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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,

3

Yunanan Mootoor,

3

Sergei A. Avdiushko,

2

Hua Zhu,

4

and Richard J. Kryscio

4

Department of Microbiology, Immunology & Molecular Genetics,

1

Department of Oral Health Practice,

2

Departments of

Neurology and Internal Medicine,

3

and Department of Statistics,

4

University 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

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

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 10␮l 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 10␮l 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

[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

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

3

than 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

3

than 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

3

versus CD4 counts of

400 cells/mm

3

;

**

,

P

0.026 for comparing CD4 counts of 201 to 399 cells/mm

3

versus CD4 counts of

400 cells/mm

3

;

[image:3.585.60.266.497.660.2]

***

,

P

0.002 for comparing

CD4 counts of

200 cells/mm

3

versus 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

c

HIV

(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. b,P0.002.

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patients with a CD4 count between 200 and 399 cells/mm

3

were 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

3

were significantly associated

with the detection of HHV-8 (

P

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

a, highest copy number detected in entire study population;ⴱⴱ, second-highest copy number detected in entire study population.

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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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Figure

TABLE 1. Demographic characteristics of the study populationa
TABLE 2. HHV genome copy numbers in saliva
TABLE 4. Relationship of oral lesions and HHVs in saliva

References

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