expertise on the effectiveness of the diosolgenerator ......an off-the-shelf surface disinfectant on...

Bionovis Hygieneinstitut OHG [email protected] Apotheker und Ärztebank FFM Siemensstraße 18, 35394 Gießen http://www.bionovis.de BLZ 500 906 07 Tel.: 0641 480937-0, Fax: 0641 480937-3 Amtsgericht Gießen (HRA 2524) Konto 0006736017

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Expertise on the Effectiveness of the

DiosolGenerator Together with Diosol-3 when Disinfecting Rooms

The DiosolGenerator System (DiosolGenerator in connection with Diosol-3) is a mobile disinfection

system for the disinfection of rooms. The device generates a fine suspensible aerosol by which

diffusing of the active ingredient in the room is achieved. The product used for disinfection is a

compound of hydrogen peroxide and silver. Due to the composition, especially due to the

hydrogen peroxide content, the product has the activity to attack organic material and has a

disinfecting effect on surfaces, even without mechanical support.

The general effectiveness of the product was examined and proven during suspension

experiments. More than 100 expertises on the effectiveness against various sorts of germs are

available.

The aim of this study was to examine the system’s capability to decontaminate or effectively

disinfect, respectively, contaminated surfaces in a room, without accompanying measures.


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Experiment Set-up and Methods

A room of 5.18 x 2.57 m (13.3 m²) floor area and a height of 2.40 m, volume approx. 32 m³,

served as test room. For the tests, the device control was set to level 30.

Performance Test

In a series of measurements in the course of an initial experiment, the constancy of the pump

performance or the disinfectant consumption of the device, respectively, was determined during

10 consecutive operating cycles. For this purpose the runtime of the timer was timed and the

weight of the consumed solution was determined. The results can be taken from Table 1. Since

the disinfectant is an aqueous solution, tap water was used for this measurement.

Indicator Test

In order to examine the disinfectant effect in the room, contaminated indicator carriers of various

materials were hanged in different parts of the room (Positioning of the indicators Sketch 1). To

simulate a drawer, two thirds of a box of 50 (width) x 47 (depth) x 10 cm (height) were covered

with a board. In the center of the bottom side of this board an indicator was hanged.

In order to examine the disinfectant effect on the bottom side, an indicator was positioned on the

bottom side of the middle board of a shelf standing beside the device.


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Indicator Test - Gap

To examine the disinfectant effect in a gap, two boards (1.0 m x 0.5 m) and distance pieces of 5

cm length simulated a gap. The indicators were placed at the top, center, and bottom on the

center line of the longer side (Attachment Sketch 2). For this purpose, one time the test set-up

was arranged on one side of the room and one time opposite the spraying direction behind the

DiosolGenerator (Results Table 4).

Countercheck - Standard Product

To examine the active effect of Diosol-3 compared with standard surface disinfectants, a test with

an off-the-shelf surface disinfectant on glucoprotamin basis (0.5 %, 1-hour-value) was carried out.

The results are listed in Table 3.

Test Materials

As test materials stainless steel, aluminum and plastic were used. The test germs were Escherichia

coli (NCTC 10538) and Staphylococcus aureus (ATCC 6535). The indicator carriers measured 100 x

10 x 2 mm. To fasten them, a hole of 4 mm dia. was drilled 10 mm from the top edge. Fixing by

means of wire straps. The positioning of the indicators can be taken from the attached sketch.


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Microbiological Process

The indicator carriers were contaminated by dipping the platelets three times for a short moment

into a fresh germ supspension which was received by inoculating an enriched thioglycolate broth

(BioMerieux order No. 42 074) and 18 hours of incubation at 36°C +1°C. Afterwards the

contaminated indicator carriers were put in sterile Petri dishes to dry in the incubation chamber for

2 to 3 hours

After a test cycle the indicator carriers were separately transferred to small sterile one-way tubes,

filled with nutrient broth (thioglycolate broth), shaken several times (3 times) for 5 minutes each

time and were then incubated for 72 hours at 36°C + 1°C. A first reading was carried out after 24

hours. If no growth was detected after 72 hours, a conclusive negative result was stated. After 24

and 72 hours, the nutrient solution of all mixtures was spread on Columbia blood plates and

checked for growth. In case of growth it was checked whether the detected germ was the test

germ. The results can be taken from the attached Table 2.

