0095-1137/06/$08.00
⫹
0
doi:10.1128/JCM.00256-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
High Prevalence of Multiple Human Herpesviruses in Saliva from
Human Immunodeficiency Virus-Infected Persons in the Era
of Highly Active Antiretroviral Therapy
Craig S. Miller,
1,2* Joseph R. Berger,
3Yunanan Mootoor,
3Sergei A. Avdiushko,
2Hua Zhu,
4and Richard J. Kryscio
4Department of Microbiology, Immunology & Molecular Genetics,
1Department of Oral Health Practice,
2Departments of
Neurology and Internal Medicine,
3and Department of Statistics,
4University of Kentucky College of Medicine and
College of Dentistry, Lexington, Kentucky 40536-0297
Received 3 February 2006/Returned for modification 27 February 2006/Accepted 6 May 2006
Human immunodeficiency virus (HIV) infection is associated with an increased risk for human
herpesvi-ruses (HHVs) and their related diseases. Methods for limiting the transmission of HHVs require a better
understanding of the prevalence and infectiousness of oral HHVs in HIV-infected patients. We performed
quantitative PCR to investigate the prevalence, quantity, risk, and correlates of salivary HHVs from 58
HIV-seropositive individuals in a case control study. HHVs were significantly more prevalent in the salivas of
HIV-seropositive persons than in those of the controls (odds ratios [ORs], 4.2 to 26.2;
P
<
0.008). In
HIV-infected patients, Epstein-Barr virus (EBV), human herpesvirus 8 (HHV-8), cytomegalovirus (CMV), and
herpes simplex virus type 1 (HSV-1) were detected in 90%, 57%, 31% and 16% of samples, respectively,
compared with 48%, 24%, 2%, and 2%, respectively, of samples from controls. Multiple HHVs were observed
in 71% of HIV-seropositive persons and only 16% of controls (OR, 13.0; 95% confidence interval, 5.29 to 32.56).
HIV-positive patients had significantly higher EBV loads than HIV-negative persons (
P
< 0.0001).
HIV-infected patients with CD4 counts above 200 cells/
l had increased probability for having HHV-8 in saliva (
P
ⴝ
0.009) compared with patients whose counts were less than 200. In contrast, HSV-1, EBV, and CMV were
detected more often when CD4 counts were low. High salivary HHV loads were detected for those (
n
ⴝ
7) with
oral lesions. These findings suggest that saliva is a potential risk factor for the acquisition of multiple HHVs,
and several host factors may function to accelerate HHV reactivation or replication in patients with HIV
infection.
Human herpesviruses (HHVs) are widely distributed
patho-gens that cause benign and malignant disease. Serological
ev-idence of infection with HHVs is found in the majority of the
world’s populations (23, 28, 32, 40, 45, 60, 64, 65, 74). Spread
of HHVs is by contact with infected secretions, usually early in
life. However, modes of transmission of select HHVs are not
fully understood (2). Of the bodily secretions and fluids
doc-umented to harbor HHVs, saliva appears important for
trans-mission of several HHVs (11, 16, 17, 33, 52, 53, 55, 56, 69). The
amount and frequency of HHVs appearing in saliva are likely
determinants of risk for transmission (48).
Herpes simplex virus 1 (HSV-1), Epstein-Barr virus (EBV),
cytomegalovirus (CMV), and human herpesvirus 8 (HHV-8)
(also called Kaposi’s sarcoma [KS]-associated herpesvirus), are
members of the
Herpesviridae
family that have common
asso-ciations with orofacial diseases in humans. They are shed in
saliva asymptomatically and simultaneously at various rates
and levels (48). The rates and viral loads detected are
influ-enced by the biological properties of the virus, method of
detection, frequency of sampling, oral health, and social
be-haviors and immunological status of the patient (46, 49). By
sensitive PCR assay, the mean detection rates for EBV (30, 31,
48, 63), CMV (15, 44), HSV-1 (15, 27, 48, 49, 67), and HHV-8
(7, 14, 36, 38, 44, 48) nucleic acids in the oral cavity of healthy
adults at a single visit are approximately 31%, 9%, 6%, and
⬍
1%, respectively. The prevalence of HHVs in the saliva of
human immunodeficiency virus (HIV)-seropositive patients
has been less often reported, with reports generally focusing on
individual prevalence, not the simultaneous presence of
HHVs. In these reports, the prevalence of HHVs appears
higher in HIV-seropositive patients (3, 4, 6, 19, 38, 44, 46, 68).
