Patent Application
20130217029
The current invention is directed to a system for collecting and analyzing airborne bacteria or biological particles; and more particularly to a system comprising an aerosol collection system for airborne bacterial or biological particle detection that can be used to prepare a sample for diagnosis or directly diagnose a sample for bacterial infection from the environment or from a patient, such as, for example, tuberculosis.
Tuberculosis (TB) is a contagious disease that causes 2 million deaths annually. Approximately 9.3 million people worldwide develop TB every year, of which some estimated 4.4 million are undiagnosed. Improving TB diagnostics would result in approximately 625,000 annually adjusted lives saved worldwide, and elimination of TB from industrialized countries. (See, e.g., Center of Disease Control and Prevention, TB elimination: Trends in tuberculosis 2008, (2009); and World Health Organization, Diagnostics for tuberculosis: global demand and market potential. (2006), the disclosures of each of which are incorporated herein by reference.)
White all common TB diagnostic devices, except X-ray, rely on sputum sample collection. Typically, three samples—more than 3 mL each—are collected per patient. This procedure is particularly difficult in some patient populations. (See, M. B. Conde, et al., American Journal of Respiratory and Critical Care Medicine, 162:2238-2240, (2000); and O. D. Schoch, et al., American Journal of Respiratory and Critical Care Medicine, 175:80-86, 2006, the disclosures of which are incorporated herein by reference.) For example, in the case of children, special procedures such as sputum induction and gastric aspiration are required; all of which are unpleasant and difficult for both healthcare providers and patients. (World Health Organization, Introduction and diagnosis of tuberculosis in children. International Journal of Tuberculosis and Lung Disease, 10(10):10911097, (2006), the disclosure of which is incorporated herein by reference.) In cases where sputum induction is not successful, a procedure known as bronchoalveolar ravage (BALI is performed using fiberoptic bronchoscopy. (N. E. Dunlap, et al., American Journal of Respiratory and Critical. Care Medicine, 161(4):1376-1395, (2000), the disclosure of which is incorporated herein by reference.) The procedure requires a medical doctor and can cause infections if the bronchoscope is not properly disinfected. Moreover, sputum samples are usually contaminated with saliva, which lowers their quality (less M. tuberculosis cells per mL) resulting in low diagnostic sensitivities. (M. Sakundarno, et al., BMC Pulmonary Medicine, 9(1):16, (2009), the disclosure of which is incorporated herein by reference.)
Each TB diagnostic test is characterized by its practicality and performance. (See, e.g., World Health Organization, Diagnostics for tuberculosis: global demand and market potential. (2006); World Health Organization, International Journal of Tuberculosis and Lung Disease, 10(10):10911097, (2006); and World Health Organization, Global tuberculosis control: Surveillance, planning, financing (2009), the disclosures of each of which are incorporated herein by reference.) Practicality refers to the time, training, resources, accessibility and cost of conducting the test. Performance refers to the sensitivity and specificity of the test. While an ideal diagnostic would be practical with high performance, current TB diagnostics inherently have a trade-off between the two. (See, WHO citations above.) These diagnostics include: radiographic methods, bacteriological culture, drug susceptibility testing culture (DST), sputum smear microscopy (SSM), and nucleic acid amplification test (NAAT). (See, WHO citations above; and M. D. Perkins and J. Cunningham, The Journal of Infectious Diseases, 196(Suppl 1):S15-27, (2007), the disclosure of which is incorporated herein by reference.) Radiology (chest X-rays) is one of the fastest diagnostics (<1 hr), but suffers from non-specificity and requires expensive equipment operated in a lab. Bacterial culture is considered the gold standard with 87% sensitivity and can detect as low as 10-100 cells/mL from sputum. Further processing of bacterial culture using DST allows the identification of specific strains of MDR-TB. However, obtaining results takes 2-6 weeks in a resource-intensive tab setting. (See, G. E. Pfyffer, et al., Journal of Clinical Microbiology, 35(9):2229-2234, (1997), the disclosure of which is incorporated herein by reference.) SSM is the most practical and widely used test because it is relatively inexpensive and simple. A sputum sample is stained for acid-fastness of M. tuberculosis. More than 10,000 cells/mL of sputum are needed for a positive identification by a microscope operator (ss+). (See, WHO, (2006) publication, cited above.) Because of backlog, these tests can take up to 6 months in some countries and require multiple visits. The SSM sensitivity is less than 60% (as low as 20% in high-risk groups) and although the specificity is high in high-prevalence groups it suffers in industrialized countries. (See, M. D. Perkins & J. Cunningham, cited above.) NAAT has a sensitivity >95% for SSM positive patients (ss+) (60-70% for ss−) with high specificity. 10-1000 cells/mL of sputum contain enough DNA to amplify, hybridize and produce a positive signal. (See, M. D. Perkins & J. Cunningham, cited above.) NAAT and bacterial culture have the highest performance and are the only two tests that can detect MDR-TB. NAAT suffers from low practicality due to sputum-based sample preparation by a lab technician and is therefore highly variable in resource-limited labs. (See, M. D. Perkins & J. Cunningham, cited above.) NAAT would satisfy priorities of developing and industrialized countries if sample preparation methods were cost-effectively automated.
