﻿Sentinel Surveillance of Influenza-Like Illness in Two
Hospitals in Maracay, Venezuela: 2006­2010
Guillermo Comach1
*, Nimfa Teneza-Mora2
, Tadeusz J. Kochel2,3
, Carlos Espino1
, Gloria Sierra1
,
Daria E. Camacho1
, V. Alberto Laguna-Torres3
, Josefina Garcia3
, Gloria Chauca3
, Maria E. Gamero3
,
Merly Sovero3
, Slave Bordones4
, Iris Villalobos5
, Angel Melchor6
, Eric S. Halsey3
1 Laboratorio Regional de Diagnostico e Investigacion del Dengue y otras Enfermedades Virales (LARDIDEV), Instituto de Investigaciones Biomedicas de la Universidad de
Carabobo (BIOMED-UC), Maracay, Venezuela, 2 Naval Medical Research Center, Silver Spring, Maryland, United States of America, 3 U.S. Naval Medical Research Unit Six
(NAMRU-6), Lima, Peru, 4 Hospital Jose
´ Mari
´a Caraban
~o Tosta, Instituto Venezolano de los Seguros Sociales, Maracay, Venezuela, 5 Hospital Central de Maracay,
Corporacio
´n de Salud de Aragua (CORPOSALUD ARAGUA), Maracay, Venezuela, 6 Direccio
´n de Epidemiologi
´a, Corporacio
´n de Salud de Aragua (CORPOSALUD ARAGUA),
Maracay, Venezuela
Abstract
Background: Limited information exists on the epidemiology of acute febrile respiratory illnesses in tropical South American
countries such as Venezuela. The objective of the present study was to examine the epidemiology of influenza-like illness
(ILI) in two hospitals in Maracay, Venezuela.
Methodology/Principal Findings: We performed a prospective surveillance study of persons with ILI who presented for
care at two hospitals in Maracay, Venezuela, from October 2006 to December 2010. A respiratory specimen and clinical
information were obtained from each participant. Viral isolation and identification with immunofluorescent antibodies and
molecular methods were employed to detect respiratory viruses such as adenovirus, influenza A and B, parainfluenza, and
respiratory sincytial virus, among others. There were 916 participants in the study (median age: 17 years; range: 1 month ­
86 years). Viruses were identified in 143 (15.6%) subjects, and one participant was found to have a co-infection with more
than one virus. Influenza viruses, including pandemic H1N1 2009, were the most frequently detected pathogens, accounting
for 67.4% (97/144) of the viruses detected. Adenovirus (15/144), parainfluenza virus (13/144), and respiratory syncytial virus
(11/144) were also important causes of ILI in this study. Pandemic H1N1 2009 virus became the most commonly isolated
influenza virus during its initial appearance in 2009. Two waves of the pandemic were observed: the first which peaked in
August 2009 and the second - higher than the preceding - that peaked in October 2009. In 2010, influenza A/H3N2 re-
emerged as the most predominant respiratory virus detected.
Conclusions/Significance: Influenza viruses were the most commonly detected viral organisms among patients with acute
febrile respiratory illnesses presenting at two hospitals in Maracay, Venezuela. Pandemic H1N1 2009 influenza virus did not
completely replace other circulating influenza viruses during its initial appearance in 2009. Seasonal influenza A/H3N2 was
the most common influenza virus in the post-pandemic phase.
Citation: Comach G, Teneza-Mora N, Kochel TJ, Espino C, Sierra G, et al. (2012) Sentinel Surveillance of Influenza-Like Illness in Two Hospitals in Maracay,
Venezuela: 2006­2010. PLoS ONE 7(9): e44511. doi:10.1371/journal.pone.0044511
Editor: Ce
´cile Viboud, National Institutes of Health, United States of America
Received April 14, 2012; Accepted August 3, 2012; Published September 11, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This study was funded by the United States Department of Defense, Global Emerging Infections Surveillance and Response System, a Division of the
Armed Forces Health Surveillance Center, WORK UNIT NUMBER: 847705.82000.25GB.B0016. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gcomach@yahoo.com
Introduction
Acute respiratory infection (ARI) remains a leading cause of
global burden of disease, and is the second most common cause of
illness worldwide, with an annual global incidence exceeding 400
million [1­3]. A prerequisite of public health planning to reduce
global disease burden from ARI is to examine data on its
epidemiology in order to better define environmental factors as
well as target populations for preventive interventions [4].
Respiratory viruses are predominant causes of ARIs, and the
epidemiology of acute viral respiratory illnesses in developed
countries with temperate climates has been well-characterized [5­
7]. In countries such as the United States, children have been
shown to carry a large burden of viral respiratory diseases [5].
Recent prospective studies, which utilized more sensitive methods
for detecting respiratory viruses such as multiplex polymerase
chain reaction (PCR), have similarly demonstrated that the highest
rates of viral respiratory infection occur among children and the
frequency of infection tends to decrease with age due to increasing
acquired immunity [8]. Respiratory syncytial virus (RSV), in-
fluenza virus, parainfluenza virus, and rhinovirus have long been
identified as common causes of ARI [9]. Recent improvements in
molecular detection techniques have allowed the identification of
multiple new respiratory viruses such as human metapneumovirus
(hMPV), human bocavirus (HBoV) and human coronavirus NL63
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[8]. While the body of literature describing the epidemiology of
acute viral respiratory diseases in developed countries has rapidly
expanded, knowledge of the distribution of these diseases in
regions such as tropical South America remains limited.
Influenza viruses are among the most impactful acute re-
spiratory pathogens in terms of morbidity and mortality. Despite
developed public health intervention programs, the estimated
annual average number of influenza-related hospitalizations in the
United States exceeds 200,000, and 36,000 deaths are attributable
to influenza infections yearly [10,11]. Information on the
contribution of influenza viruses to the global burden of disease
due to acute respiratory illness is incomplete. Data on the
epidemiology of influenza viruses in developed countries are
derived from multiple sources to include laboratory-based
surveillance, sentinel surveillance, as well as hospitalization and
outpatient records. In developing countries, where resources are
sparse, sentinel surveillance methods may be more readily
accessible and more cost-effective than laboratory-based or
population-based surveillance for determining the viral etiology
of influenza-like illness (ILI) in these regions. Better identification
of the viral causes of ILI will enable clinicians in resource-limited
settings to appropriately treat and manage patients; more
importantly, it will allow public health officials to formulate more
effective prevention and control strategies, including monitoring of
influenza vaccine efficacy in their communities [12].
