Mycobacteria and glutaraldehyde: is high-level disinfection of endoscopes possible?

0016-5107/96/4305-045155.00 + 0 GASTROINTESTINAL ENDOSCOPY Copyright © 1996 by the American Society for Gastrointestinal Endoscopy

Mycobacteria and glutaraldehyde: is high-level disinfection of endoscopes possible? Shiro Urayama, MD, Richard A. Kozarek, MD, Stanford Sumida, PhD, Shirley Raltz, MSN, R N Linda Merriam, P. Pethigal, MSN, RN Seattle, Washington

Background: High-level disinfection of endoscopes has traditionally been undertaken by manual or automatic scope cleaning plus a 10 to 20 minute soak in 2% alkaline glutaraldehyde. Mycobacteria species are less sensitive to glutaraldehyde, and a 45-minute instrument soak has recently been recommended by the manufacturer. Because of concerns over endoscope damage, need for more endoscopes, and perception that the current cleaning method is adequate, we prospectively studied mycobacteria-contaminated endoscopes at various stages of the cleaning process. Methods: All work was done under a laminar flow hood in a microbiology laboratory. Five gastrointestinal scopes were contaminated with 108 colony forming units per milliliter (CFU/mL) of Mycobacterium chelonei, an atypical mycobacterium similar in chemical resistance to Mycobacterium tuberculosis but with less infectious potential. Cultures of the sheath, biopsy channel, and elevator channel were taken at baseline, after manual cleaning, and after 10, 20, and 45 minutes of glutaraldehyde soak both before and after alcohol rinse. Results: Manual cleaning resulted in a mean of 4.7 log10 reduction in viable mycobacterial colonies. Qualitative studies of the external endoscope surface as well as the air-water valve showed no detectable organisms after a 10-minute exposure to alkaline glutaraldehyde. Conventional quantitative culture techniques of the channels demonstrated one endoscope out of five with consistent growth after a 10-minute exposure to glutaraldehyde. Following alcohol treatment, there was no significant colony growth. In contrast, a quantitative membrane filter system showed the presence of at least one mycobacterial colony in four out of five scopes after a 45-minute glutaraldehyde exposure. Conclusions: Additional studies utilizing a standardized mycobacterial species, inoculum size, and suspension characteristics are recommended to delineate adequate duration of disinfectant exposure time. (Gastrointest Endosc 1996;43:451-6.) Adequate disinfection of endoscopic equipment is necessary to prevent transmission of infectious organisms from the environment to a patient, or between patients. Currently, glutaraldehyde-based disinfecReceived June 11, 1995. For revision October 10, 1995. Accepted January 17, 1996. From the Department of Internal Medicine, Section of Gastroenterology, Virginia Mason Medical Center, Seattle, Washington. Reprint requests: R.A. Kozarek, MD, Virginia Mason Medical Center, Gastroenterology, PO Box 900, 1100 Ninth Ave. (C3-GAS, Seattle, WA 98111. VOLUME 43, NO. 5, 1996

tants are widely utilized for high-level disinfection. After new, quantitative, in-vitro testing was approved by the Environmental Protection Agency (EPA) in 1984 and adopted subsequently by the Food and Drug Administration (FDA), the product label for a glutaraldehyde-baseddisinfectant was revised. This states that high-level disinfection of semi-critical medical devices such as endoscopes requires up to 45 minutes of exposure at 25°C to ensure 100% kill of potential infectious microorganisms, particularly Mycobacterium species. 1,2 It has been noted, however, that GASTROINTESTINAL ENDOSCOPY 451

