WO2005116243A2 - Improvements to diagnostic assays that use mycobacteriophages - Google Patents
Improvements to diagnostic assays that use mycobacteriophages Download PDFInfo
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- WO2005116243A2 WO2005116243A2 PCT/US2005/014098 US2005014098W WO2005116243A2 WO 2005116243 A2 WO2005116243 A2 WO 2005116243A2 US 2005014098 W US2005014098 W US 2005014098W WO 2005116243 A2 WO2005116243 A2 WO 2005116243A2
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- the present invention is in the area of microbiological sample processing. Specifically, the present invention is directed to methods for improving the utility and performance of diagnostic assays that use mycobacteriophage to determine the presence of microorganisms that contain mycolic acid structures in their outer membranes in specimens being processed for clinical analysis. The present invention thus facilitates detection of microorganisms that contain mycolic acid structures in their outer membranes in clinical samples.
- Mycobacteriophage are bacteriophages that specifically infect mycobacteria. Such phages can be either lysogenic (i.e., virulent, causing efficient and complete lysis of the infected host), or temperate (i.e., do not cause lysis, but permit the bacillus to exist in a chronically infected state).
- Lysogenic strains of bacteriophages have been used to develop "plaque assays.”
- the principle underlying such plaque assays is that when a bacterium infected with a lysogenic bacteriophage is plated on a Petri dish with an uninfected bacterium (e.g., a helper strain) that is also susceptible to infection by the same phage, small clearings (or plaques) will form where the original bacterium resided as a result of infection and lysis of the helper strain. Diagnostic assays have been developed on this principle, and are based on the specificity of such bacteriophages to infect specific hosts.
- a Mycobacterium e.g., Mycobacterium tuberculosis
- mycobacteriophages By treating clinical samples suspected of harboring a Mycobacterium (e.g., Mycobacterium tuberculosis) with a lysogenic strain of mycobacteriophage, removing excess phage, and then plating such treated samples with the appropriate helper cells (e.g., Mycobacterium smegmatis), the occunence of plaques are diagnostic for the presence of Mycobacterium in the original clinical sample.
- helper cells e.g., Mycobacterium smegmatis
- tuberculosis is the most prevalent infectious disease in the world today, infecting approximately one-third of the world's population, and killing more people worldwide than any other single pathogen. The vast majority of these cases are in developing countries where resources are severely limited. Hence, there is a need for a rapid and inexpensive diagnostic assay to identify infected individuals quickly and accurately. Mycobacteriophage assays offer such promise.
- Diagnostic assays that use mycobacteriophage to detect the presence of mycobacteria in biological and inorganic samples must first be processed to prepare such samples for said assays.
- Cunent methods of processing biological and inorganic samples suspected of containing one or more mycobacteria, for the detection of such mycobacteria by mycobacteriophage- based assays, are constrained by the harshness of these methods.
- the contemporary methods are based primarily on the utilization of caustic acids and alkalis, such as sodium hydroxide (NaOH), sulfuric acid, and oxalic acid.
- Such specimen processing methods are necessary to decontaminate clinical specimens to alleviate contaminating pathogenic and saprophytic microorganisms that would interfere with culturing of mycobacteria; however, such methods also kill as much as 90% of the mycobacteria present, and more importantly, remove receptors from the surface of the few remaining bacilli that are essential for the infection of such mycobacteria by these mycobacteriophages. Consequently, additional preparative steps following the standard specimen processing methods are required prior to such plaque assays. For example, processed sediments must be washed to remove excess acids or alkalis. Such wash step(s) necessitate that the processed specimen be subjected to centrifugation a second time - an extremely labor intensive procedure.
- mycobacteria that have been treated with such acids or alkalis must be allowed to recuperate prior to infection: bacilli must be cultured in nutrient broth to recover from exposure to caustic agents and to restore the requisite receptors that permit infection by mycobacteriophage. This significantly lengthens the time needed to obtain the final result.
- bacilli must be cultured in nutrient broth to recover from exposure to caustic agents and to restore the requisite receptors that permit infection by mycobacteriophage. This significantly lengthens the time needed to obtain the final result.
- Procedures designed to reduce the influence of specimen processing methods on diagnostic utility would further improve the ability to conectly diagnose infections caused by bacteria containing mycolic acid structures, especially the diagnosis of mycobacterial infections, and most specially infections caused by Mycobacterium tuberculosis.
- compositions and methods of the invention preclude the need for washing processed sediments to remove caustic acids and alkalis, resulting in a significant savings in labor associated with performing such assays.
- the compositions and methods of the invention also retain the viability of microorganisms that contain mycolic acid like structures in their outer membranes to a degree that eliminates completely the need to culture such microorganisms prior to detection in said mycobacteriophage assays. This significantly decreases the time necessary to obtain a result, as compared to the mycobacteriophage assays in the art.
- compositions and methods of the invention reduces, or completely eliminates, the need to incorporate antibiotics in the media used to propagate the helper strain, thereby further reducing the expense associated with performing said mycobacteriophage assays.
- These compositions and methods are especially useful for the processing of samples for the detection of mycobacteria using mycobacteriophage plaque assays.
- Figure 1 The experimental procedure used to evaluate the consequence of exposure of Mycobacterium tuberculosis ATCC 27294 to CB- 18 in the context of a mycobacteriophage assay is shown.
- Figure 2 The experimental procedure used to evaluate the effect of the presence of CB-18 on Mycobacterium tuberculosis ATCC 27294 during infection by the mycobacteriophage D29 is shown.
- Figure 3 The experimental procedure used to evaluate the effect of combining exposure to CB-18, and carrying CB-18 into the infection buffer in the context of a mycobacteriophage assay is shown.
- Figure 4 The experimental procedure used to evaluate the consequence of exposure of Mycobacterium tuberculosis ATCC 27294 to lytic enzymes before infection in the context of a mycobacteriophage assay is shown.