However, in one study that examined multiple HHVs, rates of
detection in saliva of HIV-seropositive patients were similar to
that of the general population, except for CMV (20).
Almost 45 million people worldwide have been infected by
HIV, and prior to highly active antiretroviral therapy
(HAART), more than 75% of all HIV-infected persons
devel-oped HHV-related diseases (39, 54, 61, 66, 70). The advent of
HAART has decreased the incidence of opportunistic HHV
diseases and improved survival for those fortunate to receive
therapy (1, 9, 20, 39, 50, 59). Whether HAART has altered the
rate of HHV reemergence from latency or the ability of HHVs
to produce clinical manifestations is not well known. Clearly,
HHV-related malignancies remain a significant problem for
the HIV infected (5, 73), and laboratory assessments of HHVs
in bodily fluids that could be predictive of the development of
HHV-related diseases would be of great benefit to this
popu-lation.
* Corresponding author. Mailing address: MN 324 Oral Medicine,
College of Dentistry, University of Kentucky Medical Center, 800
Rose Street, Lexington, KY 40536-0297. Phone: (859) 323-5598. Fax:
(859) 323-9136. E-mail: cmiller@uky.edu.
2409
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This case-controlled study focused on four HHVs that
com-monly occur in the oral cavity and cause significant morbidity
in HIV-infected persons. Our aim was to determine the
prev-alence and loads of EBV, CMV, HSV-1, and HHV-8 in the
saliva of HIV-infected persons regardless of HHV serostatus
and compare these findings with age- and sex-matched
HIV-seronegative individuals during the era of HAART. Correlates
between the presence of HHVs and the degree of cellular
immunosuppression, oral health, and demographic factors in a
group of HIV-positive patients who lacked clinical evidence of
HIV-related malignancies were assessed.
MATERIALS AND METHODS
Study population and procedures.Fifty-eight HIV-seropositive subjects were recruited from the Bluegrass Care Clinic of the University of Kentucky, College of Medicine, in Lexington, Kentucky. Fifty-eight age- and sex-matched control subjects were recruited from campus volunteers and from patients in the Neu-rology Clinic at the University of Kentucky. Enzyme-linked immunosorbent assays for HIV were performed on all controls to insure that they were HIV seronegative. All subjects were 18 years or older. All controls were in good general health and did not have histories of liver or kidney dysfunction. All subjects were free of symptoms of acute illness (i.e., fever, sore throat, body aches, and diarrhea) and malignancies at the time of enrollment. Pregnancy, use of immunosuppressant medications, and antiherpetic therapy 1 week before the study enrollment date were additional exclusion criteria. The study was approved by the University Institutional Review Board, and all patients provided written informed consent as part of the study protocol.
Oropharyngeal examinations were performed by a study physician. Subjects were asked specifically about previous tonsil surgery and inspected for gum bleeding, mouth ulcers, mouth pain, dry mouth, presence of tonsillar tissue, oral mucosal lesions, and cervical lymphadenopathy. Unstimulated whole saliva (5 ml) was collected at a single visit by the method described by Navazesh (51). All samples were maintained on ice, divided into 1-ml aliquots, placed into bar code-labeled cryotubes, and frozen at⫺85°C until use. Venous blood was col-lected, processed, and analyzed for HIV serostatus using ELISA. Lymphocyte subsets were assayed via flow cytometry.
PCR primers and probes.The primers and probes used were derived from published sources, as we have previously reported (48). Briefly, HSV-1 primers and probes were designed for glycoprotein G as described by Ryncarz et al. (58). Primers and probes for EBV were directed to the BALF5 gene encoding viral DNA polymerase according to the method of Kimura et al. (35). Primers and probes for CMV were designed for glycoprotein B as described in the work of Li et al. (41). HHV-8 primers and probes were designed for HHV-8 minor capsid protein as described by White and Campbell (72). Probes for all HHVs were labeled at the 3⬘end with the quencher fluorochrome, 6-carboxy-tetramethyl-rhodamine (PE Applied Biosystems). The 5⬘end of the probes for all HHVs were labeled with the reporter fluorochrome, 6-carboxy-fluorescein. These prim-ers and probes have been found to reliably detect at least 10 copies of target HSV-1 DNA and at least 1 copy of CMV, EBV, or HHV-8 DNA in 10l purified template (48) and are specific when tested with known HHVs (i.e., cross-reac-tivity is not observed between the viral assays) (10, 35, 41, 57, 58, 72, 75).
