Patent Publication Number: US-2021177304-A1

Title: Systems and methods for detecting infections

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority under 35 U.S.C. § 119(e)(1) from U.S. Provisional Application Ser. No. 62/947,124, filed Dec. 12, 2019, the contents of which are incorporated herein by reference. 
    
    
     FIELD 
     This disclosure is related to systems and methods for collecting breath samples for use in the detection of urease respiratory colonizations and infections. 
     BACKGROUND 
     Early detection of whether a patient has a respiratory infection is important in providing suitable medical treatment for the patient and producing acceptable health outcomes. Respiratory infections not properly treated in a timely fashion can cause significant increases in length and cost of care as well as increased morbidity. Patients with community acquired and hospital associated pneumonia can suffer from both colonization and infection by virulent urease pathogens. Urease pathogens are actors in 5-15% of pneumonia patients entering hospital emergency rooms. Medical treatment for these pathogens commonly involves the use of broad spectrum antibiotics and hospital admission for observation of the resolution of the infection under antibiotic therapy. While this is appropriate treatment for 5-15% of these patients, the remaining 85-95% may unnecessarily receive exposure to broad spectrum antibiotics, a public health issue, and costly treatment in hospitals. Effectively identifying those patients who do not need broad spectrum antibiotics and hospitalization will relieve a burden on public health and patient welfare. 
     U.S. Pat. No. 9,518,972 describes methods of detecting bacterial infections by measuring  13 CO 2 / 12 CO 2  isotopic ratios of gaseous carbon dioxide in exhaled breath samples of a subject after administration of a  13 C-isotopically-labeled compound that is metabolized by the urease pathogens. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  shows a system according to one embodiment described herein. 
         FIG. 1B  shows another view of the system of  FIG. 1A . 
         FIG. 1C  shows another view of the system of  FIG. 1A  in which the sample collector is coupled to a nebulizer handset. 
         FIG. 2  shows the sample collector of the system of  FIG. 1A  connected to tubing. 
         FIG. 3  shows a top cross-sectional view of the sample collector of  FIG. 2  and an inlet valve disposed in the inlet of the sample collector. 
         FIG. 4  shows a perspective view of the sample collector of  FIG. 2 . 
         FIG. 5  shows an end view of the sample collector of  FIG. 2 . 
         FIG. 6  shows a side view of the sample collector of  FIG. 2  coupled to a nebulizer handset. 
         FIG. 7  shows a perspective view of the sample collector of  FIG. 2  coupled to the nebulizer handset. 
         FIG. 8  shows a schematic view of a system according to one embodiment, including an analyzer, a sample collector and a mouthpiece assembly. 
         FIG. 9  shows a schematic view of a system according to another embodiment. 
         FIG. 10  is a flow diagram illustrating a method of collecting a breath sample, according to one embodiment. 
     
    
    
     DETAILED DESCRIPTION 
     This description of preferred embodiments is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description of this invention. The drawing figures are not necessarily to scale and certain features of the invention may be shown exaggerated in scale or in somewhat schematic form in the interest of clarity and conciseness. In the description, relative terms such as “horizontal,” “vertical,” “up,” “down,” “top,” and “bottom” as well as derivatives thereof (e.g., “horizontally,” “downwardly,” “upwardly,” etc.) should be construed to refer to the orientation as then described or as shown in the drawing figure under discussion. These relative terms are for convenience of description and normally are not intended to require a particular orientation. Terms including “inwardly” versus “outwardly,” “longitudinal” versus “lateral” and the like are to be interpreted relative to one another or relative to an axis of elongation, or an axis or center of rotation, as appropriate. Terms concerning attachments, coupling and the like, such as “connected” and “interconnected,” refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise. 
     As used herein, use of a singular article such as “a,” “an” and “the” is not intended to exclude pluralities of the article&#39;s object unless the context clearly and unambiguously dictates otherwise. As used herein, the term “fluid” is used to describe the contents expelled by a subject during breathing and includes, predominantly, breath gases, but can also include liquids. Terms such as “fluidly connected” and “fluidly coupled” refer to a connection in which breath gases and/or liquids can be transferred between the connected or coupled components. 