Another diagnostic issue that is challenging the medical community involves hospital-acquired infections (HAIs). According to the Centers for Disease Control and Prevention (CDC), 1.7 million (9.3 per 1,000 patient-days or 4.5 per 100 admissions) annual HAIs occur in U.S. hospitals causing some 99,000 deaths a year. (See, e.g., R. M. Klevens, at al., Public Health Reports, 122, (2007), the disclosure of which is incorporated herein by reference.) In turn, it is estimated that HAIs correspond to annual medical costs exceeding $35 billion. (See, R. D Scott II, Centers for Disease Control and Prevention, 2009, the disclosure of which is incorporated herein by reference.) More and more it is being recognized that HAIs are a serious problem, both from a public health standpoint, and from the perspective of cost-containment for medical expenses.
There are three different routes of transmission for these diseases: touch, droplet, and airborne. Both droplet and airborne transmissions require the formation of an aerosol that can be inhaled to cause infections. For example, one of the most common inhaled infections is pneumonia. Moreover, while hospital acquired pneumonia (HAP) accounts for 11-15% of all HAIs, it causes a disproportionate 36% of deaths. Indeed, some 79% of HAP infections are non-device related and more than 80% are bacterial. (See, D. J. Weber, et al., Infection Control and Hospital Epidemiology, 28(12):1361-1366, (2007), the disclosure of which is incorporated herein by reference.) From this it can be estimated that nearly 160,000 HAIs and 23,000 deaths are caused by inhalation of aerosol bacteria (specifically S. pneumoniae). Furthermore, TB, and multidrug-resistant tuberculosis (MDR-TB) have also surfaced causing further problems in aerosol transmission in hospitals. (See, S. K. Sharma and A. Mohan, Chest, 130(1):261-272, (2006), the disclosure of which is incorporated herein by reference.)
Bacterial aerosol collectors/analyzers that can diagnose contaminants, either directly from a patient or in the atmosphere, have the potential to provide warnings of these infectious agents, and, thereby avoid further infection. However, a key challenge to providing a detector system with sufficient sensitivity is to collect and rapidly amplify the low-concentrations of bacterial aerosol by delivering highly concentrated analyte (such as DNA) to sensors. Conventional sensors simply do not meet the requirements needed to provide direct diagnostic of possible infection or contamination risks from aerosol sources, such as a patient's breath, cough or sneeze, or from the environment. Accordingly, a need exists to provide a bacterial and or biological collector/analyzer for detecting possible sources of infection directly from a patient and/or contamination from airborne bacterial sources.
The current invention is directed to an aerosol bacterial and or biological collector/analyzer for the diagnosis of respiratory tract infections in patients or contaminants in the atmosphere.
In one embodiment, the aerosol collection and analysis system includes:
Wherein in an embodiment where a sufficient inertial force is applied to the biological particulates to lyse the particulates, the internal components of the lysed biological particulates, such as, for example, DNA, are released.
In another embodiment, the interface comprises a full-face constant positive airway pressure mask.
