Introduction
Allergic inflammation is associated with tissue eosinophilia, which is a prominent finding in nasal mucosa of patients with allergic rhinitis (AR).1 Nasal eosinophils correlate with nasal symptom severity in adults with seasonal AR.2 In addition to reflecting inflammation within the upper airway, nasal eosinophilia is associated with sputum eosinophilia in AR patients with concomitant asthma.3 Nasal eosinophils can be objectively measured as a biomarker allergic airway inflammation3 Nasal eosinophils correlated with chronic nasal symptoms in a cross-sectional study of Finnish children and adults, however their atopic status was unknown.4 Currently, it is unknown the percentage of young children with AR that have nasal eosinophilia.
It is unknown if early skin prick testing to aeroallergens can identify children with severe AR using an objective biomarker such as nasal eosinophils. Linking the magnitude of the wheal reaction earlier in life to an objective biomarker, such as nasal eosinophilia, may be attractive in future intervention trials by identifying those children most susceptible to the later onset of severe AR symptoms. The hypothesis of this study is that specific aeroallergen wheal area(s) in the first three years of childhood is associated with allergic eosinophilic rhinitis (AER) at age four. An association between early SPT wheal area and AER reinforces a connection between early aeroallergen sensitization and childhood AR and also provides important diagnostic information for earlier diagnosis of severe AR.
Methods
Study Population
The Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS) strategy for recruiting infants at high risk for developing allergic disease have been previously published.5,6 Birth records were obtained for infants born in Greater Cincinnati and Northern Kentucky. Parents were required to live either less than 400 meters or more than 1500 meters from a major road in order to determine if early traffic-related air pollution exposures is associated with allergic disease. However, we have previously found that though traffic-related air pollution is associated with wheezing but not AR.7,8 Of those parents living within the defined area, at least one parent reporting a symptom history of allergies or asthma was required for skin testing eligibility. Symptomatic parents were invited to a screening visit and after obtaining written informed consent that was approved by University of Cincinnati Institutional Review Board, underwent skin prick testing (SPT) to 15 aeroallergens. Aeroallergens in the screening SPT panel included: eastern red cedar, American elm, maple mix, white oak, meadow fescue, timothy, short ragweed, house dust mite mix (Dermatophagoides farinae, and Dermatophagoides pteronyssinus), German cockroach, cat, dog, and four mold allergens (Alternaria alternata, Aspergillus fumigatus, Penicillium species mix and Cladosporium species) (ALK-Abelló Inc.). These symptomatic parents that were also sensitive to at least one aeroallergen were invited to enroll their infant into the CCCAPS cohort.5
Clinical visits
At age one, parent(s) brought their infants to CCAAPS for clinical evaluation. The CCAAPS clinical staff interviewed the parent(s) using questionnaires, obtaining details on both the infant's medical history and the home environmental history. The infants were examined and SPT to the same 15 aeroallergens in the parental screening panel in addition to cow's milk and hen's egg. The children returned to the CCAAPS clinics annually at two, three and four years of age for repeat physical examination, skin testing and parental interview. At the year four visit, nasal epithelial smears also were obtained.
Quantitative Skin Prick Testing
Skin prick testing was performed using bifurcated needle coated with the positive histamine dihydrochloride (10 mg/ml) control, the negative 50% glycerinated human serum albumin-saline control, or one of the 17 test panel allergens.9 Skin reactions were read after 15 minutes of skin testing. A positive reaction was noted if the diameter was ≥ 3 mm than the negative control in accordance with the most recent the American Academy of Allergy, Asthma and Immunology/and the American College of Allergy, Asthma and Immunology Allergy Diagnostic Practice Parameter.9 All wheal and flare circumferences were traced with ink pen. The ink was absorbed by Transpore tape (3M, St. Paul Minnesota) and affixed to labeled grid paper in the child's permanent record. These records were scanned and saved as true image files. The ink outlines of wheal circumferences were digitally re-traced and the enclosed area was calculated using AutoCAD (Autodesk, Inc.). For accuracy, these measurements were performed independently in duplicate by two independent individuals.
