PEDIATRICS Vol. 103 No. 5
May 1999,
p. e61
ELECTRONIC ARTICLE:
Spread of Varicella-Zoster Virus DNA to Family Members and
Environments From Siblings With Varicella in a Household
Yoshizo Asano, MD,
Tetsushi Yoshikawa, MD,
Masaru Ihira, BS,
Hiroshi Furukawa, BS,
Kyoko Suzuki, MD, and
Sadao Suga, MD
From the Department of Pediatrics, Fujita Health University
School of Medicine, Toyoake, Aichi, Japan.
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ABSTRACT |
Objective. To elucidate virus spread
from siblings with varicella to other family members and environmental
objects in a family setting before and after onset of the disease.
Materials and Methods. Among a family consisting of five
members, a boy developed varicella and the remaining two siblings
developed the disease 17 and 18 days after onset of the index case.
Swab samples from throats and hands of parents and three siblings and
samples from several sources in the environment of the house were
collected frequently before and/or after onset of the disease.
Varicella-zoster virus (VZV) DNA in the samples was examined by a
sensitive polymerase chain reaction amplification assay.
Results. In total, 108 samples from the throats and hands
of the three children with varicella, 72 such samples from parents, and
72 samples from the surfaces of several sources in the house were collected. Eight days after onset of the index case (the older boy),
VZV DNA was detected in both samples from the index case and on the
surfaces of four sources (air conditioner filter, table, television
channel push-buttons, and door handle), but not from the two other
siblings or parents. Then, it was detected once on the mother's hand
and the air conditioner filter and three times on the television
channel push-buttons by January 30, 1998, when the girl developed
varicella, 17 days after onset of the index case. The younger brother
developed the disease on January 31. Viral DNA could not be detected in
any samples obtained on January 30; however, it was detected on the
hands of the older boy and the father and in samples from the hand and
throat of the girl on January 31. Thereafter, virus DNA was detected
three times intermittently by February 13 on the hand and three times persistently in the throat of the girl. The virus DNA was detected three times between February 1 and 3 on the hand and three times between February 1 and 4 in the throat of the younger boy. It was
detected occasionally on the hands of the older boy and the parents,
and occasionally or intermittently on surfaces of four environmental
sources between February 2 and 13.
Conclusions. The present study showed the rapid and broad
contamination of the environment with the VZV DNA when the varicella
patient appeared in a family, although it does not directly mean
infectivity.
Key words:
varicella,
varicella-zoster virus,
transmission of VZV,
VZV DNA,
nested PCR.
Varicella (chickenpox) is a ubiquitous, contagious,
generalized exanthematous disease of childhood, which is the result of primary infection of varicella-zoster virus (VZV). Epidemiologic and
virologic observations suggest that the infection occurs after exposure
to aerosolized VZV or by direct contact with patients with varicella or
herpes zoster, although the mechanisms by which the virus is shed and
spread to susceptible individuals are ill-defined. It is generally
accepted that the typical case of varicella is infectious for 1 to 2 days before the appearance of rash and for 4 to 5 days thereafter.
However, the period of virus excretion from patients with varicella is
not well-understood. In this report, we examined virus spread from
siblings with varicella to other family members and environmental
objects in a family setting before and after onset of the disease using
a sensitive polymerase chain reaction (PCR) amplification assay.
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MATERIALS AND METHODS |
The family examined in the present study consisted of five
members: the father, age 36 years; mother, 36 years; older brother, 6 years; younger brother, 2 years; and sister, 5 years. They were all
otherwise healthy before the present varicella episode and had no
underlying diseases. The parents had a known history of varicella. The
father was a staff member of the central laboratory facility of our
university hospital. On the morning of January 13, 1998, the older
brother developed vesicular rashes compatible with typical varicella
accompanied by fever (number of skin lesions, 150-200; day and peak of
fever, day 1 and 38.0°C; total days of illness, 5). The remaining
sister (number of skin lesions, 25-50; total days of illness, 4) and
younger brother (number of skin lesions, 50-75; total days of illness,
4) showed signs and symptoms of varicella without fever on January 30 and 31, 1998, 17 and 18 days after onset of the index case,
respectively. All three were treated with routine doses of oral
acyclovir (80 mg/kg/d in 4 divided doses) for 5 days started within 3 days after the onset (the index case at 3 days after the onset, the
girl at 1 day, and the younger boy at 1 day). Informed consent was
obtained from the parents after the project was first thoroughly
explained. The review board of our university for the study of human
subjects approved the project of the present study.
