JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1991,
Vol. 29, No. 9
p. 1801-1803
0095-1137/91/091801-03$02.00/0 Copyright © 1991, American Society for Microbiology
Effect of Agitation of BACTEC 13A Blood Cultures Mycobacterium avium Complex
on
Recovery of
KATHY JACKSON,* AINA SIEVERS, AND BRIAN DWYER Mycobacterium Reference Laboratory, Fairfield Infectious Diseases Hospital, Fairfield, Victoria, Australia 3078 Received 26 March 1991/Accepted 29 May 1991
The effect of agitation of BACTEC 13A bottles (Becton Dickinson) on the recovery of Mycobacterium avium complex (MAC) from blood was compared with that of static incubation. A total of 265 blood specimens was inoculated in duplicate into BACTEC 13A bottles. One specimen was statically incubated at 35°C, and the other was incubated with agitation on a Gyrotory shaker at 35°C for the first 2 weeks and thereafter without shaking for up to 12 weeks. Of the 265 specimens, 77 (29.1 %) were positive in either one or both of the paired bottles. The average detection times for the shaken and nonshaken bottles were 12.7 and 15.9 days, respectively. A total of 10.4% of the specimens in the shaken bottles became positive 1 week before those in the nonshaken bottles, and 16.9% of the shaken cultures were positive more than 2 weeks before their counterparts. A further 46.8% of the agitated specimens became positive while the corresponding nonagitated cultures remained negative. When both specimens became positive at the same time, 88% of the shaken cultures had higher growth indices than their nonshaken counterparts. A further 11 paired blood cultures were taken from patients known to be infected with MAC to assess the effect of agitation of bottles on the utility of making twice-weekly readings during the first 2 weeks of incubation. Ten of the 11 sets of specimens in the shaken bottles were positive 1 or more weeks before those in the corresponding nonshaken bottles. In the remaining set, both specimens became positive on the same day; however, the growth index of the agitated culture was higher. The results indicate that agitation of BACTEC 13A blood cultures enhances the growth of MAC in blood cultures and may lead to shorter detection times.
with BACTEC 12B vials in which agitation was shown to inhibit mycobacterial growth. We decided to compare the effects of agitation of 13A blood cultures with those of static incubation on the recovery of mycobacteria from blood.
Disseminated mycobacterial infections have become increasingly common, particularly in patients with AIDS. Mycobacterium avium complex (MAC) is the most common cause of mycobacteremia in AIDS patients (4-6, 14), although other Mycobacterium species have been implicated (3, 9-11). Methods for the recovery of mycobacteria from blood have been time-consuming and have involved intensive laboratory manipulation of the specimens, which may be hazardous to laboratory personnel. The usual method has involved lysis of anticoagulated blood with sterile distilled water, saponin, or some other lysing agent, followed by concentration by centrifugation and then inoculation onto appropriate media. The Du Pont Isolator lysis-centrifugation system uses this principle and can be used for isolating and quantitating mycobacteria in blood. However, the Isolator system has the inherent disadvantage of requiring many laboratory manipulations (1, 12, 13), and so contamination may also be a problem (13). The BACTEC 13A blood culture system was developed specifically for the isolation of mycobacteria from blood (1, 8, 11-13). A 5-ml volume of blood is inoculated directly into the medium, and 0.5 ml of an enrichment fluid is then aseptically added. By incorporation of 14C-labelled palmitic acid into the medium, mycobacterial growth can be detected radiometrically by measuring the amount of 14C-labelled carbon dioxide produced after metabolism of the substrate. Introduction of the BACTEC 13A system into our laboratory has greatly reduced the work load and shortened the times of detection of positive blood cultures. The manufacturer's instructions suggest that the inoculated bottles be incubated without shaking. This is based on their unpublished studies *
MATERIALS AND METHODS Blood specimens. In this study, 265 specimens were collected in pairs from patients of our hospital between 23 April and 31 July 1990. A total of 258 specimens were taken from 111 patients infected with the human immunodeficiency virus (HIV), and 7 were taken from non-HIV-infected individuals. A further 11 paired blood cultures were taken from seven HIV-infected patients with MAC-positive blood cultures between 8 October and 12 November 1990. Up to 10 ml of blood was collected from each patient. Half of this volume was then inoculated into each of the paired bottles. Procedure. Upon receipt in the laboratory, the bottles were randomly assigned consecutive laboratory numbers. Enrichment supplement (0.5 ml) containing 15% bovine serum albumin (Becton Dickinson, Sydney, Australia) was aseptically added to each bottle. The bottles with the even laboratory numbers were then statically incubated at 35°C. The odd-numbered bottles were placed on a Gyrotory shaker (New Brunswick Company, Edison, N.J.) and were rotated at 125 rpm in a 35°C incubator for the first 2 weeks and thereafter were incubated without shaking. The agitation was slow enough to prevent frothing and therefore to reduce the occurrence of cross-contamination during reading. The first 265 pairs of blood cultures were read weekly by the BACTEC 460 TB instrument (Becton Dickinson) for 6 weeks and then once more at 12 weeks. The 11 paired specimens taken after October were read twice weekly for the first 2 weeks and weekly thereafter. The BACTEC needles were changed twice weekly, and the needle heater
