New Biocides Development - ACS Publications - American Chemical

Chapter 22

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 26, 2018 | https://pubs.acs.org Publication Date: September 7, 2007 | doi: 10.1021/bk-2007-0967.ch022

A Comparison of the Microbicidal Efficacy on Germ Carriers of Several Tertiary Amine Compounds versus ΟΡΑ and Perasafe 1,3

3

Rafael Herruzo , Maria Jose Vizcaíno-Alcaide , and Julio Rodriguez 2

1

2

Services of Preventive Medicine and Microbiology of La Paz Hospital (Madrid), and the Department of Preventive Medicine, Public Health and Microbiology of Madrid Autonomous University, Madrid, Spain 3

Several tertiary amine formulations have been marketed as high-level disinfectants (HLD). This study compares some of these formulations with two accepted H L D (ΟΡΑ and Perasafe), using various methods. 1) determination of the bactericidal effect on 52 microorganisms, using a metallic germ carrier 2) Sporicidal effect using a commercial germ carrier (3M spores) 3) Corrosion test on surgical blades plus human blood. It should be noted that, despite acting for only 10 minutes, ΟΡΑ and Perasafe (not differing from each other) are significantly more effective than all other products tested, despite their longer contact (15 and 20 minutes), both for microorganisms overall and by groups. However, as an exception, Instrunet F A showed no significant differences at 20 minutes as compared to ΟΡΑ and Perasafe at 10 minutes for Gram-negative microorganisms. On the other hand, except for this last data, the tested amines did not significantly differ in their bactericidal efficacy. On the other hand, only ΟΡΑ and Perasafe were effective against mycobacteria (though the contact time had to be increased to about 15 minutes), but did not reach the sporicidal level. Finally, only one of them (Ultradesmit) altered the metallic material in the corrosion test.

© 2007 American Chemical Society

Zhu; New Biocides Development ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

451

452


Among all disinfectants tested in this study, only OPA and Perasafe can be considered as HLD, but an action time of 1520 minutes should be recommended, and it should acknowledged that both products are not free from disadvantages.

Introduction Tertiary amines are described as surface-active agents with an increased efficacy against Mycobacteria, (1) and could therefore be used as high level disinfectants (HLD) provided their efficacy was maintained against all other bacteria and this mycobactericidal action did not only cover M. tuberculosis (as killing this bacterium alone would only imply a medium level disinfection), but also other more resistant Mycobacteria (some of them atypical) or spores (2-7). On the other hand, these tertiary amines are associated to other surfaceactive compounds, usually quaternary ammonia such as benzalkonium chloride, and the mixtures are not always similar, so that efficacy may vary between the different commercial formulas. It must therefore be verified which is most effective and whether all or some of them may be classified as HLDs. We think that to consider a disinfectant as a HLD, it cannot just be accepted that in some tests, with microorganisms in suspension, the disinfectant achieves a 5 log reduction of some Mycobacteria and other collection microorganisms (ATCC) for several reasons: 1) ATCC microorganisms and strains aged at the laboratory are always more susceptible than thosefreshlyobtained from patients, particularly when these are microorganisms R or polyR to antibiotics (e.g. we obtained with OPA the following log 10 reductions in P. aeruginosa: with ATCC >5.5, withfreshlyisolated microorganisms S to antibiotics, 4.8 loglO, and for polyR germs, 3.8 loglO). Thus, we must always include in our tests freshly isolated R and polyR microorganisms to evaluate the efficacy spectrum against such microorganisms. 2) In addition, these microorganisms are never in suspension when instruments are to be disinfected, but on the smooth or rough surface of the instrument. Suspension tests alone cannot therefore be accepted, and some germ carrier or surface test must always be included to verify efficacy in this situation (the one that will be found in clinical conditions), because differences between methods with microorganisms in suspension or on germ carriers may be of several log 10 (8,9) 3) Finally, since different mycobacteria and viruses cannot be studied in most laboratories, it is appropriate to include a sporicidal test, because if the disinfectant passes this test, efficacy can be extrapolated to other