Studies on the epidemiology of ILI in the tropical South and
Central American countries of Peru, Brazil, Ecuador, Nicaragua,
Honduras, and El Salvador have been published [13­16]. A
prospective study of adults with ILI in Sao Paulo, Brazil, revealed
that while influenza viruses were the predominant cause of ILI,
rhinoviruses and other respiratory viruses were detected in 19.6%
and 13.7% of subjects, respectively [14]. This observation
illustrated that a significant proportion of patients who are
clinically diagnosed with influenza virus infection may have
symptoms indistinguishable from other respiratory viruses. In
a prospective study of ILI in Ecuador, the regional distribution of
influenza virus infections varied; a higher detection rate of
influenza A occurred in Quito, located in the highlands where
the level of absolute humidity is lower, whereas influenza A
detection rate was lower in the coastal city of Guayaquil, which
has a more humid tropical climate [15]. Additionally, an expanded
sentinel surveillance of ILI conducted at 31 health centers and
hospitals located in 13 Peruvian cities showed that the distribution
of ILI-causing viruses varied by region [13]. These studies
illustrate the importance of conducting baseline and continuing
ILI surveillance in other countries of Central and South America
because ILI can be caused by pathogens other than influenza
viruses. The distribution of respiratory infections may vary within
the region or within a particular country, depending on climate
and topography.
In Venezuela, ARI is the primary cause of weekly notifiable
diseases registered by the National Epidemiological Surveillance
System (NESS) [17]. Nonetheless, very low numbers of respiratory
samples were collected (16,664 from 2006 to 2010) and even lower
numbers (5,167) were confirmed by the National System for
Virological Surveillance of ARI [17­21]. Furthermore, the
epidemiology, clinical characteristics, and viral causes of ILI have
been poorly characterized in Venezuela; to our knowledge, only
one longitudinal study, carried out during a limited period of time
(February 2005­July 2006) and with a low number of patients
(n = 102) with ARI, has been published [22]. The objective of this
paper is to describe the epidemiology of ILI using data from
a sentinel surveillance system at two major hospitals in Maracay,
Venezuela.
Results
General Findings
A total of 916 subjects participated in this study. Six hundred
and thirty seven (69.5%) were recruited at the Hospital Central de
Maracay (HCM). There was a slightly larger proportion of females
compared with males who enrolled in the study (55.2% vs 44.8%),
and the gender distributions at the two sites were similar. Subject
ages ranged from 1 month to 86 years with a mean age of 19.2
years (S.D. = 14.3 years) and a median of 17 years. A significant
percentage of the subjects (17.6%) were under 5 years of age, while
less than 1.0% were 60 years or older. The 15­29 year old group
was the most predominant group comprising 32.8% of the study
population, followed by the 5­14 year old age group (27.2%). The
subjects who presented to HCM were significantly older (mean
age = 21.2 years; S.D. = 13.2 years) compared with those who were
seen at the Hospital del Instituto Venezolano de los Seguros
Sociales Jose Maria Carabano Tosta (IVSS-JMCT) (mean
age = 14.4 years; S.D. = 15.6 years). A large proportion (37.8%)
of the subjects at HCM belonged to the 15­29 age group, while
nearly half of the patients (42.3%) seen at IVSS-JMCT were
younger than 5 years old. Approximately 5% of the subjects
reported having received influenza vaccination within the last
year. A small percentage of patients (2.0%) received antibiotic
treatment for their acute respiratory illness prior to enrollment in
the study. No hospitalized cases with ILI were recruited in this
study; only outpatient subjects participated. Demographic in-
formation is summarized in Table 1.
Laboratory Results
Among the 916 patient samples obtained from the sentinel
surveillance, at least one viral organism was detected by viral
isolation and/or reverse transcriptase-polymerase chain reaction
(RT-PCR) in 143 (15.6%) subjects (Table 2). Only one participant
with a co-infection was observed. Influenza viruses were the most
frequently detected organisms in this passive surveillance, consist-
ing of 67.4% (97/144) of all the viruses detected by viral isolation
and/or RT-PCR. Of the 916 samples collected, 78 (8.5%) were
positive for influenza A viruses and 19 (2.1%) for influenza B
viruses by viral isolation and/or RT-PCR. In addition, non-
influenza viruses were detected only by virus isolation; they were:
adenovirus (1.6%), parainfluenza virus (1.4%), RSV (1.2%), and
other viruses (0.8% ), which included HBoV (0.1%), enterovirus
(0.2%), hMPV (0.1%), rhinovirus (0.1%), and herpes simplex virus
(HSV, 0.2%). The only co-infection was observed in a 22 month
old toddler in whom adenovirus and HSV were isolated.
The virus etiology and detection rates varied with age (Table 2).
The most commonly detected viral pathogens in the 0­4 year old
group were influenza A (11.1%) and adenoviruses (5.6%).
Influenza A and B were the most commonly detected viruses in
the school-age group (5­14 year old: 6.8% and 4.0%, respectively)
and the late adolescent and young adult group (15­29 year-old:
9.7% and 1.7%, respectively). Influenza A was the only detectable
virus among patients who were 60 years or older.
Of the 78 influenza A cases, H3 subtype was detected in 33, and
was observed in all the age groups but in subjects aged 45­59 year
old (Table 2). Six cases had the H1 subtype (non-pandemic), and
18 influenza A viruses were isolated but not subtyped by one-step
RT-PCR; of the latter, 11 can be classified as seasonal because
they were isolated from patients samples collected before the start
of the H1N1 pandemic outbreak of 2009. Twenty-one cases of
pandemic H1N1 2009 influenza virus (pH1N1) infections were
identified in this sentinel surveillance; thirteen occurred in the 15­
Influenza-Like Illness in Maracay, Venezuela
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29 year old group. There were no cases of pH1N1 infections
among adults 45 years old or higher.
