Article

Etiology and Seasonality of Viral Respiratory Infections in Rural Honduran Children

‡Harvard University, Cambridge, MA
The Pediatric Infectious Disease Journal (Impact Factor: 2.72). 05/2012; 31(11):1113-8. DOI: 10.1097/INF.0b013e31826052eb
Source: PubMed

ABSTRACT

: Limited data are available in Honduras that describe the etiology and seasonality of respiratory infections, especially in rural outpatient settings. Better data may lead to improved therapeutic and preventive strategies. The goal of our study was to determine the viral etiology and seasonality of acute respiratory infections in a rural Honduran population of children.
: Prospective clinic surveillance was conducted to identify children < 5 years of age presenting with respiratory symptoms of < 5 days duration. We obtained data on age, sex, medical history, breastfeeding history, symptoms, risk factors, household setting, temperature, respiratory rate and chest examination findings. To assess the association between specific viruses and weather, regional meteorological data were collected. Nasopharyngeal samples were tested for 16 respiratory viruses using a multiplex polymerase chain reaction panel.
: From February 2010 through June 2011, 345 children < 5 years of age were enrolled; 17%, 23%, 30% and 31% were <6, 6-11, 12-23 and 24-60 months old, respectively. Including all clinics in the region, 44.5% of patients < 5 years of age with documented respiratory diagnoses were enrolled. At least 1 virus was identified in 75.4% children, of which 7.5% were coinfections; 13.3% were positive for parainfluenza, 11.9% for influenza, 8.1% for human metapneumovirus and 7.5% for respiratory syncytial virus. Rainfall correlated with parainfluenza (P < 0.0001), influenza (P < 0.0001), human metapneumovirus (P = 0.0182) and respiratory syncytial virus (P < 0.0001).
: These results suggest that the spectrum of viruses in ill, rural, Honduran children is similar to that in North and Central America, although the seasonality is typical of some tropical regions.

Full-text

Available from: Caitlin Dodd
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Etiology, Seasonality, and Clinical Characteristics of Viral Respiratory
Infections in Rural Honduran Children
A thesis submitted to the
Graduate School
of the University of Cincinnati
in partial fulfillment of the
requirements for the degree of
Master of Public Health
in the Department of Public Health Sciences
of the College of Medicine
by
Elizabeth P. Schlaudecker, M.D.
University of Cincinnati
May 19, 2011
Committee:
William Mase, Ph.D., Chair
Charles Schubert, M.D.
Mark Steinhoff, M.D.
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ABSTRACT:
Background: Limited data are available in Honduras describing the etiology and
seasonality of childhood acute respiratory infections (ARIs), and better data may lead to
improved therapeutic and preventative strategies.
Objective: We conducted a prospective sentinel clinic surveillance study to determine
the viral etiology of ARIs in rural Honduran children less than 5 years of age to
characterize the spectrum and seasonality of viruses associated with acute respiratory
infections.
Methods: We gathered data on age, sex, medical history, symptoms, demographics,
geographic setting, vital signs, and physical exam findings. Nasopharyngeal samples
were obtained via flocked swab and shipped to the U.S. in both universal transport
medium (UTM) on dry ice and PrimeStore
®
nucleic acid stabilizing buffer at room
temperature. Samples were tested for 14 respiratory viruses using the Luminex
Diagnostics polymerase chain reaction (PCR) respiratory viral panel (RVP ID-Tag
TM
).
Results: 267 samples were collected from February 2010 – March 2011; 13.9% were
positive for influenza, 7.9% for human metapneumovirus, 7.5% for respiratory syncytial
virus (RSV), 7.1% for parainfluenza and 2.2% for adenovirus. At least one virus was
identified in 194 (72.7%) cases, of which 16 (6.0%) were co-infections. Influenza rose
from 1.8% of isolates in February through June to 25.7% of isolates in July through
October. No cases of influenza were identified from November 2010 through February
2011. Influenza was present for 5 out of 12 months, and influenza correlated with
monthly rainfall in millimeters (R² = 0.2857). Including all tested respiratory viruses
except enterovirus/rhinovirus, the presence of a respiratory virus positively correlated
with average monthly precipitation (R² = 0.2863). UTM and PrimeStore
®
results for
influenza correlated well (K = 0.767, p<0.0001).
Conclusions: These unique results suggest that the spectrum of viruses in rural Honduran
children is similar to those found in the U.S., though the seasonality is tropical. This
region of rural Honduras demonstrated one large peak in influenza positivity prior to the
peak in the U.S., while monthly influenza results from the Pan American Health
Organization (PAHO) obtained in the capital of Honduras demonstrated two clear peaks.
Influenza and respiratory viruses in general correlated with average monthly rainfall.
PrimeStore
®
at room temperature is an effective shipping method for subsequent isolation
of influenza as compared to UTM on dry ice. Further research is needed to determine the
best methods of prevention and treatment of these viral respiratory infections.
Key words: viral respiratory infections; influenza; seasonality; Honduras
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ACKNOWLEDGEMENTS
Elizabeth Schlaudecker was supported by the Fogarty International Clinical
Research Scholars and Fellows Program, National Institutes of Health (NIH) grant 5 R24
TW007988-03. This project was completed with the support of the Cincinnati Children’s
Hospital Center for Clinical and Translational Science and Training grant UL1-
RR026314-01 NCRR/NIH.