In still another embodiment, the interconnecting tube includes at least one auxiliary compensating inlet disposed traverse to the axis of the interconnecting tube providing an outlet to the atmosphere. In one such embodiment, the at least one auxiliary compensating inlet is disposed at an angle to the interconnecting tube of greater than 90 degrees. In still another such embodiment, a filler is disposed at the outlet of the auxiliary compensating inlet. In yet another such embodiment, the filler is a high-efficiency particulate air (HEPA) filter.
In yet another embodiment, the impactor includes a converging nozzle defining the fluid path of the impactor, and a flat collection surface disposed distal to said converging nozzle and perpendicular to the flow path. In such an embodiment, the flat collection surface has an inlet orifice disposed therein and aligned with the fluid path of the converging nozzle. In another such embodiment, the sample pressure downstream (P1) of the nozzle and the sample pressure upstream (P0) of the nozzle follow the inequality P1/P0<0.53. In still another such embodiment, the distance from the nozzle to the collection surface (x), and the diameter of the nozzle (d) have the following ratio x/d=1.2. In yet another embodiment, the x/d ratio is made variable. In yet another such embodiment, the sample-facing surface of the nozzle is mirror polished.
In still yet another embodiment, the collection vessel tapers toward the second end outlet.
In still yet another embodiment, the outlet may be accessed without opening any other portion of the collection vessel. In one such embodiment, the outlet may be accessed via a syringe.
In still yet another embodiment, the distance between the first end of the collection vessel and the surface of the liquid medium is approximately 4 mm.
In still yet another embodiment, the system further includes a sensor capable of detecting DNA from the lysed sample, the sensor being disposed in fluid connection with the second end of the collection vessel.
In still yet another embodiment, the system further includes a sensor capable of detecting intact bacteria, the sensor being disposed in fluid connection with the second end of the collection vessel.
In still yet another embodiment, the interconnections between the components of the system comprise quick-disconnect couplings having valves such that once disconnected the valves automatically close.
In still yet another embodiment, the components of the system are autoclavable.
In still yet another embodiment, the components of the system are resistant to common sterilization chemicals.
In still yet another embodiment, the sample facing surfaces of the system are formed from a plastic material.
In still yet another embodiment, the sample is formed by a method selected from breathing, coughing and sneezing.
In still yet another embodiment, the liquid medium is a buffer.
In another embodiment the invention is directed to a method of collecting and analyzing an aerosol sample for bacterial infection including:
In still another embodiment, the method includes compensating the pressure of the system for the oscillating outflow from the patient.
In yet another embodiment, the method includes analyzing the internal components of the cells for bacterial contaminants in real-time.
In still yet another embodiment, the method includes withdrawing a batch sample of the internal components from the fluid medium for analysis without having to disassemble the system.
These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying data and figures, wherein:
a
1 and 1a2 provide schematics of embodiments of an aerosol biological collector/analyzer system in accordance with the current invention;
b and 1c provide schematics of embodiments of a component of an aerosol biological collector/analyzer system in accordance with the current invention;
d provides a data graph showing flow rate compensation studies for an embodiment of an aerosol biological collector/analyzer system in accordance with the current invention;
e and 1f provide schematics of an embodiment of an impactor system in accordance with the current invention, where 1e shows a schematic view of the impactor, and 1f shows a cross section along axis “B” in
a and 2b provide block-diagrams of: (a) an existing aerosol biosensor for airborne bacterial detection; and (b) an embodiment of an aerosol biological collector/analyzer system in accordance with the current invention;
a and 4b provide data graphs showing the theoretical response of conventional systems and the inventive system given a step input (a), and a puke input (b);
The current invention is directed to an aerosol biological collector/analyzer system. In particular, the current invention is directed to an airborne aerosol collection and bacterial analysis system, capable of collecting an airborne aerosol sample and preparing it for analysis via aerodynamic shock in a single-step.
A schematic of one embodiment of the aerosol collection and analysis system (10) of the instant invention is schematically depicted in
In summary, the aerosol biological collector/analyzer system of the current invention includes four basic components, including: the patient/biosensor interface (14), which includes a device capable of engaging the patient and collecting substantially all the airflow from the patient; a device for interconnecting (17) the patient/sensor interface with the impactor, which includes tubing filters, flow regulators, etc. capable of both providing an interconnection between the components and balancing the naturally oscillating input flow-rate from patient with the constant flow-rate of the pump; an impactor (20), which includes a nozzle, impactor plate, collection tube, etc. capable of separating and/or lysing the cells of any bacteria suspended in the collected sample from the patient; and a pump (not shown), which includes all necessary tubing, filters, flow regulators, etc. capable of providing a steady and sufficient flow-rate through the apparatus to the patient/biosensor interface. Although the following will describe alternative embodiments of each of the components of the biosensor system of the invention, it will be understood that these alternative embodiments are merely provided as examples, and that one of ordinary skill in the art will appreciate other modifications and alternatives to the basic aerosol biological collector/analyzer system without departing form the scope of the current invention.
With regard to the system/patient interface (14), although any device capable of providing an interconnection between the patient and the aerosol biological collector/analyzer system to collect natural breath samples may be used, such as, for example, a simple tube, in a preferred embodiment an interface designed to fit securely and fully over a patient's nose and mouth is preferred. Such a secure interface is preferable both because it allows for the patient to cough and breathe regularly during testing, but also because it prevents external atmospheric contaminates from entering the system during testing (which can lead to detection of environmental bacteria), and simultaneously prevents potential infectious agents emitted from the patient from escaping the system (which leads to system losses and loss in sensitivity and can cause infection of other patients or healthcare professionals). In one such embodiment, the interface would comprise a full-face CPAP (Constant Positive Airway Pressure) mask, such as, for example, the ComfortGel Full® from Phillips Respironics. Although not required, preferably such masks would be disposable to reduce the chances of contamination before and after use.
With regard to the interconnection (17), it should be understood that any device capable of providing suitable fluid communication between the patient/biosensor interface (14) and the impactor (20), such as, for example, a simple length of tubing, may be used in the current invention. However, in one embodiment, a device capable of balancing the naturally oscillating input flow-rate from the patient with the constant flow-rate of the pump is provided between the patient/biosensor interface and the impactor. An exemplary embodiment of such a device is shown schematically in
Although this connection may take any suitable form, in an exemplary embodiment the auxiliary compensating opening includes a tee connection, such as a Wye connection, that is angled in relation to the main interconnection at an angle of greater than 90 degrees. The angled design of the compensating tube is provided to minimize aerosol particle losses by having the airflow divert more 90 degrees as it exits through the fillers. The bend in the compensating tube prevents the particles from following the airstream because of the inertial forces on the fluids. Although only a single compensating opening (30) is shown in
Row compensation through this compensating opening is important.
As an example, providing a compensating opening comprising five side tubes at a 135 degree angle of 5 mm diameter can maintain a flow-rate of 1000 lpm. This flow-rate represents conservative peak flow-rates during a cough. Peak flow rates in male patients of up to 300 lpm during coughing have been seen. (See, e.g., Gupta J K, Indoor Air, 9:517-525 (2009), the disclosure of which is incorporated herein by reference.) Under such a system, the particle loss through the side tubes can be calculated and is 16.5%, however, the other 84.5% of particles are carried in an airstream of 1 lpm to the impactor. This results in a 1000-fold increase in particle concentration carried to the impactor with minimal loss.
Accordingly, the presence of this compensating opening has a major impact on possible flow-rates, and on the operation of the device. While inhaling and exhaling, the compensating opening will equilibrate the flow through the HEPA filter opening (32). In contrast, while a patient is coughing the compensating opening allows the system to rid itself of the excess flow-rate. Accordingly, placing such a compensating opening before the impactor allows for the compensation of the fluid flow-rate between the patient and the impactor with HEPA filtered air, increasing the possible flow-rate of the system substantially. Moreover, having the HEPA filters allows for the avoidance of contamination from the outside in and inside out.