     It can be difficult to detect respiratory infections prior to a subject becoming highly symptomatic. This can present problems in the timely treatment and care of such subjects. The systems and methods described herein overcome these difficulties and allow for the collection of breath samples from subjects for analysis of breath gases for a test marker (e.g.,  13 CO 2 ) indicating the presence of pathogens associated with infection and other health conditions. For example, the systems and methods described herein allow for the early detection of respiratory and other urease pathogen infections in pre-symptomatic and symptomatic patients and assessment of the presence and level of putative urease pathogens in the patient&#39;s respiratory system. Elevated levels of such pathogens can be associated with community acquired pneumonia (“CAP”), hospital acquired pneumonia (“HAP”), or ventilator associated pneumonia (“VAP”). Although the systems and methods described herein are well-suited to the detection of pneumonia, it should be understood that these systems and methods can additionally and/or alternatively be used to detect other infections, such as tuberculosis, cystic fibrosis, and others. 
     In various embodiments, the systems and methods described herein are configured for the collection of breath samples to allow for the detection of respiratory and systemic infections in a subject (e.g., a human patient) in conjunction with delivery of a drug to the subject. The drug may be configured to be metabolized by putative urease pathogens colonizing and/or infecting the subject. The metabolism of the drug by the putative urease pathogens produces elevations in the abundance of  13 CO 2  in the patient&#39;s breath samples. In various embodiments, the described systems and methods involve collection of one or more baseline breath sample before introduction of the drug into the subject&#39;s respiratory airway, as well as one or more breath samples collected a selected period or periods after the completion of the drug delivery. Comparing the abundance of  13 CO 2  in the post-administration sample(s) to the abundance of  13 CO 2  in the baseline sample(s) allows for the detection of urea metabolizing infections of interest. 
     As described, breath samples are measured for changes in the abundance ratio of  13 CO 2  reflective of the metabolism of  13 C urea by the urease pathogens of specific clinical interest in pneumonia patients (CAP, HAP, VAP). Two breath samples are collected for measurement and comparison in the  13 C urea breath test. The first breath sample is collected and measured to establish the baseline  13 CO 2  abundance. A second sample is collected after delivery of  13 C urea into the patient&#39;s respiratory tract. The change in breath  13 CO 2  abundance between the baseline sample and the post exposure sample reflects the presence of urease pathogens present in either colonizations or infections. Accurate measurement of  13 CO 2  abundance changes related to lower respiratory tract colonization or infection requires that breath samples represent  13 CO 2  changes at or near the anatomical location of interest (e.g., lower respiratory tract), and not be confounded by signals that may arise in other areas of the respiratory tract. In particular,  13 CO 2  signal produced by urease pathogens in the mouth and upper respiratory tract can be particularly problematic in making accurate measurements of changes in the lower respiratory tract. The breath collection systems described herein are configured to reduce or eliminate the amount of gases originating in the mouth or upper respiratory tract from the analyzed breath sample. 
     As described above, the methods described herein include administering to the subject a urea drug that includes an effective amount of a  13 C-isotopically-labeled compound that produces  13 CO 2  upon bacterial metabolism. Administration of the  13 C-isotopically-labeled compound can be achieved by any appropriate means. For example, in one embodiment, the compound is administered via a nebulizer (e.g., a mesh nebulizer or a jet nebulizer). The  13 C urea marker may also be delivered by dry powder inhaler, DPI, or metered dose inhaler (“MDI”). 
     Compositions for oral administration or inhalation of the  13 C urea drug can be in any appropriate form. Oral compositions can include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be used. Compositions for pulmonary administration may include a pharmaceutically acceptable carrier, additive or excipient, as well as a propellant and optionally, a solvent and/or a dispersant to facilitate pulmonary delivery to the subject. Sterile compositions for injection can be prepared according to methods known in the art. 