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Etiology, Seasonality, and Clinical Characteristics of Viral Respiratory
Infections in Rural Honduran Children
TABLE OF CONTENTS
ABSTRACT…………………………………………………………………….…………2
ACKNOWLEDGEMENTS……………………………………………………………….4
PURPOSE…………………………………………………………………………………7
BACKGROUND.…………………………………………………………………………7
Mortality Secondary to Pneumonia………………………………………….……7
Etiology of Acute Respiratory Infections…………………………………….……7
Influenza Burden in the Tropics and Subtropics……………………………..……9
Influenza in Honduras…………………………………...……….………………10
Diagnostic Tests for Influenza………………………...…………………………11
Transport of Respiratory Viruses………………………..………………………13
Other Respiratory Viruses in Honduras…..……………..…………….…………13
OBJECTIVES……………………………………………………………………………15
METHODS………………………………………………………………………………15
Ethical Considerations………………...…………………………………………15
Surveillance Sites……………………………………………………………...…15
Study Population……………………………………………………………....…17
Subjects……………………………………………………………..................…17
Study Design…………………………………………………………………..…18
Roles and Responsibilities…………………………………………………….…21
LABORATORY DETECTION OF RESPIRATORY VIRUSES…………………….…22
Specimen Type/Collection..…………...…………………………………………22
Laboratory Procedures………………………………………………………..…23
Records Management………………………………………………………….…24
ANALYSIS………………………………………………………………………………24
Objective 1: Etiologic Spectrum and Seasonality…………………………….…24
Objective 2: Clinical and Demographic Characteristics……………………..…26
Objective 3: Nucleic Acid Extraction/Stabilization Buffer…………….……..…26
Limitations……………………………………………………….....................…27
RESULTS…………………………………………………………………………..……28
Objective 1: Etiologic Spectrum and Seasonality…………………………….…28
Objective 2: Clinical and Demographic Characteristics……………………..…29
Objective 3: Nucleic Acid Extraction/Stabilization Buffer…………….……...…31
DISCUSSION.…………………………………………………………………….....…..31
Etiology of Acute Respiratory Infections……………………………………...…31
Seasonality of Influenza and RSV……………………………………………..…32
Clinical and Demographic Characteristics……………………..……………….33
Nucleic Acid Extraction/Stabilization Buffer…………….…………………...…34
SUMMARY.…………………………………………………………………….....….....35
REFERENCES.………...……………………………………………………….....….....36
FIGURES AND TABLES……………………………………………….....…................43
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LIST OF TABLES AND FIGURES:
Figure 1: Map of Honduras with Enlarged Map of Southern Intibuca
Figure 2: Study Design Flow Diagram
Figure 3: Luminex Diagnostics RVP ID-Tag
TM
Flow Diagram
Figure 4: Viruses Detected in Honduran Children
Figure 5: Respiratory Viruses by Month and Average Monthly Rainfall
Figure 6: Influenza vs. Average Monthly Rainfall
Figure 7: RSV vs. Average Monthly Rainfall
Figure 8: Respiratory Viruses vs. Average Monthly Rainfall
Figure 9: Distribution of Influenza Viruses under Surveillance in Honduras, 2010-2011
Table 1: Number of Viruses Detected by PCR
Table 2: Number of Influenza Detected by PCR
Table 3: Clinical Characteristics by Respiratory Virus
Table 4: Demographic Characteristics by Respiratory Virus
Table 5: Quantitative Clinical Characteristics by Respiratory Virus
Table 6: Quantitative Demographic Characteristics by Respiratory Virus
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PURPOSE:
The purpose of this project is to describe the etiologic spectrum, seasonality, and
clinical characteristics of viral respiratory infections in rural Honduran children less than
five years of age.
BACKGROUND:
Mortality Secondary to Pneumonia
Acute respiratory infections (ARIs), including pneumonia, are the leading cause
of death among children under five years of age in developing countries (Black, Morris et
al. 2003; Mulholland 2003). Approximately two million children die yearly from ARIs, a
likely conservative estimate (Bryce, Boschi-Pinto et al. 2005; Mulholland 2007). These
recent data from the World Health Organization suggest that 17% of deaths in children
under five are due to pneumonia alone. Many neonatal deaths are misclassified, and
surveillance is limited in high pneumonia-endemic regions. Additionally, over 90% of
these deaths secondary to pneumonia occur in forty countries, and two thirds occur in ten
tropical and subtropical countries (Rudan, Boschi-Pinto et al. 2008).
Etiology of Acute Respiratory Infections
Throughout the world, the etiologies of ARIs and pneumonia are largely
unknown. As demonstrated by vaccine trials and previous surveillance data, invasive
bacterial disease due to Haemophilus influenzae type b (Hib) and Streptococcus
pneumoniae plays a significant role in the etiology of pneumonia (Berman 1991; Levine,
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Lagos et al. 1999; Klugman, Madhi et al. 2003; Adegbola, Secka et al. 2005; Cutts,
Zaman et al. 2005; Brooks, Breiman et al. 2007; Rudan, Boschi-Pinto et al. 2008). This
information led to the recommendations for vaccination against Haemophilus influenzae
type b (Hib) and Streptococcus pneumoniae disease (WHO 2006; WHO 2007).
However, these two pathogens only make up approximately one half of all pneumonias in
the developing world.
It is difficult to estimate the contribution of respiratory viruses, including
influenza (Scott, Brooks et al. 2008). In the United States, respiratory syncytial virus
usually contributes to a large proportion of hospitalizations, emergency department visits,
and outpatient visits. A large surveillance study revealed
919 (18%) RSV infections in
children out of 5067 children enrolled (Hall, Weinberg et al. 2009). Another surveillance
study with 2798 enrolled children identified 191 (6.8%) parainfluenza infections,
compared with 521 respiratory syncytial viruses and 159 influenza viruses (Weinberg,
Hall et al. 2009). Throughout the world, it is estimated that anywhere from 17.8% to
65% of pediatric patients admitted to the hospital for ARIs and pneumonia are in fact
infected with viruses (Juven, Mertsola et al. 2000; Tsolia, Psarras et al. 2004; Tajima,
Nakayama et al. 2006; Arnold, Singh et al. 2008; Bonzel, Tenenbaum et al. 2008).
Among children hospitalized with ARIs and pneumonia in tropical Southern India, one
half of the infections were of viral etiology (John, Cherian et al. 1991). Even with the
best diagnostic techniques, 25%-33% of cases do not have a clear etiology (Scott, Brooks
et al. 2008). This may represent poor laboratory capabilities or the emergence of novel
agents, such as avian influenza H5N1.
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Influenza Burden in the Tropics and Subtropics
In the United States during some influenza seasons, up to 9% of all infants
younger than 6 months of age are hospitalized or seen in a clinic or emergency
department secondary to influenza (Poehling, Edwards et al. 2006). During the 2004-
2005 influenza season, the estimated influenza-related hospitalization rate was 8.6/10,000
children aged less than 5 years (Grijalva, Weinberg et al. 2007). The burden of influenza
is unknown in most developing countries (Viboud, Alonso et al. 2006), but some data
from the tropics and subtropics demonstrates incidence and hospitalization rates for
influenza that exceed those reported for temperate regions (Chiu, Lau et al. 2002;
Nascimento-Carvalho, Ribeiro et al. 2008; Zaman, Roy et al. 2008). The seasonality of
influenza virus in the tropics is variable, with some studies demonstrating year round
disease (Tsai, Kuo et al. 2001; Straliotto, Siqueira et al. 2002; Viboud, Alonso et al.
2006) and some demonstrating one or two clear peaks (de Arruda, Hayden et al. 1991;
Chew, Doraisingham et al. 1998; Alonso, Viboud et al. 2007; Gordon, Ortega et al.
2009). This is significantly different from the defined, once yearly influenza season
identified in most temperate countries.
A recent study from Managua, Nicaragua, monitored influenza-like illness (ILI)
activity from April 2005 through April 2007 (Gordon, Ortega et al. 2009). One peak of
ILI activity occurred during 2005, in June-July; and two peaks occurred during 2006, in
June-July and November-December. This study demonstrates that influenza seasonality
may vary substantially from season to season, even within the same region.
Global influenza disease burden data are incomplete, as surveillance and
laboratory capabilities are limited in developing countries (Yazdanbakhsh and Kremsner
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2009). Several sites in sub-Saharan Africa, Latin America, and Asia have recently added
influenza surveillance programs (Higgs, Hayden et al. 2008), but new technologies,
including RT-PCR (reverse transcription-polymerase chain reaction) for virus detection,
are often unavailable in low-resource settings.