Corresponding author. 1801
1802
J. CLIN. MICROBIOL.
JACKSON ET AL.
TABLE 1. AFB detection times for 40 paired positive specimens No. of positive cultures Wk no.
1 2 3 4 5 6 12
Shaken
Nonshaken
14 22 1 2 0 1 0
12 14 2 2 3 2 5
was replaced every 2 months to reduce the possibility of cross-contamination. Specimens showing a growth index (GI) reading of 20 or greater were subcultured onto egg yolk agar and Lowenstein-Jensen medium containing pyruvate, and a smear was prepared for Ziehl-Neelsen staining. The specimens were incubated at 35°C in air for up to 6 weeks. A specimen was considered positive when acid-fast bacilli (AFB) were detected on subculture. If only one of the paired specimens became positive and the other remained negative after 12 weeks of incubation, then 0.2 ml of the negative culture was subcultured onto solid medium. While the results of the subculture were pending, the inoculated blood culture bottles were stored at room temperature in the dark. For the cultures which failed to yield AFB on blind subculture, a 20-ml aliquot of the original culture was removed and centrifuged at 2,500 x g for 20 min and then the deposit was inoculated into a fresh 13A bottle, which was agitated. The new cultures were then read twice weekly by the BACTEC 460 TB instrument for 6 weeks or until they became positive, whichever occurred first. Mycobacterial isolates were identified by standard tests (7).
RESULTS
Of the 265 specimens processed between 23 April and 31 July 1990, 77 (29.1%) yielded MAC from either one or both of the paired 13A bottles. All isolates were from 27 patients infected with HIV. Of the paired specimens, 36 (46.8% of the total number of positive specimens) were positive in the shaken bottles only, 1 (1.3%) was positive only in the bottles which were not shaken, and 40 (51.9%) were positive in both bottles. Of the nonshaken cultures, 36 whose shaken counterparts yielded AFB remained negative after 12 weeks of incubation; 13 of the 36 subsequently yielded AFB after subculture to solid media even though the GIs had been below the cutoff value of 20. A further 12 cultures became positive after subculture to fresh 13A bottles and agitation. Table 1 summarizes the times required to detect AFB in the shaken and nonshaken bottles when both cultures were positive. The majority of the specimens became positive within the first 2 weeks. The average detection times for positive cultures in the shaken and nonshaken bottles were 12.7 and 15.9 days, respectively (excluding the cultures which became positive after 12 weeks of incubation). Of the 77 positive specimens, 8 (10.4% of the total number of positive specimens) in the shaken bottles were detected at least 1 week earlier and 13 (16.9%) were detected 2 or more weeks earlier than the nonshaken counterparts. A total of 57 shaken cultures (including the 36 whose statically incubated counterparts remained negative after 12 weeks) showed positive results before the corresponding nonshaken cul-
TABLE 2. AFB detection times for 11 paired specimens taken from MAC-positive patients after October 1990 No. of positive cultures
Wk no.
(reading)a
Shaken
Nonshaken
1 (first) 1 (second) 2 (first 2 (second) 3 4 5 6 12
0 1 2 4 2 2 0 0 0
0 1 0 0 1 0 0 4 5
a Two readings were taken in each of the first two weeks.
tures. Both bottles became positive simultaneously in 17 instances, although 15 (19.5%) of the agitated cultures had higher GI values than their statically incubated counterparts when detected together. One agitated culture had a lower GI value than its statically incubated counterpart, and one pair had comparable GI readings when detected on the same day. Only two agitated cultures became positive after their nonshaken counterparts. Another nonshaken specimen had a low-positive GI (43) after 12 weeks of incubation and grew MAC, whereas its shaken counterpart was negative. Table 2 summarizes the times of detection of AFB in the 11 paired specimens taken after October 1990, which were read twice weekly by the BACTEC instrument in the first 2 weeks of incubation. All the shaken cultures except one became positive before their statically incubated counterparts. For this pair, positive results were detected on the same day; however, the GI of the agitated culture (447) was greater than that of its statically incubated counterpart (134). Two specimens from one AIDS patient incidentally yielded Cryptococcus neoformans, in the agitated bottles only. No other mycobacterial species were isolated during the study period.
