Patent Grant
8614083
1. Field of the Invention
The present invention relates to a method and device for the simultaneous concentration of multiple waterborne pathogens from large volumes of liquid toxins, and more particularly to the effective concentration of small numbers of multiple waterborne pathogens from large volumes of various water matrices.
2. Description of the Related Art
Effective concentration of small numbers of multiple waterborn pathogens from large volumes of various water matrices is a serious challenge. Current commercially available technologies are not able to automatically concentrate and elute simultaneously multiple pathogens from large liquid volumes using portable equipment. Pathogens size, variability and biological differences requires the use of specific, expensive, and labor intensive concentration techniques, especially for viruses and protozoa.
Currently, the two major approaches for concentrating multiple microorganisms from large water volumes include filtration and continuous flow centrifugation. Ultrafilters, which organisms are retained by size exclusion have much smaller pore size than viruses, which allow for the simultaneous concentration of viruses, bacteria, and protozoa. The particles are kept in the retentate and thus prevent clogging of the filter surface. Several publications have described the application of hollow fiber ultrafilters for concentrating human viruses, see Bicknell, 1985, Cryptosporidium oocysts (Simmons, 2001; Kuhn and Oshima, 2002, Ferguson, 2004), and the simultaneous concentration of viruses, bacteria and protozoa (Juliano and Sobsey, 1997/1998; Morales-Morales, 2003; Hill 2005).
However, ultrafiltration methodology is cumbersome and requires further systematic evaluation for much larger volumes of water than the 100 L volumes that are currently available. Electropositive filters are not rated by pore size and were initially designed to capture viruses from large water volumes by electrostatic attraction. There have been a few publications that evaluated their efficacy to concentrate bacteria and protozoa. See Hou et al (1980) which reported substantially high retention of E. coli, Viruses (Polio, and MS2 bactenophages, and endotoxin) by using charged modified filters. Watt et al. (2002) reported low recoveries for Cryptosporidium oocysts, Giardia cysts and Polio virus from large water volumes, 14% and 17%, respectively.
Recently, two configurations of the ZetaPlus® Virosorb® 1 MDS filter were evaluated for simultaneous recovery of multiple microorganism types from tap water (Polaczky et al., 2007). High numbers of C. parvum, S. enterica and B. globigii, and bacteriophages, were spiked into 25 L of tap water samples. Low recoveries were achieved using the cartridge filter, 21%, 37% and 87%, respectively and much higher recoveries, 28%, 50%, 65%, respectively, were reported for the flat filter.
Continuous flow centrifugation (CFC) enables large scale collection of particles, such as protozoa and bacteria, through their sedimentation due to high centrifugal forces. Whitmore and Carrington, (1993) employed a bench-top centrifuge for the recovery of C. parvum oocysts from water samples.
Utilization of stationary bench-top blood cell separators with recoveries of several folds higher than the cartridge filtration was also reported (Goatcher, 1996). Substantially high recoveries (>90%) were reported for simultaneous concentration of Cryptosporidium, Giardia, Microsporidium, and bacteria from small volumes of source and potable water using a stationary centrifuge and a costly labor-intensive protocol (Borchardt and Spencer 1 996; Borchardt and Spencer, 1998; Borchardt and Spence 2002).
However, less efficient recoveries of C. parvum oocysts from 100 L were also reported by this method (Swales and Wright, 2000). In addition, very low recoveries, less than 5%) of C. parvum from 10 L of water spiked source water, using a compact continuous flow centrifuge were reported (Higgins, 2003).
The above reports demonstrate that currently suggested approaches for concentrating multiple microbe types from large water samples are limited by manually operated systems, using expensive disposables and employing tedious protocols that are not suitable for rapid response to accidental or deliberate water contamination and may complicate routine monitoring.
An earlier prototype of a modified blood separation apparatus, such as the 625B standard disposable bowl manufactured by Haemonetics, of Braintree, Mass.) efficiently concentrated Cryptosporidium oocysts, Giardia cysts, and Microsporidian spores from tap and source water samples. The basic principle underlying the Continuous Flow Centrifugation (CFC) technique has also been described (Zuckerman et al, 1999; Zuckerman and Tzipori, 2004; 2006).
However, there still exists the need for a continuous flow centrifuge that enables concentration and elution of targets from large volumes of liquids.