     The  13 C urea drug can be, for example, inhaled by the patient using a nebulizer affitted to a nebulizer handset, mouthpiece or mask. When the patient inhales from the nebulizer, breath is conducted into the patient&#39;s respiratory tract by normal breathing, and the drug is distributed to all parts of the respiratory tract. The presence of urease pathogens—that can be present in respiratory tract colonizations and infections—is not limited to the lower respiratory tract. These pathogens can also be found in the upper respiratory tract and mouth. The presence of such pathogens in the upper respiratory tract and mouth does not have the same clinical import as the presence of pathogens in the lower respiratory tract. For example, the patient can have active mouth colonization of urease pathogens that does not correlate to a lower respiratory tract infection. As a result, the metabolism of  13 C urea by urease pathogens in the mouth can produce a confounding quantity of  13 CO 2  that prevents a breath sample from providing a reliable indication of lower respiratory tract infections. As described in further detail herein, in order to more accurately understand the colonization and or infection of the lower respiratory tract, the lower respiratory tract sample may be separated, or fractionated, from the sample that originates in the mouth and upper respiratory tract. Fractionation of the exhaled breath to collect a more representative lower respiratory tract sample reduces potential confounding mouth and upper respiratory signals. Doing so produces  13 C urea breath tests that more clearly represent the presence of urease pathogens in the lower respiratory tract. 
     Any bacteria that can convert the  13 C-isotopically-labeled compound administered to the subject into  13 CO 2  can be detected using the systems and methods described herein. Examples of such bacteria include  Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium tuberculosis, Acenitobacter baumannii, Klebsiella pneumonia, Francisella tularenis, Proteus mirabilis, Aspergillus species , and  Clostridium difficile.    
     The detection apparatus for analyzing the breath samples can include near infrared diode lasers to attain field portable, battery operated δ 13 CO 2  measurement instruments with high degrees of accuracy and sensitivity. These devices and the methodologies which employ them may be used to determine δ 13 CO 2  in exhaled breath samples of subjects having, or suspected of having, a bacterial infection. The analyzer can include features and analyze the sample as described in U.S. Pat. No. 9,518,972, which is incorporated herein by reference in its entirety. 
     This disclosure provides devices and methods for collecting breath samples from subjects such that only a portion of the breath sample is retained and analyzed. Such devices and methods can be used, for example, to preferably retain portions of breath sample that originate in the lower respiratory tract and discard portions that originate in the upper respiratory tract and mouth. The devices and method for collecting breath samples may be used, for example, in subjects in which community acquired or hospital acquired pneumonia is suspected. As described further herein, the volume of breath sample retained can be selected to retain the desired portion of the subject&#39;s exhaled breath gases. 
       FIGS. 1A, 1B, 1C, and 2  show a system  100  for collecting and analyzing breath samples of a subject. The system  100  includes an analyzer  102  and a sample collector  104 . As shown, the analyzer  102  and the sample collector  104  are fluidly connected by tubing  106 . The analyzer  102  can include a spectrometer. For example, the analyzer  102  can be an AVISAR™ spectrometer distributed by Avisa Pharmaceuticals, Inc. The tubing  106  can be, for example, PVC tubing with a ⅛″ inner diameter. The analyzer  102  can include a biologic filter  103  to which the tubing  106  connects. The filter  103  can be configured, for example, to prevent biologic microparticulate from entering the analyzer  102 . As shown in  FIG. 1C , and described in more detail below, the sample collector  104  can be configured to couple to an exit port  122  of a mouthpiece  124  coupled to a nebulizer handset  121 . 
     As shown in  FIGS. 3-5 , the sample collector  104  includes a body  108  defining a breath collection reservoir chamber  110 . As will be described further herein, the reservoir chamber  110  is configured to receive a breath sample of the subject. The body  108  can be constructed of any appropriate material, such as, for example, polypropylene or other polymer. In other embodiments, the sample collector  104  is constructed from Tedlar. In some embodiments, the interior of portions of the body  108  can be coated with a hydrophilic coating to remove moisture from the breath sample in order to reduce the quantity of moisture that is introduced to the analyzer  102 . Additionally, or alternatively, the tubing  106  can also include a hydrophilic coating to reduce the amount of moisture introduced into the analyzer  102 . 
