The studies on epidemiological profile of
rotavirus infection have revealed that 20-50 percent of
hospitalizations for diarrhea among children in 0-5 years age
group are associated with this infection. The morbidity and
mortality are much in children infected with rotavirus than
previously estimated, and is responsible for two million deaths
per year in developing countries [1-3]. Rota virus genogroups
are based on the structure of specific genes: G types (G1-G14)
refer to the VP7 protein (gene 9) and P types P1A[8]-P1B[4]
refer to VP4 protein (gene 4) and are differentiated by Reverse
transcriptase polymerase chain reaction (RT-PCR). Rotaviruses
can therefore be typed and strains circulating in the community
can be monitored. The strains change in frequency of circulation
over time, and occasionally new reassortment strains are
introduced to a community [4,5]. At least 42 P-G combinations
have been recognized in human infections. Some of the vaccine
strains like G9, G8, G5 and P2A[6] are not covered by available
Rota virus vaccines that have undergone clinical trials[6]. This
study is part of an ongoing research project in a tertiary care
hospital.
Methods
A total of 300 stool samples were collected
from children under 5 years of age. These children were admitted
for symptoms of diarrhea, vomiting, abdominal pain and
dehydration due to acute gastroenteritis in Department of
Paediatrics, SRM Medical College Hospital and Research Centre,
Kattankulathur village, Tamilnadu during the period January
2009-December 2010. Immediately after their admission, we
collected 5 to 10 mL of fresh stool sample from the patients, in
a sterile container.
The laboratory request form with all
necessary details of the patient was duly filled up. An informed
consent form signed by the guardian was obtained. The
Institutional ethical committee clearance was obtained for this
study. Stool samples were stored in sterile vials containing 70
percent tryptic soya broth with 30 percent glycerol, labelled
with patient details and kept in deep freezer at –20ş
Celsius for long term use.
Three months after adequate number of stool
samples had been collected, we performed Enzyme Linked
Immunosorbent Assay (ELISA-GA Generic assays GmbH, Germany).
Rotavirus antigen is a fast enzymometric one- step immunoassay
for the qualitative determination of Rotavirus antigen,
employing a solid phase immobilized polyclonal antibody (sheep)
and murine monoclonal antibodies conjugated to horseradish
peroxidase. Both antibodies are directed against the group
specific VP6 antigen of group A rotaviruses. The test was
performed with the positive and negative control, and assay was
performed according to the manufacturers protocol. Optical
density (OD) of the solution read at 450nm is directly
proportional to the amount of rotavirus antigen bound. For
optimal results, a reference filter of 620nm wavelength was
used. Recommended cut-off value results were interpreted as
positive or negative.
Diagnostic RT PCR was done for the 47 samples
that were positive for rota viral antigen. Extraction of RNA was
performed using QIAGEN miniviral RNA kit (QIAgen Gmbh Hildem,
Germany) in accordance with manufacture’s instructions. The
double standard RNA (dsRNA) was extracted directly from faecal
sample. Prior to addition of RNA to RT-PCR master mix, sample
RNA was subjected to denaturation at 95o
C for 5min followed by incubation in ice for 2min, to separate
the Rotavirus dsRNA. Single stranded RNA was used as template
for RT-PCR to amplify the VP6 antigen coding gene
(VP6F-GACGGVGCRACTACATGGT and VP6R-CCAATTCATNCCTGGTG.) by using
INVITROGEN one step RT-PCR Kit [7].
20 samples of rotavirus antigen positive by
ELISA and RT-PCR were subjected for P and G genotyping with
appropriate primers, and partial nested RT-PCR was performed to
determine both the VP7 (G) and the VP4 (P) genotypes. For VP7
genotyping, rotaviral RNA was subjected to RT-PCR with primers
VP7-F,VP7-R followed by 35 cycles of RT- PCR (each at a
concentration of 20 µM). For VP4 genotyping, all procedures were
identical to those described above, except that RT and the first
amplification was done with appropriate primers. Genotyping was
done using multiplex PCR for (VP7) G-Type and (VP4) P-Type. The
samples were then resolved on 1 percent agarose gel to determine
the G and P types. This combined typing scheme was designed to
detect VP7 genotypes G1, G2, G4, and G9 as well as VP4 genotypes
P4 and P8.
Results
47 stool samples were positive for rotavirus
antigen by ELISA and all of these were positive by RTPCR
(positive predictive value for the test was 100 percent). The
different genotypes identified in 20 positive samples were G9P8,
G1P8, G2P4, G9P4, G1P6 and G1(P- type was not identified in 5
samples) (Table I). To the best of our knowledge,
the genotype G9P4 is a unique rotavirus strain that has not been
reported previously from India.