Real-time quantitative PCR.Real-time quantitative PCR was used for the detection and quantification of HHVs in saliva. The DNA from 1 ml of each saliva specimen was centrifuged, and the DNA was isolated from the cell pellet using the QIAamp DNA Mini kit (QIAGEN, Valencia, CA). Herpesviruses are more readily detected in the cell pellet than in the supernatant (48). Each 50-l PCR mixture contained 10l purified DNA template in a final volume consisting of 1⫻TaqMan Universal PCR master mix (PE Applied Biosystems), 900 nM primers, and 250 nM TaqMan probe. Real-time PCR was performed on an ABI
Prism 7700 sequence detection system (PE Applied Biosystems). Cycling param-eters were 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 1 min. Each PCR run contained negative controls, including reaction mixtures without DNA template as well as several specimens that were known to contain no HHV DNA, a positive amplicon control, and a 10-fold dilution series (1⫻100
to 1⫻106
genome equivalents per reaction) of either genomic HHV DNA or cloned HHV sequences. The positive control standards were HSV-1 genomic DNA; plasmid pGEM-BALF5 containing the EBV BALF5 gene, kindly provided by H. Kimura, Nagoya University, Nagoya, Japan; plasmid pCR2.1 with a cloned 254-bp fragment of CMV glycoprotein B, kindly provided by Y.-W. Tang, Vanderbilt University, Nashville, Tennessee; and plasmid pMCP, contain-ing nucleotides 47239 to 47554 of the HHV-8 genome, kindly provided by T. B. Campbell of the University of Colorado, Denver. Each specimen was analyzed in duplicate. Results were scored positive if both reactions yielded a threshold cycle value (Ct) above the limit of detection for the standards. Reactions that yielded one positive and one negative result were repeated in duplicate. Samples were scored positive only when both repeat reactions yielded positive results. Viral genome copy number results are reported as the means of the two runs.
Statistical analysis.Odds ratios (and 95% confidence intervals) to measure the association between demographics (age and gender), or risk factors (CD4/ CD8 counts, use of HAART, etc.) and the presence of a virus were based on 2⫻2 contingency tables.Pvalues associated with these odds ratios were based on chi-square statistics or Fisher’s exact test. A stepwise logistic regression model with a significance level to enter of 0.20 and a significance level to stay of 0.05 was used in the multivariate analysis. Mean titer levels were compared between controls and HIV patients using two samplettests after log transforming the viral DNA copy numbers. Statistical significance was determined at the 0.05 level. All data were analyzed with use of the SAS statistical analysis software (SAS Institute).
RESULTS
Demographic and clinical findings.
The 58 HIV-infected
individuals were demographically similar to the 58 controls
(Table 1). The majority of subjects in both groups were
Cau-casian men. None of the participants in either group had
clin-ical KS, and 47/58 (81%) of the HIV-seropositive patients were
taking HAART. The majority of HIV-positive patients were
adequately controlled as determined by HIV loads and CD4
counts. Oral lesions were noted in seven individuals; all were
HIV seropositive.
Prevalence of salivary HHVs.
HHVs were more prevalent in
[image:2.585.43.282.81.125.2]salivas from HIV-seropositive persons than healthy controls
(Fig. 1). Ninety-seven percent of HIV-positive patients had
detectable salivary HHV DNAs. In contrast, only 34 of 58
(58.6%) of the healthy controls had HHV DNAs in saliva (
P
ⱕ
[image:2.585.320.517.542.687.2]FIG. 1. Prevalence of HHVs in saliva as determined by quantitative
PCR. The chi-square statistic demonstrated that the rate of detecting
each virus in the HIV-seropositive group was significantly greater than
the rate for the controls (P
ⱕ
0.008).
