Native American (NA) children, particularly Alaska Natives and American Indians in the southwestern United States, have high rates of morbidity and mortality related to acute lower respiratory infections (ALRIs).1 , 2 Streptococcus pneumoniae or Haemophilus influenzae type b, or bronchiolitis due to respiratory syncytial virus (RSV).3–10 influenza in NA communities, even during the 2009 pandemic, have been limited to retrospective observational studies.11–15
METHODS
Study Population
Subjects for this analysis were drawn from a prospective clinical trial evaluating the effectiveness of motavizumab, an investigational monoclonal antibody directed against the RSV F protein, in preventing severe RSV-related disease in American Indian infants of the American Southwest (NCT00121108, www.clinicaltrials.gov 16 influenza A/H1N1 (H1N1pdm) within NA communities,11 , 12 influenza in relation to other respiratory pathogens in this group of children during that time period. Therefore, all children from the third and fourth enrollment seasons (2006 and 2007) who were still participating in the trial follow-up as of January 1, 2009, were included in this report.
Clinical Surveillance
Active surveillance was conducted 6 days a week at Indian Health Service clinical facilities where the study subjects primarily sought care. A nasal wash specimen was collected in the clinic or at home by a study nurse within 72 hours of any visit with a respiratory illness diagnosis, or if presenting signs/symptoms suggested a respiratory illness. Because specimen collection occurred before formal assessment of the respiratory category of the illness, nursing staff were instructed to collect specimens in children with diagnoses suggesting a lower respiratory illness (pneumonia, bronchiolitis, asthma, croup, etc.) or common presenting signs/symptoms from a prespecified list (cough, wheeze, crackles, shortness of breath, etc.). Therefore, samples were obtained from a subset of upper respiratory illnesses when the determination of lower versus upper respiratory involvement had not been made within the specimen collection window. Nasal washes were obtained by instilling 15–20 mL of lactated Ringers solution into the nasopharynx with a bulb syringe and passively collecting the fluid into a sterile specimen cup. Specimens were combined with viral transport medium, snap frozen in liquid nitrogen and stored at –70°C.
A study physician categorized each clinical visit; an MAARI episode was defined as an event in a study child with any lower or upper respiratory symptoms; the presence of an abnormality on chest examination (eg, wheeze, crackles, rhonchi); or where an acute respiratory illness diagnosis was made by the primary care provider based on clinical judgment. Study physicians then categorized the episode as an upper versus lower respiratory illness and assigned a final study diagnosis using a standardized protocol based on clinical examination findings, imaging reports, laboratory testing, clinical diagnoses and, where necessary, discussion with the provider. For study purposes, the protocol diagnosis took precedence when the assessments of the study and treating physicians were discordant. Study physicians communicated regularly with each other to discuss cases in which the final diagnosis was not straightforward, to maximize consistency and standardization across the study.
Diagnostic Testing
All specimens obtained between January 1 and December 31, 2009, were tested for this study. Total nucleic acid was extracted from 250 µL aliquots of the nasal wash samples using the easyMAG extraction platform (bioMérieux, Marcy l’Etoile, France) and eluted in 35 µL. Complementary DNA was generated from 10 µL aliquots of total nucleic acid using the Superscript II reverse transcription kit (Invitrogen, Carlsbad, CA) and analyzed by the MassTag polymerase chain reaction respiratory panel.17–21 influenza A, influenza B, human rhinoviruses (HRVs), human enteroviruses, human metapneumovirus, human parainfluenza viruses 1–4, human coronaviruses 229E and OC43, RSV A and B, adenovirus, Chlamydophila pneumoniae , H. influenzae , Mycobacterium tuberculosis , Mycoplasma pneumoniae , Legionella pneumophila and S. pneumoniae . Samples positive by MassTag polymerase chain reaction were reamplified by singleplex polymerase chain reaction and confirmed by sequencing.
