Viral Gastroenteritis Among Young Children in
Northern Jordan
by Mamdoh M. M. Meqdam,* Mohammed T. Youssef,** Laila F. Nimri,* Abdullah A. Shurman,***
Mohammed O. Rawashdeh,*** and Munier S. Al-Khdour**
^Department of Applied Biology, Jordan University of Science and Technology,
**Department of Biology, Yarmouk University, and
***Princess Basma Teaching Hospital, Irbid, Jordan
Summary
Daring the summer months of 1992 and 1993, a total of 439 diarrhoeatic fecal specimens from infants
and young children less than 3 years of age admitted to the pediatric ward of Princess Basma
Teaching Hospital, northern Jordan were tested for the presence of viruses using direct electron
microscopy (EM) and enzyme-linked immunosorbent assay (ELISA) for rotavirus. EM revealed
rotavinises in 83 (18.9 per cent) of cases, adenoviruses in five (1.1 per cent) cases, and small round
viruses in three (0.68 per cent) cases. In contrast, the ELISA assay detected rotavinises in 174 (39.6
per cent) of cases. In an evaluation of the collected diarrhoeatic fecal samples for rotavirus detected
by ELISA, a sensitivity of 95.2 per cent and a specificity of 73 J per cent was demonstrated.
Introduction
Acute viral gastroenteritis in infants and young children
causes great deal of morbidity and mortality throughout
the world. Rotavirus and adenovirus are the predominant
enteric pathogens detected in children with nonbacteria]
gastroenteritis.1'2 An estimated five million deaths are
caused by diarrhoea in children under 5 years old each
year, 20 per cent of which are due to rotavirus. In
developing countries an estimated 870,000 children die
from rotavirus diarrhoea each year which reflects an
urgent need to develop a vaccine.3 In addition to
rotavirus and enteric adenovirus, other viruses are
important as aetiologic agents of dirrrhoea in infants
and young children. Among these viruses, are the
Norwalk group,4 astroviruses,3 small round viruses,6
and coronaviruses.7 Since most enteric viruses cannot be
isolated in cell culture,8 direct visualization of them in
stool samples by electron microscopy is still the
mainstay of diagnosis.9 Commercial kits detecting
rotavirus and adenovirus in stool samples are now
available, and include the enzyme-linked immunosorbent assay (ELISA),10"13 and latex agglutination
assay."' 14 The present study was carried out to
Acknowledgements
The authors acknowledge Professor Sami Abdel-Hafez for the
critical review of the manuscript. We thank Omar Fiat for the
technical assistance during this work. This work was supported
by a grant from the Jordan University of Science and
Technology, Jordan (grant number, 28/93).
Correspondence: M. M. M. Meqdam, Department of Applied
Biology, Jordan University of Science and Technology, P.O.
Box 3030, Irbid, Jordan.
Journal of Tropical Pediatrics
Vol. 43
December 1997
determine the incidence of rotavirus and other enteric
viruses in infants and young children less than 3 years of
age with diarrhoea. Additionally, we wanted to compare
the results of virus identification using EM with those
obtained by ELISA using monoclonal antibodies.
Materials and Methods
Stool specimens
During the summer seasons of 1992 and 1993,439 stool
specimens were collected from children less than 3 years
old admitted with acute diarrhoea to Princess Basma
Teaching Hospital, a largest 360-bed hospital in northern
Jordan. All information concerning children under study
was recorded using a data collection protocol. The
collected specimens were kept at 4°C and delivered to
the laboratory on the day of collection. Stool specimens
were prepared for EM and the remaining stool was stored
at -70°C until tested for rotavirus by ELISA.
Electron microscopy
A 10 per cent stool suspension in phosphate-buffered
saline was centrifuged at 10,000 rpm at 4°C lOmin
(Sigma 2K15) to remove debris. The collected supernatant was centrifuged at 20,000 rpm at 4°C for 2 h (MSE
Europa-24 M). The pellet was suspended in two drops of
the supernatant A drop of suspension was placed on a
wax sheet and a 200-mesh FormVar-carbon coated
copper grid was floated on the surface for 3 min. The
extra fluid was absorbed by a filter paper and the grid was
floated on a drop of distilled water for 1 min. After the
excess fluid was removed, the grid was floated on a drop
of 2 per cent aqueous phosphotungstic acid (pH 6.8) for
© Oxford University Press 1997
349
M. M. M. MEQDAM ET AL
TABLE 1
TABLE 3
Results of testing 439 stool specimens by electron
microscopy and ELJSA
Comparison of electron microscopy and ELJSA for
detection of rotavirus on 439 stool specimens
EM
No. (%) of positive cases tested for
Diagnostic
method
Rotavirus
Adenovirus
Small round
viruses
EM
ELISA
83 (18.9%)
174(39.6)
5(1.1%)
ND*
3 (0.68%)
ND*
ELJSA
+
+
-
79
95
4
261
Sensitivity
Specificity
95.2
Efficiency
77.5
73.3
*ND, not done.
