Sentinel Surveillance of Influenza-Like-Illness in Two Cities of the Tropical Country of Ecuador: 2006–2010
et al. (2011) Sentinel Surveillance of Influenza-Like-Illness in Two Cities of the Tropical
Country of Ecuador: 2006-2010. PLoS ONE 6(8): e22206. doi:10.1371/journal.pone.0022206
Sentinel Surveillance of Influenza-Like-Illness in Two Cities of the Tropical Country of Ecuador: 2006-2010
Richard W. Douce 0 1
Washington Aleman 0 1
Wilson Chicaiza-Ayala 0 1
Cesar Madrid 0 1
Merly Sovero 0 1
Franklin Delgado 0 1
Mireya Rodas 0 1
Julia Ampuero 0 1
Gloria Chauca 0 1
Juan Perez 0 1
Josefina Garcia 0 1
Tadeusz Kochel 0 1
Eric S. Halsey 0 1
V. Alberto Laguna-Torres 0 1
Patricia V. Aguilar, University of Texas Medical Branch, United States of America
0 Funding: This study was funded by the United States Department of Defense Global Emerging Infections Systems Research Program, 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
1 1 Hospital Vozandes , Quito, Ecuador, 2 Hospitals Alcivar y Vernaza, Guayaquil , Ecuador , 3 Hospital Naval, Guayaquil, Ecuador, 4 United States Naval Medical Research Unit Six , Lima , Peru
Background: Tropical countries are thought to play an important role in the global behavior of respiratory infections such as influenza. The tropical country of Ecuador has almost no documentation of the causes of acute respiratory infections. The objectives of this study were to identify the viral agents associated with influenza like illness (ILI) in Ecuador, describe what strains of influenza were circulating in the region along with their epidemiologic characteristics, and perform molecular characterization of those strains. Methodology/Findings: This is a prospective surveillance study of the causes of ILI based on viral culture of oropharyngeal specimens and case report forms obtained in hospitals from two cities of Ecuador over 4 years. Out of 1,702 cases of ILI, nine viral agents were detected in 597 patients. During the time of the study, seven genetic variants of influenza circulated in Ecuador, causing six periods of increased activity. There appeared to be more heterogeneity in the cause of ILI in the tropical city of Guayaquil when compared with the Andean city of Quito. Conclusions/Significance: This was the most extensive documentation of the viral causes of ILI in Ecuador to date. Influenza was a common cause of ILI in Ecuador, causing more than one outbreak per year. There was no well defined influenza season although there were periods of time when no influenza was detected alternating with epidemics of different variant strains.
Competing Interests: Eric S. Halsey and T. Kochel are U.S. military service members, and V. Alberto Laguna-Torres, Julia Ampuero, Gloria Chauca, Juan Perez,
Josefina Garcia and Merly Sovero are employees of the U.S. Government. This work was prepared as part of their official duties.
In an effort to control influenza A by early detection of
potentially epidemic strains for vaccine production, the behavior
of influenza A on a global scale has become very important .
The timing of seasonal influenza epidemics seems to be related to
latitude, with waves of seasonal epidemics radiating from tropical
to temperate regions . In tropical climates, although there may
be a season in which influenza epidemics are more common, there
is much more geographic variation in seasonality, and influenza
seasons are less well defined . The behavior of influenza in the
tropics seems to fall into three patterns; year round activity with
one or two peaks per year, two influenza seasons per year, or
regular peaks of activity without significant epidemic activity in the
rest of the year . Although many authors have sought to link the
behavior of influenza epidemics in the tropics to environmental
factors, no dominant hypothesis seems to explain the varied
seasonal activity of influenza in the tropics .
