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Atypical mycobacteria and the xenograft valve
Atypical mycobacteria and the xenograft valve During a 4 month interval, cultures taken at the time of porcine xenograft valve implantation grew a fastidious atypical mycobacterium, Mycobacterium chelonei (Runyon's group IV), in eight of 20 patients. Initial growth occurred at 2 to 3 weeks in thioglycollate broth only; detailed biochemical and bacteriologic evaluation demonstrates altered catalase activity accounting for its initial slow growth. Only one patient has manifested clinical evidence of infection 5 months after mitral valve replacement. A large pericardial effusion required pericardiectomy. Pericardial tissue and fluid have grown M. chelonei with the same growth characteristics as the initial valve culture. It is recommended that manufacturers of xenograft valve bioprostheses maintain a quarantine of 6 weeks until cultures, smears of culture broth, and microscopic evaluation of aortic wall coupons are negative. Users of these prostheses should keep cultures for a minimum of 3 weeks. Should positive cultures result, antimicrobial therapy should be considered only with clinical evidence of infection.
Denis H. Tyras, M.D., George C. Kaiser, M.D., Hendrick B. Barner, M.D., Leonard F. Laskowski, Ph.D., and J. Joseph Marr, M.D., St. Louis, Mo.
Recent reports of favorable six-year results with implanted porcine xenograft valvular prostheses have contributed to their widespread use.': 2 Whereas procurement of porcine tissue valves is less of a problem than was encountered with human aortic valve homografts, sterility of the implantable prosthesis is still a major consideration. The purpose of this paper is to report the isolation of a fastidious atypical mycobacterium from cultures taken at the time of porcine xenograft valve implantation in eight patients. Patients and methods
Between July and November, 1976, 24 Hancock* porcine xenograft valves were implanted in 20 patients at our institution. All valves were received from the manufacturer during the period from May through August, 1976. At the time of implantation, each valve was removed aseptically from its shipping jar containing cotton wads soaked with 0.2 percent stabilized From the Departments of Surgery, Pathology (Clinical Microbiology), and Medicine (Infectious Diseases), St. Louis University School of Medicine, St. Louis, Mo. Read at the Third Annual Meeting of The Samson Thoracic Surgical Society, Colorado Springs, Colorado, June 4-7, 1977. Address for reprints: Denis H. Tyras, M.D., Department of Surgery, St. Louis University School of Medicine, 1325 South Grand Boulevard, St. Louis, Mo. 63104. *Hancock Laboratories, Inc., Anaheim, Calif. 0022-5223/78/0375-0331$00.70/0 © 1978
The C. V. Mosby Co.
glutaraldehyde. After the cotton wads were removed from the valve cusps, each valve was rinsed in three separate stainless steel basins containing sterile 0.9 percent saline. At the conclusion of the rinse, the accompanying aortic wall coupon was removed, placed in a sterile culture tube, and transported immediately to the laboratory. The specimen was placed in a tube of liquid thioglycollate medium without indicator (BBL) and incubated at 37° C. Initial cultures were held for a minimum of 2 weeks with Gram stain and subculture to blood and MacConkey agar plates were made before a final negative report. After the first positive culture, all valve cultures were held for 12 weeks before being considered to be negative. Beginning in September, 1976, a portion of aortic wall coupon was cultured before as well as after rinsing. Patients with positive cultures have been followed closely and, when possible, have undergone follow-up physical examination, chest roentgenogram, complete blood counts, echocardiography, electrocardiography, and tuberculin skin testing (intermediate and secondstrength purified protein derivative [PPD]). Results
Prior to July, 1976, none of the porcine valve cultures handled in this manner produced any growth of organisms. Beginning with a culture taken July 29, 1976, after 2V2 weeks of incubation, several cultures 331
332
The Journal of
Tyras et al.
Thoracic and Cardiovascular Surgery
Table I. Selected biochemical and culture characteristics of mycobacterial species * Species Property
MacConkey agar Arylsulfatase (3 days) Growth on 5 per cent NaCI Tellurite red'n (3 day) Catalase (68° C.) Niacin Tween 80 hydro!. (5 days) Catalase (>45 mm. foam)
M. tuberculosis
I
M. chelonei, subsp. chelonei
I
M. chelonei, subsp. abscessus
+
+ + +
+
+
+
+
d
+
±
Organism isolated
+ + + d
d d
'Modified from Buchanan RE, Gibbons NE: Bergey's Manual of Determinative Bacteriology, ed. 8, Baltimore, 1974, The Williams & Wilkins Company. td = variable depending on strain.
demonstrated anaerobic growth in the thioglycollate medium. These organisms were acid fast on staining. Including the initial positive culture, eight samples of porcine valves from different lot numbers yielded acid-fast organisms. Positive cultures were not sequential. Subculture was possible on solid thioglycollate agar after 2 weeks' incubation at 37° C. in a 5 percent carbon dioxide atmosphere. Control cultures of saline normally used for valve rinsing, of culture tubes used in transport of specimens, and of thioglycollate broth from the same batch used for porcine valve culture but inoculated with cerebrospinal fluid during routine fungal culture failed to demonstrate growth of acid-fast organisms. Moreover, cultures of aortic coupons taken before as well as after rinsing demonstrated growth of acid-fast bacilli in two patients in whom this culturing sequence was performed. Microscopic examination of stained specimens of aortic coupons in the last five positive cultures demonstrated acid-fast organisms within the specimen, although litt le cellular reaction was seen. Biochemical testing (Table I) strongly suggests that this organism is a subspecies of Mycobacterium chelonei, a member of Runyon's group IV. After multiple subcultures, if blood was added to the liquid thioglycollate broth, the organism began to grow aerobically within 5 to 6 days. At this point, catalase activity, which had been low, became high. Incubation of an aliquot of the positive culture with a piece of human aorta in different concentrations of buffered glutaraldehyde (pH 7.4) showed uniform inhibition of growth in all concentrations incubated for at least 48 hours (Table 11). If 0.5 ml. of human blood was added to the culture, only concentrations of glutaraldehyde of 0.625 and 0.8 percent were able to inhibit growth after at least I week of incubation. Lesser con-
centrations showed no inhibition of growth even after 3 weeks' incubation. Glutaraldehyde concentrations of 1.0 percent or greater showed uniform inhibition with only 24 hours' incubation (Table II). Antibiotic testing, after multiple organism subcultures, showed the organism to be sensitive only to rifampin and resistant to all other antituberculous drugs. Patient follow-up (Table III) revealed only one instance in which this organism was clinically manifest. Five months after mitral valve replacement, this patient was readmitted with lower extremity thrombophlebitis, congestive heart failure, and cardiac silhouette enlargement on chest roentgenogram. Pulmonary perfusion scans were normal. Anticoagulation and diuresis cleared both the thrombophlebitis and pulmonary congestion, but echocardiography demonstrated a large pericardial effusion. Results of intermediate and second-strength PPD skin testing were negative. Blood cultures were negative. The diagnosis of postpericardiotomy syndrome was suspected and the patient was begun on a course of corticosteroids. Pericardiocentesis was not done because of the anticoagulation. After 3 weeks of therapy, the pericardial effusion had increased slightly and the patient underwent partial pericardiectomy. The pericardium contained 1,300 m!. of clear, yellow fluid. Sections of pericardium and spun specimens of pericardial fluid demonstrated acid-fast organisms. Subsequent cultures yielded findings compatible withM. chelonei, with the same growth characteristics that had been found in the initial porcine valve culture. The patient is being maintained on rifampin therapy at the present time and remains asymptomatic. Discussion
The goals of cardiac valve replacement have been to provide satisfactory hemodynamic characteristics,
Volume 75
Atypical mycobacteria and xenograft valve
Number 3 March,1978
333
Table II. Effect of glutaraldehyde and blood on porcine valve acid-fast bacillus (AFB) isolate Control: 10 mi. thioglycollate 0.1 mi. subcultured to new thioglycollate after:
Glutaraldehyde concentrations 10 mi. (pH) 7.4 + 0.1 mi. AFB* + piece of human aorta 0.2% 0.5% 0.625% 0.8% 1.0% 1.5% 2.0% The above culture + 0.5 mi. human blood 0.2% 0.5% 0.625% 0.8% 1.0% 1.5% 2.0%
24 hr.
