Diagnosing lung infections is often challenging because of the lack of a high-quality specimen from the diseased lung. Since persons with cystic fibrosis are subject to chronic lung infection, there is frequently a need for a lung specimen. In this small, proof of principle study, we determined that PneumoniaCheckTM, a non-invasive device that captures coughed droplets from the lung on a filter, might help meet this need. We obtained 10 PneumoniaCheckTM coughed specimens and 2 sputum specimens from adult CF patients hospitalized with an exacerbation of their illness. We detected amylase (upper respiratory tract) with an enzymatic assay, surfactant A (lower respiratory tract) with an immunoassay, pathogenic bacteria by PCR, and markers of inflammation by a Luminex multiplex immunoassay. The amylase and surfactant A levels suggested that 9/10 coughed specimens were from lower respiratory tract with minimal upper respiratory contamination. The PCR assays detected pathogenic bacteria in 7 of 9 specimens and multiplex Luminex assay detected a variety of cytokines or chemokines. These data indicate that the PneumoniaCheckTM coughed specimens can capture good quality lower respiratory tract specimens that have the potential to help in diagnosis, management and understanding of CF exacerbations and other lung disease.

Diagnosing lung infections is often challenging because of the lack of a high-quality specimen from the diseased lung as illustrated by lack of a specific etiologic diagnosis in about 50% of pneumonia cases [1–3]. Persons with cystic fibrosis (CF) are subject to chronic lung infection and the associated damage to the lung is a leading cause of morbidity and mortality [4]. Common pathogens often found in CF patients include Pseudomonas aeruginosa, Staphylococcus aureus, the Burkholderia cepacian complex, Achromobacter species, and Haemophilus influenzae. With more recent comprehensive test methods the picture has become more complex with demonstration of individually distinct microbial communities [5]. Exacerbations associated with infection is an important contributor to progression of the lung disease in CF [6]. A high-quality lung specimen could provide important information on both the extent of inflammation or disease activity indicative of exacerbations and identification of the associated pathogen or pathogens. This information could help in understanding and managing exacerbations of CF lung disease.

Sputum specimens are commonly used for lung specimen but are contaminated with upper respiratory tract secretions. Additionally with highly effective CFTR modulator therapy it has become less common for patients to be able to produce sputum specimens [7]. Oropharyngeal swabs specimens can be obtained but do not offer information on inflammation or pathogens in the lung, and have not been validated for use in managing adult CF patients [8]. A variety of other methods to get lung specimens have been used including bronchiolar alveolar lavage but it is invasive, not available in all settings, costly, and includes increased risk to the patient compared to non-invasive methods [9, 10]. Exhaled breath specimens, including a recently described device that passes exhaled breath through water, have also been used to obtain specimens from the lung [11–13] In this study, we report a proof of principle study of a device (PneumoniaCheckTM ) that captures coughed droplets from the lung [14–16], to detect both bacterial pathogens and biomarkers from the lung.

Patients. The study was reviewed and approved by the Vanderbilt IRB. Adult CF patients admitted to Vanderbilt University Adult Hospital who had not undergone lung transplantation were eligible for enrollment and those who chose to participate and provided informed consent were enrolled.

Specimen Collection. Cough droplet samples were collected using the PneumoniaCheckTM device (MD Innovate, Inc., Decatur, GA) (figure 1). Patients were instructed to breathe deeply and cough into the device, continue to end expiration to exhaust the inhaled breath, and keep a tight seal around the mouthpiece. Each participant attempted 5 rounds of 10 coughs at a time (50 coughs total) at 20–30 min intervals.

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Specimen Processing. The cough device was kept at 4 °C between collections and during transport to the laboratory where it was stored at −80 °C until shipment to the testing laboratory. The specimens were shipped on dry ice with a specimen identifier but no accompanying patient information. On arrival, the devices were stored at −80 °C until tested. For testing, the devices were acclimatized to room temperature for 30 min in a Class II Biological Safety Cabinet using BSL2 biologic safety precautions, the filter removed and placed in 300 ul of the dilution buffer for the cytokine/chemokine Luminex assay described below in an Eppendorf tube (Eppendorf, Germany). The tube was vortexed for 5 min. To collect residual fluid in the membrane, the membrane was transferred to a Spin-X column, (Corning, New York) centrifuged at 10 000 rpm for 10 min, the centrifugate fluid was removed from the Spin-X column and placed in the original Eppendorf tube. The resultant specimen was divided into aliquots and stored at −80 °C for subsequent study.

DNA Extraction. After thawing, DNA was extracted from 100 ul of the stored specimen using the magnetic bead-based Kingfisher Pure DNA Blood Kit (Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions on an automated KingFisher Duo system (Thermo Fisher Scientific, Inc., USA).