Influenza in Honduras
Honduras is an impoverished country in Central America with a population of
approximately seven million people and gross national income per capita of $3420. The
under-five mortality rate per 1000 births is 42.6, and acute respiratory infections are the
leading cause of death in this age group (United Nations 2007). The number of ARIs and
pneumonia reported to the World Health Organization has been increasing consistently
over the past decade. In 1996, about 90,000 infections were reported; in 1998, reported
cases reached 98,790 (World Health Organization 2006). Acute respiratory infections
and pneumonia accounted for 22% of deaths in children under the age of five in 1991-
1992, rising to 23% in 1996. Data about hospitalizations rates are minimal, and only
limited data are available about the cause of these ARIs.
A search of the scientific literature reveals only two studies that examine the
etiology of respiratory illness in Honduras. Reyes et al. investigated children hospitalized
in Honduras and El Salvador in 1991-1992, and they suggested that overuse of antibiotics
in this area of high RSV and influenza prevalence leads to iatrogenic diarrhea (Reyes,
Hedlund et al. 1996). A more recent study by Laguna-Torres et al. analyzed etiologic
agents associated with influenza-like illness in five participating hospitals or health
centers in large urban areas in El Salvador, Honduras, and Nicaragua. Overall, influenza
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A virus made up 7.4% of isolates, while influenza B virus was present in 2.7% of cases.
In Honduran isolates alone, influenza A was present in 47 of 427 (11.0%) isolates, and
influenza B was present in 19 of 427 (4.4%) isolates (Laguna-Torres, Sanchez-
Largaespada et al. 2011). The proportions of cases caused by influenza were almost
double those of the overall rate. However, both of these studies were localized to urban
regions, failing to represent the large proportion of the population found in rural, isolated
areas. Additionally, these studies failed to account for the seasonality of influenza and
other viruses, a necessary component of immunization strategies. A further
understanding of etiology and seasonality of respiratory infections in Honduras may lead
to improved therapeutic and immunization strategies.
Diagnostic Tests for Influenza
As demonstrated by Laguna-Torres et al., influenza surveillance often includes
only major cities in developing countries due to limited laboratory capabilities in outlying
areas. These remote areas are also affected by poor infrastructure, including lack of
electrical power and refrigeration. These logistical constraints often dictate the types of
laboratory tests available for influenza surveillance. RT-PCR and viral isolation are the
preferred diagnostic methods in any setting, but rapid influenza tests are valuable for their
ease of use and lack of laboratory requirements. Historically, viral isolation by culture
has been regarded as the “gold standard” for diagnosis of influenza infection. However,
recent studies have demonstrated superior sensitivity of RT-PCR for the detection of
influenza viruses (Steininger, Kundi et al. 2002), so RT-PCR is now regarded as the new
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diagnostic standard (Weinberg, Erdman et al. 2004). Currently, many RT-PCR tests are
commercially available for influenza diagnosis.
Rapid antigen tests in the United States have a sensitivity and specificity of 45-
90% and 86-100%, respectively, compared to nucleic acid amplification (Rodriguez,
Schwartz et al. 2002; Ruest, Michaud et al. 2003; Uyeki 2003; Agoritsas, Mack et al.
2006; Poehling, Zhu et al. 2006; Grijalva, Poehling et al. 2007; Hurt, Alexander et al.
2007). In developing countries, the performance characteristics of rapid influenza tests
are not well documented. Even though rapid tests would greatly add to surveillance
capabilities, their performance may be adversely affected by unreliable infrastructure,
humid climate, and staff with little or no experience collecting respiratory samples. A
recent prospective study from Managua, Nicaragua, compared the QuickVue Influenza
A+B test to RT-PCR in children aged 2-12 years (Gordon, Videa et al. 2009). The
sensitivity and specificity of the rapid test were 68.5% and 98.1%, respectively,
comparable to performance in the United States. However, this study was conducted in a
large health center in the capital of Nicaragua, likely affording better access to reliable
infrastructure and laboratory facilities unavailable in rural Honduras.
The commercially available Luminex Diagnostics respiratory viral panel (RVP
ID-Tag
TM
) is a multiplex nucleic acid amplification test that can detect 16 different
viruses and subtypes from a single respiratory specimen. The RVP ID-Tag
TM
detects
respiratory syncytial virus, influenza A, influenza B, adenovirus, parainfluenza 1, 2, 3, 4,
enterovirus/rhinovirus, coronavirus, and human metapneumovirus. This test has a
sensitivity of 97.8% and a specicity of 96.4% (Mahony, Chong et al. 2007; Zaman, Roy
et al. 2008), and is a Federal Drug Administration (FDA) certified diagnostic test.
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Transport of Respiratory Viruses
One of the most significant difficulties of providing access to advanced diagnostic
technologies in a low-resource setting is transport of specimens under adverse conditions.
As researchers and clinicians in remote locations begin to require RT-PCR and other
molecular diagnostic procedures, specimens require transport in hot, humid climates that
may or may not have dry ice available. PrimeStore
®
Molecular Transport Medium
(MTM) is a blend of nucleic acid stabilizing reagents that can inactivate nucleases and
preserve released ribonucleic acid (RNA) at ambient temperature for downstream real-
time and traditional RT-PCR detection. A recent study shows that PrimeStore
®
MTM
effectively kills pathogens, including highly pathogenic H5 influenza virus, and preserves
the RNA at ambient temperatures (Daum, Worthy et al. 2010). It is therefore well-suited
for clinical specimens that require field collection in remote areas such as Honduras.
Other Respiratory Viruses in Honduras
As compared to the already limited knowledge regarding influenza in Honduras,
even less is known about other respiratory pathogens, such as parainfluenza and human
metapneumovirus. Reyes et al. performed tissue cultures and immunofluorescence
studies on nasopharyngeal samples from 135 hospitalized children in Honduras and El
Salvador (Reyes, Hedlund et al. 1996). They also looked for the presence of respiratory
virus-specific immunoglobulin A antibodies. Viral respiratory infections were detected
in 63 (46.7%) patients, and 20 (14.8%) patients had more than one viral infection.
Respiratory syncytial virus (RSV) or the respective IgA antibodies were found in 43
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(31.9%) patients, influenza A and B viruses in 29 (21.5%) patients, adenovirus in four
(3.0%) patients, and enterovirus in two (1.5%) patients.
More recently, Laguna-Torres et al. looked for etiologic agents in El Salvador,
Honduras, and Nicaragua (Laguna-Torres, Sanchez-Largaespada et al. 2011). At least
one viral agent was identified in 434 (24.7%) cases, and 17 (3.9%) cases were dual
infections. Influenza A virus was detected in 130 (7.4%) cases, RSV in 122 (6.9%),
adenoviruses in 63 (3.6%), parainfluenza viruses in 57 (3.2%), influenza B virus in 47
(2.7%), and herpes simplex virus 1 in 22 (1.3%). Human metapneumovirus and
enteroviruses (coxsackie and echovirus) were isolated from less than 1% of patient
specimens each. The proportions of cases caused by parainfluenza (4.2%), human
metapneumovirus (<1%), and enteroviruses (0%) were similar for surveillance in
Honduras; though RSV made up a smaller proportion (1.2%) and adenoviruses made up a
larger proportion (7.3%).
Because mortality rates for upper respiratory infections in developing countries
far exceed those of developed countries (Williams, Gouws et al. 2002), information about
prevalence of these pathogens in the developing world is crucial for the development of
effective prevention, surveillance, and treatment strategies. This study describes the
etiologic spectrum, seasonality, and clinical characteristics of viral respiratory infections
in rural Honduran children less than five years of age.
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OBJECTIVES:
Primary
1. Define the etiologic spectrum and seasonality of ARI in patients less than five
years of age presenting to a clinic in rural Honduras with symptoms of less than
five days duration using PCR diagnostics during one calendar year
Secondary
2. Define the clinical and demographic characteristics associated with specific viral
etiologies of acute respiratory infection, including age, sex, medical history,
symptoms, geographic setting, vital signs, and physical exam findings
3. Compare the effectiveness of using a nucleic acid extraction/stabilization buffer
(PrimeStore
®
) to ship specimens at room temperature to dry ice shipment
METHODS
Ethical Considerations
Prior to enrollment of the participants, the protocol was approved by the
Institutional Review Board (IRB) of both the home institution (Cincinnati Children’s
Hospital Medical Center) and the Hospital Regional del Occidente in Santa Rosa de
Copan, Honduras (IRB00001470, Ministry Health, Western Region, Region #5 IRB #1).
Surveillance Sites
Surveillance was conducted at the Clinica Hombro a Hombro (Shoulder to
Shoulder Clinic) in Santa Lucia and Centro de Salud (Health Center) in Magdalena,
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Intibucá, Honduras. The two clinics provide primary medical care, health education, and
community resources for over twenty thousand rural Hondurans. Both clinics are
operated by Shoulder to Shoulder and Hombro a Hombro, two partnering, private, non-
profit, non-governmental organizations (NGOs) formed in the United States and
Honduras, respectively. Because of this long-term, successful partnership, the
government of Honduras made these NGOs responsible for all medical care in Intibucá.
Clinica Hombro a Hombro is equipped with six examination rooms, an
emergency room, a laboratory, X-ray equipment, and a dental clinic. Four full-time
doctors and a dentist operate the clinic 24 hours per day with an ancillary staff of nurses
and assistants. The Centro de Salud is equipped with three examination rooms and is
staffed by two physicians and two nurses. Clinica Hombro a Hombro acts as a referral
center for the Centro de Salud. The Centrol de Salud had a total of 6815 patient
encounters during the study period, with 1208 patient encounters for children under the
age of 5. Clinica Hombro a Hombro had a total of 10,717 patient encounters with 1943
children under the age of 5. No other access to primary care is available in this region of
Honduras; there are no hospitals, private clinics, or government clinics in this area.
U.S. brigades visit Honduras approximately once monthly for two-week periods
to work in the Santa Lucia and Magdalena area. These brigades consist of fourth year
medical students, residents, and attending physicians from various partnering U.S.
institutions. The primary partnering U.S. institutions are the University of Cincinnati and
the University of North Carolina. Brigades are scheduled monthly throughout the
calendar year.
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Study Population
Children were recruited at the two clinics located in Santa Lucia and Magdalena,
Intibucá, Honduras (Figure 1). Santa Lucia is located in the mountainous southwestern
region of Honduras, one hour from the El Salvador border. Magdalena is approximately
two miles north of Santa Lucia. The clinics serve approximately twenty thousand
Hondurans in the area surrounding Santa Lucia. Intibucá is one of the 18 departments
(departamentos) into which the nation of Honduras is divided. Intibucá is one of the
poorest departments in Honduras, and the communities of Santa Lucia and Magdalena
have limited access to health care beyond the Centro de Salud and Clinica Hombro a
Hombro. The capital of Intibucá, La Esperanza, is approximately three hours by car from
both towns. The mountainous terrain and limited transportation hinder access to care for
Hondurans in this region.
Subjects
All children 0-5 years of age who met the study criteria and presented at the
clinics were eligible for participation. From February 2011 through February 2012, the
sites enrolled 257 children from the Clinica Hombro a Hombro and Centro de Salud.
Inclusion Criteria:
1. Children < 5 years of age
2. Patients who presented at the surveillance location and were willing to provide a
specimen
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3. Patients who presented with respiratory illness: maternal report of illness,
occurring more than 7 days after previous illness, with any one respiratory
symptom including runny nose, nasal congestion, cough, difficulty swallowing or
difficulty breathing, with fever > 37.8 degrees Celsius in children greater than 1
year of age
4. Patients with history of illness less than 5 days
Exclusion Criteria:
1. Children > 5 years of age
2. Children already enrolled within the previous 7 days
3. Children with respiratory symptoms for greater than 5 days
4. Children whose parents or guardians lacked the communication skills to
understand the consent process
All subjects received a blanket or a similar gift for their participation, a normal
compensation in this area of Honduras. The blankets were valued at approximately $1
(US dollar) each and were purchased in the community of Santa Lucia, Intibucá,
Honduras.
Study Design
The design is a prospective sentinel clinic surveillance study to estimate the viral
etiology of acute respiratory infections in a rural outpatient Honduran population of
children less than five years of age (Figure 2). This study was conducted at the Clinica
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Hombro a Hombro (Shoulder to Shoulder Clinic) and the Centro de Salud (Health
Center) in Santa Lucia, Intibucá, Honduras.
Children who presented to the clinics with axillary temperature > 37.8 degrees
Celsius or maternal history of fever and respiratory symptoms were recruited and
provided consent via a guardian. Infants less than 1 year of age were recruited without
fever, as influenza patients may present without fever in this age group. This definition
of respiratory illness with fever (Zaman, Roy et al. 2008) was modified for infants and
young children from the Centers for Disease Control and Prevention (CDC) definition of
influenza-like illness as outlined above in the inclusion criteria. Surveillance took place
between February 2010 and February 2011.
Subjects were recruited year-round during clinic hours Monday through Friday.
Clinic staff recruited patients presenting to the Centro de Salud and the Clinica Hombro a
Hombro. Over the course of one year, 257 children were recruited. See Analysis section
for sample size calculations.
All other children under 5 years of age seen in the Clinica Hombro a Hombro and
the Centro de Salud were recorded in the clinic’s computer database. Weekly queries
identified all children seen in the clinics under the age of 5. Any children under the age
of 5 with fever and respiratory symptoms per the clinic database who were not recruited
for the study were also documented in the study database.