I
48 hr.
I
72 hr.
I
1 wk.
I
2 wk.
3 wk.
-t
+AFB +AFB
+AFB(s) +AFB(s) +AFB(s) +AFB
I
+AFB(s) +AFB(s) +AFB(s) +AFB
+AFB(s) +AFB(s) +AFB +AFB
+AFB(s) +AFB(s)
+AFB(s) +AFB(s)
Original + 0.1 10.1 mi. subcultured at 24 hr. mi. AFB *
+AFB +AFB +AFB +AFB +AFB +AFB +AFB
+AFB +AFB +AFB +AFB +AFB +AFB +AFB
+AFB(s) +AFB(s)
'Turbidity = Bauer Kirby BaSo, standard. tNo growth after four weeks. t(s) = Pellicle with surface growth.
long-term mechanical stability, and a low incidence of thromboembolic complications. Among the favorable attributes of tissue valves are the low incidence of thromboembolic complications, the frequent lack of need for anticoagulation, and the central flow dynamics. Early efforts were directed toward the use of preserved aortic valve homografts. Sterilization was attempted with many techniques, including electron-beam irradiation," antibiotic solutions," beta propiolactone," ethylene oxide," and 4 percent buffered formaldehyde solution (formalin)." Procurement of cadaveric aortic valve homografts was limited not only by availability, but also by the finding that the incidence of positive cultures increased markedly from 10 percent to 30 to 40 percent when grafts were harvested sterilely more than 8 to 12 hours after death." Even when sterility could be obtained, later foIlow-up revealed high rates of premature graft failure from valve cusp deterioration, calcification, and infection." 9 Similar results were encountered with early graft deterioration of homograft dura mater and fascia lata valves .10 Heterograft (porcine) valves were more readily obtained, but treatment with buffered formalin solution again appeared to lead to early graft deterioration." Pioneering work by Carpentier, 1 using buffered
glutaraldehyde to stabilize and sterilize porcine aortic xenografts, has shown excellent stability of the graft even up to 6 years. These findings have been supported by the reports of Pipkin," Mclntosh,!" and Zuhdi'" in this country. Similarly encouraging results have been described by Ionescu" with the use of glutaraldehyde-stabilized bovine pericardial xenografts. At the present time, three manufacturers provide porcine xenograft valves prepared with buffered glutaraldehyde solution in concentrations varying from 0.2 to 0.625 percent. Glutaraldehyde stabilization of the collagen matrix of the porcine bioprosthesis has apparently also been an effective antimicrobial treatment as weIl. Control cultures are taken by the manufacturer at the time of prosthesis construction, and valves are not released to users until a quarantine period has passed with negative cultures. Prior to our recent experience there have been no published reports of positive cultures at the time of valve implantation. In fact, results have been excellent in terms of resistance to prosthesis endocarditis in patients with active bacterial endocarditis receiving porcine bioprosthesis implants.!" Careful investigation of possible sources of contamination of cultures at our institution have supported the conclusion thatM. chelonei organisms were indigenous
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Thoracic and Cardiovascular
Surgery
Table III. Atypical mycobacteria in porcine valve culture: Patient data Follow-up status
Case No.
Date of implant
7/29/76 2
8/03/76
3
8/20/76
4 5 6 7 8
8/31/76 9/08/76 10/04/76 10/08/76 10/13/76
Purified protein derivative Valve implanted
31 mm. mitral-pos. culture 25 mm. aortic-neg. culture 31 mm. mitral 31 mm. mitral (+ double coronary bypass) 29 mm. aortic 27 mm. aortic 27 mm. aortic 33 mm. mitral 27 mm. aortic
Interm.
Clinical
I
Second strength
ECG/echo
CBC
Well
Neg.
Not done
Okay
Okay
Pericarditis (see text) Well
Neg.
Neg.
See text
Okay
Okay
Okay
Okay Okay Okay Okay
Okay Okay Okay Okay
Prev. TBC,1961
Well Neg. Not done Well Not done Well Neg. IOmm. 14mm. Well Neg. Died 1O/14/76-low output syndrome
Legend: ECG/echo, Electrocardiogram/echocardiogram. CBC, Complete blood count. TBC, Clinical tuberculosis.
to material received from the manufacturer. This contention is supported by the finding of similar organisms from porcine valve cultures in at least five other institutions.": 18 Initial isolation of this organism may be extremely difficult because of its fastidious growth characteristics. Characteristically, M. chelonei is a "rapid grower" of Runyon's group IV. The organism isolated and subcultured demonstrated low catalase activity, which may account for its slow growth, Many microorganisms produce hydrogen peroxide, which inhibits growthcatalase is effective in destroying hydrogen peroxide. It is interesting to note that sensitive strains of M. tuberculosis lose catalase activity after treatment with isoniazid.!" Also, blood contains catalase and was effective in restoring rapid growth characteristics to our cultures. Atypical mycobacterial infections are not uncommon in swine, but the overwhelming majority (97 percent) of isolates have been M, avium.i? A single case of M. chelonei causing an abscess in a pig has been identified." Clinically significant infections in man due to M. chelonei have been reported. These have included thyroiditis.P pneumonitis.P cutaneous abscesses." de novo endocarditis," prosthetic valve endocarditis.": 27 and osteomyelitis, pericarditis, and mediastinitis after coronary artery bypass." To date, only one of our patients has had any clinical manifestation of M. chelonei infection, namely pericarditis. This condition has responded well to pericardiectomy and rifampin therapy. The fact that this patient received therapeutic doses of corticosteroids in the treatment of the pericardial effusion may have in some way encouraged the growth of
these organisms. All other patients remain free of clinical evidence of infection; thus, we would recommend antituberculous therapy only if clinical infection is demonstrated. It is especially important to emphasize that none of the patients has shown any evidence of endocarditis, and follow-up echocardiograms continue to show satisfactory valve function. Although 2 percent glutaraldehyde solution (pH 8.5) is mycobactericidal, evidence exists that more acidic or less concentrated solutions of glutaraldehyde are less effective;" Our studies with varying concentrations of glutaraldehyde tend to substantiate this. What must be balanced is the antimicrobial efficacy of differing concentrations of buffered glutaraldehyde with the undesirable "tanning" effects of higher concentrations. Excellent results of valve durability have been reported with the Hancock valve (0.2 percent glutaraldehyde),13, 14 the Angell-Shiley valve (0.5 percent glutaraldehydej.P" and the Carpentier-Edwards valve (0.625 percent glutaraldehyde).' Because of our findings, we strongly recommend that the manufacturer quarantine bioprosthetic valves for 6 weeks until cultures (both aerobic and anaerobic), smears of the culture, and stained representative specimens of the aortic coupon are organism free. This is part of the current protocol practiced by at least one of the manufacturers." At the time of implantation, the aortic coupon should be cultured both before and after rinsing and the cultures should be kept for a minimum of 3 weeks. Apparent lack of growth should be confirmed by stained smear of the culture broth. Should positive cultures result, close patient observation is indicated, but antimicrobial therapy should be reserved for those patients with clinical signs of active infection.
