Title: Validation of a device to diagnose pneumonia from coughed droplets Investigators: Introduction Questions

Transcription

1 Title: Validation of a device to diagnose pneumonia from coughed droplets Investigators: Larry J. Anderson, Emory Children s Center, Emory University Susan Ray, Grady Memorial Hospital (Grady), Emory University David Ku, Georgia Institute of Technology Introduction Pneumonia is the 7 th leading cause of death and associated with an estimated 500,000 hospitalizations each year in the United States and associated with 2 million deaths in children under 5 years of age worldwide 1. Previous studies have identified Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, influenza, parainfluenza and adenoviruses as important causes of community-acquired pneumonia 2. However, identification of etiologic agents is often compromised by factors such as patients presenting to medical care late in the clinical course, prior use of antibiotics and the low sensitivity of some diagnostic assays. Even with extensive diagnostic testing, between 20-45% of cases of lower respiratory tract infections are left without an identified etiology 2-6. This lack of diagnostic success can lead to ineffective therapy and greater use of empiric broad-spectrum antibiotic coverage with accompanying health risks to the patient and community from increased prevalence of pathogens resistant to antibiotics. New molecular diagnostic assays provide the sensitive and efficient tools to detect pathogens but accessing a specimen from the site of disease, the lung is problematic. The specimens that come from the site of disease, i.e. the lung, are invasive and costly, e.g. needle or open lung biopsy specimens or washes or aspirates obtained by bronchoscopy. Other specimens that are noninvasive and less costly such as sputum, throat swabs, and nasal pharyngeal swabs or aspirates contain contaminates from the upper respiratory tract which confound interpretation of diagnostic test results. Many pathogens can reside in the upper respiratory tract without causing disease and, consequently, their detection from these non-invasive specimens does not necessarily indicate that they are causing disease. We propose to evaluate a novel cough specimen collection device to detect pathogens in patients with a clinical diagnosis of pneumonia. The devise is designed to collect droplets and aerosols generated from the lung on a filter while minimizing the chance that upper respiratory tract secretions contaminate the specimen. Genomic material is then extracted from the filter and detected by polymerase chain reaction (PCR) assays. The device is handheld and users simply cough through a mouthpiece connected to a tube. In studies to date, we found that our first device design was inefficient in capturing coughed droplets. With that device, coughed droplets were likely to adhere to surfaces on the device before they reached the filter, resulting in a decreased chance of detecting pathogens. We have developed two redesigned devices that address this problem. The devices are described below. These devices are now ready for clinical evaluation. We also hypothesize that there is substantial variability in the amount of pathogen present in droplets generated by coughs. This proposal requests funding to determine which device and what methods for collecting coughed specimens provide the best balance of sensitivity and specificity. These data will be used as preliminary data, ~proof of principle, to support proposals for a comprehensive clinical evaluation of this approach to diagnosing pneumonia. Given the importance of pneumonia morbidity and mortality worldwide and the lack of good diagnostic tools for the many cases, data that suggest our approach will improve pneumonia diagnosis should generate substantial interest in funding studies to fully characterize the device at the NIH, the Biomedical Advanced Research and Development (BARDA) program, or other funding sources. Questions

2 Specific Aim 1: To determine if cough induction, e.g. inhalation of hypertonic saline, will increase the quantity of pathogens captured on the filter. We hypothesize that inducing coughs will increase the number and quality (i.e. amount of pathogen contained in droplets)of droplets coughed that can be captured on the filters. One coughed specimen, i.e. up to 10 coughs into device A (Figure 1), will be collected before and one coughed specimen collected after cough induction and nucleic acids extracted from the two filters and microbial nucleic acids detected by real time PCR. We will compare positive and negative results, and cycle threshold (CT) values for the various RT PCR assays between specimens collected with and those collected without cough induction. The results of testing on other specimens such as sputum and swabs, the history and physical examination information, and other clinical and laboratory findings will be used to determine the likely etiologic diagnosis of pneumonia and this diagnosis compared with pathogens detected on the filter. Specific Aim 2: To determine which of the two optimized devices provides the best sensitivity and specificity profile for detecting pathogens from pneumonia patients. One of the two devices is designed to divert dead space air (i.e. the first part of a cough that includes air in the mouth and trachea) from the filter and the other does not. We hypothesize that in the process of diverting the dead space air from the filter, turbulence is generated that increases the chance that droplets may stick to the sides of the tube or entrance to the balloon reducing the amount captured on the filter, i.e. decrease sensitivity. It is also possible that not diverting the upper respiratory air will increase risk of capturing mouth flora on the filter. We will compare the two devices head-to-head in the same patient and use real time PCR to detect pathogens and contaminating bacteria on the filter. Coughed specimens, or induced coughed specimens if that provides a better sample, will be collected using both devices in the same patient, the filters collected, and the nucleic acids extracted from both filters. The extracted nucleic acids will be tested by real time PCR assays for viral and bacterial pathogens, human DNA, and commensal bacteria present in mouth and throat specimens. Positive and negative results and CT values for the various pathogens and commensal bacteria RT-PCR assays will be compared for the two devices. We will use this comparison to determine which device is best able to detect pathogen nucleic acids with the lowest background signal, i.e. RT-PCR for the commensal bacteria, on the filter. Study Methodology (the study protocol has been reviewed and approved by IRBs at CDC, Emory and Georgia Tech) Patients: Patients admitted to Grady Memorial Hospital with physician diagnosed pneumonia will be candidates for study. All candidate patients who can provide specimens within 48 hours of admission and who, after being apprised of the study and risks and benefits of participation, consent will be entered into the study. Data Collection: Study patients will be administered a brief questionnaire for demographics and some clinical information. This data, as well as clinical data, laboratory test results, and X-ray results obtained during their hospitalization will be entered into a clinical database (Section IV-V in VA-CDC Data Collection Form or Grady-CDC Data Collection Form) that will be linked to the laboratory data. This data will be used to assess the likelihood the patient had pneumonia and identify any pathogens linked to the illness. Specimen Collection: Participants will be asked to provide coughed specimens as well as minimalrisk additional specimens to support interpretation of results from the cough specimen collection devices. These specimens will be collected by trained health professionals as usually done for patient evaluation and will include: 1) Nasopharyngeal and oropharyngeal swabs (one swab from each site) 2) One expectorated sputum