in-vitro q u a n t i t a t i v e t e s t s p r e c l u d e d a n initial w a s h step. I n addition, a n e x t r e m e l y large n u m b e r of o r g a n i s m s w e r e u s e d in t h e c o n t a m i n a t i o n process. More recently, H a n s o n et al. 3 u n d e r t o o k a bronchoscopic s t u d y t h a t r e v e a l e d a significant reduction of m y c o b a c t e r i a a f t e r a w a s h step alone. Total disinfection t i m e c o n t r i b u t e s to costs of endoscopic p r o c e d u r e s b e c a u s e of potential n e e d for additional endoscopes, i n c r e a s e d m a n p o w e r r e q u i r e m e n t s , a n d i n c r e a s e d endoscope t u r n a r o u n d times. Moreover, the caustic n a t u r e of t h e disinfectants t h e m selves m a y be associated w i t h s h o r t e r i n s t r u m e n t life spans. Finally, i n c r e a s i n g t e m p e r a t u r e r e q u i r e m e n t s m a y c a u s e additional safety p r o b l e m s f r o m "toxic f u m e s " produced b y g l u t a r a l d e h y d e . Accordingly, we a t t e m p t e d to a s s e s s t h e a p p r o p r i a t e disinfection t i m e for a v a r i e t y of g a s t r o i n t e s t i n a l endoscopes u s i n g c o m m o n l y e m p l o y e d cleaning methods.

MATERIALS AND METHODS Preparation of organisms Mycobacterium chelonei, subspecies abscessus, was obtained from the Washington State Microbiology Laboratory. The organism was inoculated onto slant test tubes with Middlebrook 7 H l l agar (Difco, Detroit, Mich.) and incubated for 7 days at 37°C. The subpopulation of the slant culture was suspended in 50 ml of trypticase soy broth (Becton Dickinson and Co., Cockeysville, Md.), and McFarland turbidity of 1.0 was obtained (approximately 10 s CFU/mL). Viable counts were carried out by plating on Middlebrook 7 H l l agar (Difco) and counting the colonies after incubation at 37°C for 1 week.

pendent on scope size) through the suction port by a 60 ml syringe. The endoscope was then allowed to stand for 10 minutes. The channels were aerated, and the inoculum evacuated using the syringe.

Sampling The internal channel was sampled with 40 ml of sterile tap water irrigated through the suction port. Air was subsequently injected through the port using a syringe for optimal volume collection. Samplings were collected after (1) initial inoculation, (2) manual cleaning, (3) disinfection and rinsing, and (4) alcohol irrigation and aeration. Swab samplings were also obtained from the distal endoscope shaft and from the air-water valves. Additional cultures of the elevator channel of the duodenoscopes were also done. These swabs were directly plated onto agar plates of incubation.

Cleaning A manual endoscope cleaning technique was employed. Internal channels were irrigated and thoroughly washed through twice using a wire brush in 6 L of sterile tap water with a commercial detergent (Manuklenz, Merck, St. Louis, Mo.). External surfaces were sponged thoroughly. The endoscope was subsequently rinsed in another 6 L of sterile tap water in a new basin.

Disinfection The endoscope and valves were immersed in 6 L of freshly activated 2% alkaline glutaraldehyde and the channels perfused. At Specified time periods (10, 20, and 45 minutes), endoscopes were removed to a basin with 6 L of sterile tap water for rinsing. Irrigation of the channels and scrubbing of the external surface were done. This rinsing step was repeated two more times with equal volumes of sterile water.

Alkaline glutaraldehyde solution

Use of 70% isopropyl alcohol

Two percent alkaline glutaraldehyde (Cidex, Johnson & Johnson, Arlington, Texas) was freshly made up as recommended by the manufacturer.

After disinfection and sampling, biopsy channels were irrigated with 40 mL of 70% isopropyl alcohol; 3 mL of alcohol, in turn, was injected into the duodenoscope elevator channel. Air was passed through the channels using a syringe. Then, after letting the endoscope stand for 1 minute, 40 mL of sterile tap water was irrigated and aerated through the channels and collected at the distal tip.

Endoscopes Five fiberoptic endoscopes (Olympus America Inc., Melville, N.Y.), including a colonoscope (CFIT1L), two gastroscopes (Model #GIFXQ10, GIFXQ10), and two duodenoscopes (JFIT10, FJIT20) were used. Mean instrument age was 8.8 years (range from 5.8 to 10.7 years). All instruments had been repaired within the previous 18 months. These endoscopes were leak-tested and were prepared for each individual experiment by gas sterilization with ethylene oxide.

