Introduction
Combined clinical observations and pathologic evaluations represent the standard to diagnose oral complications of immunodeficiency and are used to make treatment decisions. By comparison, the treatment of HIV relies heavily on HIV plasma viral load. Current recommendations are to initiate combination antiretroviral therapy (ART) as early as possible. Starting ART independently of CD4+ cell count is justified, in part, to prevent HIV transmission [1]. It depends on an extensive healthcare infrastructure to supply ART and to provide for early detection of HIV. In populations with limited access to ART, Kaposi Sarcoma, oral hairy leukoplakia (OHL), and oral candidiasis signify advanced HIV disease. This study asked whether the presence of herpes simplex virus-1 (HSV-1), cytomegalovirus (CMV), Epstein–Barr virus (EBV), or Kaposi's sarcoma-associated herpesvirus (KSHV) in the throat wash of persons with HIV infection correlated with immune status as measured by CD4+ cell count and plasma HIV viral load. If this was true, monitoring the levels of oral pathogens could help in the diagnosis of deteriorating immune status. As sampling the oral cavity is minimally invasive compared to blood sampling and can be done by self-collection this may present an option to reach underserved populations and to improve HIV care in resource limited settings.
Observing cutaneous Kaposi sarcoma is part of the definition of AIDS (reviewed in [2]). Kaposi sarcoma disease is associated with declining immune status in the setting of untreated HIV infection. In HIV-negative transplant recipients Kaposi sarcoma is a complication of iatrogenic immune suppression and the relative risk of Kaposi sarcoma in transplant recipients is estimated at 200-fold compared to the general population [3–5]. Oral Kaposi sarcoma in addition to cutaneous or systemic Kaposi sarcoma is considered an indicator of advanced Kaposi sarcoma disease (T1); it is associated with poor outcome in response to cytotoxic therapy. Oral Kaposi sarcoma may benefit from intralesional injections of cytotoxic agents [6]. Aspects of the AIDS Kaposi sarcoma epidemic in the US have changed with the introduction of ART. One-third of Kaposi sarcoma now develops in patients on effective ART with plasma HIV viral load below the limits of detection [7,8]. The decline of Kaposi sarcoma incidence as observed concurrent to the introduction of ART has leveled off since 2010 and Kaposi sarcoma remains the single most prevalent cancer occurring in persons with HIV today [9,10]. KSHV is the etiological agent of Kaposi sarcoma. All cases of Kaposi sarcoma are associated with prior or concurrent KSHV infection regardless of HIV status. KSHV is detected in saliva and an inhibitor of KSHV viral replication (ganciclovir) reduces oral shedding and lifetime Kaposi sarcoma risk [11–17]; whether or not established Kaposi sarcoma lesions respond to inhibitors of viral replication is unclear [18,19].
OHL is an AIDS-defining condition [20]. EBV is the etiological agent of OHL and all cases of OHL are associated with concurrent permissive EBV lytic replication in the lesion [21,22]. OHL has also been found in HIV-negative, EBV-positive organ transplant patients [23]. It tends to resolve upon immune reconstitution in the setting of HIV infection and responds to acyclovir, an inhibitor of the EBV DNA polymerase [24]. EBV is detected at high levels in saliva and an inhibitor of EBV viral replication (acyclovir) reduces oral shedding [25–27].
Oral candidiasis was the most common oral complication of HIV/AIDS prior to the introduction of ART (reviewed in [28,29]); it is still in many low and middle-income countries. Oral candidiasis can be caused by multiple Candida spp. in the setting of HIV [30]. Oral candidiasis is associated with severe immune suppression and extremely low CD4+ cell counts. Systemic therapy is indicated though studies suggest that local agents are efficacious as well [31,32].
The primary objective of the ACTG A5254 trial was to describe the prevalence of oral lesions in people with HIV infection and to test the hypothesis that after training nonprofessional oral health specialists can accurately diagnose oral lesion frequently found in this population. This work has been published, as have the details of the clinical data and socioeconomic characteristics of the study population [33]. As oral complications of HIV/AIDS were previously detected at lower CD4+ cell counts, this cohort was geared toward enrollment of persons with low CD4+ cell counts. This design allowed evaluation of associations between different pathogens in the oral cavity in participants with defined immunologic and HIV plasma viral load status.
