WO2011117202A1

WO2011117202A1 - Capture of mycobacteria

Info

Publication number: WO2011117202A1
Application number: PCT/EP2011/054267
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: GB201004709D0

Abstract

Mycobacterium is captured from a sample by treating the sample with a reagent to raise the pH of the sample to at least 10, thereby to provide a high pH sample, and a) contacting said high pH sample with a solid surface such as quaternary ammonium ion exchange beads capable of binding Mycobacteria at the pH of the high pH sample, and separating said surface bearing captured Mycobacteria from said sample or by b) contacting said high pH sample with a capture agent such as pDADMAC capable of binding Mycobacteria at the pH of said high pH sample to form a bound complex of said Mycobacteria and capture agent, and contacting said complex with a solid surface to bind said complex thereto.

Description

Capture of Mycobacteria

The present invention relates to the capture to a surface of Mycobacteria spp and to subsequent processing such as assays for their presence or identification or drug susceptibility testing.

Pathogenic mycobacteria are responsible for several severe infectious diseases in humans and animals. The mycobacteria are characterised by a hydrophobic, waxy coat comprising mycolic acid or related compounds. Mycolic acids are complex hydroxylated branched chain fatty acids,

typically having hydrocarbon chains with a chain length in the range C77-80, which causes severe problems in sample handling, causing the bacteria to clump forming cords and to float on the surface of liquids and to be resistant to centrifugation . The hydrocarbon chains may or may not contain sparse oxygenated groups such as hydroxyl, methoxy, keto or carboxyl . Pathogenic mycobacteria include

Mycobacterium tuberculosis, which is the causative agent of TB, the mycobacteria of the MAC complex (primarily M. avium and M. intracellulare) 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 . Also, other members of the Mycolata family have similar hydrophobic coat components. In some, the chain length of the hydrophobic fatty acids is shorter than in the mycobacteria, at around 50 carbon atoms, and in others around 30.

In order to diagnose mycobacterial infections such as tuberculosis, the presence of the organism must be

demonstrated by microscopy, culture or molecular methods such as PCR. Although microscopy can be done directly from the biological sample, it is more usual to first isolate and concentrate the mycobacteria from the biological specimens prior to analysis. Biological samples can include sputum, urine, blood, bronchial lavage etc. 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 eg. Pseudomonas . In culture based methods, contaminating micro-organisms may out grow and mask

Mycobacteria .

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 for thinning, sodium dodecyl sulphate, oxalic acid or trisodium phosphate. Treatment times can be 20-120 minutes. These treatments are designed to thin the sputum and kill the majority of contaminating organisms. Mycobacteria have a thick waxy coat and are more resistant to such treatments. Even so, it is estimated that up to 60% of Mycobacterium tuberculosis are killed or rendered non-viable by this treatment. In addition, because the Mycobacterium

tuberculosis and other members of the family grow so slowly, the growth of contaminating organisms that are not killed by this treatment is still a problem with a high percentage of cultures being overgrown by the fast-growing contaminants.

After treatment with the harsh decontaminants the sample is centrifuged to concentrate the mycobacteria which are then analysed by microscopy, culture or molecular amplification. This centrifugation step introduces a risk of infection to the laboratory staff as the contents of any tube that cracks or breaks during the centrifugation may be aerosolised and contaminate the environment. The centrifugation also introduces a bottle-neck in the sample processing as only a limited number of samples can be centrifuged at any one time. In addition, the centrifugation pellets all material that was rendered denatured and insoluble by the harsh decontamination procedure and very large pellets can be obtained which pose problems for microscopy or molecular methods.

Because of the problems listed above with the current decontamination and concentration approaches it would be extremely useful if the mycobacteria could be captured directly from the biological sample. It would be helpful if this procedure removed some or all of the contaminating organisms such that the chemical decontamination is not needed or could be performed with less harsh conditions.

This would also enhance the survival of the purified

mycobacteria and increase the sensitivity of subsequent tests .

