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Standard Operating ProcedureThe Kwikculture IDSX Kit for presumptive

Identification and direct (without prior isolation) Antimicrobial Susceptibility Testing

of urinary tract pathogens

Rev. 07222011

Slides 20-31 are still pictures and notes from Movie posted on YouTube http://youtu.be/G5sNoGOtU3Y

The IDSX kit is a new in-vitro diagnostic medical device used for identification and direct antimicrobial susceptibility testing of bacterial infections. It is inexpensive, designed for “point of care”, easy to use, and with an over-night result. Measuring films also provide a straight forward reading of Resistant, Intermediate or Susceptible directly from the chambers. This replaces the need to manually measure zones and consult standard interpretive charts to obtain results.

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IDSX Kit contents and description

Transparent measuring films (2) (for post-incubation placement)

Color coded Antibiotic applicators(2) with antibiotic papers(up to 14) attached under protective covers.

Loop/Needle(1) (ends bent)

IDSX Plate(1) with lid removed.

CCNABColumbia CNA agar with 5% blood

MACMacConkey agar

MHMueller-Hinton agar

MHMueller-Hinton agar

Sterile Water(2)

Antibiotic paper release rod (1)

Rayon tipped swab(1)

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IDSX Kit packaging

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Kits are stored, with lids upside down, at refrigerator temperature to maximize storage life of the agar-based media (at least 12 months). Storage in crisper is ideal location to avoid freezing and also refrigerator temperature variations.

IDSX Kit Storage

Remove dish from storage pouch and inspect plate, making sure there are no contaminants present. Retain pouch for incubation step.

Begin the IDSX Kit set-up:

IDSX Kit Storage and Set-up

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IDSX kit tools used for sample application and colony isolation

The rayon tipped sterile swab is used for applying sample to the chambers of the IDSX plate by: (1)uniformly spreading the sample over the surface of the agars in the two MH chambers [ where susceptibility testing is done] and (2) applying the sample as a streak in the CCNAB and MAC chambers [where isolation, quantitation and eventual identification are performed].

The loop/needle dilutes out the sample from the streaks in the CCNAB and MAC chambers to allow for isolated colony growth (“isolation”).

sterileswab

Loop/needle

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Multistix 10 SG Reagent Strips for Urinalysis

10 LEUKOCYTES neg. trace small mod. large2 min

9 NITRITE neg. pos.=any degree of uniform pink60 sec.

8 UROBILINOGEN(mg/dl) 0.2 1 2 4 860 sec. normal

7 PROTEIN(mg/dl) neg. trace 30 100 300 >200060 sec.

6 pH 5.0 6.0 6.5 7.0 7.5 8.0 8.560 sec.

5 BLOOD neg. trace mod. trace small mod. large60 sec. non-hemolyzed hemolyzed

4 SP.GRAVITY 1.000 1.005 1.010 1.015 1.020 1.025 1.03045 sec.

3 KETONE(mg/dl) neg. 5 15 40 80 16040 sec.

2 BILIRUBIN neg. small mod. large30 sec.

1 GLUCOSE(mg/dl) neg. 100 250 500 1000 >200030 sec.

Bayer 1 2 3 4 5 6 7 8 9 10

UA or urine dipstick

Prior to setting up the IDSX kit, a urine dipstick may be used to determine if a dilution of the specimen is warranted. A too-high of concentration of pathogens in the applied sample may cause false negative results( see “inoculum effect…”, slide 17..). Leukocyte esterase, nitrite, blood or protein in the specimen may indicate bacteria in the urine. When NITRITE is positive, suggesting a significant concentration of gram negative uropathogens, prepare a 1:10 dilution of specimen in sterile water for IDSX plate application (see second paragraph of next slide).

Preliminary determination of bacterial concentration

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Sample application to the twin MH Antimicrobial susceptibility chambers

Apply sample to one MH chamber by first dipping rayon swab in specimen to just cover the tip for 5 seconds. Without touching the edge of the sample tube, transfer this to chamber and uniformly spread over the surface. This will apply approximately 0.05ml of the 0.14ml picked up by the swab to the surface of the agar. Repeat for the other MH chamber. Antimicrobial agents will be added at a later step to these two chambers.

Do the following if a 1:10 dilution is necessary (see urine dip stick slide): Since the swab picks up 0.14ml under the above conditions, add the sample saturated swab to 1.26ml sterile water supplied with the kit and mix by swirling prior to applying as described above.