Endoscope contamination Experiments were done on three different occasions utilizing the same endoscopes but with variable disinfectant exposure times: 10, 20, and 45 minutes. Prior to each experiment, endoscopes were gas sterilized by ethylene oxide. All experiments were done under a class II exhaust protective cabinet at a temperature of 20°C by personnel wearing appropriate protective gear. All internal channels of the endoscopes were exposed and contaminated by infecting 20 to 40 ml of the inoculum (de452 GASTROINTESTINAL ENDOSCOPY

Recovery of organisms Serial dilutions using 0.5 mL of collected samples were made and double aliquots were plated on Middlebrook 7H11 agar (Difco) and incubated for 1 week at 37°C. Mycobacterial colonies were confirmed with Ziehl-Nielsen staining and counted. The arithmetic mean of double colonies was corrected for dilution to give the viable number of organisms. The remainder of the collected sample was filtered through a 0.2 ]~m Millipore membrane (Millipore Corp., Bedford, Mass.) and the membrane was plated on 7 H l l agar and incubated similarly.

Mini in-vitro experiment for microbial cluster effect Using an inoculum of 10 s CFU/mL, two separate mycobacterial suspensions in trypticase soy broth (Becton) were prepared: one with residual visible small "clumps" and anVOLUME 43, NO. 5, 1996

other with more homogeneous suspension without visible clumps. A 0.5 mL sample of each group was mixed in 4.5 mL

of freshly activated alkaline glutaraldehyde and at a specified time (5, 10, 20, 30, 45, and 60 minutes) filtered through a 0.2 pm Millipore filter followed by 75 mL of sterile saline solution and 10 mL trypticase soy broth. The quantitative suspension test was also repeated for homogeneous concentrations of 106 and 104 CFU/mL.

RESULTS The inoculum size ofM. chelonei used in this experiment ranged from 2.2 to 5.6 × l0 s CFU/mL. Figure 1 shows the decrease in viable colony counts after the manual Cleaning for individual endoscopes. To prevent an overestimation of disinfection, endoscopes were gas sterilized with ethylene oxide after each alcohol exposure and contaminated with the same inoculum concentration. Thus for each endoscope, there were three occasions to assess the effect of cleaning. The mean reduction of the viable colony count for manual cleaning alone was 4.7 log10 CFU/mL. Table 1 shows the qualitative study results of endoscope culturing at each site sampled. Utilizing the swab method, manual cleaning alone appeared to eliminate viable organisms on the external endoscope surface in two of the five instruments tested. None of the endoscope shafts had detectable organisms after a 10-minute exposure to glutaraldehyde. Air-water valves, however, had organisms detectable by swab culture on all five endoscopes after the manual cleaning step alone whereas a 10-minute glutaraldehyde soak prevented any culture growth at this site. Duodenoscope elevator channels, in contrast, yielded positive cultures after a 10-minute soak, but were negative after the addition of 70% isopropyl alcohol. All glutaraldehyde soaks in excess of 10 minutes prevented detection of viable organisms at every swab sampling site. Table 2 depicts results following glutaraldehyde exposure when conventional plating techniques were used and viable organisms were grown from serial dilutions of collected internal channel irrigant. For a 10-minute exposure, there was a single colony growth from the most dilute irrigant. However, after a 70% isopropyl alcohol treatment, no mycobacterial colony growth was noted on any of the agar plates. After a 20-minute glutaraldehyde soak, some random colony growths were noted on agar plates of the most dilute sample irrigant from four of the five endoscopes. However, these growths were not statistically significant because less than one colony per duplicate agar plate grew. At this exposure time, gastroscope #2 was the only instrument that had no colony growth. The addition of alcohol resulted in no detectable organisms from the colonoscope and other gastroscope, but this was not the case with the duodenoscopes. After a 45minute soak alone, no viable colony was detected in VOLUME 43, NO. 5, 1996

,og,0c u,m,! 1 4 31

:t

Before Cleaning

/°2 • ~ G C 1

C

After Cleaning

D2 = Duodenoscope 2, Dt = Duodenoscope 1, C = Colonoscope,G1 = Gastroscope 1, G2 = Gastroscope 2