3 3) One acute and one convalescent sera serum spun from approximately 10cc of blood collected at admission (acute-phase) and a second serum collected 2-3-weeks later (convalescent-phase) (if available). 4) One urine collected at the time of admission and one collected 2-3 weeks after admission (if available) 5) The coughed specimens The cough specimens will be collected using non-invasive handheld devices developed and provided by collaborators at Georgia Tech. Two device designs ( A and B ) will be used and evaluated in terms of sensitivity and specificity. Device A is handheld and users cough through a mouthpiece. The mouthpiece segregates oral contaminants and lung aerosols are collected on a filter. There is no bag involved with the mechanism of Device A (Figure 1). Device B is handheld and users cough through a mouthpiece. Contents from the upper airway first flow into a bag. When the bag is full, air and the associated contents from the lower airway, then flow through a filter. The droplets that contain pathogens and other molecules from the lower respiratory tract are collected on the filter. Fluid mechanics have used to create the pressure to segregate the upper and lower airways samples (Figure 2). Fig. 1 Fig 2 Each patient will be asked to either 1) cough up to 10 times into Device A and then up to 10 times into Device A after cough induction or 2) cough up to 10 times into Device A and then up to 10 times into Device B. For the second comparison, both coughs will be done after cough induction if cough induction provides a better quality sample. The device with the filters will be individually bagged and refrigerated at 4 C or frozen at -70 C until transport to CDC or Emory Children s center for laboratory testing. Laboratory Studies: The filters from each device will be removed under sterile conditions stored at - 70 o C until testing. The sputum, throat swab and urine specimens will be divided into aliquots and stored at 70 o C until testing. The specimens will be batch tested and results will not be used for managing patients. The specimen to be tested will be thawed and nucleic acid will be extracted using validated total nucleic acid extraction kits (Quiagen Inc, Valencia, CA). Initial testing will on sputum (or throat swab, if the sputum is unavailable) specimens using the TLDA card assay as described below to identify bacterial and viral pathogens potentially associated with the illness. If a potential viral or bacterial pathogen is found, the filter will be tested with the corresponding single PCR assay. The TLDA card assay makes it possible to perform individual real time PCR assays for up to 20 different pathogens and controls efficiently 7. In addition, the nucleic acid extract from each filter will be tested by Real Time-PCR for 16s bacterial ribosomal DNA, human RNP3 DNA, and a