TABLE 1. Demographic characteristics of the study population
a Serostatus(nb) Gender
Age range
(avg⫾SD) Race
HIV positive (58) 48 M; 10 W 21–61 (39.8⫾8.4) 42 C; 13 AA; 3 O HIV negative (58) 43 M; 15 W 18–75 (38.1⫾13.1) 39 C; 14 AA; 5 O
a
M, men; W, women; C, Caucasian; AA, African-American; O, other. b
n, no. of subjects.
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0.008). In the HIV-seropositive group, the most prevalent viral
DNA was EBV (90%), followed by HHV-8 (57%), CMV
(31%), and HSV-1 (16%). Detection rates in the
HIV-sero-negative group were as follows: EBV, 48%; HHV-8, 24%;
CMV, 2%; and HSV-1, 2%.
Viral loads.
Concentrations of HHV DNA in the expressors
ranged greatly from a few copies to millions and varied by HIV
serostatus and HHV type (Table 2). CMV and HHV-8 were
detected, in general, at lower genome copy numbers than EBV
and HSV-1. Samples from HIV-seropositive persons had a
significantly higher geometric mean viral genome copy number
for EBV than those from the controls (
P
⬍
0.0001). Significant
differences in the geometric mean genome copy numbers of
the other viruses (i.e., HSV-1, CMV, and HHV-B) between
groups were not observed.
Relationships with the presence of multiple HHV DNAs in
saliva.
Salivas of HIV-positive subjects (71%) were more likely to
contain multiple HHVs than those of the controls (16%;
P
⬍
0.0001). The odds of having more than one HHV in
HIV-seropositive persons was 13 times the odds of having multiple
HHVs in the controls (odds ratio [OR], 13.13; 95% confidence
interval [CI], 5.29 to 32.56). Viruses most likely to be
simulta-neously present in the saliva of HIV-seropositive persons were
HHV-8 and CMV (
P
⫽
0.0004), HHV-8 and EBV (
P
⬍
0.0001), CMV and EBV (
P
⬍
0.0001), EBV and HSV-1 (
P
⫽
0.001), and HHV-8, CMV, and EBV (
P
⫽
0.0004) (data not
shown).
Relationships with immunological characteristics and HIV
disease severity.
The presence of select HHVs correlated with
the level of cellular immunosuppression (Fig. 2). CMV was
more likely to be present when CD4 counts were less than 350
cells/mm
3than above this level (
P
⫽
0.047) and when HIV viral
loads were between 400 and 5,000 copies/ml than when they
were above 5,000 (
P
⫽
0.048). HHV-8 was more likely to be
present in saliva when CD4 counts were greater than 200
cells/mm
3than when they were less than 200 (
P
⫽
0.009).
Salivary EBV and HSV-1 loads increased with increasing HIV
loads (Spearman’s correlation, 0.29 [
P
⫽
0.028] and 0.40 [
P
⫽
0.002], respectively), and HHV-8 loads correlated with the
CD8 count (Pearson’s correlation, 0.46;
P
⫽
0.0003).
Immunological status and multiple salivary HHVs.
Table 3
shows that the CD4 count was significantly associated with the
simultaneous presence of CMV and EBV (
P
⫽
0.002) and
CMV, EBV, and HHV-8 (
P
⫽
0.003) in HIV-positive patients.
When the CD4 count was less than 200 cells/mm
3, CMV and
EBV were 18 times more likely to be present in saliva than
when the CD4 count was greater than 400 cells/mm
3(OR,
[image:3.585.42.545.81.202.2]18.12; 95% CI, 1.84 to 178.57;
P
⫽
0.007). Also, HIV-positive
FIG. 2. Relationship of CD4 count and presence of HHVs in saliva
from HIV-seropositive patients. Fisher’s exact test was performed and
revealed the following
P
values:
*
,
P
⫽
0.058 for comparing CD4
counts of
⬍
200 cells/mm
3versus CD4 counts of
ⱖ
400 cells/mm
3;
**
,
P
⫽
0.026 for comparing CD4 counts of 201 to 399 cells/mm
3versus CD4 counts of
ⱖ
400 cells/mm
3;
[image:3.585.60.266.497.660.2]***
,
P
⫽
0.002 for comparing
CD4 counts of
⬍
200 cells/mm
3versus counts of
⬎
400 cells/mm
3.