- the invention described herein uses "betaine-like" detergents as described in U.S. 5,658,749, U.S. 6,004,771, and WO 95/27076 to process clinical samples.
- there is no need to wash the sediment or pellet to remove undesirable caustic agents and there is no need to culture (pre-culture) the sample containing the mycobacteria prior to a desired plaque assay.
- pre-culture the sample containing the mycobacteria prior to a desired plaque assay.
- the invention has significant advantages in that the methods greatly reduce the labor required to prepare such samples by eliminating the need to wash processed sediments
- mycobacteriophage is meant a virus that specifically infects microorganisms of the genus Mycobacterium, as understood in the art (Hatfull, et al, Mycobacteriophages: Cornerstones of Mycobacterial Research. In: Bloom, B.R., ed. Tuberculosis: Pathogenesis, Protection and Control. Washington, D.C., American Society for Microbiology Press, (1994) pp. 165- 183; Sarkis, et al. Methods Mol. Biol 101 :145 (1998); Hatfull, G.F., Mycobacteriophages. In: Ratledge, C. and Dale, J., eds.
- mycobacteriophages include "LI”, “L5", “D29”, “TM4", “Bxbl”, “DS6A”, and “13”, as well as those that are genetically engineered, such as "phAE40", “phGS18”, and "phBD ⁇ .”
- plaque assay is meant a diagnostic assay wherein a mycobacteriophage is mixed with a prepared sample to facilitate infection of the target microorganism (e.g., Mycobacterium tuberculosis) by said mycobacteriophage. After removal of such mycobacteriophage by methods known in the art (e.g., treatment with fenous ammonium sulfate (McNerney, R., et al, Res. Microbiol. 149:487 (1998)), said sample is then mixed with helper cells and permitted to incubate for a time and at a temperature that facilitates production of a helper cell lawn as understood in the art.
- fenous ammonium sulfate McNerney, R., et al, Res. Microbiol. 149:487 (1998)
- the reproduction of phage in the context of the plaque assay results from primary infection in said sample and subsequent infection of said helper cells that form the lawn. Propagation of mycobacteriophage in the helper cell population results in small clearings or plaques as understood in the art. The observation of plaques is said to be confirmatory or diagnostic for the presence of the target microorganism in the clinical sample.
- helper strain or "helper cells” is meant a microorganism (e.g., Mycobacterium smegmatis) that is closely related to the target microorganism, and that may be infected by the mycobacteriophage used in the plaque assay.
- the function of such helper cells is to generate an opaque lawn as understood in the art, such that the occunence of a primary infection in the form of plaques on said lawn are more easily observed.
- lysogenic is meant a strain of mycobacteriophage that is capable of lysing the target microorganism and the helper cells, the latter of which is necessary to produce the characteristic clearings in the plaque assay.
- Betaine-like is synonymous with "SB-18-like” as used in WO 95/27076, incorporated herein by reference.
- Betaine-like detergents according to WO 95/27076 have the ability to disperse cords (and clumps) of mycobacteria and/or compensate buoyancy of the mycobacteria. Dispersion of mycobacteria that cord, such as, for example, Mycobacterium tuberculosis complex (MTB) organisms, facilitates detection by increasing the probability that aliquots taken for detection be representative of all the types of the whole sample.
- Betaine-like detergents that disperse cords have an alkyl chain length that is greater than 16 carbon atoms, and alkyl chains with 18-20 carbon atoms are most prefened.
- Betaine-like detergents also have the ability to facilitate collection of mycobacteria, such as, for example, Mycobacterium avium complex (MAC) organisms, that do not grow in clumps, by compensating, to some degree, the natural buoyancy of such organisms. Such compensation preferably occurs by a mechanism that involves movement of the detergent into the bacterial cell. Betaine-like detergents that compensate buoyancy preferably have an alkyl chain length greater than 12 carbon atoms, and most preferably 16-20 carbon atoms. [0019] Therefore, "betaine-like,” as used herein includes structures as described in Tables 2 and 3 of WO 95/27076, U.S. 5,658,749, and U.S.
- betaine-like is meant a zwitterionic compound of the structure shown in Table 1.
- Table 1 The Structure of Alkyl Betaines The general structure of n-alkyl betaines is shown.
- Ri is the hydrophobic alkyl chain, and ⁇ is the “linkage” connecting Ri to the cation, ⁇ .
- R and R 3 modify the cation, when required.
- R is the "bridge” that connects the cation to the anion, ⁇ .
- CB-like is meant those betaine-like detergents having a carboxylate (-COO ⁇ ) moiety as the anion (e.g., carboxybetaine-like).
- SB- like is meant those betaine-like detergents having a sulfonate (-SO 3 ⁇ ) moiety as the anion (e.g., sulfobetaine-like).
- HSB-like is meant those betaine- like detergents having a sulfonate moiety as the anion, and a hydroxyl group (- OH) in the bridge (e.g., hydroxysulfobetaine-like).
- PB-like betaine-like detergents having either a phosphate (-OPO 3 ⁇ ), phosphonate (-PO 3 ⁇ ), or a phosphinate (-PO 2 ⁇ ) moiety as the anion (e.g., phosphobetaine- like).
- StB-like betaine-like detergents having a sulfate (- OSO 3 ⁇ ) moiety as the anion (e.g., sulfatobetaine-like).
- AO-like is meant those betaine-like detergents having an oxide radical (-O ⁇ ) as the anion (e.g., amine oxide-like).
- PhB-like is meant those betaine-like detergents having a phosphonium (— P — ) moiety as the cation (e.g., phosphoniumbetaine-like).
- SoB-like is meant those betaine-like detergents having a sulphonium (— S ⁇ — ) moiety as the cation (e.g., sulphoniumbetaine-like).
- n-alkyl betaine is meant those betaine-like detergents having an ammonium (— N ⁇ — ) moiety as the cation (e.g., n-alkyl betaine-like).
- ImB-like is meant those betaine-like detergents having a imidazolinium moiety as the cation (e.g., imidazoliniumbetaine-like).