DISCUSSION
The results of this study indicate that agitation of the BACTEC 13A bottles enhances the growth of MAC in blood cultures. A total of 74% of the shaken cultures became positive a week or more before their statically incubated counterparts. Further, 19.5% of the agitated cultures gave higher GIs than the corresponding nonshaken cultures even when growth was detected simultaneously in both bottles. The data suggest that perhaps 46.8% of the blood cultures would have been classified as negative if they had not been shaken. This figure may not have been so high if the bottles had been read twice weekly as the manufacturer suggests. This was not carried out during the study due to an increased work load and a shortage of laboratory staff. However, in October and November 1990, a further 11 paired 13A blood specimens were collected from patients known to be MAC positive to assess the effects of agitation and twice-weekly readings during the first 2 weeks. As Table 2 shows, there is still a marked improvement in the times of detection of AFB in the shaken bottles compared with the nonshaken bottles. Twice-weekly reading may give the bottles a fresh supply of oxygen and carbon dioxide more frequently. However, agitation of the bottles may improve the distribution of the nutrients and gas within the medium itself. Only three statically incubated cultures became positive
VOL. 29, 1991
EFFECT OF AGITATION ON RECOVERY OF MAC BY BACTEC 13A
before their shaken counterparts. One of these became positive after 12 weeks of incubation and presumably had only small numbers of AFB present initially. Hence, random distribution of a very small inoculum into the pair of bottles could explain the lack of growth in the shaken bottle. The other two were taken from two different patients. Both of these patients had other positive specimens for which growth was enhanced by shaking. Twenty-five nonshaken cultures whose counterparts were positive were shown to contain AFB even though they consistently gave negative GIs. Some of these cultures showed a slight increase in GI value not exceeding 18, and then a decrease. The GI values of others were initially less than 12 and then steadily declined. The majority of the blood cultures became positive within the first 2 weeks of incubation (Table 1). The average detection time for the shaken bottles was slightly less (12.7 days) than those obtained by Agy et al., Kiehn and Cammarata, Strand et al., and Witebsky et al., who obtained average detection times of 14.2, 14, 23.6, and 14.8 days, respectively, with the BACTEC 13A system (1, 8, 12, 13). The shorter detection times obtained in this study may be due to the enhancement of growth through agitation; however, it is difficult to make a conclusive comparison, as the patient populations may differ a great deal between the studies. Only one other investigator reported a shorter mean detection time (11.5 days), and this was with statically incubated cultures (11). It is not surprising that agitation of the blood cultures enhances growth of MAC, as mycobacteria are aerophilic. Becton Dickinson investigated shaking of the BACTEC 12B vials and found it to be detrimental to mycobacterial growth (2). However, that study investigated only one strain each of four mycobacterial species and the effect of agitation of BACTEC 12B medium only. BACTEC 12B vials contain only 4 ml of medium, whereas the BACTEC 13A bottles contain 30 ml, i.e., a larger volume for gaseous dispersion, and they have the potential capability of providing a relative lack of oxygen in the depths of an undisturbed bottle. This study can supply evidence of the enhancement of the growth of only MAC, as no other mycobacterial species were isolated. However, MAC is the most common organism implicated in mycobacteremia in AIDS patients (4-6, 14). To determine the possible effect of growth of Mycobacterium tuberculosis in 13A medium, we experimentally inoculated two sets of bottles with two different volumes of a turbid suspension of M. tuberculosis and agitated one bottle of each set. All cultures became positive within 4 days; however, the GIs of both of the shaken cultures were higher. This suggests that shaking may enhance the growth of M. tuberculosis in a blood culture. Since the completion of the study, M. tuberculosis from an HIV-infected patient has been isolated in an agitated BACTEC 13A culture. It appears that agitation of the medium also improves the recovery of C. neoformans from blood. Cryptococcemia is a common opportunistic infection in AIDS patients, and isolation from blood is important in the diagnosis. In summary, the results of this study suggest that agitation of BACTEC 13A bottles for the first 2 weeks of incubation may enhance the growth of MAC and lead to shorter detection times. In addition, lack of agitation could cause false-negative results, particularly if cultures are read only once a week. It is therefore recommended that routine
1803
clinical laboratories agitate BACTEC 13A blood cultures for at least the first 2 weeks of incubation for improved recovery of MAC. ACKNOWLEDGMENTS We gratefully acknowledge Maria Globan, Sandra Aiuto, and Irenea Zarubova for laboratory assistance.