TABLE 2
Clinical characteristics of children with diarrhoea infected with rotavirus as demonstrated by both EM and ELJSA
Symptoms
Age
(months)
Total
patients
Total
positive
0-6
7-12
13-18
19-24
25-30
31-36
Total
112
97
58
67
34
71
439
19
18
9
17
9
7
79
M
F
Soft
Watery
10
9
7
9
5
3
43
9
9
2
8
4
4
36
4
3
3
7
3
2
22
16
14
4
8
6
7
Mucus Vomiting Dehydration
55
3 min. The grid was blotted, air dried, then examined for
virus particles using Zeiss 10CR Electron Microscope at
a magnification of 50,000. A minimal of 10 grid squares
were examined over a period of 15 min on each grid.
Enzyme-linked immunosorbent assay (ELJSA)
Specimens were tested for rotavirus specific antigens
using monoclonal antibodies by a commercial kit
according to the manufacturer's instructions (Amico
Laboratories INC, Illinois, USA). The specificity,
sensitivity and efficiency of the ELISA test were
calculated as proposed by Galen and Gambino.15
1
2
3
2
1
0
9
3
6
5
6
4
3
27
Abdominal
pain
8
3
1
1
0
0
13
Weakness
Fever
8
6
3
9
7
1
43
10
7
4
5
6
3
35
0
2
1
5
8
2
18
results show that children with rotavirus infection are
mostly associated with watery stool (70 per cent),
vomiting (34 per cent), weakness (43 per cent), and
fever (44 per cent).
Table 3 shows a comparison between rotavirus
positive results obtained by EM and ELISA where EM
was used as a standard for comparison. Both EM and
ELISA techniques detected 79 cases of rotavirus
infection, while 95 cases were detected by ELISA
assay alone. Only four stool specimens were found
positive by EM and negative by ELISA. ELISA test has a
sensitivity of 95 per cent, a specificity of 73 per cent, and
an efficiency of 77 per cent.
Results
Stool specimens used in this study were collected from
children with acute diarrhoea during the dry summer
months of May through August of 1992 and 1993.
Specimens were screened for viruses by direct EM and
by ELISA test.
Table I shows the positivity rate of 439 diarrhoeatic
infants and children as tested for viruses by EM and
ELISA. While 40 per cent of the patients were positive
for rotavirus employing the ELISA technique, only 19
per cent of them were infected with rotavirus using EM
and few cases were positive with adenovirus and small
round viruses.
The clinical data about children with diarrhoea as a
result of rotavirus infection are shown in Table 2. The
Discussion
This is the first report from Jordan about the role of
viruses as important aetiological agents for gastroenteritis in diarrhoeatic children under 3 years of age. The
samples were collected during the dry summer months
because the incidence of gastroenteritis has been
observed to be more common during these periods in
northern Jordan. In contrast, this condition can occur
anytime of the year in tropical climates and tends to be
more common in cooler months of the year in
temperature climates.4'16
The present investigation has reinforced the results
that gastroenteritis caused by rotavirus is a major child
health problem. 251718 It had been shown that human
350
Journal of Tropical Pediatrics
Vol.43
December 1997
M. M. M. MEQDAM ET AL
rota viruses were responsible for 30-50 per cent of severe
diarrhoeal illness in infants and young children in
developed, as well as in developing countries.4 The
present study showed that rotavirus infection was
diagnosed by EM in 19 per cent of the patients, which
is consistent with previous findings reported by Dennehy
et al in Rhode Island (17 per cent),19 and Donelli et al in
Rome, Italy (18 per cent).9
One-hundred-and-seventy-four (40 per cent) patients
were positive for rotavirus by ELISA test (Table 3). In
comparison with EM method, 79 cases were positive by
both ELISA and EM, and 95 cases were negative by EM.
ELISA assay proved to be an appropriate test for the
detection of rotavirus antigens in diarrhoeatic stool with
high sensitivity as 95.2 per cent which is consistent with previous reports.9"1318-20 The relatively
low ELISA specificity could be due to the fact that
direct EM method detects approximately 108 virus
particles per ml.21 Furthermore, ELISA specificity
might be higher than we estimated, as direct EM is less
sensitive when compared with immune electron microscopy22 or solid-phase immune electron microscopy.23
Fecal adenovinis infection is probably the second
most common cause of gastroenteritis in children
associated with diarrhoea as it has been reported in
several countries.2'9'15'1
In the present study adenovinis was detected in 1 per cent of the patients which is in
line with previously reported study (0.9 per cent) carried
out in Rome by Donelli et al.24 The low rate of infection
with adenovinis obtained by us could be due to the
milder illness seen in patients infected with adenovinis.
Patients infected with adenovinis showed no symptoms
that are normally seen in rotavirus infected patients like
dehydration, abdominal pain, weakness and fever. In two
patients, simultaneous rotavirus and adenovirus infections were detected in our study. Similar finding have
been reported by Kim19 and Donelli et al.9 However,
Hermann et al26 detected coinfections with rotavirus only
in patients with nonenteric adenovinis. The symptoms
shown by the two patients were similar to those produced
by patients infected with rotavirus alone.
The association of diarrhoea with small round viruses
has been documented earlier.13'27'28 However, the
infection rate with small round viruses reported in our
study is small (0.68 per cent). As small round viruses
have not been implicated as important cause of
gastroenteritis requiring hospitalization in children,4'29
a low rate of infection is expected.
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December 1997