     The body  108  further defines an inlet  112  opening into the reservoir chamber  110 . As shown in  FIGS. 2 and 3 , an inlet valve  114 , such as a one-way valve, is disposed in the inlet  112 . The inlet valve  114  controls the flow of air through the inlet  112  and into the reservoir chamber  110 . Specifically, in use, the inlet valve  114  is configured to open at the beginning of the subject&#39;s exhalation and close at the end of the exhalation. Hence, the inlet valve  114  only allows fluid to flow into the reservoir chamber  110  during an exhalation of the subject. In this way, the inlet valve  114  prevents ambient air from entering into the reservoir chamber  110 . The inlet valve  114  also prevents fluid from flowing out of the reservoir chamber  110  and through the inlet  112  during inhalation by the subject. While the inlet valve  114  is described herein as being positioned in the inlet  112  of the body  108 , in other embodiments (not shown) the inlet valve  114  is disposed in a nebulizer or nebulizer handset that the sample collector  104  is coupled to (e.g., in the exit port  122 ). The inlet valve  114  can be any type of one way valve, such as, for example, an umbrella valve. The inlet valve  114  can preferably have a low cracking (i.e., opening) pressure to reduce back-pressure exerted on the patient&#39;s breath. The inlet valve  114  is configured to close after completion of exhalation and before initiation of the subject&#39;s next inhalation to prevent the flow of fluids out of the reservoir chamber  110  and into the subject&#39;s mouth. 
     The sample collector  104  is configured such that only sample from the desired portion of the subject&#39;s exhalation is retained in the reservoir chamber  110 . For example, for purposes of identifying infections in the lower respiratory tract of the subject, the initial portion of the patient&#39;s exhalation may not be retained in the reservoir chamber  110 . This portion of the exhalation may originate from the mouth and upper respiratory tract and, therefore, may not be indicative of infections in the lower respiratory tract. 
     In order to discard the fluid from the early portion of the subject&#39;s exhalation, the sample collector  104  can include a purge aperture  116 . As shown in  FIG. 3 , the purge aperture  116  may be on the opposite portion of the body  108  from the inlet  112 . In some embodiments, the purge aperture  116  is open to the environment. In other embodiments, flow through the purge aperture  116  is restricted by a flow restrictive structure such as a filter or valve. During use, when the patient exhales, fluid flows through the inlet  112  and into the reservoir chamber  110 . The purge aperture  116  may be configured such that it introduces a low flow resistivity to allow convective flow through the purge aperture  116 . The size of the purge aperture  116  may be chosen to balance the goals of reducing passive diffusion of the sample through the purge aperture  116  while also minimizing back pressure on the patient&#39;s breathing. CO 2  has a very low diffusion constant in open air, thereby helping to retain the sample in the reservoir chamber  110  during the patient&#39;s inhalation. The cross-sectional area of the purge aperture  116  is preferably smaller than the cross-sectional area of the inlet  112  to restrict the flow of fluids out through the purge aperture  116  during breath sample collection. In some embodiments, the purge aperture  116  is circular and has a diameter of about 9 mm. In another embodiment, the purge aperture  116  has a diameter of about 7 mm and about 11 mm. 
     The volume of the reservoir chamber  110  is configured to be less than the total exhaled volume of the patient. As a result, the fluid that enters the reservoir chamber  110  at the beginning of exhalation is forced out through the purge aperture  116  as exhalation continues and more fluid flows into and through the reservoir chamber  110 . For example, in some embodiments, the volume of the reservoir chamber  110  is about 150 ml (milliliters). In another embodiment, the volume of the reservoir chamber  110  is between about 125 ml and about 175 ml. In another embodiment, the volume of the reservoir chamber  110  is between about 100 ml and about 200 ml. In another embodiment, the volume of the reservoir chamber  110  is about 300 ml. In another embodiment, the volume of the reservoir chamber  110  is between about 50 ml and 300 ml. 
     The tidal volume for a patient is typically between about 350 ml and about 700 ml. Because the volume of the reservoir chamber  110  is less than the tidal volume, the fluid from the first portion of expiration is forced out of the reservoir chamber  110  by fluid that subsequently enters the reservoir chamber  110 . Further, as the subject takes additional breaths, the fluid exhaled during the subsequent breaths displaces the fluid that is present within the reservoir chamber  110 . In these subsequent breaths, the fluid that is exhaled at the later portions of the exhalation displaces the fluid from the initial portion of the exhalation, as described above. 
