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.
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.
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.
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.
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.
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
It has been observed that the dominant strains of influenza in seasonal epidemics seem to be synchronized between the northern and southern hemispheres
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
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
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 those strains.
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 (
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 Ecuador's 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 26°C with an average high temperature of 31°C in December through April and an average low temperature of 20°C 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 15°C and a daily temperature range from 7°C to 23°C. During the year, the monthly average maximum and minimum temperatures fluctuate less than 5°C 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 (≥38°C) and cough or sore throat fewer than five days in duration, with or without general symptoms such as muscle ache, prostration, headache, or malaise
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 8–17 years old was obtained in addition to the parent's approval.
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.
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 Hank's 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 −70°C until they were delivered on dry ice to NAMRU6 in Lima, Peru, for laboratory analysis.
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 (RT-PCR) 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 RT-PCR. 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.
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 manufacturer's 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 (
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
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).
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)
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 +/− 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
Characteristics of the population | Guayaquil | Quito | ||||
Count | % | Hosp. Luis Vernaza | Hosp. Naval | Hosp. Vozandes | ||
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1702 | 100.0 | 482 | 427 | 793 | |
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Female | 788 | 46.2 | 248 | 160 | 383 |
Male | 911 | 53.5 | 234 | 266 | 408 | |
Missing | 3 | 0 | 1 | 2 | ||
Age | Mean, ±STD | 24.4±19.4 | 12.1, ±17.1 | 34.7, ±18.4 | 26.2, ±17.1 | |
Median, [range] | 23, [0–100] | 2, [0–82] | 32, [0–85] | 25, [0–100] | ||
Mode | 1 | 1 | 18 | 20 | ||
Age Range (years) | ||||||
0–2 | 278 | 16.6 | 248 | 5 | 25 | |
3–4 | 76 | 4.5 | 41 | 3 | 32 | |
5–17 | 265 | 15.8 | 48 | 33 | 184 | |
18–49 | 889 | 53.0 | 123 | 303 | 463 | |
50–64 | 102 | 6.1 | 15 | 39 | 48 | |
≥65 | 68 | 4.1 | 6 | 39 | 23 | |
missing | 24 | 1 | 5 | 18 | ||
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192 | 11.1 | 29 | 19 | 144 | |
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106 | 6.1 | 20 | 12 | 74 | |
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57 | 3.3 | 3 | 0 | 54 | |
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Saw Doctor | 503 | 29.4 | 228 | 99 | 176 |
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Antibiotics | 398 | 23.0 | 149 | 105 | 182 |
Antivirals | 2 | 0.1 | 0 | 0 | 2 | |
Antibiotics & antivirals | 4 | 0.2 | 0 | 2 | 2 | |
No treatment | 1278 | 73.8 | 333 | 294 | 651 | |
Unknown | 50 | 2.9 | 1 | 32 | 17 | |
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Influenza A | 246 | 14.3 | 48 (9.9) | 4 (.9) | 194 (23.9) |
Influenza B | 77 | 4.5 | 14 | 0 | 63 | |
Undifferentiated A or B positive | 25 | 1.5 | 0 | 4 | 21 | |
Negative | 1240 | 72.5 | 386 | 364 | 490 | |
No test | 121 | 7.2 | 34 | 55 | 25 |
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 (
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 1.191–1.874) 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 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 (
Total | Guayaquil | Quito | |||||||
Count | % | Hosp. Luis Vernaza | Hosp. Naval | Hosp. Vozandes | |||||
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Positive | 597 | 35.1 | 128 | 26.6 | 76 | 17.8 | 393 | 49.6 | |
Negative | 1105 | 64.9 | 354 | 73.4 | 351 | 82.2 | 400 | 50.4 | |
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H1N1 SOIV | 69 | 4.1 | 23 | 46 | |||||
H1N1 | 87 | 5.1 | 8 | 7 | 72 | ||||
H3N2 | 94 | 5.5 | 10 | 2 | 82 | ||||