Nasal cytology
At age four, each inferior nasal turbinate was swabbed with a separate cotton applicator. The samples were adapted from a previously published protocol.2,10,11 Cells were stained with Nasal Cytology Stain (Volu-Sol Inc.).12 Only cells with an intact nucleus and cytoplasm were counted. The number of eosinophils was counted using 40 or 100 times magnifications until a maximum of 400 cells were counted. For quality control, a second scientist counted 10 percent of samples using a random block sampling procedure of each quartile. There was no significant difference between the cell counts of each scientist.
Health outcomes
At each annual visit, the parents were asked the ISAAC validated question “In the past 12 months, has your child ever had a problem with sneezing, or a runny, or a blocked nose when he/she DID NOT have a cold or flu?”13 Allergic rhinitis (AR) was defined as a positive response to the ISAAC question, 2) a positive SPT to one of 15 aeroallergens. Allergic eosinophilic rhinitis (AER), the primary outcome of this study, was defined as: 1) a positive response to the ISAAC question, 2) a positive SPT to one of 15 aeroallergens, and 3) greater than 5% nasal eosinophils.3 These AER cases were compared to children without nasal symptoms and negative SPT to all 15 aeroallergens.
Exposure Assessments
Before age one, the CCAAPS research staff visited the infant's home. The home's general characteristics, basement, the infant's primary activity room and sleeping room were inspected for visible mold, water damage and the general state of repair of each room. In order to determine the greatest component of endotoxin, (1-3)-β-D-glucan, and indoor allergen exposure, the infant's primary activity room, a 2 m2 area of floor space, was vacuumed at a standard rate of two min/m2.14,15 The collected dust samples were filtered, desiccated and stored at -20 °C.16 The dust samples were separated for measuring house dust endotoxin (HDE, endotoxin units (EU)/mg of settled dust) and (1-3)-β-D-glucan (μg/g of dust) by the limulus amebocyte lysate assay (Associates of Cape Cod Inc.,).17 Separate aliquots of settled dust were used for analysis of major cat allergen (Fel d1), major dog allergen (Can f1), major dust mite allergen (Der f1), and major cockroach allergen (Bla g1) by monoclonal sandwich ELISA.18-21
Covariates
Other covariates previously identified in the CCAAPS cohort as relevant for AR were evaluated for model inclusion and included: ethnicity (non-African American (NAA), African-American (AA)); gender; annual household income (> $20,000, ≤ $20,000); breastfeeding duration (months); number of children in the home (≥ 2 children, <2 children); season of birth; and aforementioned environmental covariates.22 Hair cotinine levels were measured and used as an objective biomarker of tobacco smoke exposure at age two.23
Data analysis
The aeroallergen wheal area at ages one, two and three were analyzed for associations to AER using logistic regression. The 95% confidence intervals and odds ratios reported were obtained from the profile likelihood ratio. Any allergen wheal area or covariate significantly associated with AER at the α < 0.2 was further evaluated in multivariate logistic regression. Home environmental exposures (endotoxin, β-glucan, Fel d1, Can f1, Der p1, and Bla g1) were analyzed as continuous independent variables with thresholds defined by the turning points in the smooth plot that result from the use of general additive model.24 Independent exposures and covariates associated with AER at the 0.2 level were further investigated in multivariate model. Variables in the multivariate model were eliminated by the “all subsets” method of selection, with the purpose of minimizing the log likelihood ratio. A combined regression model that used informative predictors from age one, two and three regression models was developed. Analyses were performed using SAS 9.2 (SAS Institute Inc,).
Results
Subjects
CCAAPS enrolled 762 infants and 636 (83.5%) were evaluated at age four. Of these, the parents of 478 (75.2%) children consented to nasal eosinophil sampling. Children whose parents did provide consent to nasal sampling were more likely to be NAA, higher father education, higher household income, two children or less in the home and lower elemental carbon attributable to traffic (ECAT) exposure (Table E1 and E2). Of 478 children, 437 (91.4%) children had technically interpretable nasal smears and these were more likely to be from households of higher income, and lower ECAT exposure (Table E3 and E4). Complete rhinitis response, skin testing and interpretable nasal scraping data was available on 434 children.