To determine the initial germ concentration on the indicator carriers, the carriers were rinsed with

9 ml of sterile isotonic NaCl-solution. 0.5 ml of the rinsing solution were then spread evenly on

Columbia blood agar plates (BioMerieux order No 43 041) which afterwards were incubated for 24

hours at 36°C + 1°C and then counted. The respective results are listed in Table 2. To prove the

contamination directly on the indicator carriers, additional impression tests with contact plates

were carried out and evaluated.


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Results

As to the runtime, the measuring of the operation constancy showed a deviation of + 1.7 %. This

is equal to a solution quantity of +1.1 g. Concerning the consumed solution, a deviation of + 4.8

% or + 3 g, respectively, was measured.

Depending on the material, the determination of the initial germ concentration for Escherichia coli

resulted in an overall concentration on the indicator of 2.5 to 3.3 x 10³ CFU, for Staphylococcus

aureus 1.8 x 104 CFU (Table 2).

With Escherichia coli the examination of the indicators in the room showed germ growth in one of

36 test places, in the remaining places the test germ was not traceable after disinfection. With

regard to Staphylococcus aureus the test germ could be re-isolated in three of 60 test places, in

which case a positive proof was always detected in the same test place (3), Table 3. The

examination with a standard product resulted in a positive germ detection with all indicators (Table

3).

With the gap tests no germs were detected on the indicator carriers after disinfection, neither with

the setup beside nor with the setup behind the device (Table 4).

In all cases, the additional test of the indicator carriers with contact plates to detect successful

contamination showed a lawn-like growth on the carriers. A difference in the test results could

neither be stated with the various test germs nor with the different test materials although the test

germ concentration with Staphylococcus aureus tests exceeded that of Escherichia coli by abt. a

power of ten.


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Discussion

The testing of the operation constancy regarding the determination of the runtime at the chosen

instrument setting (level 30) showed a deviation of 1.7 % and a deviation of 4.8 % regarding the

pump performance. This results in a max. quantity variance of +3 g or approx. 3ml, respectively,

during one cycle. By including a safety margin in the disinfectant quantity to be applied, a good

and, for the scope of application sufficient operation constancy is achieved.

As to the microbiological tests, test germs were only detectable sporadically after disinfection.

Since preferably difficult conditions were chosen for the test places, room conditions that could

have prevented an unhindered ventilation at the test place are considered possible causes. This is

also suggested by the fact that the result was not influenced by increasing the applied disinfectant

quantity (up to 3 times the determined dose).

Since the indicators were inoculated directly from the enriched broth containing peptone, a certain

protein concentration in the test places during the tests may be assumed. Yet the germs were

almost completely eliminated without active suspension in the disinfectant. In contrast, an off-the-

shelf surface disinfectant on a glucoprotamin basis had no effect at all. The test germs could still

be detected on all indicators after disinfection.

Also with the tests in a gap of 5 cm width and a surface of 0.5 m², the test germs were completely

eliminated in the center of the gap (distance to the edges 0.25 m or 0.5 m, respectively),

irrespective of the carrier material used.


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The tested germ concentration on the indicator carriers ranged from 2.5 x 10³ up to 1.8 x 104 CFU

which equals the extensive contamination of a bacterial lawn and a concentration which in practice

is not likely to be found often. Still, test germs were only detected in very few places.

As other studies showed it is difficult or, without actively removing product residues (silver),

almost impossible to contaminate the treated surfaces a second time since the residues obviously

prevent germ growth and a re-isolation cannot be accomplished despite the application of a high

germ concentration. At least this effect occurred on stainless steel surfaces. Thus the product has

a remanence effect that is due to the silver component.

Résumé

The tests showed an adequate operation constancy which ensures that preset quantities to be

diffused are also applied in acceptable ranges of fluctuation.