453 not tested mycobacteria and viruses, with or without lipid envelope, which are known to be less resistant than spores. However, if a disinfectant does not pass the sporicidal test it should not be dismissed, because in principle HLDs do not have to be able to kill these resistance forms, but we then must test their efficacy against the more resistant mycobacteria and a substantial number (at least 30) of other bacteriafromdifferent species before classifying them as HLDs. Therefore, we will use in this study microorganisms freshly obtained from patients and other collection microorganisms. Using a surface test, we will compare several tertiary amine formulations with two disinfectants used in our country as HLDs. Such disinfectants include OPA (0.55% ortho-phthalaldehyde, highly effective against mycobacteria and thus recommended as an adequate HLD, though its efficacy against spores is very low), and Perasafe (a powder compound that is diluted in water at the time of use and releases peracetate ions at an approximate 0.25% concentration) (10-12).

Materials and Methods Materials 1)

2)

Disinfectants: - 0.55% Ortho-phthalaldehyde (Johnson & Johnson), - 1,65% Perasafe (Lab Antec Int), - 4% Korsolex-plus (didecyl-dimethyl-ammonium-chloride 13%, dodecylbis-prpylentriamin 9,2%), Lab Bode. - Instrunet FA (didecyl-dimethyl-ammonium-chloride 0,7%, diaminopropyl-dodecyl-amin 0,6%, ethanolamine 1,4%): Lab Inibsa, in two versions, one with the already diluted product and certain excipients that increase its efficacy, and another version in which product should be diluted to 5% before use), - 12% Sinaldehyd (aminopropyl-dodecylamin 9,9%, didecyl-methylpolyoxi-ethyl-ammonium-propionate 12%, Lab J Collado), - 4% Ultradesmit (dodecyl-bis-propilen-triamin 21%, didecyl- dimethylammonium- chloride 14%, Lab Steer-Pharma). 52 microorganisms: (obtained from ICU patients) from different genera including Staphylococcus, Streptococcus, Enterobacteriaceae, Pseudomonas, Candida, M. fortuitum, and M. avium complex (only against OPA and Perasafe). 2 additional collection strains, Mycobacterium smegmatis 155 mc and B. subtilis (3M) spores. Endodoncy files no. 25 Glass beads (0.25 mm in diameter) Surgical blades 2

3) 4) 5)


454 6)

Inhibitor of antiseptic action: Todd Hewitt broth (Difco) + 6% v/v Tween 80, 0.5% w/v sodium bisulphite and 0.5% w/v sodium thiosulphate.


All culture media and Tween 80 used in this research were purchased from Johnson & Johnson, Tedec-Meiji Lab and Inibsa Lab through FUAM (Fundacion Universidad Autonoma Madrid).

Methods 1) determination of the bactericidal effect using a metallic germ carrier (no. 25 endodoncy files) (13). Strains recently isolated from ICU patients or ATCC strains. Endodoncy files were contaminated with a suspension of microorganisms (10 " CFU/ml) by immersion for one hour before being left to dry (15 minutes) on a sloping sterile surface (Petri dish with no culture medium). Each file was then placed in a tube with 5 ml of the disinfectant for different times: ranging from 1-20 minutes. After this, the files were removed, placed into another tube containing 5 ml of inhibitor with 0.5 g of glass beads 1 mm in diameter, and shaken in a Vortex at 1000 rpm for 1 minute. Finally, two 0.1 ml samples of the supernatant were cultured on Mueller-Hinton plates (or on Sabouraud-dextrose for yeasts or Coletsos medium for Mycobacteria) and incubated at 37°C for 24-48 hours (or 4-15 days for Mycobacteria) to count the number of microorganisms surviving after exposure to the disinfectant. The number obtained was compared with the one obtained for the control (same method but introducing the germ carrier in sterile distilled water instead of disinfectant), and 1/100 and 1/10000 dilutions were made in order to facilitate CFU counts. The assay was performed for all the microorganisms described in the Materials and Methods section. 2) Sporicidal effect using a commercial germ carrier (3M spores) (14): B. subtilis spores were removed from the capsule in which they are supplied and the cellulose impregnated with them was used as a germ carrier that was placed in the disinfectant and left there for 10 or 20 min. Cellulose was subsequently removed and placed in the inhibitor to stop the sporicidal effect. The rest of the assay continued as in the surface test. 3) Corrosion test: This was a modification of a previously described test (14). The lancet blade was impregnated with human blood (from a blood bank) to promote deterioration of the blade and mimic a defective cleaning situation. The blade was then introduced in disinfectant and examined for deteriorated areas or global deterioration at 1,2 ,4, 8, and 24 h, and daily up to 1 week. 4) Statistical method: The log 10 reduction in the CFU count compared to the control is considered the "effect" of the disinfectant. For disinfections with no surviving organisms, in which the log 10 reduction cannot be calculated, a value of 5.5 was recorded. The minimum bactericidal effect for this rough germ carrier test is 3.5 for most bacteria and 3 for Mycobacteria (13). For the 7