Phylogenetic Analysis
Genetic analysis based on partial hemagglutinin gene
sequences (approximately 900 bp) of 27 influenza isolates is
shown in Figure 1 (See Material and Methods for GenBank
accession numbers). This analysis shows that the circulating
seasonal A/H1N1 strains in Venezuela (Figure 1A) were similar
to the ones previously described in Central and South America
[15,16] and all of these samples can be grouped with the A/
Brisbane/56/07 like 2008­2009 genotype. The pH1N1 samples
were part of only one cluster similar to previously reported
strains in Latin America [23]. Influenza A/H3N2 samples
possessed more genetic variability (Figure 1B); isolates from
2007 revealed two genotypes, A/Brisbane/10/07-like and A/
California/7/04-like, while the more recent isolates were closer
to the A/Perth/16/09-like genotype. Finally, in Figure 1C, the
genetic analysis for influenza B isolates disclosed the presence of
two different genotypes, B/Florida4/06-like and B/Malaysia/
2506/07-like, in agreement to what was previously found in the
region [13,16].
Temporal Distribution
Figures 2 and 3 illustrate the temporal distribution of ILI and
the viral etiology of ILI, respectively, from October 2006
through December 2010. ILI was observed throughout the year
with irregular peak activity occurring once or twice annually
during the months of June 2007, January 2008, January and
October 2009, and June 2010 (Figure 2). The pattern of
influenza A occurrence was variable from year to year (Figure 3).
Influenza A virus was not detected during every month of every
year and was observed during 3­6 month periods in 2007,
2009, and 2010 (4, 6, and 3 months, respectively). Lower
influenza A activity was observed in 2008, with detection only
during the months of January, March, and September. The
occurrence of peak ILI activity correlated with the detection of
influenza A virus in the subjects' respiratory samples during
those months (see Figures 2 and 3). Peak influenza B virus
activity was detected in January 2008 and December 2010.
Adenovirus was detected more frequently from December to
April. RSV infection occurred more frequently from June to
November. Parainfluenza viruses were detectable throughout the
year but without distinct seasonality.
The passive surveillance illustrated the impact of pH1N1 on
the distribution of respiratory viruses associated with ILI in
2009 (Figure 3). During the ILI peaks from 2006­2010, the
percentage of ILI cases attributable to influenza viruses ranged
from 10% to 44%. The first cases of pH1N1 in this surveillance
system were detected in July 2009, three months after the swine
origin influenza outbreak began in Mexico [24]. During the first
wave in July 2009, the monthly virus detection rate rose to
33%, and 57% (4/7) of all the viruses detected were of the
pandemic strain. The first wave simulated a typical ILI peak
activity similar to those observed in other years. However,
during the larger second wave in October 2009, the virus
positivity rate exceeded 40%, and the pandemic strain was
Table 1. Study population recruited by passive surveillance in two hospitals of Maracay, Venezuela: October 2006­ December
2010.
Characteristics of the population HCM* IVSS{ Total
No. (%) No. (%) No. (%)
Number of subjects enrolled 637 279 916
Gender
Female 344 (54.0) 162 (58.1) 506 (55.2)
Male 293 (46.0) 117 (41.9) 410 (44.8)
Age
Mean, 6STD (yrs) 21.2613.2 14.4615.6 19.2. 614.3
Median, (range in yrs) 20 (1 mo­86) 7 (1 mo­65) 17 (1 mo ­86)
0­4 43 (6.8) 118 (42.3) 161 (17.6)
5­14 196 (30.8) 53 (19.0) 249 (27.2)
15­29 241 (37.8) 59 (21.1) 300 (32.8)
30­44 126 (19.8) 30 (10.8) 156 (17.0)
45­59 25 (3.9) 18 (6.5) 43 (4.7)
$60 6 (0.9) 1 (0.4) 7 (0.8)
Influenza vaccination (self-reported) 31 (4.9) 17 (6.1) 48 (5.2)
Medical attention before enrollment 175 (27.5) 29 (10.4) 204 (22.3)
Previous treatment
Treatment 14 (2.2) 21 (7.5) 35 (3.8)
Including antibiotics 9 (1.4) 9 (3.2) 18 (2.0)
No treatment 468 (73.5) 168 (60.2) 636 (69.4)
No information 155 (24.3) 90 (32.3) 245 (26.7)
*Hospital Central de Maracay.
{
Hospital del Instituto Venezolano de los Seguros Sociales-Jose
´ Mari
´a Caraban
~o Tosta.
doi:10.1371/journal.pone.0044511.t001
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identified in greater than 73% (11/15) of the viruses detected.
Figure 3 demonstrates that pH1N1 did not completely replace
seasonal influenza A viruses. The peak pandemic activity in
October 2009 was followed by a rapid decline in the rate of
pandemic strain detection one month later. Meanwhile, seasonal
influenza A viruses remained in circulation throughout the
pandemic period comprising 27% of all the influenza A viruses
detected in October 2009. In 2010, 20% of influenza A
specimens obtained via oropharyngeal swabs were randomly
selected and tested for the pandemic strain via RT-PCR, and
no cases of pH1N1 were detected (Figure 3). However, seasonal
influenza A/H3N2 continued to be detected by our surveillance
system with monthly positivity rates ranging from 16% to 30%.
Clinical Manifestations
The clinical features of subjects in our surveillance are
summarized in Table 3. Compared with those whose respiratory
secretions tested negative, subjects in whom virus was identified
were more likely to have sore throat, headache, pharyngeal
congestion, and ear pain. There were no significant differences in
the symptoms of individuals who had seasonal influenza A when
compared with those who suffered from pH1N1 influenza, except
that a higher proportion of the latter subjects had expectoration,
myalgias, and lymphadenopathy. The symptoms of influenza A
(either seasonal or pandemic) and influenza B were clinically
indistinguishable. When compared with patients who had ILI due
to other viruses, a higher percentage of those with confirmed
influenza virus infection experienced sore throat, myalgias, and
headache.