The results of the indicator tests showed no influence of the carrier materials on the disinfectant

effect. Test germs were only traceable in a few isolated test places that were difficult to reach.

Since also by increasing the applied quantity no different results could be obtained it is to be

assumed that unfavourable air flow conditions prevented that the indicator was reached and thus

prevented any effect. In other respects the suspensibility of the aerosol leads to an adequate

diffusion of the disinfectant in the room which is sufficient to disinfect the indicators placed at the

bottom sides of surfaces. When operating the device it is, therefore, imperative to always ensure

that the surfaces to be disinfected can be reached .

No difference in the effectiveness against gram-negative or gram-positive germs could be

observed. In other expertises the effectiveness of the product against a multitude of medically and

hygienically relevant germs was proven. The effectiveness against spores which are a general


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problem with disinfectant measures is of interest because it may result in the use in case of e. g.

problems with Clostridium difficile.

Also the tests in gaps showed a good disinfectant effect under difficult conditions. In summary it

can be stated that the DiosolGenerator System is suitable to achieve a surface disinfection of all

accessible surfaces in a room by means of a generated aerosol. Also objects placed behind the

device are reached and, depending on the air flow conditions, the aerosol will enter open hollow

spaces as well.

To safeguard the disinfection measures it should be checked whether a measuring device could be

integrated which will stop the device in case of insufficient diffusion in connection with the chosen

instrument setting and which will also report the malfunction.


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Expertise on the Efficiency of the

DiosolGenerator Together with Diosol-3 on Bottom Sides of Surfaces when Disinfecting

Rooms









available.


disinfect, respectively, easily accessible surfaces in a room, also on the bottom sides, without

accompanying measures.


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served as test room. For the tests, the device was set to level 30.

Indicator Test

In order to examine the disinfectant effect, contaminated indicator carriers of various materials

were positioned at the bottom side of a board of a shelf standing at a side wall and having a depth

of 0.5 m.

Test Materials






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2 to 3 hours



time and were then incubated for 72 hours at 36°C +1°C. A first reading was carried out after 24








hours at 36°C +1°C and then counted. The respective results are listed in Table 2. To prove the




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Results

With none of 12 tests did the examination of the indicators on the bottom side of the board show

germ growth. Neither Staphylococcus aureus nor Escherichia coli could be re-isolated. The

examination with a standard product resulted in a positive germ detection with all indicators (Table

3).





power of ten.

Discussion

In no case did the microbiological examination of the disinfection effect on the bottom side of a

surface show test germs after disinfection.



completely eliminated without active suspension in the disinfectant. In contrast, an off-the-shelf

surface disinfectant on a glucoprotamin basis had no effect at all. The test germs could still be

detected on all indicators after disinfection.


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is not likely to be found often.






Résumé

With regard to the tested materials, the results of the indicator tests showed no influence of the

carrier material on the disinfectant effect. The suspensibility of the aerosol leads to a very

satisfactory diffusion of the disinfectant in the room which also suffices to disinfect the indicators

placed at the bottom sides of surfaces as was the case in this study. A pre-condition when

operating the device is to always ensure that the surfaces to be disinfected can be reached.


observed. In other expertises the efficiency of the product against a multitude of medically and


problem with disinfectant measures is of interest.


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DiosolGenerator Together with Diosol-3 in Open Drawers when Disinfecting Rooms









available.


disinfect, respectively, drawers in a room, without accompanying measures.


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Indicator Test


materials were used. To simulate a drawer, two thirds of a box of 50 (width) x 47 (depth) x 10 cm

(height) were covered with a board. In the center of the bottom side of this board an indicator

was hanged.






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Test Materials










2 to 3 hours











hours at 36°C +1°C and then counted. The respective results are listed in Table 2.


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To prove the contamination directly on the indicator carriers, additional impression tests with

contact plates were carried out and evaluated.

Results




In none of 8 test places did the examination of the indicators in a drawer show germ growth after

disinfection, as regards Staphylococcus aureus and Escherichia coli. When carrying out the test

with a standard product (glucoprotamine) the test germ could be re-isolated with regard to all

indicators (Table 3).





power of ten.