1.
2.
3.
4.
REFERENCES Agy, M. B., C. K. Wallis, J. J. Plorde, L. C. Carlson, and M. B. Coyle. 1989. Evaluation of four mycobacterial blood culture media: BACTEC 13A, Isolator/BACTEC 12B, Isolator/Middlebrook agar, and a biphasic medium. Diagn. Microbiol. Infect. Dis. 12:303-308. Anonymous. 1981. Shaking versus non-shaking experiment. Unpublished data on file at Becton Dickinson, Sydney, Australia. Chaisson, R. E., G. F. Schecter, C. P. Theuer, G. W. Rutherford, D. F. Echenberg, and P. C. Hopewell. 1987. Tuberculosis in patients with the acquired immunodeficiency syndrome. Clinical features, response to therapy, and survival. Am. Rev. Respir. Dis. 136:570-574. Hawkins, C. C., J. W. M. Gold, E. Whimbey, T. E. Kiehn, P. Brannon, R. Cammarata, A. E. Brown, and D. Armstrong. 1986. Mycobacterium avium complex infections in patients with the acquired immunodeficiency syndrome. Ann. Intern. Med. 105:
184-188. 5. Horsburgh, C. R., and R. M. Selik. 1989. The epidemiology of disseminated non-tuberculous mycobacterial infection in the acquired immunodeficiency syndrome (AIDS). Am. Rev. Respir. Dis. 139:4-7. 6. Hoy, J., A. Mijch, M. Sandland, L. Grayson, R. Lucas, and B. Dwyer. 1990. Quadruple therapy for Mycobacterium aviumintracellulare bacteremia in AIDS patients. J. Infect. Dis. 161:801-805. 7. Kent, P. T., and G. P. Kubica. 1985. Public health mycobacteriology. A guide for the level 3 laboratory. Centers for Disease Control, Atlanta. 8. Kiehn, T. E., and R. Cammarata. 1988. Comparative recoveries of Mycobacterium avium-M. intracellulare from Isolator lysiscentrifugation and BACTEC 13A blood culture systems. J. Clin. Microbiol. 26:760-761. 9. Levy-Frebault, V., B. Pangon, A. Burk, C. Katlama, C. Marche, and H. L. David. 1987. Mycobacterium simiae and Mycobacterium avium-M. intracellulare mixed infection in acquired immune deficiency syndrome. J. Clin. Microbiol. 25:154-157. 10. Rogers, P. L., R. E. Walker, H. C. Lane, F. G. Witebsky, J. A. Kovacs, J. E. Parillo, and H. Masur. 1988. Disseminated Mycobacterium haemophilum infection in two patients with the acquired immunodeficiency syndrome. Am. J. Med. 84:640642. 11. Salfinger, M., E. W. Stool, D. Piot, and L. Heifets. 1988. Comparison of three methods for the recovery of Mycobacterium avium complex from blood specimens. J. Clin. Microbiol. 26:1225-1226. 12. Strand, C. L., C. Epstein, S. Verzosa, E. Effatt, P. Hormozi, and S. H. Siddiqi. 1989. Evaluation of a new blood culture medium for mycobacteria. Am. J. Clin. Pathol. 91:316-318. 13. Witebsky, F. G., J. F. Keiser, P. S. Conville, R. Bryan, C. H. Park, R. Walker, and S. H. Siddiqi. 1988. Comparison of BACTEC 13A medium and Du Pont Isolator for detection of mycobacteremia. J. Clin. Microbiol. 26:1501-1505. 14. Wong, B., F. F. Edwards, T. E. Kiehn, E. Whimbrey, H. Donnelly, E. M. Bernard, J. W. M. Gold, and D. Armstrong. 1985. Continuous high-grade Mycobacterium avium-intracellulare bacteremia in patients with the acquired immune deficiency syndrome. Am. J. Med. 78:35-40.