Sensitivity analyses
To determine if 5% was an acceptable cut-off for the definition of AER, the eosinophil threshold was varied over a range of possible limits. Figure 1 shows number of children with AER as a function of the percentage of nasal eosinophils. Increasing the eosinophil threshold above 5% did not appreciably change the number of children with AER. In contrast, lowering the eosinophil threshold increased the number of children with AER at the risk of losing phenotype specificity. Therefore, an eosinophil threshold of 5% was used in the study. This threshold is consistent with a previously published study and represents a more specific inflammatory phenotype of AR.3 Of these 434 children, 119 (27.4%) had 5% or more nasal eosinophils. Of 119 children with 5% or more nasal eosinophils, 79 (66.4%) were sensitized to aeroallergens, while 49 (41.2%) had chronic nasal symptoms. Of the 49 children with 5% or more nasal eosinophils and chronic nasal symptoms, 38 (77.5%) had AER, representing 8.8% of the entire sample. The comparison group for AER includes children without AR.
Figure 1. Sensitivity analysis with variation of the threshold for defining allergic eosinophilic rhinitis.
Unadjusted analyses
The allergen wheal areas were analyzed for associations with AER after correcting for multiple comparisons. At ages one and two, no allergen wheal area was significantly associated with AER. At age three, Penicillium (p=0.04), maple (p= 0.02) and elm (p=0.11) wheal area met criteria for further analysis (p<0.2) in the multivariable model with AER.
As shown in Table 1, the unadjusted odds ratio of the children characteristics which predicted AER and were included in the multivariable model included gender (OR 2.81; 95% CI, 1.34-6.46; p=0.009), mothers' education level (OR 0.37; 95% CI, 0.17-0.76; p=0.01), breastfeeding duration (OR 0.55; 95% CI, 0.27-1.08; p=0.09). The unadjusted odds ratio of environmental exposures meeting the inclusion criteria were only low, medium and high house dust Fel d1 levels (Table 2). ECAT showed a non-significant protective association. After further investigation, it was determined that a positive SPT at ages two and three were acting as intervening variable, thus necessitating removal from further analysis.
Table 1. Unadjusted children characteristics to Allergic Rhinitis with high eosinophils (AER) compared to the combined phenotypes other than AR at age four.
A total of 434 children had complete questionnaire, SPT and nasal cytology at age four.
| Covariates | Frequency (%) | Allergic Eosinophilic Rhinitis (AER) n=38/434 (8.8) | All phenotypes other than AER n=335/434 (77.2) | OR (95% CI) |
|---|---|---|---|---|
|
| ||||
| Gender: | ||||
| Female | 193 (44.5) | 9 (4.7) | 156 (80.8) | 2.81 |
| Male | 241 (55.5) | 29 (12.0) | 179 (74.3) | (1.34, 6.46)† |
|
| ||||
| Race: | ||||
| NAA | 345 (79.5) | 31 (9.0) | 268 (77.7) | 0.90 |
| AA | 89 (20.5) | 7 (7.9) | 67 (75.3) | (0.35, 2.03) |
|
| ||||
| Mother's Education: | ||||
| College graduate | 213 (50.7) | 27 (12.7) | 161 (75.6) | 0.37 |
| Some college/trade | 207 (49.3) | 10 (4.8) | 162 (78.3) | (0.17, 0.76)† |
|
| ||||
| Father's Education | ||||
| Some college/trade | 298 (71.1) | 29 (9.7) | 232 (77.9) | 0.71; |
| HS diploma or less | 121 (28.9) | 8 (6.6) | 90 (74.4) | (0.29, 1.55) |
|
| ||||
| Household Income: | ||||
| ≥ $20 K | 354 (84.7) | 33 (9.3) | 273 (77.1) | 0.69 |
| < $20 K | 64 (15.3) | 4 (6.3) | 48 (75.0) | (0.20, 1.83) |
|
| ||||
| Season of Birth: | ||||
| Winter | 151 (34.8) | 11 (7.3) | 116 (76.8) | -- |
| Spring | 90 (20.7) | 6 (6.7) | 73 (81.1) | 0.87; (0.29, 2.38) |
| Summer | 83 (19.1) | 8 (9.6) | 65 (78.3) | 1.30; (0.48, 3.37) |
| Autumn | 110 (25.4) | 13 (11.8) | 81 (73.6) | 1.69; (0.72, 4.04) |
|
| ||||
| Breastfeeding duration (months): | ||||
| ≥4 | 202 (46.5) | 23 (11.4) | 153 (75.7) | -- |
| <4 | 232 (53.5) | 15 (6.5) | 182 (78.5) | 0.55 (0.27, 1.08)* |
P<0.2
P <0.05
P<0.003
Table 2. Unadjusted environmental exposure association to Allergic Rhinitis with high eosinophils (AER) compared to the combined phenotypes other than AR at age four.