Swab samples from throat and hand and samples from several sources in
the environment of the house
including a door handle, table,
television channel push-button, and air conditioner filter, collected
by scrubbing the surface of the object using cotton swabswere
immersed immediately into sterile tubes containing 1 mL of Roswell Park
Memorial Institute (RPMI)-1640. The samples were collected frequently
before and/or after onset of the disease (Table
1). Three hundred µL of culture medium
was used for extraction of the virus DNA. We used highly sensitive
nested PCR to detect VZV DNA. One µg of DNA extracted from each
sample was subjected to PCR. Nested PCR was conducted for amplification of VZV DNA using two primer sets (VZV1:
5'-TCCAGATGATATATGAGCGAT-3'/VZV2: 5'-CAGGCTTTAGGAATCAAGAC-3', VZV3:
5'-TCGTCGAAATTGTAAATACA-3'/VZV4: 5'-GCCTATCTGGATACGATTAC-3'), which
amplified a 203-base-pair fragment located in gene 5 of the VZV DNA.
Reactions consisted of 50 µM of KCl, 10 mM of Tris (pH 8.3), 1.5 mM
of MgCl2, 200 µM of deoxynucleotide triphosphates (dATP,
dCTP, dGTP, and dTTP), 0.5 mM of each primer, and 2.5 U of
Taq DNA polymerase (Ampli Taq Gold; Perkin-Elmer, Norwalk, CT). A Perkin-Elmer Cetus model 9600 DNA thermocycler (Norwalk, CT) was programmed as follows: 10 minutes at 95°C, 30 cycles of 1 minute at 94°C, 2 minutes at 57°C, and 3 minutes at 72°C, and 10 minutes at 72°C. Samples were stored at 4°C until the products could be analyzed. Aliquots of Tris HCl-EDTA buffer were
processed as a negative control, and appropriate precautions were taken
during sample preparation and performance of PCR to avoid
cross-contamination. All specimens except for samples obtained from the
air-conditioner filter were analyzed using a primer to the 
-globin
gene to confirm the absence of residual PCR inhibitors in the sample.
The specificity of the primers for VZV DNA was demonstrated by sequence
analysis of the second PCR product. The sensitivity limit of our
PCRas assessed by amplification of serial dilutions of plasmid
pVZ, which contains our second PCR product amplified from the
positive controlwas 100 copies of VZV genome.
View this table:
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TABLE 1
Detection of Varicella-Zoster Virus DNA by Nested
Polymerase Chain Reaction Assay in Samples From Siblings With
Varicella, Household Members, and Their Home*
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RESULTS |
In total, 108 samples from the throats and hands of the three
children with varicella, 72 such samples from parents, and 72 samples
from the surfaces of several sources in the house were collected. Table
1 summarizes the results of the PCR assay. Eight days after onset of
the index case (the older boy), VZV DNA was detected in both samples
from the index case and on the surfaces of four sources (air
conditioner filter, table, television channel push-buttons, and door
handle), but not from the two other siblings or parents. Then, it was
detected once on the mother's hand and the air conditioner filter and
three times on the television channel push-buttons by January 30, 1998, when the girl developed varicella, 17 days after onset of the index
case. The younger brother developed the disease on January 31. Viral
DNA could not be detected in any samples obtained on January 30;
however, it was detected on the hands of the older boy and the father
and in samples from the hand and throat of the girl on January 31. Thereafter, virus DNA was detected three times intermittently by
February 13 on the hand and three times persistently in the throat of
the girl. The virus DNA was detected three times between February 1 and
3 on the hand and three times between February 1 and 4 in the throat of
the younger boy. It was detected occasionally on the hands of the older
boy and the parents, and occasionally or intermittently on surfaces of
four environmental sources between February 2 and 13.
