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Egyptian Journal of Medical Microbiology Volume 26 / No.2 / April 2017 27-36
Egyptian Journal of Medical Microbiology
27
ORIGINAL ARTICLE
Comparison of Reverse Transcription-PCR and Viral Culture for
detection of Respiratory Syncytial Virus in Young Children: Relation
to Epidemiological and Clinical Findings
1Wafaa A. Zahran, 1Amal F. Makled, 1Ahmed A. Salama*, 1Ghada R. El-Hendawy and
2Hassan S. Bader
1Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Egypt
2Department of Pediatrics, Faculty of Medicine, Menoufia University, Egypt
ABSTRACT
Key words:
Respiratory syncytial virus,
LRTIs,
Tissue culture,
RT-PCR
*Corresponding Author:
Ahmed Attia Salama
Department of Medical
Microbiology and Immunology,
Faculty of Medicine, Menoufia
University, Egypt
Email:ahmed3attia@yahoo.com;
Tel: 01006245429
Background: RSV is the most frequently identified agent responsible for LRTIs especially
bronchiolitis worldwide. Multiple methods are used for the laboratory diagnosis of viral
infections, including viral culture, antigen detection, nucleic acid detection, and serology.
Morbidity and mortality of RSV infection are higher in infants suffering from concomitant
disease as prematurity, congenital heart disease and others. Objectives: To compare the
value of Reverse-Transcription Polymerase Chain Reaction (RT-PCR) and Conventional
Viral Culture in diagnosis of RSV, to determine the incidence of RSV in pediatric
respiratory illness and patients’ findings and to determine the mortality rate of RSV
infection with concomitant disease. Methodology: This study was conducted on Ninety
seven children (30 females and 67 males aged from 0 month up to 5 Years and were
presenting with clinical pictures suggesting of LRTI (acute bronchiolitis or pneumonia).
All patients were subjected to history taking, complete medical examination and thorough
laboratory and radiological investigations. Tissue culture and RT-PCR were done on
nasopharyngeal aspirates collected from all of these patients and the results of the two
methods were compared with patient's demographic data, clinical and radiological
findings to determine the best method for detection of RSV. Results: RSV was detected in
68.04% and 30.9% of LRTI cases by using RT-PCR and tissue culture respectively and
there was a highly significant statistical difference (P value= 0.0001).The highest
isolation rate of RSV (56.7% and 40.9%) was in children with age from 0-6m by tissue
culture and RT-PCR respectively. The incidence of RSV was decreasing by increasing the
age of the patients. By both techniques for RSV isolation, RSV was higher in males than
females but there was no statistically significant difference. Sixty–six positive RSV cases
detected by RT-PCR were distributed as the following in both seasons; 43.94% in
January, 31.82%, in December, 22.73% in February and only 1.51%, in November. RSV
was isolated from 92.7% of 61 cases of bronchiolitis by RT-PCR and the presence of
hyperinflated chest is significantly higher among positive RSV cases (P value=0.001).
Mortality rate in positive RSV patients with comorbidities was 25%. Conclusion: RT-PCR
was more sensitive than tissue culture in diagnosis of RSV, especially in older age group
cases. Mortality rate was higher among positive RSV who had concomitant diseases.
INTRODUCTION
RSV is a globally ubiquitous respiratory pathogen
of the Paramyxoviridae family. It is a major cause of
hospitalizations, morbidity and mortality among
children due to lower acute respiratory tract infections
that manifested as bronchiolitis or pneumonia 1
. Acute
bronchiolitis is characterized by tachypnea, wheezing,
suprasternal or intercostal retraction, cyanosis or apnea,
and even respiratory failure. Risk factors for severe
bronchiolitis include prematurity, cardiovascular
disease, chronic pulmonary disease and
immunodeficiency 2.
RSV is a highly communicable but humans are the
only known reservoir. Its incubation period varies from
two to eight days. The virus spreads from respiratory
secretions through close contact with infected persons
via respiratory droplets or contact with contaminated
surfaces or objects. Infection by RSV may confer partial
immunity so individuals may be infected repeatedly
with the same or different strains of RSV 3.
Infection with RSV is a clearly identified as a
winter virus, usually occurring within the period from
October to March with most infections occurring in a
relatively short epidemic of about six weeks 4. In ‘high-
risk’ children (especially having cardiac abnormalities
and multiple co-morbidities), the mortality rate of RSV
Zahran et al. / Comparison of Reverse Transcription-PCR and Viral Culture, Volume 26 / No. 2 / April 2017 27-36
Egyptian Journal of Medical Microbiology
28
infected children is about 3%2. RSV-associated
mortality is highest in developing countries, but RSV
can have a significant burden on the cost of care and the
economy of all countries 5.
Multiple methods are used for the laboratory
diagnosis of RSV infections, including viral culture,
antigen detection, nucleic acid detection, and serology 6.
For faster diagnosis, the isolation of RSV in cell culture
has been replaced by antigen detection-based assays and
new sensitive molecular techniques such as RT-PCR 7.
However, cell culture method continues to be valuable
because it allows the amplification of small amounts of
virus that are present in a specimen, providing isolates
for subtyping and further analysis. Moreover, it is less
likely to diagnose false epidemics and permits the
recovery of several additional agents that may be
present in a specimen 8.