Methods
Study design
Detailed study design, patient population and sample collection were previously published [33]. In brief, ACTG A5254 is a cross-sectional study that enrolled HIV-1-infected adults 18 years or older with or without prior ART from five locations in the US and one location in Haiti between 2009 and 2012. Institutional Review Boards or Ethics Committees of each participating institution approved the study, and each patient gave written informed consent.
As previously reported [33], there were 128 (42%) cases of oral candidiasis, 39 (13%) cases of OHL, and 31 (10%) cases of oral Kaposi sarcoma in the A5254 cohort. Oral candidiasis was most common in stratum A (A: 66, B: 18, and C/D: 14%, P value for the Fisher's exact test across strata <0.001). OHL seemed less common in stratum B (A: 24 (15%); B: 5 (5%); C/D 10 (17%), P value for the Fisher's exact test across strata <0.05). Oral Kaposi sarcoma was most common in stratum A (A: 29 (18%); B: 2 (2%); C/D (0%), P value for the Fisher's exact test across strata <0.001). HSV-1 and CMV oral lesions could not be discerned from oral lesions of nonviral origin. In sum, oral candidiasis and oral Kaposi sarcoma were more common in participants with lower CD4+ cell count overall and within the context of immunodeficiency-associated higher HIV viral load (comparing stratum A and B).
Throat wash collection
A 5-min unstimulated whole saliva flow rate was recorded and collected [34]. A 1-min oral rinse/throat wash using 10 ml of sterile saline was collected afterwards. Both saliva and throat wash specimens were frozen in aliquots at −80°C at the site laboratory, and banked. Only soluble throat wash was used for the current study.
Candida spp. analysis
Before the throat wash was processed further 2.5 ml sterile saline was aliquoted and processed for Candida spp. detection by culture. A culture was defined as positive, and confirming the clinical diagnosis of oral candidiasis, among individuals with clinical features of oral candidiasis and a number of colony forming units.
CD4+ cell count and plasma HIV viral load
Phlebotomy was performed at the time of the visit and CD4+ cell count and plasma HIV-1 viral load were measured. Plasma HIV-1 viral load was measured using the Abbott real-time HIV-1 quantitative PCR assay on 0.6–1.0 ml input volume (sensitivity of 40 copies/ml, linear range 1.6 log copies/ml to 7.0 log copies/ml, specificity >99% as reported by the manufacturer).
Throat wash herpesvirus load (cytomegalovirus, Epstein–Barr virus, Kaposi's sarcoma-associated herpesvirus, herpes simplex virus-1)
Real-time quantitative PCR was used to detect multiple herpesviruses as described previously [29]. The assay measures KSHV, CMV, EBV, and HSV-1 independently. This was important, as we expected multiple herpesviruses be present at widely differing levels in the same sample, which may have an impact on the sensitivity of multiplexed PCR. Herpesvirus load were categorized either as present or absent (based on endpoint result regardless of viral load) and as copies/ml if more than 500 copies/ml, which was the lower limit of the linear range for this assay.
Data availability
Analyses were performed by the Statistical Data Analysis Center for the ACTG, which is located within the Center for Biostatistics in AIDS Research, Harvard School of Public Health, Boston, Massachusetts, USA. This is where the datasets are stored. Consistent with National Institutes of Health (NIH) regulations and ACTG policy, the data are publically available. Requests for data are to be submitted to [email protected]. Data are deidentified prior to distribution. Additional analyses were performed at the University of North Carolina Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina, USA.
Statistical procedures
Sample characteristics were summarized using proportions for categorical variables, and mean, median with 1st and 3rd quantiles (Q1 and Q3) for continuous variables. The results were presented for all the strata, and for each CD4+ cell count/plasma HIV-1 viral load stratum separately (strata C and D were combined). The frequency of oral candidiasis was confirmed by a positive culture (≥1 colony unit count/ml) was computed and the difference across strata using the Fisher's exact test was explored. CD4+ cell count and log10 (plasma HIV-1 viral load) was compared among participants across strata using the Kruskal–Wallis test. Log10 (viral load) of individual herpesviruses in saliva was also calculated and summarized across strata for participants with detectable herpesviruses. The frequency of virus-detectable cases was computed by CD4+ cell count/plasma HIV-1 viral load stratum, and the difference across strata was compared using the Fisher's exact test. Logistic regression was used to model the relationship between the presence of herpesvirus oral shedding and HIV-1 viral load in plasma controlling for CD4+ cell count, oral candidiasis, and sites. A detailed description of the methods of diagnosis of oral manifestations of disease in this study has been reported previously [33]. The model yielded adjusted odds ratios with 95% confidence intervals.