Volume75 Number3 March, 1978
We wish to acknowledge the valuable work of Norma Jean Frank, who performed much of the biochemical and microbiological testing; Dr. Sidney Tolod, who performed the glutaraldehyde studies; and Patricia Henderson, who helped greatly in preparation of the manuscript. REFERENCES
2 3
4
5 6
7 8
9
10
II
12
13
14
15
16
Carpentier A, Deloche A, Reiland J, Fabiani IN, Forman J, Camilleri JP, Soyer R, Dubost C: Six-year follow-up of glutaraldehyde-preserved heterografts. J THORAC CARDIOVASC SURG 68:771, 1974 Zuhdi N: The porcine aortic valve bioprosthesis. A significant alternative. Ann Thorac Surg 21:573, 1976 Bowman FO Jr, Maim JR, Harris PD, et al: Further evaluation of aortic valve homografts sterilized by electron-beam energy. Circulation 49, 50:Suppl 1:57, 1969 Rittenhouse EA, Sands MP, Mohri H, et al: Sterilization of aortic valve grafts for transplantation. Arch Surg 101:1, 1970 Barratt-Boyes BG: Homograft aortic valve replacement in aortic incompetence and stenosis. Thorax 19: 131, 1964 Ross D: Homotransplantation of the aortic valve in the subcoronary position. J THORAC CARDIOVASC SURG 47:713, 1964 Zerbini EJ, de Oliviera SA, Pileggi F, et al: Early results of aortic valve homograft replacement. Chest 55:32, 1969 Angell WW, Stinson EB, IBen AB, Shumway NE: Multiple valve replacement with the fresh aortic homograft. J THORAC CARDIOVASC SURG 56:323, 1968 Wallace RB, Londe SP, Titus JL: Aortic valve replacement with preserved aortic valve homografts. J THORAC CARDIOVASC SURG 67:44, 1974 Ionescu MI, Pakrashi BC, Mary DAS, Bartek IT, Wooler GH: Long-term evaluation of tissue valves. J THORAC CARDIOVASC SURG 68:361, 1974 Buch WS, Kosek JC, Angell WW: Deterioration of formalin-treated aortic valve heterografts. J THORAC CARDIOVASC SURG 60:673, 1970 Pipkin RD, Buch WS, Fogarty TJ: Evaluation of aortic valve replacement with a porcine xenograft without longterm anticoagulation. J THORAC CARDIOVASC SURG 71:179, 1976 McIntosh CL, Michaelis LL, Morrow AG, Itscoitz SB, Redwood DR, Epstein SE: Atrioventricular valve replacement with the Hancock porcine xenograft. A fiveyear clinical experience. Surgery 78:768, 1975 Zuhdi N, Hawley W, Voehl V, et al: Porcine aortic valves as replacements for human heart valves. Ann Thorac Surg 17:479, 1974 Ionescu MI, Tandon AP, Mary DAS, Abid A: Heart valve replacement with the Ionescu-Shiley pericardial xenograft. J THORAC CARDIOVASC SURG 73:31, 1977 Magilligan DJ Jr, Quinn EL, Davilla JC: Bacteremia, endocarditis, and the Hancock valve. Presented at the Thirteenth Meeting of the Society of Thoracic Surgeons, San Francisco, Calif., Jan. 24-26, 1977
Atypical mycobacteria and xenograft valve
335
17 Center for Disease Control Morbidity and Mortality Weekly Report: Isolation of mycobacteria species from porcine heart valve prostheses. 26:42, 1977 18 Platt M: Personal communication, May, 1977 19 Middlebrook G: Isoniazid resistance and catalase activity of tubercle bacilli. A preliminary report. Am Rev Respir Dis 69:471-478, 1954 20 Thoen CO, Jarnigan JL, Richards WD: Isolation and identification of mycobacteria f rom porcine tissues. A three-year summary. Am J Vet Res 36:1383, 1975 21 Thoen CO: Personal communication, 1977 22 Gutman LT, Handweger S, Zwadyk P, et al: Thyroiditis due to Mycobacterium chelonei. Am Rev Respir Dis 110:807, 1974 23 Dreisin RB, Scoggin C, Davidson PT: The pathogenicity ofMycobacteriumJortuitum and Mycobacterium chelonei in man. A report of seven cases. Tubercle 57:49, 1976 24 Inman PM, Beck A, Brown AE, et al: Outbreak of infectious abscesses due to Mycobacterium abscessus. Arch Dematoll00:141, 1969 25 Geraci JE, Anderson MW, Karlson AG: Endocarditis due to a rapidly growing chromogenic mycobacterium. Mayo Clin Proc 43: 124, 1968 26 Altmann G, Horowitz A, Kaplinsky N, et al. Prosthetic valve endocarditis due to Mycobacterium chelonei. J Clin Microbioll:531, 1975 27 Repath F, Seabury JH, Sanders CV, et al: Prosthetic valve endocarditis due to Mycobacterium chelonei, South Med J 69: 1244, 1976 28 Jauregui L, Arbulu A, Wilson F: Osteomyelitis, pericarditis, mediastinitis, and vasculitis due to Mycobacterium chelonei. Am Rev Respir Dis 115:699, 1977 29 Collins FM, Montalbine V: Mycobactericidal activity of glutaraldehyde solutions. J Clin Microbio14:408, 1976 30 Angell WW, Angell JD, Sywak A: Selection of tissue or prosthetic valve. A five-year prospective, randomized comparison. J THORAC CARDIOVASC SURG 73:43, 1977 31 Written communication from Hancock Laboratories, Inc., February, 1977
Discussion DR. WILLIAM W. ANGELL San Jose, Calif.