- RevB-like is meant those betaine-like detergents wherein the alkyl chain is covalently attached to the anion, as opposed to the cation (e.g., reverse betaine-like).
- cAB-like is meant those betaine-like detergents wherein the alkyl chain is covalently attached to the bridge, as opposed to either the cation or the anion (e.g., c-alkyl betaine-like).
- CB-18 is meant N-(3-carboxypropyl)-N,N-dimethyl-l- octadecanaminium, inner salt.
- CB-18 is also known as N,N-dimethyl-N-(n- octadecyl)-N-(3-carboxypropyl) ammonium inner salt, or C ⁇ 8 - carboxypropylbetaine.
- CB-18 has been assigned the CAS ® No. 78195-27-4.
- SB-18 N-octadecyl-N,N-dimethyl-3-ammonio-l- propane-sulfonate (CAS ® No. 13177-41-8).
- SB-16 N-hexadecyl-N,N-dimethyl-3-ammonio-l- propane-sulfonate (CAS ® No. 2281-11-0).
- SB-14 N-tetradecyl-N,N-dimethyl-3-ammonio-l- propane-sulfonate (CAS ® No. 14933-09-6)
- SB-12 N- dodecyldecyl-N,N-dimethyl-3-ammonio- 1 -propane-sulfonate (CAS ® No.14933-08-5).
- mycolic acid structures chemical compounds that can be described as a ⁇ -hydroxy acid substituted at the ⁇ -position with a moderately long aliphatic chain, as understood in the art (Goren, M.B. Bad. Rev. -? ⁇ 5:33-64 (1966), incorporated herein by reference). The term is synonymous with “mycolic acid- like structures.” Mycolic acid structures are also collectively termed “mycolic acids.” Additional tables of representative mycolic acid structures, including some that are unsaturated, cyclopropanoid, methoxylated and ketonic acids, may also be found, for example, in Lederer, E. Chem. Phys, Lipids 7:294-315 (1967); Lederer, E.
- Mycolic acid structures are acid-stable molecules. Examples of classes of microorganisms that contain mycolic acid structures in their outer membranes would be Mycobacterium, Nocardia, Corynebacterium, and Rhodococcus, as understood in the art.
- Good buffer an aqueous solution containing a chemical compound that resists pH changes as understood in the art (Beynon, R.J., et al, Buffer Solutions, The Basics. ERL Press, New York (1996), incorporated herein by reference). Such buffers serve to stabilize the hydronium ion concentration of aqueous solutions that are used in the biological, biomedical and biochemical arts. There are numerous Good buffers useful in the methods and compositions of the invention.
- Such Good buffers are named more because of the original description of a series of buffers by Good, N.E., et al, Biochemistry 5:467-477 (1966) (incorporated herein by reference) that were considered to possess attractive qualities for use in the biological, biomedical and biochemical arts, than for the fact that they were "good” choices for use in the biological, biomedical and biochemical arts.
- Good buffers useful in the methods of the invention include those that are carboxylic acid- based, alcohol-substituted amines, and sulfonic acid-based, and especially those sulfonic acids that are alcohol-substituted, cyclohexyl-substituted, morpholino-substituted, and piperazine-substituted.
- contaminant is meant a living or detectable microorganism, for example, a bacterium, a fungus or mold, or yeast, as understood in the art (Manual of Clinical Microbiology 6 th Edition, Munay, P.R. et al, eds. ASM Press, Washington, D.C. (1995), incorporated herein by reference), that is present at a detectable level in a preparation and is other than a desired microorganism that is of interest that contain mycolic acid structures in its outer membrane.
- lytic enzyme is meant an enzyme, as understood in the art, and as described in U.S. 5,985,593 (incorporated herein by reference), that has enzymatic activity against the components of the outer membrane, cell wall, capsid or capsular structures of contaminating microorganisms. That is to say that the substrates of such lytic enzymes are present in the components of the outer membrane of such contaminants. Lytic enzymes are said to have “lytic activity.” Such lytic activity in the methods of the invention serves to destabilize the structural integrity of such contaminant.
- the outer membrane is an essential aspect of structural integrity and/or viability of the contaminant, destabilizing said outer membrane matrices causes an inherent change in the ability of the contaminants to remain physically intact, and/or to continue to survive (e.g., maintain viability).
- treatment or “treating” is meant to incubate specimens with, expose specimens to, or otherwise cause the specimen to come in contact with enzymes, proteins, chemicals, or inert substrates under conditions that serve to reduce the complex nature of specimens (e.g., to liquefy specimens, solubilize components, and/or remove inhibitors).
- enzymes such as for example, proteases, glycosidases, and/or DNase's serves to cleave, degrade or digest proteins, polysaccharides, and/or DNA, respectively, in the specimen matrix as understood in the art (i.e., "enzymatic digestion") to liquefy specimens and solubilize such digested components.
- DTT dithiotheritol
- NALC N-acetyl-L- cysteine
- BME ⁇ -mercaptoethanol
- Cross linked polymers such as Sephadex ® , cellulose, or ion exchange resins, as understood in the art, can be used to either remove components of the specimen matrix by chromatographic means, or clarify the specimen matrix by physically separating soluble from insoluble components, or both (i.e., "purify" the specimen).
- Specimen is meant a material from which a sample can be obtained for a desired analysis. Specimens include, but are not be limited to, biological samples and inorganic samples.
- biological sample is meant a sample derived from, or taken from, a specimen of biological origin, such as a specimen taken from an animal (including human) or plant.
- Biological samples can be derived from, or taken from any part of the biological organism.
- Biological samples include, but are not limited to any biological liquids and solids, for example, expectorated matter (for example, sputum, saliva and phlegm), bronchial lavages and analogous respiratory washings, feces, tissue samples including skin samples, gastric aspirates, urine, tears, perspiration, blood and cerebral spinal fluid (CFS).