Results
Oral disease was associated with severe immunodeficiency
The salient features of the A5254 study design were as follows: A5254 was a single-time-point, cross-sectional study, which enrolled its first participant in October 2009 and its last participant in September 2012 [33]. All participants were evaluated by oral health specialists, and throat wash samples were taken. Participants were initially enrolled across four strata. We combined participants for two strata (C and D) with CD4+ cell counts more than 200 cells/μl regardless of HIV viral load to obtain a more balanced design (Table 1). Stratum A encompassed N = 157 participants with a CD4+ cell count of 200 cells/μl or less and plasma HIV-1 viral load more than 1000 copies/ml. Of these, 99 (63%) were enrolled in Haiti and 58 (37%) in continental USA. In total, 88% of participants in stratum A were black, nonLatino. The proportion of black, nonLatino was significantly larger than 60 and 47% in stratum B and C/D, respectively (P value for the Fisher's exact test across strata <0.001). The majority (93%) of participants from Haiti were enrolled only in stratum A. Stratum B encompassed N = 91 participants with CD4+ cell count of 200 cells/μl or less and a plasma HIV-1 viral load of 1000 copies/ml or less. Stratum C/D encompassed N = 59 participants with CD4+ cell count at least 200 cells/μl regardless of HIV plasma viral load. The majority of participants were men in stratum B (85%) and C/D (68%), significantly larger than 55% in stratum A (P value for the Fisher's exact test across strata <0.001). The median age was 44 (Q1–Q3: 38–51) with participants in stratum B being somewhat older compared to stratum A and C/D.
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Table 1: Demographics and baseline characteristics.
Ninety-nine (32%) participants were not on combination ART at study entry. Almost all patients in stratum B were on ART. In total, 52% of patients in stratum A also reported being on ART, yet more than 90% of those did not recount a prior AIDS defining illness (data not shown). Presumably this reflects participants, who only recently started on ART.
Immune status-associated shedding in the oral cavity differs among different herpesviruses
HSV-1 was only detectable in stratum A participants (8%; Table 2). The median (Q1, Q3) of HSV-1 viral load (log10 copies/ml) for all virus-positive samples was 4.25 (3.90, 4.40). CMV was detectable in throat wash of 50% of stratum A participants, 15% in stratum B, and 10% in stratum C/D. There was a significant association between the presence of CMV and stratum (P < 0.001). The median (Q1, Q3) of detectable CMV viral load (log10 copies/ml) was 3.60 (3.40, 3.90). KSHV, the etiologic agent of Kaposi sarcoma, was detectable in throat wash of 3% of stratum A participants, 4% of stratum B participants, and 7% of stratum C/D participants. There was no association between the presence of KSHV and stratum. Overall the median (Q1, Q3) of detectable KSHV viral load (log10 copies/ml) was 3.35 (3.20, 3.70). EBV was detectable in throat wash of 87% of stratum A participants, 77% of stratum B participants, and 83% of stratum C/D participants. There was no significant association between the presence of EBV and stratum. Overall the median (Q1, Q3) of detectable EBV viral load (log10 copies/ml) was 4.60 (4, 5). We found no evidence for an association of EBV viral load or CMV viral load with Candida spp. burden.
Table 2: Herpesvirus loads by stratum.
The overall frequency of HSV-1 or KSHV-positive sample was very low (4%), thus the study was inadequately powered to detect possible associations of HSV-1 or KSHV with Candida spp. burden, that is, colony count units. EBV was detectable in 83% of samples and CMV in 32% and their viral loads spanned a large range (Fig. 1). Evidence for an association between EBV viral load or CMV viral load with Candida spp. burden was not statistically significant (P
= 0.98, P = 0.60; data not shown). No significant association was found between EBV and CMV viral load in the oral cavity (P = 0.63; data not shown), suggesting that independent factors foster oral replication and shedding of these two herpesviruses.
