WO2011117201A1

WO2011117201A1 - Detection of mycobacteria

Info

Publication number: WO2011117201A1
Application number: PCT/EP2011/054266
Authority: WO
Inventors: Christopher John Stanley; Stuart Mark Wilson
Original assignee: Microsens Medtech Limited
Priority date: 2010-03-22
Filing date: 2011-03-21
Publication date: 2011-09-29
Also published as: GB201004710D0

Abstract

Mycobacteria cell wall components are captured by contacting with a capture reagent, e.g. poly-diallyldimethyl ammonium chloride (pDADMAC) or polybrene, which binds them and is itself either bound to a solid surface, or is present in solution, said method then further comprising capturing said components to a surface by binding said capture reagent to said surface.

Description

Detection of Mycobacteria

The present invention relates to the detection of

Mycobacteria by an improved method.

Pathogenic mycobacteria are responsible for several severe infectious diseases in humans and animals, they include

Mycobacterium tuberculosis, which is the causative agent of TB, the mycobacteria of the MAC complex (primarily M. avium and M. intracellular^~e) which are opportunistic pathogens in AIDS patients, M. paratuberculosis, which causes bowel inflammation, M. leprae causing leprosy, M. kansasii , M.

marinum, M. fortuitum complex, and many others. There are also many other non-pathogenic mycobacteria, including M. smegmatis .

The Mycobacteria are characterised by a cell membrane that contains a number of components, including lipoarabinomannan (LAM) and mycolic acids. LAM is a complex branched chain carbohydrate linked to a lipid.

Biological samples in which Mycobacteria can be found include sputum, urine, blood, bronchial lavage, milk, and faeces. One of the most common specimen types delivered for diagnosis is sputum. Sputum presents unique problems for bacteriology. Sputum is heterogeneous in nature and can be bloody,

purulent, and viscous. It can also be contaminated with other micro-organisms e.g. Pseudomonas . Commonly, sputum is thinned and at the same time decontaminated by the use of various pre-treatments . These treatments include the use of 0.25-0.5 M sodium hydroxide for decontamination with or without N-acetyl L-cysteine, sodium dodecyl sulphate, oxalic acid or trisodium phosphate. Treatment times can be 20-120 minutes . After treatment with the harsh decontaminants the sample is generally centrifuged to concentrate the Mycobacteria which are then analysed by microscopy, culture or molecular amplification . In the past the use of immunoassay detection methods for

Mycobacteria in sputum or in other biological fluids has not been successful. Recently the performance of a commercial ELI SA kit for LAM present in the urine of TB or TB/HIV co- infected patients has been reported (Reither et al, BMC Infectious Diseases 2009, 9:141) . LAM present in the blood stream of an infected patient passes through the kidneys and is detectable in the urine. In the LAM ELISA the sample preparation procedure involves boiling the urine to

sterilise, followed by centrifugation to remove debris. The supernatant containing LAM is then assayed by ELISA. It is believed that the boiling procedure improves the performance of the LAM assay. LAM ELISAs have also been developed for use in other sample types such as sputum or blood but these are not widely used.

It has been reported (WO 2008/065047) that certain binding agents including poly diallyldimethyl ammonium chloride (p- DADMAC) bind Mycobacteria and they can be either captured directly to p-DADMAC coated surfaces or can be captured to a surface indirectly. It is believed that p-DADMAC binds to the hydrophobic mycolic acids in the outer membrane of the Mycobacterial cell. We have now discovered that similar binding systems can be used to bind LAM or LAM containing bacterial fragments. Description of the invention

The present invention provides a method for the capture from a sample of Mycobacteria cell wall components, which method comprises contacting a sample containing said

components with a capture reagent, which capture reagent binds said components, said capture reagent either being a solid surface and capturing said components thereto, or being present in solution, said method then further comprising capturing said components to a surface by binding said capture reagent to said surface.

The cell wall components may be insoluble (e.g. able to be separated by centrifugation) or may be soluble.

The capture reagent is non-immunological . The capture reagent may be a soluble reagent and preferably has both a hydrophobic character and a polar character. The hydrophobic character may assist its binding to lipid containing

components in cell walls of Mycobacteria. However, binding to cell wall materials may also be through a mixture of hydrophobic and ionic interactions or purely ionic

interactions. Its polar character may participate in binding to solid surfaces, as may the hydrophobic character,

depending on the nature of the solid surface. The capture reagent may itself be a solid surface having the requisite cell wall component binding ability. The invention includes a method for the binding of components of the Mycobacterial cell wall by polyionic compounds or polyionic surfaces.