In this study, we aimed to assess the value of RT-
PCR and conventional viral culture in diagnosis of
RSV, to study the epidemiology of RSV in pediatric
respiratory illness and patients’ findings and to
determine the mortality rate of RSV infection with
concomitant disease in our locality.
METHODOLOGY
Study population and selection of patients
This study was conducted at the Microbiology and
Immunology Department, Faculty of Medicine,
Menoufia University in collaboration with the Pediatric
Department, Faculty of Medicine, Menoufia University
during the period from October 2013 to April 2014
(season 1) and from October 2014 to April 2015 (season
2).
Ninety seven children (30 females and 67 males
aged from 0 month up to 5 Years were presenting with
clinical pictures suggesting of LRTI (acute bronchiolitis
or pneumonia).
Medical records were reviewed for detailed
demographic, clinical, and laboratory data, radiographic
images and underlying conditions of the patients. All
the clinical symptoms and signs were recorded on a
standardized form on hospitalization of the patients.
The study protocol was approved by local ethics
committee of the Menoufia University. Informed
consent was obtained from parents of patients before the
beginning of the study.
Specimen collection
Nasopharyngeal aspirate specimens were collected
by inserting a tube attached to a mucus extractor into
one nostril and applying gentle suction by suction
apparatus. These aspirated contents were suspended in
virus transport medium “Dulbecco′ s minimal essential
medium (DMEM)”. The specimens were divided into
two aliquots; one of them was transported to
Microbiology laboratory to be inoculated on tissue
culture for RSV detection without delay and the other
one was kept at -80C for RT-PCR testing.
RSV inoculation and isolation in cell culture: (Nunes
and Moura, 2006) 9
The specimens received in DMEM were vortexed
for 30 seconds, 8 drops of antibiotic solution were
added to samples with a Pasteur pipette before the
growth medium was aspirated and 0.2ml sample
inoculum was inoculated into duplicate tubes of HEp-2
cells with confluent monolayer. After addition of
maintenance medium, the tubes were incubated at 37C.
Four uninoculated cell culture tubes were included as
cell culture controls.
Microscopic examinations for cytopathic effect
(CPE) were performed daily for 10- 14 days using
inverted microscope. The culture was considered
positive when giant cells and syncytia were observed
(fig.1). When CPE reached third (+3) or fourth degree
(+4) the cell culture tubes were removed from the
incubator and the cell associated virus was released by
repeated freezing and thawing for three times. Then, the
suspension was diluted 1/1000 and amplified by another
two successive passages. Samples were followed up for
2 weeks before they were considered as negative.
Reversed- transcription polymerase chain reaction
(RT-PCR):
(A) RNA Extraction:
This was carried out by the use of the Thermo
Scientific Gene JET Viral DNA and RNA
Purification Kit # K0821 supplied by Thermo Fisher
Scientific Inc, California, USA for the extraction and
purification of genomic RNA from viral strains. This
was achieved through the following steps;
1. The sample was lysed by incubation with lysis
solution and proteinase K (they inactivate both
RNases and DNases, ensuring protection of viral
nucleic acids against degradation) under
denaturating conditions at elevated temperatures
(56C).
2. The lysed sample was transferred to a spin column
where released viral nucleic acids immediately bind
to the silica-based filter in the presence of
chaotropic salts. The remaining lysate was removed
by centrifugation.
3. The remaining contaminants were removed by
using Wash Buffer 1 and 2, whereas pure nucleic
acids remain bound to the membrane.
4. Pure viral nucleic acids were released from the spin
column filter using Eluent. The purified nucleic
acids were ready for subsequent use in downstream
applications.
On starting a new procedure, we always used a
freshly prepared mixture of carrier RNA and lysis
solution. To calculate the correct quantity of carrier
RNA and lysis solution required to process multiple
specimens, the table supplied in the extraction kit was
used.
Zahran et al. / Comparison of Reverse Transcription-PCR and Viral Culture, Volume 26 / No. 2 / April 2017 27-36
Egyptian Journal of Medical Microbiology 29
(B) cDNA synthesis, amplification and detection:
(Nikfar et al., 2013) 10
PCR was done using RT-PCR kit according to the
following protocol:
1st step PCR was done by using (Perkin-Elmer Gene
Amp PCR System 9600 thermal cycler): 5ul of
extracted RNA was combined with 1 ul of G1 and G2
primers, 1.25 ul of RT buffer, 0.5 ul of verso enzyme
mix and volume was completed to 25ul by DEPC water.
1st step RT-PCR thermal cycling program:
Temp Time Number of
cycles
cDNA synthesis 50C 15 min 1 cycle
Verso inactivation 95C 2 min 1 cycle
Denaturation 95C 20 sec 35-45 cycles
Annealing 56C 30 sec
Extension 72C 1 min
Final extension 72C 5 min 1 cycle
2nd cycle RT- PCR: 5 ul of 1st cycle product was
combined with 12.5 ul of Master mix, 1 ul of G3 andG4
primers and volume was completed to25 ul. The PCR
was performed for 35 cycles ,initially for 5 min at 95C
followed by, 1 min at 95C, followed by 1 min at 72C,
1 min at 95C and finally 5 min at 72 C for one cycle.
The expected final PCR product was 326bp (fig. 2).