CCNAB MAC

MH MH

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Sample application to MAC and CCNAB chambers to provide for colony isolation

Following the addition of organisms to the MH chambers, as described in the previous slide, sample is now added to the MAC and CCNAB chambers. The swab is dipped in sample and a single streak is made across the MAC chamber (see “1” at left) at a distance of approximately 5mm from the edge by holding the swab at a 45 degree angle from the plane of the chamber. Re-dip swab and repeat for the CCNAB chamber. (The CCNAB step locations are a mirror image of the MAC isolation steps.) Allow streaks to dry one to two minutes and then effect an isolation using the loop/needle: Using the loop end, spread as shown at “2” using 8 to 10 passes within the shaded area boundary. Repeat for the CCNAB using the same loop end. Now use the bent needle end to complete the process for the MAC and CCNAB as shown at “3” also using 8 to 10 passes per chamber. This should result in isolated colonies.

CCNAB MAC

MH MH

MAC

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The antimicrobial papers come loaded on two applicators. Pictured at left is an example of applicators with antibiotic papers, attached only at corners, before and after removal of white protective covers. The colored applicator is inserted in the left MH chamber (directly below the red CCNAB chamber). The clear applicator is inserted in the right MH chamber. The release rod shown in right picture is used to apply the papers to the two chambers. The rod is poked into each of the holes down to the yellow stop and then applicators are removed from the chambers. Check to make sure all papers are seated on the agar.

Addition of antimicrobial susceptibility papers

CCNAB MAC

MH MH

top

bottom

top

bottom

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CCNAB MAC

F

left right

Example of Applicator placements of a UTI antibiotic set

CPD = CefpodoximeCF = CephalothinCC = ClindamycinAMC = Amoxicillin/ ClavulanateFOX = CefoxitinAM = Ampicillin

SXT = Trimethoprim/ SulfamethoxizoleF/M = NitrofurantoinTZP = Piperacillin/ TazobactamAN = AmikacinGM = GentamicinCIP = CiprofloxicinD = Doxycycline

Incubation1. Cover plate with lid, return to pouch and tape

closed.2. Place plate in incubator upside down. 3. incubate at 35C for 12 to 24 hours.

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Analyzing results following 12-24 hr incubation

The IDSX kit is a point of care system providing a next day result. The sample is put immediately on the plate, with or without dilution, following specimen collection. This avoids any growth that could take place using a transport medium. This then provides an accurate picture of species populations (restated next paragraph) at the culture site. The antimicrobial susceptibility test is called “direct” because antimicrobial agents are applied at the first incubation and results are evident by the next day

Direct antimicrobial susceptibility test

A detailed discussion of the above 5 points follow on the next 5 slides:

The incubation reveals the following:

(1) Is there a pure culture or are multiple species present?: This is determined by colony characteristics (see next slide), selective growth on MAC or CCNA, as well as other spot tests (see below). The results provide a picture of the relative quantities of the different bacterial species that may be present in the specimen when not a pure culture. This in turn, allows for the determination of which organism(s) is/are significant in terms of predominance.

(2) The Identification to gram (+) and gram (-) levels

(3) Display of Isolated colonies for identification and ability to repeat antimicrobial susceptibility testing when inoculums are too light(<105 cfu/ml) or too heavy(>108 cfu/ml).

(4) Colony forming units (cfu) per ml is determined. See slide #15

(5) Following the placement of transparencies, a result of resistant, intermediate or susceptible is visually determined for each antibiotic tested without the need to measure zones and consult Standard Interpretive Charts. This allows for targeted prescribing with the most prudent choice of antimicrobial agent.

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(1) Is there a pure culture on the plate or are there several different species present?

Each colony originates from a single bacterium. Each species of bacterial or fungal organism exhibits a fairly unique colony. See table to right for a listing of various characteristics that brings about uniqueness.

For example, in regards to the “effect of the colony on culture medium” with respect to hemolysis, observe the hemolytic reactions on the blood containing CCNAB medium . Beta hemolysis is a clear zone around the colony, that results from the complete hemolysis of red blood cells (rbc’s). Alpha hemolysis is a greening of the agar surrounding a colony due to partial hemolysis of rbc’s, and gamma hemolysis is the term for non-hemolytic colonies.

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(2) Identification to gram (+) and gram (-) levels: MAC supports the growth of gram negative bacteria and gram positive bacteria are inhibited. CCNA supports the growth of gram positive bacteria and gram negative organisms are inhibited.

(3) The isolated colonies on the MAC and/or CCNA allow for further identification. The pictures below show two simple spot tests of the many that are available:

A sample of a colony placed in 3% hydrogen peroxide forms bubbles of oxygen when the enzyme catalase is present.(If possible, sample a colony from the MH chamber since red blood cells also contain catalase)

The cytochrome oxidase test produces a purple color when a gram negative colony contains the oxidase enzyme. If a microscope is available, cellular morphology may be observed (see next slide).

