SAMPLES

The present invention relates to a composition for use in the collection and pre-analysis storage of sputum samples. The present invention further relates to a system for the collection and storage of sputum samples prior to analysis. BACKGROUND OF THE INVENTION

The pre-analytical phase in the laboratory is where most errors occur and represents the most labour intensive, least standardized aspect of the process. Further, analysis of samples does not always occur immediately after the sample has been collected and therefore, effective storage of the sample may be required that stores the collected sample in an appropriate form to be further analysed at the appropriate time. Thus, the processing of collected samples is of paramount importance to allow effective downstream analysis, especially when using automated processing techniques and systems.

This presents a challenge for any sample collection and storage system. However, when working with sputum samples, the viscosity of the samples can reduce reproducibility on growth media and also preclude the use of automated pre-analytical systems. Automated processing, such as the Kiestra Total Lab Automation System (TLA) requires a liquefied sample in a standardised container, to minimise the level of manual processing required prior to automated processing. Liquification of viscous sputum samples is currently carried out by immersing the collected sputum sample in a solution of DTT (Dithiothreitol, also known as Cleland ^' s reagent) or N-acetyl-L-cysteine-sodium hydroxide (NALC/NaOH). However, both these reagents are highly caustic in solution and are also relatively unstable. Therefore, the reagents are unpleasant and potentially dangerous to use and have a relatively short shelf- life. Further, although DTT and NALC/NaOH are often used for mycobacteria (which causes tuberculosis), it is lethal for many other bacterial types and therefore cannot be used for sputum samples intended for microbiological processing to potentially detect the presence of other bacteria. For example, DTT is lethal for Haemophilus influenzae.

There is therefore a need for a system for the collection, liquefication and storage of collected sputum samples prior to onward automated processing and analysis that avoids the use of potentially dangerous caustic reagents. SUMMARY OF THE INVENTION

The present invention seeks to address the problems of the prior art. Accordingly, a first aspect of the present invention provides a composition for the preparation and pre-microbiological analysis of a sputum specimen, wherein the composition comprises a mucolytic agent having the following formula:

or salts thereof, where:

Ri is hydrogen, or an unsubstituted alkyl group having 1 to 3 carbon atoms;

each R ₂ and R ₃ is independently hydrogen, an alkyl group having 1-3 carbon atoms, a phenyl or a benzyl group each of which is optionally substituted with one or more of substituents Wa;

each R« and R ₅ is independently hydrogen, a halogen, a cyano group, a nitro group, a hydroxy!, an alkyl group having 1 -6 carbon atoms, a phenyl, a benzyl group, an N(Rg) ₂₍ or a -COR10 group, wherein each alkyl, phenyl, or benzyl group is optionally substituted with one or more of substituents WE;

each Re and R?, is independently hydrogen, a 1 - 2 carbon alkyl group, an aryl group, a heterocyclic group, a heteroaryl group, a-COR _t i group, a -COORn group, a -CO

MHR11 groyp, a -CO-NHR group, a -SOrRn group, or a -(CH ₂ ) _n -Ri2 group, wherein each alkyl, aryl heterocylic or heteroaryl group is optionally sybstiluted with one or more of substituents W ₃ ; and each K« s independently hydrcgen or -CC-K-?.

wherein:

each R§ isMepeno nlly hyttoaen a I - ' ? i:a«:xm alkyl group, an aryl ij- up, a heterocyclic g-oup a eterparyi grojp. a -COH,. aroup. a -CQOH-, croup. a-CO NHRr grr.up, i CO NHR,, rjroi.p. a SO- Π ₍ · group, cr a ! h^n F,_ r..-e$up winro n is an integer ranging from ^; 1 - ^' ? where-; ^* each alkyl, aryl heterocyc ic o ^* ^eiercaryf group is oal nally sjbslluted w.ih crie o' no e of s-bstituefils W ₃ :

each Rio isindcp ndc": y h ciogen a l 1? RU grsu it nhor/ ·.:· tv izyl group which are optional y subsltluieo witn ere or mrj-e of sjfastituents W.