It has been observed that the dominant strains of influenza in
seasonal epidemics seem to be synchronized between the northern
and southern hemispheres . There are two competing
hypothesis to explain how the tropics are involved in this
phenomenon. One hypothesis is that influenza strains constantly
migrate around the world, passing through the tropics . The
other hypothesis is that there is constant latent viral activity in the
tropics that seeds the hemispheric seasonal epidemics and that the
viral genetic diversity of influenza A is generated in the tropics and
is transmitted to the temperate regions . Understanding
influenza activity in the tropics may help predict the global
behavior of specific strains and aid in the selection of vaccine
Influenza like illness (ILI) is a clinical syndrome that seeks to
identify patients with acute respiratory infection more likely to have
influenza as a cause of their illness . In the tropical part of Latin
America, sentinel surveillance  and population-based studies 
have described the etiology and incidence of ILI as well as
characterized the circulating influenza viruses in Peru, Central
America [11,12] and Brazil . The molecular characteristics of
adenovirus subtypes in some South American countries  have
also been reported. Review of the international literature reveals
very few studies published about acute respiratory infections in
Ecuador [15,16,17,18]. Only one manuscript was published during
the 2009 H1N1 pandemic, describing a high case fatality rate
(16.6%) among patients hospitalized in Quito with acute respiratory
distress syndrome . Comparatively, there remains a notable lack
of published data regarding etiologic agents and burden of
respiratory disease, and ILI in particular, in Ecuador.
Since 2006, a collaborative network was established in Ecuador
with the support of the United States Naval Medical Research
Unit Six (NAMRU6). Data on antiviral resistance patterns 
and adenovirus  obtained in Ecuador during the period
reported in this paper has been reported previously.
The objectives of this study were to identify the viral agents
associated with ILI in Guayaquil and Quito, describe what strains
of influenza were circulating in the region along with their
distribution in time, and perform molecular characterization of
Materials and Methods
The eligible study population included every patient with ILI,
regardless of age, who sought attention or was hospitalized in
participating health centers between July 2006 and June 2010 and
agreed to participate in the study. Participants (outpatients or
inpatients) were recruited when reporting to any of the
participating hospitals (Figure 1). In Guayaquil, participants were
enrolled in the Hospital Naval of Guayaquil, a 130-bed hospital
that serves a population of around 20,000 military personnel and
their families who have rights to the Naval Hospital services, and
in the Emergency Department (ED) of Hospital Luis Vernaza, an
836-bed general hospital which is the major acute care hospital of
Guayaquil and receives adults with major trauma and other major
illnesses. Some of the patients reported from Hospital Luis
Vernaza were referred from Hospital Alcivar, a private, general
100-bed teaching hospital; Hospital Francisco Icaza Bustamante
which is a pediatric hospital; and Hospital Daniel Rodriguez,
which specializes in infectious diseases of adults. In Quito, the
cases were enrolled in the ED of Hospital Vozandes Quito, a
75-bed teaching hospital with an 8-bed intensive care unit and a
20-bed ED, which receives approximately 4,000 patients per
month and manages major trauma as well as urgent care.
At each site, trained medical personnel were responsible for
properly identifying and classifying patients with ILI.
Hospitalization was noted if the patient spent at least one night in the hospital
or health center.
Ecuador is divided by the Andes Mountains into four different
regions: coastal, highlands, rainforest and the Galapagos Islands.
Although Ecuadors equatorial location results in very little
seasonal temperature change, there are a wide variety of biomes
from tundra to tropical rainforest. Guayaquil is a coastal city of 3.3
million located at 4 m altitude, latitude 02.15 S and longitude
79.52 W. The average temperature is 26uC with an average high
temperature of 31uC in December through April and an average
low temperature of 20uC in July through October. The humidity
ranges from 70% in December to 81% in February with an
average of 76%. The capital city of Quito has a population of 1.8
million and is located at 2,800 m altitude, latitude 0.15 S and
longitude 78.35 W. It has a subtropical highland climate with an
average temperature of 15uC and a daily temperature range from
7uC to 23uC. During the year, the monthly average maximum and
minimum temperatures fluctuate less than 5uC and the average
relative humidity ranges from 65% in August to 82% in March.
An ILI case was defined as any person with a sudden onset of
fever ($38uC) and cough or sore throat fewer than five days in
duration, with or without general symptoms such as muscle ache,
prostration, headache, or malaise [9,11]. Severe Acute
Respiratory Infection (SARI) was defined as any case of ILI that was
accompanied with shortness of breath or difficulty breathing and
required hospitalization .