In other applications distinct from sample processing it might also be useful to bind the mycobacteria to a solid surface to allow easy concentration or manipulation of the organisms e.g. capture and washing of the mycobacteria from a phage solution to remove exogenous non-infecting phage or capture and transfer of the mycobacteria from one solution to another .

Methods of capturing mycobacteria to solid surfaces have previously been proposed, including the use of bound phage or phage derived binding peptides immobilised on beads and acting as capture agents (Stratmann et al; J Clin Microbiol. 2002 November; 40(11): 4244-4250) and including isolation of M. paratuberculosis from milk by the use of antibody coated beads (Grant I. R. et al; Appl Environ Microbiol. 1998 Sep; 64(9) :3153-8) . However, such a method may be too expensive for extensive use, especially in less developed countries, and may be over specific in that not all desired bacteria will be captured and involves protein-based molecules that are susceptible to proteases, denaturation and harsh

chemicals .

According to Hetland G. et al . , Immunology 1994, 82, 445- 449, it is possible to coat latex microbeads with BCG by incubation of the beads with cultured and separated bacteria. However, this is unlikely to be effective to capture

efficiently such bacteria from a biological sample containing other hydrophobic organisms or materials.

As disclosed in WO2008/065047, poly diallyldimethyl ammonium chloride -DADMAC) binds mycobacteria to carboxylic acid micro-beads. Without being bound by the following theory, we believe that the backbone chain of the p-DADMAC hydrophobically interacts with the waxy coat of the

mycobacteria and the positive charge in the backbone of p- DADMAC can also interact with negative charges on the surface of the mycobacteria, the p-DADMAC then interacts ionically through its pendant quaternary ammonium groups with the carboxylic acids of the micro-beads. We have also observed that p-DADMAC coated surfaces such as plastics and glass can bind mycobacteria directly.

Thus mycobacteria can be either captured directly to p- DADMAC coated surfaces or can be captured to a surface indirectly. However, in the methods described there, sputum is decontaminated with NaOH and is then neutralised before being captured to DADMAC coated beads in the presence of a detergent at a relatively low ionic strength (50mM) . The detergent is used to prevent clumping of the beads. In principle, it would be desirable to simplify this capture procedure .

The present invention now therefore provides in a first aspect a method for the capture of Mycobacterium from a sample comprising treating the sample with a reagent to raise the pH of the sample to at least 10, thereby to provide a high pH sample, and a) contacting said high pH sample with a solid surface, optionally bearing a capture agent, capable of binding

Mycobacteria at the pH of the high pH sample, and separating said beads bearing captured Mycobacteria or b) contacting said high pH sample with a capture agent capable of binding Mycobacteria at the pH of said high pH sample to form a bound complex of said Mycobacteria and capture agent, and contacting said complex with a solid surface to bind said complex thereto.

The pH of said high pH sample is preferably at least 12. Accordingly, suitable pH raising reagents comprises an alkali, which may for instance be sodium hydroxide, potassium hydroxide, or lithium hydroxide, sodium hydroxide being preferred .

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.

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. Preferably, said capture reagent comprises cationic groups which remain positively charged at the pH of said high pH sample. 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 or semi-solid sample such as faeces or 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 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 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 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.

Optionally, and especially where the sample is sputum, said pH raising reagent further comprises a sputum thinning reagent. The most convenient of these is generally N-acetyl cysteine .

This acts as a mild reducing agent and probably

denatures S-S cross links in proteins in the sample. Other reagents for thinning sputum that can be used include sodium dichloroisocyanurate, sodium hypochlorite, mercaptoethanol , and 1 , 4-dithioerythritol or 1 , 4-dithiothreitol .