One of the unique features of the system of the instant invention is that it consists of an impactor system (20), which can both collect intact cells and/or lyse cells. That is, the impactor system is capable of passing bacterial cells in air through a shock and either separating them from the gaseous sample intact, or lysing the cells and collecting fragments, simultaneously. However, the DNA itself does not fragment with one pass through the shock. (See, e.g., K. Teshima, et al., Shock Waves, 4(6):293-297, (1995), the disclosure of which is incorporated herein by reference.) Although any suitable aerodynamic impactor capable of both passing bacterial particulates, such as, for example, cells intact and/or generating shockwaves sufficient to lyse bacterial cells may be used with the biosensor of the instant invention, in one embodiment, as shown in
In such an impactor system, an aerodynamic shock sufficient to lyse cells is created by operating the impactor nozzle at sonic velocity when χ=P1/P0<0.53 (where, P1 is the pressure downstream and P0 is the pressure upstream of the nozzle). Recent computational and experimental studies have shown that E. coli bacteria passing through an aerodynamic shock of this magnitude reach critical decelerations, which causes their break-up. (See, Example 2, below, and P. R. Sislian, et al., Chemical Engineering Science, 64(9):1953-1967, (2009) & P. R. Sislian, et al., Chemical Engineering Science, 65(4):1490-1502, (2010), the disclosures of each of which are incorporated herein by reference.) These studies show that, operating at these conditions, more than 90% of cells are broken up to spill their internal components for subsequent collection or detection, allowing for the collection and preparation of the bacterial aerosol samples in a single step. In addition, the simultaneous break-up and collection in one device eliminates the need to build microfluidic components for cell lysis, greatly simplifying the device.
Although each of the components of the impactor may take a form such that the combination of elements is capable of producing a suitably strong shock in the aerosol sample to lyse any bacterial cells produced by the patient. In particular, the impactor should be provided with a nozzle (34) that creates the impinging flow necessary for inertial particle collection. In turn, the nozzle should preferably be positioned in relation to the orifice (38) of the collection stage such that the components meet the dimensional requirement: x/d=1.2, where x is the distance from the nozzle exit to the collection tube entrance, and d is the diameter of the nozzle exit. Moreover, the nozzle and orifice need to be perfectly aligned and have a tolerance of at least 1%. To reduce air-flow friction, the nozzle may be mirror-polished.
As described above, and shown schematically in
Although any sample collector (24) suitable for gathering a sample for either direct or later analysis may be integrated with the impactor (20), preferably the collector is designed to both contain a buffer medium for preserving the sample and also to decelerate the sample bacterial particles before collection. In one embodiment, for example, the collector has the following features:
Although suitable collection media are well-known in the art, such as, for example, Phosphate Buffered Saline (PBS) buffer, and may be used in conjunction with the current invention, it will be understood that the instant invention also allows for the integration of a real-time sensor, such as, for example, Fluidigm's Access Array™ System and others, which can identify DNA from internal components specific to a particular bacteria, such as, for example, M. tuberculosis bacterium. Such sensors can be integrated with the collection module described above to provide real-time sensing of bacteria, or other biological components of the respiratory tract.
The above discussion has not focused on the materials to be used in constructing the aerosol biological collector/analyzer system of the instant invention. It will be understood that preferably, the device should be isolated from the external environment, and so designed to avoid external contamination, which can lead to detection of environmental bacteria, and also to avoid sample leakage, which can lead to system losses and loss in sensitivity, and which can also lead to spread of disease. For example, the use of quick-disconnect couplings, such as, for example, an Acetal® Quick-Disconnect Coupling Plug, with valves on both plug and socket between components, would allow the lower chamber of the impactor to be isolated both before and after use. Such an arrangement would reduce contamination both from the outside in and inside out, and is preferred to help control contamination before and after use.
In addition, the device should be contamination free, and therefore should be designed to withstand standard disinfection apparatus used on other medical devices. Accordingly, all materials chosen should be autoclavable or should be purchased sterile. Moreover, to minimize the loss of sample, the electrostatic properties of the materials used should be evaluated to avoid collecting charged biological aerosol. In particular, since bacteria are usually negatively charged particles, plastics that do not hold static charges would be a preferred material for parts that might interact with the sample.