If the child met inclusion criteria and was eligible for the study, a parent or guardian
of the child was approached to obtain informed consent for the child’s participation.
Following informed consent, there were four parts to data collection:
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1. Parents or guardians were asked a series of brief questions via questionnaire:
a. Honduran identification number
b. Name
c. Municipality
d. Aldea (i.e. village)
e. Age and/or Date of Birth
f. Sex
g. History of child’s illness and symptomology, including duration of
symptoms
h. Past medical history
i. Breastfeeding history
j. Number and age of household children
k. Environmental smoke exposure (i.e. wood stoves or smokers in the
household)
l. History of influenza-like illness in mother during pregnancy
m. Educational status and age of caregiver
n. Family member working in the United States
o. Childhood illness exposure (i.e. school attendance)
2. Nasal and pharyngeal swabs were collected and stored in the clinic freezer.
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3. The clinician evaluating the patient recorded current illness data (i.e. temperature,
respiratory rate, lung exam findings, pulse oximetry, concurrent otitis or
pharyngitis).
4. Chart abstractions were performed to record:
a. Demographic data
b. Past medical history
c. Height and weight (i.e. nutritional status)
5. In order to direct diagnostic and treatment recommendations for a subset of
subjects, some specimens were tested inside the clinic with a commercial rapid
influenza test (BinaxNOW
®
) for immediate clinical information.
Roles and Responsibilities
Data were collected by the principal investigator and two research coordinators
trained specifically in human subjects research as dictated by the Collaborative
Institutional Training Initiative (CITI). Clinical information was obtained from the
principal investigator and clinic doctors and other medical personnel participating in
patient care at the Clinica Hombro a Hombro and Centro de Salud. His or her case
identification number identified each study participant for analysis.
All demographic, clinical, and laboratory data was stored in a study computer
database within the Hombro a Hombro clinic. The data were cross-referenced with
recent demographic data obtained by Shoulder to Shoulder within the clinics’ catchment
area. Meteorological data was obtained retrospectively from nearby weather stations
located at airports in Santa Rosa de Copan and La Esperanza. Rainfall data was obtained
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from water collection equipment at a nearby Hombro a Hombro clinic in Concepcion,
Intibucá, Honduras.
LABORATORY DETECTION OF RESPIRATORY VIRUSES:
Specimen Type/Collection
Nasal and pharyngeal swabs were obtained and processed for nucleic acid
amplification tests. One swab was placed into the nasopharynx as per package
instructions to collect epithelial tissue and absorb secretions. This swab was
subsequently placed into UTM. A second swab was placed into the opposite
nasopharynx and then into PrimeStore
®
buffer. A subset of patients had a third swab
obtained nasally for immediate rapid influenza antigen testing. Nasopharyngeal swabs
for rapid influenza antigen testing were transported to the Clinica Hombro a Hombro
laboratory for immediate analysis.
The original nasopharyngeal swabs were then separated into aliquots and stored in
the laboratory freezer (-80 degrees Celsius). The freezer in Honduras was supported by a
back-up generator in the event of a power failure. With Centers for Disease Control
(CDC) clearance, specimens were shipped bimonthly to Cincinnati Children’s Hospital
Medical Center (CCHMC) via two methods: on dry ice or via PrimeStore
®
buffer.
Samples were stored in the United States until laboratory tests were performed.
Nasopharyngeal specimens were thawed per protocol for analysis.
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Laboratory Procedures
Nasopharyngeal swabs for rapid influenza antigen testing were transported to the
Clinica Hombro a Hombro laboratory for immediate analysis. As per the rapid influenza
antigen testing package insert, refrigerated samples were tested within 24 hours of sample
collection.
Beginning with sample collection in February 2010, samples were tested for
various respiratory viruses using the commercially available Luminex Diagnostics
respiratory viral panel (RVP ID-Tag
TM
), a multiplex nucleic acid amplification test
(NAAT). The respiratory viral panel (RVP) from Luminex Diagnostics is a multiplex
nucleic acid amplification test that can detect 16 different viruses and subtypes from a
single respiratory specimen (Figure 3). The RVP detects respiratory syncytial virus,
influenza A, influenza B, adenovirus, parainfluenza 1, 2, 3, 4, enterovirus/rhinovirus,
coronavirus, and human metapneumovirus. This test has a sensitivity of 97.8% and a
specicity of 96.4% (Mahony, Chong et al. 2007; Zaman, Roy et al. 2008), and is a
Federal Drug Administration (FDA) certified diagnostic test.
Samples that tested positive for influenza A, non-H3N2, were subsequently tested
via real-time RT-PCR for pandemic H1N1. Samples have been retained in a secured
freezer at CCHMC until these analyses are completed. No personal identifiers are
included in this repository of samples, but a case identification number links the
repository to an individual’s questionnaire.
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Records Management
Data were entered into a computerized database at the Clinica Hombro a Hombro.
The database and the questionnaires were kept in a locked area of the clinic. Only study
personnel have access to these data.
ANALYSIS:
Objective 1: Etiologic Spectrum and Seasonality
We investigated the etiologic spectrum, clinical characteristics, and seasonality
during one calendar year of influenza and other viral respiratory infections via Luminex
Diagnostics polymerase chain reaction (PCR) respiratory viral panel (RVP ID-Tag
TM
).
Patients less than five years of age presenting to clinics in a rural Honduran village with
acute respiratory infections of less than five days duration were recruited for analysis.
Primary analysis using SPSS
®
provided descriptive epidemiologic data on seasonal
distribution and seasonal variations for the major viral pathogens in this population of
rural Honduran children under the age of five years. Viral pathogens tested by the
Luminex RVP ID-Tag
TM
included influenza A, influenza B, respiratory syncytial virus
(RSV), adenovirus, parainfluenza 1, 2, 3, 4, enterovirus/rhinovirus, coronavirus, and
human metapneumovirus. We compared the seasonal distribution of the major viral
pathogens in this area.
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Sample Size Considerations
This primary analysis is a descriptive study of a dichotomous variable, positive
versus negative influenza virus determined by Luminex RVP ID-Tag
TM
. With an
expected proportion of disease of 20% (Reyes et al., 1996; Gordon et al., 2009), a total
width of the confidence interval of 0.10, and a confidence level of 95%, the estimated
sample size is 246.
Statistical Assessment
Etiologies of respiratory illness in enrolled participants were described with
summary statistics.
1. Denominators:
Denominators included all children under five presenting to the Clinica Hombro a
Hombro and Centro de Salud between February 2010 and March 2011. Some of
these children were clinic patients under the age of five documented in the clinic
database but not recruited for the study.
2. Numerators:
Numerators included all children meeting eligibility requirements with laboratory-
confirmed viral respiratory disease. Data were stratified by age group, gender, and
other subpopulations.