I would like to thank Dr. Tyras for making his manuscript available to me well in advance of this meeting and to compliment him on his significant contribution emphasizing the importance of guaranteed sterility with the use of tissue valves. Unlike the heat-sterilized and ethylene oxide-sterilized prosthetic valves, tissues shipped in aqueous solution present significant technical problems. Many years ago, we had a significant similar experience with the use of antibioticsterilized homograft valves. Because of several cases of iatrogenic bacterial endocarditis related to the implantation of contaminated homograft valves, we have been highly sensitive to accurate and effective sterilization and culture tech-
The Journal of Thoracic and Cardiovascular Surgery
336 Tyras et al.
niques in order to guarantee sterility at the time of implantation. I should point out first that there are two issues related to Dr. Tyras' work. The first of these is the bactericidal efficacy of glutaraldehyde solutions, and the second, and perhaps most important, is our ability to analyze properly the sterility of valves at the time they leave the factory and to subsequently guarantee that sterility at the time of implantation into the patient. We are all familiar with the sterilizing capacity of glutaraldehyde, 2 percent in concentration, as the active agent produced under the brand name of Cidex, The action of glutaraldehyde on organisms present on prosthetic surfaces is similar to its effect as a tanning agent of mammalian collagen. This sterilizing effect is primarily related to the free aldehyde groups which react with the primary amino acid groups and subsequently reduce to form secondary amines. These are extremely stable bonds and serve to fix the organisms, rendering them nonviable. In general, glutaraldehyde is most rapidly effective against the bacterial vegative phases and less effective against those forms with resistant cell walls, such as the Bacillus subtilis spore and mycobacteria, which have a high lipid content in the cell wall. In addition to concentration, the efficacy of glutaraldehyde is related to the duration of exposure, pH, temperature of incubation, and, very importantly, to the character and composition of the glutaraldehyde solution. Unfortunately, the polymerization of glutaraldehyde serves to reduce the number of active aldehyde groups and thereby effectively reduces the concentration of the active sterilizing component. Thus it is essential that the composition of the glutaraldehyde solution be known and that significant polymerization not be present. Using the purified glutaraldehyde solutions made at Shiley Laboratories, we have seen no positive cultures with the use of 0.5 percent glutaraldehyde. These solutions have been checked weekly against Bacillus subtilis spores since January of 1976, so that more than 70 tests have been run against the standard solution with a zero incidence of positive cultures. We have seen positive cultures from clinical valves after processing at the factory. Re-exposure in each of these thirteen instances has led to negative cultures, and in no case are we aware of a positive culture at the time of valve implantation. This is supported by in vitro laboratory tests, which show the alkaline-buffered glutaraldehyde solution to reduce mycobacteria to zero by the extrapolation of this curve. Incidentally, during processing, these valves are exposed to a 5 percent glutaraldehyde solution which may materially assist in sterilization. The second issue, and perhaps the most vital, is the determination of proper sterility testing for aqueous solutions. Somewhat to my embarrassment, it was not until Mark O'Brien, who pioneered the work with formalin-treated heterografts, suggested the use of millipore filtration that we discovered it to be a standard USP test for aqueous pharmaceuticals. It is not clear to me why this same guideline is not applied to heart valves in aqueous soltuion. Millipore filtration or membrane filtration involves subjecting all of the
valve solution with its contained microscopic and macroscopic particles, including tissue coupons, to standard aerobic and anaerobic culture methods. The filtration has two benefits: First, it removes all bacteria both in solution and attached to or embedded in particles in the container and concentrates them on the filter. Second, it removes all traces of inhibitory aqueous-soluble bactericidal or bacteriostatic agents. Thus, the filter is left with as complete a sample as is obtainable from the valve. We went to the extent of rinsing the whole valve and culturing the whole valve, mincing adjacent portions of tissue along with the solution, in order to obtain every possible organism present. We found that the solutions were an excellent sample and that any viable organisms present would be contained in the filter after the solution had been passed through it. Using this method, we eliminated iatrogenic contamination with homograft valves, and we believe this to be the procedure of choice for quality control of sterility with the commercially produced tissue valves. I have three questions for Dr. Tyras. I first wish to ask whether millipore filtration methods were used either by the manufacturer in his clinical bacteriology of the valve solutions or in the in vitro tests that were used to determine the presence of viable organisms after exposure to the three concentrations of glutaraldehyde. My second question is this: Since the manufacturer's valves were exposed to a 0.2 percent solution in the absence of blood, and since this in vitro test system would show that this was an effective sterilizing method after 48 hours of exposure, can you explain how viable organisms could have been present after more than 3 weeks of exposure to the 0.2 percent solution. My third question relates to the technique of the in vitro tests conducted specifically. What were the temperatures of exposure, the concentration of microorganisms, the length of assay exposure time, and the concentrations of glutaraldehyde after exposure to both the tissue and blood, as they will markedly reduce effective concentration by tying up active aldehyde groups? The authors are certainly to be congratulated for their effort in identifying a problem which can be clinically devastating and for initiating some investigations related to adequate sterilization and testing methods essential to the proper use of glutaraldehyde-treated heterograft valves.
DR. TYRAS(Ciosing) I would like to thank Dr. Angell for his remarks and for further clarifying some of the problems that exist in assuring sterility of the bioprosthesis. In our in vitro tests with the differing concentrations of glutaraldehyde, the size of the inoculum was by the BauerKirby turbidity standard of 107 to 108 organisms per millilter; we used 0.1 ml., and thus our inoculum was in the order of 106 to 107 organisms. How can the organism still exist despite what appears to be adequate concentrations of glutaraldehyde? There are a couple of possibilities. One is that the activity of the glutaraldehyde may be very important. As Dr. Angell has demonstrated, the amount of polymer that exists may
Volume 75
Atypical mycobacteria and xenograft valve
Number 3
337
March,1978
appreciably affect the germicidal action of glutaraldehyde. We have limited knowledge of precisely what each manufacturer does to control the polymer content of the glutaraldehyde solution. Also, though the valve is not shipped in blood, blood cells and blood protein still reside within the aortic coupon and, in most commercially available bioprostheses, in the rim of myocardium that is along the right coronary cusp. Thus a protein coagulum can exist and, Dr. Laskowski assures me, can adequately protect microorganisms, specifically mycobacteria, from the germicidal action of glutaraldehyde. The glutaraldehyde used in the in vitro testing was commercially produced Cidex that was diluted fresh with sodium and potassium phosphate buffers to the desired concentration. The incubation was carried out at room temperature and, after subculturing, the cultures were placed at 37°. The polymer content of the glutaraldehyde was not tested, and Dr. Angell's suggestion that this be looked at in a more definitive fashion is well taken. [Slide] Dr. Tascon of Hancock Laboratories provided me with some culture data from their laboratory. In a sample of
5,800 cultures with 87 positive cultures for mycobacteria from their laboratory, they had a biphasic culture response. There were two strains. One grew rapidly and appeared an average of II days after culturing. The other grew somewhat more slowly and appeared an average of21.6 days after initial incubation. According to these data, if cultures are held for zero days, naturally the probability of missing a positive culture is I. If they are held for slightly more than 3 weeks, the probability of a positive culture escaping detection is in the neighborhood of 2 per 1,000. For a commercial manufacturer using just conventional culture techniques, 2 per 1,000 is an unacceptably high rate. This is why we would recommend something in the order of 6 weeks of quarantine unless special procedures, such as Dr. Angell has suggested, can be shown to be effective in order to shorten the needed quarantine interval. With 6 weeks of incubation, the probability of a positive culture escaping detection is in the order of I in ten million. Thus we feel a 6 week quarantine is a reasonable precaution that all manufacturers should take.