- CFS cerebral spinal fluid
- Any animal species may be used as a source for such samples, including but not limited to ruminant animals (such as members of the bovine family (bulls, ox, buffalo, cattle, cows, etc.) or members of the ovine family (sheep, etc.)), pigs, fish, members of the avian family (birds), badgers, deer, elk, cats and dogs.
- ruminant animals such as members of the bovine family (bulls, ox, buffalo, cattle, cows, etc.) or members of the ovine family (sheep, etc.)
- pigs, fish members of the avian family (birds)
- badgers deer
- elk members of the avian family
- cats and dogs include a specimen taken from a processed or an unprocessed food source.
- processed or unprocessed food sources include, for example, a specimen derived from meat, diary products (especially, for example, eggs, cheese and milk), plants and processed food
- Food sources also include animal feed, for example, cattle feed, silage, hay, alfalfa bales, and food samples from pastures.
- animal feed for example, cattle feed, silage, hay, alfalfa bales, and food samples from pastures.
- biological sample is also intended to include specimens taken from a cell culture source (such as monocyte or fibroblast cultures).
- inorganic sample is meant a sample derived from, or taken from, a non-biological specimen, such as, for example, from an environmental source such as soil, mud, sludge, water, sawdust and air.
- sample that is "derived from" a specimen or extract thereof is meant a sample that is directly taken from or otherwise indirectly prepared from such specimen or extract thereof.
- antibiotic is meant a compound that has a deleterious effect on the viability, integrity, or competence of a contaminant, as understood in the art (see: Munay, P.R. et al, eds. Manual of Clinical Microbiology, ASM Press, Washington, D.C. (1995) pp.1281-1307, 1385-1404, and 1405-1414; Kucers, A. et al, The Use of Antibiotics 4 ed. J.B. Lippincott Co. Philadelphia, PA (1987); and Lorian, V. ed. Antibiotics in Laboratory Medicine 2 nd Edition, Williams & Wilkins, Baltimore, MD, all incorporated herein by reference).
- antibiotic is synonymous with "antimicrobial,” as used herein.
- a sample or clinical specimen that is suspected of harboring a microorganism that contains mycolic acid structures, especially the mycobacteria is first processed with any of the methods of U.S. 5,658,749, U.S. 6,004,771, and WO 95/27076 to the sediment or button (pellet) stage.
- the resulting sediment or button is then "pre-cultured," that is, it is resuspended in nutrient broth, such as, for example 7H9 media, and then allowed to incubate for a time and at a temperature that will allow such microorganisms, and especially mycobacteria, to initiate the cellular processes associated with replication, or allow the mycobacteria to replicate.
- the resulting broth is then analyzed for the presence of such microorganisms, and especially, mycobacteria, by any mycobacteriophage-based plaque assay. In this embodiment washing of the processed sediment is avoided.
- the sample or clinical specimen that is suspected of harboring a microorganism that contains mycolic acid structures, and especially, mycobacteria is processed with any of the betaine-like detergents to the sediment or button (pellet) stage.
- the resulting sediment or button is then resuspended in nutrient broth, but NOT allowed to incubate for a time and at a temperature that will allow the mycobacteria to initiate the cellular processes associated with replication, or allow the mycobacteria to replicate.
- the resulting broth is then analyzed for the presence of such microorganisms, and especially, mycobacteria, by any mycobacteriophage- based plaque assay.
- both washing of the processed sediment is avoided, and the pre-culture step is avoided.
- the sample or clinical specimen that is suspected of harboring a microorganism that contains mycolic acid structures, and especially, a mycobacteria is processed with any of the methods taught in U.S. 5,658,749, U.S. 6,004,771, and WO 95/27076 to the sediment or button (pellet) stage.
- the resulting sediment or button is then resuspended in a buffer, preferably a Good buffer. No washing of the sediment or pre-culture of the processed sample is performed.
- the resulting buffer suspension is then analyzed by any mycobacteriophage-based plaque assay.
- the resuspension buffer that is chosen in this embodiment is such that its composition has been optimized for use in conjunction with the mycobacteriophage-based assay used to analyze the sediment. While the buffer used to process such samples with any of the betaine-like detergents does not necessarily have to be the same buffer used to resuspend the button, in a variation of this embodiment the buffer used to process such samples with any of the betaine-like detergents is the same as the buffer used to resuspend the button.
- divalent metal cations such as Mn +2 , Mg +2 , and/or Ca +2 may be used to supplement the resuspension buffer to further facilitate infection by a desired mycobacteriophage.
- divalent metal cations such as Mn +2 , Mg +2 , and/or Ca +2 may be used to supplement the resuspension buffer to further facilitate infection by a desired mycobacteriophage.
- This is the most prefened embodiment because it is the least labor intensive, generates the clinical diagnostic result in the shortest period of time, reduces the quantity of broth required (i.e., reduces the cost of the assay), and reduces the dependence of the assay on antibiotics (i.e., further reducing the cost of the assay) because contaminants are not given the opportunity to replicate prior to any mycobacteriophage-based plaque assay, and the helper cells (e.g., M. smegmatis) can more efficiently compete for nutrients in the culture media.
- the helper cells
- lytic enzymes can be used to treat samples or processed sediments as taught in U.S. 5,985,593. Such lytic enzyme treatment can be performed after processing with any of the betaine- like detergents, but before infection with any of the mycobacteriophage assays. Alternatively, lytic enzyme treatment can be performed after both processing with a betaine-like detergent and the infection with any mycobacteriophage, but before plating with helper cells.
- the purpose of treating with the lytic enzymes of U.S. 5,985,593 is to reduce the complexity of the specimen in regard to contaminating microorganisms, thereby reducing complications associated with contamination during pre-culture or helper cell lawn development.
- treatment of specimens with other chemicals enzymes, proteins, cellulose, and/or cross linked polymers in the form of beads or fibers can be used to reduce the complex matrix of the specimen itself.