TABLE 2. HHV genome copy numbers in saliva
Virus Subject group (na) Median (range) Geometric mean Standard error Pvalueb
HSV-1
HIV
⫺
(1)
20,516
20,517
NA
cHIV
⫹
(9)
14,948 (1,254–82,806)
12,777
6,228
EBV
HIV
⫺
(28)
1,759 (18–114,614)
1,775
926
⬍
0.0001
HIV
⫹
(52)
308,875 (16–53,247,808)
128,028
78,058
CMV
HIV
⫺
(1)
762
762
NA
HIV
⫹
(17)
1,501 (89–9,584)
1,180
355
HHV-8
HIV
⫺
(14)
172 (5–2224)
167
92
0.42
HIV
⫹
(33)
217 (5–1,087,543)
311
138
a
n, no. of subjects. b
Logarithm was taken of viral loads for each virus, and a two-samplettest was performed to determine if the mean viral loads were different between the groups. All values reported are per ml of saliva.
c
NA, not available due to low frequencies.
TABLE 3. Relationship of CD4 count and presence of
multiple salivary HHVs in HIV-positive patients
CD4 count (na
)
No. of patients with presence ofb:
CMV and EBV
CMV and HHV-8
EBV and HHV-8
CMV, EBV, HHV-8
HSV-1, CMV, EBV, HHV-8
⬍
200 (13)
5*
0
2
1
0
200–399 (15)
0
0
5
7*
1
⬎
400 (30)
1
0
12
2
0
an, no. of patients. bⴱ,P⫽0.002.
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[image:3.585.301.541.629.709.2]patients with a CD4 count between 200 and 399 cells/mm
3were 12 times more likely to have the simultaneous presence of
CMV, EBV, and HHV-8 in saliva than patients with a CD4
count greater than 400 cells/mm
3(OR, 12.25; 95% CI, 2.11 to
70.92;
P
⫽
0.003).
Risk factors for HHVs.
Several factors (i.e., age, race,
gen-der, CD4 and CD8 cell count, CD4/CD8 ratios, HIV viral
loads, HAART use, presence of tonsils, bleeding gums, oral
lesions, and abnormalities of the mouth and/or neck) were
assessed for their association with the detection of HHV
DNAs in saliva, first in univariate analysis and then in
multi-variate models. In the unimulti-variate analyses, HIV-positive
pa-tients were significantly more likely to shed HHVs than the
controls (OR, 4.15 [95% CI, 1.87 to 9.19] for HHV-8, 9.29
[95% CI, 3.45 to 24.98] for EBV, 10.47 [95% CI, 1.28 to 85.53]
for HSV-1, and 26.19 [95% CI, 3.34 to 205.22] for CMV). No
other demographic (age, race, gender) or clinical risk factors
were significantly related to the odds of shedding these viruses.
To investigate why HHV shedding was related to HIV disease,
a number of clinical and immunological related factors were
examined using the HIV-positive patients only. A summary of
multivariate analyses follows.
Patients with HIV loads over 5,000 copies/ml were
signifi-cantly more likely to shed HSV-1 than patients with HIV loads
under 5,000 (adjusted OR, 14.3; 95% CI, 2.7 to 100).
Interme-diate HIV loads (in the range of 400 to 5,000 copies/ml) had a
significantly larger chance of shedding CMV than patients with
loads below 400 (adjusted OR, 7.08; 95% CI, 1.21 to 41.46). In
contrast, patients whose HIV loads were below 400 copies/ml
were more likely to express HHV-8 and EBV than those with
HIV loads over 400 (adjusted OR, 4.08; 95% CI, 1.23 to 13.59)
or between 400 and 5,000 copies/ml, respectively (adjusted
OR, 0.11; 95% CI, 0.01 to 0.97).
CD4 count was predictive of one HHV in saliva. An
increas-ing CD4 count significantly protected patients from expressincreas-ing
EBV in saliva (adjusted OR, 0.998; 95% CI, 0.997 to 1.00).
Oral lesions.
Seven HIV-positive patients had oral lesions
(Table 4). Two had hairy leukoplakia of the tongue, one had an
aphthous-like ulcer on the labial mucosa, and the remainder
had erythematous patches of the tongue and palate consistent
with erythematous candidiasis. All salivas from these patients
were positive for EBV, and none were positive for HSV-1.
CMV was detected in highest copy number for the patient
having an erythematous lesion of the palate. EBV was detected
in high copy number for both patients with hairy leukoplakia.
