Affiliations of authors: M. Andreoni, E. Nicastri, L. Ventura, L. Ercoli, L. Sarmati, G. Rocchi, Infectious Diseases Institute, University of "Tor Vergata," Rome, Italy; G. El-Sawaf, University of Alexandria, Egypt; G. Rezza, Centro Operativo AIDS, Istituto Superiore di Sanità, Rome; B. Ensoli, Laboratory of Virology, Istituto Superiore di Sanità, Rome.
Correspondence to: Massimo Andreoni, M.D., Department of Public Health and Cellular Biology, University "Tor Vergata," Via di Tor Vergata 135, 00133 Rome, Italy (e-mail: andreoni{at}uniroma2.it).
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ABSTRACT |
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INTRODUCTION |
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Among HIV-infected individuals, homosexual men appear to be at higher risk of HHV-8 infection than intravenous drug users, individuals with hemophilia, and women (8,9). These data and the detection of HHV-8 DNA in semen (17) suggest that the virus may be sexually transmitted.
The extent to which HHV-8 is a ubiquitous infectious agent is still unclear. It is not known whether HHV-8 may also be transmitted by routes similar to those of other herpesviruses and whether the mode of transmission may differ according to geographic and socioeconomic settings. Studies of blood donors have shown little or no infection among U.S. blood donors and have shown intermediate and high prevalence rates among Italian blood donors and Ugandan blood donors, respectively (7). Studies of children conducted in the United States have failed to detect HHV-8 infection, as defined by viral sequences in the peripheral blood mononuclear cells (18), or have shown a low prevalence or the absence of anti-lytic-phase antibodies with no anti-latent-phase antibodies (9,10,18). In contrast, HHV-8 DNA sequences have been detected in a high proportion of Japanese children (19). To our knowledge, limited data are available for children from developing countries (20), where the prevalence of HHV-8 in adults is high (7,9).
In industrialized western countries, the findings reported above suggest that HHV-8 is mainly transmitted through sexual intercourse, where most infections have been detected in homosexual men; yet these findings also suggest that other routes of transmission may play a role, especially in developing countries, where the infection appears to be widespread.
To test the hypothesis that HHV-8 infection may be acquired largely before an individual reaches a sexually active age, we evaluated the prevalence of anti-lytic and anti-latent HHV-8 antibodies in Egyptian children. To better evaluate the epidemiologic pattern, we also studied the seroprevalence of HHV-8 and other herpesviruses that are known to be widespread and are usually acquired early in life in developing countries.
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MATERIALS AND METHODS |
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Serum samples were taken from 246 apparently healthy individuals recruited at the Medical Research Institute of the University of Alexandria, Egypt, where they were attending a vaccination program. Consecutive children, adolescents, and young adults (<25 years old) attending the vaccination program were recruited until approximately 40 individuals recruited for each age group was reached. Demographic and clinical information on the participants was recorded on standardized forms. All participants were HIV seronegative.
Serologic Assays
Antibodies to lytic and latent antigens of HHV-8 were detected by using two different immunofluorescence assays (IFAs) based on the BCBL-1 (body cavity B-cell lymphomas) cell line (obtained through the acquired immunodeficiency syndrome [AIDS] Research and Reference Reagent Program, Division of AIDS, National Institutes of Health). The BCBL-1 cells were grown in RPMI-1640 medium with 10% heat-inactivated fetal calf serum, antibiotics (penicillin at 100 U/mL and streptomycin at 100 µg/mL), and 5 x 10-5 M 2-mercaptoethanol.
For the IFA to anti-lytic antigens, BCBL-1 cells were treated for 48 hours with phorbol 12-myristate 13-acetate at 20 ng/mL. Ten microliters of a cell suspension (4 x 106 cells/mL) was smeared on slides, air-dried at room temperature, and then fixed in methanol/acetone, 1 : 1 (vol/vol), at -20 °C for 10 minutes.
