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Enhancing recovery of Francisella tularensis from blood
DIAGNMICROBIOLINFECTDIS 1988;11:117-119
117
NOTES
Enhancing Recovery of Francisella tularensis from Blood Brent W. Reary and Stephen A. Klotz
Francisella tularensis m a y be isolated with the BACTEC 480 blood culture system even though bottles may not meet the established criteria for recognizing positive blood cultures. We describe three proven bacteremic cases in which growth indices were not positive, gram stain of broth showed no microorganisms but F. tularensis grew on chocolate agar culture.
Isolation of F r a n c i s e l l a t u l a r e n s i s from blood is u n c o m m o n due in part to the microorganism's requirement of cystine for growth that is lacking from m a n y commercial m e d i a (Weaver et al., 1985). The introduction of radiometric blood culture systems such as the BACTEC 460 System (Johnston Laboratories, Towson, MD) appears to be associated with the more frequent isolation of this microorganism from blood (Centers for Disease Control, 1983; Klotz et al., 1987; Provenza et al., 1986). Tularemia is e n d e m i c in our region, and confirmation of the diagnosis is often sought b y serological tests that are more often than not nondiagnostic on first examination (Klotz et al., 1987). Therefore, any method that will hasten the diagnosis may be advantageous to the patient. We describe a m e t h o d that has proved useful in establishing the diagnosis in two patients not otherwise suspected of having tularemia. Although the BACTEC system is extremely sensitive, growth of F. t u l a r e n s i s m a y occur in the m e d i u m but not achieve the p r e d e t e r m i n e d Growth Index (GI) used as the threshold to denote positive blood cultures. The manufacturer r e c o m m e n d s that aerobic bottles, 6B BACTEC, be treated as potential positives if the GI is ->30 and anaerobic bottles, 7D BACTEC, that be considered suspicious if the GI is ->20. In addition, both bottle types should be treated as potentially positive and subcultured if a) there is an increase in GI > 10 between two consecutive readings or b) a bottle shows visible signs of growth, such as turbidity, discoloration, sedimentation, or gas production. However, unless specifically asked to do so, laboratory personnel routinely do not subculture aerobic bottles with a GI of <30. Recent experience with blood cultures from nine bacteremic patients with tularemia has led us to modify these criteria. From three of the bacteremic patients, eight blood cultures grew F. t u l a r e n s i s on subculture, but only three of the bottles met the criteria for definite or even suspicious growth as established by the manufacturer
From the Department of Pathology (B.W.R.), Phelps County Regional Medical Center, Rolla, Missouri, and the Department of Medicine and Ophthalmology (S.A.K.), Veterans Administration and Louisiana State University Medical Centers, Shreveport, Louisiana. Address reprint requests to: Stephen A. Klotz, Department of Medicine, VA Medical Center, 510 East Stoner, Shreveport, LA 71101-4295. Received July 5, 1988; revised and accepted October 3, 1988.