Statistical Analysis
Data were analyzed using Stata 11.0 (StataCorp, College Station, TX). Healthcare provider records were reviewed by a study physician. MAARI episodes were categorized as outpatient or inpatient ALRI as appropriate, and diagnoses were classified by major clinical syndrome (Table 1 ). All clinical data were recorded on paper forms and submitted to the trial sponsor (MedImmune, Gaithersburg, MD), after which a cleaned, locked data set was returned to the study investigators for further analysis. Clinical episodes from which multiple nasal washes were collected were reviewed and molecular testing results were consolidated if clinically compatible. Illness incidence rates were calculated based on the subjects under active surveillance, and were compared using rate ratios and exact binomial 95% confidence intervals. Person-time at risk for each participant was calculated from January 1, 2009, through the date of study withdrawal/completion or December 31, 2009, whichever occurred first. Pathogen-specific incidence rates were adjusted for the proportion of episodes sampled. Associations between diagnostic categories or clinical syndromes and individual pathogens or pathogen combinations were examined using the 2-sample test of proportions, χ2 analysis or Fishers exact test, as appropriate. Baseline characteristics (Table 2 ) were evaluated as risk factors for lower respiratory tract disease or hospitalization using multiple logistic regression. The median ages between groups were compared using the Mann–Whitney test. A P value of <0.05 was considered statistically significant.
TABLE 1: Guidelines for Clinical Syndrome Categorization of Acute Respiratory Illness Episodes
TABLE 2: Demographic, Clinical and Environmental Factors Among Study Subjects*
Human Subject Research Issues
The study was approved by the Navajo and White Mountain Apache tribes, as well as the institutional review boards of the Johns Hopkins Bloomberg School of Public Health, the Navajo Nation and the Phoenix Area Indian Health Service.
RESULTS
Incidence of MAARI
At the start of the analysis period, 915 children (median age at start on January 1, 2009: 16.9 months) were under active follow-up; 260 (28%) enrolled in 2006, and 655 (72%) enrolled in 2007 (Table 2 ). The 2 cohorts were well balanced at enrollment in the parent trial, with the exception of having a family history of asthma or hay fever. A high proportion of subjects was breastfed in infancy (86%), had young siblings at home (66%), was exposed to secondhand smoke (19%) or wood-burning or coal-burning stoves (62%) in the home or had been hospitalized before enrollment in the parent trial (12%). Although two thirds of subjects lived with other young children, formal day-care attendance (3%) was low.
Of the 915 subjects under surveillance, 669 (73%) experienced a total of 1476 MAARI episodes, resulting in an incidence rate of 1756 episodes per 1000 child-years (Table 3 ). Of these episodes, 329 were ALRI managed on an outpatient basis, whereas 66 were ALRI requiring hospitalization, resulting in incidence rates of 391 and 79 episodes per 1000 child-years, respectively. Rates of respiratory illness were higher in the younger cohort compared with the older cohort for all categories (P < 0.05 for all). Subjects experienced 2.2 MAARI episodes on average (range 0–11), whereas 246 (27%) experienced none. Repeat visits accounted for 27% and 12% of inpatient and outpatient ALRI episodes, respectively (Table 4 ). Of the 240 children who experienced an outpatient ALRI, 25 (10%) also experienced an inpatient ALRI episode. Episodes occurred in 2 waves during 2009 (Fig. 1 ). The first wave, peaking in mid to late winter, represented the second or third winter respiratory season experienced by the 2 cohorts, whereas the second wave occurred throughout the autumn, after a proportion of the older cohort had completed follow-up.
FIGURE 1: MAARIs by date of onset among participants (N = 915), January 1 to December 31, 2009, including inpatient acute lower respiratory illnesses (black), outpatient acute lower respiratory illnesses (gray) and all other MAARIs (white).
TABLE 3: Episodes and Incidence Rates* of Medically Attended Acute Respiratory Infections, Outpatient Acute Lower Respiratory Illnesses and Inpatient Acute Lower Respiratory Illnesses, by Seasonal Cohort
TABLE 4: Number of Times a Child Was Seen, According to Visit Type
Of the 1476 episodes, 989 (67%) were diagnosed as upper respiratory infection or cough, whereas pneumonia, bronchiolitis and other wheezing illnesses accounted for 23% (Table 5 ). Among outpatient ALRI, bronchiolitis was seen in one fifth (21%), whereas pneumonia accounted for more than one quarter (28%) and other wheezing illnesses accounted for one third (32%). Pneumonia was diagnosed in a majority (59%) of ALRI hospitalizations, followed by bronchiolitis and other wheezing illnesses.