A total of 434 children had complete questionnaire, SPT and nasal cytology at age four. Not shown are the allergic rhinitis without high nasal eosinophils (n=61).
| Environmental Exposure | Number (%) | Allergic Eosinophilic Rhinitis (AER) n=38/434 (8.8) | All phenotypes other than AER n=335/434 (77.2) | OR (95% CI) |
|---|---|---|---|---|
|
| ||||
| HDE (EU/mg dust)<230 | 388(89.4) | 32 (8.3) | 300 (77.3) | N.C. |
| 230-640 | 39 (9.0) | 6 (15.4) | 29 (74.4) | N.C. |
| ≥ 640 | 7 (1.6) | 0 (0) | 6 (85.7) | N.C. |
|
| ||||
| β-glucan (μg/g dust) | ||||
| <60 | 272 (62.7) | 24 (8.8) | 205 (75.4) | 1.00; (0.98, 1.02) |
| 60-170 | 116 (26.7) | 10 (8.6) | 95 (81.9) | 1.01; (0.97,1.03) |
| ≥33.12 | 46 (10.6) | 4 (8.7) | 35 (76.1) | 1.00; (0.98, 1.02) |
|
| ||||
| Fel d1 (μg/ml) <4.1 | 76 (17.5) | 3 (4.0) | 65 (85.5) | 3.38;(0.95,18.67)* |
| 4.1-148.4 | 230 (53.0) | 20 (8.7) | 179 (77.8) | 0.24; (0.04, 1.09)* |
| ≥ 148.4 | 128 (29.5) | 15 (11.7) | 91 (71.1) | 1.64; (0.92 2.93)* |
|
| ||||
| Der p1 (μg/ml) < 54.6 | 337 (77.7) | 33 (9.8) | 257 (76.3) | 1.03; (0.79, 1.34) |
| ≥ 54.6 | 97 (22.4) | 5 (5.2) | 78 (80.4) | 0.66; (0.28, 1.37) |
|
| ||||
| Can f1 (μg/ml) < 0.74 | 210 (48.4) | 18 (8.6) | 161 (76.7) | 5.60; (0.03, >999) |
| 0.74-9.03 | 96 (22.1) | 12 (12.5) | 72 (75.0) | 0.19; (<0.01,19.78) |
| 9.03-221.4 | 95 (21.9) | 7 (7.4) | 75 (79.0) | 1.15; (0.33, 4.06) |
| ≥ 221.4 | 33 (7.6) | 1 (3.0) | 27 (81.8) | 0.61; (0.28, 1.31) |
|
| ||||
| Bla g1 (μg/ml) < 0.07 | 414 (95.4) | 37 (8.9) | 321 (77.5) | 0.58; (0.16, 1.35) |
| ≥ 0.07 | 20 (4.6) | 1 (5.0) | 14 (70.0) | 1.65; (0.87, 5.34) |
|
| ||||
| Year 2 Cotinine | ||||
| (ng/mg hair) <0.11 | 301 (69.4) | 26 (8.6) | 235 (78.1) | 1.02; (0.45, 2.31) |
| 0.11-0.67 | 116 (26.7) | 12 (10.3) | 86 (74.1) | 0.98; (0.61, 1.56) |
| ≥0.67 | 17 (3.9) | 0 (0) | 14 (82.4) | N.C. |
|
| ||||
| Year 1 ECAT exposure (μg/m3) | ||||
| ≤ 0.32 | 298 (68.7) | 31 (10.4) | 226 (75.8) | -- |
| > 0.32 | 136 (31.3) | 7 (5.2) | 109 (80.2) | 0.47; (0.19, 1.04)* |
|
| ||||
| Children in home at 12 months | ||||
| ≥ 2 child | 141 (32.5) | 9 (6.4) | 113 (80.1) | 1.64 |
| < 2 child | 293 (67.5) | 29 (9.9) | 222 (75.8) | (0.78, 3.78) |
|
| ||||
| Stays in daycare-like facility for ≥ 8 hours during 1st year. | ||||
| No | 277 (64.9) | 28 (10.1) | 214 (77.3) | 0.53 |
| Yes | 150 (35.1) | 8 (5.3) | 116 (77.3) | (0.22, 1.14) |
|
| ||||
| No. colds at 12 months: | ||||
| < 7 colds | 396 (91.2) | 35 (8.8) | 307 (77.5) | 0.94 |
| ≥ 7 colds | 38 (8.8) | 3 (7.9) | 28 (73.7) | (0.22, 2.83) |
P<0.2
Adjusted analysis
Table 3 shows the adjusted odds ratio of allergen wheal area and covariates (children characteristics and environmental exposures). At age three, Penicillium (aOR 1.18; 95% CI, 1.06-1.32; p=0.002) and maple (aOR 1.07; 95% CI, 1.01-1.13; p=0.02) wheal areas were significantly associated with AER. In addition, elm showed a borderline association with AER (aOR 1.06; 95% CI, 0.98-1.14; p=0.11). In comparison to the binary (positive or negative) measure of aeroallergens, the Penicillium wheal area was more precise (smaller confidence intervals) and more significantly associated with AER. The two tree measures also were more precise (smaller confidence intervals) compared to the binary values but the significance levels were comparable.