Said capture of the Mycobacteria is preferably carried out at an ionic strength of at least 0.3M, e.g. from 0.5 to 1.0M. This contrasts with what is shown in WO2008/065047. It is found that beads bearing captured Mycobacteria are not prone to clumping at high ionic strength and high pH, even in the absence of detergent, which is preferably not present. Non-Mycobacteria organisms that may be present will generally be rendered non-viable by the alkali treatment described above and in addition, not being sufficiently hydrophobic will not bind to the capture reagent, e.g. to p-DADMAC coated surfaces, thus giving a degree of selectivity of the type of organism captured.

In either aspect of the invention, said surface is suitably provided by beads. These may be of micro or nano dimensions. Suitably they are paramagnetic for easy

separation from liquid media. They may have a carboxylic acid polymer surface or a surface characterised by sulphate or phosphate groups.

The captured Mycobacterium, may be any of those referred to above.

The invention includes a method for the detection of a Mycobacterium, comprising capturing said Mycobacterium to a surface by a method as described, optionally washing said captured Mycobacterium, and detecting said captured

Mycobacterium on said surface or after removal therefrom.

The detection method used may be any appropriate method. For mycobacteria in general and M. tuberculosis in

particular, these will microscopic detection, e.g. by acid fast staining, optionally preceded by culturing to multiply said Mycobacteria, PCR - polymerase chain reaction, TMA - transcription mediated amplification, SDA - strand

displacement assay, or other amplification and detection methodologies directed to the nucleic acids of the organism itself, and phage based methods including FASTPlaqueTB where Mycobacterium infecting phage is added and allowed to enter the cells, phage that is left outside the cells is killed and after further incubation to release phage from the cells, the presence of the released phage is detected by infecting a further microorganism. Also included are immunological detection methods such as use of an antibody or immunological binding partner (including fragments of antibodies having immunological binding properties) which may have binding specificity for LAM. LAM ( lipoarabinomannan) is a complex branched chain carbohydrate linked to a lipid. LAM is essentially exclusive to Mycobacteria and anti-LAM antibodies can bind whole bacteria such as M. tuberculosis (Glatman-

Freedman et al; Jnl Clin Microbiol, Nov 1996, p 2795-2802) . An anti-LAM Elisa is available from Inverness Medical

Innovations (Clearview ™) .

Where intact cells are not required for the detection method, it may be preferable to lyse captured cells to increase accessibility of cell components to the detection system, such as by mechanical lysis disruption methods or by ultrasonic treatment. Mechanical disruption may be carried out using lysis beads in a Disruptor Genie™ or the like.

The materials, or selected key materials, needed for the practice of the Mycobacterium detection methods described above may be provided in kit form. Accordingly, the invention includes a Mycobacterium assay kit comprising either (a) a soluble capture reagent having both a hydrophobic character whereby the capture reagent is capable of binding a

Mycobacterium to be detected by hydrophobic interaction therewith and a polyionic character, a substrate having a surface for capturing said Mycobacteria to said surface by binding said capture reagent to said surface by polar

interaction between said surface and said capture reagent, or (b) a capture reagent coated on and thus immobilised upon a solid surface, said capture reagent having both a hydrophobic and polyionic character whereby the capture reagent is capable of binding a Mycobacterium to be detected, or else a solid surface capable of directly binding Mycobacteria under high pH conditions, a strong base for raising the pH of a Mycobacterium sample to at least 10,

and at least one of:

- phage capable of infecting said Mycobacterium;

- primers for carrying out an amplification of genomic nucleic acid of said Mycobacterium or said phage;

- a culture medium for culturing said micro-organism;

- an acid fast stain for visualising said Mycobacterium for microscopic inspection;

- an antibody for binding said Mycobacterium such as an anti- LAM antibody or binding fragment thereof; or

-a detection reagent for use in detecting a metabolite produced upon culture of said Mycobacterium.

In accordance with the invention described above, the sample may also be a gaseous, e.g. air, sample having

Mycobacteria entrained therein. Such a sample may be bubbled into a capture reagent solution to bind the Mycobacteria to the capture reagent.

A solid surface may be a microscope slide or beads as described above.