Discussion
There is scarce information about the epidemiology of acute
febrile respiratory illness in Venezuela and, to our knowledge, only
one longitudinal study has been published [22]. This investigation,
however, was limited to few number of subjects (n = 102) recruited
during a short period of time (17 months) and did not describe the
transmission seasonality of the viral infections. Thus, our study is
the first to fully describe the epidemiology and viral etiology of ILI
in Venezuela and provides baseline levels of ILI activity in a typical
highly-populated urban city.
Our study demonstrated that influenza viruses are a main cause
of ILI at HCM and IVSS in Maracay in agreement with findings
reported by Venezuelas NESS [17­21]. Nevertheless, the rate of
confirmed influenza virus infections found in our surveillance
(10.6%, 97/916; Tables 2 and 3) during the study period was
lower than the one (27.7%) reported by the NESS for the same
period [17­21]. These differences in detection rates may be
attributable to different strategies for capturing ARI patients,
especially those with influenza, used by the NESS and by this
study protocol (Flores E, Director of Epidemiology, Corposalud
2011, personal communication). The NESS randomly selected
a sample of ARI cases (including those with influenza) with
emphasis on severe hospitalized cases, whereas in our protocol we
recruited ILI subjects in an outpatient setting where the majority
had symptoms that were not severe. On the other hand, the
percentage of influenza viruses (not including pH1N1) detected in
our study during a similar period of time, but in different years
(February 2007­ July 2008: 22 of 38, 57.9%; data not shown), was
much higher than the one reported by Valero et al. (February
2005­ July 2006: 7 of 46, 15.2% ) [22]. Two causes may have
Table 2. Age distribution of febrile respiratory viral infections detected by passive surveillance in two Hospitals of Maracay,
Venezuela: October 2006­December 2010.
Age group (in years)
0­4 5­14 15­29 30­44 45­59 $60 Total
Viral Pathogen No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%)
Adenovirus 9*(5.6) 3 (1.2) 0 2 (1.3) 1 (2.3) 0 15 (1.6)
Influenza virus A:
H3{ 9 (5.6) 8 (3.2) 7 (2.3) 8 (5.1) 0 1 (14.3) 33 (3.6)
H1{ 3 (1.9) 2 (0.8) 1 (0.3) 0 0 0 6 (0.7)
Seasonal not subtyped 3 (1.9) 2 (0.8) 4 (1.3) 1 (0.6) 1 (2.3) 0 11 (1.2)
pH1N11 2 (1.2) 3 (1.2) 13 (4.3) 3 (1.9) 0 0 21 (2.3)
Not subtyped|| 1 (0.6) 2 (0.8) 4 (1.3) 0 0 0 7 (0.8)
Influenza virus B 4 (2.5) 10 (4.0) 5 (1.7) 0 0 0 19 (2.1)
Parainfluenza virus 6 (3.7) 1 (0.4) 3 (1.0) 3 (1.9) 0 0 13 (1.4)
RSV" 8 (5.0) 0 1 (0.3) 2 (1.3) 0 0 11 (1.2)
ORV** 2 (1.2) 2 (0.8) 0 3 (1.9) 0 0 7 (0.8)
Positives 47 (29.2) 33 (13.3) 38 (12.7) 22 (14.1) 2 (4.7) 1 (14.3) 143 (15.6)
Negatives 114 (70.9) 216 (86.7) 262 (87.3) 134 (85.9) 41 (95.3) 6 (85.7) 773 (84.4)
Total 161 249 300 156 43 7 916
*One mixed infection with herpes simplex virus in a 22 month old toddler.
{
Influenza A/H3 subtype.
{
Influenza A/H1 subtype (non-pandemic).
1
pH1N1: Pandemic (H1N1) 2009 influenza virus.
||
Influenza A isolated but not subtyped by RT-PCR (unknown subtype).
"
Respiratory syncytial virus.
**ORV: Other respiratory viruses, which includes human metapneumovirus, human bocavirus, herpes simplex virus, and enterovirus.
doi:10.1371/journal.pone.0044511.t002
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accounted for the significant differences found in both studies: a)
the collection, preservation and further processing of respiratory
samples, and b) the type of cells and IFA reagents used for virus
isolation and identification.
The proportion of influenza cases was significantly different
when comparing non-pandemic and pandemic periods. Before the
H1N1 2009 pandemic, the NESS [21] detected influenza virus in
64.7% of subjects in whom a virus was isolated; a similar
proportion to the 55% (data not shown) found in our study.
During the height of the pandemic (from July through Dec 2009),
the NESS confirmed 97% of all ARI cases with a virus as having
either influenza A or B virus compared with 87% observed in our
study.
The predominance of influenza viruses as etiological agents of
ILI in Maracay, Venezuela, is consistent with observations of
surveillance studies in other tropical Central and South American
countries [13,15,16]. While the overall virus detection rate (143/
916, 15.6%; Table 2) was lower compared with other studies, the
positive rates for influenza A (8.5%) and influenza B (2.1%) in this
surveillance were comparable to those observed in a similar study
in El Salvador, Honduras, and Nicaragua [16]. Our findings were
also consistent with a study in Indonesia, which identified
influenza A or B in 11% of all respiratory samples using viral
isolation and RT-PCR [25]. Our findings were consistent with
those from a prospective study of outpatient children in northern
Taiwan, in which influenza A or B were isolated in 12.2% of
subjects with ILI, using Madin-Darby canine kidney (MDCK) cell
cultures and hemagglutinin inhibition assay for antigen detection
[26]. However, our detection rate for all respiratory viruses, as well
as influenza A and B viruses, was generally lower compared to
other studies conducted in South America. In an expanded
sentinel surveillance study in Peru, which utilized a similar
methodology for viral detection as our study, the virus positivity
rate was 42.1%, and influenza A and B rates were 25.1% and
9.7%, respectively [13]. In a prospective surveillance study of ILI
in two Ecuadorian cities using similar methods, at least one virus
was detected in 35% of the participants; influenza A was detected
by PCR in 21.6% while 6.4% tested positive for influenza B [15].