TABLE 5: Number and Incidence Rates* of Clinical Syndrome Categories by Visit Type
Detection of Viruses and Bacteria
A total of 699 respiratory specimens were collected from 639 (43%) of the 1476 MAARI events, including 295 (30%) of the 984 upper respiratory infection episodes, 236 (72%) of the 329 outpatient ALRI episodes and 59 (89%) of the 66 ALRI hospitalizations. Samples were collected a median of 4 days (interquartile range 2–6 days) after illness onset. Molecular diagnostic testing detected ≥1 potential pathogens in 88% of episodes overall, including 91% of outpatient ALRI and 93% of inpatient ALRI (Table 6 ). Virus detection decreased in frequency as the number of days between illness onset and sample collection increased (P = 0.001). Among the respiratory viruses, HRV and RSV were most commonly detected across all categories, with RSV predominant among the more severe episodes (eg, 44% of ALRI hospitalizations). RSV episodes were seasonal, peaking in early February, and ending by early May (Fig. 2 ). HRV was detected in roughly one quarter of episodes, with winter and fall waves similar to those observed for MAARI overall. HRV was detected among outpatient and inpatient ALRI cases in similar proportions; of these, less than one fifth were associated with HRV alone. Influenza , parainfluenza (PIV; particularly type 3), and human metapneumovirus (hMPV) were detected at comparable rates (8–13%). S. pneumoniae was detected in 60% of episodes, with a smaller proportion positive for H. influenzae (all types; 19%) and C. pneumoniae (3%).
FIGURE 2: MAARIs by date of onset and visit type associated with RSV including inpatient acute lower respiratory illnesses (black), outpatient acute lower respiratory illnesses (gray) and all other MAARIs (white).
TABLE 6: Number (%) and Incidence Rates* of Pathogens Detected, by Visit Type
Among influenza detections, episodes during the first 3 months of the year were associated with seasonal influenza A(H1N1) and influenza B viruses, whereas pandemic influenza A(H1N1) (H1N1pdm) was first identified in late May (Fig. 3 ). Although the first spring wave was largely absent in this population, H1N1pdm ultimately accounted for more than half of all influenza detections, including 11 (64%) of the 22 ALRI cases and all 3 illnesses requiring hospitalization.
FIGURE 3: MAARIs associated with influenza by subtype and illness category. Seasonal influenza A acute lower respiratory illnesses (light gray) and other MAARIs (white), influenza B acute lower respiratory illnesses (heavy stripe) and other MAARIs (light stripe), and H1N1pdm acute lower respiratory illnesses (dark gray) and other MAARIs (medium gray).
Lower respiratory illnesses were more likely to be associated with RSV compared with upper respiratory illness (detected in 25% versus 11%, P < 0.001) whereas inpatient status was associated with the detection of RSV (P = 0.029), enterovirus (P < 0.001) and adenovirus (P < 0.001) when compared with all outpatient MAARI. By contrast, the detection of influenza virus (including H1N1pdm), rhinovirus or pneumococcus did not increase the risk of either lower respiratory tract involvement or hospitalization. It is notable that all 5 inpatient episodes associated with adenovirus involved the detection of an additional virus (RSV in 4, HRV in 1), and only 1 of the 6 enterovirus-associated hospitalizations involved its detection as a single pathogen.
Approximately one third of episodes were associated with only 1 pathogen across all visit types; the largest proportion demonstrated 2 pathogens (Table 7 ). Detection of ≥3 pathogens was more frequently seen among hospitalized episodes (29%) than among MAARI episodes overall (17%; P = 0.030). S. pneumoniae was identified in combination with a virus in 48–58% of episodes, yet HRV was the only virus specifically associated with its presence (P = 0.028 for all MAARI; P = 0.034 for all ALRI; P = 0.145 for outpatient ALRI; P = 0.069 for inpatient ALRI). Influenza virus, RSV and rhinovirus were negatively associated with each other (P < 0.05 for all by pairwise combination).
TABLE 7: Multiple Pathogen Detections, by Visit Type
The frequencies of pathogens according to diagnostic category among ALRI were unevenly distributed (Table 8 ). As expected, RSV was prominently seen among pneumonia and bronchiolitis cases, whereas rhinovirus was more frequent in other wheezing illnesses and pneumonia. Influenza and parainfluenza cases were notable for pneumonia and nonspecific ALRI, but were seen in a considerable proportion of wheezing illnesses as well. In contrast with previous reports,22 , 23 24
TABLE 8: Pathogen Detection (Number, % Positive Among Those tested) by Diagnostic Category Among Outpatient and Inpatient ALRI Episodes
Risk Factor Analyses
Subjects who experienced ≥1 MAARI (n = 669) were more likely to have been younger at the start of the study period compared with those who had not (n = 246; median age 16.5 versus 18.4 months, P = 0.0002). These 2 groups were otherwise similar in terms of baseline characteristics. Among subjects who had an MAARI, those who experienced lower respiratory involvement (n = 273) were younger (P = 0.0001), were more likely to be male (P = 0.017), to have had an ALRI as an infant before enrollment in the trial (P = 0.034) and to have a family history of an atopic condition (P = 0.001) compared with those who had not had ALRI (n = 396), by multiple logistic regression. Among the children with ALRI, those who required hospitalization (n = 58) were more likely to have been younger (P = 0.017), or to have a history of eczema (P = 0.014), compared with subjects who were managed as outpatients (n = 215), by multiple logistic regression. The effects of motavizumab on bronchiolitis and RSV will be reported separately.
