Patent Publication Number: US-10787715-B2

Title: Methods for culture and identification of mycrobacterium avium subspecies in crohn&#39;s disease

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The instant application is a Divisional of application Ser. No. 14/803,511, filed Jul. 20, 2015, the contents of which are incorporated by reference herein in their entirety. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy is named “6219-40004_Sequence_Listing_ASCII.txt” and is 2,108 bytes in size. 
     FIELD OF THE INVENTION 
     The invention generally relates to the field of inflammatory diseases and, more particularly, to methods and cultures used for diagnosing Crohn&#39;s disease. 
     BACKGROUND 
     Crohn&#39;s disease is a chronic inflammatory disorder involving the gastrointestinal tract. Crohn&#39;s disease is currently considered an idiopathic autoimmune conditions, and immune suppressants are generally used in the treatment of the disease (1). In the past 60 years, many investigators have sought a pathogen(s) causing the disease, and various bacteria and viruses have been isolated and reported (2-4). For many years,  Mycobacterium avium  subspecies  paratuberculosis  (MAP), a known pathogen in Johne&#39;s disease, a chronic wasting condition in cattle and sheep (5) has been suspected to cause Crohn&#39;s disease (6). For example, U.S. Pat. No. 7,488,580 issued to Naser, discloses a method of diagnosing inflammatory bowel disease (including Crohn&#39;s disease) by detecting MAP. The role of MAP in Crohn&#39;s disease is controversial (7, 8) but two meta-analyses have concluded that a majority of patients with Crohn&#39;s disease have evidence of MAP infection (9, 10). 
     SUMMARY 
     MAP is a notoriously slow grower under the routine cultural conditions (6, 11). Therefore, the inventors sought to develop a more rapid culture method for MAP and/or other bacteria from the blood of Crohn&#39;s patients. As a result of this effort, the inventors have cultured  Mycobacterium avium  subspecies  hominissuis  (MAH) from the blood of a Crohn&#39;s patient and, to their knowledge, are the first to report MAH isolated from a Crohn&#39;s patient. The significance of this finding is discussed. Successful isolation and identification of MAH from the blood of a Crohn&#39;s patient validated the culture methods, media preparation and analytic processes. 
     Aspects of the invention include methods to culture and identify the presence of  Mycobacterium avium  subspecies from the blood of Crohn&#39;s patients. Aspects of the invention include culturing and identifying a  Mycobacterium avium  subspecies  hominissuis  (MAH) from the blood of a patient with Crohn&#39;s disease using novel methods, in addition to culture and identification of  Mycobacterium avium  subspecies  paratuberculosis  (MAP). A test case involved an individual with a diagnosis of Crohn&#39;s disease for two years. The individual had been treated with corticosteroids and Humira injection for six months. A blood specimen from the individual was prepared and cultured in a specialized media for four weeks, and there was visible bacterial growth in the liquid culture media. PCR (polymerase chain reaction) analysis of the bacterial growth and subsequently direct sequencing of the PCR amplicons confirmed the presence of MAH. The test case thus resulted in a first case of MAH isolated from the blood of a patient of Crohn&#39;s Disease, and successful culture and identification of MAH validated the culture media, methods and analytic processes. 
     Aspects of the invention provide many improvements over the method described in U.S. Pat. No. 7,488,580 issued to Naser. For example, Naser discloses a method that detects MAP, whereas aspects of the invention are instead directed to detecting MAH. Specifically, the PCR primers described in Naser are not capable of being used to detect MAH, and can only be used to detect MAP. Moreover, Naser employs nested PCR; in contrast, implementations of the invention may be practiced using direct sequencing. Further, Naser cultures a sample of blood consisting of the buffy coat, whereas aspects of the invention culture a sample made up of blood cells and fragments that remain after removing plasma and lysing the red blood cells, as well as sediments after centrifugation. Even further, aspects of the invention use a different culture medium than that disclosed by Naser. Specifically, Naser only uses a single medium. In contrast, embodiments of the invention may be practiced by culturing the sample with three different media including a liquid medium, a solid induction medium, and a solid maintenance medium. 
     In an aspect of the invention, there is a method of diagnosing Crohn&#39;s disease in patients, comprising: obtaining a sample from an individual; determining the presence or absence of  Mycobacterium avium  subspecies  hominissuis  (MAH) in the sample; and diagnosing the individual with Crohn&#39;s disease based on the determining the presence of MAH in the sample. 
     In another aspect of the invention, there is a composition usable as a culture medium, comprising: one of Middlebrook 7H9 and Middlebrook 7H10; Yeast extract; Glycerol; Sucrose; Tween 80; Mycobactin J; Oleic acid; and NAD. 
     In another aspect of the invention, there is a method of serology testing, comprising: serology testing plasma of an individual to identify a specific antibody against  Mycobacterium avium  subspecies  paratuberculosis  (MAP), wherein the serology testing is performed using a 12-well glass-slide for antibody titration. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
       The present invention is described in the detailed description which follows, in reference to the noted plurality of drawings by way of non-limiting examples of exemplary embodiments of the present invention. 
         FIGS. 1 a -1 d    show histologic features of colonic biopsy (AFB, acid-fast stain) in accordance with aspects of the invention. 
         FIG. 2  shows acid-fast stain of the blood culture (Oil×1000) in accordance with aspects of the invention. 
         FIG. 3  shows gel electrophoresis of PCR results of MAH isolate in accordance with aspects of the invention. 
         FIG. 4  shows modified ELISA assay using slide format for patient testing in accordance with aspects of the invention. 
         FIG. 5  shows PCR primer sequences in accordance with aspects of the invention (SEQ ID NOS 1-8, respectively, in order of appearance). 
         FIG. 6  shows steps of a method in accordance with aspects of the invention. 
     