     The body  108  further defines an outlet  118 . The tubing  106  is connected to the outlet  118  to allow the flow of fluid from the reservoir chamber  110  to the analyzer  102 . In some embodiments, the analyzer  102  includes a spectrometry chamber that is at a pressure that is, in use, less than the pressure within the reservoir chamber  110 . For example, the spectrometry chamber may be at a pressure of about 75 to 375 Torr. As a result, the fluid flows from the reservoir chamber  110  to the spectrometry chamber through the tubing  106 . In other embodiments, the sample collector  104  is directly coupled to the analyzer  102 . 
     In some embodiments, as shown in  FIGS. 6-7 , the sample collector  104  is configured to couple to a nebulizer handset  121  (e.g., to the exit port  122  of the mouthpiece  124  of the nebulizer handset  121 ). A nebulizer  120  is also coupled to the nebulizer handset  121  to allow for the delivery of a drug to the subject. The subject may breathe using the nebulizer handset  121  such that the drug is delivered from the nebulizer  120  during an inhalation phase of the subject&#39;s respiratory cycle. As the subject exhales, the fluid expelled from the subject&#39;s respiratory tract flows through the mouthpiece  124  of the nebulizer handset  121 , through the exit port  122 , and into the sample collector  104  where fluid from the desired portion of the exhalation is retained, as described above. In some embodiments, the nebulizer handset  121  is an AEROGEN ULTRA sold by Aerogen of Galway, Ireland and the nebulizer  120  is an AEROGEN SOLO sold by the same company. The geometry of the sample collector  104  can be configured to accommodate the nebulizer handset  121  and the nebulizer  120 . For example, as shown in  FIG. 7 , the portion of the body  108  near the purge aperture  116  can be concave to allow access to the nebulizer  120 . 
     It should be understood that the sample collector  104  need not be connected to the nebulizer handset  121 . In some embodiments, the sample collector  104  is connected to a dedicated mouthpiece or mask with appropriate valve(s) to control the flow into the reservoir chamber  110 , as described herein. 
     The rate of transport of breath gases from the reservoir chamber  110  to the analyzer  102  can be controlled to ensure that the pressure within the reservoir chamber  110  is maintained within a desired range that prevents the flow of ambient air into the reservoir chamber, which would result in dilution of the sample. In other words, the pressure in the reservoir chamber is maintained at or above ambient air pressure. The rate of emptying of the reservoir chamber  110 , and thereby the pressure in the reservoir chamber  110 , can be controlled by controlling the flow into the analyzer  102  (e.g., by controlling the pressure in the spectrometry chamber or using a variable restriction valve) as well as through appropriate selection of the length and diameter of the tubing  106 . This may prevent cracking of the body  108  and opening of the inlet valve  114 . In some embodiments, fluid is drawn continuously over a nine second period. This may equate to breath sample entering the analyzer  102  at a rate of 33.3 ml/second. This rate can be modified to optimize system performance if different reservoir volumes are selected, or if total sample volume required by the spectrometer is changed. During the sample collection period, the patient may exhale 3-5 breaths. As a result, the sample that is analyzed represents a blend of the gas exhaled during these breaths. With each breath, the portion of breath sample analyzed preferably originates from the lower respiratory tract as a result of the arrangement of the purge aperture  116 , as described above. 
     In some embodiments, as shown in  FIG. 8 , the inlet  112  of the sample collector  104  is connected to a mouthpiece assembly  150  as opposed to a nebulizer handset  121 . In this dedicated breath collection assembly, the mouthpiece assembly  150  includes a mouthpiece  152 , an inspiration valve  154 , and an exhalation valve  156 . Each of the valves  154 ,  156  are one-way valves. The inspiration valve  154  can be configured to allow the flow of ambient air into the mouthpiece during inspiration. The exhalation valve  156  is configured to allow for the passage of exhalation fluids through the mouthpiece  152  and into the reservoir chamber  110 . 
     In such embodiments, when the subject breathes in, the pressure within the mouthpiece  152  may decrease, thereby causing the inspiration valve  154  to open to allow ambient air to flow into the mouthpiece  152 . When the subject breathes out, the pressure within the mouthpiece  152  increases, thereby closing the inspiration valve  154  and opening the exhalation valve  156  to allow fluid to flow into the reservoir chamber  110 . As described above, the volume of the reservoir chamber  110  in conjunction with the purge aperture  116  leads to only the desired portion of the exhalation to be retained in the reservoir chamber  110 . 