Seasonality Analysis
Data were used to describe the seasonal distribution of respiratory viral infections.
These were graphically depicted, and the correlation coefficient (R
2
) was used to test for
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a statistically significant relationship between the observed distribution of viral
respiratory disease and meteorological variables such as rainfall.
Objective 2: Clinical and Demographic Characteristics
We performed a descriptive analysis of clinical and demographic characteristics
associated with specific viral etiologies of acute respiratory infection. These secondary
analyses compared subjects based on characteristics including age, sex, symptomology,
past medical history, breastfeeding history, number and age of household children,
environmental smoke exposure, history of influenza-like illness in mother during
pregnancy, educational status and age of caregiver, U.S. job status of family member,
childhood illness exposure, severity of illness, clinical exam findings, and geographic
location.
Statistical Assessment
Demographic and clinical characteristics of enrolled participants were described
with summary statistics (mean, median, and standard deviation). Binomial and
categorical variables were described with percentages of the total number of subjects in
that category.
Objective 3: Nucleic Acid Extraction/Stabilization Buffer
We assessed the effectiveness of a nucleic acid extraction/stabilization buffer to
detect influenza in specimens shipped at room temperature in comparison to specimens in
universal transport media shipped on dry ice. The laboratory-confirmed results from
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specimens shipped via PrimeStore™ nucleic acid extraction/stabilization buffer were
compared to the results from specimens shipped on dry ice. This information will assist
in further microbiological assessments in the developing world.
Statistical Assessment
The nucleic acid extraction/stabilization buffer was compared to the gold
standard, universal transport media on dry ice shipment, in secondary analyses. The
effectiveness of detecting influenza with this shipment method was described with
concordance statistics (Cohen's Kappa).
Limitations
Even though the Clinica Hombro a Hombro and Centro de Salud serve a large
catchment area for ill child care, we could not confirm that all subjects with respiratory
disease were presenting to the clinics for health care. Some ill children may have been
unable to travel to the clinic due to severity of illness, and some families may not have
sought care for their children if the illness was particularly mild. We do not have data on
the number of ill children who do not make it to the Clinica Hombro a Hombro and
Centro de Salud for treatment.
This study was also limited by the lack of a control group. The Clinica Hombro a
Hombro does not have offer immunizations and other routine well child care, so we were
unable to find a sufficiently large group of matched healthy children to compare to the
study subjects. The study sample size was not powered to detect differences between
subjects with one respiratory virus versus another respiratory virus.
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RESULTS:
From February 15, 2010 to March 9, 2011, we collected 267 nasopharyngeal
samples from symptomatic children less than five years of age presenting to the clinics.
Of these 267 samples, 55.1% were from males and 44.9% were from females. The
average age of the children sampled was 19.94 months (SD = 14.33, range 18 days - 58
months). Of the 267 subjects, 178 (67%) were under 24 months of age and 98 (37%)
were under 12 months of age.
Objective 1: Etiologic Spectrum and Seasonality
Etiologic Spectrum
Of the 267 samples, viruses were detected in 194 samples (72.7%) (Table 1,
Figure 4). Coinfections were identified in 15 samples (5.6%). Enterovirus/rhinovirus
was most commonly identified, in a total of 82 samples (30.7%). Influenza was
identified in 37 samples (13.9%). Of these 37 samples, 8 samples were identified as
influenza A (3.0%), 21 as influenza A, H3 (7.9%), and 8 as influenza B (3.0%) (Table 2).
Metapneumovirus was identified in 21 samples (7.9%), and RSV was identified in 20
samples (7.5%). Of these 20 samples, 9 samples (3.4%) demonstrated RSV A and 13
samples (4.9%) demonstrated RSV B. Parainfluenza was identified in 19 samples
(7.1%), of which 4 (1.5%) were parainfluenza 1, 14 (5.2%) were parainfluenza 3, and 1
(0.4%) was parainfluenza 4. Six samples (2.2%) yielded adenovirus.
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Seasonality
Influenza peaked in the months of August and September 2010. Influenza rose
from 1.8% of isolates in February through June to 25.7% of isolates in July through
October. No cases of influenza were identified from February 2010 through May 2010
and November 2010 through March 2011. Influenza was therefore present five out of 12
months (June 2010 through October 2010). Rainfall data obtained from a nearby village,
Concepción, revealed a peak in rainfall from July to September 2010 (Figure 5). Two
viruses demonstrated a positive correlation with rainfall: influenza (Figure 6) and RSV
(Figure 7). Influenza correlated with the rainy season (R² = 0.2857) more than RSV (R²
= 0.1684). With the inclusion of influenza, RSV, metapneumovirus, parainfluenza, and
adenovirus, the percentage of respiratory viruses by month correlated overall with rainfall
in millimeters by month (R² = 0.2863) (Figure 8).
Objective 2: Clinical and Demographic Characteristics
We performed a descriptive analysis of clinical (Table 3) and demographic
(Table 4) characteristics associated with specific viral etiologies of acute respiratory
infection. We also described quantitative variables for each virus category with mean,
median, and standard deviation, both for clinical (Table 5) and demographic (Table 6)
characteristics.
In participants with a respiratory virus diagnosed by PCR, the most common
symptoms reported by the parents were cough (89.2% of participants), fever (82.5%), and
runny nose (79.4%). The most common clinical findings reported by the physician in
participants with a PCR positive respiratory virus were rhinorrhea (40.7%), wheezing
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(27.8%), and rhonchi (9.3%). Though all parents reported a recent fever at the time of
recruitment, the average temperature of all participants with viruses recorded at the clinic
was less than 100 degrees Fahrenheit (or 37.8 degrees Celsius). Parents reported a
history of asthma in 16.5% of subjects with respiratory viruses by PCR, while physicians
reported a history of asthma in only 5.7% of subjects. Parents also reported a maternal
influenza-like illness in 9.8% of subjects with respiratory viruses, though none were
reported in subjects with influenza by PCR. Results for each respiratory virus are
reported in Table 3.
In participants with respiratory virus diagnosed by PCR, we analyzed several
demographic variables, including gender, children born at home, smoke exposure, and
subjects with family members working in the U.S. Among participants with a respiratory
virus diagnosed by PCR, 43.8% were female and 19.1% of subjects were born at home.
Very few children sleep in the room where food is prepared (3.1%), but 91.2% of
subjects live in homes where wood is used to prepare wood and only 50% of stoves are
vented. Out of subjects with respiratory viruses, 11.3% are exposed to cigarette smoke.
Almost half of the subjects (48.5%) have at least one family member working in the
United States. Results for each virus are reported in Table 4.
Other clinical data are summarized (mean, median, and standard deviation) in
Table 5: fever recorded in the clinic, respiratory rate, oxygen saturation by pulse
oximetry, weight, and height. Quantitative demographic characteristics are summarized
(mean, median, and standard deviation) in Table 6: age in months, number of people
living in the household, rooms in the subject’s home, average monthly income, maternal
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age, highest school grade completed by mother, and highest school grade completed by
anyone in the household.