- Such treatment(s) can be performed either before or after processing with any of the betaine-like detergents, but before infection with any of the mycobacteriophage assays.
- Such treatments serve to degrade or denature proteins (e.g., actin, fibrin, and immune complexes), glycoprotein's (e.g., mucins), or nucleic acids (e.g., DNA) present in the entangled mesh that comprises clinical specimens (e.g., the mucus present in respiratory specimens), or reduce the amount of insoluble material present in clinical specimens by solubilization or clarification.
- Such treatments can take place before or after processing with any of the betaine-like detergents, but before infection with any of the mycobacteriophage assays. Alternatively, such treatments can be performed simultaneously with processing with a betaine- like detergent.
- Mycobacteriophages have also been genetically engineered to include reporter genes such as luciferase or ⁇ -galactosidase to develop chemiluminescent and colorimetric plaque assays, respectively (WO 93/16172; WO 94/25572; U.S. 5,968,733; U.S. 6,225,066; U.S. 6,300,061).
- Cunent diagnostic methods in tuberculosis rely on smear microscopy, culture, nucleic acid amplification, and/or serological methods. Smear microscopy is rapid and inexpensive, but lacks sensitivity and specificity (Burdash, N.M., et al, Jour. Clin. Microbiol. 4:190-191 (1976); Aber, V.R, et al, Tubercle. 61:123-133 (1980); Lipsky, B.A., et al, Rev. Infect. Dis. 6:214- 222 (1984); Munay, P.R., et al, Ann. Intern. Med. 92:512-513 (1980)).
- Serological methods such as skin testing (e.g., PPD and Mantoux), are more rapid, but also lack sensitivity and cannot distinguish between latent and active infections, nor can these tests distinguish between individuals vaccinated with BCG or exposed to M. tuberculosis. Therefore, diagnostic assays that utilize mycobacteriophage fill an important niche in tuberculosis testing: these assays can be engineered for a high degree of specificity, identify patients with active disease, and results can be obtained within several days. In addition, these tests require minimal technical expertise and can be performed in developing countries where the tests are needed most for a reasonable cost.
- NALC/NaOH N-acetyl-L- cysteine
- the NALC facilitates liquefaction of the specimen, while the NaOH kills most contaminants, but at the expense of viability: in excess of 90% of the bacilli are killed in this step by exposure to these agents (Yajko, D., et al, Jour. Clin. Microbiol 33:1944-1947 (1995); Burdz T.V.N., et al, Diagn. Microbiol. Infect. Dis. 47:503-509 (2003)).
- the specimen is then subjected to centrifugation and the resulting sediment (e.g., pellet or "button") is used as the source of the sample that is to be assayed for the presence of the desired microorganism that contains mycolic acid structures.
- microorganisms prior to the methods of the invention, in order to assay with a mycobacteriophage assay, such microorganisms must be subjected to a recovery period that involves culture.
- microorganisms that contain mycolic acid structures can be incubated in nutrient broth for a time and at a temperature that allows recovery of bacilli and regeneration of such receptors prior to any method that comprises detection of microorganisms with mycolic acid structures by a mycobacteriophage-based plaque assay, it is not required and in a preferred embodiment, such culture or "pre-culture" is not performed.
- the methods of the invention allow the artisan to eliminate such wash steps and/or pre-culturing of said microorganisms. This is a significant advantage in regard to enabling mycobacteriophage assays to be used as a means for rapid, large-scale screening for tuberculosis. While Jacobs et al. (WO 93/16172; WO 94/25572; U.S. 6,225,066; U.S. 6,300,061) teach that such mycobacteriophage assays can be performed "...either directly or after culture", they provide no guidance as to how such mycobacteriophage plaque assays might be accomplished in the absence of culture. To the contrary, Jacobs, et al.
- 6,004,771, and WO 95/27076 could be modified in such a way that eliminated the need to culture microorganisms prior to being tested in such mycobacteriophage assays. Indeed, the inventor was further surprised to discover that such betaine-like detergent methods could also be used in such a way as to eliminate the requirement for any secondary wash step, thereby further reducing the time and labor associated with performing mycobacteriophage assays. Such discoveries led the inventor to realize that additional savings were also plausible.
- propagation of mycobacteriophage in bacilli that have been exposed to CB-18 for up to three hours appears to be unaffected. While an overnight incubation in nutrient broth generated slightly more robust results, these results clearly indicated that the receptors necessary for infection are mostly intact, if not completely intact following exposure to CB-18.
- the plaque assay can be performed in such a manner as to avoid any culture step prior to such mycobacteriophage assays (e.g., pre-culture).
- the second reason such results were surprising was that Sarkis, et al, Methods Mol. Biol 101 :145 (1998) teach that detergents interfere with the ability to infect mycobacteria.
- White, A. et al, Am. Rev. Tuberc. 77:134 (1958) teach specifically that Tween 80 interferes with the ability to propagate the mycobacteriophage D-29 in M. tuberculosis at concentrations above 0.0012%) (i.e., ⁇ l ⁇ M).
- Example 2 it is shown that the presence of CB-18 during infection of M. tuberculosis ATCC 27294 at concentrations as high as 20 ⁇ g/ml (i.e., 52 ⁇ M) does not interfere with the assay. This is further surprising since Thornton, CG., et al, Jour. Clin. Microbiol. 36:2004-2013 (1998) teach that when CB-18 is present at 20 ⁇ g/ml in the culture media, the growth characteristics of the M. tuberculosis type strain ATCC 27294 are significantly affected. These results suggest that washing sediments processed with the methods of U.S. 5,658,749, U.S. 6,004,771, and WO 95/27076 may not be necessary prior to any mycobacteriophage plaque, thereby significantly reducing such a labor intensive step.
- Example 3 combines the concepts outlined in Examples 1 and 2: M. tuberculosis can be incubated in the presence of 1 mM CB-18 for 90 minutes and immediately taken for infection so long as the concentration of CB-18 carried into the infection media or buffer is below approximately 40 ⁇ g/ml.