Data collection and management
Data on gender, age, lost work or school days, previous
treatments, medical attention before enrollment, influenza
vaccination status, and travel in the last seven days 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 and epidemiological week (EW) 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 city. Monthly reports of enrolled ILI participants and
laboratory results were sent to the Ministry of Health. Regular
personnel training in protocol procedures and semi-annual site
visits were conducted as part of the strategy to improve sampling,
storage, and shipping procedures. Enrollment was limited to
patients seen in the emergency departments or admitted to a
clinical service of Hospital Vozandes in Quito and Hospital Naval
in Guayaquil. The patients enrolled in Hospital Luis Vernasa in
Guayaquil were also referred from Clinica Alcivar, Hospital
Francisco Icaza Bustamante and Hospital Daniel Rodriguez.
This ILI surveillance protocol was approved as a less than
minimal risk research by the Naval Medical Research Center
Institutional Review Board (IRB; Protocol NMRCD.2002.0019)
in compliance with all applicable U.S. Federal regulations
governing the protection of human subjects, and authorization
was given to perform the study using an information sheet
approved and stamped by the IRB. In view of the fact that this
protocol is a surveillance protocol with no intervention planned,
involving routine care of patients with upper respiratory infections,
with no perceived risk to the patient, in 2006, the IRB of Hospital
Vozandes in Quito also approved the use of verbal consent. On an
annual basis thereafter it was approved by the administration of
said hospital. This was also approved by the other Ecuadorian
institutions involved. The content of the information sheet was
explained to all potential study participants; samples were taken
only after the verbal consent was obtained. Verbal consent from
children 817 years old was obtained in addition to the parents
Disclaimer: The views expressed in this article are those of the
authors and do not necessarily reflect the official policy or position
of the Ministry of Health of Ecuador, Department of the Navy,
Department of Defense, nor the U.S. Government. The study
protocol was approved by the Ministry of Health of Ecuador and
the Naval Medical Research Center Institutional Review Board
(Protocol NMRCD.2002.0019) in compliance with all applicable
Federal regulations governing the protection of human subjects.
The corresponding author had full access to all data in the study
and final responsibility for the decision to submit this publication.
Sample collection. Two types of samples were obtained for
diagnostic testing: a nasal swab for the Rapid Influenza Test (RIT;
QUICKVUE Influenza test; Quidel, San Diego, CA) and an
oropharyngeal swab for viral isolation. The RIT was processed on
site, and the results were provided to the patient. Oropharyngeal
swabs were placed in Universal Transport Media (UTM;
consisting of modified Hanks balanced salt solution
supplemented with bovine serum albumin, cysteine, gelatin,
sucrose and glutamine acid. Phenol red is used to indicate pH.
Vancomycin, amphotericin B and colisitin are included in the
medium to inhibit growth of competing bacteria and yeast) and
stored at 270uC until they were delivered on dry ice to NAMRU6
in Lima, Peru, for laboratory analysis.
Virus isolation and identification. For virus isolation,
patient specimens were inoculated into four commercial cell
lines from the American Type Culture Collection (ATCC):
Madin-Darby canine kidney (MDCK), African green monkey
kidney (Vero76 and VeroE6) and rhesus monkey kidney
(LLCMK2). Upon the appearance of a cytopathic effect (CPE)
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 a direct
fluorescence antibody (DFA) assay. 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, 3 and 4), and respiratory
syncytial virus (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 human metapneumovirus (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.
Cases were further tested for influenza A and B viruses using a
one-step reverse transcriptase-polymerase chain reaction
(RTPCR) with the influenza primers described below. The viral
etiology of cases was determined based on the isolation of virus
(CPE and fluorescent antibody positive) or a positive result by
RTPCR. Real time-PCR was applied for all ILI samples between
May 20 and September 30, 2009, for detection of the pandemic
influenza A (H1N1) 2009 virus.
RNA extraction and RT-PCR. For the genetic analyses of
influenza viruses, viral RNA extraction was performed from the
supernatant of infected MDCK cells using a QIAamp Viral RNA
kit (QIAGEN; Valencia, CA) following the manufacturers
protocol. The one-step RT-PCR was performed 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-AAGCAGGGGAAAATAAAA-39) and H1R-1193
(59-GTAATCCCGTTAATGGCA-39); for H3 influenza A viruses: H3F-7
(59-ACTATCATTGCTTTGAGC-39) and H3R-1184
(59-ATGGCTGCTTGAGTGCTT-39); for influenza B viruses: BHAF-36
(59-GAAGGCAATAATTGTACT-39) and BHAR-1140
(59-ACCAGCAATAGCTCCGAA-39). Five ml of the extracted RNA was
added to 20 uL of master mix containing the enzyme mixture
(Superscript III RT/Platinum Taq), 26 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 two 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 five
In order to check for the resistance to antiviral agents, around
10% of the influenza isolates were studied by sequencing
fragments of their matrix and neuraminidase genes, as previously
described  to verify if mutations conferring resistance were
The RT-PCR products 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 manufacturers
instructions. Sequences were analyzed and edited using the
Sequencer 4.8 software (Applied Biosystems; Foster City, CA).