    
    
     DETAILED DESCRIPTION 
     The particulars shown herein are by way of example and for purposes of illustrative discussion of the embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show structural details of the present invention in more detail than is necessary for the fundamental understanding of the present invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the present invention may be embodied in practice. 
     The invention generally relates to the field of inflammatory diseases and, more particularly, to methods and cultures used for diagnosing Crohn&#39;s disease. According to aspects of the invention, Crohn&#39;s disease may be diagnosed in a patient by: taking a sample from a patient having symptoms of Crohn&#39;s disease; detecting the presence or absence of MAH in the sample; and diagnosing Crohn&#39;s disease in the patient based on detecting the presence of MAH in the sample. In embodiments, the detecting the presence or absence of MAH in the sample is performed using a novel culture method that uses novel recipes (e.g., compositions) of three different culture media. In implementations, the culture method involves taking a whole blood sample from the patient, removing the plasma proteins from the blood, lysing the red blood cells, and only using the cellular particles from the blood with the culture media. 
     Methods: 
     In the test case described herein, an individual with a history of Crohn&#39;s disease for two years had been in and out of hospital for flares and currently on Humira twice a month for the past six months. The individual&#39;s symptom was not fully controlled and a visit was to work up for worsening of abdominal pain and cramping. Colonoscopy was performed by the gastroenterologist, and active disease was seen during the procedure. A random colon biopsy shows chronic active colitis with cryptitis, crypt abscesses and increased lymphocytic infiltrate in the lamina propria. A blood culture was performed in the specialized media based on the Middlebrook 7H9 with supplements of OADC and mycobactin J as the following, and one species of MAH was isolated from the liquid media. The identity of the MAH was confirmed by PCR of IS1245, 16S rDNA and the direct DNA sequencing of the PCR amplicons. 
     Media Preparation: 
     There are three types of media developed in our own lab using the Middlebrook 7H9 and 7H10 (Fisher Scientific/BD Biosciences) as the basic components: liquid maintenance media or liquid media, solid maintenance media and solid induction media (plates and/or slants). The recipes of the three different media are as the following in Table 1. In embodiments, for each respective medium, the constituents listed in Table 1 are mixed in water (e.g., Mili-Q water). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 (weight/volume or volume/volume) 
               
            
           
           
               
               
               
            
               
                 Liquid Media 
                 Solid Induction Media 
                 Solid Maintenance Media 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Middlebrook 7H9 
                 0.47%  
                 Middlebrook 7H10 
                 1.9% 
                 Middlebrook 7H10 
                 1.9% 
               