One aspect of the present invention is to provide an automated portable continuous flow centrifuge that enables complete automation of the concentration and elution of the targets from large volumes of liquids.
Immediate and anticipated uses and/or commercial applicability of the continuous flow centrifuge of the present invention include: the simultaneous concentration of viruses, protozoa, bacteria, bacterial spores, algae, and toxins from large volumes of liquids such as water, wastewater, milk, wine, beverages and other liquids; on line monitoring of microbial quality of liquids; and on line monitoring of biothreat agents in liquids
The device is simple to operate, rapid, robust and portable. The device can also be easily upscaled to concentrate larger volumes. The process of sample concentration and elution is completely automated and the resulting concentrate is small in volume and presentable to a variety of detection methods.
In accomplishing these and other aspects of the present invention, there is provided a method for the simultaneous concentration of multiple toxins from large volumes of water. The method includes the steps of providing a disposable separation centrifuge bowl, the centrifuge bowl including a positively charged material at it's inner core. A large water sample contaminated with toxins from a group consisting of protozoa, bacteria, bacterial spores, and toxins is delivered to the centrifuge bowl. A centrifugal force is applied to the separation bowl. The water sample is concentrated to remove large particles of the toxins in the bowl due to the centrifugal forces. The concentrated water sample is passes through the positively charged inner core to capture any remaining concentrated targets by electrostatic forces and the concentrated targets are eluted.
There is further provided a device for concentrating multiple pathogens from large liquid samples. The device includes a centrifuge bowl and a core including a positively charged material located within the centrifuge bowl. An inlet located at one end of the bowl receives the liquid sample containing the pathogens. The core has a passageway and the liquid sample flows through the core and the concentrated pathogens are attracted and adhere to the positively charge material.
These and other objects, features, aspects, and advantages of the present invention will become more apparent from the following detailed description of the preferred embodiment relative to the accompanied drawings, in which:
The automated portable concentrator of the present invention simultaneously concentrates protozoa, bacteria, bacterial spores, and toxins from large volumes of liquids. After completion of the concentration cycle it elutes the concentrated targets and delivers the concentrate to disposable sterile bags that can be presented in the field to various detection kits or can be sent to a lab for analysis. The concentrate could also be presented to a variety of detection methods.
As will be described in further detail herein, the portable continuous flow device includes a disposable bowl that simultaneously concentrates multiple pathogens from the same sample, and an automated portable continuous flow centrifuge that accommodates the modified bowl and a disposable tubing harness and allows for sample concentration and elution from large water volumes.
Referring to
Housing 20 includes a base 21 and a top deck 24 to which centrifuge 12 and pump 14 are mounted. Motor control units (not shown) to control the centrifuge and pump speeds are also mounted within the base. Pluralities of pinch valves 16, 18 are also mounted on the top deck. For example, two large valves 18 and three small valves 16 are provided to control the tubing flow. The PLC control and pneumatic module for the pinch valves can also be mounted to the inside of the top deck.
As shown in
Referring to
As described above, a plurality of pneumatic valves 16, 18 are provided. Referring to
Referring to
The CFC is powered by and an Astrodyne MK75s-24, which has and input voltage range of 90-264 VAC and an output voltage of 25 VDC. A relay is used for communication between the PLC and the motor controllers and can be a 70-782EL8-1 socket relay, manufactured by Magnacraft of Milwaukee, Wis., and is an eight pin logic style DIN panel with an elevator terminal or module compatible relay. It has a normal voltage rating of 300 volts and a normal current rating of 12 amps.
An EMI filter, such as that manufactured by Filter Concepts of Santa Ana, Calif., is designed to effectively attenuate differential and common mode noise from switch mode power conversion and regulation circuits subject to FCC Class B or CISPRJEN Class A conducted EMI limits. Maximum attenuation is achieved at 150 kHz and above. The filter is also available with an A-circuit option to enhance its performance in applications with high source impedance.