- the inventor further recognized that because of the physio-chemical flexibility of betaine-like detergents as processing reagents, processing with any of the methods of U.S. 5,658,749, U.S. 6,004,771, and WO 95/27076 can be modified in such a way that the composition of the buffer used to process such specimens is the same buffer used to perform the infection in any such mycobacteriophage plaque assay.
- the betaine-like detergents can be used in practically any of the Good buffers, within a broad pH range, and at virtually any ionic strength.
- the buffer used to process such clinical samples in preparation for analysis by any such mycobacteriophage plaque assay can be matched in a novel way to optimize both processing of such specimens with any of the betaine-like detergents, and performance of the mycobacteriophage plaque assay.
- Use of a solution(s) to process clinical samples for detection of microorganisms with mycolic acid structures, especially mycobacteria, that is the same as, or identical (or matched with any desired detection assay - that is, compatible with and useful for a desired detection assay) was hereinbefore unimaginable because such processing methods utilized caustic acids and alkalis. Such caustic reagents would have to be neutralized and/or removed in some fashion prior to performing any diagnostic assay.
- the methods of the invention allow, for the first time, the processing reagent to be matched in terms of pH and composition, with the needs of the detection assay (i.e., any such mycobacteriophage plaque assay).
- a consequence of eliminating pre-culture of microorganisms prior to assay in any mycobacteriophage plaque assay is the reduction of contaminants that can interfere with such plaque assays.
- the specimen can be subjected to enzymatic decontamination with lytic enzymes either before infection with mycobacteriophage, during infection with mycobacteriophage, or following infection with mycobacteriophage.
- the lytic enzyme cocktail contains lysozyme or lyticase, and in a highly prefened embodiment, at least both lysozyme and lyticase.
- the enzyme cocktail contains lytic enzyme-containing extracts of Trichoderma and/or Cytophaga, in addition to lysozyme and/or lyticase, but most especially lytic enzyme- containing extracts of both Trichoderma and Cytophaga in addition to lysozyme and lyticase.
- Lytic enzyme-containing extracts of Lysobacter may be used in place of Cytophaga extracts, if desired.
- the enzyme cocktail contains lytic enzyme-containing extracts of both Trichoderma and Lysobacter in addition to lysozyme.
- lytic enzyme preparations of Micromonospora can be used alone or combined with other lytic enzyme preparations.
- the lytic enzymes can be either natural or recombinant, and in a purified or unpurified form.
- the specimen can be subjected to treatments with proteins, enzymes, chemicals, and/or inert substrates before infection with mycobacteriophage.
- Example 5 shows that one of the most significant problems with applying mycobacteriophage assays to clinical specimens is that the level of inhibition encountered is severe.
- the most common specimens analyzed for diagnosing tuberculosis are respiratory specimens (e.g., sputum, alveolar lavage, bronchial washings, etc.).
- Mucus is a complex matrix of DNA, cellular debris, and filamentous actin derived from lysed neutrophils and leukocytes, all entangled within a gelatinous matrix of mucin, (Fuloria, M., et al, Respir. Care 45:868-873 (2000)). This matrix must be reduced, degraded, or eliminated in order for the mycobacteriophage assays to perform optimally. Proteins, enzymes, or chemicals which target each of the matrix components would be useful means to reduce the complexity of the specimen matrix.
- Proteins such as gelsolin have been shown to liquefy sputum by cleaving actin filaments (Vasoncellos, C.A., et al, Science 263:969-971 (1994)).
- Gelsolin is a protein involved in the rearrangement of the cytoskeleton; other proteins, such as the ADF/cofilin family of proteins (Maciver, S.K., et al, Genome Biol. 3:1-12 (2002)) might also be useful in conjunction with the methods of the invention.
- the chemical swinholide A (Bubb, M.R., et al, Jour. Biol. Chem.
- a cytotoxin isolated from a marine sponge also cleaves actin filaments, and as such would be expected to be useful in the methods of the invention.
- Enzymes that digest nucleic acids, such as DNA might also be used to facilitate liquefaction of sputum.
- the recombinant form of human DNase I rhDNase
- rhDNase the recombinant form of human DNase I
- Nucleases such as DNase I from human or bovine sources are currently commercially available, and would be expected to be useful in the methods of the invention.
- liquefy sputum involves the use of chemical reducing agents such as DTT, NALC, or BME. Such reagents reduce disulfide bonds between mucins.
- hypertonic saline e.g., l%-7% NaCl
- Robinson, M., et al, Thorax 52:900-903 (1997) can also be used to liquefy sputum.
- Example 6 suggests that the most inhibitory component(s) of the specimen matrix are associated with the insoluble, precipitated material that forms the button (pellet).
- methods that serve to clarify or physically separate such precipitated material from that portion of the processed pellet that will be assayed with any such mycobacteriophage assay are important adjuncts to protein, enzyme and/or chemical treatments.
- Methods involving the use of inert matrices in the form of beads or fibers would include those products involved in chromatographic methods, such as for example, gel filtration beads, or ion exchange resins.
- gel filtration medias useful in the methods of the invention are the Sephadex ® products, which are dextran polymers cross linked with epichlorohydrin.
- Cellulose powders by Whatman such as the CC31, CC41, CF1, and CF11 powders, and most especially the CDR (cell debris remover) powder are all useful in the methods of the invention.
- Anion exchange resins useful in the methods of the invention are those wherein cellulose beads or fibers are modified with diethylaminoethyl (DEAE) tertiary amine groups. Examples of such anion exchange resins manufactured by Whatman would include DE23, DE32, DE51, DE52, DE53, and QA52.
- Cation exchange resins useful in the methods of the invention are those wherein cellulose beads or fibers are modified with carboxylate or phosphate groups.
- Examples of such cation exchange resins manufactured by Whatman, and modified with carboxylic acids would include CM23, CM32, CM52, and examples of such cation exchange resins modified with phosphates would include PI and Pl l.