               
                 Yeast extract 
                 0.1% 
                 Yeast extract 
                 0.1% 
                 Yeast extract 
                 0.1% 
               
               
                 Glycerol 
                 0.5% 
                 Glycerol 
                   3% 
                 Glycerol 
                 0.5% 
               
               
                 Sucrose 
                 0.2% 
                 Sucrose 
                  20% 
                 Sucrose 
                 0.2% 
               
               
                 Tween 80 
                 0.05%  
                 Tween 80 
                 0.05%  
                 Tween 80 
                 0.05%  
               
               
                 Mycobactin J 
                  2 μg/ml 
                 Mycobactin J 
                  2 μg/ml 
                 Mycobactin J 
                  2 μg/ml 
               
               
                 Oleic acid 
                 0.1% 
                 Oleic acid 
                 0.1% 
                 Oleic acid 
                 0.1% 
               
               
                 NAD 
                 20 μg/ml 
                 NAD 
                 20 μg/ml 
                 NAD 
                 20 μg/ml 
               
               
                   
               
            
           
         
       
     
     All the media are prepared by dissolving the various components in the Mili-Q water, and autoclaved at 121° C. for 15 minutes. The media are cooled to 55° C. before OADC (oleic acid, albumin, Dextrose, catalase, NaCl from BD Bioscience Inc, 10%) and the antibiotics mix (Azlocillin 10 μg/ml, polymyxin B 10 μg/ml, amphotericin B 10 μg/ml) are added. Media plates and/or slant tubes are made. The media is stored at 4° C. refrigerator for minimally 24 hours before use. 
     As used herein, Middlebrook 7H9 is a liquid growth medium comprising: Ammonium Sulfate; L-Glutamic Acid; Sodium Citrate; Pyridoxine; Biotin; Disodium Phosphate; Monopotassium Phosphate; Ferric Ammonium Citrate; Magnesium Sulfate; Calcium Chloride; Zinc Sulfate; and Copper Sulfate. As used herein, Middlebrook 7H10 is a solid growth medium comprising: Ammonium Sulfate; Monopotassium phosphate; Disodium phosphate; Sodium Citrate; Magnesium Sulfate; sodium; Zinc Sulfate; Copper Sulfate; L-Glutamic Acid (sodium salt); Ferric Ammonium Citrate; Pyridoxine Hydrochloride; Biotin; Malachite Green; and Agar. Further, as is understood by those of skill in the art, Tween 80 is another name for Polysorbate 80, and NAD stands for Nicotinamide adenine dinucleotide. 
     Culture Method: 
     The basic outlines of the methods for culture and identification of bacterial growth are the following in a stepwise manner. The detailed description of the methods is followed after the outlines. 
     1: Transfer 4 ml blood from the purple/lavender-top vacutainer tube to a sterile 15-ml centrifuge tube, and spin the blood in a centrifuge at 6000 g for 10 minutes at room temperature. 
     2: Remove the plasma using a sterile pipette and store plasma in a 2-ml microfuge tube at −20° C. for antibody titration later. 
     3: Add 10-ml red blood cell lysis buffer (ammonium chloride 0.8%, sodium bicarbonate 0.08% and disodium EDTA 0.037%) to the cells at room temperature, and resuspend the cells by turning the capped tube up and down several times. 
     4: Spin the tube at 6000 g for 10 minutes in a centrifuge, and discard the supernatant to a biohazard container. 
     5: Add 4-ml liquid media to the centrifuge tube, and resuspend the cell pellet by turning the capped tube up and down. 
     6: Remove two aliquots of 100 μl the resuspended cells in liquid media, and plant them in one solid induction plate/slant and one solid maintenance plate/slant. 
     7: Incubate all the culture tube and plates/slants at 37° C. for 2 weeks without additional CO2. The liquid culture tube, solid plates/slants are examined visually every week. The culture media are kept at 37° C. for minimally 12 weeks. 
     8: After the 2 week incubation, an aliquot of 100 μl liquid culture is removed by a sterile pipette, and the culture is transferred to a microfuge tube. The cells are collected by centrifugation at 12000 g for 5 minutes. Discard the supernatant. 
     9: Resuspend the cells with 500 μl acetone. In embodiments, the cells are fully resuspended at this step. 
     10: Collect cells by centrifugation at 12000 g for 5 minutes. Discard the supernatant. 