The present invention also provides for a sterile, disposable set 58 (
Referring to
As mentioned above, the portable equipment uses disposable set 58 that uses ⅜″ outside diameter by ¼″ inside diameter large tubing, 3/16″ outside diameter by ⅛ inside diameter small tubing, bowl 60 and two plastic bags (100 ml for the viruses and 400 ml for the protozoa/bacteria). During the concentration process, the large diameter tubing delivers the liquid samples to the pump and into the disposable bowl. The large particles (protozoa, bacteria, spores, suspended material and debris) are sedimented, the “cleaner” liquid which contains negatively charged viruses/toxins is forced through the core and viruses and toxins are captured by strong electrical force. Then, the large tubing directs the waste water from the machine. During the automated elution process, the small tubing directs the protozoa/bacteria buffer into the bowls residual and also delivers the virus buffer into the core. The large bag initially accommodates the protozoa/bacteria buffer and after the elution it contains the protozoa/bacteria concentrate. The small bag contains the virus buffer and retains the virus concentrate.
Referring to
As described above, disposable continuous flow centrifuge bowl 60 has a positive charged matrix for virus capturing, simultaneously with bacteria, protozoa and toxins. The positive charged core allows for simultaneous concentration of protozoa, bacteria, spores, toxins and viruses, from large water volumes.
Two different techniques were used to prepare the charged component. The first configuration was prepared as follows: the outer glass fiber covers of a 12″×9.5″ sheet of ViroCap viral filter manufactured by Argonide Co. of Sanford, Fla., were gently peeled away, removing them from the inner membrane filter. This inner membrane filter was then cut into strips, placed into a blender, and shred to a consistency of “cotton”. The same was repeated with an equal volume of tissue paper. An equal weight of 15 grams each of ViroCap viral filter “cotton”, and tissue “cotton” were then added to one bag containing 10 grams of aluminum hydroxide Al(OH)3 nano-ceramic fibers Boehmite R0608 by Argonide Co. The bag was then sealed and mixed thoroughly, so as to evenly distribute all materials.
The out flow chamber of the core was then packed with the final product of the filter material prep. To further ensure a homogenous mix, the volume of filter material prep for each core, approximately 4 grams, were teased apart and mixed manually before being inserted into the core. A 6″ thin plastic ruler was used to pack the prepped material into the core out flow chamber, a consistent and form an optimal homogenous, dense but fluffy filter matrix. A fiber cover support, for example, Hollytex 3267 spunbond polyester, Haemonetics part #50996-00, made by Ahlstrom Technical Specialties of Mt. Holly Springs, Pa., was cut to fit to seal the top of the out flow chamber, securing the filter material prep within the out flow chamber.
A second configuration involved the preparation of a slurry composed of 1 g Pyrex fiber glass wool, 8 micron pore size, borosilicate such as Aldrich CLS3950 manufactured by Sigma of St. Louis, Mo., 1 g of nano-ceramic Boehmite fibers powder, for example, R0608 of Argonide Corp., of Sanford, Fla.) and 1 g of cellulose microcrystalline powder (Aldrich 435236 by Sigma), 1 g of paper pulp, and 5 ml of DI water which was mixed thoroughly, and was poured into the inner space of the bowl's insert. The insert was then placed in a dry oven overnight at 70° C. Both types of modified cores were then sent to Haemonetics and used in the final assembly of the HS plasma aphaeresis bowls.
The present invention also encompasses a continuous flow centrifugation methodology which allows for the simultaneous concentration and processing of protozoa, bacteria, viruses, and toxins presented in large water volumes. The overall process involves the following steps: a) concentration; and b) elution. In the concentration step, the peristaltic pump delivers a water sample through the bowl's inlet port 68, the large particles such as protozoa, bacterial spores/cells and other suspended material is sedimented due to the strong centrifugal forces and the much cleaner water sample is forced to flow through the designated passages in the bottom of the inner core, where as the negatively charged viruses are captured by the strong electrostatic attraction, eliminates clogging issues that are common in standard filtration components.
During elution protozoa/bacteria are dislodged by the addition of detergent and agitation, and the viruses which are trapped in the virus matrix are released using a protein buffer which inverts the negative charge. Both the virus and protozoa/bacteria concentrates are delivered to a sterile bag. The bags could be processed in the field by using portable detection kits or delivered to the laboratory.
Referring to
Currently, a system which simultaneously concentrates and recovers multiple types of microorganisms from large water volumes is not commercially available. A continuous flow centrifugation methodology which allows for the simultaneous concentration and processing of protozoa, bacteria, and viruses presented in large water volumes was developed according to the present invention.
Several stages of experimentation led to the development of the system. In order to test the method recovery efficiency, Cryptosporidium oocysts, Bacillus anthracis spores and MS2 bacteriophages were simultaneously spiked in 10-50 L tap and turbid surface water samples, resulting in mean recoveries of 40%, 35%, and 50% respectively.
