Human herpesvirus type 6 is a widespread beta-herpesvirus that was initially isolated in 1986 from patients with lymphoproliferative disorders , and initially characterized as a human T-lymphotropic virus . It was officially classified as a member of the Herpesvirales order, Herpesviridae family, Beta-herpesvirinae subfamily and Roseolovirus genus, together with human herpesvirus 7 (HHV-7), a closely related herpesvirus discovered in 1990 . HHV-6 infects a wide range of human cells in vitro, but it preferentially replicates in activated CD4+ T lymphocytes which are infected through their CD46 receptor . The HHV-6 is ubiquitous being detected in all human populations around the world, as reviewed by (Stone et al., 2014) . As a whole, HHV-6 infection is detected in more than 90% of adult populations in developed countries, although the seroprevalence may reveal significant differences according to geographic location, age, and sensitivity and specificity of serologic assays . The HHV-6 infection is usually acquired very early in life, between 6 months and 2 years of age, following the loss of protective maternal antibodies . Saliva is assumed to be the main vehicle for virus transmission, as supported by the frequent detection of HHV-6 in saliva and salivary glands . Congenital infection following intrauterine transmission occurs in about 1% of children, and perinatal transmission has also been described . The congenital infection is mainly linked with chromosomally integrated HHV-6 in mothers .
Primary HHV-6 infection has been found to be the cause of febrile illness, roseola infantum, in 10% to 45% of infants. A population-based study indicated that 40% of HHV-6 infection occurs by age 12 months and 77% by age 24 months. It was more common in females and children with older siblings . The peak incidence of the virus is in the spring and fall seasons  . Three stages were recognized in the natural history of HHV-6 infection; the first is represented by acute primary infection in infants . Detectable viremia is generally considered the hallmark of a systemic active infection and the whole blood is the most valuable specimen for detecting such viremia by real-time PCR  . The second one occurs in healthy children and adults; the virus replicates in the salivary glands and is secreted in saliva without inducing any obvious pathology, remains latent at least in lymphocytes and monocytes, and persists in various tissues, possibly with a low-level replication . The third stage occurs infrequently, typically in immunocompromised persons, and is linked to reactivation of virus from latency or reinfection  .
Anti-HHV-6 IgG as a marker of past or latent infection, different studies had yielded variable results. Among healthy Thai children aged 0 to 12 years, the anti-HHV6 IgG using an ELISA method was 88.10% . In Italian people, seropositivity of 83.7%, 92.6% and 63.6% were found in subjects aged 3 months - 6 years, 6 - 18 years and over 18 years respectively . In Greece HHV-6 seropositivity among blood donors was 78.7% and the seroprevalence did not differ between males and females or among different decade age groups . In another study on blood donors, 34% were positive for IgG anti-HHV6 and with titers ranged from 1:40 to 1:160 . It has been affirmed that Antibody responses after exanthem subitum were well correlated with clinical recovery from the disease . This study was conducted to explore the anti-HHV-6 IgG positivity rate among apparently healthy infants and to figure out the effect of certain risk factors.
2. Subjects and Methods
This is a part of larger cross sectional study conducted in Diyala province during the period from August 2017-July 2018. A total of 180 apparently healthy (clinically they were free from any sign or symptoms) infants were included. They were collected from those attending for routine vaccination. Their ages ranged between 6 - 24 months. They consist of 100 males with mean age ± SD 15.05 ± 6.42 months and 80 females with mean age ± SD 15.56 ± 6.66 months. Human privacy was respected by obtaining parental consent. Venous blood samples were collected aseptically from each participants. Sera were separated and tested for the anti-HHV-6 IgG (Sunlong Biotech, China) by Enzyme Linked Immunosorbant assay (ELISA) technique. Human privacy was respected by taken the parents consent. Furthermore, the study was permitted by the Ethical Commity in the College of Medicine-Diyala University. Statistical analysis was done using SPSS version 23 and P value < 0.05 was considered significant. The frequency distributions for selected variables were done first. To measure the strength of association between 2 variables, such as the presence of certain explanatory variable (or risk factor) and a positive antibody test the odds ratio (OR) was used. The statistical significance of the measured OR is assessed by a special χ2 formula. The relative risk is the real measure of association between exposure to a certain factor and having positive antibody. The calculated OR is the best estimate for the real measure of relative risk (RR). The 95% confidence interval of an estimate gives an idea about the expected range the parameter in the target population with 95% confidence.
The results found that the overall positivity rate of anti-HHV6 IgG among apparently healthy infants was 43.9%. The distribution according to age groups found that the highest positivity rate was among children 19 - 24 months (59.6%), followed by the age group 13 - 18 months (53.3%), and lastly among the age group 7 - 12 months (31.2%). However, the difference was statistically insignificant (Table 1).
The anti-HHV6 positivity rate was slightly higher in males compared to females (44.0% versus 43.8%) and the difference was statistically insignificant (P = 0.973), Table 2.