Said capture reagent is preferably polymeric and

preferably comprises a hydrocarbon chain or chains bearing multiple polar sites, which may be spaced along said chain or may connect hydrocarbon chains. Optionally, said capture reagent comprises cationic groups which remain positively charged even at high pH such as may be used to decontaminate a sample (e.g. pH ≥ 10, for instance 12-14) . Suitably these are quaternary ammonium groups. A preferred capture reagent is poly- diallyldimethyl ammonium chloride (pDADMAC) .

The molecular weight of the poly-DADMAC may be in the range of less than 100,000 (very low), 100,000 - 200,000 (low), 200,000 - 400,000 or 500,000 (medium) or over 500,000 (high) .

An alternative said capture agent is polybrene, i.e. 1 , 5-dimethyl-l , 5-diazaundecamethylene polymethobromide (or hexadimethrine bromide)

Both of these reagents have a methylene chain of 4-6 units bearing spaced quaternary ammonium groups.

The sample may be a fluid sample such as sputum

(preferred) , urine, blood (including blood components such as plasma or serum), bronchial lavage, etc. or may be a solid sample such as a tissue biopsy, e.g. a skin sample, which may be homogenised and which preferably is treated to extract or disperse micro-organisms into a liquid to produce a fluid sample.

For appropriate solid surfaces the capture reagent may preferably be sufficiently hydrophobic in character to bind hydrophobically to plastics, e.g. to the polystyrene

microplates usually employed to bind proteins, or

alternatively may be able to bind to glass or a glass like surface, either by polar interaction or by being sufficiently hydrophobic in character to bind hydrophobically to the surface, which may suitably be such as might be found in microscope slides or cover slips. But it should be

sufficiently hydrophilic in character that it will be soluble in an aqueous medium, preferably in even a high pH aqueous medium. It is therefore soluble in the admixture with the sample and any other materials used.

Optionally, the solid is such that its surface by itself has the ability to bind the Mycobacteria cell wall components without the use of a separate capture agent coated on the surface. This may be achieved by incorporating suitable ionic groups such as quaternary ammonium groups into the polymer structure of a plastics material, as in quaternary ammonium ion exchange beads, which may be produced in

accordance with e.g. US4207398.

Thus, the preferred compounds are polycationic

quaternary amines such as p-DADMAC or hexadimethrine bromide that remain positively charged at the high pHs (>12) used for sample decontamination (and thinning of sputum samples) .

Alternatively polyanionic compounds such as dextran sulphate can be used. Preferably the polyionic compounds may be bound to the surface of a magnetic bead, or the bead itself may have a surface that is positively or negatively charged. The beads may also be non-magnetic and of high density to promote rapid settling in solution. In a preferred embodiment a sample containing intact Mycobacterial cell wall material, e.g. fragments of cells, or soluble components derived from such cells, is incubated with the polyionic compound-coated beads. This procedure can preferably be used to concentrate cell components from large sample volumes e.g. 1 to 10 ml of thinned sputum or 1 to 100 ml (e.g. 1 - 10 ml) of urine, thus greatly increasing the sensitivity of the detection method.

Capture may take place under near neutral pH conditions described in WO2008/065047, preferably in the presence of detergent as described there, or may take place under high pH conditions as generated by a decontaminating agent like sodium hydroxide. Generally, no detergent need then be used. Suitable high pH decontaminating agents include other

alkalis, such as KOH and LiOH.

Optionally, following binding the beads can be washed to remove sample contaminants prior to detection and, when high pH capture conditions are used, to adjust the pH to something more suited to the intended detection method.