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Discussion








is not likely to be found often. Still, test germs could not be detected in any of the test places.





a remanence effect which is due to the silver component.

Résumé


effect. The suspensibility of the aerosol leads to an adequate diffusion of the disinfectant which

obviously suffices also to disinfect indicators placed in open drawers. A pre-condition when

operating the device is to ensure that the surfaces to be disinfected can be reached.


observed. In other expertises the effectiveness of the product against a multitude of medically

and hygienically relevant germs was proven. The effectiveness against spores which are a general

problem with disinfectant measures is of interest.


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DiosolGenerator Together with Diosol-3 in Narrow Spaces (Gap) when Disinfecting

Rooms









available.


disinfect, respectively, contaminated surfaces in a narrow gap, without accompanying measures.


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Indicator Test


materials were hanged in different parts of the room (Positioning of the indicators Sketch 1).

Indicator Test - Gap

To examine the disinfectant effect in a gap, two boards (1.0 m x 0.5 m) and distance pieces of 5

cm length simulated a gap. The indicators were placed at the top, center, and bottom on the

center line of the longer side (Attachment Sketch 2). For this purpose, one time the test set-up

was arranged on one side of the room and one time opposite the spraying direction behind the

DiosolGenerator (Results Table 4).


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Test Materials










2 to 3 hours




hours. If no growth was detected after 72 hours, a conclusive negative result was stated.


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After 24 and 72 hours, the nutrient solution of all mixtures was spread on Columbia blood plates

and checked for growth. In case of growth it was checked whether the detected germ was the test





hours at 36°C +1°C and then counted. The respective results are listed in Table 2. To prove the



Results




With the gap tests no germs were detected on the indicator carriers after disinfection, neither with

the setup beside nor with the setup behind the device (Table 4).


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neither be found with the various test germs nor with the different test materials although the test


power of ten.

Discussion






With the tests in a gap of 5 cm width and a surface of 0.5 m², the test germs were completely

eliminated in the center of the gap (distance to the edges 0.25 m or 0.5 m, respectively),

irrespective of the carrier material used.



is not likely to be found often. Still, with this test no test germs were detected in any of the test

places after disinfection.


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Résumé


effect. The suspensibility of the aerosol leads to an adequate diffusion of the disinfectant in the

room and far into narrow gaps. Even under difficult conditions the tests in a gap showed a good

disinfectant effect. A pre-condition when operating the device is to always ensure that the surfaces

to be disinfected can be reached.


observed. In other expertises the effectiveness of the product against a multitude of medically and


problem with disinfectant measures is of interest because, among others, it may result in the use

with Clostridium difficile.

In summary it can be stated that the DiosolGenerator System is suitable for the disinfection of all

accessible surfaces in a room by means of a generated aerosol. Also objects placed behind the

device are reached and, depending on the air flow conditions, the aerosol will enter open hollow

spaces as well.


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Following Attachment is Intended for the Expertises Mentioned Below:

- Expertise on the Effectiveness of the DiosolGenerator Together with Diosol-3

when Disinfecting Rooms

- Expertise on the Effectiveness of the DiosolGenerator Together with Diosol-3 in Narrow Spaces

(Gap)


- Expertise on the Effectiveness of the DiosolGenerator Together with Diosol-3 in Open Drawers


- Expertise on the Effectiveness of the DiosolGenerator Together with Diosol-3 on Bottom Sides of

Surfaces when Disinfecting Rooms

Bionovis Hygieneinstitut Dr. Käflein & Kruff OHG [email protected] Deutsche Apotheker- u. Ärztebank Siemenstr. 18, 35394 Gießen http://www.bionovis.de BLZ 500 906 07 Telefon 0641-480 93 70 Fax 0641-480 93 73 Amtsgericht Gießen (HRA 2524) Kto.-Nr. 0006736017