8


455


sporicidal test, the threshold established in UNE standards to consider a product as "sporicidal" of 4 log 10 is applied. The effects of the different products were compared using analysis of variance with Bonferroni's or Dunnett's correction depending on whether or not homogeneity of variances was found. The "final effect" at the maximum time considered in the comparison of all amines and the two reference disinfectants, that is, 10 min for OPA and Perasafe and 20 min for all others, was also compared.

Results The first comparison was performed in order to determinate the compared speed of kill microorganisms between a classic tertiary amine (Korsolex) and the two most effective reference products used in this study (OPA and Perasafe). Table 1 shows the bactericidal effect of these three products at between 1 and 15 minutes against 27 microorganisms. Overall, it should be noted that they all have a greater efficacy against Gram-positive organisms, followed by Gramnegative organisms (particularly at 1-5 min), and show a less effective action against Mfortuitum. The good efficacy of OPA against almost all microorganisms tested should be noted, as it rapidly increases from 1 min, and total killing of the inoculum is achieved at 10 min. For M. fortuitum, however, a slower increase in efficacy is seen, and 15 min are required to achieve a substantial mycobactericidal effect (approximately 4 log 10). Perasafe acts similarly, with no significant differences from OPA between the trends for the different microorganisms, and even the lower efficacy of both disinfectants against M. fortuitum almost overlaps. The only difference that can be stressed is maybe the greater efficacy of Perasafe at short times (1-5 min) against Gram-positive organisms, but no significant differences are seen in the tested time points overall. However, 4% Korsolex consistently has a lower bactericidal effect as compared to the other two products, and at the longest time tested does not achieve total killing of the microbial inoculum in any case, though it achieves the minimum efficacy of 3.5 log 10 against Staphylococcus and NFB other than P. aeruginosa. Korsolex does not also reach the 3 log 10 threshold against M. fortuitum, and the bactericidal trend also expresses a "depletion" of efficacy against mycobacteria, since little increase is seen in log 10 reduction even when exposure time is greatly increased. Table 2 simultaneously compares all 7 products in a determinate point of time: 10 minutes, Perasafe and OPA and tertiary amines were evaluated at two different times, since some manufacturers recommend 15 minutes and others, 20 minutes. The following results must be noted: OPA and Perasafe completely kill the microbial inocula of Gram-positive



2 4 1.9

2.6 5 2.9

3.1 2.6 1.1

2.6 3.34 0.8

S. coag- (2) and S. faecalis (2) OPA Perasafe 4% Korsolex

Klebsiella (2), Enterobacter (2), and Serratia (2) OPA Perasafe 4% Korsolex

E. coli (2) and P. mirabilis (2) OPA Perasafe 4% Korsolex

/'

MRSA (2) and MSSA (2) OPA Perasafe 4% Korsolex

Microorganisms and Products

4.4 3.8 2

4.8 4.6 2.1

5 5.5 3.5

4 5.5 3.4

5'

time points

5.5 4.4 2.5

5.5 4.9 4.5

5.5 5.5 3.9

5.5 5.5 3.8

10'

5.5 4.7 3.2

5.5 5.1 3.9

5.5 5.5 5

5.5 5.5 3.9

15'

Table 1: Speed of the bactericidal effect between 1 and IS minutes of 3 disinfectants against various microorganisms: mean loglO reduction at each time point.