Vol. 29, No. 9
p. 1801-1803
0095-1137/91/091801-03$02.00/0 Copyright © 1991, American Society for Microbiology
Effect of Agitation of BACTEC 13A Blood Cultures Mycobacterium avium Complex
on
Recovery of
KATHY JACKSON,* AINA SIEVERS, AND BRIAN DWYER Mycobacterium Reference Laboratory, Fairfield Infectious Diseases Hospital, Fairfield, Victoria, Australia 3078 Received 26 March 1991/Accepted 29 May 1991
The effect of agitation of BACTEC 13A bottles (Becton Dickinson) on the recovery of Mycobacterium avium complex (MAC) from blood was compared with that of static incubation. A total of 265 blood specimens was inoculated in duplicate into BACTEC 13A bottles. One specimen was statically incubated at 35°C, and the other was incubated with agitation on a Gyrotory shaker at 35°C for the first 2 weeks and thereafter without shaking for up to 12 weeks. Of the 265 specimens, 77 (29.1 %) were positive in either one or both of the paired bottles. The average detection times for the shaken and nonshaken bottles were 12.7 and 15.9 days, respectively. A total of 10.4% of the specimens in the shaken bottles became positive 1 week before those in the nonshaken bottles, and 16.9% of the shaken cultures were positive more than 2 weeks before their counterparts. A further 46.8% of the agitated specimens became positive while the corresponding nonagitated cultures remained negative. When both specimens became positive at the same time, 88% of the shaken cultures had higher growth indices than their nonshaken counterparts. A further 11 paired blood cultures were taken from patients known to be infected with MAC to assess the effect of agitation of bottles on the utility of making twice-weekly readings during the first 2 weeks of incubation. Ten of the 11 sets of specimens in the shaken bottles were positive 1 or more weeks before those in the corresponding nonshaken bottles. In the remaining set, both specimens became positive on the same day; however, the growth index of the agitated culture was higher. The results indicate that agitation of BACTEC 13A blood cultures enhances the growth of MAC in blood cultures and may lead to shorter detection times.
with BACTEC 12B vials in which agitation was shown to inhibit mycobacterial growth. We decided to compare the effects of agitation of 13A blood cultures with those of static incubation on the recovery of mycobacteria from blood.
Disseminated mycobacterial infections have become increasingly common, particularly in patients with AIDS. Mycobacterium avium complex (MAC) is the most common cause of mycobacteremia in AIDS patients (4-6, 14), although other Mycobacterium species have been implicated (3, 9-11). Methods for the recovery of mycobacteria from blood have been time-consuming and have involved intensive laboratory manipulation of the specimens, which may be hazardous to laboratory personnel. The usual method has involved lysis of anticoagulated blood with sterile distilled water, saponin, or some other lysing agent, followed by concentration by centrifugation and then inoculation onto appropriate media. The Du Pont Isolator lysis-centrifugation system uses this principle and can be used for isolating and quantitating mycobacteria in blood. However, the Isolator system has the inherent disadvantage of requiring many laboratory manipulations (1, 12, 13), and so contamination may also be a problem (13). The BACTEC 13A blood culture system was developed specifically for the isolation of mycobacteria from blood (1, 8, 11-13). A 5-ml volume of blood is inoculated directly into the medium, and 0.5 ml of an enrichment fluid is then aseptically added. By incorporation of 14C-labelled palmitic acid into the medium, mycobacterial growth can be detected radiometrically by measuring the amount of 14C-labelled carbon dioxide produced after metabolism of the substrate. Introduction of the BACTEC 13A system into our laboratory has greatly reduced the work load and shortened the times of detection of positive blood cultures. The manufacturer's instructions suggest that the inoculated bottles be incubated without shaking. This is based on their unpublished studies *
MATERIALS AND METHODS Blood specimens. In this study, 265 specimens were collected in pairs from patients of our hospital between 23 April and 31 July 1990. A total of 258 specimens were taken from 111 patients infected with the human immunodeficiency virus (HIV), and 7 were taken from non-HIV-infected individuals. A further 11 paired blood cultures were taken from seven HIV-infected patients with MAC-positive blood cultures between 8 October and 12 November 1990. Up to 10 ml of blood was collected from each patient. Half of this volume was then inoculated into each of the paired bottles. Procedure. Upon receipt in the laboratory, the bottles were randomly assigned consecutive laboratory numbers. Enrichment supplement (0.5 ml) containing 15% bovine serum albumin (Becton Dickinson, Sydney, Australia) was aseptically added to each bottle. The bottles with the even laboratory numbers were then statically incubated at 35°C. The odd-numbered bottles were placed on a Gyrotory shaker (New Brunswick Company, Edison, N.J.) and were rotated at 125 rpm in a 35°C incubator for the first 2 weeks and thereafter were incubated without shaking. The agitation was slow enough to prevent frothing and therefore to reduce the occurrence of cross-contamination during reading. The first 265 pairs of blood cultures were read weekly by the BACTEC 460 TB instrument (Becton Dickinson) for 6 weeks and then once more at 12 weeks. The 11 paired specimens taken after October were read twice weekly for the first 2 weeks and weekly thereafter. The BACTEC needles were changed twice weekly, and the needle heater
