Rotaviruses are the most common cause of acute childhood diarrhea
in both developed and developing countries ().
Around 125 million rotavirus-induced diarrhea episodes occur annually
worldwide, resulting in 500,000 to 600,000 deaths ().
Epidemiological surveys performed on a global scale ()
observed incidences of 12 to 71% (average 34%) of rotavirus identification
among children less than three years old suffering from acute diarrhea.
Data reviewed from Brazil demonstrates variation in gastroenteritis
incidence associated with rotavirus in children treated at clinics
or hospitals, in the order of 12 to 42% ().
Type A Rotaviruses are classified into G genotypes and P according
to variations antigens caused by the structural proteins VP7 and
VP4, respectively. There is a global predominance of samples characterized
as G1P ().
The worldwide impact of rotavirus infection has resulted in the
development of vaccination strategies capable in reducing morbidity
and mortality. The tetravalent vaccine (RotashieldTM) was suspended
due to a potential (and controversial) association with intussusception
New vaccine candidates have been examined in recent studies ().
The current study assessed occurrence of re-infection by rotavirus
and mapped prevalent genotypes with the intention of contributing
to the development of safe and effective prevention procedures and
also to the planning of Rotavirus A induced diarrhea control activities.
Patients and methods
Two hundred and seven children with acute diarrhea where studied.
They were recruited from those treated at the walk-in centers and
infirmaries of the Pediatrics and Childcare Department of the Universidade
Federal de Goiás Medical Faculty and the Hospital Materno
Infantil de Goiânia - Goiás, during the period between
July 2000 and October 2002. Inclusion criteria were the presence
of acute diarrhea, defined as three or more liquid or semi-liquid
evacuations per day. Those responsible for the children who met
the criteria had the study explained to them and, agreeing to the
child's participation, signed an informed consent for approved by
the Ethics Committee of the Hospital das Clínicas at FMUSP.
A total of 46 of the children with acute diarrhea who were identified
as having rotavirus in fecal samples and whose guardians gave consent
to participate in outpatients follow-up were selected for a one-year,
prospective study. An equal number of children, who did not have
acute diarrhea and had no evidence of rotavirus in their fecal samples,
were paired for age and sex and enrolled as the control group, observing
the same ethical principles as before.
Both groups were observed for diarrhea episodes, associated or not
with symptomatic or asymptomatic rotavirus infections, for the period
of the study by means of fecal samples. Samples were taken for rotavirus
identification on day zero (recruitment to study), and monthly thereafter
for one year (in addition to samples taken during acute diarrhea
episodes ) from children from both the Rotavirus A positive group
and the control group. Rotaviruses were identified in fecal samples
using polyacrylamide gel electrophoresis (PAGE) ()
and by combined enzyme immunoassay for rotavirus and adenovirus
Rotavirus A genotypes were identified by polymerase chain reaction
and by Nested-PCR for differentiation between VP4 proteins (P genotype
P) and VP7 (G genotypes). The first amplification reaction (RT-PCR)
employed the consensus primers 9con1/9con2 (G genotyping)
and 4con2/4con3 (P genotyping). In the sequential Nested-PCR
reaction a mixture of specific primers was used to identify the
genotypesG1, G2, G3, G4, G5, G8, G9 and G10, and another mixture
to identify genotypes P, P, P, P and P. After amplification,
the products obtained from each fecal sample by RT-PCR and
Nested-PCR were examined in agarose gel, viewed with an ultraviolet
transilluminator and analyzed in comparison with a standard molecular
Statistical analysis was performed using Fisher's exact test, with
the level of rejection of the null hypothesis set at 5% (alpha error
0.05), with a 95% confidence interval.
Two hundred and seven children with acute diarrhea were studied.
Of these, 77 (37.2%) were positive for Rotavirus A when the first
sample was collected and 46 of these were followed up at outpatients.
Similarly, 46 children who had not exhibited acute diarrhea on recruitment
and had not presented rotavirus in their first fecal samples were
paired for age and sex, making up the control group. A total of
766 fecal samples were collected over the year for both groups;
six children from each group were excluded after abandoning follow-up.
It proved possible to identify G genotypes in 61 (79.2%) of the
77 Rotavirus A samples. The G1 genotype was the most prevalent,
occurring in 38 (62.3%) of the genotyped samples, followed by genotype
G9, occurring in 21 (34.4%) samples and G4 in two samples (3.4%).