(1)

OPA Perasafe 4% Korsolex

M. fortuitum


Other N F B s (4)


P. aeruginosa S (2) and P. aeruginosa polyR (2)

1.5 1 0.9

3.3 3 2.5

3.1 2.6 2.2

2.1 17 \A

4.5 4 2.9

4.3 3 2.6

2.95 3 2A

5.5 4.8 3.8

5.5 4.2 2.9


3.9 3.8 2.3

5.5 5.1 4.3

5.5 4.8 3.1

in

458 Table 2: Bactericidal effect between 10 and 20 minutes of 7 disinfectants against various Gram positive microorganisms: mean loglO reduction at each time point:


w J wt J J. Microorganisms and Products

^

time points jy

^Q'

S. epidermidis (3) (Non-dil)Instrunet FA 5%Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA

5.2 * 5.2 *

2.8 2.9 4.2 3.3 3.9

3.9 3.7 4.8 4 4

S. aureus (3) (non-dil)Instrunet FA 5% Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA

4.5 * 4.5 *

2.2 1.5 2.4 2.5 3.7

2.5 3 2.5 3.4 3.9

S. faecalis (3) (non-dil)Instrunet FA 5% Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA

4.3 * 4.3 *

4.3 * 2.6 3.2 3.1 2.9

4.3 * 3.5 3.5 3.6 3.1

K. pneumoniae (3) (non-dil)Instrunet FA 5% Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA

5.2 * 52^

5.1 2.7 3.3 3 2.6

5.1 3.1 3.7 3.2 2.9

*= inoculum is completely killed Table 2 cont: Bactericidal effect between 10 and 20 minutes of 7 disinfectants against various microorganisms: mean loglO reduction at each time point:


459 Table 2. Continued. Bactericidal effect between 10 and 20 minutes of 7 disinfectants against various Gram negative microorganisms: mean loglO reduction at each time point:


Microorganisms and Products P. mirabilis (3) (non-dil)Instrunet FA 5%Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA E. cloacae (3) (non-dil)Instrunet FA 5% Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA P. aeruginosa (3) (non-dil)Instrunet FA 5%Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA C. albicans (3) (non-dil)Instrunet FA 5%Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA

-

-

3.4 1.2 2.7 2.9 1

5.1 * 3.3 3.6 3.3 1.2

4.4 3.1 2.8 3.4 2

5 3.5 3.3 3.7 2.6

4.9 3.7 3.8 4.7 3

5.2* 3.8 4 4.8 3.6

2.7 1.2 1.4 3.9 1.8

3.2 1.5 3.1 4 2.6

5.1 * 5.1 *

-

5.2* 5.2*

-

5.2* 5.2*

-

4* 4*

*= inoculum is completely killed Table 2 cont: Bactericidal effect between 10 and 2 0 minutes of 7 disinfectants against various microorganisms: mean log 10 reduction at each time point: Continued on next page.


460


Table 2. Continued. Bactericidal effect between 10 and 20 minutes of 7 disinfectants against various Mycobacteria and one spore: mean loglO reduction at each time point: Microorganisms and Products

^

time points

^

M. fortuitum Non-dil)Instrunet FA 5%Instrunet FA 4%Korsolex 12%Sinaldehyd 4% Ultradesmit Perasafe OPA

3.3 3.1

1.6 1 1.7 1.2 0.6 3.5* 3.5*

1.8 1.4 2.2 1.5 1.2

4 4.7*

2.1 1.7 2.7 3 2.5 4.7* 4.7*

3.9 2.7 2.8 3.5 3.2

2

M. smegmatis mc 155 (non-dil)Instrunet FA 5% Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA M. avium-complex (non-dil)Instrunet FA 5%Instrunet FA 4%Korsolex 12%Sinaldehyd 4%Ultradesmit Perasafe OPA

4.9 4.9*

4.9* 4.9*

B. subtilis spores (ATCC) (Non-dil)Instrunet FA 5% Instrunet FA 4% Korsolex 12% Sinaldehyd 4% Ultradesmit Perasafe OPA

2.9 L2

0.7 0.2 0.3 0.3 0.3

-

1.3 1.3 0.9 1.2 0.6

*= inoculum is completely killed Table 2 cont.: Bactericidal effect between 10 and 20 minutes of 7 disinfectants against various microorganisms: mean loglO reduction at each time point:



461 cocci, Gram-negative bacilli, and yeasts, while for M. smegmatis and M. avium complex they reach more than 4 log 10, but for M. fortuitum they barely exceed the minimum threshold of 3 loglO. On the other hand, they show a lower efficacy against spores, with reductions of just over 1 log 10 for OPA and almost 3 loglO for Perasafe. Overall, these are the most effective products tested, but there are no significant differences between them. As regards tertiary amines, we should note that the formulation of Instrunet FA requiring no dilution was more effective than the one that should be diluted before use. A comparison of this undiluted formula to all other amines shows the following: The most effective products against S. epidermidis are Korsolex and Ultradesmit, that reach the 3.5 loglO threshold in 15 minutes, while against S. aureus only the latter formula reaches this critical point from 15 minutes. The opposite occurs with S. faecalis, since Ultradesmit does not exceed a 3.5 log 10 reduction in 20 minutes, unlike all other tertiary amines. Against Gram-negative microorganisms, Instrunet FA is most effective, achieving over 5 log 10 in 20 minutes, while all other product barely reach the required threshold, except against P. aeruginosa, which is the Gram-negative microorganism most susceptible to these tested product. Against yeasts, only Sinaldehyd achieves 3.5 loglO, both a 15 and 20 minutes. All preparation, except Korsolex and Instrunet FA, are effective in 20 minutes against M. smegmatis, but none of them achieves 3 log 10 against M fortuitum. Due to such low efficacy for the latter microorganism, the effect of these amines against M. avium complex was not tested. Finally, all amines are poorly sporicidal in 20 minutes, as they only kill 0.61.3 loglO. Table 3 compares statistically the bactericidal effects of the used products against microorganisms, overall and grouped as Gram-positive microorganisms (S. epidermidis, S. aureus, E. faecalis, and C. albicans), Gram-negative bacteria (P. aeruginosa, Proteus, Enterobacter, and Klebsiella), Mycobacteria (M fortuitum and M. smegmatis or, for OPA and Perasafe, also including M. avium complex), and B. subtilis spores. subtilis spores It should be noted that, despite acting for only 10 minutes, OPA and Perasafe (not differing from each other) are significantly more effective than all other products tested, despite their longer contact (15 and 20 minutes), both for microorganisms overall and by groups. However, as an exception, Instrunet FA showed no significant differences at 20 minutes as compared to OPA and Perasafe at 10 minutes for Gram-negative microorganisms. On the other hand, except for this last data, the tested amines did not significantly differ in their bactericidal efficacy.



0.65

0.95 0.83 1.05 0.97

4.67

3.47 4.07

2.81 3.38

3.04 3.47

2.46 2.85

Perasafe (10 min)

Instrunet FA undiluted (15 min) (20 min)

5% Korsolex (15 min) (20 min)

12% Sinaldehyd (15 min) (20 min)

4% Ultradesmit (15 min) (20 min)

1.06 0.99

1.23 1.16

0.88

SD

4.63

Mean

All micro organisms

(10 min)

OPA

Products

3.07 3.4

3.2 3.75

2.8 3.47

3 3.47

4.5

4.5

Mean

Gram+

0.86 0.6

0.52 0.27

1.07 0.88

0.81 0.71

0.46

0.46

SD

2.15 2.57

3.5 3.75

3.15 3.65

0.79 0.91

0.74 0.66

0.45 0.26

0.69 0.07

0.04

5.17

4.45 5.1

4.3

0.04

5.17

1.8 2.35

1.9 2.37

2.2 2.5

1.85 2.85

4

Mean

SD

Mean

Gram-

Mycob

Table 3. Statistical comparison of the bactericidal effect of different amines versus OPA and Perasafe (loglO reduction in 10,15, or 20 minutes, depending on the product. Mean and SD).


