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Background Serologic studies indicate that human herpesvirus 6 (HHV-6) infects 90 percent of children by two years of age. Little is known about the acquisition, virologic course, and clinical manifestations of HHV-6 infection.
Methods We prospectively studied a cohort of 277 children from birth through the first two years of life to define the pattern of acquisition of HHV-6. The children's saliva was tested weekly for HHV-6 DNA with the use of the polymerase chain reaction. Parents maintained a daily log of signs and symptoms of illness in their children.
Results Primary HHV-6 infection occurred in 130 children, with cumulative percentages of 40 percent by the age of 12 months and 77 percent by the age of 24 months. The peak age of acquisition was between 9 and 21 months. The acquisition of HHV-6 was associated with female sex (adjusted hazard ratio, 1.7; 95 percent confidence interval, 1.2 to 2.4) and having older siblings (adjusted hazard ratio, 2.1; 95 percent confidence interval, 1.4 to 2.9). Among 81 children with a well-defined time of acquisition of HHV-6, 93 percent had symptoms, and 38 percent were seen by a physician. None had seizures. As compared with children who had other illnesses, those with primary HHV-6 infection were more likely to have fever (P=0.003), fussiness (P=0.02), diarrhea (P=0.03), rash (P=0.003), and roseola (P=0.002) and were more likely to visit a physician (P=0.003).
Conclusions The acquisition of HHV-6 in infancy is usually symptomatic and often results in medical evaluation. Roseola occurs in a minority of patients, and febrile seizures are infrequently associated with primary HHV-6 infection. Older siblings appear to serve as a source of HHV-6 transmission.
Methods
This clinical research was conducted in accordance with guidelines for human experimentation specified by the institutional review board of Children's Hospital in Seattle. The institutional review boards of all involved hospitals approved the protocol. Between April 1997 and August 2001, pregnant women were recruited from obstetricians' offices. After the women had provided written informed consent, their infants were followed from birth until two years of age.
Parents were taught to collect saliva by placing five sterile, precut filter-paper strips (Sno strips) into their child's mouth until saturated6 and to wash their hands before collection and avoid touching the collection end of the strips to minimize contamination. Strips were then air-dried, bagged, and mailed to the laboratory weekly. Infants' serum (1 ml) was collected when blood was being drawn for other purposes.
Demographic information (e.g., sex and race or ethnic group) was collected by means of a standardized questionnaire at enrollment. Parents completed a daily symptom report for their child that offered the following choices: no symptoms, fever (temperature >38.0°C), cough, rhinorrhea, vomiting, diarrhea, rash (generalized), fussiness (above the baseline level for the child), seizure, and a visit to a physician (for illness). Roseola was defined post hoc as a febrile illness with a generalized rash on defervescence,7 with symptoms that lasted for at least two days and the appearance of rash on the day of or the day after defervescence. Throughout the study, parents were questioned about whether and how long their child breast-fed and regularly attended child care in a group setting or a playgroup (defined as scheduled group activities with other young children at least weekly).
Polymerase Chain Reaction
Saliva specimens were tested for HHV-6 DNA by means of the polymerase chain reaction (PCR) as previously described.5,8 Briefly, DNA was extracted from the collection ends of the five Sno strips. Qiagen ATL Buffer and Qiagen protease were added to each sample and incubated overnight. Then, Qiagen AL Buffer was added to each sample and the sample was incubated for 10 minutes. The rest of the procedure followed Qiagen's single-column extraction protocol except that the sample was run through the spin column three times at an initial spin speed of 8000 rpm. DNA was eluted into 100 µl of Qiagen AE Buffer, and 20 µl of the solution was tested for HHV-6 DNA with the use of a real-time quantitative fluorescent-probe PCR assay as previously described.5,8 The detection of 1 copy of HHV-6 DNA per reaction, or 80 copies per milliliter of saliva, was considered to indicate positivity. HHV-6 DNA identified by PCR was then further typed as A or B.5
Serologic Studies
IgM and IgG antibodies against HHV-6 were detected by means of Western blotting with the use of a method adapted from Black and colleagues9 and Yamamoto and colleagues.10 Purified HHV-6 antigen (ABI) was separated on sodium dodecyl sulfate–polyacrylamide gel by electrophoresis and then transferred to polyvinylidene fluoride (PVDF) strips. Three PVDF strips were incubated with each serum sample: strip 1 was incubated with test serum in 4 percent goat serum, and strips 2 and 3 were incubated with test serum pretreated with IgG–rheumatoid factor adsorbent (Meridian Bioscience). Horseradish-peroxidase–labeled goat antihuman secondary antibodies (Chemicon) were then added to the strips. Immunoreactive bands were visualized with the use of a chemiluminescent substrate sensitive to horseradish peroxidase (Pierce) as described previously.11 Seroconversion was defined by the detection of serum IgM at any age or by the detection of IgG in a child who had previously undetectable levels of IgG or who was at least 18 months of age.