TABLE I Age Distribution of Children With Different Genotypes of Rotavirus in Stool Samples
Genotype
|
Total no.
|
Age group
|
|
of cases |
< 1 yrs |
1-2 yrs |
2-3 yrs |
3-5 yrs |
G9P8 |
1 |
– |
1 |
– |
– |
G1P8 |
7 |
3 |
2 |
1 |
1 |
G2P4 |
5 |
2 |
– |
1 |
2 |
G9P4 |
1 |
– |
1 |
– |
– |
G1P6 |
1 |
1 |
– |
– |
– |
G1* |
5 |
3 |
1 |
– |
1 |
Total |
20 |
9 |
5 |
2 |
4 |
*P-not able to identify.
Discussion
The majority of the children who tested
positive for rotaviral antigen had clinical features of vomiting
and moderate dehydration. Rotavirus antigen detection by ELISA
and RTPCR for routine screening is labour intensive and has high
cost implications. Since the number of virus particles will
decrease beyond acute phase of gastroenteritis, we collected
stool samples within 3 days after onset of symptoms (to avoid
false negative results).
The G9P4-non vaccine G serotype was
identified in a one and half year old child. This child was
admitted with severe dehydration due to diarrhea, vomiting and
fever. Other routine laboratory investigations were not
significant. The child was treated with parentral fluids and
recovered within 3 days.
Previous studies done in India, during
1990-1991 in New Delhi had G1P8 strain as the predominant
strain. It was documented that during the period of 1992-1993,
G9P11 strain had emerged. In 2000-2007, a new strain of
rotavirus namely G12 was identified [8]. This seems to indicate
a notable relative shift in the prevalence of circulating
viruses, which should be monitored over the coming years. In the
same manner, while analyzing the emergence of new variants in
Tamilnadu, South India, G1 strain were found in Vellore during
1983-1985 and G1-G2 P4-P8 in Chennai during 1995-1998 [9].
Currently there are two Rota virus vaccines
available. One of them is a monovalent Rotavirus G1P8 vaccine
while the other one is a pentavalent Rota Virus vaccine covering
G1, G2, G3, G4 and P8. This study showed a case of G9P4
infection in a child. This serotype is not a part of the vaccine
serotypes. This study revealed the considerable serodiversity
among human rotaviruses in this geographical region and hence
emphasizes the need to give protection with multivalent rota
virus vaccines.
Acknowledgment: The authors thank Dr
Savithri MD, Director, Govt. King Institute of Preventive
Medicine, Guindy, Chennai for granting permission to utilise the
molecular testing facility.
Funding: None; Competing
interests: None stated.
What This Study Adds?
• A unique G9P4 non-vaccine genotype
Rotavirus infection in a child caused acute
gastroenteritis.
|
References
1. Parashar UD, Bresee JS, Gentsch JR, Glass
RI. Rotavirus.Emerg Infect Dis. 1998;4:561-70.
2. Kim JS, Kanf JO, Cho SC, Jang YT, Min SA,
Park TH, et al. Epidemiological profile of rotavirus
infection in the Republic of Korea: Results from prospective
surveillance in the Jeongeub District, 1 July 2002 through 30
June 2004. J Infect Dis. 2005;192:S49-56.
3. Fang ZY, Wang B, Kilgore PE, Bresee JS,
Zhang LJ, Sun LW, et al. Sentinel hospital surveillance
for rotavirus diarrhea in the Peoples Republic of China, Aug
2001-Jul 2003. J Infect Dis. 2005;192:S94-9.
4. Hoshino Y, Jones RW, Ross J, Honma S,
Santos N, Gentsch JR, et al. Rotavirus serotype G9 trains
belonging to VP7 gene phylogenetic sequence lineage 1 may be
more suitable for serotype G9 vaccine candidates than those
belonging to lineage 2 or 3. J Virol. 2004;78:7795-802.
5. Iturriza Gomara M, Green J, Brown DWG,
et al. Seroepidemiological and molecular surveillance of
human Rotavirus infections in the UK. London: Public Health
Laboratory Service; 2000.
6. Gentsch JR, Laird AR, Beilfelt B, Griffin
DD, Banyai K, Ramchandran M, et al. Serotype diversity
and reassortment between human and animal rotavirus strains:
Implications for rotavirus vaccine programs. J Infect Dis. 2005;
192:S146-59.
7. Gouvea V, Glass RI, Woods P, Taniguchi K,
Clark HF, Forrester B, et al. Polymerase chain reaction
amplification and typing of rotavirus nucleic acid from stool
specimens. J Clinic Microbiol. 1990;28: 276-82.
8. Bahl R, Ray P, Subodh S, Shambharkar P,
Saxena M, Parashar U, et al. Incidents of severe of
rotavirus diarrhea in New Delhi, India, and G and P types of the
infecting rotavirus strains. J Infect Dis. 2005;192:S114-S9.
9. Brown DWG, Mathan MM, Mathew M, Martin R,
Beards GM, Mathan VI. Rotavirus epidemiology in Vellore, South
India: Group, subgroup, serotype and electrophore type. J Clinic
Microbiol. 1988;26:2410-4.
10. Ruiz-Palacios GM, Perez-Schael I,
Velazquez FR, Abate H, Breue T, Clemens SA, et al. Safety
and efficacy of an attenuated vaccine against severe
gastroenteritis. N Engl J Med. 2006;354:11-22.
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