SD

ND ND

ND ND

ND ND

ND ND

ND

ND


For Mycobacteria, the efficacy order is: 1. OPA and Persafe: No significant differences between them 2. All other products testes: No significant differences between them

For Gram-negative microorganisms, the efficacy order is: 1. OPA and Perasafe: No significant differences between them 2. Instrunet FA undiluted: Not different from OPA and Perasafe in 20. 3. Korsolex and Sinaldehyd: No significant differences between them 4. Ultradesmit

For Gram-positive microorganisms, the efficacy order is: 1. OPA and Perasafe: No significant differences between them 2. All amines: No significant differences between them

According to the analysis of variance: For the microorganisms overall, the efficacy order is: 1. OPA and Perasafe: No significant differences between them 2. Instrunet FA undiluted 3. Sinaldehyd 4. All others: No significant differences between them

ND = non determined (only n=3)



464 Thus, we can group the 7 products tested in 3 categories. OPA and Perasafe would be the most effective, followed by Instrunet FA (undiluted) as an amine somewhat more effective than the others, and then all other products tested. Finally, Figure 1 shows the "most favorable" mean effect of those obtained, i.e. 10 min for OPA and Perasafe or 20 min for all other disinfectants. Breakpoints are plotted, and a visual assessment can be made of what the statistical analysis suggests: among amines, only the undiluted formula of Instrunet FA shows significant differences from the others (due to its greater efficacy against Gram-negative germs), but they are overall similar. However, they all are less effective that OPA and Perasafe. Moreover, this greater efficacy is achieved by OPA and Perasafe after only 10 min, as compared to 20 min with amines. They did not alter the lancet blades with human blood in 7 days (equivalent to more 500 continuous disinfections of 20 min each in the case of amines and to more 1000 for OPA and Perasafe), except for Ultradesmit, which oxidizes the blade in one point (about 1 mm ) after the first day. 2

Discussion When the different microorganisms S or R to antibiotics (freshly isolated from patients) are tested on rough germ carriers, it may occur that a disinfectant considered as a HLD does not exceed the threshold required by this surface test against the specific microorganism, although such microorganism should theoretically be killed by that disinfectant to maintain its HLD label. For this we must verify the efficacy in a high number of microorganisms, but there is always a possibility that such decreased efficacy occurs in a certain number of them. We think that this problem should be addressed by proposing that the number of microorganisms for which efficacy is decreased does not exceed 0.5% when they are randomly selected from non-ICU patients, or 1% if they come from ICU patients (microorganisms probably selected by antibiotic therapy). Such cases would result in disinfection failure, but it should be taken into account that this would require another concomitant failure in washing before disinfection. However, this does not mean that an infection will necessarily occur, because microorganisms must previously overcome antagonism by the endogenous flora and immune system of the patient in whom instruments with such failed disinfection are introduced. Nonetheless, we must avoid that risk as much as possible. Efficacy of the different tertiary amines is quite similar when globally considered, and the optimum time of use is 20 min, as there are significant differences between 15 and 20 min. However, if one of them should be chosen, the most effective appears to be the Instrunet FA solution requiring no dilution, probably because of its excipients, that greatly increase its pH and place the formulation in a better position to achieve the best performance among all ammonia compounds used.



465

0 Lev

G-

G+

Myc

Esp

microorganisms grouped Figure 1. Global comparison between disinfectants (OPA and Perasafe, 10 min versus various tertiary amines, 20 min)