Figure 1. Reduction of viable mycobacterium after manual cleaning step for endoscopes. any endoscope with or without subsequent alcohol irrigation. Table 3 shows the result of the same experiment using another recovery system for the organism. In this case, the remaining internal channel irrigant was passed through a membrane filter and the filter itself was plated onto an agar plate. After a 10-minute exposure to glutaraldehyde, four out of the five endoscopes showed at least one colony growth. In the colonoscope and duodenoscope #1, this was true even after the alcohol step. After a 20-minute exposure, the membrane filter system detected viable organisms in three out of five endoscopes after glutaraldehyde disinfection. Following alcohol exposure, organisms were detected on a single endoscope (duodenoscope #1). Finally, after a 45-minute soak, glutaraldehyde failed to eradicate mycobacteria in four out of the five instruments. Subsequent alcohol irrigation, however, decreased culture positivity to one out o f five instruments (colonoscope). A mini-experiment utilizing two different inoculum suspensions was undertaken in ex-endoscope fashion. The summary is listed on Table 4. In a suspension with visible clumping and an inoculum concentration of 10 s CFU/mL, six colonies were detected on the membrane filter after a 30-minute glutaraldehyde exposure. On the other hand, only one colony was visible on the filter at 10 minutes if a homogeneous inoculum suspension with the same initial concentration was used. High-level disinfection (eradication of mycobacteria) was obtained after 20 minutes with this inoculum size if a homogeneous suspension was used. Furthermore, at lesser inoculum sizes of the homogeneous suspension, i.e., at 10 6 a n d 10 4 CFU/mL, a 5-minute exposure was adequate for 100% kill of the mycobacterial population.

DISCUSSION Prior to 1984, testing of high-level disinfectants was done utilizing a method developed by the Association of Official Analytical Chemists (AOAC). 1,2,4 This testing method came under attack because of lack of G A S T R O I N T E S T I N A L E N D O S C O P Y 453

Table la. Swab cultures of external surface and air-water valves After cleaning

Endoscope

10 m i n (-A)

SO

SV

SO

+ +

+ + +

. . .

Colonoscope Gastroscope #1 Gastroscope #2

10 m i n (+A)

SV

SO

. . .

20 m i n (-A)

SV

. .

. . .

SO

. .

SV

. .

.

20 m i n (+A) SO

. .

.

. .

.

45 m i n (-A)

SV . .

.

SO . .

.

SV

. .

.

45 m i n (+A) SO

SV

. .

.

- A , W i t h o u t 70% isopropyl alcohol; +A, with 7 0 % isopropyl alcohol; +, positive viable colonies; - , negative viable colonies; SO, swab c u p rare: outer surface o f endoscope; SV, swab culture: air-water valves.

Table lb. Swab cultures of external surface, air-water valves, and elevator channel After cleaning

Endocope

Duodenoscope #1 Duodenoscope #2

10 rain (-A)

10 m i n (+A)

SO

SV

SE

SO

SV

SE

SO

+ -

+ +

+ +

-

-

+ +

. .

SV . .

20 m a n (-A) SE

.

SO .

.

.

SV

. .

SE

. .

20 m i n (+A) SO

. .

SV

.

.

45 m i n (-A) SE

.

.

SO

. .

SV

SE

.

.

. .

45 m i n (+A)

.

.

.

SO . .

SV

SE

.

A, W i t h o u t 70% isopropyl alcohol; +A, with 70% isopropyl alcohol; +, positive viable colonies; - , negative viable colonies; SO, swab culture: outer surface o f endoscope; SV, swab culture: air-water values; SE, swab culture: elevator channel.

Table 2. Viable Mycobacteriumcounts using conventional culture techniques Endoscope

After cleaning

10 rain (-A)

10 m i n (+A)

20 m i n (-A)

20 m i n (+A)

Colonoscope Gastroscope #1 G a s t r o s c o p e #2 Duodenoscope #1 Duodenoscope #2

9.5 x 103 2.0 x 103 4.0 xl03 1.4 x 10 4 2.4 x 10 4