The automated portable concentrator was challenged with small numbers of Cryptosporidium oocysts, Bacillus anthracis spores and MS2 bacteriophages simultaneously spiked in 10 L tap and turbid surface water samples, resulting in mean recoveries of 40%, 35%, and 50% respectively. The following is the summary of the results for 10 L of tap water.
Tap water samples were obtained from the Division of Infectious Diseases, Cummings School of Vetinary Medicine. This tap water was supplied by the North Grafton Water Utility and is initially collected from a local ground water source. Turbidity of the sample was measured at 0.002 NTU by a DRT-15CE Tubidimeter, such as that manufactured by HF Scientific of Fort Meyers, Fla. Surface water samples were then collected from a near-by pond. The samples were autoclaved and the mean turbidity post sterilization was measured at 3 NTU.
Ten liters of each water matrix was transferred to sterile, disposable plastic cubitainers. The cubitainer was placed on a stir plate and stirred while being seeded with an aliquot of each representative organism and refilled with none liters of water once one liter of the original sample remained to be concentrated.
Prior to concentration the disposable harness is assembled in the machine according to the schematic layout of
After concentration, the residual liquid is subject to a bacteria and protozoa elution cycle. 5 ml of elution buffer is delivered through the inlet port of the bowl from the designated PVC bag. The bacteria/protozoa elution buffer is a 5×PBS and 0.06% of a tween 80 solution is used as a detergent to dislodge bacteria and protozoa that have compacted to the walls of the bowl during the concentration process.
Once the elution buffer has been added, the centrifuge spins to a speed of 7000 rpm and then is immediately braked until it comes to a complete stop to dislodge the bacteria and protozoa. The cycle of spinning and braking is repeated ten times with a 10 second interval between each cycle. Upon completion of the final spin and brake cycle the 275 mL eluate is pumped from the inlet port of the bowl and returned to the PVC bag that originally contained the elution buffer. The centrifuge is then spun at 9000 rpm for 1 minute to extract any residual liquid from the out-flow chamber of the core, which is subsequently pumped from the inlet to the rest of the eluate. The entire volume, approximately 300 mL, is split into two separate volumes of 150 mL for analysis of bacteria and protozoa.
Following the bacteria and protozoa elution cycle, 10 mL of virus elution buffer is pumped into the out-flow chamber of the core through the outlet port of the bowl from the designated PVC bag. The virus elution buffer is a beef extract, glycine and Tween 80 protein solution (Scientific Methods, Granger, Ind.) used to neutralize the positive charge of the filter material inside of the core and dislodge the virus that were adhered during the concentration process.
After addition of the elution buffer, the filter material inside the core is saturated for 5 minutes at which point the centrifuge is spun for 1 minute at 9000 rpm to extract the liquid from the out-flow chamber of the core into the bowl. A second volume of 10 mL of virus buffer is pumped into to out-flow chamber and the process is repeated. The cycle ends by pumping the entire volume of virus eluate, approximately 20 mL, from the inlet port to the PVC bag that originally contained the virus elution buffer.
Certain microorganisms were used. C. parvum oocysts were chosen for use as representative protozoa. Water samples were spiked with one of two commercially available products. EasySeed, produced by Biotechnology Frontiers, LTD, NSW, Australia, consists of 100 flow-sorted; gamma irradiated C. parvum oocysts and Giardia lamblia cysts with a cell count standard deviation of less than 2.5 for each microorganism. Samples were spiked with EasySeed by decanting the contents of each vial as prescribed by the manufacturer. The second spiking suspension was produced by the Wisconsin State Laboratory of Hygiene, Madison, Wis., and consists of 100 C parvum oocysts in 10 mL of reagent grade water and 0.01% Tween 20 with a cell count standard deviation of less than 2.5. The contents of one 10 mL tube was poured into the water sample and the tube was rinsed with 2 mL of 0.01% Tween 20 PBST followed by two 2 mL rinses of reagent grade water seeded the water samples. Each rinse was shaken vigorously for 30 seconds before being decanted into the water sample. Enumeration of the protozoa eluate from spiked water samples was performed as described by Method 1622 (USEPA, 2001). Dynabeads anti-Cryptosporidium Kit (Invitrogen Dynal AS, Oslo, Norway) was employed for selective separation of oocysts from water sample concentrates using immunomagnetic separation according to the manufacturers instructions. Crypt-A-Gb fluorescent monoclonal antibodies (Waterborne, Inc., New Orleans, La.) were used against C. parvum oocysts for detection and enumeration with a fluorescent microscope (Olympus Optical CO., Tokyo, Japan). The recovered numbers were multiplied by a factor of 2.