Sequencing and phylogenetic analysis. The nucleotide
sequences were aligned using the Clustal program in the Mac
Vector software package (Mac Vector Inc.; Cary, NC), and
phylogenetic analyses were performed using the neighbor joining
and maximum likelihood algorithms implemented in the
Phylogenetic Analysis using MEGA software (version 4) .
For the neighbor joining analyses, the HKY85 distance was used
and bootstrap values were calculated based on 1000 replicates to
place confidence values on groupings within trees.
Data from the case report forms was entered into a database
created in Microsoft Office Access 2003. Proportions were
calculated with their respective 95% confidence intervals (CI)
and were compared using a chi-squared test (X2). Continuous data
were described with mean +/2 standard deviation (STD),
medians and modes. P values,0.05 were considered statistically
significant. Analyses were conducted using Epi-Info 3.5 and SPSS
software version 17.0 (SPSS Inc. Chicago, IL).
A total of 1,702 participants were enrolled in this study: 793 at
Hospital Vozandes (Quito), 482 at Hospital Luis Vernaza
(Guayaquil), and 427 at Hospital Naval (Guayaquil). Of the total,
788 (46.2%) were female. All enrolled participants provided a
respiratory specimen. Overall, the median age of participants was
24 years old (range 0 to 100 years), with 354 under the age of 5,
and 265 between the ages 5 to 17. Only 4% of the patients in this
study were over 65 years old. As seen in Table 1, the ages were not
equally distributed between hospitals. Most of the patients from
Hospital Vozandes and Hospital Naval were young adults, while
most of the patients reported from Hospital Vernaza were less
than three years old.
Treatments taken by the patients that were registered on the
case report forms included a variety of generic and commercial
names. When it was possible to identify the medicines, they were
coded into broad groups. Before enrollment, 503 patients reported
having sought medical care and 398 (23%) patients took
antibiotics. The antibiotics taken included: beta-lactams (31%),
quinolones (8%), macrolides (8%), aminoglycosides, tetracyclines,
trimethoprim/sulfamethoxazole and chloramphenicol. Most of
these patients took combinations of drugs, some with multiple
antibiotics, antipyretics, antihistamines, and cold medicines.
Of the patients between 5 and 65 years of age, 640 (37.6%)
participants reported having already lost part or a full day of work
or school at the time of enrollment, with an average of 1.4 days
lost. A total of 57 (3.3%) patients were hospitalized, of whom nine
(15.8%) were younger than five years of age. One hundred and six
(6.1%) participants reported having received the influenza vaccine
within six months prior to enrollment and only two of 57 (3.5%)
hospitalized patients reported having received the flu vaccine
(Table 1). A respiratory virus was isolated from 33 of the ILI
participants who reported receiving vaccination, including 26
(25% of the participants reporting vaccination) with influenza A
and 4 (3.8%) with influenza B.
Of the 1,702 enrolled participants, 35% (95% CI: 32.4 to 36.9)
were positive for at least one respiratory virus. The specimens
submitted on the first day of symptoms were 1.5 times (Odds
Ratio = 1.494, 95%CI 1.1911.874) more likely to have positive
results compared with samples taken on subsequent days. Of the
patients with confirmed viral infections, 486 of 551 (88.2%)
presented before the fourth day of symptoms. A total of 617 viral
agents were identified from 597 participants. The most common
viral agent detected was influenza A virus, which was isolated or
identified by PCR from 373 (21.6%) participants. Influenza B
Positive rapid test
Characteristics of the population
Total patients enrolled
Travel (last 7 days)
Medical attention before enrollment
Antibiotics & antivirals
Undifferentiated A or B positive
virus was identified from 110 (6.4%) participants and
parainfluenza viruses were isolated from 35 (2.1%), while adenovirus was
isolated from 34 (2.0%). Only 16 (0.9%) and 15 (0.9%) of the
participants were culture positive for RSV and enterovirus
respectively (Table 2). Human metapneumovirus was not detected
in this study.