Preferably, the captured Mycobacteria, either captured directly or indirectly, are not harmed by this capture and remain viable. Thus the invention can be used for drug susceptibility testing of the Mycobacteria. In one aspect, the Mycobacteriacan be exposed to a drug in such a way as to allow the drug to affect the Mycobacteria. Subsequently, the Mycobacteriacan be captured in any of the ways described herein and then can be investigated for viability, optionally after neutralisation, using any number of previously

described methods which might include microscopy using viability stains, phage based methods, culture-based methods or PCR-based methods. In another aspect, the Mycobacteriacan be first captured in any of the ways described herein then subsequently exposed to a drug in such a way as to allow the drug to affect the Mycobacteria. Subsequently, the

Mycobacteriacan then be investigated for viability using any number of described methods which might include microscopy using viability stains, phage based methods, culture-based methods or PCR-based methods . The drugs used may include those commonly used to treat tuberculosis such as rifampicin, streptomycin, isoniazid, ethambutol, pyrazinamide, and ciprofloxacin. A kit as described above may include one or more drugs for drug susceptibility testing.

The invention will be further described and illustrated by the following Example. In the example, M. tuberculosis was used as a representative model organism for the mycobacterium genus .

EXAMPLE 1 : Concentration of Mycobacterium tuberculosis from sputum under conditions of high pH.

Rationale .

In this example, the Mycobacterium tuberculosis (TB) was concentrated from sputum in high pH conditions using beads (BioMag Amine beads, Bangs Laboratories, catalogue number BM546) coated with poly diallyl dimethyl ammonium chloride (pDADMAC) . The presence of TB on the beads was then

confirmed by acid fast microscopy without intermediate culturing .

Method

For comparison, sputum samples were thinned with sodium hydroxide, N-acetyl cysteine then split and one half

processed by the bead capture and the other half processed by routine laboratory procedure which included centrifugation concentration of the bacilli and auramine staining of the deposits . The bead capture protocol

1. The sputum was thinned following standard laboratory procedure by adding an equal volume of 0.5 M NaOH, 2% (w/v) N-acetyl cysteine.

2. Samples were left for 10 min.

3. 2 ml of the thinned sputum was then added to 1 ml of 4.5 M NaCl and 80μ1 of the pDADMAC coated beads (about 5 mg of beads) was added and incubated for 10 min. (note: the sample was not neutralised prior to this step and the capture occurred at high pH measured at 14 using pH paper) .

4. The beads were then collected using a magnet and the supernatant removed.

5. The beads were resuspended in 1 ml PBS containing 0.75M NaCl and again collected on a magnet.

6. The supernatant was removed and the beads resuspended in 50μ1 PBS before spreading onto a glass microscope slide and drying .

7. The slide was stained with auramine 0 for acid fast mycobacteria using a standard protocol.

Results

10 sputum samples were tested. 6 of the samples were

positive by microscopy by both methods (i.e. the traditional centrifugation concentration method and the bead capture method) . 4 samples were negative by microscopy by both methods. There was, therefore, 100% correlation between the two methods . Discussion

This example demonstrates that the TB can be captured from sputum under alkali conditions and that the method is

comparable in efficacy to the traditional method of

concentration by centrifugation but substantially more convenient. High salt conditions also contribute to thinning of the sputum and stop the beads from clumping. Salt is therefore a substitute for detergent which was used

previously to achieve the same result.

Example 2 : Further investigation of detection of TB in sputum by p-DADMAC-coated magnetic beads

Method

Sputum samples frozen in an equal volume of NALC were

received from the WHO TDR sputum bank. Samples were added to 2 ml sodium hydroxide and extracted using p-DADMAC-coated magnetic beads. The beads were analysed by auramine

microscopy and, after elution of the antigens by lysis disruption, by LAM-specific ELISA.

Results

* readings after subtraction of conjugate control blanks Anumbers given are numbers of bacilli per microscopy field Discussion

There was broad agreement between the numbers of bacilli seen by microscopy and the LAM ELISA signal. Sample 5 is of interest as microscopy and culture were negative but the ELISA was positive; the patient was designated as clinical TB.