B. anthracis Sterne, a kanamycin resistant strain, was selected as representative bacteria. This strain of B. anthracis was constructed by the replacement of particular RNA coding sequences with an omega element, Q-kan, conferring kanamycin resistance. The stock suspension of B. anthracis was kept at −80° C. Each week during testing a spiking suspension was produced by diluting 100 μL of the stock suspension into 50 mL of maximum recovery diluent (Oxoid Ltd., Basingstoke, Hampshire, England) to an estimated concentration of 30 colony-forming units (cfu) per mL. Water samples were spiked by pipetting 1 mL of the spike suspension to the sample while stirring. The spike dose was quantitated by pipetting 1 mL of the spiking suspension to 50 mL of reagent grade water, vortexing and vacuum filtrating through a 0.45 μm, 47 mm membrane (Millipore, Billerica, Mass.). Post filtration, the membranes were placed on LB agar plates (Acros Organics, N.J.) containing kanamycin (Fisher Scientific, Fair Lawn, N.J.), inverted and incubated overnight at 37° C. After incubation, colony-forming units with the typical appearance of B. anthracis, opaque grey and white formations, were counted and recorded. To quantitate the recovery of B. anthracis from spiked water samples, the bacteria eluate was vacuum filtrated and incubated in the same manner. The recovered numbers were multiplied by a factor of 2.
Eschericlzia coli ATCC 15597: E. coli (American Type Culture Collection, Manassas, Va.) was used as host bacteria for MS2 bacteriophage. Freeze dried samples were purchased from ATCC, re-hydrated with 0.850 mL of LB broth (Acros Organics, N.J.) and 1 50 mL of sterile glycerol, aliquoted to a volume of 50 μL and stored at −80° C. To propagate the bacteria one 50 μL aliquot was diluted in 10 mL of LB broth and incubated overnight at 37° C. and 200 RPM on a shaking incubator. After incubation 1.5 mL of sterile glycerol was added to the host bacteria broth, which was subsequently aliquoted to a volume of 200 μL and stored at −80° C. for no longer than one month. To quantitate the recovery of MS2 bacteriophage from spiked water samples and to enumerate the spike dose of phage LB agar plates were prepared with a field of actively growing E. coli to spot the phage on. Prior to preparation of the plates 800 μL of LB broth was added to one 200 μL aliquot of overnight grown E. coli and incubated for two hours at 37° C. After incubation, the host bacteria broth was spread evenly over an LB agar plate and the excess aspirated off. Serial dilutions of phage were than spotted onto the plate immediately after the E. coli had air-dried.
Results:
Although there have been major advancement in methodologies and equipment aimed at detecting waterborne pathogens, it is imperative that no matter how good the detection method is, an efficient sample concentration is required due to the small numbers of the target microorganisms. Moreover, most of the cutting edge detection technologies utilize portable detection equipment for field testing, but lack of an efficient portable sample concentrator restricts its use. Among the several approaches for an efficient sample concentration, continuous flow centrifugation appears to be very promising.
Although the present invention has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. It is preferred therefore, that the present invention be limited not by the specific disclosure herein, but only by the appended claims.
This application is a 35 U.S.C. §371 National Stage of International Application No. PCT/US2008/066894 filed on Jun. 13, 2008, which designates the United States, and which claims the benefit of priority under 35 U.S.C. §119(e) of U.S. provisional application No. 60/934,417, filed Jun. 13, 2007, the contents of which are incorporated herein by reference in its entirety.
This invention was made with United States Government support under Grant No. RD83300301 awarded by the Environmental Protection Agency, and Grant No. W911NF-07-C-0030 awarded by the Army Research Office. The United States Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2008/066894 | 6/13/2008 | WO | 00 | 12/3/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/157337 | 12/24/2008 | WO | A |
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| Number | Date | Country | |
|---|---|---|---|
| 20100216215 A1 | Aug 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60934417 | Jun 2007 | US |