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DISCUSSION |
VZV is a highly contagious agent that leads to outbreaks of
infection in closed populations.1 Transmission is thought to occur by aerosol spread, direct contact with an infected individual, or both. To identify the mode of transmission of VZV, it is important to isolate the virus from various samples obtained before and after
onset of the disease. However, isolation of the virus from clinical
samples other than vesicles is difficult and insensitive, so, instead,
a sensitive PCR amplification assay was used in the present study.
Consequently, positive PCR findings reflect the presence of VZV DNA but
not necessarily the presence of infectious viral particles.
In the present study, the viral DNA could be detected in only 1 of 42 samples obtained during the 3 days before onset of the two secondary
cases with varicella. Although it is generally believed that the
typical case of varicella is infectious for a few days before
appearance of the rash, the actual amount of VZV excreted from the
patients just before the onset may be smaller than expected. Alternatively it might have been possible to detect the viral DNA if a
more sensitive PCR assay had been used in the present study. However,
after the onset of the two cases with varicella, viral DNA was more
frequently detected in cases and among family members and on the
surface of several sources in the house. This finding would suggest
that the amount of virus excreted from the patients during the eruptive
stage is greater than that of the preeruptive phase of varicella. Of
interest in understanding the pathophysiology and mode of transmission
of varicella is that the virus DNA was detected occasionally or
persistently or intermittently in family members and on several
environmental surfaces for ~2 weeks after the onset of the disease.
The positive finding in the throat of the two secondary cases during
the acute stage of the disease indicates a possible source of
transmission of the virus. This is further supported by a report in
which higher amounts of viral DNA were detected in the saliva of all
acute varicella patients tested during the first 3 days after
onset.2 However, the positive findings detected in the
convalescent stage of the disease might indicate the presence of VZV
DNA but not the infectivity. Another interesting finding is the
detection of viral DNA on the surface of the air conditioner filter.
This evidence strongly suggests that transmission of the virus can occur by aerosol spread, because family members could not directly touch the filter. This is supported by the findings of Sawyer et
al,3 whose PCR analysis of air samples from hospital rooms
of patients with varicella revealed viral DNA in 82% of the samples
1.2 to 5.5 meters from the patient's bed for 1 to 6 days after onset
of rash. However, the significance of the positive findings other than
in the throat of the two secondary cases with varicella, such as on the
air conditioner filter, is not clear because the VZV DNA detected in
these situations could represent noninfectious VZV DNA, and not viable
virus particles. Levin et al4 demonstrated that VZV could
be recovered from a laboratory coat or human skin (0.1% to 0.3% of
input VZV) or from a stethoscope (19% of input VZV) as late as 30 minutes after inoculation, suggesting the heat-unstable nature of the
virus in the natural environment. Moreover, how long noninfectious VZV
DNA can be detected by PCR is not clear. Although VZV has an extremely
heat-unstable nature, comparison between the results of virus isolation
and presence of VZV DNA by PCR is another important issue for future
study. It is also of interest to determine the origin of the virus DNA detected by the present PCR assay. The virus is present in the fresh
skin lesions, in upper and lower respiratory tract, and in other
organs5 so that, theoretically, several routes would exist
for dissemination of the virus. The relative role of airborne spread as
contrasted with direct contact also remains unresolved.
Finally, because the three children with varicella in the present study
were treated with routine doses of oral acyclovir for 5 days under the
health insurance policy in Japan, the data obtained from this study
should be compared with those from nontreated children.
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ACKNOWLEDGMENTS |
This work was supported in part by a grant from Fujita Health
University and by a Grant-in-Aid for Scientific Research, the Ministry
of Education, Science, and Culture, Japan.
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FOOTNOTES |
Received for publication Sep 28, 1998; accepted Dec 17, 1998.
Reprint requests to (Y.A.) Department of Pediatrics, Fujita
Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
E-mail: yasano{at}fujita-hu.ac.jp
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ABBREVIATIONS |
VZV, varicella-zoster virus;
PCR, polymerase chain
reaction.
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Pediatrics (ISSN 0031 4005). Copyright ©1999 by the American Academy of Pediatrics
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