Corresponding author. 1801
1802
J. CLIN. MICROBIOL.
JACKSON ET AL.
TABLE 1. AFB detection times for 40 paired positive specimens No. of positive cultures Wk no.
1 2 3 4 5 6 12
Shaken
Nonshaken
14 22 1 2 0 1 0
12 14 2 2 3 2 5
was replaced every 2 months to reduce the possibility of cross-contamination. Specimens showing a growth index (GI) reading of 20 or greater were subcultured onto egg yolk agar and Lowenstein-Jensen medium containing pyruvate, and a smear was prepared for Ziehl-Neelsen staining. The specimens were incubated at 35°C in air for up to 6 weeks. A specimen was considered positive when acid-fast bacilli (AFB) were detected on subculture. If only one of the paired specimens became positive and the other remained negative after 12 weeks of incubation, then 0.2 ml of the negative culture was subcultured onto solid medium. While the results of the subculture were pending, the inoculated blood culture bottles were stored at room temperature in the dark. For the cultures which failed to yield AFB on blind subculture, a 20-ml aliquot of the original culture was removed and centrifuged at 2,500 x g for 20 min and then the deposit was inoculated into a fresh 13A bottle, which was agitated. The new cultures were then read twice weekly by the BACTEC 460 TB instrument for 6 weeks or until they became positive, whichever occurred first. Mycobacterial isolates were identified by standard tests (7).
RESULTS
Of the 265 specimens processed between 23 April and 31 July 1990, 77 (29.1%) yielded MAC from either one or both of the paired 13A bottles. All isolates were from 27 patients infected with HIV. Of the paired specimens, 36 (46.8% of the total number of positive specimens) were positive in the shaken bottles only, 1 (1.3%) was positive only in the bottles which were not shaken, and 40 (51.9%) were positive in both bottles. Of the nonshaken cultures, 36 whose shaken counterparts yielded AFB remained negative after 12 weeks of incubation; 13 of the 36 subsequently yielded AFB after subculture to solid media even though the GIs had been below the cutoff value of 20. A further 12 cultures became positive after subculture to fresh 13A bottles and agitation. Table 1 summarizes the times required to detect AFB in the shaken and nonshaken bottles when both cultures were positive. The majority of the specimens became positive within the first 2 weeks. The average detection times for positive cultures in the shaken and nonshaken bottles were 12.7 and 15.9 days, respectively (excluding the cultures which became positive after 12 weeks of incubation). Of the 77 positive specimens, 8 (10.4% of the total number of positive specimens) in the shaken bottles were detected at least 1 week earlier and 13 (16.9%) were detected 2 or more weeks earlier than the nonshaken counterparts. A total of 57 shaken cultures (including the 36 whose statically incubated counterparts remained negative after 12 weeks) showed positive results before the corresponding nonshaken cul-
TABLE 2. AFB detection times for 11 paired specimens taken from MAC-positive patients after October 1990 No. of positive cultures
Wk no.
(reading)a
Shaken
Nonshaken
1 (first) 1 (second) 2 (first 2 (second) 3 4 5 6 12
0 1 2 4 2 2 0 0 0
0 1 0 0 1 0 0 4 5
a Two readings were taken in each of the first two weeks.
tures. Both bottles became positive simultaneously in 17 instances, although 15 (19.5%) of the agitated cultures had higher GI values than their statically incubated counterparts when detected together. One agitated culture had a lower GI value than its statically incubated counterpart, and one pair had comparable GI readings when detected on the same day. Only two agitated cultures became positive after their nonshaken counterparts. Another nonshaken specimen had a low-positive GI (43) after 12 weeks of incubation and grew MAC, whereas its shaken counterpart was negative. Table 2 summarizes the times of detection of AFB in the 11 paired specimens taken after October 1990, which were read twice weekly by the BACTEC instrument in the first 2 weeks of incubation. All the shaken cultures except one became positive before their statically incubated counterparts. For this pair, positive results were detected on the same day; however, the GI of the agitated culture (447) was greater than that of its statically incubated counterpart (134). Two specimens from one AIDS patient incidentally yielded Cryptococcus neoformans, in the agitated bottles only. No other mycobacterial species were isolated during the study period.