Table 3. Adjusted odds ratio (aOR) and 95% confidence intervals of allergen wheal area at each year associated to allergic eosinophilic rhinitis at age four.
Gender and mother's education were included in age three model to improve model fit.
| Age Three | |||
|---|---|---|---|
| Allergen Wheal Area aOR; 95% CI; P-value |
Allergen Binary value aOR; 95% CI; P-value |
||
| Allergens | Elm | 1.06; (0.98, 1.14); 0.11 |
2.93; (0.68, 10.92); 0.12 |
| Maple | 1.07; (1.01, 1.13); 0.02 |
3.72; (1.11, 11.53); 0.03 |
|
| Penicillium | 1.18; (1.06, 1.32); 0.002 |
4.29; (0.99, 15.86); 0.03 |
|
These three informative allergen wheal areas at age three were summed and showed a significant linear relationship to AER (OR 1.1; 95% CI, 1.02-1.09; p=0.003). These summed wheal areas were compared to children that were negative for all three allergens. As shown in Figure 2, each percentile showed a dose dependent increased risk of AER. Children with a sum of all three allergen wheal areas in the 25th percentile, in the > 25th and < 75th and in the 75th percentile had an increased risk of AER (OR 3.6; 95% CI, 1.12-10.47; p=0.03), (OR 4.1; 95% CI, 1.78-9.01; p=0.0001) and (OR 10.7; 95% CI, 3.43-34.20; p<0.0001) respectively. Male gender (OR 2.5 95% CI, 1.07-6.44; p=0.04) and higher mother's education (OR 0.38; 95% CI, 0.16-0.87; p=0.03) were also significantly associated with AER.
Figure 2. Odds ratios of developing allergic eosinophilic rhinitis at age four by percentiles of total of important allergen wheal areas* from multivariate regression model at age three.
*Allergen wheal areas that were included in the final multivariate model at age 3 includes: Penicillium, Maple, and Elm.
Discussion
Previously, little has been reported about the etiology and significance of nasal eosinophilia in children. In a small study of twenty adolescent Italian children with perennial AR, nasal eosinophils were significantly associated with nasal total symptom score showing a significant inverse association with nasal airflow.25 A larger study of 160 Chinese preschool children with perennial AR found a significant association of nasal eosinophil grade with total nasal symptom score.26 Our study is the first to demonstrate that early aeroallergen sensitization is associated with children with AR that also have a high number of nasal eosinophils (AER) at age four in a large North American cohort. Of the 434 children in this study, 38 (8.8%) had AER.
