Technical Support

Assay Protocols: Training Video Tests 1 & 2

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Do’s and Don’t’s – UTI Tests 1 & 2:

The following provides guidance/pointers for current and new customers when running tests 1 and 2:

General:

  • Set heater block to 37 degrees Celsius
  • Use a multi-channel timer (eg 4-channel)
  • Use a different disposable tip for each sample addition, even for multiple additions of the same sample
  • Be consistent with your technique to achieve better and consistent results e.g.
    • After addition of Reagent 2 from the Reagent Delivery Device (RDD) gently mix (half a second) and, immediately, place in the luminometer read chamber
  • Gauge the number of RDD’s you are likely to use in one day then remove them from refrigerated storage and allow to equilibrate to ambient temperature (at least 20 mins). It is possible to take from refrigerated storage and place directly into the dry bath incubator but better if the RDD’s are allowed to gradually equilibrate.
  • Allow all tubes to reach 37 degrees Celsius after placing them in the dry bath incubator (10 mins minimum)
  • Sample addition:
    • Urine samples used in this technology must be fresh from the patient (maximum 1-2 hours from sampling). They must not be refrigerated (or frozen) at any stage prior to testing
    • All sample additions (both tests) must be 25uL; the tests and cut-off levels are determined using 25uL
    • Use a fresh sterile disposable tip for each sample addition – not just different patient urine samples but for each sample addition for the same patient e.g. antibiotic sensitivity additions
    • With your 25uL pipette, depress the plunger , hold and dip just beneath the liquid surface and release to take up the sample.
    • Place the tip surface just beneath the Reagent 1 surface and depress the pipette plunger 3 times to wash the sample into the Reagent 1 mix. Holding the pipette plunger in the depressed position, lift away from the liquid surface – this will ensure you do not remove any liquid from the sample /Reagent 1 mix
  • After each sample or reagent addition use gentle agitation to ensure effective mixing
  • The ITL “Lumini” luminometer is designed to capture the “peak signal output” – the actual reading time (eg 20 secs, 40 secs) may differ from sample to sample. This can be due to a number of reasons - for example, different causal bacteria behave differently due to cell membrane differences. The reading obtained will then be interpreted by the software to provide a definitive result.
  • Gentle agitation of tubes is good technique and ensures mixing of sample and reagent:
    • After adding urine sample to Reagent 1
    • After addition of antibiotic disks
    • After adding Reagent 2 from the Reagent Delivery Device prior to reading the luminometer output signal
  • If you are presented with a “dirty” urine which is cloudy, contains flocculent, detritus, cast material, visible blood - you may want to consider:
    • Filtering 1ml of urine to provide a better quality sample (recommend Sysmex Celltrics disposable 10um) – this will remove unwanted material but allow bacteria (if present) to flow through the filter membrane
    • Increasing incubation times to allow Reagent 1 to work more efficiently (the 5 minute incubation time is a “minimum” recommendation; incubation can, if desired, be carried out on Test 1 for up to 60mins
    • A combination of the above two recommendations
  • Test&Treat recommends use of Positive and Negative Control material to ensure all equipment and reagents are performing as required

Test 1 (Detection):

  • Uses 25uL urine sample
  • Please ensure Test 1 is carried out within 1 to 2 hours or as soon as possible from collection:
    • Do NOT refrigerate or freeze the sample
  • The 5 min incubation time (37 degrees Celsius) is a minimum time. Test 1 works by removing host cell and any free ATP in the urine, thereby leaving (if present) any bacterial cells for detection. You may vary the incubation time (up to 60 mins) according to the visual quality of the sample ie a “dirty” sample may require longer for Reagent 1 to do its work
  • If the result obtained is equivocal (eg borderline POS/NEG cut off; or you are presented with a “dirty” sample (presence of visual blood, abnormal leucocytes, cast material, detritus) carry out a “Confirmatory Test” using the same reagents but incubating longer (at 37 degrees Celsius):
    • If you elect to run Test 1 for eg 40mins as a Confirmatory test

When you compare the Confirmatory test to the 5min Test 1:

      • If the signal output decreases this represents removal of more host cell ATP and is NEGATIVE for bacterial infection (UTI)
      • If the signal increases (usually significantly) this is due to presence of increased bacterial metabolic activity: (POSITIVE UTI)
    • As a guideline you can use the following criteria to declare a POSITIVE/NEG result, following Test 1 + Confirmatory test:

  1. Compare signal output of Test 1 with Confirmatory Test signal output. If the comparison indicates:
        • Signal output of Confirmatory test is >80% higher than Test 1
        • Confirmatory test signal is above the provided POS/NEG cut off

Result is POSITIVE

      • If the comparison indicates:
        • Little or no change in signal (<20% increase)
        • Signal output for Confirmatory Test is lower than Test 1

Result is NEGATIVE

Test 2 (Antibiotic sensitivity):

  • pre-incubation (5 mins) prior to addition of antibiotics is mandatory – this initial incubation “wakes up” any bacteria in stationary phase to kick-start metabolic activity. This reduces overall assay time to result

  • Best results will be achieved if all tubes are gently agitated every 10-15 mins or so (timing not critical) – this will ensure optimum contact between bacteria and target antibiotic

  • Use one antibiotic disk ejector for each antibiotic; label each with a permanent marker pen – this will avoid cross contamination between antibiotics

  • Incubation times stated are minimum incubation times following addition of antibiotic disks. For example:
    • 30 mins contact time for cidal antibiotics is a minimum incubation time – extending the incubation time (up to 90 mins) will enhance results
    • 90 mins contact time with Trimethoprim/sulfamethoxazole is, again, a minimum incubation time – this type of antibiotic is inhibitory to bacterial growth and metabolism and takes longer for the antibiotic to inhibit folate metabolism and produce the difference between the Control and Test tubes (in an organism which is Susceptible)

  • replace RDD’s on to all tubes after addition of antibiotics to prevent unnecessary evaporation

  • Whilst tubes are incubating try to get into the habit of gently agitating all tubes every 10 to 15 mins or so to ensure antibiotics are mixing with bacteria

  • Trimethoprim/sulfamethoxazole is an inhibitory antibiotic (not cidal and therefore does not actively kill bacteria); as a result the action of Trimethoprim/sulfamethoxazole requires a longer incubation time to produce definitive results. Minimum incubation time 90mins. This can be increased to 120 – 150mins
  • The ITL “Lumini” luminometer compares the signal reduction of a sample, when incubated with a chosen antibiotic, with a “Control’ tube – a tube incubated for the same length of time but which does not contain an antibiotic ie bacteria present are uninhibited by antibiotic action
    • A signal reduction greater than 25% indicates susceptibility
    • A signal reduction less than 25% indicates a level of resistance
    • If you are running a panel of antibiotics, the bar-chart provided by the Lumini will indicate the action (kill rate) of each antibiotic in comparison with the Control
  • Test&Treat Test 2 (Antibiotic Sensitivity) provides a quantitative assessment and comparison of the antibiotic(s) tested

Control material (Positive & Negative)

Positive (POS) and Negative (-ve or NEG) controls are provided as a check to ensure all equipment and reagents are performing as required:

  • Frequency of running Controls may be recommended by your Practice or Regulatory body. As a guideline Test&Treat recommends running Controls (POS and NEG) at least once per week

How to use:

  • POS and NEG Control tubes are accompanied by Reagent Delivery devices (RDD’s) – please note you must use the appropriately labelled RDD (POS or NEG) for each Control
  • Screw the labelled RDD onto the matching Control tube, align the notches then depress the RDD fully to dispense the RDD liquid
  • The resulting liquid has a milky appearance – this is normal
  • Agitate each Control tube (eg using a Vortex mixer) then incubate, in a dry bath incubator, overnight at 37 degrees Celsius – this allows the freeze-dried material to re-invigorate and provide the correct result using the Test&Treat Tests 1&2
  • Once activated use each Control (POS and NEG) as you would a sample, in both Tests 1&2
  • After following the above recommendation, if the Controls do not produce the expected result please contact your Supplier and/or Test&Treat for advice
  • Please ensure all materials are within labelled expiry date

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