Primers (Qiagen Germany):
For 1st cycle
G1: CCA TTC TGG CAA TGA TAA TCT C
G2: GTT TTT TGT TTG GTA TTC TTT TGC GA
For 2nd cycle
G3: CGG CAA ACC ACA AAG TCA CAC
G4: GGG TAC AAA GTT AAA CAC TTC
Statistical analysis
Computer SPSS program version 17 was used. The
results were expressed by applying Chi-square test. p <
0.05 was considered to be significant.
RESULTS
This study was carried out on 97 children (patient
group); 67 males and 30 females, their ages ranged from
0-5 years (mean±SD;9.32.31 years old).They were
admitted to Pediatrics Department, Faculty of Medicine,
Menoufia University during the period from October
2013 to April 2014 and the period from October 2014 to
April 2015. They were diagnosed at the time of
admission as lower respiratory tract infections.
Nasopharyngeal aspirate specimens were collected and
transported to Microbiology laboratory to be
immediately inoculated on tissue culture for RSV
detection and for RT-PCR testing (Fig. 1&2).
RSV was detected in 30.9% and 68.04% of cases
by using tissue culture and RT-PCR respectively. There
was a highly significant (p<0.0001) difference between
the two methods for detection of RSV (Table 1). The
specificity and sensitivity of tissue culture method were
calculated in relation to PCR as a gold standard method
used; Tissue culture showed sensitivity of 41%,
specificity 90.0%, positive predictive value 90.0%,
negative predictive value 42.0% (Table 2).
The highest isolation rate of RSV (56.7% and
40.9%) was in children with age from 0-6 months by
tissue culture and RT-PCR respectively. The incidence
of RSV by tissue culture and RT-PCR was decreasing
by increasing the age of the patients but without
significant difference. By both techniques for RSV
isolation, RSV was higher in males than females but
there was no significant difference between positive and
negative RSV patients by both tissue culture and RT-
PCR as regarding the gender (Table 3). Fifty specimens
were collected in the 1st season (winter 2013-2014) and
47 in the 2nd season (winter 2014-2015, where sixty–
six positive RSV cases were detected by RT-PCR were
distributed as the following in both seasons; 29
(43.94%) in January, 21(31.82%), in December, 15
(22.73%) in February and only 1(1.51%), in November
(Fig. 3).
The severity of symptoms was decreasing with
increasing age of the patients because higher number of
patients suffering from clinical symptoms was at
younger age < 1 year compared to number of patients at
older age > 1 year (Table 4).
In positive RSV cases by RT-PCR, cough was the
most predominant symptom (72.7%), followed by
wheezing (66.75), rhinorrhea (57.6%), fever (42.4%)
and cyanosis (9.5%). On the other hand, in the positive
RSV cases by tissue culture, rhinorrhea and wheezing
were the predominant symptoms (85.7%), followed by
cough in (66.7%) and fever (60 %). As regard
respiratory distress; grade I was the most predominant
in RSV positive cases by RT-PCR (74.2%) and tissue
culture (80%) (Table 5). The clinical diagnosis of the
positive cases for RSV; the virus was isolated from 61
cases of bronchiolitis (92.4%) by RT-PCR and from 28
cases (93.3%) by tissue culture, while 7.6% and 6.7 %
of positive RSV cases had pneumonia that was detected
by RT-PCR and tissue culture respectively (Table 5).
Table (6) showed that the presence of hyper
inflated chest was significantly higher among positive
RSV cases (P value=0.001) while there was non-
significant difference between positive RSV and
negative RSV cases regarding increased
bronchovascular markings.
As shown in table (7), 16 out of 97 patients of
LRTIs had comorbidities, all of them had RSV detected
by RT-PCR. Mortality rate in these patients was 25% (4
patients). Percentage of death was higher among
positive RSV who had comorbid congenital heart
disease 60% (3 patients) followed by prematurity 25%
(one patient).
Zahran et al. / Comparison of Reverse Transcription-PCR and Viral Culture, Volume 26 / No. 2 / April 2017 27-36
Egyptian Journal of Medical Microbiology
30
Table 1: Distribution of RSV among 97 LRTI patients by using tissue culture and RT-PCR.
Method of detection Positive RSV Percentage Negative RSV Percentage P value
Tissue culture 30 30.9 67 69.1
RT-PCR 66 68.04 31 31.9
0.0001
Table 2: Validity of tissue culture in relation to PCR results.
Sensitivity Specificity PPV NPV Accuracy
Tissue culture: 41% 90.0% 90.0% 42.0% 57.0%
Table 3: Age and gender-related RSV among 97 LRTI patients by using tissue culture and RT-PCR.
RSV-positive
by tissue culture RSV-positive
by RT-PCR
Demographic data
No. (30) % No. (66) %
P value
Age
- 0-6 months
- 6-12 months
- 12-18months
- 18-24 months
- >24 months up to 5 years
17
10
2
1
0
56.7%
33.3%
6.7%
3.3%
0%
27
19
10
8
2
40.9%
28.8%
15.15%
12.12%
3.03%
0.28
Gender
- Males
- Females
23
7
76.7%
23.3%
51
15
77.3%
22.7%
0.84
Table 4: Clinical symptoms associated RSV-positive LRTI patients by RT-PCR according to age.