Although the above discussion has focused on an aerosol biological collector/analyzer system for analyzing aerosol samples, the current invention is also directed to a method of analyzing aerosol samples. As shown in the flow-chart in
Although these are the basic steps in the process, it should be understood that other steps may be included to comport with the operation of the device described in the above discussion. For example, in one embodiment, the method would also include compensating the system pressure for the natural oscillation in the out and inflow of breath from the patient, and from any over-pressures caused by coughs and/or sneezes.
In terms of the analysis itself, the analysis could be done via a batch method, by withdrawing individual samples from the liquid medium. Alternatively, the analysis could be accomplished in real-time by interconnecting an analyzer in-line with the liquid medium.
In this section several examples of how aerosol biological collector/analyzer systems operate or could be implemented are provided. In addition, a comparison of the performance of an aerosol biological collector/analyzer system made in accordance with the current invention versus a conventional system is provided. The person skilled in the art will recognize that additional embodiments according to the invention are contemplated as being within the scope of the foregoing generic disclosure, and no disclaimer is in any way intended by the foregoing, non-limiting examples.
Turning to a comparison of the operation of the instant invention with that of a conventional aerosol collection and analysis system, it should be understood at the outset that access to the internal components (ICs) of cells, mainly DNA, is necessary for most detection methods. (See, e.g., N. Bao and C. Lu., Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems, pages 817-831. Springer, (2008), the disclosure of which is incorporated herein by reference.) In conventional biosensor systems, as shown in a block-diagram in
In short, existing systems (
The goal in both cases is to detect changes in the concentration of airborne S. pneumoniae (xg1) through a fast and concentrated release of cellular DNA (yl). The dynamics of the processes required for the method shown in
where G and L are the gas and liquid sampling flow-rates, respectively; the subscripts g and l refer to gas and liquid phases, respectively; x is the number concentration of bacteria; yl is the number concentration of DNA that feeds to purification steps (not shown in
A number of assumptions have been made in this calculation. For example, it is assumed for the purposes of this calculation that there is one DNA molecule per cell. All concentrations are normalized (non-dimensional) relative to a critical concentration of bacteria in air that will cause infection (i.e., xg1>1 causes infection). The break-up of cells is assumed to be a first order reaction in a continuous stirred tank reactor (CSTR) with a reaction constant k. (See, e.g., J. A. Asenjo. Separation Processes in Biotechnology. Marcel. Dekker, New York, 1st edition, (1990), the disclosure of which is incorporated herein by reference.)
The system (52) used in the experiments is shown schematically in
Finally, system dynamics for the inventive system is expressed as follows:
where ƒ is the fraction of cells that remain intact and ηd is the collection efficiency of DNA from air. In the gas phase dead volume is approximately 0. Therefore, the break-up is a steady state process that decreases the response time of inventive system. This enhances the theoretical response of the system. EQs. 1 & 2 are of the form z′=Az+Bw, where w and z are a time-dependent input and output, respectively.
Table 2, below provides a set of parameters used in simulations of EQ. 1 and EQ. 2. For the system in
MATLAB® code was used for assessing the time response (yl(t)) of the existing systems (
In all three scenarios, the parameters ƒ, L and V1 are fixed. The value of ƒ was obtained from previous work while the values of L and V1 were set to the existing system's specifications. The first scenario of G and ηd corresponds to values shown in italics in Table 2. As shown in
As shown, the existing system produces a maximum signal at around 5 mins after the input signal consistent with experimentally reported values of 15 min for full detection cycles using currently employed systems. Accordingly, the time-savings are significant given requirements to get below 1 min detection in some applications. (See, e.g., J. Vitko, Technical. Report, The National. Academies Press, (2005), the disclosure of which is incorporated herein by reference.) What is more, the increase in the signal allows more flexibility with the limits of detection (LOD) of current sensors.
Studies have been conducted using the experimental apparatus described in Example 1, above, to show that airborne bacteria passing through an aerodynamic shock breaks-up by experiencing a relative deceleration because of sharp changes in the gas velocity. An aerodynamic shock is created by operating the impactor nozzle at sonic velocity when χ=P1/P0<0.53 (P1 is the pressure downstream and P0 is the pressure upstream of the nozzle). Uncontrolled instabilities in the form of waves on the surface of the bacterium perpendicular to the direction of acceleration cause bacterial cells to break-up. The critical acceleration ac is given by:
where σ is the surface tension of the bacterium, dp is the diameter of the bacterium, and ρp is the density of the bacterium. (See, e.g., D. D. Joseph, et al. International Journal of Multiphase Flow, 25(6-7):1263-1303, (1999); Sislian (2009) & Sislian (2010), the disclosure of each of which are incorporated herein by reference.)
