METHODS AND DEVICES

Abstract

A method of determining the presence of a threshold concentration of leukocyte cells in a sample, the method comprising (i) contacting the sample with a leukocyte detection means comprising (a1) an indicator compound; and (ii) examining the leukocyte detection means to determine whether leukocytes are present.

Claims

1. A method of determining the presence of a threshold concentration of leukocyte cells in a sample, the method comprising (i) contacting the sample with a leukocyte detection means comprising (a1) an indicator compound; and (ii) examining the leukocyte detection means.

2. A method according to claim 1 wherein the sample is selected from peritoneal dialysis effluent and fluid drained from the peritoneal cavity present due to ascites.

3. A method according to claim 1 wherein the sample is peritoneal dialysis effluent.

4. A method according to 1, wherein the indicator compound (a1) is a tetrazolium compound.

5. A method according to 1, which further involves analysing the sample for the presence of microorganisms wherein step (i) further comprises contacting the sample with: (II) a first reporting means comprising: (a2) an indicator compound; and (d1) media and/or nutrients that support or encourage microbial growth.

6. A method according to claim 1, wherein step (i) further comprises contacting the sample with (III) a second reporting means wherein the second reporting means comprises: (a3) an indicator compound; (d2) media and/or nutrients that support or encourage microbial growth; and (e) a selection factor.

7. A method according to claim 5, wherein the indicator compound (a2) in the first reporting means is a water soluble tetrazolium salt.

8. A method according to claim 5 wherein the media and/or nutrients that support or encourage microbial growth (d1) in the first reporting means is Wilkins Chalgren media.

9. A method according to claim 5 wherein the first reporting means further comprises an electron mediator.

10. A method according to claim 1, wherein the leukocyte detection means further comprises an electron mediator.

11. A method according to claim 10 wherein the electron mediator is 1-Methoxy-5-methylphenazinium methylsulfate (mPMS).

12. A method according to claim 6, wherein the selection factor inhibits the growth of Gram positive microorganisms.

13. A method according to claim 12 wherein the selection factor is vancomycin.

14. A method according to 1, wherein the leukocyte detection means further comprises one or more antibacterial agents.

15. A method according to claim 5, wherein the first reporting means reporting means further comprises a leukocyte inhibiting agent.

16. A method according to 1, wherein step (ii) involves examining the leukocyte detection means.

17. A device for detecting a threshold concentration of leukocytes in a sample, the device comprising a channel arranged to receive the sample wherein the sample comprises (a 1) an indicator compound and a buffer.

18. A device according to claim 17 which is also useful for detecting and/or identifying microorganisms that may be present in the sample, said device comprising three channels that are arranged to receive the fluid and wherein: a first channel contains a first reporting means comprising: (a2) an indicator compound; and (d1) media and/or nutrients that support or encourage microbial growth; and a second channel contains a second reporting means comprising: (a3) an indicator compound; (d2) media and/or nutrients that support or encourage microbial growth; and (c) a selection factor which selectively inhibits growth of microorganisms; and a third channel contains a leukocyte detection means comprising: (a 1) an indicator compound; and (b) a buffer.

19. A device according to claim 18 wherein each of the first and second channels further comprises an electron mediator and the third channel further comprises a buffer.

20. The device according to claim 18 wherein the channels are retained within a casing and said casing has viewing windows for observing the contents of the channels.

Description

DETAILED DESCRIPTION

[0319] The present invention will now be described in detail with reference to the following figures which show:

[0320] FIG. 1: represents a perspective view from top and two sides of a device which may be used according to the invention.

[0321] FIG. 2: represents a perspective view of channels within the device of FIG. 1.

[0322] FIG. 3 shows a channel comprising a leukocyte detection means before incubation and after 10 hours of incubation.

[0323] FIG. 4: is a photograph of tubes containing reporting means and no microorganisms (control) or between 10.sup.4 and 10.sup.6 CFU/mL of Staphylococcus aureus (SA) or Pseudomonas aeruginosa (PA). The upper row of tubes did not contain any selection factor whereas the lower row contained vancomycin.