Information for authors Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: "The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journal, and has not been previously published. " Authors will be consulted, when possible, regarding republication of their material.
Denis H. Tyras, M.D., George C. Kaiser, M.D., Hendrick B. Barner, M.D., Leonard F. Laskowski, Ph.D., and J. Joseph Marr, M.D., St. Louis, Mo.
Recent reports of favorable six-year results with implanted porcine xenograft valvular prostheses have contributed to their widespread use.': 2 Whereas procurement of porcine tissue valves is less of a problem than was encountered with human aortic valve homografts, sterility of the implantable prosthesis is still a major consideration. The purpose of this paper is to report the isolation of a fastidious atypical mycobacterium from cultures taken at the time of porcine xenograft valve implantation in eight patients. Patients and methods
Between July and November, 1976, 24 Hancock* porcine xenograft valves were implanted in 20 patients at our institution. All valves were received from the manufacturer during the period from May through August, 1976. At the time of implantation, each valve was removed aseptically from its shipping jar containing cotton wads soaked with 0.2 percent stabilized From the Departments of Surgery, Pathology (Clinical Microbiology), and Medicine (Infectious Diseases), St. Louis University School of Medicine, St. Louis, Mo. Read at the Third Annual Meeting of The Samson Thoracic Surgical Society, Colorado Springs, Colorado, June 4-7, 1977. Address for reprints: Denis H. Tyras, M.D., Department of Surgery, St. Louis University School of Medicine, 1325 South Grand Boulevard, St. Louis, Mo. 63104. *Hancock Laboratories, Inc., Anaheim, Calif. 0022-5223/78/0375-0331$00.70/0 © 1978
The C. V. Mosby Co.
glutaraldehyde. After the cotton wads were removed from the valve cusps, each valve was rinsed in three separate stainless steel basins containing sterile 0.9 percent saline. At the conclusion of the rinse, the accompanying aortic wall coupon was removed, placed in a sterile culture tube, and transported immediately to the laboratory. The specimen was placed in a tube of liquid thioglycollate medium without indicator (BBL) and incubated at 37° C. Initial cultures were held for a minimum of 2 weeks with Gram stain and subculture to blood and MacConkey agar plates were made before a final negative report. After the first positive culture, all valve cultures were held for 12 weeks before being considered to be negative. Beginning in September, 1976, a portion of aortic wall coupon was cultured before as well as after rinsing. Patients with positive cultures have been followed closely and, when possible, have undergone follow-up physical examination, chest roentgenogram, complete blood counts, echocardiography, electrocardiography, and tuberculin skin testing (intermediate and secondstrength purified protein derivative [PPD]). Results
Prior to July, 1976, none of the porcine valve cultures handled in this manner produced any growth of organisms. Beginning with a culture taken July 29, 1976, after 2V2 weeks of incubation, several cultures 331
332
The Journal of
Tyras et al.
Thoracic and Cardiovascular Surgery
Table I. Selected biochemical and culture characteristics of mycobacterial species * Species Property
MacConkey agar Arylsulfatase (3 days) Growth on 5 per cent NaCI Tellurite red'n (3 day) Catalase (68° C.) Niacin Tween 80 hydro!. (5 days) Catalase (>45 mm. foam)
M. tuberculosis
I
M. chelonei, subsp. chelonei
I
M. chelonei, subsp. abscessus
+
+ + +
+
+
+
+
d
+
±
Organism isolated
+ + + d
d d
'Modified from Buchanan RE, Gibbons NE: Bergey's Manual of Determinative Bacteriology, ed. 8, Baltimore, 1974, The Williams & Wilkins Company. td = variable depending on strain.
demonstrated anaerobic growth in the thioglycollate medium. These organisms were acid fast on staining. Including the initial positive culture, eight samples of porcine valves from different lot numbers yielded acid-fast organisms. Positive cultures were not sequential. Subculture was possible on solid thioglycollate agar after 2 weeks' incubation at 37° C. in a 5 percent carbon dioxide atmosphere. Control cultures of saline normally used for valve rinsing, of culture tubes used in transport of specimens, and of thioglycollate broth from the same batch used for porcine valve culture but inoculated with cerebrospinal fluid during routine fungal culture failed to demonstrate growth of acid-fast organisms. Moreover, cultures of aortic coupons taken before as well as after rinsing demonstrated growth of acid-fast bacilli in two patients in whom this culturing sequence was performed. Microscopic examination of stained specimens of aortic coupons in the last five positive cultures demonstrated acid-fast organisms within the specimen, although litt le cellular reaction was seen. Biochemical testing (Table I) strongly suggests that this organism is a subspecies of Mycobacterium chelonei, a member of Runyon's group IV. After multiple subcultures, if blood was added to the liquid thioglycollate broth, the organism began to grow aerobically within 5 to 6 days. At this point, catalase activity, which had been low, became high. Incubation of an aliquot of the positive culture with a piece of human aorta in different concentrations of buffered glutaraldehyde (pH 7.4) showed uniform inhibition of growth in all concentrations incubated for at least 48 hours (Table 11). If 0.5 ml. of human blood was added to the culture, only concentrations of glutaraldehyde of 0.625 and 0.8 percent were able to inhibit growth after at least I week of incubation. Lesser con-
centrations showed no inhibition of growth even after 3 weeks' incubation. Glutaraldehyde concentrations of 1.0 percent or greater showed uniform inhibition with only 24 hours' incubation (Table II). Antibiotic testing, after multiple organism subcultures, showed the organism to be sensitive only to rifampin and resistant to all other antituberculous drugs. Patient follow-up (Table III) revealed only one instance in which this organism was clinically manifest. Five months after mitral valve replacement, this patient was readmitted with lower extremity thrombophlebitis, congestive heart failure, and cardiac silhouette enlargement on chest roentgenogram. Pulmonary perfusion scans were normal. Anticoagulation and diuresis cleared both the thrombophlebitis and pulmonary congestion, but echocardiography demonstrated a large pericardial effusion. Results of intermediate and second-strength PPD skin testing were negative. Blood cultures were negative. The diagnosis of postpericardiotomy syndrome was suspected and the patient was begun on a course of corticosteroids. Pericardiocentesis was not done because of the anticoagulation. After 3 weeks of therapy, the pericardial effusion had increased slightly and the patient underwent partial pericardiectomy. The pericardium contained 1,300 m!. of clear, yellow fluid. Sections of pericardium and spun specimens of pericardial fluid demonstrated acid-fast organisms. Subsequent cultures yielded findings compatible withM. chelonei, with the same growth characteristics that had been found in the initial porcine valve culture. The patient is being maintained on rifampin therapy at the present time and remains asymptomatic. Discussion
The goals of cardiac valve replacement have been to provide satisfactory hemodynamic characteristics,
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Atypical mycobacteria and xenograft valve
Number 3 March,1978
333
Table II. Effect of glutaraldehyde and blood on porcine valve acid-fast bacillus (AFB) isolate Control: 10 mi. thioglycollate 0.1 mi. subcultured to new thioglycollate after:
Glutaraldehyde concentrations 10 mi. (pH) 7.4 + 0.1 mi. AFB* + piece of human aorta 0.2% 0.5% 0.625% 0.8% 1.0% 1.5% 2.0% The above culture + 0.5 mi. human blood 0.2% 0.5% 0.625% 0.8% 1.0% 1.5% 2.0%
24 hr.