HHV-8 was detected in highest copy number for the patient
having an ulcer of the labial mucosa and in low copies for two
patients with nonulcerative oral lesions. The presence of oral
lesions was not associated with immunological parameters or
HIV loads (data not shown).
DISCUSSION
This study represents the first report of the prevalence,
loads, risks, and correlates of multiple salivary HHVs in
HIV-seropositive patients who lacked clinical evidence of KS during
the HAART era. Our analyses demonstrate that HSV-1, EBV,
CMV, and HHV-8 were more prevalent and EBV loads were
significantly higher in the saliva of HIV-positive patients than
in that of matched healthy controls. The risk for salivary HHV
shedding related to immune status but not demographic
find-ings. Immunosuppression increased the likelihood for the
in-dividual presence of HSV-1 and CMV (CD4 count less than
400 cells/mm
3) and the simultaneous presence of CMV with
EBV (CD4 count less than 200 cells/mm
3). In contrast, CD4
counts greater than 200 cells/mm
3were significantly associated
with the detection of HHV-8 (
P
ⱕ
0.009).
HSV-1, EBV, CMV, and HHV-8 were more prevalent in
HIV-infected patients than in the age- and sex-matched
con-trols. A 4- to 26-fold-increased odds of HHV shedding in the
saliva of HIV seropositive patients was observed. The
frequen-cies of detection in the HIV-positive and HIV-negative groups
are consistent with those previously reported, indicating that
HHVs are frequently shed asymptomatically in the saliva of
HIV-infected patients who take HAART (3, 4, 6, 19, 37, 38, 44,
68). The higher prevalence in the HIV-positive group may
reflect altered health status or differences in herpesvirus
sero-positivity between the groups, particularly for CMV.
In prior studies of HIV-infected patients without KS or
AIDS, the detection rates in saliva for HSV-1/HSV-2 were
24% (4, 20), EBV, 71% to 93% (6, 18, 24, 42, 43), CMV, 17%
to 77% (19, 20, 25, 44), and HHV-8, 0% to 57% (3, 4, 11, 13,
36–38, 56, 68, 71). Most prior studies examined for individual
HHVs in saliva, not their simultaneous presence, and oral
lesions may have been present or not reported (44). In this
cohort, 88% of the HIV-positive patients were free of
demon-strable oral lesions. Thus, these findings help clarify the
prev-alence of HHV shedding in the saliva of HIV-positive patients
who are asymptomatic, not taking antiherpes medications, and
in large part lacking oral mucosal lesions. Interestingly, all
patients who had oral lesions had EBV DNA in their saliva.
Salivary EBV loads were high for the two HIV-positive
pa-TABLE 4. Relationship of oral lesions and HHVs in saliva
Oral lesion Clinical diagnosis
Viral genome copy no.a
CD4 count CD4/CD8 ratio
HSV-1 EBV CMV HHV-8
Ulcer, mandibular labial mucosa
Aphthous
0
24,042
144
1,087,543*
332
0.1
Erosion, lateral palatal vault
Erythematous candidiasis
0
6,588,386
9,584*
0
42
0.05
Erythema, midline soft palate
Erythematous candidiasis
0
121
751
0
24
0.03
Erythema, dorsum of tongue
Median rhomboid glossitis
0
19
0
0
171
0.2
Erythema, dorsum of tongue
Median rhomboid glossitis
0
3,041,076
0
27
344
0.5
White patches, bilateral, tongue
Hairy leukoplakia
0
1,127,921
677
365
0.2
White patch, dorsolateral, tongue
Hairy leukoplakia
0
41,486,288**
0
0
161
0.1
aⴱ, highest copy number detected in entire study population;ⴱⴱ, second-highest copy number detected in entire study population.
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[image:4.585.42.543.83.179.2]tients who had hairy leukoplakia, and the highest observed
viral copy numbers of CMV and HHV-8 were from patients
with oral lesions. These findings suggest that interactions with
HHVs may contribute to the persistent nature of certain
mu-cosal lesions.