An IFA for anti-latent antigens was performed on isolated nuclei of BCBL-1 cells. Briefly, cells were washed twice with phosphate-buffered saline (PBS), resuspended in 10 mL of buffer containing 5% citric acid, and layered on 15 mL of 0.88 M sucrose in 5% citric acid. Nuclei were pelletted by centrifugation at 5000g for 5 minutes at 4 °C, washed with PBS until the pH of the suspension was 7.4, and resuspended in PBS containing 0.25% fetal calf serum to a concentration of 1.5 x 106 nuclei/mL. Twelve microliters of the suspension was then smeared on slides, air-dried at room temperature, and fixed in methanol/acetone at -20 °C for 10 minutes. For IFA, fixed smears were preblocked by incubation with PBS containing 3% fetal calf serum for 30 minutes in a humidified chamber and then incubated sequentially for a 45-minute period at 37 °C with the test serum diluted 1 : 10 (in PBS containing 1% glycine and 2% fetal calf serum) and for a 45-minute period at 37 °C with fluorescein isothiocyanate-conjugated goat anti-human immunoglobulin antibodies. Titrations were done by 1 : 4 serial dilutions. An inverse titer of 10 or more was considered positive. All microscopic examinations were evaluated by investigators blinded to the status of the specimens.
Quantitative detection of antibodies to Epstein-Barr virus (EBV) viral cap antigen, cytomegalovirus (CMV), and HHV-6 were done by immunoenzymatic assays (viral cap antigen immunoglobulin G enzyme-linked immunoabsorbent assay and CMV immunoglobulin G enzyme-linked immunoabsorbent assay, Gull Laboratories, Inc., Salt Lake City, UT; Milenia HHV-6 immunoglobulin G, Diagnostic Products Corporation, Los Angeles, CA).
Italian blood donors and patients with KS were included as reference groups.
Data Analysis
The prevalence of antibodies directed against herpesviruses was calculated after stratifying by age (<1, 1-3, 4-6, 7-9, 10-12, and >12 years). The participants were also stratified by anti-lytic HHV-8 antibody titer (<1 : 10 or negative, 1 : 10, 1 : 50-1 : 200, and >1 : 400 dilution) and by age. The odds ratio (with 95% confidence intervals) of having anti-latent HHV-8 antibodies for individuals with different anti-lytic titers was also calculated.
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RESULTS |
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In the study population, anti-HHV-6 antibodies were largely detected in the first year of life; then an almost 50% decrease in the seroprevalence was observed, with a symmetric dramatic increase after the children reached 10 years of age. Anti-CMV and anti-EBV antibodies had a similar pattern of increase; at least 70% of the children were exposed to these viruses within their 3rd year of life, and seroprevalence for CMV approached 100% in late childhood.
The distribution of anti-lytic antibody titers to HHV-8 by age group is reported in Table 2. The proportion of children with high titers (>1 : 400 dilution) of
anti-lytic antibodies was higher in the age group of 1-3 years compared with older individuals.
Participants with high anti-lytic titers were more likely to have anti-latent antibodies than those
with low-intermediate titers. Anti-latent antibodies were detected in 33.3% (seven of 21)
of children with high titers and in 15.1% (eight of 53) and 16.7% (six of 36) of
those with intermediate (1 : 50-1 : 200 dilution) and low (1 : 10 dilution) titers, respectively.
Although the risk of having anti-latent antibodies was 2.68 times higher among those participants
with high anti-lytic titers than among the other HHV-8-positive participants, the difference was
not statistically significant (95% confidence interval = 0.81-8.83).
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DISCUSSION |
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We do not know the extent to which our study population is representative of all children of the urban area of Alexandria. However, we recruited a convenience sample of consecutive children who were equally distributed across the age groups attending the vaccination program and in the process tried to avoid any selection mechanism likely to introduce possible bias; thus, the results can be considered potentially generalizable.