© 1988 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
0732-8893/88/$3.50
118
B.W. Reary and S.A. Klotz
TABLE 1. Growth Indices for Aerobic Bottles (BACTEC 6B) from Which
F. tularensis Was Isolated on Blind Subculture ° Day 1
Day 3
Day 4
Day 5
Day 6
Day 7
Patient 1 Culture 1 2 3 4 5
10 9 4 6 5
9 9 4 4 5
10 10 6 5 5
9 6 6 5 2
16 17 6 7 4
30 28 12 9 13
Patient 2 Culture 1
6
4
5
5
6
11
Patient 3 Culture 1 2
23 18
17 14
32 15
m
m
m
aMaximal GI are provided. The reading routine was to obtain two readings on days 1 and 2 of culture and one reading each day thereafter until day 7. Cultures 1 and 2 of patient 3 were subcultured on day 5 and found to be positive;therefore, no further GI readings were obtained. (Table 1). Cultures 1 and 2 of Patient 1 and Culture 1 of Patient 3 met the criteria for a potentially positive culture. The five other cultures, i n c l u d i n g the only blood culture obtained from Patient 2, did not meet the criteria for a suspicious culture. All anaerobic bottles showed stable or decreasing GI readings over the 7-day period. Nevertheless, eight of the n i n e bactermic patients were detected by the BACTEC 460 system. Gram stains were performed on all bottles but no microorganisms were demonstrated. However, b l i n d subculture was performed on to blood and chocolate agar (Remel Inc., Leneka, KS); the latter is s u p p l e m e n t e d with cystine. After overnight i n c u b a t i o n at 35°C in a 5% CO2 atmosphere, the chocolate agar plates showed small grayish colonies, and the blood agar plates demonstrated faint growth. Gram stains revealed small gram-negative coccobacilli that resembled Haemophilus species microscopically. Organisms were tested to rule out more c o m m o n l y encountered species. The isolates were forwarded to the state laboratory for confirmation as F. tularensis by fluorescent antibody tests. All patients had clinical syndromes compatible with tularemia but only Patient 2 demonstrated serological evidence of the disease (a convalescent tularemia agglut i n i n titer of >1:320). Patients 1 and 3 had no serological evidence of tularemia. Furthermore, Patient 1 died from his illness, which is consistent with previous observations that patients with positive blood cultures for F. tularensis have a more severe manifestation of the disease and poorer prognosis (Centers for Disease Control, 1983; Evans et al., 1985; Fulkerson et al., 1979; Kaiser et al., 1985; Klotz et al., 1987; Ludmerer and Kissane, 1984; Provenza et al., 1986). Radiometric blood cultures containing F. tularensis and showing only small incremental changes in the GI readings at days 5, 6, and 7 may be a more widespread p h e n o m e n o n than presently recognized. It is interesting to note that growth indices for Coccidioides immitis were demonstrated to be nondiagnostic for 6B BACTEC bottles, which, however, contained visible mycelial elements (Ampel and Weiden, 1988). Based u p o n our experience, we believe that blood cultures from patients believed to have tularemia should be b l i n d l y subcultured to chocolate agar on day 7 regardless of the GI reading. This is particularly true of those aerobic bottles i n
Notes
119
w h i c h GI readings show a rise at the end of 7 days of culture. It may also be advisable to count the bottles at 10 and 14 days of culture, w h i c h m a y further enhance the n u m b e r of positive bottles. In nonradiometric blood culture systems such as the BD blood culture bottle (Becton-Dickinson, Cockeysville, MD) F. tularensis is occasionally isolated from blood (J.H. Riley, Ft. Smith, AK, personal communication). Perhaps these blood culture systems should also be b l i n d l y subcultured onto agar m e d i u m s if they are taken from patients in w h o m the suspicion of tularemia is high. This paper was presented in part at the Annual Meeting of the American Society for Microbiology, Las Vegas, Nevada, 1985. Fluorescent antibody staining of F. tularensis was performed by the Missouri Department of Health, Jefferson City, Missouri.
REFERENCES Ampel NM, Weiden MA (1988) Discrepancy between growth of Coccidioides immitis in bacterial blood culture media and a radiometric growth index. Diagn Microbio| Infect Dis 9:7. Centers for Disease Control (1983) Tularemic pneumonia Tennessee. Morbid Mortal Weekly Rep 32:363-369. Evans ME, Gregory DW, Schaffner W, McGee ZA (1985) Tularemia: A 30-year experience with 88 cases. Medicine 64:251-269. Fulkerson W, Spiekard A, Davis BW (1979) Opportunistic infections in chronic lymphocytic leukemia. South Med J 72:1487-1488. Kaiser AB, Rieves D, Price AH, Gelfand MR, Parrish RE, Decker MD, Evans ME (1985) Tularemia and rhabdomyolysis. JAMA 253:241-243. Klotz SA, Penn RL, Provenza JM (1987) The unusual presentations of tularemia. Bacteremia, pneumonia and rhabdomyotysis. Arch Int Med 147:214. Ludmerer KM, Kissane JM (eds) (1984) Fever, leukopenia, acute renal failure, and death in a 65 year old man. Am J IVied 77:117-124. Provenza JM, Klotz SA, Penn RL (1986) Isolation of Francisella tularensis from blood. J C/in Microbiol 24:453-455. Weaver RE, Hollis DG, Bottone EJ (1985) Gram negative fermentative bacteria and Frencisella tularensis. Manual of Clinical Microbiology. Eds. EH Lennette, A Balows, WJ Hausler, Jr., and HJ Shadomy, Washington D.C.: American Society for Microbiology, pp 316-319.