More than one virus was detected in 19 participants. Of 29
isolates of HSV, eight were coinfections. Of 34 isolations of
adenovirus, five were coinfections. Of 15 isolates of enterovirus,
four were coinfections. The only time rhinovirus was isolated it
was found as a coinfection with enterovirus. PCR revealed viral
sequences for bocavirus (hBoV), in four patients, and three of them
The distribution of viral isolates according to age group can be
seen in Figures 2 and 3. The diversity of viruses isolated in patients
with ILI was greater in patients less than five years old. Three of
the participants with bocavirus were children less than three years
old. Parainfluenza, RSV and adenovirus were more commonly
identified in children less than five years old than in persons five
years old or older (X2, p,0.001). In this study, only 4% of the
patients were over the age of 65 and the diversity of viral isolates
shown in the elderly is less reliable.
The antiviral resistance pattern was determined by amino acid
comparison to published sequences in 67 influenza isolates
randomly selected from samples obtained in this study. All 14
isolates of influenza B were sensitive to oseltamivir and resistant to
*A total of 617 viral agents were found from 597 participants, co-infections were detected in 19. One person had three viruses detected.
hBoV is human bocavirus. HSV is herpes simplex virus. RSV is respiratory syncitial virus.
Hosp. Luis Vernaza
Viral agents detected*
Herpes simplex virus (HSV)
Respiratory syncitial virus (RSV)
Influenza A - HSV
Influenza B - HSV
Adenovirus - Parainfluenza 3
Adenovirus - Enterovirus
Influenza A- Adenovirus
Adenovirus - RSV - hBoV
Influenza A- Enterovirus
Influenza B- Enterovirus
Enterovirus - Rhinovirus
Influenza A - RSV
Influenza B - hBoV
amantidine. In all 35 isolates of influenza A/H3N2 as well as in
seven isolates of the pandemic (H1N1) 2009 virus, there was
evidence of resistance to amantadine (predicted by the S31N
mutation on the M2 gene) and sensitivity to oseltamivir. The
resistance pattern for the 14 isolates of seasonal influenza A/H1N1
was more complex. One of 14 strains of influenza A/H1N1 had
mutations suggesting resistance to amantadine and three of 14
strains indicators of resistance to oseltamivir. Before 2008, most
isolates of A/H1N1were sensitive to both amantadine and
oseltamivir , but in 2008 a strain possessing the Y274H
Figure 3. Viral culture results by age group in Quito. All patients in this chart were enrolled in Hospital Vozandes Quito.
mutation on the neuraminidase gene was detected. Between 2008
and the arrival of the 2009 pandemic strain, 99% of all A/
H1N1isolates had mutations commonly associated with resistance
to oseltamivir (data not shown).
H1N1 influenza A viruses. The genetic analysis of the HA
gene of 16 H1N1 isolates from Ecuador showed that two genetic
variants circulated in the country before the pandemic: 1) A/
Solomon Islands/03//06-like 20062007 and 2) A/Brisbane/59/
07 like 20082009 (Figure 4).
Only A/Solomon Islands/03/06-like 20062007 was found
from the end of 2006 until January 2007. In 2007, no
H1N1 influenza viruses were identified between February
and November. In December 2007, A/Brisbane/59/07-like
20082009 appeared and became the most common
circulating strain for H1N1 viruses in both Quito and Guayaquil.
This genetic variant does not include the recommended
2008 vaccine strain for the Southern Hemisphere (A/Solomon
H3N2 influenza A viruses. The HA gene of 35 H3N2 viral
isolates from Ecuador was genetically analyzed. Figure 5 shows the
resultant phylogenetic tree from these isolates and clearly shows
that they group with two genetic variants: 1)
A/Brisbane/10/07like 2007 and 2) A/Perth/16/09-like 20082009. The color code
each year shows that until 2007, all isolates circulating in Ecuador
grouped with the A/Brisbane/10/07 like 2007 strain and that in
2008 a change in the circulating strain took place and the A/
Perth/16/09 like 20082009 became predominant in both regions
of the country after the second half of the year. We have included
the phylogenetic analyses of isolates from Peru (Iquitos and
Tumbes) to show that this was also the case in other countries of
South America. This genetic variation does not group with the
2008 vaccine strain for the Southern Hemisphere (A/Brisbane/
10/07-like 2007). However, in 2009, the A/Perth/16/09 like
20082009 genotype was utilized in the vaccine and it did group
with the circulating isolates in Ecuador.