According to the type of feeding, the results found that mixed feeding (55.3%) and bottle feeding (42.7%) were insignificantly higher than the breast feeding (34.4%), Table 3.
The anti-HHV6 IgG positivity rate among infants who had previous hospitalization was lower than that of children with negative history (26.9% versus 46.8%), but the difference was failed to reach the levels of statistical significant, Table 4.
The results in Table 5 revealed that infants with currently positive family history had significantly higher anti-HHV6 IgG positivity rate compared to those children with negative history (62.2% versus 35.8%, P = 0.006). On the other hand,
Table 1. Anti-HHV6 IgG positivity rate by age groups.
Table 2. Anti-HHV6 IgG positivity rate by gender.
Table 3. Anti-HHV6 IgG positivity rate by type of feeding.
Table 4. Anti-HHV6 IgG positivity rate according to previous hospitalization.
Table 5. Anti-HHV6 IgG positivity rate according to family history.
infants with positive past family history were also had higher anti-HHV6 IgG positivity rate compared to its negative counterpart, but the difference was failed to reach the levels of statistical significance (50.0% versus 35.8%, P = 0.141).
Like other herpesviruses, HHV-6 causes a primary infection at early infancy life followed by latency and persistence of the virus in various body tissues with frequent reactivation under immunocompromised conditions . During the second stage, latently infected children and adults clinically appear healthy while the virus replicates in the salivary glands and is secreted in saliva without inducing any obvious pathology, and persists possibly with a low-level replication . As the virus persists inside the body, the infants or children are seroconverted from anti-HHV6 IgM to IgG antibodies which last for the life representing the serological marker of past infection  .
In the present study which was conducted on apparently healthy infants aged 6 - 24 months, the serum anti-HHV6 IgG was 43.9% as detected by ELISA technique. In this regard, different studies had yielded variable results. However, generally the result of the current study is low compared to other studies  . This low anti-HHV 6 IgG positivity rate may be due to multi-factorials; for instance, it was found that infection with HHV 6 A variant provoked low anti-HHV 6 IgG titers that cannot be detected by serological procedures compared to HHV 6 B . Furthermore, the IgG subclasses, as it was documented that anti-HHV6 IgG was restricted to IgG1, whereas anti-HHV-7 IgG subclasses included two groups, one restricted only to IgG1 and the other to IgG1 and IgG3 . Additionally, the causative agent of primary infection of the clinically suspected roseola infantum cases may be other than HHV6  . It is worth to remember that clinically, the roseola infantum is closely similar to other five clinical conditions among infants and children and thus it is called the sixth disease   . Lastly, the sensitivity and specificity of the employed laboratory test may affect the study outcomes  .
Although it is insignificant, the results also found that the highest anti-HHV 6 IgG positivity rate was among those 19 - 24 months (59.6%). Again in this regard, variable results had been obtained, but generally are consistent with the present results in that the anti-HHV 6 IgG positivity rate is gradually increased by infants or children age  . Moreover, Ongradi et al. (1999)  reported that all infants had by the age of 1 month antibodies to HHV-6, which decreased with age to the lowest level at the age of 3 to 6 months and then increased and reached the maximum level by 1 to 2 years and the prevalence was almost stable after 3 years, affirming that antibody responses after exanthem subitum were well correlated with clinical recovery from the disease and the level of antibody was well correlated with indirect immunofluorescence assay and the neutralization test. Additionally, it was concluded that age is the important factor associated with HHV-6 infection, while sex, socioeconomic status, number of children in the family and child rearing place has no association .
Regarding the association of gender with the anti-HHV6 IgG positivity rate, the insignificant higher positivity rate among males obtained in this study is consistent with (Farshadmoghadam et al., 2014) . On the contrary, Zerr et al., (2005)  found that the acquisition of HHV-6 was associated with female sex. Traditionally, in our community, parents, the older family members and relatives are more attracted to male infants, usually hold them and kiss them several times. Undoubtedly, these unhealthy practices are efficiently responsible for the transmission of HHV 6 to infants through the saliva or other oral fluids  .
A fascinating finding was that the anti-HHV 6 IgG positivity rate was significantly higher among family siblings who had a current positive case. These results is seems logic and consistent with most previous studies  . It has been documented that the risk of HHV 6 infection increase by the number of siblings and the frequency that the child received parental saliva when being kissed . Certainly it is well documented that the HHV 6 is intensively found in saliva and oral fluids, even in healthy population . Probably the most prominent limitations of this study were the limited age group included and limited number of participants due to expensiveness of laboratory material as this study was personally funded.
In conclusion, about one-half of apparently healthy infants up to two years of Diyala population have anti-HHV6 IgG antibodies and the presence of intrafamilial primary HHV-6 positive case is markedly associated with the increased rate of anti-HHV6 IgG among siblings.
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