466 Efficacy may however be considered from a qualitative viewpoint, that is, evaluating whether the level required to consider them as HLDs is reached. Such level implies the ability to kill microorganisms more susceptible than spores, including all Mycobacteria. This is very difficult to assess, since it depends on the method selected, and even the disinfectant most widely accepted as HLD, 2% glutaraldehyde, is not affective in 20 min against M avium-intracellulare, but requires an increase in exposure time to 45 min. Nevertheless, certain Mycobacteria, including at least one that is more resistant than M. tuberculosis such as M. fortuitum, should at least be considered in a germ carrier test, because microorganisms to be disinfected in HLDs will actually be on various germ carriers with a smooth of rough surface. In our case, there is not much difference in using any of both germ carriers if we establish different efficacy thresholds, that is, if the European germ carrier test (using a smooth glass plate) requires a 4 log 10 reduction and our test (using rough metal and plastic) only requires a 3 loglO reduction (13) From this viewpoint, tertiary amines used in this study should not be classified as HLDs both based on tests with other microorganisms more susceptible than Mycobacteria and on their action against Mycobacteria, and only OPA and Perasafe meet the requirements to be considered HLDs: 1) more than 3 loglO Mycobacteria killed, and 2) among ICU microorganisms, less than 1% show more survivors than the critical threshold, 3.5 log 10 (0% for both these products). These products could participate in a HLD "process" if an additional stage was added to it, consisting of flooding with 100 ml of product before leaving the endoscope channels filled and starting the disinfection process proper (9) Thus, most disinfection controls conducted at our hospital have been negative (despite use of a disinfectant not having a high efficacy against Mycobacteria and which could not be adequately considered as a high level disinfectant), provided all other steps have been adequate, but in a minimum percentage (less than 2% in our samples) this does not occur for several reasons, and disinfection may then fail. Hence, disinfectants achieving by themselves an appropriate reduction of microorganisms, regardless of the rest of the disinfection process, should be used. We therefore think that among all disinfectants analyzed, only OPA and Perasafe should be included in high level disinfection protocols at our hospitals, because they do not only kill all tested microorganisms, but can also kill M avium-intracellulare (11,12) though it should be reminded that OPA cannot be used in urology and stains the areas where it is poured (clothes, etc.), and Perasafe is difficult to dilute if the water used is not at an adequate temperature (about 40°C), and both this and its permanence in the disinfection tray allows for peracetic evaporation, and the resulting smell has been sometimes poorly accepted by the exposed health care personnel. An additional problem reported for this product is an impaired articulation of instruments such as biopsy forceps, probably due to oxidation of this point of instruments. We also think that after 10 minutes both products are too near the "non efficacy" level with the M. fortuitum used, and could have failed the test if a



467 more resistant microorganism had been used. However, in the study where exposure time was increased to 15 minutes (Table 1), an increased efficacy was shown as compared to 10 minutes, allowing for a substantial safety margin with both Perasafe and OPA. This agrees with some guidelines (15-20) that recommend a time longer than 10 minutes for HLD with OPA, and 15 min was also the time recommended by us for Perasafe in a prior publication (14) Efficacy on spores has been tested in order to extrapolate conclusions to other untested microorganisms, that is, if a sporicidal effect had been obtained (>4 loglO reduction) (21), we could infer that the products would also be effective against other untested Mycobacteria particularly resistant to certain disinfectants (such as glutaraldehyde, the reference HLD product, which is however more effective against mycobacteria, killing them in 25 min, while it takes 3-10 h to have a sporicidal effect) o against viruses (Enterovirus, Coronavirus, Myxovirus, etc, that may be present in endoscopes) (22,23). However, such extrapolation is not possible because of the low sporicidal power of most tested products, although Perasafe may approach this induction, particularly when used with an exposure time of 15 instead of 10 minutes. Finally, there are other factors related to the two products we consider HLDs to be taken into account, namely: 1) changes in the material on which they act (they both appear to be similar in this regard, except for the abovementioned cases of biopsy forceps), and 2) price, which is in principle higher for OPA, but may actually be the opposite, taking into account Perasafe should be discarded daily, while OPA may act for up to 14 days, and also has an adequate indicator that would allow for product disposal before the expected data if it has degraded more than usual with use. This is a control mechanism that should be required for any disinfecting solution that is claimed to last for longer than one day. To conclude, among all disinfectants tested in this study we only can include OPA and Perasafe as high level disinfectants, but recommending an action time of 15-20 minutes and taking into account that they are notfreefrom disadvantages (staining and avoidance of use in urology for OPA, and a strong smell of vinegar, occasional oxidation of instruments, and need to discard solution every day, which may ultimately make the process much more expensive for Perasafe). Thus, research must be continued in this field to obtain products that come closer to the characteristics of the ideal HLD.

References 1.

2.

Rossmoore HW. Nitrogen compounds. . In: Disinfection, Sterilization and Preservation. Block SS Ed. Fifth Ed. Lippincott Williams and Wilkins. Philadelphia, 2001 p: 383-413. Lanzardo M , Rubin J. Micobactericidal disinfection. In: Disinfection, Sterilization and Preservation. Block SS Ed. Fifth Ed. Lippincott Williams and Wilkins. Philadelphia, 2001 p: 513-528.


468 3. 4.


5.

6. 7. 8.

9.

10.

11.

12.

13.

14.

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