Cough Rhinorrhea Fever Wheezing Cyanosis Cerpitations Respiratory distress
Symptom
Age group
Months + - + - + - + - + - + - + -
0-6 months 20 7 24 3 20 7 22 5 4 23 4 23 26 1
6-12 months 15 4 13 6 7 12 15 4 1 18 4 15 18 1
12-18months 5 5 1 10 2 8 5 5 1 9 4 6 9 1
18-24 months 3 5 0 8 1 7 3 5 0 8 1 7 6 2
>24 months up
to 5 years 0 2 0 2 0 2 1 1 0 2 2 0 2 0
Table 5: Comparison between positive RSV cases by RT-PCR and tissue culture regarding clinical symptoms.
Positive RSV by RT-PCR (66) Positive RSV by Tissue culture (30)
No. % No. %
Symptoms
- Cough
- Rhinorrhea
- Fever
- Wheezing
- Cyanosis
48
38
28
44
6
(72.7%)
(57.6%)
(42.4%)
(66.7%)
(9.5%)
20
26
18
26
0
(66.7%)
(85.7%)
(60%)
(85.7%)
(0%)
Observed signs
Respiratory distress
- Grade I (Tachypnea)
- Grade II (chest wall retraction)
- Coarse Crepitation
Clinical diagnosis
- Bronchiolitis
- Pneumonia
49
12
15
61
5
74.2%
18.18%
22.72%
92.4%
7.6%
24
6
1
28
2
80%
20%
3.33 %
93.3%
6.7%
Zahran et al. / Comparison of Reverse Transcription-PCR and Viral Culture, Volume 26 / No. 2 / April 2017 27-36
Egyptian Journal of Medical Microbiology 31
Table 6: Comparison between radiological findings among cases of LRTI positive and negative RSV by RT-PCR.
Total RSV Negative (n=31) RSV Positive (n=66) P-value
% No. % No. % No.
Item
Hyperinflated chest:
60 52 21.1 7 68.3 45
Present 0.001
40 45 78.9 24 31.7 21
Absent
Increased bronchovascular markings:
96.7 94 100 31 95.1 63
Present 0.83
3.3 3 0 0 4.9 3
Absent
Table (7): Comparison between alive and dead cases regarding the presence of co -morbidities.
Alive (No.=93) Dead (No.=4)
Co - morbidities: Total
(97) No. % No. %
Chi square P value
Present:
Prematurity
Congenital heart disease
Down "s syndrome
Chronic lung disease
Malignant disease
16
4
5
2
2
3
12
3
2
2
2
3
12.9
75
40
100
100
100
4
1
3
0
0
0
100.0
25
60
0
0
0
21.12 0.0001
Absent: 81 81 87.1 0 0.0
Fig. 1: HEp-2 cell culture showing characteristic RSV infection CPE
(giant cells and syncytia formation: Arrows 1&2) x400 magnification and stained by safranin T.
Fig.2: Agarose gel electrophoresis analysis of RT-PCR amplification products:
Lane 1: molecular weight marker
Lanes 3, 4, 6, 8 and 10 were positive samples (RSV appeared as a band at 326 b.p.)
Lanes 2, 5, 7 and 9 were negative samples.
Zahran et al. / Comparison of Reverse Transcription-PCR and Viral Culture, Volume 26 / No. 2 / April 2017 27-36
Egyptian Journal of Medical Microbiology
32
Fig. 3: The distribution of 66 isolated positive cases for RSV by RT-PCR during the
2 seasons of 2013-2014 and 2014-2015
DISCUSSION
RSV is recognized as the most frequent agent
responsible for LRTIs in children, where bronchiolitis is
the signal illness for RSV 11. In infants and young
children, RSV infections progress from upper to lower
respiratory tract infections in approximately 40% of
infected children 12. RSV infections are associated with
significant diseases burden in children in term of
hospitalization, related complications, and even
mortality 13.
In this study, there was a significant (p<0.0001)
difference between two previous methods for detection
of RSV. Thirty out of 97 (30.9%) from nasopharyngeal
samples showed RSV-like CPE (syncytia formation) in
HEP-2 cells, while 68.04% of them (66 specimens)
were positive for RSV by RT-PCR. RSV viability may
be lost during processing of specimens or infectivity
may be compromised by the presence of secretory
antibodies in respiratory secretions capable of blocking
virus adsorption 14.
Similar results were observed in other studies that
were done by Falsey et al (15, 16) in (2002) and in (2003)
as they found that 37% and 34% of nasopharyngeal
samples were positive for RSV by tissue culture
respectively. A higher result of RSV detection by tissue
culture was reported by Gueudin et al.,17 (45.3%) and
Popow-Kraupp and Aberle 18 (46%). On the other hand,
lower results of positive RSV by tissue culture were
demonstrated by many authors. Torres and vicente 19
found that only 142 out of 776 (18%) nasopharyngeal
aspirates of children were positive for RSV, Templeton
et al.,20 found that the incidence of RSV was 19%. Also,
Jonathan 14 found that 11 out of 100 specimens (11%)
were positive for RSV by tissue culture. The lowest
result (9.8%) was obtained by Khalil et al., 21 as 22
samples out of 224 showed RSV- like CPE in HEP-2
cells.
Viral culture procedure is a slow method for
diagnosis of RSV, as it takes 3 to 10 days from
inoculation on HEp-2 cells to produce results.
Therefore, the majority of cases may be discharged
from the hospital before the culture results became
available 6.
In the present study, RT-PCR was done as a
confirmatory test for viral culture. Nested RT-PCR is a
diagnostic test as it was proved to be more reliable
regarding time, sensitivity and specificity 22. Another
advantage is the avoidance of viral neutralization or
inactivation by antiviral products within respiratory
secretions that can inhibit viral growth in culture 23.