[0324] FIG. 5: the top row of photographs shows the appearance of an uninfected effluent bag and images of channels 1 and 2 (first and second reporting means) after 0 and 8hrs incubation with the PD effluent from the effluent bag; and the bottom row of photographs shows the appearance of an effluent bag which a clinician suspected was infected and images of channels (removed from the device casing) after 0 and 8hrs incubation with the PD effluent from the suspect effluent bag.

[0325] FIG. 6 is a schematic diagram showing possible outcomes for each channel and what this is indicative of, in terms of infection.

[0326] FIG. 1 is a perspective view from top and two sides of a device 1 according to the present invention. The device comprises an opaque plastic casing 2 which has transparent viewing windows 3, 4, 5 in the top face. A first viewing window 3 is aligned over a first channel 30 contained within the casing, the second viewing window 4 is aligned over a second channel 40 contained within the casing, and the third viewing window 5 is aligned over a third channel 50 contained within the casing. The viewing windows 3, 4, 5 are position such that the contents of the channels may be observed by a user of the device. In use fluid is introduced via inlet 6 and flows through tubing (not shown in FIG. 1) into the channels (also not shown in FIG. 1) which contain the reporting means.

[0327] FIG. 2 is a perspective view of the channels 30, 40, 50 contained within the casing 2 shown in FIG. 1. Tubing communicates fluid to the first channel 30, second channel 40 and third channel 50 which in this embodiment are bags with a transparent upper face. The tubing includes one-way valves, 31, 41, 51, 61 which prevent back flow of the fluid and reporting means up the tubing. The reporting means within the first channel 30 comprises one or more capsules containing WST9, mPMS Wilkins Chalgren media, and polyvinyl pyrrolidone (PVP) filler. The reporting means within the second channel 40 comprises one or more capsules containing WST9, mPMS, Wilkins Chalgren media, PVP filler and vancomycin is also provided impregnated on a filter disc. Each of the capsules dissolves when fluid is introduced into the channels. The components of the reporting means effectively combine when the fluid is introduced into the channels. The WST9 in the first channel is reduced to dark coloured formazan if microorganisms are present in the fluid and WST9 in the second channel is reduced to dark coloured formazan if microorganisms are present in the fluid which are resistant to vancomycin.

[0328] The leukocyte detection means 50 comprises in one or more capsules MTT and MES buffer.

EXAMPLES

[0329] The inventors realised that there were no commercially available products that were small and simple which may be used for detecting high levels of leukocytes and microbial contamination of fluids, in particular, PD effluent.

[0330] Initial proof of principle experiments were conducted to establish whether or not devices could be created that could distinguish/select between microorganisms and also for which the threshold for triggering of the leukocyte detection means and reporting means could be controlled.

[0331] 1. Preparation of Components of Reporting Means

[0332] Reporting means comprising WST-9, mPMS and PVP were tested in the presence of eight different species of bacteria. It was established that 10.sup.5 CFU/mL of all bacteria were able to reduce the tetrazolium to provide a colour change within 8 or 10 hours at 37 C.

[0333] Capsule Filling

[0334] An empty capsule was weighed as a blank. The tapped density of each active component (i.e. WST-9, mPMS and vancomycin) with PVP excipient was established by weighing the amount of active and adding it to a size 5 capsule. PVP excipient was added and compressed into the capsule until full. The capsule was weighed again to establish the mass of excipient required to fill a size 5 capsule for each active component. This was repeated 5 to give an average weight of a capsule for each active and excipient.

[0335] 100 mass of each active and excipient was prepared and mixed thoroughly to generate a homogenous mixture of powders with the active evenly distributed throughout the excipient. This was used to fill 100 capsules using a Feton Fastlock Kit.