Real Time PCR. Previously published oligonucleotide sequences [14, 17, 18] were used to detect S. aureus (LOD 10 fg), H. influenzae (LOD 5 fg), P. aeruginosa (LOD 100 fg), Streptococcus pneumoniae (LOD 100 fg), and Streptococcus mitis (LOD 50 fg). The individual pathogen-specific real time PCRs were performed in duplicate on the Applied Biosystems 7500 (Applied Biosystems, Foster City, CA ) platform in a 96-well format using the TaqMan™ Gene Expression Master Mix (Cat no 4370048, Thermo-Fisher Inc, USA) with 20 ul PCR mix containing 250 nM of the forward and reverse primers and 100 nM of Taqman probe plus 10 ul of PCR master mix 5 ul of CF specimen DNA, and nuclease free water. The PCR thermal cycling conditions were UNG incubation at 50 °C for 2 min, AmpliTaq Gold®, UP enzyme activation at 95 °C for 10 min, and 45 cycles of denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 1 min. The cycle threshold (CT) for a positive test was determined and the average of duplicate values used for analysis.

Biomarker detection. Cytokines and chemokines were detected with a 25-plex human cytokine/chemokine kit (Invitrogen/Fisher Scientific/Life Technologies, Carlsbad, CA) according to the manufacturer's instructions.

Amylase. Amylase was determined by with the enzyme activity assay, Amylase Activity Assay (MilliporeSigma, Burlington, MA) according to the manufacturer's instructions.

Surfactant A. Surfactant A was detected with the Surfactant Protein A (SP-A) ELISA Kit (Antibodies-online Inc., Limerick, PA) according to the manufacturer's instructions with modifications to account for smaller sample volumes.

Ten CF patients were enrolled, consented, and had cough specimens collected. All patients had been admitted to the hospital for a CF exacerbation. The study subjects were 19–54 years of age (median age of 28) and 6 (60%) were female. Two study subjects also provided sputum specimens. The surfactant A and amylase test results support the pulmonary origin of the captured droplets with minimal upper respiratory tract contamination (table 1) for all but one patient. Amylase coming from saliva indicates contamination with upper respiratory tract secretions while surfactant A secreted by lung cells indicates lower respiratory tract secretions. The one patient, patient 7, had an amylase level of 8455 mU/ml and ratio of 640. The amylase levels for the other patient cough specimens were zero or low giving a ratio of amylase/surfactant protein A <50. In comparison, the two sputum specimens had amylase levels and amylase/surfactant protein A ratios over 100 times higher than the coughed specimens except for patient 7. Note that the surfactant protein A level was at the lowest limit of detection for most cough specimens.

Table 1. PCR CT values and surfactant A and Amylase levels.

 CT valuePg/mlmU/mlRatioPt#-SpecS. aurPse. aS. pneS. mitSurAAmyAmy/SurA1-PneuCkNegNegNegNeg2.3Neg 2-PneuCkNeg38.7Neg26.02.3Neg 3-PneuCk37.237.9NegNeg8.028035.084-PneuCkNeg37.0NegNeg2.6Neg 5-PneuCkNeg38.642.027.72.32410.386-PneuCkNeg37.1Neg21.12.220.937-PneuCkNeg30.1NegNeg13.28455640.038-PneuCkNegNegNeg30.72.32711.649-PneuCkNegNegNeg25.92.3Neg 9-SputumNeg27.6Neg42.0256.71727 2576728.0710-SputumNeg36.935.0Neg230.9310 5781344.98

Pt# = patient number; Spec = Specimen type; PneuCk = Coughed PneumoniaCheckTM specimen; S.aur = Staphylococcus aureus; Pse.a = Pseudomonas aeruginosa; S.pne = S. pneumoniae; S.mit = Streptococcus mitis; SurA = surfactant protein A; Amy = amylase; Neg = below limit of detection; mU/ml = milliunits of amylase activity per milliliter.

Bacterial DNA from 1 to 3 species was detected in all specimens except for patient 1. The CT values were relatively high indicating a relatively small amount of bacterial DNA captured in the coughed specimens. Note that the pattern of detected DNA was different between the coughed specimen and sputum specimen for patient 9 suggesting a different specimen source. Presumably the coughed specimen came primarily from the lung while the sputum specimen was heavily contaminated by upper respiratory tract secretions.

We next sought to determine if the biomarkers potentially indicative of disease in the lung could be detected in the PneumoniaCheckTM specimen from CF patients hospitalized with exacerbations. Our results demonstrate that the device captured coughed specimens do contain detectable level of cytokines and chemokines with distinct patterns among the specimens (table 2) including IL-8 and TNF-a in some patients. IL-8 and TNF-a are two of the proinflammatory cytokines associated with exacerbations of CF disease [19]. Note that table 2 gives the subset of cytokines and chemokines which had levels greater than the lower limit of quantitation for three or more of the 12 specimens.

