms of frequency and severity in a structured and reproducible manner. This represents an important advance in the study of this common problem.

Tapering of sedation and analgesia is often used to try to minimise withdrawal but this takes no account of the pharmacokinetics of many of these drugs which are subject to longish half-lives (2-3 hours in the case of morphine and midazolam). This is also made more complex by context-sensitive half-lives, particularly in highly lipid soluble drugs such as fentanyl. Morphine and midazolam have active metabolites , morphine-6-gluconuride and 1-hydroxy midazolam both of which are excreted via the kidney which is often a problem in the ICU. Again, this only serves to compound the problem and muddy the waters with 'tapering'.

The effects of disturbed physiology on drug clearance should be anticipated in the critically ill, but in neonates and particularly preterm babies the clearance of many sedative drugs is also prolonged by immature physiology, often markedly. Morphine clearance may only be about 30% of that seen in children over one year of age when adult rates are usually attained [START_REF] Anand | Morphine pharmacokinetics and pharmacodynamics in preterm and term neonates: secondary results from the NEOPAIN trial[END_REF]. This study also showed that clearance is decreased in preterm babies and varies with Post Menstrual Age (PMA) in a non-linear fashion. It also showed that in those born preterm, clearance remained at a lower level when compared to the same PMA of term babies, at least for the time window of this study; i.e. there was no acceleration in maturation of clearance occurring as a result of birth.

Metabolites of morphine are morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) both of which are mostly renally excreted. Although M3G is the predominant metabolite and is pharmacologically inert in terms of both analgesia and respiratory depression, about 15% of morphine is metabolised to M6G which has analgesia and respiratory depression effects of the same order as morphine itself. Formation of these metabolites and their elimination via the kidney are dependent on maturation of both the liver and the kidney respectively [START_REF] Bouwmeester | Developmental pharmacokinetics of morphine and its metabolites in neonates, infants and young children[END_REF] and where this is probably best described in terms of post menstrual age rather than post-natal age [START_REF] Rhodin | Human renal function maturation: a quantitative description using weight and postmenstrual age[END_REF][START_REF] Sumpter | Pediatric pharmacology in the first year of life[END_REF].

As in neonatal practice, attempts have been made either to forestall withdrawal or treat it with other classes of drugs. Recently the use of α 2 -agonist drugs such as dexmedetomidine or clonidine have been used in PICUs. Apart from one small prospective study [START_REF] Ambrose | Intravenous clonidine infusion in critically ill children: dosedependent sedative effects and cardiovascular stability[END_REF], these drugs have been introduced into practice in an uncontrolled fashion and a multicentre trial is currently underway in PICUs in the UK comparing the use of either clonidine or midazolam for intravenous sedation in a combination with morphine. These α 2 -agonist drugs have been used to wean patients off 'conventional' sedation with opioids and benzodiazepines but are now being reported to be associated with withdrawal symptoms themselves [START_REF] Darnell | Withdrawal from multiple sedative agent therapy in an infant: is dexmedetomidine the cause or the cure?[END_REF][START_REF] Greenberg | Clonidine withdrawal in a 3 month old premature male infant[END_REF].

Clonidine is being used more frequently to treat or preempt withdrawal symptoms [START_REF] Jenkins | Current United Kingdom sedation practice in pediatric intensive care[END_REF], but the pharmocokinetics of this drug also vary with age, where neonates demonstrate only approximately 30% of the clearance seen in older children [START_REF] Potts | Clonidine disposition in children; a population analysis[END_REF].

Once withdrawal is identified or anticipated, a practical strategy to deal with this needs to be adopted. Now that we have validated withdrawal scoring systems for the paediatric population such as the Withdrawal Assessment Tool -1 (WAT-1) (37) and the Sophia Observation Withdrawal Symptoms Scale (SOS) [START_REF] Ista | Construction of the Sophia Observation withdrawal Symptomsscale (SOS) for critically ill children[END_REF], the presence of withdrawal clinical features can be detected and tracked more reliably and reproducibly.

In the past it has been usual to taper sedation to allow a gradual re-setting of receptors and enzyme systems with a view to preventing decompensatory withdrawal phenomena appearing. Some of these tapering schedules may last 1-2 weeks [START_REF] Franck | The Withdrawal Assessment Tool-1 (WAT-1): an assessment instrument for monitoring opioid and benzodiazepine withdrawal symptoms in pediatric patients[END_REF] or up to as much as 5-6 weeks using subcutaneous infusions [START_REF] Tobias | Subcutaneous dexmedetomidine infusions to treat or prevent drug withdrawal in infants and children[END_REF] or oral substitute medications, eg methadone [START_REF] Tobias | Tolerance, withdrawal, and physical dependency after long-term sedation and analgesia of children in the pediatric intensive care unit[END_REF]. However, evidence on the length of time needed to taper varies. A randomised study in a PICU population, utilising methadone to wean from opioids, found no differences between the group weaned over 5 days compared to the group weaned over 10 days [START_REF] Berens | A prospective evaluation of opioid weaning in opioiddependent pediatric critical care patients[END_REF] .

Additionally, in a study on withdrawal scoring that used a 'tapering' regimen, symptoms took a median of three days to develop [START_REF] Franck | Opioid and benzodiazepine withdrawal symptoms in paediatric intensive care patients[END_REF], whereas in the UK-wide sedation survey, where sedation was stopped in an uncontrolled manner, these symptoms appeared much sooner [START_REF] Jenkins | Current United Kingdom sedation practice in pediatric intensive care[END_REF]. There may be something to be said for abrupt cessation of these drugs and dealing with withdrawal symptoms either pre-emptively or swiftly once they occur, rather than delaying what may be inevitable anyway and subjecting these children to prolonged weaning protocols that may not be necessary. Now that reliable systems of withdrawal scoring exist, such a proposal could be tested more reliably.

Addiction in Clinicians

It is not just the patients that are vulnerable to agents used in anesthesia or intensive care. A recent survey in the United States showed that of the academic anesthesiology departments contacted, 18% had recorded one or more incidents of propofol abuse or 'diversion' [START_REF] Wischmeyer | A survey of propofol abuse in academic anesthesia programs[END_REF]. Of the individuals that had been identified, 28% had died as a result of the propofol abuse. Propofol causes tolerance [START_REF] Ypsilantis | Tolerance to propofol's sedative effect in mechanically ventilated Rabbits[END_REF] and has psychotropic properties that lend themselves to abuse [START_REF] Wilson | The abuse potential of propofol[END_REF] and this has been corroborated in human volunteers [START_REF] Zacny | Propofol at a subanesthetic dose may have abuse potential in healthy volunteers[END_REF]. Clearly, anesthesiologists and their immediate co-workers have greater access to such drugs than other physicians and this results in greater drug abuse in this group [START_REF] Berge | The anesthesiology community's approach to opioid-and anesthetic-abusing personnel: time to change course[END_REF]. However, what may be less well appreciated is that in a study utilising mass spectrometry in operating room environments, aerosolized fentanyl was been detected in the air of an operating room, in patients' expiratory circuits and in the headspace above sharps boxes. Aerosolized propofol was also detected in the expirations of a patient undergoing transurethral prostatectomy [START_REF] Mcauliffe | Second-hand exposure to aerosolized intravenous anesthetics propofol and fentanyl may cause sensitization and subsequent opiate addiction among anesthesiologists and surgeons[END_REF]. Given the potential of these drugs to exert effects at minute nanomolar concentrations [START_REF] Li | Nanomolar propofol stimulates glutamate transmission to dopamine neurons: a possible mechanism of abuse potential?[END_REF], it may be that occupational exposure is a real hazard for addiction developing in operating room staff. There needs to be institutional recognition of several aspects to this. Tracking of drugs with known addictive potential should be rigorous and such drugs should have safe and irreversible disposal. This should now probably include propofol and other drugs will undoubtedly emerge that have addictive properties. Lastly, the inherent

  and intensive care staff to developing addiction is not solely due to their obvious access to these drugs but may also be due to subliminal as well as overt occupational environmental exposure.

Pediatric Anesthesia