Influenza B viruses. Phylogenetic analyses based on the HA
sequence of 23 influenza B virus isolates revealed the presence of
two strains in Ecuador: B/Malaysia/2506/07-like and B/Florida/
4/06-like. In 2006, only the B/Malaysia/2506/07-like strain
circulated in the country. Later, in 2007 and 2008 both strains
cocirculated in both regions of Ecuador although the vaccine strain
used until 2008 belonged to solely the B/Malaysia/2506/07-like
genotype. However, the most recent influenza B virus isolates from
2009 belong to the B/Florida/04/06 genotype, which also
includes the vaccine strain for the Southern Hemisphere (Figure 6).
The distribution of influenza A and B cases over time in
Guayaquil and Quito is seen in Figures 7 and 8. During this study,
there were six outbreaks of influenza in Quito. In periods of
increased influenza activity before the pandemic there were one or
two strains of influenza A circulating with influenza B. The first
period of increased activity at the end of 2006 was dominated by
influenza A/H1N1/Solomon Islands/3/06-like strains that lasted
around 10 weeks. After a period of reduced activity, a second
outbreak began in Quito and lasted from the eleventh through the
30th week (April through August) of 2007. This outbreak was
predominantly influenza A/Brisbane/10/07- (H3N2)-like strains.
Around week 47 (November) of 2007, a third outbreak began and
lasted for about 20 weeks and was mostly influenza A//Brisbane/
59/07 (H1N1)-like strains. About 10 weeks later the fourth
epidemic began in 2008, which was mostly influenza B/Florida/
4/2006-like strains. During these Quito outbreaks there was a
smattering of cases identified in Guayaquil until the end of 2008
and beginning of 2009, when more cases were documented in
Guayaquil and there were only a few cases documented in Quito
in an epidemic of A/Perth/16/09- (H3N2)-like strains that lasted
for about 12 weeks. There was then a period with little influenza
activity until week 20 of 2009 when the influenza A(H1N1)
pandemic began in Guayaquil and a large wave of pandemic
influenza activity started in Quito around week 28 and lasted until
the first weeks of 2010. The age group most affected by the
pandemic was the group between 5 and 14 years old regardless of
place of origin (X2, p,0.001). During the pandemic, specimens
from 368 participants were tested by RT-PCR, confirming 70
cases of p(H1N1)2009 (23 from Guayaquil and 47 from Quito). At
the time of publication, none of the influenza A isolates from the
post pandemic period had been subtyped.
Of the 57 hospitalized patients, 52 (3.1%) had either cough or
shortness of breath with fever, meeting the criteria  for severe
acute respiratory infection (SARI). Of the patients with SARI, 15
(28.8%) had positive cultures for influenza A. Of the patients with
SARI, three had asthma, 4 had chronic obstructive pulmonary
disease, one had congestive heart failure, one had a myocardial
infarction, and six had other commorbidities including diabetes
mellitus, leukemia, and Kikuchi disease. Only two patients were
reported to have died in the study. They were admitted with
circulatory collapse and shortness of breath. Both had symptoms
for less than a week and no pathogen was isolated.
Analysis of the symptoms and combinations of symptoms on the
case report forms did not reveal differences in symptom complexes
according to which viruses were isolated. This was due to the
inclusion criteria. This paper studied a single symptom complex,
ILI, which may only be a part of the clinical spectrum of disease
caused by the viruses isolated.
The results of this study are consistent with other studies of
influenza in tropical countries  where there is little seasonal
variation in climate, and it is more difficult to define an influenza
season [4,22,23]. During the four year study period, although
influenza virus was isolated in each of the twelve months, there
were often one or two month periods during a specific year when
no influenza activity was detected. Some years had more than one
period of increased activity. As in other countries, except during
the 2009 H1N1 pandemic, two to four genotypes of influenza
circulated at the same time, consistent with multiple introductions
of influenza viruses [7,24]. Over the four year study period, the
monthly pattern of laboratory confirmed cases was similar to
Singapore , Hong Kong  and Nicaragua , with more
than one outbreak per year with little activity in between.