Objective 3: Nucleic Acid Extraction/Stabilization Buffer
We assessed the effectiveness of a nucleic acid extraction/stabilization buffer to
detect influenza in specimens shipped at room temperature in comparison to specimens in
universal transport media (UTM) shipped on dry ice. The laboratory-confirmed results
from specimens shipped via PrimeStore™ nucleic acid extraction/stabilization buffer
were compared to the results from specimens shipped on dry ice. Using Cohen’s Kappa
coefficient, we found a significant correlation between influenza PCR results obtained
from samples shipped with UTM on dry ice and samples shipped via PrimeStore
®
(K =
0.767, p<0.0001).
DISCUSSION:
Etiology of Acute Respiratory Infections
These unique descriptive findings are the first data regarding the etiology of acute
respiratory infections in rural Honduras. Influenza was the most prevalent virus
throughout the year, contrary to previous studies demonstrating a high prevalence of RSV
(Reyes, Hedlund et al. 1996). This high rate of disease cannot be attributed to the 2009-
2010 pandemic H1N1 influenza epidemic, because only a small proportion (1.5%) of the
influenza A isolates identified were not H3. Further analyses will confirm that these
influenza A isolates are pandemic H1N1.
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This study identified a fairly large proportion of respiratory disease caused by
human metapneumovirus (7.9%). This virus was also surprisingly more common than
RSV, which is usually assumed to be the most prevalent respiratory virus in young
children. This proportion of metapneumovirus may be attributable to limited testing
capabilities prior to the availability of respiratory PCR panels. Further studies and
extended surveillance are necessary to determine the presence of influenza, human
metapneumovirus, and RSV over subsequent years.
Parainfluenza and adenovirus were present in much smaller numbers. They
tended to occur in small clusters that can be attributed to the closed nature of this rural
community. The individuals in this community have limited access to large cities in
Honduras and other surroundings areas. The presence of different viruses may vary year
to year based on exposure to outside communities.
Seasonality of Influenza and RSV
These results demonstrated a clear peak of influenza in the months of July
through October, coinciding with the rainy season in this region of Honduras. This
pattern differs from those observed in many tropical regions, where influenza circulates
throughout the year. Influenza was present for 5 out of 12 months of the study period in
this area of rural Honduras. This seasonality differs from that of urban Honduras, where
influenza circulates throughout the year with two clear peaks. Respiratory virus data
from the capital of Honduras, Tegucigalpa, is publicly available on the Pan American
Health Organization website (http://www.paho.org) and was obtained from the 2010
epidemic disease database (Figure 9). This is consistent with data from other tropical
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countries and may be explained by regular traffic between urban settings around the
globe.
The correlation between influenza and rainfall, as well as RSV and rainfall, may
be due to close living quarters and increased transmission of respiratory viruses. This is
similar to seasonality in the United States, where crowding and exposure to other children
increase influenza and RSV circulation in the winter. Some have speculated that the
seasonality of influenza is mediated by vitamin D levels (Juzeniene, Ma et al. 2010), but
further research is needed to confirm these findings and to assess the need for vitamin D
supplementation.
Clinical and Demographic Characteristics
For participants with positive PCR tests for respiratory viruses, the most common
symptoms reported by the parents were cough, fever, and runny nose. The most common
clinical findings reported by the physician were rhinorrhea, wheezing, and rhonchi.
These findings were much more common than difficulty breathing and retractions,
suggesting that subjects had more upper respiratory symptoms and were not severely ill.
Though all parents reported a recent fever at the time of recruitment, the average
temperature of all participants with viruses recorded at the clinic was less than 37.8
degrees Celsius. This suggests that parents were overestimating their children’s
temperatures in this setting. We are currently analyzing follow-up data from the clinics
to determine recovery time and severity of illness.
Parents reported a history of asthma in 16.5% of subjects with respiratory viruses
by PCR, while physicians only reported a history of asthma in 5.7% of the same subjects.
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It is difficult to explain this discrepancy, as many children received bronchodilators as
part of their therapy in spite of these numbers. Parents were possibly concerned about
symptoms of asthma in their children, or physicians may have overlooked some of the
medical history in their evaluations. We are awaiting further analyses of bronchodilator
use in the subjects.
Parents also reported a maternal influenza-like illness in 9.8% of subjects with
respiratory viruses, though none were reported in subjects with influenza by PCR. This
suggests that maternal influenza illness may have provided protection against influenza in
the infants. Previous research has demonstrated protection against influenza up to one
year after maternal influenza immunization (Zaman, Roy et al. 2008). Further research is
needed to identify the immunological mechanisms of protection during this time period.
Nucleic Acid Extraction/Stabilization Buffer
PrimeStore™, a nucleic acid extraction/stabilization buffer designed to store and
transport samples at room temperature, was an effective method for PCR detection of
influenza in comparison to specimens in universal transport media (UTM) shipped on dry
ice. The laboratory-confirmed influenza results from specimens shipped via
PrimeStore™ correlated significantly with samples shipped with UTM on dry ice (K =
0.767, p<0.0001). This technology may therefore overcome one of the most significant
difficulties of providing access to advanced diagnostic technologies in a low-resource
setting. As researchers and clinicians in remote locations begin to require RT-PCR and
other molecular diagnostic procedures for influenza and other pathogens, PrimeStore™
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may be a viable option for clinical specimens that require field collection in remote areas
such as Honduras.
SUMMARY:
Acute respiratory infections affect millions of children worldwide and are the
leading cause of death in Honduran children under the age of five. While epidemiological
data is available from industrialized nations, minimal diagnostic information is known
about respiratory disease in other parts of the world. Current data suggests that viruses
such as influenza cause more hospitalizations in the tropical and sub-tropical settings than
in temperate areas. With this study, we present descriptive data regarding the etiology of
disease in rural Honduran children under five with acute respiratory infections. These
unique results suggest that the spectrum of viruses in rural Honduran children is similar
to those found in the U.S., though the seasonality is tropical. We also demonstrated that a
nucleic acid stabilizing buffer stored at room temperature is an effective shipping method
for influenza samples as compared to UTM on dry ice. Data from this study will
hopefully guide further research into prevention and treatment of acute viral respiratory
diseases, including improved therapeutic options and prevention with immunization.
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Figure 1:
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Figure 2:
Figure 3:
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Figure 4:
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Figure 5:
Figure 6:
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Figure 7:
Figure 8:
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Figure 9:
Source: Epidemic disease database, 2010. Pan American Health Organization (PAHO).
http://www.paho.org.