Unfortunately, the applicability of RT-PCR on wide
scale will be difficult due to its high costs 22. Studies
comparing molecular diagnostic assays to virus isolation
in cell culture and to antigen detection assays have
demonstrated superior sensitivity for nucleic acid tests
which leads to an approximately two-fold increase of
detection rates in infants with respiratory illness 1.
Similar results were obtained by Tabatabai et al., 4
as RSV infection was detected by RT-PCR in 134 out of
242 samples (55.4%) that were collected from
hospitalized LRTIs children. Also, Azkur et al., 2
detected that RSV was the most common virus (45.5%)
identified by RT-PCR in patients of bronchiolitis. A
higher result was demonstrated in a study done in Saudi
Arabia, by Akhter et al., 24 where RSV was identified in
83% of LRTIs patients. On the other hand, lower results
were reported by many researchers; Fattouh et al., 25 in
Zahran et al. / Comparison of Reverse Transcription-PCR and Viral Culture, Volume 26 / No. 2 / April 2017 27-36
Egyptian Journal of Medical Microbiology 33
Egypt, (16.4%), Popow- Kraupp and Aberle 18 (22%),
Nikfar et al., 10 in Iran, (9%), Yadav et al., 23 in Delhi, in
India (12.3%). Also, Khalil et al., 21 in Sudan reported
that 19.6% were positive for RSV (44 out of 224
patients) and Yadav et al., 13 noticed that RSV was
detected in only 10.8% of their patients. Low results in
the previous studies might be attributed to the lack of
going off some cases for specialized clinics because of
their mild symptoms and signs and considerable
distance between villages and hospitals 21. Moreover,
the difference in RSV epidemiology in the world may
be related to differences in climate conditions,
environmental factors and severity of its epidemics from
one year to another 10.
Primary RSV infection commonly occurs within
the first year of life 26 and the risk of RSV infection
decreases with increasing age 4. This fact has been
confirmed in this research as the highest percentage of
positive RSV by RT-PCR and tissue culture was in age
< 1 year (69.7% and 90% respectively). In the study of
Tabatabai et al.,4 age group analysis revealed that
infants below one year of age had the highest RSV
infection rates (77.6 %). Also, Khalil et al.,21 noted
reduction in the number of children infected with RSV
with increasing age as RSV infection was 68.2% in
children < 2years and 31.8 % in children 2-5 years.
Moreover, Yadav et al.,13 showed in their study that
RSV was demonstrated in much higher percentage
(72%) in age group 2-6 months.
In this study, higher age group cases were better
diagnosed by RT-PCR rather than by tissue culture
where; 20 cases (30.30%) and 3 cases (10%) of the
positive RSV patients aged from 1 year to 5 years were
positive for RSV by RT-PCR and tissue culture
respectively. In accordance of this result, Popow-
Kraupp and Aberle, 18 showed that the best method for
detection of RSV was RT-PCR in children older than 1
year. Previous studies demonstrated that there was a
reduction in the efficacy of RSV detection with
increasing age of the patient (27, 28, 29, 30). The better
detection of RSV by RT-PCR in children of older age
group can be explained as young children experiencing
primary infection usually shed large quantities of RSV
over a prolonged period of time. With the increasing
number of subsequent infections, virus is shed in lower
quantities for shorter times due to the presence of
secretory and humoral virus-specific antibodies
influencing the degree of viral replication (18, 31).
As regards to gender, predominance of males was
evident in positive RSV group (77.3% by RT-PCR and
76.7% by tissue culture) with a significant difference
between males and females who had RSV infection
(p=0.01). Male sex was reported in some studies as a
risk factor for RSV infection 32. RSV infection was
confirmed in 47 male vs. 44 female and 57 male vs. 43
female by Savić et al.,33 and Nikfar et al.,10 respectively.
In the study of Tabatabai et al., 4, males were
representing 61.9% and females were 38.1% of positive
RSV cases. Also, Khalil et al., 21 reported that RSV was
detected in 12.9% male and 6.7% female patients, using
RT-PCR technique and there was no significant
difference.
Infection with RSV occurs in seasonal outbreaks,
which arise annually, peaking during the winter in
temperate climates and during the rainy season in
warmer climates, although exceptions to this pattern
occasionally occur 10. In the United States, the annual
epidemics usually begin in November, peak in January
or February, and end in May 34. This study confirmed
the seasonal character of RSV infections with an
increasing in hospitalization rate of LRTIs and an
increased isolation rate of RSV from November to
February. The incidence of positive RSV cases in this
study was 1.51%, 31.82%, 43.94% and 22.73% in
November, December, January and February
respectively in both seasons (2013-2014 and 2014-
2015). This result was in agreement with that reported
by Cui et al., 35 in China and Savic et al., 33 in Serbia.
Also, Yadav et al., 13 observed a rising in RSV infection
towards winter season, where 13 positive RSV cases
were detected from November to February months
compared to only one case in August and October. In
contrast, Breiman et al., 36 in Kenya and Oladokun et al.,
5 in South Africa showed that RSV cases were recorded
from January until October with most cases occurring in
May.