[0336] Preparation of a Vancomycin Filter Disc

[0337] 6 mm Whatman filter discs were prepared by soaking in a stock of vancomycin in water. These were dried as a single sheet at 37 C. for 18 hours or until completely dry. The final concentration of antibiotic on each disc was 256pg.

[0338] 2. Assembly of a Device according to the Invention

[0339] Device for detecting microbial contamination of PD effluent from an Automated peritoneal dialysis (APD) machine (Baxter) were produced by:

[0340] 3. Fabrication of Channels and Loading them with Reporting Means [0341] 1. White PVC material is overlaid with transparent PVC, both cut to 93 mm70 mm. [0342] 2. The longest edges are welded and two channels are created by welding the middle of the bag. [0343] 3. At the top edge two tubes (3.0 mm diameter, 25 mm length) are inserted, one into each channel, which are secured by sealing around the tubes. [0344] 4. Capsules are inserted between the PVC sheets that will form the first channel and the bottom edge is sealed. [0345] 5. Capsules and a vancomycin filter disk are inserted between the PVC sheets that will form the second channel and the bottom edge is sealed. [0346] 6. Capsules are inserted between [0347] 7. White PVC one way check valves are inserted into each of the tubes [0348] 8. Two flexible PVC tubes are secured to each valve. The two tube are joined by a PVC Y connector from which a single tube, 1 meter in length, is attached.

[0349] 4. Assembly of the Device [0350] 9. The bag/channel assembly is inserted into a casing comprising a thermoformed blister tray and the assembly is secured in place by pressing the valves into preformed recesses in the blister tray. [0351] 10. The tube is fed through a hole in the right side of the blister tray. [0352] 11. At the end of the tube a valve in attached followed by a male luer lock that is compatible with the peritoneal dialysis consumables from which effluent is sampled. [0353] 12. The luer lock is protected by a cap. [0354] 13. The blister pack is sealed with an opaque Tyvek lid which has two transparent windows through which the bag channels can be visualised. [0355] 14. The blister tray and tubing is sealed into a PET/PE/Tyvek peel pouch and packaged into boxes.

[0356] 5. Testing Channels for use in Devices according to the Present Invention

[0357] Dialysis effluent samples were obtained from peritoneal dialysis patients by filling their peritoneum with dialysate for a minimum of two hours. After this dwell time, fluid was drained from the peritoneum and a sample was tested by filling the channels of the device with 16mL per channel. The channels were then incubated at 37 C. for 8 hours, after which the result was read.

[0358] A leukocyte detection channel according to the invention was also prepared. This contained MTT buffered to pH 6.5 with MES. The PD effluent samples were tested using this channel. A first sample from a well-patient of clear fluid and independently confirmed to have below the threshold concentration of leukocyte cells was tested along with a cloudy sample of an unwell patient independently confirmed to contain high concentration of leukocytes. FIG. 3 shows in the top row the sample of the well patient initially and after incubation for 8 and 10 hours. In the bottom row the sample from the unwell patient is shown.

[0359] FIG. 5 shows photographs of first and second channels of two exemplary tests performed on PD effluent bags from a clinic where the patients were undergoing automated peritoneal dialysis (APD).

[0360] The top row of photographs shows the appearance of the channels from PD effluent collected from a patient that seemed well. Photographs were taken of the channels after 0 and 8hrs incubation at 37 C. and it can be seen that the reporting means within the channels have not been activated after 8 hours incubation. A photograph was also taken on the effluent bag and it can be seen that the fluid is relatively clear. These results suggested that the patient was not suffering from peritonitis and the clinician subsequently confirmed that the patient remained well.