Not typed | 123 | 7.2 | 24 | 38 | 61 | ||||
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Parainfluenza 1 | 9 | 0.53 | 4 | 1 | 4 | ||||
Parainfluenza 2 | 10 | 0.59 | 5 | 2 | 3 | ||||
Parainfluenza 3 | 15 | 0.88 | 9 | 1 | 5 | ||||
Parainfluenza 4 | 1 | 0.06 | 0 | 1 | 0 | ||||
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19 | 1.2 | 6 | 4.7 | 1 | 1.3 | 12 | 3.0 | |
Influenza A - HSV | 5 | 5 | |||||||
Influenza B - HSV | 3 | 3 | |||||||
Adenovirus - Parainfluenza 3 | 2 | 2 | |||||||
Adenovirus - hBoV | 1 | 1 | |||||||
Adenovirus - Enterovirus | 1 | 1 | |||||||
Influenza A- Adenovirus | 1 | 1 | |||||||
Adenovirus - RSV - hBoV | 1 | 1 | |||||||
Influenza A- Enterovirus | 1 | 1 | |||||||
Influenza B- Enterovirus | 1 | 1 | |||||||
Enterovirus - Rhinovirus | 1 | 1 | |||||||
Influenza A - RSV | 1 | 1 | |||||||
Influenza B - hBoV | 1 | 1 |
*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.
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 were coinfections.
The distribution of viral isolates according to age group can be seen in
This chart includes patients enrolled in Hospital Luis Vernaza and Hospital Naval in Guayaquil.
All patients in this chart were enrolled in Hospital Vozandes Quito.
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 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
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 2006–2007 and 2) A/Brisbane/59/07 like 2008–2009 (
Numbers indicate bootstrap values. The legend indicates the geographical origin of the strains: an asterisk denotes cases from the coast of Ecuador (Guayaquil) and the rest come from the Andean region (Quito). The year of isolation is coded by color: 2006 (pink), 2007 (blue), 2008 (green) and 2009 (red). Arrows indicate the recommended vaccine strain for the Southern Hemisphere for each year of the study period.
Only A/Solomon Islands/03/06-like 2006–2007 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 2008–2009 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 Islands/03/06).
The HA gene of 35 H3N2 viral isolates from Ecuador was genetically analyzed.
Numbers indicate bootstrap values. The legend indicates the geographical origin of the strains: an asterisk denotes cases from the coast of Ecuador (Guayaquil) and the rest come from the Andean region (Quito). The year of isolation is coded by color: 2006 (pink), 2007 (blue), 2008 (green) and 2009 (red). Arrows indicate the recommended vaccine strain for the Southern Hemisphere for each year of the study period.
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 co-circulated 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 (
Numbers indicate bootstrap values. The legend indicates the geographical origin of the strains: an asterisk denotes cases from the coast of Ecuador (Guayaquil) and the rest come from the Andean region (Quito). The year of isolation is coded by color: 2006 (pink), 2007 (blue), 2008 (green) and 2009 (red). Arrows indicate the recommended vaccine strain for the Southern Hemisphere for each year of the study period.
The distribution of influenza A and B cases over time in Guayaquil and Quito is seen in
Each bar represents the number of positive cultures per epidemiologic week from throat swabs patients reported as enrolling in Hospital Luis Vernaza or Hospital Naval in Guayaquil, Ecuador. The symbols above the bar chart represent what strains were circulating in Guayaquil and correspond with the cases of influenza isolated in Guayaquil in the phylogenetic trees in
Each bar represents the number of positive cultures per epidemiologic week from throat swabs from patients enrolled in the emergency department of Hospital Vozandes in Quito, Ecuador. The symbols above the bar chart represent the strains of influenza circulating in Quito, and correspond with the influenza cases on the phylogenetic trees in
Of the 57 hospitalized patients, 52 (3.1%) had either cough or shortness of breath with fever, meeting the criteria
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
It has been suggested that tropical zones may function as mixing pools for viruses from around the world
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
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
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
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
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
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
Although the percentage of patients with ILI in the tropics with rhinovirus has been found to be as high as 24.8%
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 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 Vásconez 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.