RSV infection in children almost always causes
clinical manifestations that can vary widely in severity,
depending on the patient’s age, co morbidities,
environmental exposures, and history of previous
infections. Typically, the infection starts with signs and
symptoms of mucosal inflammation and irritation of
upper respiratory tract (congestion, rhinorrhea and
sneezing). After few days, lower respiratory tract
clinical manifestations appear in the form of cough and
increased work of breathing 22. In this study, evidence of
respiratory tract infection in positive RSV was cough
(72.7%), wheezing (66.7%), rhinorrhea (57.6%), fever
(42.4%) tachypnea (74.2%) and chest wall retraction
(18.18%). These findings were similar to that reported
by Nikfar et al., 10 who found that cough was present in
77.7% of cases, followed by fever in 55.5% of cases.
Those authors observed that 100%, 88.8%, and 22.2%
of their positive RSV cases had tachypnea, wheezing
and cyanosis respectively.
In the present study, the clinical symptoms were
more severe among positive RSV cases which were
detected by tissue culture than by RT-PCR; as
rhinorrhea (%85.7), wheezing (%85.7), cough (%66.7),
and fever (%70). These results agreed with the fact that,
as far as RSV is concerned higher viral loads seem to
correspond with a more severe clinical course of the
disease 18. Also, Buckingham et al., 37 and DeVincenzo
et al., 38 demonstrated that the higher quantities of the
RSV titres or loads in nasal aspirates correlated with
more severe symptoms and signs in their patients.
Zahran et al. / Comparison of Reverse Transcription-PCR and Viral Culture, Volume 26 / No. 2 / April 2017 27-36
Egyptian Journal of Medical Microbiology
34
Bronchiolitis is the most prominent sign for clinical
diagnosis of patients with RSV infection especially in
infants 39. The present study further supported that
finding and showed that positive RSV cases detected by
RT-PCR and tissue culture had bronchiolitis with a
higher prevalence (92.4% and 93.3% respectively).This
was in agreement with Nikfar et al., 10; as 100% of
positive RSV children whose ages less than one year
had bronchiolitis. Also Tabatabai et al., 4 showed that,
the majority of positive RSV children presented with
bronchiolitis.
In this study, only 7.2% and 6.7% of positive RSV
case detected by RT-PCR and tissue culture respectively
had pneumonia. In contrast, a higher result was reported
by Goto-Sugai et al., 39 (33.3%) and Khalil et al., 21
(53.3%) This variation may be due to the fact that,
children with acute bronchiolitis are at higher risk of
developing pneumonia 21.
Radiological findings of positive RSV positive
patients were obtained and compared with negative
RSV patients, where hyperinflated chest was more
significantly (p<0.001) present in 68.3%. There was no
significant difference between positive and negative
RSV cases regarding increased bronchovascular
markings. These findings were similar to that reported
by Aboul –Ftouh et al., 40, as hyperinflated chest was
found in 68% of RSV positive with bronchiolitis. Also,
Savic et al., 33 showed that hyperinflation was the most
common radiographic finding, while infiltrations and
consolidations were less prevalent.
Morbidity and mortality rates in RSV infections are
higher in premature infants and in those with chronic
lung disease (e.g., bronchopulmonary dysplasia, cystic
fibrosis, and interstitial lung diseases) or
hemodynamically significant congenital heart disease 12.
In the present study, all of the 16 cases had different
concomitant diseases were positive for RSV by RT-
PCR, four out of them died (25%). One child (25%) was
premature and 3 (75%) were suffering from congenital
heart disease. No death occurred in positive RSV
patients without co-morbidities. Savic et al., 33 and
Azkur et al., 2 noted that the mortality rate in positive
RSV with concomitant diseases was 16.7% and 3.2%
respectively. Increasing in cases of death in children
infected with RSV in presence of other concomitant
disease may be the result of complications and greater
length of hospital staying 2. Therefore, in most countries
with developed healthcare system passive immunization
with palivizumab, recombinant monoclonal antibody is
performed in specially defined groups 41.
CONCLUSIONS
RT-PCR was more sensitive than tissue culture in
diagnosis of RSV with increasing age. RSV was found
to be the major cause of LRTI in infants and young
children. RSV was the major cause of LRTI in infants
and young children especially those below 6 months.
The incidence was decreasing by increasing the age.
RSV was a main cause of acute bronchiolitis from
November to February. Mortality rate was 25% among
positive RSV who had concomitant diseases.
RECOMMENDATION
Passive immunization with specific monoclonal
antibody (palivizumab) during RSV season should be
considered especially in children with concomitant
diseases.
REFERENCES
1. Aziz T A.G, Hamid Z A and Bayati
(2016):Detection of respiratory syncytial virus in
hospitalized children with acute respiratory tract
infections in Kurdistan region in Iraq. G.J.B.B,
(3):335-339.
2. Azkur D, Özaydın E, Dibek-Mısırlıoğlu E, Vezir
E, Tombuloğlu D, Köse G, Kocabaş CN (2014):
Viral etiology in infants hospitalized for acute
bronchiolitis. Turk J Pediatr. 56(6):592-596.
3. Oshansky CM, Zhang W, Moore E, Tripp RA
(2009): The host response and molecular
pathogenesis associated with respiratory syncytial
virus infection. Future Microbiol. 4(3):279-297.
4. Tabatabai J, Prifert C, Pfeil J, Grulich-Henn J,
Schnitzler P (2014): Novel respiratory syncytial
virus (RSV) genotype ON1 predominates in
Germany during winter season 2012-13. PLoS
One. | www.plosone.org. 9(10):e109191.