The lower virus detection rates found in our study is unclear
because we used the same operative protocol described in the
mentioned studies [13,15]. Nevertheless, the different skills to
collect respiratory tract specimens from ILI patients, by the health
personnel employed in their studies and ours may have accounted
for the lower virus detection rates found in our study.
While influenza viruses were observed to be the most prevalent
viral pathogen, adenovirus, parainfluenza virus, and RSV were
Figure 1. Phylogenetic trees of influenza viruses circulating in Maracay. This figure shows the phylogenetic relationship of the HA gene
segment within influenza A/H1N1 (A), influenza A/H3N2 (B) and influenza B (C) viruses. Phylogenetic trees were constructed by the neighbor-joining
method and bootstrap analysis to determine the best-fitting tree for the gene. For the comparison, we have included strains reported from GenBank.
Only bootstrap values over 90% are shown.
doi:10.1371/journal.pone.0044511.g001
Figure 2. Influenza-like illness detected by passive surveillance in Maracay, Venezuela: October 2006­December 2010.
doi:10.1371/journal.pone.0044511.g002
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important causes of ILI at the two hospitals (Table 2). In
Venezuela, the NESS reported RSV as the most frequently
detected non-influenza respiratory virus followed by parainfluenza
virus and rhinovirus [17­21]. The study performed in Zulia,
Venezuela, also reported RSV as the most common detected virus,
followed by adenovirus, parainfluenza virus and influenza viruses
[22]. As previously mentioned, the differences in the detection
rates may be attributed to the different procedures used by the
NESS, other Venezuelan researchers. [22] and us. These viral
pathogens were also frequently detected in other surveillance
studies in Central and South America [14­16].
The highest detection rate of respiratory viruses was observed in
the 0­4 year old group (29.2%; Table 2). The rate of viral
positivity generally decreased with age as acquired immunity
increased in older subjects. A slight increase can be seen between
the 30­44 year old group (14.1%) and 60 years or older group
(14.3%). Subjects who were 60 years old or older comprised less
than 1% of the study population, and thus, the prevalence in this
age group may be unreliable. Contrary to our study, the other
study from. Venezuela had higher detection rates in both the 0­6
and $41 year old groups (48.2% and 57.1%, respectively) [22].
On the other hand, our findings are consistent with observations in
the Tecumseh Study, a community-based surveillance in Michi-
gan, in which children were shown to have the highest rate of viral
respiratory diseases [5,6]. The Tecumseh study further illustrated
the variable impact of influenza viruses among the different age
groups depending on the influenza virus subtype. For example,
influenza A/H3N2 affected a wide range of age groups while
influenza A/H1N1 and influenza B virus infections occurred more
frequently among older children and young adults [5,6]. Our
findings were similar being seasonal influenza A/H1N1 viruses
and influenza B detected primarily in children and young adults,
and seasonal influenza A/H3N2 found in all age groups.
As expected, pH1N1 was the most common influenza A subtype
identified among the subjects with ILI in 2009 (Figure 3). In 2009,
37.5% (Figure 3) of ILI cases were due to pH1N1; this detection
rate was lower than the 52.2% detection rate reported by the
Venezuela's NESS [21]. Nevertheless, this finding was similarly
demonstrated in respiratory illness surveillance networks in other
tropical and temperate South American countries such as
Guatemala [27], Peru [28], Argentina [29], and Brazil [30]. In
2010, non-pandemic influenza viruses continued to circulate in
Venezuela, and pH1N1 was not detected in our surveillance study,
suggesting that pH1N1 did not displace seasonal influenza A
viruses. During the same year, 1.6% of the ARI cases reported to
Venezuela's NESS were due to pH1N1 [21]. Infection with
pH1N1 may have stimulated immunity among the residents of this
community during its initial arrival in 2009. This observation
suggests that the circulating pH1N1 in 2010 may not have
significantly mutated relative to the strain in 2009, so that
antibodies stimulated by the natural infection during its initial
arrival may have still been highly efficient in protecting the
Figure 3. Monthly distribution of acute febrile respiratory viral infections by different viruses detected through a passive
surveillance in two health centers of Maracay.
doi:10.1371/journal.pone.0044511.g003
Influenza-Like Illness in Maracay, Venezuela
PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44511
community from another wave of pH1N1 outbreak. In 2010, the
infection rate of influenza A/H3 was higher than the rates
observed during previous years, further illustrating the lack of cross
protection between pH1N1 and influenza A/H3.
Our study shows that hMPV and HBoV were not commonly
associated with ILI, based on the low detection rates observed in
our surveillance. In a study from Ecuador which utilized methods
similar to those employed in our study, a low detection rate for
HBoV (0.2%) was reported [15]. The latter study, as well as
another from Central America which used methods similar to
ours, reported rare detection of hMPV (,0.2%) [15,16]. In
contrast, a prospective study of ILI among Brazilian adults, which
utilized viral isolation and RT-PCR testing on respiratory samples,
detected rhinoviruses in 19.6% of patients [14]. Although
rhinoviruses are typically associated with milder illness, they can
contribute to the misdiagnosis of influenza based on clinical case
definition alone. A cohort study of Vietnamese children hospital-
ized for acute febrile respiratory illness, which applied multiplex-
PCR assays on respiratory samples, revealed slightly higher
prevalence rates of HBoV (2%) and hMPV (5%) infections [31].
It is important to note that our method of identification (culture on
three cell lines), compared to molecular diagnostic methods,
substantially lacked sensitivity for detecting rhinoviruses, hMPVs,
and HBoVs.