The critical acceleration for two different test bacterial aerosols are shown in Table 3 below, which shows the cell properties for E. coli and B. atropheus and critical shock properties needed to induce bacterial break-up. The values are reported for 0.5 μm spores.
E. coli
B. atropheus
Previous calculations predict the break-up of both E. coli and B. atropheus spores when compared to the ac in Table 3. (See, Sislian, (2010), cited above.) Experimentally measured values of ƒ (see Example 1, above) are shown in
The experimental setup provided in Example 1, can also be used to measure the collection efficiency (ηd) of the system. In such an experiment, the bacterial suspension would be aerosolized using a capillary nebulizer (TR-30-A1, Meinhard Glass Products) at a Nitrogen flow-rate of 0.2 mL/min. The suspension concentration and flow-rate will be controlled to produce single bacterial cells in the aerosol.
E. coli will be used as the test aerosol because (1) it does not require biosafety chambers and (2) is easily cultured and washed. In addition, S. pneumoniae is a vegetative bacterium expected to have an ac similar to E. coli vs. B. atropheus spores and hence a similar value of ƒ. The ds-DNA (double stranded) in our samples will be stained with PicoGreen fluorescent dye (P11495, Life Technologies) using the protocols provided by the manufacturer and in other work. (See, W. Martens-Habbena and H. Sass, cited above.)
Quantitative data in the form of mass concentration of ds-DNA (as low as 25 pg/mL) can be obtained using a spectrofluorometer (Q32857, Life Technologies) at an excitation wavelength of 480 nm and emission at 520 nm. Purified E. coli ds-DNA solution of known concentration will be stained at different dilutions to obtain a standard curve of emission intensity vs. mass concentration. The ds-DNA will be measured for the following samples from different points in the setup (see
where the numerator is the total extracted ds-DNA and the denominator is the total number of ds-DNA before the shock; φ is the fraction of particles lost in the system before the impactor and is equal to 0.088 Å} 0.029; φ is a property of the experimental setup and not that of the impactor; ƒ is described in Example 1.
In this experiment, PG3 is measured as a control to assess the effect of nebulization; PG1 is measured to determine the extracellular ds-DNA (=PG2−PG1) in the starting suspension. The bacterial suspension will be washed to reduce the amount of extracellular ds-DNA (<1% of PG2) to reduce errors in the calculation of ηd, which is the collection efficiency of ds-DNA from broken-up cells only. PG5 is measured to ensure that the extracellular ds-DNA is higher than the starting extracellular ds-DNA.
The collection efficiency of bacterial particles (η) has been previously calculated computationally. (See, Sislian, (2010), cited above.) The steady-state Navier Stokes equations with a standard k—turbulence model are used to solve the gas dynamics. The particle dynamic equations are one-way coupled to the gas dynamic equations at low aerosol concentrations. Stochastic particle tracking is used to account for the turbulence in the impactor. Although η does not factor into EQ. 2, it is a function of geometry and operating condition of the impactor like ηd. The difference is in the particle sizes: bacteria are 1 μm while DNA particles are 10 nm.
Low-pressure impactors (Hering design) have been used previously to collect nanoparticles. (See, S. V. Hering, et al., Environmental Science and Technology, 12(6):667-673, (1978) & S. V. Hering, et al., Environmental Science and Technology, 13(2):184-188, (1979), the disclosures of each of which are incorporated herein by reference.) The particle collection efficiencies for 10 nm particles have been shown to increase with increasing x/d and decreasing χ, consistent with the trends in amax. (See, O. Abouali and G. Ahmadi, Journal of Nanoparticle Research, 7(1):75-88, (2005), the disclosure of which is incorporated herein by reference.)