Statistical Analysis
SPSS (version 11.5) and SAS (version 8.02) statistical-software programs were used for analyses. On the basis of observed patterns of salivary HHV-6 DNA, primary HHV-6 infection was defined as a positive PCR result followed within 28 days by at least one positive test at any level and within 42 days by at least one finding of 800 copies of HHV-6 per milliliter or more, by a positive PCR result followed within 28 days by a finding of at least 800 copies per milliliter, or by an initial result of at least 800 copies per milliliter followed by a finding of at least 800 copies per milliliter at the next measurement. This strategy ensured that children with fewer follow-up data had to have relatively higher levels of HHV-6 DNA to be counted as infected. Positive specimens that did not meet the definition of primary infection were excluded (treated as missing). In the case of 13 children (15 specimens) the final specimens were positive but the date of acquisition could not be determined; for these children, follow-up data were included only through the date on which the last negative specimen was obtained.
The age at the time of primary HHV-6 infection was analyzed with the use of Kaplan–Meier survival analysis and Cox regression. The time of acquisition was calculated as the midpoint between the last negative and the first positive saliva specimen. (Estimates of acquisition rates were nearly identical when the time of the first positive specimen was used for the time of acquisition.) Data on children who did not acquire HHV-6 were censored at the time of the last negative saliva specimen.
Factors associated with the acquisition of HHV-6 were examined by means of Cox regression with backward elimination for the final model selection. Because information on the use of breast-feeding or participation in a child-care group or a playgroup was not available for some children, sensitivity analyses were performed in which missing values were designated as either "yes" or "no." None of these transformed variables were significantly associated with the acquisition of HHV-6. Children who acquired HHV-6 were followed significantly longer than those who did not. Survival analysis was based on the assumption that children who discontinued follow-up before the acquisition of HHV-6 did so for reasons unrelated to their risk of acquisition. We find this assumption plausible and do not suspect that there was bias in our evaluation of risk factors for HHV-6.
Analyses of symptoms associated with primary HHV-6 infection included only the 81 children for whom the time of acquisition of HHV-6 was well defined — that is, only those whose last negative test and first positive test for salivary HHV-6 were no more than 14 days apart. No descriptive characteristics were found to differ significantly between the 81 children who were included in the analysis and the 49 children who were excluded. To identify HHV-6–associated symptoms while taking into account PCR sampling frequency, we evaluated the period spanning from two weeks before to one week after the first detection of HHV-6 DNA for symptoms.
To determine whether symptoms were associated with HHV-6 acquisition, we compared 80 of the 81 children for whom the time of acquisition was well defined with 80 children age-matched to the day, drawn at random from the 147 children who had no apparent acquisition of HHV-6 and for whom there were complete symptom data at the age that the corresponding patient had primary HHV-6 infection. Differences were examined with the use of conditional logistic regression.
To determine which symptoms were associated with primary HHV-6 infection rather than other infectious illnesses, we compared symptoms that occurred during the acquisition of HHV-6 with those occurring during the closest episode of illness before and after acquisition in the 81 children with a well-defined time of HHV-6 acquisition. Generalized estimating equations with a logit link were used to test the hypothesis that symptoms during primary HHV-6 infection differed from those during other illnesses.
The duration of symptoms occurring during primary HHV-6 infection and at other, control times in the 81 children with a well-defined time of acquisition was compared with the use of the Wilcoxon signed-rank test and a case-crossover design.12 Control episodes were random episodes of illness during a period other than that in which primary HHV-6 infection occurred. For each symptom, only children who had the symptom in both periods were included in the analysis.
To identify correlates of febrile primary HHV-6 infection as compared with afebrile infection, we performed multivariate logistic regression using data from the 81 children with a well-defined time of acquisition of HHV-6. All reported P values are two-sided.