Fig. 1: Association between very high EBV and CMV levels in throat wash.The data are separated by stratum. The three panels represent the three study strata A, B, and C/D. The top panel shows the measurements for stratum A (high HIV viral load, CD4+ ≤200 cells/μl), the middle panel stratum B (low HIV viral load, CD4+ ≤200 cells/μl), and the bottom panel stratum C/D (CD4+ >200 cells/μl). Shown in each panel is log10EBV copies/ml on the horizontal axis compared to log10 CMV copies/ml on the vertical axis. To aid visualization the threshold was set at 3 × log10 so that participants with detectable viral load lower than 3 × log10 were coded as 3 × log10, and participants with detectable viral load higher than 6 × log10 were coded as 6 × log10. Last, ART status is reflected in the shape. Black circles are participants not on ART and shown as triangles are participants who self-reported being on ART, though we do not know the time of therapy initiation. ART, antiretroviral therapy; CMV, cytomegalovirus; EBV, Epstein–Barr virus.
We used logistic regression to evaluate the relationship between CMV shedding in oral cavity and HIV plasma viral load. (Table 3). The odds of detecting CMV was 1.31 times higher with one unit increase in log10(HIV viral load) when controlling for oral candidiasis, CD4+ cell count, and sites (95% confidence interval 1.04–1.65, P = 0.02), suggesting a significant association between detection of CMV in throat wash and HIV viral load in plasma. The association between the presence of EBV in throat wash and HIV viral load in plasma was marginally significant with odds ratio 1.26 (95% confidence interval 0.99–1.61, P = 0.06; data not shown). CMV and HSV-1 were more often detected in stratum A than in stratum B, which had a similar level of immunodeficiency as ascertained by CD4+ cell count but suppressed HIV replication.
Table 3: Logistic regression model exploring the association between detection of cytomegalovirus in throat wash and HIV viral load in plasma controlling for CD4+ cell count, oral candidiasis, and sites.
Discussion
Oral complications of HIV/AIDS manifest primarily in stages of advanced HIV disease. OHL was one of the first described clinical manifestations of AIDS and oral Kaposi sarcoma became widely recognized during the early AIDS epidemic in the US and Europe. CMV-associated diseases, in particular retinitis, pneumonia, and encephalitis also are part of the clinical manifestations that signify end-stage AIDS, as is HSV-1 disease. The etiological agent for Kaposi sarcoma is KSHV and the etiological agent for OHL is EBV. CMV, EBV, KSHV, and HSV-1 are present in oral secretions and transmitted via the oral route [12,35–37]. CMV plasma viral load is closely linked to immune suppression, whether by HIV or iatrogenic drug regimen [35,38]. A close association between plasma viral load and disease burden also holds true for EBV-associated posttransplant lymphoproliferative disease and some EBV-positive lymphoma [39,40], but not always for KSHV and Kaposi sarcoma [41]. A5254 found that oral shedding of HSV-1 and CMV, but not of EBV and KSHV, were correlated with immune deficiency.
The viral load of each of the four herpesviruses did not correlate with each other and not always with immune status. EBV was shed consistently independent of immune status, and independent of CMV viral load (Figure 1). EBV oral viral loads were much higher than those of the other herpesviruses consistent with prior work [25,35,37,42]. Our single point detection rate for KSHV was low and not associated with immune status, whereas oral Kaposi sarcoma disease was. This lack of association may be the result of sampling limitations as outlined below or it may be because of the fact that for KSHV, like for other herpesviruses, the majority of shedding occurs in the absence of overt lesions. KSHV viral loads in the oral cavity tended to fluctuate, necessitating the need to sample frequently over extended periods to detect virus [36,43]. Longitudinal sampling of the same person and detailed biopsies may be needed to establish associations among the sporadically shed herpesviruses (HSV-1, KSHV), lesion phenotype, and clinical parameters. HSV-1 shedding and disease has been associated with overall immunodeficiency and more directly with HIV replication [35,44]. In the A5254 trial, HSV-1 was only detected in the oral cavity of participants in stratum A, that is, in the setting of high HIV viral loads and CD4+ less than 200 cell/μl, though we did not have enough events to establish a robust association. This may be because of the previously described episodic nature of HSV-1 shedding [43,44].