each Hi a-d K..-> is ir.dopendorr. y *iydro¾cn. a '-12 carbon alkyl group, an ary: g-ouD a icorocyc-Lc group, a clcroaryl group, a L-T group or a M group, wherein each alky :¾ryi ctc^uy'ic iv heitj-onryl group is optionally subet luted wth one c note Of substitu ntS W-i, -L-is a diva erl 'inkc Cro a-d I is bioiog'cat soeaes or a suraee to which tho rc .ic ng agent is lin ed and is a rnnrtvc greuD or a anncer moiety SAn ar-u

eaeh !·· Γ; -dep-rarc^ _f 'C0Gi>\ a ^* - ^* 2 z^bcr r.-ky qr ^" .,p, a- :ry p'=gi> :.· hetero yclic g'ftiip, or a hotc onty g:.iup A-h rn-n ach alkyl, aryi hGlnrc r,y nlic or heteroaryl group- is optionally substiltied win one or more of substituents W .

wheren:

W. s one or noio suhai Le^'s selected *rcn» halogen, ar MD j c .p i 0), oyono «mgp, n !rc 3 reus hydr xyl, unsubstihJted alky! grouo lavr g ^* -3 carto ^* atoms, ha egen- substiluiea alkyl grojp hav ng 1-3 carbon atoms, or„nc-bciu:ad slkcty a-aup having

I 3 en TO- atoms; and

Wi s one ty more subsi,Le ^r - ^* .£ selected ^r, cm ha gon, ar exo group .0}. cyan? grou , n :rc grouo, hydroxyl, oplionally suDsUluloo a!kyl grc p hav ng 1-8 carbon ¾oms, unsubstitutad alkyl group having 16 narbcn atoms, hydroxyl-subs: "uted alky! g^-uo havirg 14} a-bon atom? group havlnj 1-H camin aton? urt$ub$titutad alkoxy group having 1-B cardan atoms, alkenyl grojp hav ng∑ ^» 6 carbon atoms; alkynyl group having ? f> Rvtan atoms, aS-i-memfcr-r mnycic ring, wl-nrain one u- two nng a-tuHis ary oplionally replaced with -CO- ard ivh«c ^* i may co-tam one or two double DO ids. a- a'y group laving 6-14 carbon ring atoms a Dhenyl group., a benzyl :ii ^' L- L ^' . ' U c- T Toer πι "*-!*.. 'o c i . .iL' ^" j ^r .) ^" and wherein - - f - t-.vo r - f; - rl- - n r rye n :, rmn- 1 v ··:. p n with i-1.v n ττ, Γ - - - - - ? - -· c two douh!e iwtdr or ;:=. Mwriaryl gn .p li v ^r, g 1-3 hcku aioms (N 0 oi S), ~G<¾R« g-ouD -CONlHi _f Jtyowp, -OCON^-.!? group, -NiBisw group. a-SOr-OH^ prcua - i;c;HJ.„ OR, _» group ,CH _: i,, N _: P _lr .^. each Ri.i and F,. is independent hydrogen, an antubsiluted alk * ly o lavmg 1-6 Ciirfcw iiloms an . ^, nr^.bsr,- cd qr l nroi-p gv ng 54 exrooi iUoms. an yn tetituicd pfceryl g-OL , ar ..n substituted group, an unsubstHulec 6- or c- rerbei ring heterocyclic g'oim · -3 hottiroaioms and wf-orwi -jne ur two ππι; carbon a-u ly rep ^' need with C J< a-d whicn -rr.y coT.ar. cne or two doub c bonds, or a unsubstitui u h ½r a ^* yl gicrnp ha ing 1-3 -u?:eioalcns with ih© motion that ^rt o R. ₄ is r ugen.

^• .nrys" ottwrthan a hydrogen,

Compounds of the above formula are known as dithioamine compounds. One example of a dithioamine is dithiobutylamine (DTBA).

In one embodiment, the mucolytic agent is selected from the group consisting of DTBA and DTBA hydrochloride (DTBA.HCI).

Dithiobutylamine (DTBA) has the formula:

(2S)-2-amino-1,4-dimercaptobutane

Dithiobutylamine hydrochloride (DTBA.HCI) has the formula: (2S)-2-amino-1 ,4-dimercaptobutane hydrochloride; or

Sputum is typically understood to be a mixture of saliva and mucus coughed up from the respiratory tract. Examination of a sputum sample from an individual permits the

identification of any bacterial infection present and the level of infection present. This then allows an appropriate treatment regime to be identified to either clear or manage the bacterial infection. As a sputum sample has high viscosity, it is necessary to reduce the viscosity to allow onward automated processing of the sample. Dithiobutylamine hydrochloride (DTBA.HCI) is a stable white crystalline powder which is soluble in water. It is commonly used as an agent for reducing disulphide bonds in protein/peptide research in order to keep proteins in an unfolded state while carrying out various biophysical studies. Dithiobutylamine hydrochloride, as used in the present invention, provides significant advantages over the traditionally used Dithiothreitol (DTT) as, at physiological pH, DTT is a slow reducing agent. Further, DTT is highly caustic and relatively unstable in solution. Thus, it can be hazardous to use and has a limited shelf-life.