I
48 hr.
I
72 hr.
I
1 wk.
I
2 wk.
3 wk.
-t
+AFB +AFB
+AFB(s) +AFB(s) +AFB(s) +AFB
I
+AFB(s) +AFB(s) +AFB(s) +AFB
+AFB(s) +AFB(s) +AFB +AFB
+AFB(s) +AFB(s)
+AFB(s) +AFB(s)
Original + 0.1 10.1 mi. subcultured at 24 hr. mi. AFB *
+AFB +AFB +AFB +AFB +AFB +AFB +AFB
+AFB +AFB +AFB +AFB +AFB +AFB +AFB
+AFB(s) +AFB(s)
'Turbidity = Bauer Kirby BaSo, standard. tNo growth after four weeks. t(s) = Pellicle with surface growth.
long-term mechanical stability, and a low incidence of thromboembolic complications. Among the favorable attributes of tissue valves are the low incidence of thromboembolic complications, the frequent lack of need for anticoagulation, and the central flow dynamics. Early efforts were directed toward the use of preserved aortic valve homografts. Sterilization was attempted with many techniques, including electron-beam irradiation," antibiotic solutions," beta propiolactone," ethylene oxide," and 4 percent buffered formaldehyde solution (formalin)." Procurement of cadaveric aortic valve homografts was limited not only by availability, but also by the finding that the incidence of positive cultures increased markedly from 10 percent to 30 to 40 percent when grafts were harvested sterilely more than 8 to 12 hours after death." Even when sterility could be obtained, later foIlow-up revealed high rates of premature graft failure from valve cusp deterioration, calcification, and infection." 9 Similar results were encountered with early graft deterioration of homograft dura mater and fascia lata valves .10 Heterograft (porcine) valves were more readily obtained, but treatment with buffered formalin solution again appeared to lead to early graft deterioration." Pioneering work by Carpentier, 1 using buffered
glutaraldehyde to stabilize and sterilize porcine aortic xenografts, has shown excellent stability of the graft even up to 6 years. These findings have been supported by the reports of Pipkin," Mclntosh,!" and Zuhdi'" in this country. Similarly encouraging results have been described by Ionescu" with the use of glutaraldehyde-stabilized bovine pericardial xenografts. At the present time, three manufacturers provide porcine xenograft valves prepared with buffered glutaraldehyde solution in concentrations varying from 0.2 to 0.625 percent. Glutaraldehyde stabilization of the collagen matrix of the porcine bioprosthesis has apparently also been an effective antimicrobial treatment as weIl. Control cultures are taken by the manufacturer at the time of prosthesis construction, and valves are not released to users until a quarantine period has passed with negative cultures. Prior to our recent experience there have been no published reports of positive cultures at the time of valve implantation. In fact, results have been excellent in terms of resistance to prosthesis endocarditis in patients with active bacterial endocarditis receiving porcine bioprosthesis implants.!" Careful investigation of possible sources of contamination of cultures at our institution have supported the conclusion thatM. chelonei organisms were indigenous
The Journal of
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Tyras et al.
Thoracic and Cardiovascular
Surgery
Table III. Atypical mycobacteria in porcine valve culture: Patient data Follow-up status
Case No.
Date of implant
7/29/76 2
8/03/76
3
8/20/76
4 5 6 7 8
8/31/76 9/08/76 10/04/76 10/08/76 10/13/76
Purified protein derivative Valve implanted
31 mm. mitral-pos. culture 25 mm. aortic-neg. culture 31 mm. mitral 31 mm. mitral (+ double coronary bypass) 29 mm. aortic 27 mm. aortic 27 mm. aortic 33 mm. mitral 27 mm. aortic
Interm.
Clinical
I
Second strength
ECG/echo
CBC
Well
Neg.
Not done
Okay
Okay
Pericarditis (see text) Well
Neg.
Neg.
See text
Okay
Okay
Okay
Okay Okay Okay Okay
Okay Okay Okay Okay
Prev. TBC,1961
Well Neg. Not done Well Not done Well Neg. IOmm. 14mm. Well Neg. Died 1O/14/76-low output syndrome
Legend: ECG/echo, Electrocardiogram/echocardiogram. CBC, Complete blood count. TBC, Clinical tuberculosis.
to material received from the manufacturer. This contention is supported by the finding of similar organisms from porcine valve cultures in at least five other institutions.": 18 Initial isolation of this organism may be extremely difficult because of its fastidious growth characteristics. Characteristically, M. chelonei is a "rapid grower" of Runyon's group IV. The organism isolated and subcultured demonstrated low catalase activity, which may account for its slow growth, Many microorganisms produce hydrogen peroxide, which inhibits growthcatalase is effective in destroying hydrogen peroxide. It is interesting to note that sensitive strains of M. tuberculosis lose catalase activity after treatment with isoniazid.!" Also, blood contains catalase and was effective in restoring rapid growth characteristics to our cultures. Atypical mycobacterial infections are not uncommon in swine, but the overwhelming majority (97 percent) of isolates have been M, avium.i? A single case of M. chelonei causing an abscess in a pig has been identified." Clinically significant infections in man due to M. chelonei have been reported. These have included thyroiditis.P pneumonitis.P cutaneous abscesses." de novo endocarditis," prosthetic valve endocarditis.": 27 and osteomyelitis, pericarditis, and mediastinitis after coronary artery bypass." To date, only one of our patients has had any clinical manifestation of M. chelonei infection, namely pericarditis. This condition has responded well to pericardiectomy and rifampin therapy. The fact that this patient received therapeutic doses of corticosteroids in the treatment of the pericardial effusion may have in some way encouraged the growth of
these organisms. All other patients remain free of clinical evidence of infection; thus, we would recommend antituberculous therapy only if clinical infection is demonstrated. It is especially important to emphasize that none of the patients has shown any evidence of endocarditis, and follow-up echocardiograms continue to show satisfactory valve function. Although 2 percent glutaraldehyde solution (pH 8.5) is mycobactericidal, evidence exists that more acidic or less concentrated solutions of glutaraldehyde are less effective;" Our studies with varying concentrations of glutaraldehyde tend to substantiate this. What must be balanced is the antimicrobial efficacy of differing concentrations of buffered glutaraldehyde with the undesirable "tanning" effects of higher concentrations. Excellent results of valve durability have been reported with the Hancock valve (0.2 percent glutaraldehyde),13, 14 the Angell-Shiley valve (0.5 percent glutaraldehydej.P" and the Carpentier-Edwards valve (0.625 percent glutaraldehyde).' Because of our findings, we strongly recommend that the manufacturer quarantine bioprosthetic valves for 6 weeks until cultures (both aerobic and anaerobic), smears of the culture, and stained representative specimens of the aortic coupon are organism free. This is part of the current protocol practiced by at least one of the manufacturers." At the time of implantation, the aortic coupon should be cultured both before and after rinsing and the cultures should be kept for a minimum of 3 weeks. Apparent lack of growth should be confirmed by stained smear of the culture broth. Should positive cultures result, close patient observation is indicated, but antimicrobial therapy should be reserved for those patients with clinical signs of active infection.