4 bacterial commensal. These assays will provide information on the quality of the coughed specimen and an indication of the presence of contaminating material from the upper respiratory tract. We will also perform individual PCR assays for other pathogens, e.g. Mycobacterium tuberculosis, if indicated by clinical and laboratory findings from patient s medical record. Briefly, the RT-PCR assays will be performed on the Applied Biosystems 7900HT real-time PCR platform in a 96-well format using the AgPath-IDTM 123 One-Step Kit (Applied Biosystems, Foster City, CA) PCR reaction/enzyme reagents. The TaqMan Low Density Array (TLDA) cards (Applied Biosystems,135 Foster City, CA) are 384-well microfluidic cards with eight ports which each contain 48 connected wells. Primers and probe for each assay are preloaded and dried onto the designated duplicate wells. Samples are loaded through the 8 ports and the card is centrifuged twice at 336 g for 1 min to distribute the sample to all 48 connected wells. The card is then sealed and placed in the thermal cycler. The respiratory pathogen TLDA card is designed to run seven samples and a negative control sample on one card and test for the following pathogens: influenza A, influenza A- H1, influenza A-H3, influenza B, respiratory syncytial virus, human metapneumovirus, human parainfluenza viruses 1-3, rhinoviruses, enteroviruses, parechoviruses, adenoviruses, Legionella pan-species, Legionella spp./legionella pneumophila, Haemophilus influenza, Streptococcus pneumonia, Streptococcus pyogenes, Mycoplasma pneumonia, Chlamydophila pneumonia, Bordetella pertussis target I, and Bordetella pertussis target II. Data Analysis: The accuracy of the diagnosis of pneumonia and the quality of the specimen collected will be assessed for each patient. We will analyze results for all patients but will focus our assessment of sensitivity and specificity of the devices on results from patients with a discharge diagnosis of pneumonia and a good quality specimen (the patient was able to provide a good cough and the specimen was collected within 48 hours of admission and less than 48 hours after initiation of antibiotic treatment). The results of testing extracted nucleic acids from the filter including pathogen RNA or DNA, commensal bacteria DNA, and host DNA will be compared with other information on likely etiology of the pneumonia, and compared to results of testing on sputum and other throat swab specimens. We will use the ratio of PCR signal for pathogens to commensal bacteria on the filter compared to sputum and throat swab specimens to determine if a pathogen detected on the filter is likely from oral contamination or the lung. We expect that the ratio of the CT value for pathogen to that for the commensal bacteria will be significantly higher on the filter than for the throat swab specimen, if the pathogen detected on the filter comes from the lung. Anticipated results: We anticipate that the revisions to the devices and addition of cough induction will improve the consistency with which we can detect pathogens on the filter. In studies to date, we have results on 9 coughed specimens with device B that met our criteria for evaluable specimens, i.e. discharge diagnosis of pneumonia, good coughs into the device, and specimen collected within 48 hours of administration antibiotics and hospital admission. These data suggest the device is close to being able to improve our ability to diagnose pneumonia. One of 9 was positive for a pathogen, rhinovirus, and one was positive for RNP3 human DNA indicating droplets were captured on the filter. The etiology of all cases was uncertain and all received antibiotics between 10 and 48 hours before the coughed specimen was obtained. The coughed specimen was collected between 24 and 48 hours after antibiotic administration for 4 patients. We anticipate that inducing coughs and the redesigned devices will make it possible to efficiently capture a high percentage of coughed droplets and improve our ability to diagnose the etiology of pneumonia by 25% to 50%. Budget: $12, Study coordinator to enroll and consent patients and collect data and specimens at Grady Memorial Hospital. This will support enrollment of ~32 study patients, 16 for the assessing the value of cough induction and 16 for comparing the two devices.

5 $ Cough induction by respiratory therapy for ~32 study patients. $ Reagents for the nucleic acid extraction and RT PCR reagents for the coughed, sputum and throat swab specimens. The real time PCR assays will include those for pathogens, the commensal bacteria, and RNP3 human DNA for the filter specimen, throat swabs, and sputum specimens from the study patients. $ Fabrication of the devices to be used in the study External sponsorship: The proposal studies will guide direction for clinical studies to determine the sensitivity and specificity of this approach to diagnose the etiology of pneumonia. It will help determine the type of device, method of specimen collection, and clinical setting and type of patient most likely to benefit from the method. It also will provide the preliminary data need to submit proposals for the next level of clinical evaluation. Based on these studies we will submit proposals for clinical evaluation of the optimized approach to NIAID and BARDA this spring. Given the morbidity and mortality associated with pneumonia and the need for better diagnostics, data that demonstrate the potential value of this approach should make future studies attractive to the funding agencies. References 1. Pinner RW, Teusch SM, Simonsen L, Klug LA, Graber JM, Clarke MJ, Berkelman RL. Trends in infectious diseases mortality in the United States. JAMA 1996;275: Bartlett JG, Breiman RF, Mandell LA, File TM, Jr. Community-acquired pneumonia in adults: guidelines for management. The Infectious Diseases Society of America. Clin Infect Dis 1998;26: Woodhead MA, Macfarlane JT, McCracken JS, Rose DH, Finch RG. Prospective study of the aetiology and outcome of pneumonia in the community. Lancet 1987;1: Porath A, Schlaeffer F, Lieberman D. The epidemiology of community-acquired pneumonia among hospitalized adults. J Infect 1997;34: File TM, Jr., Tan JS. Incidence, etiologic pathogens, and diagnostic testing of communityacquired pneumonia. Curr Opin Pulm Med 1997;3: Bochud PY, Moser F, Erard P, Verdon F, Studer JP, Villard G, Cosendai A, Cotting M, Heim F, Tissot J, Strub Y, Pazeller M, Saghafi L, Wenger A, Germann D, Matter L, Bille J, Pfister L, Francioli P. Community-acquired pneumonia. A prospective outpatient study. Medicine (Baltimore) 2001;80: Kodani M, Yang G, Conklin LM, Travis TC, Whitney CG, Anderson LJ, Schrag SJ, Taylor TH, Jr., Beall BW, Breiman RF, Feikin DR, Njenga MK, Mayer LW, Oberste MS, Tondella ML, Winchell JM, Lindstrom SL, Erdman DD, Fields BS. Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens. J Clin Microbiol 2011;49: