Annals of Tropical Paediatrics (2001) 21, 59–65
Detection of adenovirus infection in children in Jordan
MAMDOH M. MEQDAM, GYEYATH NASRALLAH* & ABDULLAH AL-SHURMAN†
Departments of Applied Biology, *Clinical Laboratories Sciences and †Pediatrics,
Princess Rahma Hospital, Jordan University of Science and Technology, Irbid, Jordan
(Accepted August 2000)
Summary Between November 1997 and May 1998, 350 nasopharyngeal aspirates (NPA) were obtained
from children admitted to the Respiratory Disease Unit at Princess Rahma Hospital, northern Jordan who
were clinically diagnosed as suffering from respiratory tract infections. NPA were investigated for the
presence of adenovirus using shell vial (SV) culture assay, conventional culture (CC) assay, and direct
immunouorescence assay (DFA). Of the 350 NPA, adenoviruses were detected in 54 (15.4%) by the
combined techniques used. SV identied 34 (63%), CC 48 (89%) and DFA 30 (56%). Most virus
isolations were in children aged 1– , 5 years old and were associated with pneumonia in 39% and
bronchopneumonia in 32%. SV assay showed a sensitivity and specicity of 68.8% and 99.7%, respectively,
for detecting adenovirus from NPA. These results emphasize that CC assay is still important for the
diagnosis of adenovirus, although SV and DFA are superior diagnostic assays.
Introduction
World Health Organization (WHO) data indicate that respiratory tract infection (RTI) is
one of the major causes of morbidity and mortality in infants and children.1–3 In many countries, including Jordan, acute respiratory
infection (ARI) accounts for nearly one-third
of all deaths in children less than 5 years of
age.4,5 ARI kills an estimated four million children every year in developing countries and
most of these deaths are caused by pneumonia.6 In those countries, the rate of infection is very high, particularly in infancy and
early childhood.7
Adenovirus is known to infect persons by
aerosols or eye contact, but it also spreads by
Reprint requests to: Dr Mamdoh M. Meqdam, Department of Applied Biology, Jordan University of Science
and Technology, PO Box. 3030, Irbid 22110, Jordan.
Fax: 1 962 2 709 5019; e-mail: meqdam@just.edu.jo
faecal/oral routes, especially where hygiene is
poor. During an incubation period of 5–10
days, the virus replicates in pharynx, conjunctivae and small intestine. It is recognized that
all adenovirus serotypes are endemic in the
community and that for unknown reasons
some can cause explosive outbreaks of disease,
usually respiratory but also involving the eye.8
Adenoviruses are responsible for a signicant number of clinical respiratory illnesses.
Upper respiratory disease caused by adenoviruses includes colds, pharyngitis and tonsillitis and occurs mainly in infants and young
children. Information about adenoviruses
causing respiratory disease has been gathered
in a series of reports to the WHO and evaluated over a 10-year period (1967–76),9 during
which time adenoviruses accounted for 13% of
a total 135,702 reported cases. This was second only to inuenza A, which accounts for
28% of reported cases.
ISSN 0272-4936 print/ISSN 1465-3281/online/01/010059-07 Ó 2001 The Liverpool School of Tropical Medicine
Carfax Publishing
DOI: 10.1080/02724930020028939
60
M. M. Meqdam et al.
Stratication by age of reported adenovirus
cases revealed the following: 22% , 1 year,
42% 1–4 years, 18% 5–14 years, 7% 25–29
years, 1% . 60 years. Other studies indicate
that approximately 10% of childhood pneumonia is probably caused by adenoviruses.10
Other adenovirus-related lower respiratory illnesses include bronchitis and bronchiolitis.11
Adenoviruses are stable and do not need
refrigeration during transport. They can be
cultured and isolated in a variety of cell lines.
Suitable cell lines include Hep-2, Hela, A549
and PMK.12–14 The infected cells become
swollen, rounded and refractive and, clustering together, form grape-like structures which
subsequently become detached from their support.10 Conrmation of infection is usually
achieved by uorescent assay, enzyme immunoassay or haemagglutination-inhibition .14
The purpose of this study was to determine
the incidence of RTI caused by adenovirus
and to evaluate the different diagnostic assays,
shell vial (SV) culture assay, conventional culture
(CC)
assay
and
direct
immunouorescence assay (DFA), in terms of
sensitivity, specicity, predictive value and
agreement.
al.,15 nasopharyngeal aspirates (NPA) were
collected using a sterile infant feeding tube
connected to a vacuum pump. The tube was
introduced through each nostril, yielding
about 0.5–1 ml of aspirate. The tube was
placed into separate, tightly capped sterile
containers labelled with the child’s name,
identication number and the collection date
and time. The container was transported to
the laboratory in wet ice.
Specimen processing
Specimens were processed according to the
method described by Isenberg,16 as follows.
The contents of the infant feeding tube were
emptied into an Ependorff tube by ushing
with a mixture of 1.5 ml of PBS (pH 7.4)
including 200 ll penicillin, 0.2 mg streptomycin and 10 lg amphotoricin B per ml. The
specimen was vortexed for 30 s to dislodge
cells from mucus and centrifuged at 400 3 g
for 10 min using a Hettich centrifuge. The
supernatant was aspirated and transported to
another Ependorff tube for inoculation of cell
culture. The pellet was used for rapid detection of adenovirus antigens by uorescence
assay.
Patients and methods
Patients
This study involved 350 children less than 13
years old admitted with respiratory tract illness
to the Respiratory Diseases Unit in the Princess Rahma Hospital. Those with congenital
and persistent respiratory disease were excluded. Princess Rahma Hospital is a Ministry
of Health paediatric teaching hospital administered by the Faculty of Medicine of Jordan
University of Science and Technology. It is
the only paediatric hospital in northern Jordan
and serves Irbid city and suburbs. All information concerning patients was collected on a
special proforma.
Preparation of cell pellet for immunouorescence
staining
The cell suspension was prepared from the
pellet by washing the pellet twice in PBS
(pH 7.4) for 10 min to decrease the mucus
viscosity. The sediment was then resuspended
in 0.5 ml PBS. Twenty ll of cell suspension
was spotted per well on an eight multi-well
slide (5-mm diameter). The slide was dried by
hotplate at 40°C and xed with cold acetone
at 4°C for 10 min. The slide was then stored
in an airtight box at 2 70°C until used for
staining.
Direct immunouorescence staining
Specimen collection
Using the method described by Meziere et
Ten ll of mouse monoclonal isothiocyanatelabelled anti-RSV antibody (Chemicon, USA)
Detection of adenovirus in Jordan
was added to each well. The slide was incubated at 37°C for 30 min in a humid chamber
and then rinsed for 10–15 seconds in PBS
(pH 7.4). After mounting in phosphatebuffered glycerol (20–80% v/v), the slide was
read using a uorescence microscope (Nikon,
Japan) at 3 20–40 magncation. Specimens
yielding less than an average of 10 columnar
epithelial cells per well were rejected. RSVinfected and uninfected human epithelial cells
(Hep-2) were stained as positive and negative
controls, respectively, in each trial. The presence of one positive columnar epithelial cell
was required to consider a specimen positive
for adenovirus antigen.
Virus isolation
Conventional culture (CC) assay. Specimen inoculation was done according to the method
described by Isenberg,16 as follows. A conventional culture tube with a semiconuent
monolayer of Hep-2 cells was inoculated by
aspirating the old growth medium (GM) and
delivering 0.2 ml of specimen supernatant to
each tube. After adsorption at 37°C for
60 min, 1 ml fresh maintenance medium
(MM) was added and the incubation continued for up to 14 days. The culture was maintained by replacing the old MM with fresh
MM every other day. Culture tubes were examined daily for evidence of viral replication
and development of CPE. When seen, CPE
were harvested and prepared for immunouorescence staining.
Shell vial (SV) culture assay. Shell vial assay
was performed according to the method described by Olsen et al.,13 as follows. A shell vial
tube with semiconuent monolayers of Hep-2
cells was inoculated by aspirating the old GM
and delivering 0.2 ml of specimen supernatant
to each tube. The specimen was centrifuged
(700 3 g) for 50 min (Hettich, Germany). After centrifugation, 1 ml of MM was added to
each vial and incubated for 4 days. SV tubes
were examined daily. When the SV tube
showed toxic effects or contamination, the
61
specimen was decontaminated and the culture
repeated. At the end of the incubation period
(regardless of the presence of CPE), the Hep2 monolayers were harvested from the bottom
of the SV tubes by scraping into a small volume of PBS, pH 7.4 by rubber policeman, and
20 ll of scraped cells were spotted onto each
well of eight-well slides and prepared for immunouorescence staining.
Data analysis
Groups were compared by means of the v2 test
using Minitab version 11.
Results
A total of 350 specimens were tested for the
presence of adenovirus by SV, CC or DFA.
Table I shows the number positive for adenovirus by the assays used. The number positive for adenovirus by SV, CC and DFA was
54 (15.4%). Adenovirus was more frequently
isolated with CC (88.9%) than with SV
(63.0%) or DFA (55.6%).
The age and sex distributions of the positive
and negative cases of adenovirus are shown in
Table II. The children’s ages ranged from 1
month to 13 years (mean 7 months). Data
showed that adenovirus was highly associated
with children aged 1– , 5 years (p , 0.001).
Table III shows the ndings in children
positive for adenovirus. Adenovirus infection
was associated with pneumonia in 38.9% and
with bronchopneumonia in 31.5%. In this
study, 75.9% of hospitalized children with
adenovirus had received at least one type of
antibiotic.
The frequency of signs and symptoms in the
children positive for adenovirus is shown in
Fig. 1. Cough and wheezing were the most
prominent, 81.5% and 77.8%, respectively.
Comparison of SV with CC and DFA for
isolation of adenovirus is shown in Tables IV
and V. The sensitivity and specicity of SV
compared with CC were 68.8% and 99.7%,
respectively.
62
M. M. Meqdam et al.
TABLE I. Number of adenoviruses positive by SV, CC or DFA assays in
nasopharyngeal aspirates of 350 children
No. of specimens positive by
SV
CC
DFA
No. of positive adenovirus (%)
54 (15.4)
34 (63.0)
48 (88.9)
30 (55.6)
TABLE II. Age and sex distribution of children positive for adenovirus
by SV, CC or DFA assay
Age
(yrs)
No. of children
(M:F)
%
No. of positive
children (M:F)
%
,1
1– , 5
5–13
236 (126:110)
102 (60:42)
17 (11:6)
67.4
29.1
4.9
19 (12:7)
33 (18:15)*
2 (1:1)
8.1
32.4
11.8
Total
350
54
* p , 0.001.
TABLE III. Observations in 54 children positive for
adenovirus by SV, CC or DFA assay
Observations
Clinical diagnosis
Bronchiolitis
Pneumonia
Bronchopneumonia
Other respiratory infection
(bronchitis, croup,
upper respiratory tract)
No. (%) of
positive children
7 (13.0)
21 (38.9)
17 (31.5)
9 (16.7)
Duration of hospitalization (days)
1–3
4–6
7–9
23 (42.6)
22 (40.7)
3 (5.6)
No. of antibiotics received
0
1
2
3
13 (24.0)
26 (48.1)
9 (16.7)
6 (11.1)
Discussion
This is the rst study in Jordan to examine the
role of adenovirus as an aetiological agent of
RTI in children under 13 years of age. The
nasopharyngeal aspirates were collected during the winter (November 1997 to May 1998)
when RTI is more prevalent in northern Jordan.17
The infection rate of adenovirus varies from
region to region and from one year to another,8,10,18 and in this study it was higher than
expected (15.4%). The rate of infection with
adenovirus in children hospitalized during the
winter months in Saudi Arabia was very low
(0.7%).19 The reported infection rate has
ranged from 2 to 4% in hospitalized children
in various developing countries18 and a 6-year
study revealed an only slightly higher infection
rate of 8.2% among 1,416 sick children.20 Our
ndings differ but agree with the reported
infection rate of 14.3% among 169 hospitalized children in Argentina.21 A similar study of
80 hospitalized children reported that the
most important viral agent of RTI was RSV
followed by adenovirus.22 In addition to these
two studies, data from WHO show that 13%
of a total of 135,702 cases with respiratory
illnesses were associated with adenovirus9 and
another study reports an adenovirus infection
rate of 18% among 96 children hospitalized
with respiratory illnesses.23
Our results indicate that adenovirus infection occurs mainly in children aged 1– , 5
years, in agreement with other reports.21,22
Detection of adenovirus in Jordan
63
FIG . 1. The frequency of signs and symptoms in 54 children with adenovirus infection.