Volume75 Number3 March, 1978
We wish to acknowledge the valuable work of Norma Jean Frank, who performed much of the biochemical and microbiological testing; Dr. Sidney Tolod, who performed the glutaraldehyde studies; and Patricia Henderson, who helped greatly in preparation of the manuscript. REFERENCES
2 3
4
5 6
7 8
9
10
II
12
13
14
15
16
Carpentier A, Deloche A, Reiland J, Fabiani IN, Forman J, Camilleri JP, Soyer R, Dubost C: Six-year follow-up of glutaraldehyde-preserved heterografts. J THORAC CARDIOVASC SURG 68:771, 1974 Zuhdi N: The porcine aortic valve bioprosthesis. A significant alternative. Ann Thorac Surg 21:573, 1976 Bowman FO Jr, Maim JR, Harris PD, et al: Further evaluation of aortic valve homografts sterilized by electron-beam energy. Circulation 49, 50:Suppl 1:57, 1969 Rittenhouse EA, Sands MP, Mohri H, et al: Sterilization of aortic valve grafts for transplantation. Arch Surg 101:1, 1970 Barratt-Boyes BG: Homograft aortic valve replacement in aortic incompetence and stenosis. Thorax 19: 131, 1964 Ross D: Homotransplantation of the aortic valve in the subcoronary position. J THORAC CARDIOVASC SURG 47:713, 1964 Zerbini EJ, de Oliviera SA, Pileggi F, et al: Early results of aortic valve homograft replacement. Chest 55:32, 1969 Angell WW, Stinson EB, IBen AB, Shumway NE: Multiple valve replacement with the fresh aortic homograft. J THORAC CARDIOVASC SURG 56:323, 1968 Wallace RB, Londe SP, Titus JL: Aortic valve replacement with preserved aortic valve homografts. J THORAC CARDIOVASC SURG 67:44, 1974 Ionescu MI, Pakrashi BC, Mary DAS, Bartek IT, Wooler GH: Long-term evaluation of tissue valves. J THORAC CARDIOVASC SURG 68:361, 1974 Buch WS, Kosek JC, Angell WW: Deterioration of formalin-treated aortic valve heterografts. J THORAC CARDIOVASC SURG 60:673, 1970 Pipkin RD, Buch WS, Fogarty TJ: Evaluation of aortic valve replacement with a porcine xenograft without longterm anticoagulation. J THORAC CARDIOVASC SURG 71:179, 1976 McIntosh CL, Michaelis LL, Morrow AG, Itscoitz SB, Redwood DR, Epstein SE: Atrioventricular valve replacement with the Hancock porcine xenograft. A fiveyear clinical experience. Surgery 78:768, 1975 Zuhdi N, Hawley W, Voehl V, et al: Porcine aortic valves as replacements for human heart valves. Ann Thorac Surg 17:479, 1974 Ionescu MI, Tandon AP, Mary DAS, Abid A: Heart valve replacement with the Ionescu-Shiley pericardial xenograft. J THORAC CARDIOVASC SURG 73:31, 1977 Magilligan DJ Jr, Quinn EL, Davilla JC: Bacteremia, endocarditis, and the Hancock valve. Presented at the Thirteenth Meeting of the Society of Thoracic Surgeons, San Francisco, Calif., Jan. 24-26, 1977
Atypical mycobacteria and xenograft valve
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17 Center for Disease Control Morbidity and Mortality Weekly Report: Isolation of mycobacteria species from porcine heart valve prostheses. 26:42, 1977 18 Platt M: Personal communication, May, 1977 19 Middlebrook G: Isoniazid resistance and catalase activity of tubercle bacilli. A preliminary report. Am Rev Respir Dis 69:471-478, 1954 20 Thoen CO, Jarnigan JL, Richards WD: Isolation and identification of mycobacteria f rom porcine tissues. A three-year summary. Am J Vet Res 36:1383, 1975 21 Thoen CO: Personal communication, 1977 22 Gutman LT, Handweger S, Zwadyk P, et al: Thyroiditis due to Mycobacterium chelonei. Am Rev Respir Dis 110:807, 1974 23 Dreisin RB, Scoggin C, Davidson PT: The pathogenicity ofMycobacteriumJortuitum and Mycobacterium chelonei in man. A report of seven cases. Tubercle 57:49, 1976 24 Inman PM, Beck A, Brown AE, et al: Outbreak of infectious abscesses due to Mycobacterium abscessus. Arch Dematoll00:141, 1969 25 Geraci JE, Anderson MW, Karlson AG: Endocarditis due to a rapidly growing chromogenic mycobacterium. Mayo Clin Proc 43: 124, 1968 26 Altmann G, Horowitz A, Kaplinsky N, et al. Prosthetic valve endocarditis due to Mycobacterium chelonei. J Clin Microbioll:531, 1975 27 Repath F, Seabury JH, Sanders CV, et al: Prosthetic valve endocarditis due to Mycobacterium chelonei, South Med J 69: 1244, 1976 28 Jauregui L, Arbulu A, Wilson F: Osteomyelitis, pericarditis, mediastinitis, and vasculitis due to Mycobacterium chelonei. Am Rev Respir Dis 115:699, 1977 29 Collins FM, Montalbine V: Mycobactericidal activity of glutaraldehyde solutions. J Clin Microbio14:408, 1976 30 Angell WW, Angell JD, Sywak A: Selection of tissue or prosthetic valve. A five-year prospective, randomized comparison. J THORAC CARDIOVASC SURG 73:43, 1977 31 Written communication from Hancock Laboratories, Inc., February, 1977
Discussion DR. WILLIAM W. ANGELL San Jose, Calif.