Results
Of the 277 infants enrolled in the study, 46 percent were girls, 80 percent were white, and 52 percent had at least one sibling (Table 1). The median follow-up was 60 weeks (range, 1 to 113), and a median of 33 saliva samples per child (range, 1 to 106) were available for testing. Follow-up was longer for children who were infected with HHV-6 (median, 79 weeks; range, 14 to 113) than for those who were not (median, 39 weeks; range, 1 to 107; P<0.001 by the Mann–Whitney U test). Similarly, more samples were available from children who became infected with HHV-6 (median, 51; range, 5 to 106) than from those who did not become infected (median, 16; range, 1 to 105). At least one sample was available from 219 infants within the first four weeks after delivery (79 percent).
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Primary HHV-6 infection, defined by the onset of persistent salivary levels of HHV-6 DNA, occurred in 130 children. The incidence of infection was highest between 9 and 21 months of age (Figure 1), with a cumulative incidence of 40 percent by 12 months of age and 77 percent (95 percent confidence interval, 67 to 88) by 24 months of age. In three infants, HHV-6 DNA was detected during the first week of life, suggesting in utero infection. In an additional six infants, the first sample was also positive for HHV-6 DNA; however, they were all older than two weeks of age at the time. Hence, whether they acquired HHV-6 in utero or during the perinatal period is unclear. These nine children were not included in analyses of symptoms, because the timing of acquisition was imprecise.
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A total of 183 serum specimens were obtained from 159 children for HHV-6 antibody testing. Serologic results were available near the time of primary HHV-6 infection in the case of 7 of the 130 children who were identified as having HHV-6 infection on the basis of salivary PCR. In all seven, serum HHV-6 IgM and IgG were both detected. An eighth child had evidence of seroconversion in the absence of detection of salivary HHV-6. In this two-year-old child, HHV-6 IgM was detected, but serum HHV-6 IgG and salivary HHV-6 DNA were not; follow-up samples were not available.
Factors Associated with HHV-6 Acquisition
Female sex and having at least one older sibling were independently associated with the acquisition of HHV-6 (Table 1). The age of the mother at the time of delivery, breast-feeding, and playgroup or child-care attendance were not significantly associated with the acquisition of HHV-6. In addition, the season did not appear to be associated with acquisition. Of the 130 children who acquired HHV-6, 33 (25 percent) did so during the fall, 38 (29 percent) during the summer, 30 (23 percent) during the spring, and 29 (22 percent) during the winter (P=0.68). To evaluate further the potential of seasonality, we also compared the risk of acquisition of HHV-6 over time according to the season of birth. Examination of resulting Kaplan–Meier plots showed no significant difference among the four survival curves (P=0.64).
Clinical Manifestations of Primary HHV-6 Infection
Of the 81 children with a well-defined time of acquisition of HHV-6, 93 percent had symptoms. Fever, fussiness, and rhinorrhea were present in most children at the time of HHV-6 acquisition (in 57 percent, 69 percent, and 65 percent, respectively), whereas cough, diarrhea, and rash occurred less frequently (in 33 percent, 26 percent, and 31 percent, respectively). None of the 81 children had seizures at the time of acquisition. Roseola, a clinical syndrome that is relatively specific for HHV-6 infection, occurred in 19 of the 81 children (23 percent). The onset of roseola followed the initial detection of salivary HHV-6 by less than one week in 11 of these children (58 percent), by one to two weeks in 6 (32 percent), and by no more than three weeks in 2 (11 percent). Thirty-one children with primary infection (38 percent) were evaluated by a physician for an illness near the time of acquisition of HHV-6.
To determine whether the acquisition of HHV-6 was significantly associated with symptoms, we compared children who had well-defined primary HHV-6 infection with age-matched controls who never acquired HHV-6. Children with primary HHV-6 infection were more likely than controls to have fever, fussiness, rhinorrhea, diarrhea, rash, and roseola and were more likely to have seen a physician for illness (Table 2).
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Factors Associated with Fever during HHV-6 Acquisition
Because fever occurred in only 57 percent of children who acquired HHV-6 and because fever is a clinical sign often prompting medical evaluation in young children, we explored correlates of febrile primary HHV-6 infection. Children with febrile HHV-6 infection were more likely to be older than those who were afebrile: 43 of 46 children with fever were older than six months, as compared with 23 of 35 without fever (93 percent vs. 66 percent, P=0.004). The association between febrile infection and age remained significant after adjustment for covariates (adjusted odds ratio, 7.2; 95 percent confidence interval, 1.7 to 30.7) (see Table 1 in the Supplementary Appendix, available with the full text of this article at www.nejm.org).