DISCUSSION
The results of this study indicate that agitation of the BACTEC 13A bottles enhances the growth of MAC in blood cultures. A total of 74% of the shaken cultures became positive a week or more before their statically incubated counterparts. Further, 19.5% of the agitated cultures gave higher GIs than the corresponding nonshaken cultures even when growth was detected simultaneously in both bottles. The data suggest that perhaps 46.8% of the blood cultures would have been classified as negative if they had not been shaken. This figure may not have been so high if the bottles had been read twice weekly as the manufacturer suggests. This was not carried out during the study due to an increased work load and a shortage of laboratory staff. However, in October and November 1990, a further 11 paired 13A blood specimens were collected from patients known to be MAC positive to assess the effects of agitation and twice-weekly readings during the first 2 weeks. As Table 2 shows, there is still a marked improvement in the times of detection of AFB in the shaken bottles compared with the nonshaken bottles. Twice-weekly reading may give the bottles a fresh supply of oxygen and carbon dioxide more frequently. However, agitation of the bottles may improve the distribution of the nutrients and gas within the medium itself. Only three statically incubated cultures became positive
VOL. 29, 1991
EFFECT OF AGITATION ON RECOVERY OF MAC BY BACTEC 13A
before their shaken counterparts. One of these became positive after 12 weeks of incubation and presumably had only small numbers of AFB present initially. Hence, random distribution of a very small inoculum into the pair of bottles could explain the lack of growth in the shaken bottle. The other two were taken from two different patients. Both of these patients had other positive specimens for which growth was enhanced by shaking. Twenty-five nonshaken cultures whose counterparts were positive were shown to contain AFB even though they consistently gave negative GIs. Some of these cultures showed a slight increase in GI value not exceeding 18, and then a decrease. The GI values of others were initially less than 12 and then steadily declined. The majority of the blood cultures became positive within the first 2 weeks of incubation (Table 1). The average detection time for the shaken bottles was slightly less (12.7 days) than those obtained by Agy et al., Kiehn and Cammarata, Strand et al., and Witebsky et al., who obtained average detection times of 14.2, 14, 23.6, and 14.8 days, respectively, with the BACTEC 13A system (1, 8, 12, 13). The shorter detection times obtained in this study may be due to the enhancement of growth through agitation; however, it is difficult to make a conclusive comparison, as the patient populations may differ a great deal between the studies. Only one other investigator reported a shorter mean detection time (11.5 days), and this was with statically incubated cultures (11). It is not surprising that agitation of the blood cultures enhances growth of MAC, as mycobacteria are aerophilic. Becton Dickinson investigated shaking of the BACTEC 12B vials and found it to be detrimental to mycobacterial growth (2). However, that study investigated only one strain each of four mycobacterial species and the effect of agitation of BACTEC 12B medium only. BACTEC 12B vials contain only 4 ml of medium, whereas the BACTEC 13A bottles contain 30 ml, i.e., a larger volume for gaseous dispersion, and they have the potential capability of providing a relative lack of oxygen in the depths of an undisturbed bottle. This study can supply evidence of the enhancement of the growth of only MAC, as no other mycobacterial species were isolated. However, MAC is the most common organism implicated in mycobacteremia in AIDS patients (4-6, 14). To determine the possible effect of growth of Mycobacterium tuberculosis in 13A medium, we experimentally inoculated two sets of bottles with two different volumes of a turbid suspension of M. tuberculosis and agitated one bottle of each set. All cultures became positive within 4 days; however, the GIs of both of the shaken cultures were higher. This suggests that shaking may enhance the growth of M. tuberculosis in a blood culture. Since the completion of the study, M. tuberculosis from an HIV-infected patient has been isolated in an agitated BACTEC 13A culture. It appears that agitation of the medium also improves the recovery of C. neoformans from blood. Cryptococcemia is a common opportunistic infection in AIDS patients, and isolation from blood is important in the diagnosis. In summary, the results of this study suggest that agitation of BACTEC 13A bottles for the first 2 weeks of incubation may enhance the growth of MAC and lead to shorter detection times. In addition, lack of agitation could cause false-negative results, particularly if cultures are read only once a week. It is therefore recommended that routine
1803
clinical laboratories agitate BACTEC 13A blood cultures for at least the first 2 weeks of incubation for improved recovery of MAC. ACKNOWLEDGMENTS We gratefully acknowledge Maria Globan, Sandra Aiuto, and Irenea Zarubova for laboratory assistance.