The present study is an investigation of early aeroallergen sensitization, measured by allergen wheal area in predicting AER in high-risk children. These findings are the first to demonstrate that as early as age three, aeroallergen wheal areas are significantly associated to AER at age four. Our results indicate that especially Penicillium and maple wheal areas (with elm wheal of borderline significance) at age three are most predictive of AER. Those children with the largest sum of these three informative wheal areas had an approximate 11-fold increase in risk of AER. At ages one and two, however, after controlling for relevant covariates, no significant associations were observed with allergen wheal areas and AER at age four, indicating that in this cohort earlier aeroallergen sensitization is less informative for AER at age four. Our group has previously reported that Penicillium is the most prevalent measureable fungal species in both the indoor and outdoor environment.27 Recent evidence suggests that Penicillium is important to allergic respiratory disorders. In a murine model of asthma, Penicillium extract induced more vigorous inflammatory response in bronchoalveolar lavage fluid compared to house dust mite extract.28 In Puerto Rican inner-city children, Penicillium mold count in the child's bedroom was associated with increased frequency of asthma symptoms although no tests of sensitization were performed.29 Another study revealed that Penicillium sensitization increases with age among asthmatic children.30 More recent studies have demonstrated that the presence of Pencillium in the air was associated with infant wheezing.31 In school age asthmatic children, Penicillium sensitization and exposure increased the risk of wheezing and asthma severity score.32 Penicillium sensitization frequency in our cohort also increased at ages one (n=8), two (n=12) and three (n=25) respectively. Less is known regarding how maple allergen exposure and sensitization contributes to allergic respiratory disease. Maple is a predominant tree species and a major aeroallergen (a sensitizer in >50% of patients with springtime seasonal AR) in the greater Cincinnati and Northern Kentucky area.33 Maple pollen levels were correlated with asthma hospital admissions in Canada, and levels were positively correlated with asthma hospital admissions in Portugal.34,35 We showed in previous work that tree sensitivity at age one was associated with AR at age three.22 Our association of Penicillium and maple sensitization with AER, a possible severe AR phenotype, warrants further investigation.
The strengths of this prospective study of early childhood allergic disease are its repeated annual health interviews, physical examinations, allergen skin testing and use of eosinophils to measure AR severity. The nasal eosinophils provide an objective biomarker of a more specific phenotype of AER. This phenotype may be useful as a severe phenotype for future intervention trials especially for high risk children i.e. those born to atopic parents. The large sample size at age four allowed for investigation of less prevalent outcomes, such as AER. The use of wheal areas as a continuous measurement increased the power to detect association to AER.
As with all studies, there were limitations. The children were skin tested to relevant aeroallergens for the Greater Cincinnati metropolitan area and therefore may limit the generalizability to other regions. The importance of different sets of aeroallergen skin test results in other regions will need to be determined. Children whose parents consented to nasal scraping were more often NAA. However, when analyzing if participating children providing interpretable nasal eosinophils differed from the larger cohort, there was no significant difference in race. Also this study did not measure eosinophils at earlier ages. Therefore no assumptions can be made regarding if earlier testing might be predictive of earlier AER.
In summary, this study found that only a few age-specific allergen wheal areas were associated with age four AER. None were predictive prior to age three, but Penicillium and maple wheal areas at age three were significantly associated with AER at age four. Hence, at least as early as three years old may be an important time to begin SPT children especially from high risk families. Children with the largest sum of wheal areas for Penicillium, maple and elm at age three were at greatest risk of AER at age four. Linking the severity of early childhood allergen sensitization to a severe AR phenotype is useful for defining future clinical and study groups for longitudinal observation and intervention studies.
Supplementary Material
Table E1: Children characteristic differences among who consented to nasal epithelium scraping and those not consented.
Table E2: Environmental exposure differences among children who were consented to nasal epithelium scraping and those not consented.
Table E3- Demographic characteristic differences among children with interpretable (non-censored) nasal samples compared to those children without interpretable nasal smear information.
Table E4- Environmental exposure differences among children with interpretable (non-censored) nasal samples compared to those children without interpretable nasal smear information.
Acknowledgments
We would like to thank Stacy Langemeyer, BS who contributed in the collection, quantifying and storing the nasal eosinophils.
Supported by: National Institute of Environmental Health Services Grant ES 11170 and ES 10957; National Institute of Allergy and Infectious Disease AI 60515 T32
Footnotes
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Associated Data
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Supplementary Materials
Table E1: Children characteristic differences among who consented to nasal epithelium scraping and those not consented.
Table E2: Environmental exposure differences among children who were consented to nasal epithelium scraping and those not consented.
Table E3- Demographic characteristic differences among children with interpretable (non-censored) nasal samples compared to those children without interpretable nasal smear information.
Table E4- Environmental exposure differences among children with interpretable (non-censored) nasal samples compared to those children without interpretable nasal smear information.