After capture of Mycobacterial cell fragments or soluble components from a sample the bound material can be released into solution from the bead surface by standard techniques such as heating, addition of detergents or the addition of chaotropic salts. Alternatively the beads can be disrupted by a mechanical lysis process or ultrasound treatment for example to release the surface coating. After optional removal of the residual beads by magnetic capture,

sedimentation, filtration, or centrifugation the supernatant containing released material can then be detected using an immunoassay such as a specific ELISA or a lateral flow device or mass spectrometry or GC-MS . An ELISA method based on microplates, e.g. 96 well microplates, or adapted for a lateral flow format is preferred as this is most suited to use in the routine TB testing laboratory. As an alternative to elution the bead coated with cells fragments or soluble components can be used directly in an ELISA or lateral flow device, in the latter case the bead diameter may need to be smaller than the pore size of the porous material from which the device is constructed to allow unimpeded flow of the beads .

It has been found that where there may be intact

Mycobacterial cells as well as, potentially, a wide range of cellular fragments of varying sizes bound to the polyionic compound-coated beads, a mechanical lysis disruption method to release the bound material increases the signal in the subsequent ELISA detection assay. It is believed that the mechanical disruption process lyses intact cells, disperses the cellular components and breaks up structures such as lipid rafts. This process may make the cellular components more accessible to binding by polyionic compounds or

polyionic surfaces and also increases the effective molar concentration of components in the sample.

The materials, or selected key materials, needed for the practice of binding Mycobacterial cell fragments and soluble cellular components and the subsequent detection method may be provided in kit form. Accordingly, the invention includes a micro-organism assay kit comprising either (a) a soluble capture reagent capable of binding Mycobacteria cell wall components, a substrate having a surface for capturing said cell wall components to said surface by binding said capture reagent to said surface, or (b) a capture reagent coated on and thus immobilised upon a solid surface, said capture reagent being capable of binding Mycobacteria cell wall components to be detected, and an immunological binding partner for binding said cell wall components. The assay kit may comprise at the least a Mycobacterial capture reagent immobilised upon a solid surface, preferably para-magnetic beads. Kits may further contain decontamination reagents, wash buffers and a detection system which is preferably an ELISA or more preferably a lateral flow device that detects the cellular components directly or after elution from the solid surface.

The invention will be further illustrated and explained by the following examples. EXAMPLES

Example 1. Demonstration of binding of the Mycobacterial cellular component LAM present in sputum by p-DADMAC coated beads and subsequent detection with a commercially available LAM ELI SA

Method

Sputum samples were scored by direct fluorescent microscopy. The samples were thinned using a mixture of NaOH and N- acetylcysteine (NALC) and the thinned sputum was extracted using p-DADMAC coated magnetic beads, which are further described in WO 2008/065047. 80μ1 of the pDADMAC coated beads (about 5 mg of beads) was used for 1 ml of the thinned sputum at a pH of 14 at a high ionic strength provided by addition of an equal volume of 3 M NaCl and incubated for 10 min. The beads were collected using a magnet, washed in 1ml PBS buffer containing 0.5% Tween 20, collected using the magnet again and then resuspended in 0.1 ml PBS containing 0.5% Tween 20. The magnetic beads were boiled for 5 min, then the extracts were eluted by adding the magnetic beads to 50μ1 of lysis beads (lysis matrix B beads, Fischer Scientific Inc.) and disruption for 5 min at 2800 rpm on a Disruptor Genie (Scientific Industries) . 100 μΐ of each eluate was analysed directly by a commercially available microplate LAM ELISA (Clearview, Invernesss Medical Innovations Inc.). Results Microscopy Absorbance

score 450nm

+++ 1.26

Neg 0.12

++ 1.18

++ 0.54

Neg 0.16

Discussion

The ELISA performed on the sputum extracted by the p-DADMAC- coated beads was able to differentiate smear positive from smear negative sputum.

Example 2. Investigation of the p-DADMAC-coated magnetic beads/ ELISA combination to detect soluble cellular

components such as LAM

Method

A smear-positive and a smear-negative sputum were thinned and centrifuged at 13,000 x g for 5 min to remove intact cells and larger fragments. The supernatants and the resuspended pellets were extracted using p-DADMAC-coated beads, generally as in Example 1. Extracted antigens were eluted from the beads by a lysis disruption process as described in Example 1 and analysed by the LAM-specifc ELISA.

Results

Sample analysed Absorbance

450nm* Microscopy positive sputum

2.05

supernatant

Microscopy positive sputum

0.78

pellet

Microscopy negative sputum

0.00

supernatant

Microscopy negative sputum

0.07

pellet

* readings after subtraction of conjugate control blanks Discussion

The p-DADMAC-coated beads are able to extract LAM antigens from both the soluble supernatant and the pellet of smear- positive sputum. The ELISA signal of the extracted

supernatant is higher than the signal from the pellet

suggesting that a large proportion of the detectable LAM in sputum is in a soluble form.