It is important when considering automated microbiological analysis of a sputum sample that the viscosity of the sample is reduced. Otherwise, the automated processing will not be effective and provide reliable, repeatable results. To obtain effective liquification of sputum samples, it is necessary to use a mucolytic agent to dissolve or break-down the mucus in the sputum sample. The mucolytic agent used in the composition of the present invention acts to reduce or prevent the formation of disulphide bonds.

In one embodiment, the mucolytic agent is sterile. Preferably, the phosphate buffered saline solution is sterile. It is to be appreciated that the composition of the present invention may be provided in a non-sterile form and subsequently autoclaved by the user prior to use. However, if the composition is provided in a sealed vial and has already undergone procedures to ensure the sterility of the composition within the sealed vial then it is more convenient for the user who can simply proceed with adding the collected sputum sample to the vial without the requirement, expense, man-power or delay of having to carry out an autoclaving step to sterilise the contents of the vial prior to use.

In a further embodiment, the mucolytic agent is solubilised in phosphate buffered saline. This provides a stable environment within which the DTBA.HCI can be solubilised to DTBA which is active in reducing disulphide bonds in proteins. Solubilised DBTA is a more effective reducing agent than DTT as it works over four times faster than DTT and does not possess the distinctive foul odour of DTT.

Preferably, the solubilised mucolytic agent has a neutral pH in the range of from 6.5 to 7.5 and preferably from 6.8 to 7.2, and more preferably a neutral pH of 7. The mucolytic agent used in the composition of the present invention has enhanced mucolytic properties due to the presence of two active thiol groups which are present in the molecule at neutral pH.

In a preferred embodiment, the mucolytic agent is present at a concentration of greater than 0.5mM and more preferably at a concentration of around 0.52mM. A second aspect of the present invention provides a system for the preparation of a sputum specimen for onward automated processing, wherein the system comprises: a collection device defining a chamber therein; and

a composition according to a first aspect of the present invention received within said chamber.

Thus, the system may be supplied to a user with the composition already provided within the collection device. A sputum sample may then be introduced into the collection device and liquefied by the DBTA, ready for onward automated processing.

The collection device may be designed specifically for the automated processing apparatus to be used. Preferably, the container comprises a vial. Such a vial may be dimensioned so as to be received within a selected automated processing apparatus. For example, where the automated procession apparatus is the Kiestra Total Lab Automation System (TLA), the vial is preferably a cylindrical tube of dimensions 82mm x 13mm with a cap of dimensions 14mm to 16mm. It is important that the vial is compatible with automatic decapping and recapping, as is carried out during the automated processing steps.

In a preferred embodiment, the vial is Kiestra Total Lab Automation System (TLA) compatible.

In one embodiment, the chamber and composition are sterile. This provides the advantages mentioned above with respect to immediate use of the system by a user rather than having to first sterilise the vial and composition contained therein.

Preferably, the collection device is re-sealable. This allows a sample to be introduced into the vial, preferably under sterile conditions to prevent ingress of any additional bacteria from the environment into the composition contained within the vial, with the vial subsequently being sealed from the environment, thus maintaining the integrity of the vial contents. Thus, any bacteria detected will be due to bacterial infection within the sputum sample rather than as a result of bacterial infection from the environment contaminating the results.

A third aspect of the present invention provides a method of preparation of a sputum sample for automatic processing for the detection of bacterial infection, the method comprising the steps of a. providing a system according to a second aspect of the present invention; b. adding a sputum sample into the composition within the chamber of the collection device;

c. agitating the collection device to reduce the viscosity of the composition

containing the sputum sample; and

d. providing the sealed collection device containing the dissolved sample to an automated processing system.

The automated processing system may be used to identify the presence of one or more bacteria within the sputum sample and optionally the level of bacterial infection. This can then be used to identify an appropriate treatment for the provider of the sputum sample to clear or manage the bacterial infection.