1.
2.
3.
4.
REFERENCES Agy, M. B., C. K. Wallis, J. J. Plorde, L. C. Carlson, and M. B. Coyle. 1989. Evaluation of four mycobacterial blood culture media: BACTEC 13A, Isolator/BACTEC 12B, Isolator/Middlebrook agar, and a biphasic medium. Diagn. Microbiol. Infect. Dis. 12:303-308. Anonymous. 1981. Shaking versus non-shaking experiment. Unpublished data on file at Becton Dickinson, Sydney, Australia. Chaisson, R. E., G. F. Schecter, C. P. Theuer, G. W. Rutherford, D. F. Echenberg, and P. C. Hopewell. 1987. Tuberculosis in patients with the acquired immunodeficiency syndrome. Clinical features, response to therapy, and survival. Am. Rev. Respir. Dis. 136:570-574. Hawkins, C. C., J. W. M. Gold, E. Whimbey, T. E. Kiehn, P. Brannon, R. Cammarata, A. E. Brown, and D. Armstrong. 1986. Mycobacterium avium complex infections in patients with the acquired immunodeficiency syndrome. Ann. Intern. Med. 105:
184-188. 5. Horsburgh, C. R., and R. M. Selik. 1989. The epidemiology of disseminated non-tuberculous mycobacterial infection in the acquired immunodeficiency syndrome (AIDS). Am. Rev. Respir. Dis. 139:4-7. 6. Hoy, J., A. Mijch, M. Sandland, L. Grayson, R. Lucas, and B. Dwyer. 1990. Quadruple therapy for Mycobacterium aviumintracellulare bacteremia in AIDS patients. J. Infect. Dis. 161:801-805. 7. Kent, P. T., and G. P. Kubica. 1985. Public health mycobacteriology. A guide for the level 3 laboratory. Centers for Disease Control, Atlanta. 8. Kiehn, T. E., and R. Cammarata. 1988. Comparative recoveries of Mycobacterium avium-M. intracellulare from Isolator lysiscentrifugation and BACTEC 13A blood culture systems. J. Clin. Microbiol. 26:760-761. 9. Levy-Frebault, V., B. Pangon, A. Burk, C. Katlama, C. Marche, and H. L. David. 1987. Mycobacterium simiae and Mycobacterium avium-M. intracellulare mixed infection in acquired immune deficiency syndrome. J. Clin. Microbiol. 25:154-157. 10. Rogers, P. L., R. E. Walker, H. C. Lane, F. G. Witebsky, J. A. Kovacs, J. E. Parillo, and H. Masur. 1988. Disseminated Mycobacterium haemophilum infection in two patients with the acquired immunodeficiency syndrome. Am. J. Med. 84:640642. 11. Salfinger, M., E. W. Stool, D. Piot, and L. Heifets. 1988. Comparison of three methods for the recovery of Mycobacterium avium complex from blood specimens. J. Clin. Microbiol. 26:1225-1226. 12. Strand, C. L., C. Epstein, S. Verzosa, E. Effatt, P. Hormozi, and S. H. Siddiqi. 1989. Evaluation of a new blood culture medium for mycobacteria. Am. J. Clin. Pathol. 91:316-318. 13. Witebsky, F. G., J. F. Keiser, P. S. Conville, R. Bryan, C. H. Park, R. Walker, and S. H. Siddiqi. 1988. Comparison of BACTEC 13A medium and Du Pont Isolator for detection of mycobacteremia. J. Clin. Microbiol. 26:1501-1505. 14. Wong, B., F. F. Edwards, T. E. Kiehn, E. Whimbrey, H. Donnelly, E. M. Bernard, J. W. M. Gold, and D. Armstrong. 1985. Continuous high-grade Mycobacterium avium-intracellulare bacteremia in patients with the acquired immune deficiency syndrome. Am. J. Med. 78:35-40.