Discussion
Our prospective, population-based study yielded information on the clinical and virologic aspects of primary HHV-6 infection. We found that most children with primary HHV-6 infection had symptoms and almost half visited a physician, whereas roseola accounted for a minority of cases and seizures were not reported. Salivary levels of HHV-6 DNA were relatively low early during primary infection, increased in the subsequent weeks, and remained at high levels during the months of follow-up. Having siblings, but not attending group child care, was associated with the acquisition of HHV-6, suggesting that intimate contact is probably involved in the transmission of the virus.
The cumulative incidence of primary HHV-6 infection in this population was 77 percent by two years of age. The majority of children with primary HHV-6 infection had symptoms such as fever, fussiness, diarrhea, rash, and roseola. In contrast to the results of studies performed in emergency departments,2,13 in which fever was an inclusion criterion, we found that 43 percent of children with primary HHV-6 infection were afebrile, 7 percent were asymptomatic, and children older than six months were more likely than younger children to have fever with primary infection. Also in contrast to emergency department–based studies, in which seizures occurred in as many as 13 percent of children with primary HHV-6 infection,2 no child in our study had a seizure near the time of acquisition of HHV-6. Despite this finding, primary HHV-6 infection appears to cause a clinical illness that is relatively worrisome to parents, and perhaps physicians, since 20 percent of the emergency visits for fever were due to primary HHV-6 infection2 and substantially more children with primary HHV-6 infection than children with other types of illnesses saw a physician.
We used a new, nonserologic method to detect the acquisition of the virus that was based on the development of sustained, high levels of salivary HHV-6 DNA. Salivary shedding appeared to follow the onset of symptoms, and initial values tended to be low, rising and then plateauing over time. A salivary test for HHV-6 could be useful in the diagnosis of primary HHV-6 infection if combined with other approaches such as a plasma test for HHV-6 DNA.
Monitoring this cohort of infants for HHV-6 infection from a single geographic region required intensive parental involvement without remuneration. It is possible that in other populations, epidemiologic and pathologic features of HHV-6 could be different and that factors such as breast-feeding or group child care could play a role in acquisition. It is also possible that our method of testing saliva may have missed some cases of HHV-6 infection. The amount of saliva collected on Sno strips is small and less than that obtained by expectoration.6 The reduction of salivary viral levels to below the limit of detection in a number of children in the first weeks after their initial positive specimen may indicate that weekly sampling with Sno strips missed the onset of infection in some children. When developing a method to screen infants for this study, we were aware that the sensitivity of Sno-strip samples was less than that of expectorated samples from adults.6 This lower sensitivity was balanced, however, by a lower frequency of PCR inhibition. In addition, the use of Sno strips enabled specimens to be collected by the children's parents. Though limited, the serologic data and data we obtained regarding the onset of viral shedding in children with HHV-6–associated roseola support the usefulness of our methods for determining the approximate time of infection.
Primary HHV-6 infection frequently results in visits to physicians. Better diagnostic methods and an effective, safe therapy may improve care for the large number of children who acquire this infection during their first two years of life.
Supported by grants (AI001679 and AI-30731) from the National Institutes of Health.
Dr. Morrow reports having received consulting or lecture fees from Novartis, Focus Technologies, and Bio-Rad Laboratories.
We are indebted to the families who participated in this study, without whose dedication and attention to detail the study could not have been undertaken, and to Amanda Allpress, whose organizational skills were very much appreciated by both the families and the research team.
Source Information
From the Departments of Pediatrics (D.M.Z., L.M.F.), Laboratory Medicine (A.S.M., L.M.F., R.A.M., L.C.), Medicine (A.W.), and Epidemiology (A.W.), University of Washington; Children's Hospital (D.M.Z., L.M.F., M.P.R., R.B.); and the Programs in Biostatistics (A.S.M.) and Infectious Diseases (S.S.S., M.-L.H., L.N., L.C.), Fred Hutchinson Cancer Research Center — all in Seattle.
Address reprint requests to Dr. Zerr at Children's Hospital, 8G-1, 4800 Sand Point Way, NE, Seattle, WA 98105, or at zerr{at}u.washington.edu.
References
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