To date, few studies have reported HHV loads in the saliva
of HIV-infected patients. We found that salivary HHV loads
varied greatly within the population, and HSV-1 loads (i.e.,
geometric mean) tended to be at least 10-fold higher than
CMV and HHV-8 loads for both seropositive and
HIV-negative patients. EBV loads were the highest of the four
HHVs examined in saliva of HIV-positive persons (i.e., about
2 logs higher than loads for healthy controls). This is consistent
with data in a previous report (6) and suggests that EBV not
only is more prevalent but replicates more freely in the oral
cavity of HIV-positive patients. Although EBV replicates in
oropharyngeal tissue, we did not observe a correlation with
the presence of pharyngeal tonsils. This may be due to the
presence of additional replication sites, such as palantine
and lingual tonsillar tissue in the oral cavity, the recruitment
of EBV-infected lymphocytes to sites of chronic periodontal
inflammation, or other undetermined factors. Unlike the
findings of others, we did not observe a correlation of EBV
load (or any HHV load) with the number of CD4
⫹cells in
the peripheral blood (43). However, EBV and HSV-1 loads
increased with increasing HIV loads, and the HHV-8 load
correlated with CD8 count. HHV-8 has been detected in
CD8
⫹T cells (26, 62), which raises the possibility that the
saliva of HIV-infected patients contains HHV-8-infected T
cells or the cytokines they secrete may enhance HHV-8
replication.
HAART is known to diminish HHV-related diseases (29),
but its effects on asymptomatic HHV shedding have not been
fully explored. Gandhi et al. reported a trend toward increased
HHV-8 salivary shedding with HAART use (21), but few other
reports exist. In our study, 81% of HIV-positive patients were
taking HAART, yet 97% of HIV-positive individuals had at
least one HHV DNA detected in saliva and 71% had at least
two HHVs in saliva. HHV loads and prevalence profiles were
similar whether patients were taking HAART or not (data not
shown). These data, combined with our previous findings (48),
suggest that HAART, with its targeted specificity, does not
significantly influence the rate or load of asymptomatic
shed-ding of HHVs in saliva. Thus, control of HHV-related diseases
is most likely through HAART’s indirect effect on immune
reconstitution.
Immune function helps regulate the clinical appearance of
HHVs and the development of HHV-related diseases. In this
study, HSV-1, EBV, and CMV were detected individually
more often in saliva at lower CD4 counts (
⬍
400 cells/mm
3),
and CMV and EBV were detected simultaneously significantly
more often when CD4 counts were
⬍
200 cells/mm
3. In
con-trast, HHV-8 was more likely to be present alone or together
with CMV and EBV when CD4 counts were above 200 cells/
mm
3(
P
⫽
0.003). Higher prevalence of HSV-1, EBV, and
CMV in saliva at low CD4 counts has been previously observed
(19, 25). Likewise, the finding that HHV-8 is more likely to be
present in saliva at higher CD4 counts is consistent with the
recent findings of Gandhi et al. (21). Together, these findings
suggest that CD4 cells can contribute to the control of select
oral HHVs (i.e., HSV-1, EBV, and CMV), possibly within
germinal centers of oropharyngeal tissues, where CD4 cells
form cognate interactions with B cells (22, 34). In contrast, the
presence of CD4 and CD8 cells may enhance HHV-8
replica-tion in other cell types as suggested by Gandhi et al. (21). This
could be biologically significant in that HHV-8 may rely on
cytokines produced by immune cells or the presence and
in-teractions of CD4 and CD8 cells for efficient replication. These
interactions could be in contrast to the regulation of HSV-1,
CMV, and EBV replication provided by cytokines (i.e.,
inter-feron) and the immune response at oropharyngeal and more
distant latent sites (8, 12, 47).
In summary, our observations suggest that EBV and HHV-8
expression in saliva is a frequent event in HIV-infected
indi-viduals in the presence of HAART therapy. The frequency and
viral loads detected are suggestive that HHVs in persons with
adequately controlled HIV infection could be transmitted by
contact with saliva, and risk for transmission may be greater
during certain stages of HIV infection. These data increase our
understanding of the correlates between asymptomatic HHV
shedding and health status and provide a basis for future
stud-ies that can investigate if salivary levels of HHVs are useful for
monitoring disease states and risk for progression of
HHV-related malignancies.
ACKNOWLEDGMENTS
This work was supported by grants DE15070-01, DE014142, and
RR02602 from the National Institutes of Health.
We thank K. Westberry and the General Clinical Research Core
(GCRC) nursing staff for excellent technical assistance and R.
Dana-her for helpful discussions.
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