It has been suggested that tropical zones may function as mixing
pools for viruses from around the world . A large genetic
diversity expected from a year long influenza reservoir of multiple
influenza clades was not evident, but that may be due to the
limited size of this study. Between periods of increased influenza
activity there was a period of time in which H1N1 was not
detected in Ecuador, and when H1N1 reappeared the viral
genotype was different. Instead of constant endemic influenza
activity with many strains, there seemed to be a succession of
epidemics that passed through Ecuador, temporally related to
seasonal epidemics in both hemispheres, consistent with the
hypothesis that influenza A epidemics start in Ecuador when
strains are introduced from a different geographical region.
However, a large study of the molecular epidemiology of influenza
in the United States  showed that the genetic diversity in a
locality is strongly associated with the number of isolates sampled
from that locality. More extensive sampling is necessary to be sure
that there is not continual latent influenza activity in Ecuador.
Several factors may influence the fact that prevalence of
influenza among the patients enrolled was higher in Quito than in
Guayaquil as documented by the percentages of positive rapid test
results and influenza cultures. The same methods used in this
study have been applied in other tropical countries [9,11]. In Peru,
2192 of 6835 participants (34.8%) were culture positive for
influenza A or B, while in three countries of Central America, only
177 of 1756 (10.1%) participants were positive. The percentage of
positive influenza culture results in Quito (43%) was significantly
higher than that encountered in these other studies, while the
percentage of positive influenza cultures in Guayaquil (15%) was
lower than the average, but within the limits of the other cities
studied. It has been shown that the percentage of positive samples
increases during influenza epidemics as opposed to culturing ILI
patients in times when influenza is not known to be circulating
[26,27], which may explain in part the differences between study
sites. In Quito, samples were obtained in only one hospital and
(before the pandemic) the number of patients enrolled reflected the
number of patients in the ED with ILI, whereas in Guayaquil
samples were submitted from a larger population of patients.
Furthermore, there were a much larger percentage of patients less
than two years old enrolled in Guayaquil. In this surveillance
study, the method of patient enrollment did not generate reliable
data for inference of incidence or burden of influenza. Comparing
the rates of culture positivity between Quito and other sites would
be more reliable if incidence could be compared.
Although this study was not designed to compare differences in
incidence or burden of influenza, the findings suggest that there
are differences between the coast (Guayaquil) and the highlands
(Quito) in the etiology of ILI with a trend towards more influenza
in Quito than Guayaquil. In warm humid environments, contact
appears to be more important for influenza transmission, but
aerosol transmission may become important in cool dry
environments as shown by studies of guinea pigs separated in cages that
demonstrate the risk of airborne infection can be calculated by
measuring environmental factors like absolute humidity  and
temperature . Absolute humidity is the amount of water vapor
present in a unit volume of air. Although the relative humidity is
similar between Quito and Guayaquil, Quito has a lower average
temperature and, due to the difference in altitude, a lower average
air pressure and, consequently, a lower absolute humidity than
Guayaquil. Further studies are necessary to determine if altitude
affects the burden of influenza in tropical climates.
Primary infection with HSV is a well known cause of
gingivostomatitis and pharyngitis in children and young adults.
Whether or not reactivation of HSV infection is associated with
recurrent pharyngitis has been debated, but asymptomatic salivary
virus excretion has been documented . More than a third of
the HSV isolates in this study were co-infections, and 57% of the
patients with HSV without coinfection were over 10 years of age.
In our study, although HSV was the most common viral isolate in
the age group over 65, this may just reflect asymptomatic carriage
as opposed to a primary cause of ILI.
The clinical significance of infection with human bocavirus is
still being elucidated. Three of four patients with human bocavirus
were children less than 2 years of age with coinfecting viruses. This
is typical of other studies that show that human bocavirus is
frequently a coinfection with influenza, rhinovirus and enterovirus,
and that most people have immunity to human bocavirus, RSV,
rhinovirus and human metapneumovirus by age five .