Table 2. Cytokines and chemokines in coughed specimens.

Pt#-SpecIL-1RAIL-2RIL-7IL-8IL-15TNF-aMIP-1bIP-10MIGMCP-11-PneuCk1845.5568.41144.24*17.18114.05207.8151.44321.75104.4876.912-PneuCk294.18Neg185.3131.36Neg25.54*14.63171.81*16.09*7.643-PneuCk*203.17Neg63.43*0.60Neg18.37*11.3023.15NegNeg4-PneuCk2062.7162.74NegNeg119.02536.6736.57270.72302.2488.945-PneuCk1656.73115.83NegNeg93.72371.0824.25201.66225.1573.816-PneuCk979.43Neg*35.65*3.23*31.9840.7930.47104.02Neg*23.357-PneuCk1252.74Neg163.782405.72Neg33.03*7.88161.99*8.20181.228-PneuCk*146.10Neg144.2459.22Neg18.37*11.30155.48Neg*18.669-PneuCk294.18Neg110.56*22.04Neg25.54*7.88129.65Neg*7.6410-PneuCk**108 084*5.38*43.58**10 451Neg33.0363.06116.8229.71595.729-Sputum**108 084*53.66125.06**10 451*18.7582.7136.57168.53200.88738.810-Sputum**108 08474.06104**10 451*25.531972.0736.57142.5241.95645.14

Pt# = patient number; Spec = Specimen type; PneuCk = Coughed PneumoniaCheckTM specimen; Values are pg/ml*=below limit of quantitation; **=above the limit of quantitationIL-1RA = interleukin-1 receptor antagonist protein; IL-2R = interleukin-2 receptor; IL-7 = interleukin 7; TNF-a = Tumor necrosis factor alpha; MIP-1b = Macrophage Inflammatory Protein 1b; IP-10 = Interferon gamma-induced protein 10 (CXCL10); MIG = monokine induced by gamma interferon (CXCL9); MCP-1 = Monocyte chemoattractant protein-1 (CCL2).

This study shows that PneumoniaCheckTM collected specimens may be of help in understanding and managing CF disease and other lung diseases such as bronchiectasis, chronic obstructive pulmonary disease, and pneumonia. The amylase and surfactant protein A levels support the conclusion that most of the coughed specimens were primarily from the lung with little upper respiratory contamination indicating that the detected bacterial DNA is likely from the lung. Though we and others have previously demonstrated detection of bacterial DNA and viral RNA with this device, it has not previously been shown that cytokines and chemokines can be detected with PneumoniaCheckTM [14, 16]. This study demonstrates that cytokines and chemokines and other biomarkers can be detected in PneumoniaCheckTM specimens, which indicates an additional use for the PneumoniaCheckTM collected specimens, i.e. to inform the state of disease activity in the lung of CF and other lung diseases.

Other methods to obtain specimens from the CF lung such as sputum, broncho-alveolar lavage, and exhaled breath specimens have also been described [9–13]. The advantages of the PneumoniaCheckTm device for collecting lung specimens is that it is non-invasive, easy for most patients to use, amenable to use in the home, and of low cost. This is of particular pertinence in the current era of widely available highly effective CFTR modulator therapies. These therapies improve mucociliary clearance and many patients so treated can no longer easily produce the sputum often used to guide management [7]. The PneumoniaCheckTm specimen does not rely on sputum production and, thus, should be able to provide a high-quality lung specimen in patients on CFTR modulator therapy. This study demonstrates the potential value of this coughed specimen, but further study is needed to address the study's limitations which include small sample size, too few sputum and upper respiratory tract specimens for comparison, and a surfactant A assay that is borderline sensitive enough for this coughed specimen. Additionally, the high PCR CT values, i.e. >37 for most of the PneumoniaCheckTm Staphylococcus aureus and Pseudomonas aeruginosa positive specimens, are near the limit of detection which raise the possibility that clinically important bacteria may be missed. Increasing quantity of specimen captured by the device or sensitivity of the PCRs would help ensure clinically important bacteria are not missed.

In summary, the data from this limited study suggest that PneumoniaCheckTM can provide non-invasive specimens helpful for understanding CF and other lung diseases and its use merits further study.

All data that support the findings of this study are included within the article (and any supplementary files).

Drs Ku and Anderson (through Centers for Disease Control and Prevention, Atlanta, GA) are co-inventors on a Georgia Institute of Technology, Atlanta, GA on a patent on PneumoniaCheckTM.

This study was performed in accordance with the Nurenberg Code. This human study was approved by Vanderbilt University IRB—approval: IRB #171384.