Our study shows that patients with pH1N1 infections were
more likely to have myalgias, productive cough with expectora-
tion, and lymphadenopathy than with those infected with seasonal
influenza A virus (Table 3). In contrast, clinical manifestations in
Guatemalan subjects hospitalized for pH1N1 and seasonal in-
fluenza A infections did not significantly differ [27].
Our study had limitations worth noting. Data collection at
only two hospitals in an urban area limits our ability to
generalize our findings to the population. The low virus
detection rates may be attributable to variations in the skills
of the health staff employed to collect the respiratory specimens.
The sampling method may have a significant effect on the
proportion of respiratory viruses identified, and nasopharyngeal
washes may yield higher sensitivity over nasopharyngeal or
oropharyngeal swabs [32]. Study participants were exclusively
seen in the outpatient setting, thus limiting our ability to
examine the impact of respiratory viruses in hospitalized cases.
A large proportion of ILI cases were not associated with any
pathogen, and the impact of bacteria on this clinical syndrome
cannot be determined from this study, since the respiratory
samples were not cultured for bacteria. Two methods of
detection were used for identification of influenza (PCR and
culture) whereas only one method of detection was used for the
other viruses (culture). Twenty-two percent (9/40) of the
respiratory samples which were positive for influenza viruses
by RT-PCR were negative by viral isolation illustrating that
viral detection by culture underestimated the true prevalence.
Viral culture may not be the ideal way of isolating organisms
such as RSV, hMPV, HBoV and rhinoviruses leading to
significant underestimation of their detection rates [33]. Despite
these limitations, our study contributes information on the
distribution and etiology of ILI at two hospitals in Maracay,
Venezuela. This knowledge can serve as a baseline for future,
more expansive population-based surveillance studies of in-
fluenza and other respiratory viruses in this region.
Table 3. Signs and symptoms of patients with acute febrile respiratory infections detected by virus identified in two health centers
of Maracay, Venezuela: October 2006­December 2010.
Total
No Vi
´rus
Detected
Virus
Detected
Seasonal
Influenza A Influenza B p(H1N1) Other viruses**
N = 916 N = 773 N = 143 N = 50 N = 19 N = 21 N = 46
Signs/Symptoms No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%)
Cough 880 (96.1) 749 (96.9) 131 (91.6) 46 (92.0) 18 (94.7) 20 (95.2) 40 (87.0)
Malaise 864 (94.3) 730 (94.4) 134 (93.7) 45 (90.0) 18 (94.7) 21 (100.0) 43 (93.5)
Rhinorrhea 861 (94.0) 729 (94.3) 132 (92.3) 48 (96.0) 16 (84.2) 17 (81.0) 42 (91.3)
Sore throat 178 (19.4) 135 (17.5) 43 (30.1)* 21 (42.0) 4 (21.1) 9 (42.9) 8 (17.4){
Expectoration 536 (58.5) 459 (59.4) 77 (53.8) 23 (46.0) 7 (36.8) 18 (85.7){ 23 (50.0)
Myalgias 499 (54.5) 433 (56.0)* 66 (46.2) 20 (40.0) 8 (42.1) 16 (76.2){ 15 (32.6){
Headache 388 (42.4) 309 (40.0) 79 (55.2)* 30 (60.0) 9 (47.4) 15 (71.4) 19 (41.3){
Wheezing 342 (37.3) 310 (40.1)* 32 (22.4) 9 (18.0) 6 (31.6) 3 (14.3) 14 (30.4)
Shortness of breath 267 (29.1) 224 (29.0) 43 (30.1) 12 (24.0) 9 (47.4) 9 (42.9) 12 (26.1)
Pharyngeal congestion 132 (14.4) 81 (10.5) 51 (35.7)* 21 (42.0) 4 (21.1) 6 (28.6) 15 (32.6)
Asthenia 71 (7.8) 59 (7.6) 12 (8.4) 3 (6.0) 3 (15.8) 2 (9.5) 2 (4.4)
Ear pain 55 (6.0) 37 (4.8) 18 (12.6)* 8 (16.0) 0 2 (9.5) 3 (6.5)
Diarrhea 53 (5.8) 44 (5.7) 9 (6.3) 2 (4.0) 1 (5.3) 2 (9.5) 3 (6.5)
Conjunctival injection 36 (3.9) 18 (2.3) 18 (12.6) 7 (14.0) 2 (10.5) 2 (9.5) 5 (10.9)
Lymphadenopathy 18 (2.0) 12 (1.6) 6 (4.2) 0 0 3 (14.3){ 1 (2.2)
Abdominal pain 14 (1.5) 9 (1.2) 5 (3.5) 3 (6.0) 0 2 (9.5) 0
*Statistically significant differences (p,0.05) between patients with ILI in whom virus was detected vs. virus was not detected.
{
Statistically significant differences (p,0.05) between patients with seasonal influenza A and pH1N1.
{
Statistically significant differences (p,0.05) between patients with ILI ­ due to influenza viruses and ILI ­ due to viruses other than influenza viruses.
**Other viruses: human metapneumovirus, human bocavirus, herpes simplex virus, respiratory syncytial virus, adenovirus, parainfluenza virus, and enterovirus.
doi:10.1371/journal.pone.0044511.t003
Influenza-Like Illness in Maracay, Venezuela
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Materials and Methods
Study Sites
Maracay is located in the central northern region of Venezuela
(10u 159 N, 67u 399 W), approximately 27 miles from the
Caribbean coast (Figure 4). The climate is tropical with two
seasons: dry (December­April) and rainy (May­November). The
monthly averages for temperature, relative humidity and rainfall
are 25.1uC (range = 23.4uC­27.6uC), 75% (range = 66%­82%)
and 56 mm (range: 0 mm­187 mm), respectively. Maracay is
comprised of two main urban municipalities, named Girardot and
Mario Bricen
~o Iragorry, and had an estimated total population of
677,359 in 2010. The HCM and the IVSS-JMCT are located
approximately 4 miles apart. Both are major county hospitals and
referral health centers with adult and pediatric departments
including emergency services and 241 (IVSS-JMCT) to 433
(HCM) hospitalization beds. They provide services to people in
Maracay as well as those from the adjacent city of Aragua and
three neighboring states.