I would like to thank Dr. Tyras for making his manuscript available to me well in advance of this meeting and to compliment him on his significant contribution emphasizing the importance of guaranteed sterility with the use of tissue valves. Unlike the heat-sterilized and ethylene oxide-sterilized prosthetic valves, tissues shipped in aqueous solution present significant technical problems. Many years ago, we had a significant similar experience with the use of antibioticsterilized homograft valves. Because of several cases of iatrogenic bacterial endocarditis related to the implantation of contaminated homograft valves, we have been highly sensitive to accurate and effective sterilization and culture tech-
The Journal of Thoracic and Cardiovascular Surgery
336 Tyras et al.
niques in order to guarantee sterility at the time of implantation. I should point out first that there are two issues related to Dr. Tyras' work. The first of these is the bactericidal efficacy of glutaraldehyde solutions, and the second, and perhaps most important, is our ability to analyze properly the sterility of valves at the time they leave the factory and to subsequently guarantee that sterility at the time of implantation into the patient. We are all familiar with the sterilizing capacity of glutaraldehyde, 2 percent in concentration, as the active agent produced under the brand name of Cidex, The action of glutaraldehyde on organisms present on prosthetic surfaces is similar to its effect as a tanning agent of mammalian collagen. This sterilizing effect is primarily related to the free aldehyde groups which react with the primary amino acid groups and subsequently reduce to form secondary amines. These are extremely stable bonds and serve to fix the organisms, rendering them nonviable. In general, glutaraldehyde is most rapidly effective against the bacterial vegative phases and less effective against those forms with resistant cell walls, such as the Bacillus subtilis spore and mycobacteria, which have a high lipid content in the cell wall. In addition to concentration, the efficacy of glutaraldehyde is related to the duration of exposure, pH, temperature of incubation, and, very importantly, to the character and composition of the glutaraldehyde solution. Unfortunately, the polymerization of glutaraldehyde serves to reduce the number of active aldehyde groups and thereby effectively reduces the concentration of the active sterilizing component. Thus it is essential that the composition of the glutaraldehyde solution be known and that significant polymerization not be present. Using the purified glutaraldehyde solutions made at Shiley Laboratories, we have seen no positive cultures with the use of 0.5 percent glutaraldehyde. These solutions have been checked weekly against Bacillus subtilis spores since January of 1976, so that more than 70 tests have been run against the standard solution with a zero incidence of positive cultures. We have seen positive cultures from clinical valves after processing at the factory. Re-exposure in each of these thirteen instances has led to negative cultures, and in no case are we aware of a positive culture at the time of valve implantation. This is supported by in vitro laboratory tests, which show the alkaline-buffered glutaraldehyde solution to reduce mycobacteria to zero by the extrapolation of this curve. Incidentally, during processing, these valves are exposed to a 5 percent glutaraldehyde solution which may materially assist in sterilization. The second issue, and perhaps the most vital, is the determination of proper sterility testing for aqueous solutions. Somewhat to my embarrassment, it was not until Mark O'Brien, who pioneered the work with formalin-treated heterografts, suggested the use of millipore filtration that we discovered it to be a standard USP test for aqueous pharmaceuticals. It is not clear to me why this same guideline is not applied to heart valves in aqueous soltuion. Millipore filtration or membrane filtration involves subjecting all of the
valve solution with its contained microscopic and macroscopic particles, including tissue coupons, to standard aerobic and anaerobic culture methods. The filtration has two benefits: First, it removes all bacteria both in solution and attached to or embedded in particles in the container and concentrates them on the filter. Second, it removes all traces of inhibitory aqueous-soluble bactericidal or bacteriostatic agents. Thus, the filter is left with as complete a sample as is obtainable from the valve. We went to the extent of rinsing the whole valve and culturing the whole valve, mincing adjacent portions of tissue along with the solution, in order to obtain every possible organism present. We found that the solutions were an excellent sample and that any viable organisms present would be contained in the filter after the solution had been passed through it. Using this method, we eliminated iatrogenic contamination with homograft valves, and we believe this to be the procedure of choice for quality control of sterility with the commercially produced tissue valves. I have three questions for Dr. Tyras. I first wish to ask whether millipore filtration methods were used either by the manufacturer in his clinical bacteriology of the valve solutions or in the in vitro tests that were used to determine the presence of viable organisms after exposure to the three concentrations of glutaraldehyde. My second question is this: Since the manufacturer's valves were exposed to a 0.2 percent solution in the absence of blood, and since this in vitro test system would show that this was an effective sterilizing method after 48 hours of exposure, can you explain how viable organisms could have been present after more than 3 weeks of exposure to the 0.2 percent solution. My third question relates to the technique of the in vitro tests conducted specifically. What were the temperatures of exposure, the concentration of microorganisms, the length of assay exposure time, and the concentrations of glutaraldehyde after exposure to both the tissue and blood, as they will markedly reduce effective concentration by tying up active aldehyde groups? The authors are certainly to be congratulated for their effort in identifying a problem which can be clinically devastating and for initiating some investigations related to adequate sterilization and testing methods essential to the proper use of glutaraldehyde-treated heterograft valves.
DR. TYRAS(Ciosing) I would like to thank Dr. Angell for his remarks and for further clarifying some of the problems that exist in assuring sterility of the bioprosthesis. In our in vitro tests with the differing concentrations of glutaraldehyde, the size of the inoculum was by the BauerKirby turbidity standard of 107 to 108 organisms per millilter; we used 0.1 ml., and thus our inoculum was in the order of 106 to 107 organisms. How can the organism still exist despite what appears to be adequate concentrations of glutaraldehyde? There are a couple of possibilities. One is that the activity of the glutaraldehyde may be very important. As Dr. Angell has demonstrated, the amount of polymer that exists may
Volume 75
Atypical mycobacteria and xenograft valve
Number 3
337
March,1978
appreciably affect the germicidal action of glutaraldehyde. We have limited knowledge of precisely what each manufacturer does to control the polymer content of the glutaraldehyde solution. Also, though the valve is not shipped in blood, blood cells and blood protein still reside within the aortic coupon and, in most commercially available bioprostheses, in the rim of myocardium that is along the right coronary cusp. Thus a protein coagulum can exist and, Dr. Laskowski assures me, can adequately protect microorganisms, specifically mycobacteria, from the germicidal action of glutaraldehyde. The glutaraldehyde used in the in vitro testing was commercially produced Cidex that was diluted fresh with sodium and potassium phosphate buffers to the desired concentration. The incubation was carried out at room temperature and, after subculturing, the cultures were placed at 37°. The polymer content of the glutaraldehyde was not tested, and Dr. Angell's suggestion that this be looked at in a more definitive fashion is well taken. [Slide] Dr. Tascon of Hancock Laboratories provided me with some culture data from their laboratory. In a sample of
5,800 cultures with 87 positive cultures for mycobacteria from their laboratory, they had a biphasic culture response. There were two strains. One grew rapidly and appeared an average of II days after culturing. The other grew somewhat more slowly and appeared an average of21.6 days after initial incubation. According to these data, if cultures are held for zero days, naturally the probability of missing a positive culture is I. If they are held for slightly more than 3 weeks, the probability of a positive culture escaping detection is in the neighborhood of 2 per 1,000. For a commercial manufacturer using just conventional culture techniques, 2 per 1,000 is an unacceptably high rate. This is why we would recommend something in the order of 6 weeks of quarantine unless special procedures, such as Dr. Angell has suggested, can be shown to be effective in order to shorten the needed quarantine interval. With 6 weeks of incubation, the probability of a positive culture escaping detection is in the order of I in ten million. Thus we feel a 6 week quarantine is a reasonable precaution that all manufacturers should take.
Information for authors Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: "The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journal, and has not been previously published. " Authors will be consulted, when possible, regarding republication of their material.