Example 3. Investigation of the p-DADMAC ligand-coated magnetic beads/ ELISA to detect soluble LAM spiked into urine

Method

A BCG culture was centrifuged at 13,000 x g for 5 min and

50μ1 of the supernatant spiked into 2 ml urine. The spiked urine and a non-spiked control urine were extracted using p- DADMAC-coated magnetic beads at neutral pH in the presence of detergent as in WO2008/065047. The extracts were eluted from the beads by lysis disruption and analysed by LAM-specific ELISA.

Results Sample analysed Absorbance

450nm*

BCG culture supernatant added

1.03

to urine

Urine control 0.00

* readings after subtraction of conjugate control blanks Discussion

The p-DADMAC-coated magnetic beads were able to extract the soluble BCG derived LAM antigen that had been spiked into urine .

Example 4. Investigation of differently coated beads to extract soluble LAM antigen from urine.

Method

1. A BCG culture was centrifuged at 13,000 x g for 5 min and 50μ1 of the supernatant spiked into 4 ml urine. 2. The spiked urines and non-spiked control urines were extracted at neutral pH for 10 min using variously coated or non-coated paramagnetic beads.

3. The beads were captured and washed with 1 ml PBS.

4. The beads were resuspended in ΙΟΟμΙ PBS, 5% (w/v) BSA, 0.5% (v/v) Tween20.

5. The extracts were eluted from the beads by lysis

disruption .

6. 100 μΐ of each eluate was analysed directly by the LAM- specific ELISA. Results

* Signal after subtraction of conjugate blank Conclusion

Coating the beads with either p-DADMAC or dextran sulphate improved the signal compared to non-coated amine beads.

In this specification, unless expressly otherwise indicated, the word 'or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator 'exclusive or' which requires that only one of the conditions is met. The word 'comprising' is used in the sense of 'including' rather than in to mean 'consisting of . All prior teachings

acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

Claims

Claims :

A method for the capture from a sample of Mycobacteria cell wall components, which method comprises contacting a sample containing said components with a capture reagent, which capture reagent binds said components, said capture reagent either being bound to a solid surface and

capturing said components thereto, or being present in solution, said method then further comprising capturing said components to a surface by binding said capture reagent to said surface.

2. A method as claimed in claim 1, wherein said capture

reagent comprises a long hydrocarbon chain bearing multiple polar sites.

3. A method as claimed in claim 2, wherein said multiple

polar sites are spaced along said chain. 4. A method as claimed in any preceding claim wherein said capture reagent is cationic.

A method as claimed in claim 4, wherein said capture reagent is poly-diallyldimethyl ammonium chloride

(pDADMAC) .

A method as claimed in claim 4, wherein said capture reagent is polybrene. 7. A method as claimed in any preceding claim, wherein the cell wall components prior to said capture are present in said sample in solution.

8. A method as claimed in any preceding claim, wherein said sample is sputum, urine, blood, plasma, serum, tissue or faeces.

9. A method as claimed in any preceding claim, wherein said cell wall components comprise LAM. 10. A method as claimed in any preceding claim, wherein said surface is provided by beads.

11. A method as claimed in claim 9, wherein following capture of said components to said beads, the components are eluted from the beads.

12. A method as claimed in claim 10, wherein said elution is conducted by mechanical lysis. 13. A method as claimed in any preceding claim, wherein said components are detected after capture to said surface, and optionally following removal from the surface, by an immunological detection method. 14. A method as claimed in claim 11, wherein said components are detected by a LAM Elisa.

15. A micro-organism assay kit comprising either (a) a

soluble capture reagent capable of binding Mycobacteria cell wall components, a substrate having a surface for capturing said cell wall components to said surface by binding said capture reagent to said surface, or (b) a capture reagent coated on and thus immobilised upon a solid surface, said capture reagent being capable of binding Mycobacteria cell wall components to be detected, and an immunological binding partner for binding said cell wall components.

16. A kit as claimed in claim 15, wherein said capture

reagent is poly-DADMAC or polybrene coated on beads.