Study Population
The study population included every outpatient with ILI,
regardless of age, who sought attention at the two study sites
between October 2006 and December 2010, and agreed to
participate in the study. At each site, trained medical personnel
were responsible for properly identifying and classifying patients
with ILI.
Case Definition
Each person with ILI was asked to enroll in the study. A person
was defined as having ILI if he or she had a sudden onset of fever
($38uC) and either cough or sore throat for less than five days in
duration, with or without general symptoms such as myalgias,
prostration, headache, or malaise [13].
Data Collection and Management
Data on gender, age, previous treatments, medical attention
before enrollment, influenza vaccination status, and days of work/
school lost at the time of acute illness were collected utilizing a case
report form (CRF) from all participants who met the case
definition criteria. Temporal distribution of the results were
recorded by month during the study period, taking into account
the number of ILI cases identified and the number of confirmed
cases of influenza A and B in each study site. Monthly reports of
enrolled ILI participants and laboratory results were sent to the
Venezuelan Ministry of Health. Regular personnel training in
protocol procedures were conducted as part of the strategy to
improve sampling, storage,and shipping procedures.
Ethical Management
This protocol was approved as less than minimal risk research
by the Naval Medical Research Center (NMRC), Silver Spring,
Maryland. Institutional Review Board (IRB; Protocol
NMRCD.2002.0019) authorization was given to perform the
study using an information sheet approved and stamped by the
IRB. As this was part of clinical care and routine surveillance
Figure 4. Map of study sites in Venezuela.
doi:10.1371/journal.pone.0044511.g004
Influenza-Like Illness in Maracay, Venezuela
PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44511
benefiting the ministry of health, verbal consent was obtained from
all participants. This method of consent was accepted by the
NMRC IRB as well as the Venezuelan institutions involved. A
verbal consent was approved by both IRBs following CIOMS and
45CFR46 (the Common Rule), 1) it was a minimal risk study, and
2) local health installations would not require a written consent for
the procedures required in this study. Additionally, this document
included all the information that a written consent would require;
a copy was provided to each study subject; and study personnel
responsible for administering the process of informed consent were
trained at each site on human research protection issues and were
certified through the Collaborative Institutional Training Initiative
(CITI). Finally, the frequent monitoring visits conducted by the
study team found no problems in the process nor complaints from
study participants.
Laboratory Analysis
Sample collection. Nasal (807 of 916, 88.1%) or oropha-
ryngeal (109 of 916, 11.9%) swabs were obtained from each
subject for viral isolation and identification. The swabs were
placed in viral transport media and stored at ­70uC until they
were delivered on dry ice to NAMRU-6 in Lima, Peru
´, for
laboratory analysis. Duplicate swabs were processed and analyzed
at the Laboratorio Regional de Diagnostico e Investigacion del
Dengue y otras Enfermedades Virales/Instituto de Investigaciones
Biomedicas de la Universidad de Carabobo (LARDIDEV/
BIOMED-UC) in Maracay, Venezuela, following the same
operative protocols used in NAMRU-6.
Virus isolation and identification. Nine-hundred and
sixteen nasal or oropharyngeal swabs were processed for viral
isolation and identification following the procedure described by
Laguna-Torres et al. [13]. Briefly, patient specimens were
inoculated onto four cell lines: Madin-Darby canine kidney
(MDCK; ATCCH Number CCL-34), African green monkey
kidney (Vero76; ATCCH NumberCRL-1587) and VeroE6
ATCCH Number CRL-1586), and Rhesus monkey kidney
(LLC-MK2 ATCCH Number CCL-7). Upon the appearance of
cytopathic effect or after ten days of culture (or thirteen days in the
case of Vero cells), the cells were spotted onto microscope slides.
Cell suspensions were dried and fixed in chilled acetone for 15
minutes. Virus isolates were identified using direct fluorescence
antibody (DFA) assays. The Respiratory Virus Screening and
Identification Kit (D3 DFA Respiratory Virus Diagnostic Hybrids;
Athens, OH) was utilized for the identification of adenoviruses,
influenza A virus, influenza B virus, parainfluenza viruses (types 1,
2, and 3), and RSV. The D3 DFA Herpes Simplex Virus (HSV)
identification kit and the D3 IFA Enterovirus ID kit (Diagnostic
Hybrids; Athens, OH) were utilized for the identification of HSV
(both HSV-1 and HSV-2) and enteroviruses, respectively. For
isolation of hMPV, we used Vero E6 and LLC-MK2 cell lines. For
detection of hMPV antigens by direct fluorescence assay, we used
an anti-hMPV mouse monoclonal antibody from Diagnostic
Hybrid (Athens, OH). All assays were performed following the
manufacturers' instructions. HBoV was identified using the
methods described by Salmon-Mulanovich, et al [34]. Since
duplicate swab samples were analyzed in different laboratories
(LARDIDEV/BIOMED-UC and NAMRU-6) with the same
diagnostic kit and standard operating procedures, a virus isolation
result was considered positive if the specific virus was isolated and
identified at either site.
A subset of 254 specimens (222 nasals and 32 oropharyngeals)
was analyzed by one-step RT-PCR and/or Real Time RT-PCR
in order to sub-type influenza A/H1N1 (including pH1N1),
influenza A/H3N2, and influenza B viruses. One-step RT-PCR
and/or Real Time RT-PCR were not used to identify non-
influenza viruses because the specific primers and protocols were
available only for sub-typing influenza viruses; thus, only virus
isolation was used to detect and identify non-influenza viruses. Of
the 254 specimens, 48 (18.9%) were randomly selected from
repository samples collected before the 2009 pandemic and tested
for influenza A sub-typing by one-step RT-PCR and/or Real
Time RT-PCR. At the onset of the 2009 pandemic, 150 of 254
(59.1%) respiratory samples were tested for pH1N1 by Real Time
RT-PCR at the request of the Venezuelan Ministry of Health.