     In another embodiment, shown in  FIG. 9 , a sample collector  200  includes a first body  202  to collect the sample of interest, a second body  204  to collect fluid from the first part of exhalation (i.e., from the mouth and upper respiratory tract), and a valve apparatus  206 . The valve apparatus  206  includes a tube  207 , a first diverter valve  208  and a second sample valve  210 . Each of the valves  208 ,  210  are disposed within, or coupled to, the tube  207 . The tube  207  defines a lumen for the passage of the fluid from the breath of a subject. Each of the first body  202  and the second body  204  is in fluid communication with the lumen of the tube  207 . During the first part of exhalation, the valves  208 ,  210  are configured such that the fluid from the exhalation flows from the lumen of the tube  207  into the second body  204 . At the desired time, the valves  208 ,  210  are reconfigured such that fluid from the latter part of the exhalation flows from the lumen of the tube  207  into the first body  202 . As a result, only the desired portion of the exhalation fluid is retained in the first body  202 . This can be ensured by selecting the valves  208 ,  210  such that the first valve  208  has a lower cracking (i.e., opening) pressure than the second valve  210 . Because the first valve  208  has a lower cracking pressure than the second valve  210 , breath will enter the second body  204  until the second body  204  is filled. At that point, the first valve  208  will close, thereby allowing air to pass through the tube  207 , through the second valve  210 , and into the first body  202 . The sample from the first body  202  can be transported to the analyzer  102  for analysis. For example, the sample can travel through tubing coupled to the first body  202  as samples are collected, similar to the tubing  106  described above with reference to  FIGS. 1A-1C . Alternatively, the breath sample can first be collected in the first body  202  and subsequently introduced to the analyzer  102  by connecting the first body  202  to the analyzer  102  (either directly or through tubing) after collection of the breath samples is complete. 
     As noted above, the selective collection of breath gases in the first body  202  may be accomplished through appropriate selection of the cracking pressure of the valves  208 ,  210 . Alternatively, the opening and closing of the valves  208 ,  210  can be operated manually to collect the desired portion of exhalation gases. Alternatively, or additionally, the valve apparatus  206  can include sensors to sense patient breathing or pressure or flow changes within the valve apparatus  206  and/or the first body  202  or second body  204 . In this way, the valves  208 ,  210  can be automatically operated to collect the desired portion of the exhalation. For example, the sensor can communicate with a microcontroller that controls the position or configuration of the valves  208 ,  210  (i.e., whether the valves are opened or closed). 
     In another aspect,  FIG. 10  illustrates a method of collecting and analyzing a breath sample. At step  302 , a breath sample is collected. At step  304 , a first portion of the breath sample is discarded. The first portion of the breath sample is expelled during a first portion of the subject&#39;s expiration. At step  306 , a second portion of the breath sample is passed to an analyzer. The second portion of the breath sample is expelled during the second portion of the subject&#39;s expiration and the second portion occurs subsequent to the first portion. 
     The first portion of the breath sample preferably includes fluid that originates from the patient&#39;s mouth and upper respiratory tract and the second portion of the breath sample preferably includes fluid that originates in the lower respiratory tract. For example, the first portion of the breath sample (i.e., the portion that is discarded) can include breath gases from the first 17%-93% of the duration of the patient&#39;s tidal volume. 
     The methods described herein can include capturing and analyzing breath samples before and after administration of a drug (e.g., a  13 C urea drug). The samples collected prior to administration of the drug serve as a baseline to which the post-administration samples can be compared. Both the pre-administration and post-administration samples can be fractionated, as described herein, such that the samples preferably include breath gases that originate from the lower respiratory tract. As described above, the breath samples, both before and after drug administration, can include fluid from one or more than one exhalations by the subject. The method can also include comparing the concentration of  13 CO 2  in the breath samples collected before and after administration of the drug. 
     It will be understood that the foregoing description is of exemplary embodiments of this invention, and that the invention is not limited to the specific forms shown. Modifications may be made in the design and arrangement of the elements without departing from the scope of the invention.