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Table 1:
Virusdetected Total Percent
Novirusdetected 73 0.273
Adenovirus 6 0.022
Enterovirus/Rhinovirus 82 0.307
InfluenzaA 4 0.015
InfluenzaA,H3 18 0.067
InfluenzaB 7 0.026
Influenzacoinfections 8 0.030
Metapneumovirus 21 0.079
Parainfluenza1 4 0.015
Parainfluenza3 13 0.049
Parainfluenza4 1 0.004
RSVA 9 0.034
RSVB 13 0.049
Othercoinfections 8 0.030
Total 267 1.000
Table 2:
Virusdetected Total Percent
InfluenzaA 4 0.015
InfluenzaA,Enterovirus/Rhinovirus 3 0.011
InfluenzaA,H3 18 0.067
InfluenzaA,H3,Enterovirus/Rhinovirus 1 0.004
InfluenzaA,H3,Metapneumovirus 1 0.004
InfluenzaA,H3,RSVB 1 0.004
InfluenzaA,RSVA 1 0.004
InfluenzaB 7 0.026
InfluenzaB,Metapneumovirus 1 0.004
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    • "Several studies have shown that some respiratory viruses circulate seasonally, with a typically increase of the viral incidence in colder months, mainly in temperate regions [12], but also in subtropical regions151617. In tropical regions the results are more difficult to interpret, with several studies indicating higher viral circulation in rainy seasons [14,192021, while others show that respiratory viruses are prevalent year-round [22]. In Salvador for instance, a tropical city in the northeast of Brazil, the presence of viral infections was significantly associated with precipitation during the rainy season in patients with community-acquired pneumonia [19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Objective: To evaluate the oscillations on the viral detection in adenotonsillar tissues from patients with chronic adenotonsillar diseases as an indicia of the presence of persistent viral infections or acute subclinical infections. Study design: Cross-sectional prospective study. Setting:Tertiary hospital. Methods: The fluctuations of respiratory virus detection were compared to the major climatic variables during a two-year period using adenoids and palatine tonsils from 172 children with adenotonsillar hypertrophy and clinical evidence of obstructive sleep apnoea syndrome or recurrent adenotonsillitis, without symptoms of acute respiratory infection (ARI), by TaqMan real-time PCR. Results: The rate of detection of at least one respiratory virus in adenotonsillar tissue was 87%. The most frequently detected viruses were human adenovirus in 52.8%, human enterovirus in 47.2%, human rhinovirus in 33.8%, human bocavirus in 31.1%, human metapneumovirus in 18.3% and human respiratory syncytial virus in 17.2%. Although increased detection of human enterovirus occurred in summer/autumn months, and there were summer nadirs of human respiratory syncytial virus in both years of the study, there was no obvious viral seasonality in contrast to reports with ARI patients in many regions of the world. Conclusion: Respiratory viruses are continuously highly detected during whole year, and without any clinical symptomatology, indicating that viral genome of some virus can persist in lymphoepithelial tissues of the upper respiratory tract.
    Full-text · Article · Jul 2014 · International Journal of Pediatric Otorhinolaryngology
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    • "All PIV types were detected in our study, with a predominance of PIV3 as in several previous studies575859 . The prevalence of influenza virus (11.9%) was similar to that observed in other countries (10% to 13%) after the pH1N1 pandemic [17]. Among the influenza viruses, A(H1N1) pdm09 and type B represented 57.3% and 42.7% of cases, respectively. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Surveillance of influenza-like illness (ILI) in Central Africa began only recently, and few data are therefore available on the circulation of influenza virus and other respiratory viruses. In Gabon, a Central African country, we established a surveillance network in four major towns in order to analyze cases of ILI among patients who visited health centers between March 2010 and June 2011, and to determine the viral etiology. Methods Nasal swabs were sent for analysis to the Centre International de Recherches Médicales de Franceville, where they were screened for 17 respiratory viruses in a multiplex real-time reverse transcription polymerase chain reaction for all pathogens according the following pairs: adenovirus/parainfluenza virus 4, respiratory syncytial virus/human metapneumovirus, parainfluenza virus 1/parainfluenza virus 2, pandemic influenza virus A/seasonal influenza virus A (H1N1, H3N2)/seasonal influenza virus B, human coronaviruses 229E/OC43, human coronaviruses NL63/HKU1, rhinovirus/human parechovirus, and enterovirus/parainfluenza virus 3. Results We analyzed a total of 1041 specimens, of which 639 (61%) were positive for at least one virus. Three-quarters of the patients were children under five years old. We therefore focused on this age group, in which 68.1% of patients were positive for at least one virus. The most common viruses were adenoviruses (17.5%), followed by parainfluenza viruses (PIVs) 1–4 (16.8%), enteroviruses (EV) (14.7%), respiratory syncytial virus (RSV) (13.5%), and influenza virus (11.9%). The prevalence of some viruses was subject to geographic and seasonal variations. One-third of positive samples contained more than one virus. Conclusions Like most studies in the world, the virus PIVs, EV, RSV, Influenza virus, HRV were predominant among children under five years old in Gabon. An exception is made for adenoviruses which have a high prevalence in our study. However adenoviruses can be detected in asymptomatic persons. These finding gave a better knowledge of the circulation and the seasonality of the viruses involved in ILI in Gabon.
    Full-text · Article · Jul 2014 · BMC Infectious Diseases
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    [Show abstract] [Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory infection in children less than 5 years of age. The impact of non-RSV respiratory virus coinfection on the severity of RSV disease is unknown. This hospital-based prospective study was conducted in Nagasaki, Japan, on all children less than 5 years of age with acute respiratory infection (ARI) who had undergone a rapid RSV diagnostic test between April 2009 and March 2010. Thirteen respiratory viruses were identified from nasopharyngeal swab samples using a multiplex polymerase chain reaction; polymerase chain reaction-positive samples were considered as confirmed respiratory virus infections. The cases were classified into 3 categories (pneumonia, moderate-to-severe nonpneumonic ARI and mild ARI) according to the findings of the chest radiograph and the hospitalization records. Among 384 cases enrolled, 371 were eligible for analysis, of whom 85 (23%) were classified as pneumonia cases; 137 (37%) as moderate-to-severe nonpneumonic ARI cases and 162 (40%) as mild ARI cases. RSV was detected in 172 cases (61.6%), and 31 cases (18.0%) had double or triple infections with other respiratory viruses. RSV infection was more frequently observed in pneumonia cases (odds ratio [OR]: 2.3; 95% confidence interval [CI]: 1.31-3.9) and moderate-to-severe nonpneumonic ARI cases (OR: 2.95; 95% CI: 1.82-4.78) than in mild ARI cases. The association with moderate-to-severe nonpneumonic ARI cases was stronger with RSV/non-RSV respiratory virus coinfection (adjusted OR: 4.91; 95% CI: 1.9-12.7) than with RSV single infection (adjusted OR: 2.77; 95% CI: 1.64-4.7). Non-RSV respiratory virus coinfection is not uncommon in RSV-infected children and may increase the severity of RSV disease.
    Full-text · Article · May 2013 · The Pediatric Infectious Disease Journal
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