The variable magnitude of the increase in HHV-8 seroprevalence suggests that most primary infections occur during the second year of life. This finding is consistent with the high proportion of 1- to 3-year-old children with high antibody titers, which may be representative of a recent infection. Anti-lytic antibody prevalence tends to stabilize after children reach the age of 10 years. However, due to the small number of participants who were more than 12 years old, no conclusions about the increase of HHV-8 seroprevalence in adolescents and young adults can be made.
The Egyptian pattern appears to differ from that determined in studies conducted in the United States that showed a low prevalence of anti-lytic antibodies (9) and the absence of either anti-latent antibodies to HHV-8 (9,10) or anti-lytic and anti-latent antibodies and DNA sequences (18) in children. The difference observed in the results of studies carried out in children from Egypt and the United States can be compared with serologic studies among adults, reporting higher prevalences of HHV-8 in some developing areas, such as Central Africa compared with some developed areas of the world, where a small proportion of blood donors, or none at all, was found to be seropositive (7,9,10).
With regard to routes of transmission, findings (8,9,14) by others suggest that in industrialized countries, HHV-8 infection is likely to be acquired after adolescence, during the sexually active phase of life, and it is mainly transmitted through sexual intercourse. Our findings suggest that in developing countries, the infection appears to be acquired early in life, as a possible consequence of transmission in the family and in community settings. It has been recently reported that infectious virus and viral RNAs may be detected in the saliva of patients without KS (23), and it has been suggested that HHV-8, like EBV, undergoes lytic replication in oropharyngeal cells (24). However, it may be possible that HHV-8 is less transmissible through the saliva than other herpesviruses, such as EBV and CMV; for this reason, nonsexual transmission may play an important role only when particular environmental conditions prevail (i.e., in the presence of overcrowding and poor hygiene and primarily in areas where the background HHV-8 prevalence in the general population is high). It is thus possible that the impact of specific transmission modalities may vary across different geocultural and socioeconomic contexts.
Comparison with other widespread herpesviruses may provide additional support for these hypotheses. In Egypt, the existence of environmental conditions favoring the spread of infections requiring nonsexual intimate contact is confirmed by a seroepidemiologic pattern that is typical of underdeveloped and tropical areas (25) and population groups of low socioeconomic level (26), characterized by a high prevalence of viral capsid antigen antibodies in early childhood. At the same time, HHV-8 seroprevalence appears to be lower than that of other herpesviruses that are mainly transmitted through saliva, such as HHV-6, EBV, and CMV.
Another aspect of our study that merits consideration is the relationship between HHV-8 and KS. If an association, suggested by longitudinal studies, is assumed, we would expect that, in a determined geographic area or population group, a higher KS incidence would reflect a higher HHV-8 seroprevalence. To date, the paradigm of this ecologic association has been confirmed by studies showing a parallel increasing level of HHV-8 prevalence and KS incidence from the United States to Mediterranean countries, such as Italy, to sub-Saharan Africa (7). In contrast, Egypt appears to be a country with a low incidence of KS. KS represents less than 1% of all the cancers diagnosed in the country and a minority of all skin tumors (27). Thus, the high prevalence of HHV-8 in Egyptian children is not consistent with the low incidence of KS, suggesting that other cofactors play an important role in the pathogenesis of KS in persons who have no known causes of immunosuppression, such as HIV infection or organ transplants. The hypothesis of different virus variants with different pathogenic spectrums should also be investigated (28). However, the possibility that KS incidence in Egypt is underestimated cannot be ruled out, and studies conducted among Egyptian transplant recipients suggest that the relative frequency of KS is slightly higher than in other international series (29). For this reason, caution is needed in interpreting the apparent dissociation between the presence of HHV-8 and KS rates.
In conclusion, our data show that HHV-8 infection is widespread among Egyptian children, suggesting that sexual activity is not the only route of transmission in developing countries. The high prevalence of individuals exposed to the virus is in apparent contrast with the low incidence of KS reported in Egypt. Other cofactors might be involved in the pathogenesis of KS in HIV-negative individuals.
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NOTES |
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