A presumptive identification can be made to the genus level for three of the most common gram positive bacteria:

Staphylococcus: catalase positive, non-hemolytic, alpha hemolytic or beta-hemolytic (see slide 13). Colonies are generally larger than other types of gram positive species. Staph. aureus has colonies that are golden in color (generally following a 24hr.incubation), beta-hemolytic and are catalase positive.Streptococcus: catalase negative, and generally alpha or beta hemolytic colonies.Enterococcus: catalase negative and non-hemolytic colonies.

and Enterococcus

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(4) Approximating the concentration of colony forming units/ml)

104 105106

107

108An approximation can be made as to the concentration of organisms present in the final sample. Compare your result to these pictures. This example shows various concentrations of E.coli. Plates were incubated at 35C for 15 hours. Multiply the matched approximate concentration by 10 if a 1:10 dilution was made from patient’s specimen.

(5) Finding which Antimicrobial agents are effective(two examples shown)

The films labeled “left” are dropped into the left MH chambers, and “right” films are dropped into the right MH chambers. A determination is made as to resistant, intermediate or susceptible for the antibiotics tested by observing the bacterial growth margins relative to the resistant (R) or susceptible (S) arcs. If the margin is less than or equal to the R arc, the species is resistant to the antimicrobial agent. If the margin is greater or equal to the S arc, the species is susceptible to that agent. When the margin falls between the R and S arc, the species has intermediate susceptibility(I). 16

E.ColiATCC 25922

Staph. aureusATCC 25923

Transparencies placed

Transparencies placed

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The culture is significant when the bacterial concentration on the plate is greater or equal to 104cfu/ml of 1 type only. Also significant with mixed cultures where one species predominates with a count greater or equal to 104cfu/ml and 10-times the cfu/ml of other species present. However it will be necessary to repeat the test to get a valid antimicrobial susceptibility result when the inoculum concentration falls outside of the range indicated below.

When there are two species present of similar concentration greater than 104 cfu/ml this may also be significant. Perform a subculture of each species to new IDSX kits.

If there is a mixed culture with more than two species present and with none predominating in number, the result is most likely not significant.

SIGNIFICANT CULTURES

If there is less than 105 cfu/ml or greater than 108 cfu/ml in the final sample applied, there may be an “inoculum effect” with false positive or false negative susceptibilities respectively. Re-culture the species of interest using isolated colonies (predominating or pure culture) as described below.

INOCULUM EFFECT AND SIGNIFICANT ANTIMICROBIAL SUSCEPTIBILITY

RETESTING using isolated coloniesEach kit comes packaged with 2 vials of sterile water. Using sterile swab, pick up one larger colony or 2 to 3 small like colonies from a 15-18 hour incubation and suspend, using a swirling motion, in first vial of sterile water. Then make a one to ten dilution of this by transferring the swab to the second vial of sterile water and swirl again. This prepares an inoculum of approximately 107 cfu/ml. Apply this dilution to the plate as previously described above. Continue with set up and incubation. OTHER TYPES OF SPECIMENSThis method can be easily adapted for other specimens obtained from various body sites such as soft tissue infections, lower respiratory tract infections, eye infections etc. IDSX kits can be tailor made using different sets of antimicrobial agent papers.

Significance; Inoculum effect; retesting; and other types of culture sites

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Options for IDSX kit disposal

Tape lid to the plate and dispose of in red-bagged biomedical waste container along with other kit components.

Autoclave kit components, at 121°C (250°F) for 15 minutes. Cool to room temperature and dispose in regular trash.

Treat kit components with a fresh 50%(v/v) solution of household bleach* in water. Wear gloves. Treat plate by adding approximately 10ml of the 50% Clorox solution to each plate chamber. Allow solution to remain in chambers for 10 minutes and then carefully pour into a flushable toilet. The solid media is then easily removed: Tip the plate upside down at a 45° angle over toilet; and run the loop from the kit around the inside edge of each chamber to dislodge the media to the toilet bowl. Flush several times to complete the disposal. The empty plates and other treated components may then be placed in the regular trash.

Household bleach (such as Clorox) has an available chlorine content of 5.25%. Hazard: Do not mix with other chemicals such as cleaners, acid or ammonia-containing products. To do so may release hazardous gases. Note: Not harmful to septic systems.

IDSX kit contact information

Kwikculture LLC, 50N. Highway 89, North Salt Lake, Utah, 84054, USA

Read Taintor, pres./CEO.