- CM23, CM32, CM52, and examples of such cation exchange resins modified with phosphates would include PI and Pl l.
- gel filtration, cellulose, and ion exchange resin equivalents that are commercially available. All would be expected to be useful in the methods of the invention.
- such inert beads or fibers can be employed in a simple format when used in conjunction with centrifugation tubes containing adapters that have been modified to include a frit of approximately 5 microns to approximately 60 microns.
- Such centrifugation tubes useful in the methods of the invention can be designed to collect approximately 0.5 ml, such as the VectaSpin Micro tubes (Whatman), the Spin-X ® tubes by Corning Life Sciences (U.S.A.), or the Nanosep ® devices by Pall Gelman (U.S.A.).
- Betaines useful in conjunction with the methods of the invention include the sulfobetaines and carboxybetaines, for example, the highly purified (e.g., research grade) "SB"-series of detergents.
- C ⁇ 2 -amido (CAS ® No. 4292-10-8), C H -amido (CAS ® No. 59272-84-3), C, 6 -amido (CAS ® No. 32954-43-1), and C ⁇ 8 -amido (CAS ® No. 6179-44-8).
- the C ⁇ 8 - amido (CAS ® No. 6179-44-8) is of undefined structure because the alkyl is the "iso" form, which suggests that it branches in some undefined manner.
- amidopropyl carboxymethylbetaines in which the alkyl chain is derived from coconut oil, and differences are due to the method of preparation.
- CAS ® Numbers 61789-39-7 and 61789- 40-0 Two examples in this category include CAS ® Numbers 61789-39-7 and 61789- 40-0.
- An example of cococarboxymethylbetaine would be CAS ® No. 68424- 94-2.
- Other natural oil carboxymethyl derivatives include: ricinamidopropyl carboxymethylbetaine (CAS ® No. 71850-81-2), and Tallow bishydroxyethyl glycinate (CAS ® No. 70750-46-8). There are also several carboxymethylbetaines that have been tested for which no CAS ® Number has been given.
- wheat germ oil-amidopropyl carboxymethylbetaine Schercotaine WOAB: Scher Chemicals, Inc., Clifton, NJ
- babassuamidopropyl carboxymethylbetaine Croda, Inc., Parsippany, NJ
- soyamidopropyl carboxymethylbetaine Chembetaine S: Chemron Corp., Paso Robles, CA
- canloamidopropyl betaine Hetaine CLA: Heterene, Inc., Patterson, NJ.
- nitrogen in the amide linkage is the quaternary nitrogen (e.g., the linkage ( ⁇ ) is a carbonyl).
- the nitrogen in the amide linkage is the quaternary nitrogen (e.g., the linkage ( ⁇ ) is a carbonyl).
- C ⁇ CAS ® No.
- carboxybetaines that utilize an ethyl bridge
- carboxyethylbetaine R 4 ⁇ CH 2 CH 2 —
- C ⁇ 2 CAS ® No. 16527-85-8
- C ] 3 CAS ® No. 132621-79-5
- C, 4 CAS ® No. 69725- 38-3
- C ⁇ 6 CAS ® No. 42416-43-3
- C, 8 CAS ® No. 30612-73-8
- carboxyethylbetaine in which R 2 and R 3 are hydrogen atoms, under the appropriate conditions, would be CAS ® No. 1462-54-0 (C ⁇ 2 -beta alanine).
- carboxy betaines that utilize a pentyl bridge
- C 12 CAS ® No. 76392-97-7
- C 16 CAS ® No. 73565-98-7
- carboxybetaine examples in which a benzyl group is used as the bridge function (R 4 ⁇ -CH 2 — H - ).
- C ]2 examples one in which the carboxy function is in the 4, or para, position (CAS ® No. 71695-31-3), and one in which the carboxy function is in the 2, or ortho, position (CAS ® No. 71695-34- 6).
- C ⁇ 6 examples one in which the carboxy function is in the 4, or para, position (CAS ® No. 71695-33-5), and one in which the carboxy function is in the 2, or ortho, position (CAS ® No. 71695-35-7).
- Most commercially available betaines are used to manufacture detergents, shampoos, cosmetics, and other toiletries. These betaines are derived primarily from natural oils such as coconut oil, tallow, wheat germ, babassu oil, castor oil, canola oil, soy bean oil, and rapeseed oil.
- cocoamidopropyl hydroxypropylsulfobetaine (CAS ® No. 68139-30-0)
- cocoamidopropyl carboxymethylbetaine (CAS ® No. 61789-37-9 and CAS ® No. 61789-40-0)
- cococarboxymethylbetaine (CAS ® No. 68424-94-2). All these betaine-like detergents are useful in conjunction with the methods of the invention.
- TAPS N-tris(Hydroxymethyl)methyl-4-aminobutanesulfonic acid
- TABS N-(2-Acetamido)-2-aminoethanesulfonic acid
- ACES N-(2-Acetamido)-2-aminoethanesulfonic acid
- HEPES N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)
- HEPBS N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)
- Another group of modified sulfonic acids that would be useful as buffers in the methods of the invention would be those that are substituted with a cyclohexyl moiety; these would include: 3-(Cyclohexylamino)-2- hydroxy-1-propanesulfonic acid (CAS ® No.
- composition and method of the invention are useful for the preparation of any sample suspected of containing a microorganism having mycolic acid structures in its outer membrane.
- microorganisms include microorganisms having corynomycohc acid in their outer membrane (such as, for example, Corynebacterium diphtheria); microorganisms having nocardomycohc acid in their outer membrane (for example, Nocardia aster oides); and microorganisms having mycolic acid in their outer membrane (for example, Mycobacterium tuberculosis) (see also Funke, G. et al, Clin. Micro. Rev. 70:125-159 (1997) for further discussions on coryneform bacteria (incorporated herein by reference)).
- composition and method are useful for the preparation of a sample to be assayed for the presence of any desired Mycobacterium group or complex or Mycobacterium species.