     11: Resuspend the cells in 200 μl sterile TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6) by vortexing the tube, and heat the resuspended cells at 95° C. for 15 minutes. 
     12: Chill the tubes on ice, and use the content directly for PCR analysis. 
     13: If there are colonies on the solid plates/slants, a single colony can be picked by a sterile toothpick, and resuspended in 200 μl TE buffer. The bacteria in TE buffer is heated at 95° C. for 15 minute and it is directly used for PCR analysis. 
     DETAILED DESCRIPTION OF THE METHOD ABOVE 
     One 4-ml purple/lavender-top tube (EDTA or sodium citrate) of blood is transferred to 15-ml centrifuge tube and centrifuged at 6000 g for 10 minutes at room temperature to separate the plasma and the cellular components including red blood cells, white blood cells, platelets and any particles. The plasma is removed from the centrifuge tube by using sterile long tip pipette, transferred to a 2 ml Eppendorf tube and stored at −20° C. freezer for MAP antibody titration using the whole cell extract of commercial strain of MAP (MAP Dominic, ATCC cat. #43545). Sterile red blood cell lysis buffer (ammonium chloride buffer containing 0.8% ammonium chloride, 0.08% sodium bicarbonate, 0.037% disodium EDTA) is added to the centrifuge tube in 4× volume (10 ml), and the cellular blood components are resuspended in the red cell lysis buffer by turning the capped tube up and down until no visible clumps are present. The nucleated cells are collected by centrifugation at 6000 g for 10 minutes, and the lysed red cells in the supernatants are emptied to a biohazard container. The nucleated cell pellet is resuspended in a 4 ml liquid maintenance media described above. Two aliquots of the resuspended nucleated cells in liquid media are removed with sterile pipette and planted on two separate solid media plates (or slants), one for induction of cell wall deficient form of MAP/MAH (spheroplasts) on the solid induction media and one for solid maintenance media. All blood culture media are incubated at 37° C. incubator with 80% humidity without additional CO2. After 2 weeks, an aliquot (0.1 cc) of liquid culture is removed and the cellular components of the culture including the nucleated cells from the blood during the 2 weeks culture period is collected by centrifugation at 12000 g for 5 minutes. The cultured material is washed with PBS buffer (pH 7.4) once and resuspended with 100% acetone. The cultured material is collected by centrifugation at 12000 g for 5 minutes, and resuspended in 200 μl TE buffer (10 mM tris, 1 mM EDTA, pH 7.6). The resuspended bacteria is heated to 95° C. for 15 minutes and it is directly used for PCR analysis. Alternatively, proteinase K (6 units) and 0.5% SDS (final concentration) are added and incubated at 65° C. overnight. The genomic DNA is isolated by phenol/chloroform/isoamyl-alcohol extraction and alcohol precipitation as described elsewhere (12). The genomic DNA was resuspended in 200 μl TE buffer, and 1 μl genomic DNA is used for PCR amplification in 50 μl volume using primers specific for IS900, IS901, IS1245, and 16s rDNA ( FIG. 5 )(7, 13-15). The PCR amplification is performed using 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 45 seconds for 40 cycles using the PCR Core-kit (Sigma Aldrich). The PCR amplicon is visualized on 1.2% agarose gel electrophoresis. The PCR amplicons are subjected to direct DNA sequencing analysis by Eurofins Genomics sequencing services, Louisville, Ky. and the amplified DNA sequences are submitted for BLAST analysis against Genebank nucleotide sequences at NCBI (http://blast.ncbi.nlm.nih.gov/). 
     The liquid and solid media cultures are kept for 12 weeks with visual examination every week. Visible growth of liquid culture or bacterial colonies on the solid media are re-examined by PCR analysis and subsequent DNA sequencing analysis. 
     Comment on the Above Method: 
     Aspects of the method described herein are different from other published methods for blood culture. Traditionally in the hospital setting, blood culture is to directly add the whole blood to blood culture media bottle or plates commercially available from various vendors. The whole blood contains the blood cells and platelets as well as the serum/plasma proteins including immunoglobulins and antibodies that may inhibit the bacterial growth. An embodiment of a method described herein removes the plasma proteins, lyses the red blood cells, and only uses the cellular particles from the blood. An embodiment of a method described herein is also superior to culturing the buffy coat layer of the blood, since aspects of the inventive method include collecting the cellular particles from the lysed red blood cells. Some microorganisms grow in the red blood cells, such as Lymes disease, and an embodiment of a culture method described herein can capture the microorganisms from the red blood cells as well as white blood cells and platelets or any microorganisms in the plasma. 