The use of antibiotics in the treatment of ILI has been shown
generally to increase side effects and to not shorten the duration of
illness . The patients enrolled in this study who had sought
medical care before being evaluated in the ED were more likely to
have taken antibiotics. The antibiotics used included potentially
toxic injections of antibiotics such as amikacin, gentamicin, and
beta-lactams as well as chloramphenicol. Aside from the potential
for serious toxicity, drugs such as macrolides may cause nausea
and potentially increase the cost of the illness associated with ILI.
This study illustrates the need for education of the public and the
medical community about the proper use of antibiotics.
There was an age bias in the population studied in Quito, due to
the fact that parents were reluctant to consent to viral cultures on
small children. The patient populations served by the different
hospitals in Guayaquil also may have introduced an age bias.
It has recently been shown that for the detection of respiratory
viruses such as influenza and RSV, nasopharyngeal wash is more
sensitive than oropharyngeal swabs, which is more sensitive than a
pharyngeal swab [33,34]. However for other nonviral pathogens,
the relative sensitivity of these three sampling methods depends on
the organism being detected . The sensitivity of viral detection
could have been higher with nasopharyngeal wash.
Since RSV is a temperature labile virus, freezing the samples for
transport probably also reduced the sensitivity of RSV detection.
Nucleic acid amplification has consistently been shown to be more
sensitive for RSV than culture . Human metapneumovirus is
closely related to RSV and was not found in this study, probably for
the same reason. The number of isolates of RSV and
metapneumovirus is probably underestimated in this study, mainly due to the
methods employed for transport and viral detection.
Although the percentage of patients with ILI in the tropics with
rhinovirus has been found to be as high as 24.8% , the cell
lines and PCR used in this study were not designed for the
detection of rhinovirus.
During the influenza A (H1N1) pandemic of 2009, our
surveillance system was overwhelmed by the number of patients
with ILI. Several factors, including emergency measures imposed by
the Ecuadorian Ministry of Health, a temporary lack of culture
media for the study, and an increase in the work load, all
contributed to reduce the portion of ILI patients who were cultured.
During the pandemic, in Ecuador as well as in Peru , the 2009
pandemic strain displaced all the other influenza viruses. After
September 2009, not all the isolates were screened for the pandemic
strain, but the volume of patients in the ED of Hospital Vozandes
continued to be elevated until the first week of 2010 (data not
published). In early 2010, there was less influenza activity, but
p(H1N1)2009 was still being detected by the Ecuadorian Ministry of
Health in the area (data not published). The number of cultures
taken and isolates reported here does not adequately reflect the
disease burden especially during the pandemic.
The emergence of the 2009 pandemic H1N1 influenza A virus
has awakened interest in various aspects of respiratory disease
surveillance in the community, has demonstrated the impact of
these infections on different populations, and has emphasized the
need for strengthening health networks responsible for community
care. During the 2009 pandemic, the Ecuadorian Department of
Health improved its laboratory facilities and dedicated more
resources to influenza surveillance and local clinicians developed
more experience in recognizing influenza. Perhaps the increased
focus on influenza during future years will help to shed light on
whether Ecuador is a repository of genetic diversity where genetic
reassortment may be found or simply another stopover for strains
of influenza migrating between hemispheres.
We would like to express our gratitude to all personnel working at sentinel
centers in Ecuador for supporting this surveillance study. We thank Dr.
Brett Forshey who provided the map and the professional staff of the
Virology Department of NAMRU-6 for invaluable laboratory and
technical support in the execution of the study. We would also like to
thank Dr. Juan Martin Moreira and members of the department of
epidemiology as well as Dr. Nancy Vasconez and the members of the
department of the national program of immunization of the Ecuadorian
Ministry of Health for their comments and insight as to the behavior of
acute respiratory infections in Ecuador.
Conceived and designed the experiments: RWD VAL TK. Performed the
experiments: MS JG. Analyzed the data: RWD VAL JA TK JP ESH JC
MS. Contributed reagents/materials/analysis tools: JP MS JA. Wrote the
paper: RWD VAL. Principal investigator: VAL RD. Final approval: ESH.
Data acquisition: WC-A WA CM FD MR. Local analysis: RWD WC JP
JA. Critical revision: JA ESH. Senior Laboratory Supervisor: GC.
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