Email readtaintor@comcast.net

Telephone 801-637-9703

Fax 801-936-0354

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Intellectual property informationUS 7,018,828 issued 3/28/06US 7,262,021 issued 8/28/2007US 7,960,136 issued 6/14/201112/423,807, Pending (publication date 04/22/2010)

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Still pictures and notes from movie posted on YouTube: http://youtu.be/G5sNoGOtU3Y

KWIKCULTURE LLC IS SEARCHING FOR A COMPANY TO LICENSE THEIR IDSX KIT TECHNOLOGY. IT IS A RAPID, IN VITRO ANTIMICROBIAL SUSCEPTIBILITY TEST KIT APPARATUS AND METHOD WITH PATENT PROTECTION.

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Following is an example of Kwikculture's IDSX kit, in this case, used to analyze a urine specimen.

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Packaging of the IDSX kit

Kwikculture’s IDSX kit comes complete with everything needed to perform an antibiotic susceptibility test.

The pouch holding the IDSX dish is opened. Pouch is retained for the upcoming incubation step.

IDSX inner packaging.

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Sample application (as a lawn) to the twin MH antimicrobial susceptibility chambers

The sterile, rayon tipped swab is dipped into the specimen sample.

The swab is transferred to the first Mueller Hinton chamber where the sample is uniformly spread over the surface of the agar to form an eventual lawn of growth.

The above process is then repeated for the other Mueller Hinton chamber.

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Sample application (as a streak) to the MAC and CCNAB chambers.

The sample is now added as a single streak to the Columbia CNA agar.

The addition of sample is then repeated for the MacConkey agar.

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Dilution of the streaks in the MAC and CCNAB chambers for colony isolation

The streaks are allowed to dry for one to two minutes and then an isolation is performed using the loop/needle tool.

Lightly run the loop end of the tool back and forth across the initial streak in the MacConkey chamber.

Repeat this spreading technique now for the Columbia CNA chamber.

Rotate the tool now to the bent needle end. Further dilute to isolate by passing the tool back and forth perpendicularly across the 2nd spreadings in each of the two chambers.

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Addition of antimicrobial susceptibility test papers

The next step of the method is the placement of antibiotic papers on the twin Mueller Hinton agars.

The pair of applicators are removed from the inner envelope and un-taped.

The protective papers are removed from each of the two applicators.

Applicators are now re-joined together and inserted into the twin Mueller Hinton chambers.

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Seating the papers and final steps to incubation

The antibiotic paper release rod is now removed from its storage tube. Note the yellow stop to assure proper vertical placement.

The rod is now used to dislodge the papers from the applicators to the agar surface.

Empty applicators are now removed from the chambers.

Papers are now gently seated onto the agar surfaces using the release rod.

The lid is now placed on the IDSX dish.

The bottom of the dish is now labeled with necessary information.

The dish is now returned to its pouch, and incubated at 35*C overnight.

The IDSX dish with antibiotic papers in place is nearly ready for incubation.

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Determining results using the IDSX measuring transparencies.

Following a 17 hour incubation, the dish is removed from the incubator.

Transparencies are removed from package and oriented for placement in the chambers.

Low-tack paper strips are used to pick-up and place the transparencies into the chambers.

The Incubated dish is removed from its pouch and observed.

Measuring transparencies provide a direct susceptibility reading right from the chambers.

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Add measuring transparencies to incubated dish and read results directly from dish

Qualitative and quantitative analysis, concurrent with susceptibility is now possible within 12 to 20 hours.

The “right” labeled transparency is now placed into the right Mueller Hinton chamber.

Next, the “left” labeled transparency is placed.

The IDSX kit allows a direct readout of antibiotic susceptibility instead of having to manually measure zones and consult interpretive tables to obtain a result.

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10 cfu/ml4 10 cfu/ml6

10 cfu/ml8

10 cfu/ml5

10 cfu/ml7

Determining results: Quantitative, Qualitative, and Susceptibility

Quantitative Using the panel of standard concentrations above, one can approximate the cfu/ml of the sample added to the dish. The example to the left is approximately 106 cfu/ml. Knowing the concentration of bacteria allows the user to repeat the assay when inoculums are too light (<105 cfu/ml) or too heavy (>108 cfu/ml). See “inoculum effect” discussed on slides 6 and 17.

QualitativeThe example shows a pure culture of a lactose fermenting gram negative organism ( based on colony morphology and growth on MAC and inhibited growth on CCNA).

SusceptibilitySusceptible to SXT, F/M,TZP, AN, GM, CIP, D, FOX, AMC, CPD, AM; Intermediate to CF; Resistant to CC.

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