P genotypes were identified in 39 (50.6%) of the 77 Rotavirus A
samples. The P genotype was the most prevalent, identified in
23 (59.0%) of the genotyped samples . The P genotype was observed
in three (7.7%) samples. A total of 16 samples were positive for
more than one P genotype , with samples being identified with the
pairings P+P (23.1%), P+P (7.7%) and P+P (2.6%).
It was possible to correlate G and P genotypes in a total of 18
samples (23.4%), with a predominance (p < 0.05) of samples characterized
as G1P (77.8%) compared with G9P (11.1%), G4P (5.6%) and
G1P (5.6%) (Figure 1).
of 18 samples of rotavirus A identified with genotypes G and P during the
whole period of collection.
The 40 children in the Rotavirus A (+) follow-up group had an
average age of 19.2 months. Nineteen patients were male and 21 were
female. Three hundred and eighty samples were collected from this
group over the observation period. A total of 51 diarrhea episodes
were observed (an average of 1.3 episodes per child per year). Rotavirus
was not identified in any of the samples collected from any of the
children in this group during the follow-up period.
The control group, also composed of 40 children, had an average
age of 19.7 months. Twenty children were male and 20 were female.
Three hundred and eighty-six fecal samples were collected from the
control group. A total of 48 diarrhea episodes were observed in
this group, with an average of 1.2 episodes per child per year.
Rotavirus was identified in the feces of two of the children in
this group, associated with diarrhea, giving an incidence of 0.05
rotavirus infections per child per year.
From the total of 207 samples collected from children with acute
diarrhea, positive Rotavirus A results made up 37.2%. In general,
similar data is observed in published literature ().
Genotype G1 predominated among the genotyped samples, although elevated
percentages of G9 were also detected and only two samples were identified
as G4. A number of different have described a predominance of samples
defined as G1-G4 ().
In contrast, studies also demonstrate an increasing level of identification
of unusual serotypes, such as genotype G9, occurring in 18.1% of
infections by rotavirus in Australia ().
This fact highlights the possibility that new and emerging serotypes
will have an impact on vaccination strategies, resulting in wider-ranging
vaccines that cover genotypes other than G1-G4.
In terms of G and P genotypes in association, there was a predominance
of samples defined as G1P. On a global scale the G1P genotype
stands out as the most prevalent (),
which could give a basis for monovalent human vaccines specific
to G1P (RotarixTM - Glaxo Smithkline and Avant Imunotherapeutics,
Inc, Needham, MA, USA), including in our country.
This prospective study of children with Rotavirus A and of the control
patients found no evidence of reinfection by rotavirus, which is
in contrast with a number of different follow-up studies that obtained
variable levels of rotavirus reinfection (),
which levels were higher in studies that followed patients from
and in studies following restricted communities, such as indigenous
populations or children at day-care centers (),
In contrast with this, a wide-ranging study performed in Finland
did not identify, in common with our observations, rotavirus reinfection.
It is possible that this fact could be related to a possible protective
effect conferred by natural rotavirus infections that had occurred
prior to the one-year observation period.
Another relevant factor was the detection of two symptomatic rotavirus
infections during the observation of the control patients. The incidence
of infections by rotavirus observed by some authors presents a variation
of 0.07 to 0.8 episodes per child per year ()
during observation of children from birth to two or three years
of age. Such levels are greater than those observed in the current
study, in the order of 0.05 episodes of rotavirus infections per
child per year in the control group, which could be explained by
the older age group since the control group was paired for age with
a group of children already infected by rotavirus. Based on a study
of serological prevalence ()
and on the estimate that virtually all children have been infected
by rotavirus at least once by the age of five (),
it is possible that a percentage of the children in the control
group already had a certain level of protection resulting from previous
infections by rotavirus that were not detected by this study.
This study intends to contribute to knowledge about infections by
rotaviruses of group A in children, by means of vigilance of the
G and P genotypes prevalent in the region. Thus, it intends to provide
support for future research into prophylaxis in our country, since
further studies should be performed in the continued vigilance of
those genotypes prevalent in our country, towards the definitive
implementation of effective preventative measures in the field of
We are grateful for the financial support afforded exclusively by
the Research Support Foundation (Fundação de Apoio
à Pesquisa - UFG) and CAPES.