Arrangement

Diosol-3 Test

Sketch 1

Height: 2.4m

5.1

8m

Shel

f

Device

Cover plate

2.57m 4

7.0

cm

Drawer (50x47x10)cm

Bionovis Hygieneinstitut Dr. Käflein & Kruff OHG [email protected] Deutsche Apotheker- u. Ärztebank Siemenstr. 18, 35394 Gießen http://www.bionovis.de BLZ 500 906 07 Telefon 0641-480 93 70 Fax 0641-480 93 73 Amtsgericht Gießen (HRA 2524) Kto.-Nr. 0006736017

C

C

V

Sketch 2 Test in a Gap Diosol-3

Indicators

50

cm

25cm

10

0cm

Ind

icators

5cm


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DiosolGenerator Test Protocol Table 1: Test to determine the accurate dosage Date of test: 2007/01/15 Experimentation: Dr. Käflein Testing solution: Water Instrument setting: 30

Measuring cycle

Runtime of the

device

Weight (g) before afterwards

Difference in weight (g)

1st measuring*

3056

2999

57

2nd measuring

2997

2936

61

3rd measuring

2936

2870

66

4th measuring

2870

2807

63

5th measuring

1’55”

2807

2742

65

6th measuring

1’56”

2742

2682

60

7th measuring

1’53”

2682

2621

61

8th measuring

1’55”

2621

2556

65

9th measuring

1’54”

2556

2494

62

10th measuring

1’55”

2494

2430

64

11th measuring

1’54”

3179

3118

61

12th measuring

1’54”

3118

3054

64

13th measuring

1’55”

3054

2990

64

14th measuring

1’54”

2990

2926

64

15th measuring

1’55”

2926

2864

62

Mean values

1’54”

63

* Values printed in italics were not rated

Result: Mean value required solution (in g): 63 g +3 g (+4.8 %); Runtime: 1’54” +2” (+1.7 %)


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DiosolGenerator Test Protocol Table 2: Determination of the indicator germ concentration

Indicators: Strips of material with hole 100 x 10 mm

Test organisms: Staphylococcus aureus ATCC 6535, Escherichia coli NCTC 10538

Experimentation: 2007/07/16, Dr. Käflein

Test organism

Test material

Number of

colonies (CFU)

Allover

concentration (CFU)

E. coli NCTC 10538

AL ES KS

186 200 140

3.3 10³ 3.6 10³ 2.5 10³

S. aureus ATCC 6535

AL ES KS

1000 1000 1000

1.8 104 1.8 104 1.8 104

Dilution factor

18 (9 ml rinsing solution / 0.5 ml spread on plates

Contact plate examination

AL/ES/KS

lawn

lawn


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Table 4: Tests in a gap

Product: Diosol-3 Device: DiosolGenerator

Test places

E. coli

Indicator: ES (CFU)

S. aureus

Indicator: AL (CFU)

beside the device top center bottom

0/0 0/0 0/0

0/0 0/0 0/0

behind the device top center bottom

0/0 0/0 0/0

0/0 0/0 0/0

Conrols

+

+

Explanation: ES = stainless steel, KS = plastic, AL = aluminum CFU = Colony Forming Units Lawn = number of colonies not countable


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DiosolGenerator Test Protocol Table 2: Determination of the indicator germ concentration Indicators: Strips of material with hole 100 x 10 mm Test organisms: Staphylococcus aureus ATCC 6535, Escherichia coli NCTC 10538 Experimentation: 2007/07/16, Dr. Käflein

Test organism

Test material

Number of

colonies (CFU)

Allover

concentration (CFU)

E. coli NCTC 10538

AL ES KS

186 200 140

3.3 10³ 3.6 10³ 2.5 10³

S. aureus ATCC 6535

AL ES KS

1000 1000 1000

1.8 104 1.8 104 1.8 104

Dilution factor

18 (9 ml rinsing solution / 0.5 ml spread on plates

Contact plate examination

AL/ES/KS

lawn

lawn

Explanation:

ES = stainless steel, KS = plastic, AL = aluminum CFU = Colony Forming Units Lawn = number of colonies not countable


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DiosolGenerator Test Protocol Table 3: Study to Determine the Effectiveness Product: Diosol-3, Device: DiosolGenerator Test period: May - August 2007