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 of Mycobacterium from a sample comprising treating the sample with a reagent to raise the pH of the sample to at least 10, thereby to provide a high pH sample, and a) contacting said high pH sample with a solid surface capable of binding Mycobacteria at the pH of the high pH sample, and separating said suface bearing captured

Mycobacteria from said sample or b) contacting said high pH sample with a capture agent capable of binding Mycobacteria at the pH of said high pH sample to form a bound complex of said Mycobacteria and capture agent, and contacting said complex with a solid surface to bind said complex thereto.

A method as claimed in claim 1, wherein said solid surface capable of binding Mycobacteria at the pH of the high pH sample is a solid surface bearing a capture agent .

A process as claimed in claim 1 or claim 2, wherein the pH of said high pH sample is at least 12.

A process as claimed in any preceding claim, wherein said pH raising reagent comprises an alkali.

A process as claimed in claim 4, wherein said alkali is sodium hydroxide, potassium hydroxide, or lithium hydroxi

6. A method as claimed in any preceding claim, wherein said capture reagent comprises a hydrocarbon chain bearing multiple polar sites.

7. A method as claimed in claim 3, wherein said multiple polar sites are spaced along said chain. 8. A method as claimed in any preceding claim wherein said capture reagent and/or said solid surface comprises cationic groups which remain positively charged at the pH of said high pH sample. 9. A method as claimed in claim 8, wherein said cationic groups are quaternary ammonium groups .

10. A method as claimed in claim 8, wherein said capture

reagent is poly- diallyldimethyl ammonium chloride

(DADMAC) .

11. A method as claimed in any one of claims 1 to 9, wherein said capture agent is polybrene. 12. A method as claimed in any preceding claim, wherein said sample is a sputum sample.

13. A method as claimed in claim 12, wherein said pH raising reagent further comprises a sputum thinning reagent.

14. A method as claimed in claim 13, wherein said sputum

thinning reagent comprises n-acetyl cysteine.

15. A method as claimed in any preceding claim, wherein said capture of the Mycobacteria is carried out at an ionic strength of at least 0.3M. 16. A method as claimed in claim 15, wherein said ionic

strength is from 0.5 to 1.0M.

17. A method for detecting Mycobacteria in a sputum sample comprising capturing said Mycobacteria on a solid surface by a method as claimed in any preceding claim and

detecting said Mycobacteria.

18. A method as claimed in claim 17, wherein said

Mycobacteria are detected by acid fast staining and visualisation or by PCR.

19. A method as claimed in any preceding claim, wherein said solid surface is provided by beads. 20. A method as claimed in claim 17, wherein the viability of the captured micro-organism is determined.

21. A method as claimed in claim 20, wherein the captured

micro-organism is treated with a drug and the viability of the micro-organism is determined to establish whether the drug affects the viability of the micro-organism.

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

soluble capture reagent having both a hydrophobic

character whereby the capture reagent is capable of binding a micro-organism to be detected by hydrophobic interaction therewith and a polyionic character, a substrate having a surface for capturing said micro^¬ organisms to said surface by binding said capture reagent to said surface by polar interaction between said surface and said capture reagent, or (b) a solid surface having both a hydrophobic and polyionic character whereby the solid surface is capable of binding a Mycobacterium to be detected, a strong base for raising the pH of a

Mycobacterium sample to at least 10,

and at least one of:

- phage capable of infecting said micro-organism;

- primers for carrying out an amplification of genomic nucleic acid of said micro-organism or said phage;

- a culture medium for culturing said micro-organism;

- an acid fast stain for visualising said micro-organism for microscopic inspection;

- an antibody for binding said micro-organism; or

-a detection reagent for use in detecting a metabolite produced upon culture of said micro-organism.

A kit as claimed in claim 22, wherein the kit further comprises one or more drugs potentially able to affect the viability of said Mycobacterium.