5. Oladokun R, Muloiwa R, Hsaio N-Y, Vally-Omar
Z, Nuttal J and Eley B (2016): Clinical
characterisation and phylogeny of respiratory
syncytial virus infection in hospitalised children at
Red Cross War Memorial Children’s Hospital,
Cape Town. BMC Infectious Diseases. 16:236.
6. Bawage S S, Tiwari P M, Pillai S, Dennis V, and
Singh S R (2013): Recent Advances in Diagnosis,
Prevention, and Treatment of Human Respiratory
Syncytial Virus. Hindawi Publishing Corporation
Advances in Virology. Article ID 595768, 26
pages http://dx.doi.org/10.1155/2013/595768.
7. Chen YW, Huang YC, Ho TH, Huang CG, Tsao
KC, Lin TY (2014): Viral etiology of bronchiolitis
among pediatric inpatients in northern Taiwan with
emphasis on newly identified respiratory viruses. J
Microbiol Immunol Infect. 47(2):116-121.
8. Hall CB and McCarthy CA (2004):Respiratory
syncytial virus. In GL Mandell, JE Bennett, R
Dolin (eds), Principles and Practice of Infectious
Diseases, 6th ed., Churchill Livingstone,
Philadelphia, p. 2008-2026.
Zahran et al. / Comparison of Reverse Transcription-PCR and Viral Culture, Volume 26 / No. 2 / April 2017 27-36
Egyptian Journal of Medical Microbiology 35
9. Nunes IF and Moura FE (2006): Isolation of
respiratory syncytial virus from nasopharyngeal
aspirates stored at -20oC from one to fifteen
months after collection. Mem Inst Oswaldo Cruz.
101(4):451-453.
10. Nikfar R, Shamsizadeh A, Makvandi M,
khoshghalb A (2013): Detection of Respiratory
Syncytial Virus in Hospitalized Children With
Acute Lower Respiratory Tract Infections, Using
RT PCR in Ahvaz, Iran Arch Pediatr Infect Dis.
1(3): 118-121.
11. Broadbent L, Groves H, Shields MD, Power UF
(2015): Respiratory syncytial virus, an ongoing
medical dilemma: an expert commentary on
respiratory syncytial virus prophylactic and
therapeutic pharmaceuticals currently in clinical
trials. Influenza Other Respir Viruses. 9(4):169-
178.
12. Piedimonte G and Perez MK (2014): Respiratory
syncytial virus infection and bronchiolitis. Pediatr
Rev. 35(12):519-530.
13. Yadav K, Ramachandran VG, Das S and Shah D
(2016): Respiratory Syncytial Virus – Detection,
Prevalence and Usefulness in Clinical
Management: A hospital based study.
Int.J.Curr.Microbiol.App.Sci. 5(2): 86-91.
14. Jonathan N (2006): Diagnostic utility of BINAX
NOW RSV – an evaluation of the diagnostic
performance of BINAX NOW RSV in comparison
with cell culture and direct immunofluorescence.
Annals of Clinical Microbiology and
Antimicrobials, 5:13.
15. Falsey AR, Formica MA and Walsh EE (2002):
Diagnosis of respiratory syncytial virus infection:
comparison of reverse transcription-PCR to viral
culture and serology in adults with respiratory
illness. J Clin Microbiol. 40(3):817-820.
16. Falsey AR, Formica MA, Treanor JJ, Walsh EE
(2003): Comparison of quantitative reverse
transcription-PCR to viral culture for assessment
of respiratory syncytial virus shedding. J Clin
Microbiol. 41(9):4160-4165.
17. Gueudin M, Vabret A, Petitjean J, Gouarin S,
Brouard J, Freymuth F (2003): Quantitation of
respiratory syncytial virus RNA in nasal aspirates
of children by real-time RT-PCR assay. J Virol
Methods. 109(1):39-45.
18. Popow-Kraupp T and Aberle JH (2011): Diagnosis
of respiratory syncytial virus infection. Open
Microbiol J. 5(2): 128-134.
19. Torres G and Vicente M (1992): Detection of
respiratory syncytial and adenovirus in
nasopharyngeal aspirates: comparison of cellular
cultures and immunofluorescence. Rev Med Chil.
120(4):415-419.
20. Templeton KE, Scheltinga SA, Beersma MF,
Kroes AC, Claas EC (2004): Rapid and sensitive
method using multiplex real-time PCR for
diagnosis of infections by influenza a and
influenza B viruses, respiratory syncytial virus,
and parainfluenza viruses 1, 2, 3, and 4. J Clin
Microbiol. 42(4):1564-1569.
21. Khalil S.O, Enan K.A, Ali. Y. H, Salim B, Watt
I.M.E, P. J, Zardis M, and P. R. Lambden (2015):
Detection and Molecular Characterization of
Respiratory Syncytial Virus (RSV) in Children
with Respiratory Signs in Khartoum State, Sudan
2011-2012
22. White DA, Zar HJ, Madhi SA, Jeena P, Morrow B,
Masekela R, Risenga S, Green R (2016): Acute
viral bronchiolitis in South Africa: Diagnostic
flow. S Afr Med J. 106(4):25-26.
23. Yadav K, Ramachandran VG, Das S, and Shah D
(2013): One Step RT- PCR for Detection of
Respiratory Syncytial Virus in Children with
Lower Respiratory Tract Infection. Research and
Reviews: Journal of Medical and Health Sciences,
2 (4)49.