The automated processing system used may be the Kiestra™ Total Lab Automation System (TLA) or any other suitable automated processing system known to the skilled person.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagrammatic illustration of the chemical formula for dithiobutylamine hydrochloride; and

Figures 2A to 2D shows the Kiestra technology digital images resulting from the automated processing of sputum samples dissolved in a composition according to a first aspect of the present invention. Figure 2 A shows Staphylococcus aureus on chocolate blood agar; Figure 2B shows Staphylococcus aureus on blood agar; Figure 2C shows mixed growth of upper respiratory flora, Staphylococcus aureus and haemolytic Streptococcus; and Figure 2D shows Streptococcus pneumoniae on blood agar.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Anonymised clinical samples were split and processed via a routine non-automated methodology including streaking and manual incubation and in parallel through a separate work stream involving utilising the sigma SP liquid collection device and Kiestra pre- analytical InoqulA FA automation and automated incubation with digital reading using the following protocol:

Sample collection and processing: Sputum samples were collected from patients in wide mouth screw cap specimen pots of 60 ml in size. The samples were then processed as follows: a) A first set of ten of the collected sputum samples were added to Oxoid Sputasol SR0233A (a mucolytic agent), vortexed for 30 seconds and left at room temperature for 15 minutes before being manually spread, using a 10μΙ loop, on blood agar, chocolate agar and MacConkey agar. The agar plates were then incubated for 24 hours in a 5% C0 ₂ enriched atmosphere or under normal aerobic conditions and colony formation assessed visually. The CO2 enrichment improves the recovery of Streptococcus pneumoniae, a bacterium commonly present in respiratory infections and detectable in sputum samples.

b) A second set of ten sputum samples were collected from the same patient as for a) above, and added into vials containing composition X comprising dithiobutylamine hydrochloride in phosphate buffered saline solution at neutral pH, vortexed for 30 seconds and left at room temperature for 15 minutes before being processed automatically using the Kiestra™ InoqulA FA (blood, chocolate and MacConkey agar) and incubated on the Kiestra™ Compact Readers. An automated assessment of plate coverage and single cell colony count was carried out using digital imaging after 48 hours.

Composition X:

DTBA.HCI 0.09g/l

Na2HP04.7H20 1 .64g/l

NaH2P04 0.47g/l

NaCI 8.77g/l

H20 was then added to make the solution up to a final volume of 1 litre.

Each vial has a capacity of 5ml. 1 ml of composition X was provided to each vial and 1 ml of sputum sample then added to composition X prior to the vortexing step and subsequent incubation at room temperature prior to onward automated processing.

This automated processing method was repeated for three different batches of the dithiobutylamine hydrochloride in buffered saline solution at neutral pH, to ensure reproducibility of the results achieved.

The results of a) and b) were compared to assess the effectiveness of the automated process relative to the traditional manual process. Results

Table 1 Number of discrete colonies per plate using manual processing

Table 2 Number of discrete colonies per plate using automated processing

Analysis of results As can be seen from the results obtained, significantly more single colonies were observed using the automated procedure, and there is clear evidence that the whole surface area of the plate was utilised. This results in an increase in the number of single colonies available for secondary testing. See Figures 2A to 2D. The images of the plates in Figures 2A and 2B demonstrate good provision of single colonies using automated processing. This is particularly important for onward identification of the colony bacterial type.

The images of the plates in Figures 2C and 2D show that the use of the composition of the present invention to fluidise the sputum samples prior to automated processing of the sample using the Kiestra system results in good utilisation of the whole surface area of the plate, which has a consequential increase in the number of single colonies available for further testing. This is an important benefit as it increases the possibility of correct identification of the causative organism potentially causing illness in the subject providing the sputum sample. Further, there is greater consistency in the number of colonies identified in batches A1 to A3 than seen using the traditional manual processing method.

As can be seen from Table 2, the results demonstrate that the novel mucolytic agent used in the collection of sputum samples for batches A1 , A2 and A3 is providing effective emulsification of sputum and mucus within the samples resulting in a homogeneous suspension that allows easier, more consistent and reproducible plating and streaking of specimens than the traditional manual processing method.

Conclusions

The novel mucolytic agent containing dithiobutylamine hydrochloride in buffered saline solution at neutral pH coupled with automated sample processing provides a more standardised approach to sputum and mucus sample handling. Further, the results are both consistent and reproducible.

A further specific benefit of using dithiobutylamine hydrochloride rather than other traditional mucolytic agents is that dithiobutylamine hydrochloride is non-caustic and more stable in solution than traditional mucolytic agents and therefore is less hazardous to users during the sample collection stage of the process as well as having a longer shelf-life, thereby being more cost-effective.