CMV in the oral cavity was closely linked to immune suppression in the setting of HIV. This is consistent with prior studies [35,38]. In addition, we found that CMV viral load in the oral cavity was independently associated with HIV viral load in the setting of immunodeficiency. This suggests a relation between CMV and HIV replication above and beyond immune status. It may be feasible to develop CMV saliva viral load into a biomarker for immunodeficiency analogous to CMV plasma viral load in transplant patients. Such an assay would be minimally invasive and lend itself repeat sampling as well as self-sampling.
The study had a number of limitations, such as the cross-sectional nature of sample collection, which did not allow us to distinguish primary infection from reactivation. This design also does not allow us to make inferences about persistence. As the primary objective of A5254 was to evaluate oral examination by nonoral health specialists [33], we did not include a blood draw for research purposes. Hence, we can only infer the underlying seroprevalence for each of the herpesviruses based on published reports in these populations. For CMV, seroprevalence in MSM, and in the general population has been consistently estimated at least 80%, levels for EBV and HSV-1 are estimated at 90 and 67% in the general population globally and consistently higher among HIV+[45,46]. For KSHV, the data are more variable [47–49]. Most recently, the ACTG Longitudinal Linked Randomized Trials study estimates a seroprevalence of 38% in ACTG clinic attendants [50]. Last, a large proportion of patients in stratum A enrolled in Haiti, whereas few of the other strata enrolled at this location because of the state of HIV care on the island. This represents a potential confounder, which was included in the multivariable logistic regression model that demonstrated a direct association between CMV oral viral load and HIV systemic viral load.
In sum, we find that HIV-induced deficiency in the adaptive immune response, as ascertained here by CD4+ cell counts, does not elevate oral shedding among herpesviruses equally. These findings are consistent with a model in which local trigger/pathogen pairings synergize for herpesvirus replication, infection, reactivation, transmission, and disease. In the case of CMV one of the triggers may be HIV replication itself. If we were to understand the forces that govern virus reactivation in the oral cavity, it would open up new avenues of intervention, prophylaxis, and diagnosis.
Acknowledgements
D.P.D. designed the study and wrote the manuscript. K.T. designed the assay, generated the data and conducted primary analysis. H.C. designed and conducted the statistical analysis. A.L. conducted the statistical analysis. M.K.S. designed the assay, generated the data and conducted primary analysis. T.A.W. designed the assay, generated the data and conducted primary analysis. S.N. conducted the statistical analysis. J.W-C. designed the study. M.G. designed the study, generated primary data and analysis. C.H.S. designed the study. J.A.A. designed the study.
The list of sites for the A5254 protocol has been published and is as follows: Jean William Pape, MD, Patrice Sévère, MD, Rode Secours, MD, Daphné Bernard, MD and Maria Linda Aristhomène, RN – Les Centres Gheskio (Gheskio-INLR) CRS (Site 30022), Caroline Shiboski, DDS, MPH, PhD, Sivappiriyai Veluppillai, DDS, Amanda Hutton Parrott, DPT, NP, and Jay Dwyer, RN, -UCSF AIDS CRS (Site 801) J.A.A., MD, Karen Cavanagh, RN, Alexander Ross Kerr DDS, MSD, Sonal S Shah DDS, and Manley Lammarre RDH – New York University HIV/AIDS CRS (Site 401) J.W-C., DDS, PhD, Jonathan Oakes BA, D.P.D., Ph.D. and Lauren Patton DDS – Chapel Hill CRS (Site 3201), Jeffrey Lennox, MD, Dale Maddox, RN and David A. Reznik, DDDS – The Ponce de Leon Ctr. CRS (Site 5802) Emory University HIV/AIDS CTU Michael Lederman MD, Jane Baum RN, M.G., PhD, Nancy Isham, and Richard Jurevic – Case CRS (Site 2501).
The project described was supported by Award Number AI068636 from the National Institute of Allergy and Infectious Diseases and supported by National Institute of Mental Health (NIMH), National Institute of Dental and Craniofacial Research (NIDCR). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the NIH. Further analysis and assay development was supported by public health service grants DE018304 and DE023946 to DPD and AI068634 for the Statistical and Data Management Center for the AIDS Clinical Trials Group.
Conflicts of interest
There are no conflicts of interest.
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