TABLE IV. Comparison of SV, CC and DFA for
diagnosing adenovirus in nasopharyngeal aspirates
of 350 children
Possible
result
1 /1
1 /2
2/1
2/2
SV/CC
SV/DFA
CC/DFA
33
1
15
301
24
7
11
308
27
3
21
299
Adenovirus is responsible for a signicant
amount of clinical respiratory illness. Approximately 10% of childhood pneumonia is probably caused by adenovirus. Other lower
respiratory illnesses related to adenoviruses include bronchiolitis and bronchitis.9,10,24–26 A
signicant association was found between adenovirus and pneumonia in children older than
1 year. 21 Our study supports these ndings in
that adenovirus was identied in 38.9% children with pneumonia and in 31.5% with bronchopneumonia.
This study shows that 41 (76%) of our
children received at least one type of antibiotic
after admission. This exceeds the proportion
reported in the literature. In one study, 61% of
children were given antibiotics despite the fact
that 35% of them had a conrmed viral aetiology.22 The frequent use of antibiotics in our
patients might reect the fact that the Princess
Rahma Hospital has no facility to test for viral
infection and doctors might give antibiotics to
avoid secondary bacterial infection.
Wide variation in the sensitivity of SV for
TABLE V. Percentage of sensitivity, specicity, predictive value and agreement of SV,
CC and DFA in nasopharyngeal aspirates of 350 children
Predictive value
Assays compared
SV/CC
SV/DFA
CC/DFA
Sensitivity
Specicity
1
2
Agreement
68.8
68.6
56.3
99.7
97.8
99.9
97.1
77.4
90.0
95.3
96.6
93.4
95.4
94.9
93.1
64
M. M. Meqdam et al.
detecting adenovirus has been reported. One
study found that SV was approximately 80%
sensitive for detecting adenovirus26 while in
another study SV assay using Hep-2 cell lines
was only 52% sensitive at 24 hours for specimens previously known to be positive.22 Furthermore, only 10 of 21 adenovirus isolates
were detected by SV assay after 4 days incubation.27 In one study comparing SV with traditional tube cultures using A-549 cell lines for
detecting adenovirus, 77% of specimens were
positive in SV at 2 days and 100% at 5 days
post-inoculation, and most specimens were
eye swabs.28 It has been reported that SV
works well when specimens contain relatively
large amounts of the virus but are insensitive
when specimens contain small amounts of the
virus.13 This might explain the high rate of
detection of adenovirus in the earlier study.28
Furthermore, the average time to detect adenovirus in specimens positive by SV was 4 days
compared with 8 days for specimens negative
in shell vial and positive only in CC.13 In the
present study, 33 (61 %) of 54 adenovirus
isolates were detected by both SV and CC.
However, 15 (28%) samples were positive by
CC and negative by SV. Although SV assay is
highly sensitive for detecting viruses, its sensitivity is only fairly good (69%) for detecting
adenovirus. Based on our ndings, it might be
necessary to extend the incubation period of
SV for isolating adenovirus or simply to use
the CC assay. The variation in SV sensitivity
for adenovirus could be owing to the use of
different types of specimen; for example,
throat or eye swabs yield higher numbers of
adenovirus than nasopharyngeal specimens.1,23
Low titres of viruses in specimens, especially
adenovirus, make detection difcult.1
Our ndings explain the importance of selecting the right laboratory assay to diagnose
viral infection when managing children hospitalized with RTI. Positive laboratory diagnosis
of respiratory viruses might persuade physicians to stop unnecessary antibiotic therapy
because of its cost and side-effects and to
shorten the duration of hospitalization; it
might also reduce the need for other diagnostic tests.
Acknowledgment
Financial support for this research was provided by Jordan University of Science and
Technology, grant no. 63/97.
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