After the peak of the second wave of the pandemic, the percentage
of respiratory samples tested by Real Time RT-PCR was reduced
to 22% (56 of 254).
One-step RT-PCR was performed according the procedure and
influenza primers described below. Real Time RT-PCR was
carried out using procedures (CDC protocols CDC REF.# I-007-
05 Version 2007, CDC REF.# LB-013, R-1 and CDC REF.# I-
007-05 Version 2009: Swine Influenza) and materials provided by
the Influenza Division of the Centers for Disease Control and
Prevention, U.S.A. (Stephen Lindstrom,personal communication).
These protocols are available from CDC upon request. For the
purpose of this study, an ILI case with a confirmed viral
respiratory infection was one in which viral isolation and/or
RT-PCR identified a virus.
RNA extraction and one-step RT-PCR. Viral RNA
extraction was performed from the supernatant of infected
MDCK cells using a QIAamp Viral RNA kit (QIAGEN;
Valencia, CA) following the manufacturer's protocol. The one-
step RT-PCR was performed following a procedure described
previously [13] with primers that amplified the hemagglutinin
(HA) gene of influenza A and influenza B viruses using the
SuperScript III One-Step RT-PCR System kit (Invitrogen; San
Diego, CA). The following primers were used for the amplification
of H1 influenza A viruses: H1F-6 (59-AAGCAGGGGAAAA-
TAAAA-39) and H1R-1193 (59-GTAATCCCGTTAATGGCA-
39); for H3 influenza A viruses: H3F-7 (59-ACTAT-
CATTGCTTTGAGC-39) and H3R-1184 (59-ATGGCTGCTT-
GAGTGCTT-39); for influenza B viruses: BHAF-36 (59-GAAGG-
CAATAATTGTACT-39) and BHAR-1140 (59-
ACCAGCAATAGCTCCGAA-39). Five ml of the extracted
RNA was added to 20 ml of master mix containing the enzyme
mixture (SuperScript III RT/Platinum Taq), 2X reaction mixture
(containing 0.4 mM of each dNTP and 3.2 mM of Mg2SO4) and
20 mM of each primer. Cycling conditions included a reverse
transcription step at 50uC for 30 minutes and a denaturation step
at 94uC for 2 minutes. Cycling conditions of the PCR were 40
cycles of 94uC for 15 seconds, 52uC for 30 seconds, and 68uC for
75 seconds, followed by a final incubation step at 68uC for 5
minutes.
DNA sequencing and phylogenetic analysis. For confir-
mation of serotype and genotype of the circulating influenza
viruses, 27 samples were sequenced. As part of the Respiratory
Surveillance Protocol in Venezuela, these 27 viruses were
randomly selected from approximately 10% of the positive
samples. Only the HA gene region of influenza viruses was
analyzed routinely for genotyping The one-RT-PCR products
amplified with the primers described before were purified using
Centri-Sep Columns (Princeton Separation; Englishtown, NJ) and
sequenced using the BigDye Terminator v. 3.1 Cycle Sequencing
Kit (Applied Biosystems; Foster City, CA) following the manu-
facturers' instructions. Sequences were analyzed and edited using
the Sequencer 4.8 software (Applied Biosystems; Foster City, CA).
Phylogenetic trees were constructed by the neighbor-joining
method and bootstrap analysis to determine the best-fitting tree for
Influenza-Like Illness in Maracay, Venezuela
PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44511
the gene using MEGA software (version 4). The statistical
significance of the tree topology was tested by bootstrapping
(1,000 replicas). Pairwise distances between and within the
genotypes at the nucleotide level were calculated with Kimura 2
parameters and with Poisson correction at the amino acid level
with MEGA software. Genbank accession numbers are listed in
Table S1.
Statistical Analysis
Information on the CRFs was entered into a database created in
Microsoft Office Access 2003. The chi square and Fisher exact
tests were used to compare means and associations using SPSS
software version 10.0 (SPSS Inc.; Chicago, IL) and R version 2.8.0
(R Development Core Team; Vienna, Austria).
Supporting Information
Table S1 Genbank accession numbers of DNA se-
quences from 27 Influenza viruses isolated in two health
centers of Maracay, Venezuela: October 2006­December
2010.
(XLS)
Acknowledgments
We would like to express our gratitude to all personnel working at the two
sentinel health centers (HCM and IVSS-HJMCT) in Venezuela for
supporting this surveillance study. We thank Mr. Eduardo Guerra, the
Informatics Superior Technician of LARDIDEV/BIOMED-UC, for his
irreplaceable technical work in Venezuela. We also thank the professional
staff of the Virology Department of NAMRU-6 for invaluable laboratory
and technical support in the execution of the study.
Disclaimer: The views expressed in this article are those of the authors
and do not necessarily reflect the official policy or position of the
Department of the Navy, Department of Defense, nor the U.S.
Government. The corresponding author had full access to all data in the
study and final responsibility for the decision to submit this publication.
Additional Copyright Statement: Nimfa Teneza-Mora, Tadeusz J.
Kochel, and Eric S. Halsey are U.S. military service members, and V.
Alberto Laguna, Josefina Garcia, Gloria Chauca, Maria E. Gamero, and
Merly Sovero are employees of the U.S. Government. This work was
prepared as part of their official duties. Title 17 U.S.C. 1 105 provides that
``Copyright protection under this title is not available for any work of the
United States Government.'' Title 17 U.S.C. 1 101 defines a U.S.
Government work as a work prepared by a military service members or
employees of the U.S. Government as part of those persons' official duties.
Author Contributions
Conceived and designed the experiments: G. Comach TJK VALT.
Performed the experiments: DEC G. Chauca MEG MS. Analyzed the
data: G. Comach NTM TJK CE VALT JG ESH. Contributed reagents/
materials/analysis tools: G. Comach TJK GS DEC G. Chauca MEG MS
SB IV AM. Wrote the paper: G. Comach NTM TJK CE GS DEC VALT
JG ESH. Revised the article critically for important intellectual content:
TJK CE GS DEC VALT JG EHS SB IV AM.
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