- Mycobacterium species include a mycobacterium complex such as M. tuberculosis (MTB) complex, M. avium (MAC) complex, MAIS complex and M. fortuitum complex, as well as fast growing and slow growing mycobacteria including specified and unspecified photochromogens, nonphotochromogens, scotochromogens, and especially M. africanum, M. asiaticum, M. avium, M. bovis, M. bovis (BCG), M. butyricum, M. chelonae, M.
- M. tuberculosis (MTB) complex M. avium (MAC) complex
- MAIS complex M. fortuitum complex
- fast growing and slow growing mycobacteria including specified and unspecified photochromogens, nonphotochromogens, scotoch
- M. kansasii, M. marinum, M. simiae and M. asiaticum are examples of photochromogens.
- M. scrofulaceum, M. szulgai, M. xenopi, M. gordonae and M. flavescens are examples of scotochromogens.
- M. avium, M. intracellulare, M. gastri, M. malmoense, M. terrae and M. triviale are all examples of nonphotochromogens.
- M. africanum, M. avium, M. bovis, M. haemophilum, M. intracellulare, M. kansasii, M. malmoense, M. marinum, M. microti, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. tuberculosis, and M. xenopi are all examples of slow-growing (requiring more than seven days) mycobacterial species.
- M. chelonei, M. flavescens, M. fortuitum, M. gordonae, M. leprae, M. neoaurum, M. phlei, M. smegmatis, M. terrae, and M. ulcerans are all examples of rapid-growing (requiring less than seven days) mycobacterial species.
- M. tuberculosis, M. africanum, M. bovis, M. bovis (BCG), and M. microti are the members of the Mycobacterium tuberculosis complex (MTB).
- M. avium and M. intracellulare are the members of the Mycobacterium avium complex (MAC); there are at least three distinct sero logic groups of M. avium, and more than 25 serovars of M. intracellulare.
- Examples of the diseases and conditions in which the various mycobacterial species are of heightened importance in testing include especially the causative agents of tuberculosis (M. tuberculosis complex) and leprosy (M. leprae (human leprosy) and M. lepraemurium (rodent leprosy)).
- Mycobacterium avium complex bacteria are important bird pathogens.
- M. avium has also been isolated from AIDS patients who are afflicted with a mycobacterial superinfection (Nightingale, S.D. et al, Jour. Infect. Dis. 165: 1082- 1085 (1992)).
- M. bovis is of importance in veterinary medicine.
- fortuitum is a soil bacterium that has been isolated from lesions in animals and humans.
- M. intracellulare is opportunistic and is especially seen in patients infected with the AIDS virus.
- M. paratuberculosis is of interest in the diagnosis of Crohn's disease (regional ileitis) in humans.
- Mycobacterium kansasii is a rare but devastating agent, generally associated with pulmonary disease.
- Mycobacterium marinum infects cold-blooded animals and fish; it has also been isolated from superficial granulomas on the extremities of humans.
- Mycobacterium paratuberculosis is the causative agent of Johne's disease in cattle; it is very slow growing and cultures must be held for 16 weeks before it can be assured that they are negative. M. ulcerans is also of interest in human medicine. Many of the above and others have been discussed by Wayne, L.G. et al, Clin. Micro. Rev. 5:1-25 (1992), and Falkinham, O. Clin. Micro. Rev. 9:177-215 (1996) and are incorporated herein by reference.
- Detecting the presence of organisms containing mycolic acid structures in their outer membranes in the sample prepared by the method of the invention can be accomplished using any of the mycobacteriophage kits manufactured by Biotec Laboratories Ltd. (Ipswich, Suffolk, U.K.).
- Other variations of the plaque assay wherein colorimetric detection using ⁇ - galactosidase, or chemiluminescent detection using luciferase (the so-called "Bronx-Box", for example (Hazbon, et al., Jour Clin. Microbiol.
- Plaque formation Plaque formation of following ; infection following overnight CB-18 in Tris buffer and pre-culture in FPTB ( ⁇ g/ml) no pre- •culture nutrient broth a b a b 0 211 229 >300 >300 5 171 191 >300 >300 10 204 186 >300 >300 20 181 198 >300 >300 40 0 7 >300 >300
- Table 4 Plaque formation Plaque formation Replicate following processing in following processing in Tris buffer, no pre- CB-18, no pre-culture, culture, and infection in and infection in Tris- FPTB nutrient broth CaCl 2 buffer
- a 0.5 MacFarland stock of M. tuberculosis was manufactured as described by Thornton, C.G., et al, Jour. Clin. Microbiol. 36:2004-2013 (1998), and 200 ⁇ l portions were transferred to two 50 ml conical tubes (Figure 4).
- One tube contained 50 mM Tris-HCl pH 7.5 @ 25°C, 66 mM NaCl, and 0.05%> NALC (i.e., the control), while the other tube contained the fully diluted (i.e., 1-fold) enzyme mixture. Both tubes were incubated at 37°C for 60 minutes. At the end of the incubation period a 500 ⁇ l aliquot from each tube was serially diluted 400-fold into FPTB nutrient broth. Duplicate 1 ml portions of each 4,000-fold dilution were assayed.
- FASTPlaqueTB assay was examined. Eight random, discarded respiratory specimens were collected from the Microbiology Department at Quest Diagnostics in Baltimore, Maryland. To each specimen was added 5 ml of CB-18 processing buffer (100 mM Tris-HCl, pH 7.5 @ 25°C, 66 mM NaCl, 2 mM CB-18, and 0.5%> NALC). Specimens were incubated for 90 minutes at room temperature, diluted with sterile water to 50 ml, and then subjected to centrifugation for 20 minutes at 3,000 x g at 25°C All sediments were resuspended in 2 ml of sterile water.
- CB-18 processing buffer 100 mM Tris-HCl, pH 7.5 @ 25°C, 66 mM NaCl, 2 mM CB-18, and 0.5%> NALC.
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