DISCUSSION
Here we provide, for the first time, prospective data regarding the major presentations and pathogens associated with acute respiratory illness from a cohort of young American Indian children. Collected through a full calendar year, these findings document a wide spectrum of respiratory pathogens, including the emergence of pandemic influenza , in this population known to be at high risk for respiratory illness. These data indicate high rates of medical attendance beyond infancy for respiratory disease, particularly involving the lower respiratory tract. Our rates of ALRI hospitalizations exceed estimates of 17–20 per 1000/year derived from retrospective database analyses.2 , 25 26 27 S. pneumoniae and H. influenzae type b,28
We demonstrate the continued dominance of RSV in the years following infancy, responsible for a large proportion of pneumonia, bronchiolitis and other acute wheezing illnesses. We also confirm the high prevalence of HRV among these children, although it is unclear if HRV was the causative pathogen in all cases, as multiple detections were common. An additional virus was detected in approximately one third of HRV ALRI episodes, compared with 17% of ALRI cases positive for hMPV, 18% of those with influenza and 24% of those with RSV. Nevertheless, HRV may play at least a contributing role, as codetection of HRV with bacteria was common, and among ALRI cases and hospitalizations, was statistically associated with S. pneumoniae .
Consistent with prior reports,14 , 15 influenza -related respiratory illness in our population compared with US children in general.29 , 30 influenza season was mild in terms of severity and extent, whereas pandemic influenza affected children disproportionately worldwide.31–33 influenza -related hospitalizations in this cohort. Nevertheless, seasonal H1N1 and B viruses contributed almost as much to the total influenza burden in terms of outpatient ALRI episodes (42%) and overall MAARI visits (44%), and within a shorter span of time. These unexpectedly comparable proportions of seasonal and pandemic influenza cases may be because young age is an independent risk factor for influenza -related complications, and the study cohort was younger at the start of 2009, when seasonal viruses were in circulation. Moreover, the spring wave of H1N1pdm was comparatively mild in this population, and our surveillance did not capture the fall wave experiences of the full 2006 cohort, as up to 30% of this group (representing 8% of the total cohort) had completed follow-up by September 1, 2009.
Similar to prior studies conducted in NA communities, pneumococcal colonization within this cohort exceeded rates found among similar age groups in the general US population.34–40 influenza and S. pneumoniae .41 , 42 S. pneumoniae in nasopharyngeal samples was associated with more severe H1N1pdm illness.20 43–46 S. pneumoniae and did not measure colonization density, which may be more closely associated with disease.47 , 48 49 , 50 51–53
Our study was subject to limitations. First, our study did not include microbial testing in asymptomatic subjects, thus limiting our ability to draw causal inferences between the pathogens detected in the nasal wash and the clinical illness observed in the child. Second, clinical syndromes were assigned according to diagnoses made by the treating provider, and could only be standardized and confirmed at the level of medical record review by our study physicians. Third, we report data only from a single year, focusing on children in a restricted age range. Finally, the detection of bacteria or other pathogens in upper respiratory samples may not always be correlated with lower tract disease, thus limiting our ability to identify potential associations with coinfections or clinical outcomes.
In summary, we present the first comprehensive assessment of the clinical diagnoses and pathogens associated with acute respiratory illness among young American Indian children. These findings demonstrate the prevalence of numerous respiratory pathogens, including RSV, rhinovirus and pneumococcus in this community, and indicate that seasonal and pandemic influenza imposed a considerably higher burden compared with the general US population. Our observations offer valuable insights into the complex picture of respiratory infections and provide critical evidence to support prevention and control efforts that can be applied to epidemiologically similar communities throughout the world.
ACKNOWLEDGMENTS
The authors express their appreciation to the children and adults from the Navajo, White Mountain Apache and San Carlos Apache communities who participated actively in this study and the Indian Health Service physicians who cared for them. The authors gratefully acknowledge the dedicated efforts of the Center for American Indian Health field staff who collected these data over many years. The authors also gratefully acknowledge the work by MedImmune staff on the parent clinical trial.
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