[0361] The bottom row of photographs shows the appearance of the channels from PD effluent collected from a patient who had started to feel unwell. Photographs were also taken of the channels after 0 and 8hrs incubation at 37 C. A photograph was also taken on the effluent bag and it can be seen that the fluid did appear to be cloudy. The figure shows that the reporting means within both the first and second channels had activated. This suggested that the patient was suffering from peritonitis and that it was likely to be caused by a Gram ve organism (Vancomycin in the second channel failed to inhibit reporter activation). Two days later the clinician confirmed that the patient had a Gram ve infection when he received confirmation from the hospital testing laboratory. It will be appreciated that the device accurately and quickly (2 days quicker than routine laboratory testing) identified an infection and also the type of infection. This has the great advantage that clinicians may use the device to make informed and early decisions about treatment for peritonitis. This in turn improves the outcome for the patient, saves the health service money and also has the advantage that patient who can have infections identified and treated at an early stage have a better chance of being maintained on PD (rather than needing to be transferred to HD).

[0362] 6. Use of Devices according to the Invention in a Clinical Setting.

[0363] A protocol was established, and followed, for when devices according to the invention were used to monitor for microbial contamination of PD effluent in conjunction with an Automated peritoneal dialysis (APD) machine (Baxter).

[0364] Users of the device were instructed to: [0365] 1. Wash and dry hands thoroughly and use aseptic technique to reduce the risk of contaminating the sampling device [0366] 2. Open the device packaging [0367] 3. Ensure the clamp on the effluent sample line is closed [0368] 4. Remove the cap from the sample line [0369] 5. Remove the red cap from the device. Save the cap. [0370] 6. Connect the device to the sample line with a twist action until locked [0371] 7. Position the device on the floor at the position of the drain bag [0372] 8. Follow the instructions on the Baxter machine when you start your treatment [0373] 9. At the point of Initial drain, wait for 40 seconds [0374] 10. Open the clamp on the sample line and allow the effluent to fill the device. This should take no more than 2 minutes. [0375] 11. When the device is full, close the clamp on the sample line. [0376] 12. Disconnect the device from the sample line and re-cap the connectors [0377] 13. Ensure the incubator is switched on at the plug [0378] 14. Take the sampling device to the incubator and place inside. [0379] 15. Close the door and press start [0380] 16. The device will heat to 37 C. and will stay incubated at this temperature for 10 hours, after which it will cool to 4 C. (fridge temperature). [0381] 17. You can view the device after a minimum of 10 hours. [0382] 18. If you are not ready to view the device immediately, keep the device in the incubator with the door closed which will keep the result fixed. If you remove the device it must be read within 1 hour.

[0383] 7. Illustration of how to Read Device

[0384] FIG. 6 provides a schematic view of possible results that may be obtained when using a three channel device according to the invention wherein the device contains:

[0385] Channel 9 (first reporting means):

[0386] WST-9

[0387] mPMS

[0388] PVP

[0389] Channel 2 (second reporting means):

[0390] WST-9

[0391] mPMS

[0392] PVP

[0393] Vancomycin

[0394] Channel 3 (leukocyte detection means):

[0395] MTT

[0396] MES

[0397] 8. Further Example Device of the Invention

[0398] A further example of the invention was prepared comprising the following components in amounts to provide the concentrations specified assuming 16 mL of PD effluent is provided to each channel.

TABLE-US-00001 Final concentration Substance mg/mL Channel 1 Wst-9 0.3778 (first reporting means) mPMS 0.008415 Wilkins Chalgren Broth 11.6 Sodium bicarbonate 0.31 Digitonin 0.08 PVP >0.8 Channel 2 Wst-9 0.3778 (second reporting means) mPMS 0.008415 Wilkins Chalgren Broth 11.6 Sodium bicarbonate 0.31 Digitonin 0.08 Vancomycin 0.016 PVP >0.8 Channel 3 Wst-9 0.3778 (leukocyte detection means) mPMS 0.0168 MES hydrate 4.881 Sodium bicarbonate Meropenem 0.016 Ciprofloxacin 0.008 Vancomycin 0.008 PVP >2.5

[0399] FIG. 7 shows photographs of channels of the above device (example 8) when exposed to a variety of clinical samples. In this figure when we say that bacteria or leukocyte cells are present or not present we mean that they are present above the threshold concentration or not present above the threshold concentration.