The collection efficiency (ηd) can be calculated using the developed FLUENT code. In this simulation 10,000 DNA particles will be released right after the shock instead of the inlet impactor and counted as they enter the opening of the collection tube (
In order to reach Gηd=266 L/min, the threshold for a viable impactor, both G and ηd have to be sufficiently large. In fact, G has to be greater than Gηd=266 L/min since ηd<1. In one embodiment, G may be improved by operating multiple one-nozzle geometries such as those described in Example 1, above, in parallel. In order for the nozzles to be parallel, the gas flow structures should not interact, thereby having each nozzle act as if it had no neighbors. This ensures that ηd and ƒ stay constant. For example, with a nozzle diameter d=1 mm (9.4 L/min per nozzle), 36 nozzles are needed to reach a G=325 mL/min. If the computed separation were 3 mm (to be determined by simulation of two nozzles) arranged in a square, a 2.1 cm×2.1 cm area is needed. This does not change the overall dimensions of the impactor. The only change will be in the pumping power requirements, which can be easily estimated by multiplying the downstream pressure by the flow-rate.
The following discussion will use tuberculosis (TB) as an example to describe the advantages of the aerosol biological collector/analyzer system of the instant invention. However, it should be understood that the basic concepts described are equally applicable to other bacterial diseases. TB is among the leading causes of disease and death worldwide, which, in 2008 caused an estimated 1.3 million deaths. According to the World Health Organization (WHO), one third of the world's population is currently infected with TB and someone is newly infected every second. In developing countries, Sputum Smear Microscopy (SSM) is the most common diagnostic approach. SSM typically has a specificity of 0.97, a sensitivity of 0.51, and a response time of 2-7 days (See, e.g., Keeler, E., et al., Nature Reviews: Diagnostics, 61006:49-57, (2006), the disclosure of which is incorporated herein by reference.) The slow response time results in a loss of patient follow-up. Because the collection of sputum is a multistep process, some adult samples are diluted with saliva and contain insignificant amounts of M. tuberculosis bacterium. Therefore, a method that can provide better specificity, sensitivity, response time and sample collection will greatly enhance the diagnosis of a curable disease.
In addition, obtaining sputum from children, who account for some 11% of the TB positive population, is difficult and demands special procedures such as expectoration, gastric aspirates, and sputum induction; all of which are unpleasant for both health care providers and the children. Moreover, the quality of these samples, whenever obtainable, is also lower than that obtained from adults. By eliminating the multistep process of sputum collection, the airborne bacterial detection system of the instant invention also reduces variability in the quality of samples collected from adult patients. Furthermore, patients in underdeveloped areas often do not have the means to return to a clinic for follow-up (10-15% of all patients). By combining the collection of the sample and the detection of the M. tuberculosis DNA, the device of the instant invention will be able to obtain TB readings on the order of minutes to hours, which eliminates the loss of follow-up, resulting in an estimated 95,000 Annual. Adjusted Lives Saved (AALS). (See, again, Keeler, (2006), cited above.) In addition, the biosensor will also provide better sensitivity and specificity compared to the standard SSM technique. The total potential of AALS using the new device is 625,000, a significant improvement over current methods.
Again, using TB as an example, a typical. TB patient produces approximately 3000 and 40,000 infectious droplets containing M. tuberculosis with each cough or sneeze, respectively. As depicted in
In summary, an aerosol biological collector/analyzer system has been described that shows dramatically improved performance over conventional systems in every measurable facet. It is well-accepted that the performance of such biosensor systems is assessed by: (1) time and efficiency to extract and deliver ICs to the sensor, (2) interference with post-lysis steps, (3) cost and (4) ability to directly interface with a patient. The first point is addressed in the discussion provided above in Examples 1 to 3. In particular, the inventive device collects directly into the impactor without another chamber being present between the patient and the impactor. This allows adequate concentrations of bacteria to be collected.
This description of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form described, and many modifications and variations are possible in light of the teaching above. The embodiments were chosen and described in order to best explain the principles of the invention and its practical applications. This description will enable others skilled in the art to best utilize and practice the invention in various embodiments and with various modifications as are suited to a particular use. The scope of the invention is defined by the following claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2011/042854 | 7/1/2011 | WO | 00 | 4/30/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61361804 | Jul 2010 | US |