     An embodiment of a method described herein has been used successfully to isolate MAH and other cell wall deficient bacteria from the blood of Crohn&#39;s patients. 
     Serology Testing Design: 
     In accordance with aspects of the invention, the serology testing is to identify the presence or absence of circulating antibody against  Mycobacterium avium  subspecies  paratuberculosis  (MAP) using the whole cell extract antigens prepared from the standard human MAP isolate (MAP Dominic, ATCC #43545). The whole cell extract of MAP Dominic strain is prepared as described (16). The MAP strain is cultured using the liquid media for 4-8 weeks, and the mycobacteria are collected by centrifugation at 6000 g for 10 minutes. The cell pellet is washed once with PBS, and resuspended in 100% acetone. The cell pellet again is collected by centrifugation at 6000 g for 10 minutes, and resuspended in 1% SDS in sterile water as described (16). The total whole cell extract is stored at −20° C. freezer before using to coat the 96-well titration plate or 12-well custom made glass-slide for antibody titration as below. 
     The titration using the 96-well plate format is performed as described elsewhere (6). Utilization of 12-well glass slide format is new, and it is easier to wash the slides in Copland jar than the 96-well plate. The basic antigen coating, blocking, and washing using PBS buffer with Tween 20 are identical to those described (12). 
     Result: 
     The random colon biopsy shows chronic active colitis characterized by the presence of cryptitis, crypt abscesses and increased lymphocytic infiltrate. In addition, there are increased eosinophils within the lamina propria ( FIG. 1 a - c   ). Small loosely formed granuloma are identified. Acid fast stain is performed, and there appears to be small oval/rod acid fast bacteria within the histiocytes and the lamina propria ( FIG. 1 d   ). The histologic changes are compatible with Crohn&#39;s disease. After 4 weeks culture, a visible growth within the liquid media is present, and acid fast stain (Ziehl-Neelsen stain) reveals a spheroplast form of bacteria with partial acid-fast features ( FIG. 2 ). PCR analysis of the isolate using primers in  FIG. 5  revealed the presence of IS1245, and 16S rDNA ( FIG. 3 ). PCR primers for 16S rDNA and IS1245 produced distinct bands ( FIG. 3 ), and the amplicon sequence using 16s rDNA primers matched both MAP (100%, GenBank: CP010114.1) and MAH (100% GenBank AP012555.1), but the amplicon sequence using IS1245 primers only matched MAH (99%, GenBank AP012555.1). PCR using primers from IS900 was negative, indicating the isolate was not MAP. Thus, in embodiments, PCR is used to determine the presence of an MAH gene, and thus indicate MAH in the blood (sample). Based on a positive indication of MAH in the sample, the patient may be diagnosed with Crohn&#39;s diseases and treated accordingly. In addition to PCR, serology may be used to determine that there is an antibody in patient&#39;s circulation now against MAP/MAH, which may indicate the past exposure, and no MAP/MAH in the blood. There is cross-reactivity for MAP and MAH, i.e., both will react with the same antibody. Current serology cannot distinguish MAH from MAP. It is also possible that a patient may have positive PCR and not serology, or vice versa. 
     Serology testing of the patient&#39;s plasma using both the 96-well format and the glass-slide format showed a specific antibody against MAP/MAH whole cell antigen with a titer of 1:8. The design of the glass-slide ELISA format is showed in  FIG. 4 . The advantage of glass-slide format for ELISA or modified ELISA is that the slide holds much less reagent volume (20 μl in contrast to 100 μl) than the 96-well plate well. The slides are much easier to wash using a Copland jar with the washing buffer than a 96-well plate. In a clinical setting, the glass-slide format save testing time, and generate results faster. 