24. Akhter J, Al Johani S, Dugaishm F, Al Hefdhi R
(2008): Etiology of Respiratory Viral Infections
using Rapid Virus Isolation Methods at a Tertiary
Care Center in Riyadh, Saudi Arabia. Saudi Pharm
J. 17(2):177-180.
25. Fattouh AM, Mansi YA, El-Anany MG, El-Kholy
AA, El-Karaksy HM (2011): Acute lower
respiratory tract infection due to respiratory
syncytial virus in a group of Egyptian children
under 5 years of age. Ital J Pediatr. 37:14.
26. Shobugawa Y, Saito R, Sano Y, Zaraket H, Suzuki
Y, Kumaki A, Dapat I, Oguma T, Yamaguchi M,
Suzuki H (2009): Emerging genotypes of human
respiratory syncytial virus subgroup A among
patients in Japan. J Clin Microbiol. 47(8):2475-
2482.
27. Henkel JH, Aberle SW, Kundi M, Popow-Kraupp
T (1997): Improved detection of respiratory
syncytial virus in nasal aspirates by seminested
RT-PCR. J Med Virol. 53(4):366-371.
28. Kuypers J, Wright N, Morrow R (2004):
Evaluation of quantitative and type-specific real-
time RT-PCR assays for detection of respiratory
syncytial virus in respiratory specimens from
children. J Clin Virol. 31(2):123-129.
29. Kuypers J, Wright N, Ferrenberg J, Huang ML,
Cent A, Corey L, Morrow R (2006): Comparison
of real-time PCR assays with fluorescent-antibody
assays for diagnosis of respiratory virus infections
in children. J Clin Microbiol. 44(7):2382-2388.
30. Liao RS, Tomalty LL, Majury A, Zoutman DE
(2009): Comparison of viral isolation and
multiplex real-time reverse transcription-PCR for
confirmation of respiratory syncytial virus and
influenza virus detection by antigen
immunoassays. J Clin Microbiol. 47(3):527-532.
31. Centers for Disease Control & Prevention [CDC]
(2014). Respiratory Syncytial Virus: Infection and
Zahran et al. / Comparison of Reverse Transcription-PCR and Viral Culture, Volume 26 / No. 2 / April 2017 27-36
Egyptian Journal of Medical Microbiology
36
Incidence. Retrieved from:
http://www.cdc.gov/rsv/about/infection.htm.
32. Jadallah R.S (2011): Molecular Epidemiology of
Respiratory Syncytial Virus (RSV) In Southern
Palestine in Partial Fulfillment of the
Requirements for the Degree Masters of Science
September 2011.
33. Savić N, Janković B, Minić P, Vasiljević Z, Sovtić
A, Pejić K, Sarajlija A, Gazikalović S (2011):
Clinical characteristics of respiratory syncytial
virus infection in neonates and young infants.
Vojnosanit Pregl. 68(3):220-224.
34. McGuiness CB, Boron ML, Saunders B, Edelman
L, Kumar VR, Rabon-Stith KM (2014):
Respiratory syncytial virus surveillance in the
United States, 2007-2012: results from a national
surveillance system. Pediatr Infect Dis J.
33(6):589-594.
35. Cui B, Zhang D, Pan H, Zhang F, Farrar J, Law F,
van Doorn HR, Wu B, Ba-Thein W (2015): Viral
aetiology of acute respiratory infections among
children and associated meteorological factors in
southern China. BMC Infect Dis. 15:124.
36. Breiman RF, Cosmas L, Njenga M, Williamson J,
Mott JA, Katz MA, Erdman DD, Schneider E,
Oberste M, Neatherlin JC, Njuguna H, Ondari
DM, Odero K, Okoth GO, Olack B, Wamola N,
Montgomery JM, Fields BS, Feikin DR (2015):
Severe acute respiratory infection in children in a
densely populated urban slum in Kenya, 2007-
2011. BMC Infect Dis. 15:95.
37. Buckingham SC, Bush AJ, Devincenzo JP (2000):
Nasal quantity of respiratory syncytical virus
correlates with disease severity in hospitalized
infants. Pediatr Infect Dis J. 19(2):113-117.
38. DeVincenzo JP, El Saleeby CM, Bush AJ (2005):
Respiratory syncytial virus load predicts disease
severity in previously healthy infants. J Infect Dis.
191(11):1861-1868.
39. Goto-Sugai K, Tsukagoshi H, Mizuta K, Matsuda
S, Noda M, Sugai T, Saito Y, Okabe N, Tashiro
M, Kozawa K, Tanaka R, Morita Y, Nishina A,
Kimura H (2010): Genotyping and phylogenetic
analysis of the major genes in respiratory syncytial
virus isolated from infants with bronchiolitis. Jpn J
Infect Dis. 63(6):393-400.
40. Aboul-Ftouh D.S (2002): Respiratory Syncytial
Virus A cause of ACUTE BRONCHIOLITIS in
Egyptian Infants. THESIS submitted for partial
fulfillment of M.Sc. degree in pediatrics, Cairo
University.
41. Janković B, Minić P, Maglajlić-Djukić S,
Prekajski N (2009): Recommendations for the
prevention of respiratory syncytial virus in high-
risk pediatric groups. Vojnosanit Pregl.
66(10):827-829.