Test place

E. coli S. aureus

Diosol-3

Gluco- protamin

Diosol-3

KS

ES

AL

KS

ES

AL

ES

Instrument setting

30 60 90

front left (1) 0/0 0 0 0/0 0/0 + 0/0 0 0

front right (2) +/0 0 0 +/0 0/0 + 0/0 0 0

rear left (3) 0/0 0 0 0/0 0/0 + +/0 + +

rear right (4) 0/0 0 0 0/0 0/0 + 0/0 0 0

below shelf (5) 0/0 0 0 0/0 0/0 + 0/0 0 0

drawer IS (6) 0/0 0 0 0/0 0/0 + 0/0 0 0

positive controls + + + + + + + + +

Explanation:

ES = Stainless steel, KS = plastic, AL = aluminum + = germ growth, IS = inside


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The Retentivity Effect of Diosol-3

Dear Mr Nass,

as agreed, I carried out tests on the retentivity effect of Diosol-3 on surfaces. These tests showed

the following results:

The test surface (white coated chipboard) was sprayed with Diosol-3 on three days running and

wiped with a disposable tissue to ensure that the product was spread evenly. Afterwards, two

small areas (measuring 5 cm x 5 cm) were marked for the application of the germs. Germs from a

germ suspension made with an isotonic NaCl-solution (germ concentration > 104 CFU/ml) were

spread on the entire test surface by means of a sponge. The test organisms used were

Staphylococcus aureus (ATCC 6548) and Escherichia coli (DSM 1103). Contact agar plates were

used to prove that the smeared surfaces were contaminated.

After having applied the germ suspension to the test surfaces there was a drying phase of 18

hours. The next day a re-isolation of the applied germs was attempted with contact plates. This

cycle was continued over a period of 14 days.

The following table shows the detectable germs on the surfaces:


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Table: Germs Identified on Surfaces Treated with Diosol-3

Date Contaminated Surface Remarks

with S. aureus with E. coli

02/18/09 Diosol-3 spray application

02/19/09 Diosol-3 spray application

02/20/09 Contamination check

no growth no growth subsequent germ

application by means

of a sponge

02/21/09 no growth no growth subsequent germ


of a sponge



of a sponge



of a sponge

02/27/09 sporadic growth of

micrococci and

spore-forming bacilli

no growth of S.

aureus

sporadic growth of

micrococci and


no growth of E. coli

subsequent germ


of a sponge


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Date Contaminated Surface Remarks

with S. aureus with E. coli


micrococci and


no growth of S.

aureus

sporadic growth of

micrococci and



subsequent germ


of a sponge


micrococci and


no growth of S.

aureus

sporadic growth of

micrococci and



subsequent germ


of a sponge


micrococci and


no growth of S.

aureus

sporadic growth of

micrococci and


1 CFU E. coli

subsequent germ


of a sponge


micrococci and


2 CFU S. aureus

sporadic growth of

micrococci and


3 CFU E. coli

subsequent germ


of a sponge


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As can be taken from the table of results test germs could be re-isolated sporadically after 10 or

11 days, respectively, showing a beginning loss of effectiveness. Considering the relatively high

germ concentration in the contamination solutions one can probably still assume, at this point of

time, an inhibition effect on the applied germs due to the silver residues.

To be able to make exact statements on the interdependency between applied germ

concentrations and loss of effectiveness as well as the influence of protein loads further studies

under accordingly defined conditions would be necessary.

The results at hand suggest that an antibacterial effect can be assumed for a duration of at least

one week after a surface was treated twice. The studies made to date could not determine to

what extent a parallel cleansing would lessen the disinfectant effect. It may be, however, assumed

that the silver layer will only be removed gradually by conventional household detergents, the

more so as other studies showed that contamination of pretreated surfaces is only possible if they

were cleaned with specific cleaners.

In case you should be interested that the antibacterial effect of Diosol-3 on surfaces should be

investigated further we would, of course, be prepared to carry out respective studies.

expertise on the effectiveness of the diosolgenerator ......an off-the-shelf surface disinfectant on...

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