       FIG. 6  shows steps of a method in accordance with aspects of the invention. Step  601  includes obtaining a sample from an individual. This may include obtaining a blood sample, such as a whole blood sample. Step  602  includes preparing the sample. In embodiments, the preparing includes removing plasma proteins from the sample; and lysing red blood cells of the sample. Step  603  includes culturing the prepared sample, e.g., after removing the plasma proteins and lysing the red blood cells. The culturing may include one or more of the steps described herein, e.g., using liquid and solid media. Step  604  includes determining the presence or absence of MAH in the (prepared) sample. In embodiments, the determining includes using PCR analysis to detect the presence of MAH gene in the sample. For example, the PCR primers described herein may be used to detect the presence of IS1245 and 16S rDNA in an isolate of the cultured sample. The determining may also include determining that the isolate is not MAP, e.g., using PCR to determine that primers from IS900 are negative. Step  605  includes serology testing of the plasma that was removed from the sample to determine the presence of a specific antibody against MAP/MAH. Step  606  includes diagnosing the individual with Crohn&#39;s disease based on the determining the presence of MAH in the sample, e.g., based on the determining. Step  607  includes treating the patient for Crohn&#39;s disease based on the diagnosing. The treating may include conventional and/or heretofore developed treatments for Crohn&#39;s disease. The treating may include providing the patient with an anti-MAH effective amount of an anti-MAH compound and a pharmaceutically acceptable excipient therefor. The anti-MAH compound may include an antibiotic, and the effective amount may be an amount sufficient to eliminate MAH from registering positive in a blood test. 
     Discussion 
     MAP has been isolated from Crohn&#39;s patients for the last 3 decades and there are conflicting data about the presence of MAP in Crohn&#39;s patients (8, 17). MAH is one of the closest members of  Mycobacterium avium  subspecies to MAP, in additional to  Mycobacterium avium  subspecies  avium  (MAA) and silvaticum (MAS)(18). MAH is present in the environment such as soil and water, and it is a known pathogen in animals such as pig, dog, deer and horse (18-20). There are reports of MAH isolates in human from the lymph nodes with lymphadenitis (21). There is no report to date of MAH from Crohn&#39;s patients to our knowledge. There is a report of MAH isolated from gastrointestinal tract of deer (22). The presence of MAH in human Crohn&#39;s patient raises questions of the role of MAH in the Crohn&#39;s disease process. It is possible that MAH is a secondary infectious agent in the patients with immune suppressants (opportunistic pathogen). It is equally possible that MAH is the pathogenic agent in this patient of clinically Crohn&#39;s disease. In fact, MAH is a more common isolate from cancer patients, but the clinical manifestation of MAH in cancer patients are mostly respiratory/pulmonary (23). Regardless the role of MAH in pathogenesis of Crohn&#39;s patient, the presence of MAH in the blood of these patients indicates a Mycobacteremia, and such a condition will require antibiotic treatment of the patients. 
     Genetically there are multiple gene allelic mutations susceptible for Crohn&#39;s disease, and there is a significant overlap of genetic susceptibility loci to Crohn&#39;s disease and those to mycobacteria infections (24). These genetic loci susceptible to mycobacterial infections include  Mycobacterium tuberculosis  and  Mycobacterium leprosy . Other mycobacterial infections are possible due to the host genetic changes of Crohn&#39;s disease. It remains possible MAH is one of the causative agents of Crohn&#39;s disease. 
     It is noted that the foregoing examples have been provided merely for the purpose of explanation and are in no way to be construed as limiting of the present invention. While the present invention has been described with reference to an exemplary embodiment, it is understood that the words which have been used herein are words of description and illustration, rather than words of limitation. Changes may be made, within the purview of the appended claims, as presently stated and as amended, without departing from the scope and spirit of the present invention in its aspects. Although the present invention has been described herein with reference to particular means, materials and embodiments, the present invention is not intended to be limited to the particulars disclosed herein; rather, the present invention extends to all functionally equivalent structures, methods and uses, such as are within the scope of the appended claims. 
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