117
NOTES
Enhancing Recovery of Francisella tularensis from Blood Brent W. Reary and Stephen A. Klotz
Francisella tularensis m a y be isolated with the BACTEC 480 blood culture system even though bottles may not meet the established criteria for recognizing positive blood cultures. We describe three proven bacteremic cases in which growth indices were not positive, gram stain of broth showed no microorganisms but F. tularensis grew on chocolate agar culture.
Isolation of F r a n c i s e l l a t u l a r e n s i s from blood is u n c o m m o n due in part to the microorganism's requirement of cystine for growth that is lacking from m a n y commercial m e d i a (Weaver et al., 1985). The introduction of radiometric blood culture systems such as the BACTEC 460 System (Johnston Laboratories, Towson, MD) appears to be associated with the more frequent isolation of this microorganism from blood (Centers for Disease Control, 1983; Klotz et al., 1987; Provenza et al., 1986). Tularemia is e n d e m i c in our region, and confirmation of the diagnosis is often sought b y serological tests that are more often than not nondiagnostic on first examination (Klotz et al., 1987). Therefore, any method that will hasten the diagnosis may be advantageous to the patient. We describe a m e t h o d that has proved useful in establishing the diagnosis in two patients not otherwise suspected of having tularemia. Although the BACTEC system is extremely sensitive, growth of F. t u l a r e n s i s m a y occur in the m e d i u m but not achieve the p r e d e t e r m i n e d Growth Index (GI) used as the threshold to denote positive blood cultures. The manufacturer r e c o m m e n d s that aerobic bottles, 6B BACTEC, be treated as potential positives if the GI is ->30 and anaerobic bottles, 7D BACTEC, that be considered suspicious if the GI is ->20. In addition, both bottle types should be treated as potentially positive and subcultured if a) there is an increase in GI > 10 between two consecutive readings or b) a bottle shows visible signs of growth, such as turbidity, discoloration, sedimentation, or gas production. However, unless specifically asked to do so, laboratory personnel routinely do not subculture aerobic bottles with a GI of <30. Recent experience with blood cultures from nine bacteremic patients with tularemia has led us to modify these criteria. From three of the bacteremic patients, eight blood cultures grew F. t u l a r e n s i s on subculture, but only three of the bottles met the criteria for definite or even suspicious growth as established by the manufacturer
From the Department of Pathology (B.W.R.), Phelps County Regional Medical Center, Rolla, Missouri, and the Department of Medicine and Ophthalmology (S.A.K.), Veterans Administration and Louisiana State University Medical Centers, Shreveport, Louisiana. Address reprint requests to: Stephen A. Klotz, Department of Medicine, VA Medical Center, 510 East Stoner, Shreveport, LA 71101-4295. Received July 5, 1988; revised and accepted October 3, 1988.
© 1988 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
0732-8893/88/$3.50
118
B.W. Reary and S.A. Klotz
TABLE 1. Growth Indices for Aerobic Bottles (BACTEC 6B) from Which
F. tularensis Was Isolated on Blind Subculture ° Day 1
Day 3
Day 4
Day 5
Day 6
Day 7
Patient 1 Culture 1 2 3 4 5
10 9 4 6 5
9 9 4 4 5
10 10 6 5 5
9 6 6 5 2
16 17 6 7 4
30 28 12 9 13
Patient 2 Culture 1
6
4
5
5
6
11
Patient 3 Culture 1 2
23 18
17 14
32 15
m
m
m
aMaximal GI are provided. The reading routine was to obtain two readings on days 1 and 2 of culture and one reading each day thereafter until day 7. Cultures 1 and 2 of patient 3 were subcultured on day 5 and found to be positive;therefore, no further GI readings were obtained. (Table 1). Cultures 1 and 2 of Patient 1 and Culture 1 of Patient 3 met the criteria for a potentially positive culture. The five other cultures, i n c l u d i n g the only blood culture obtained from Patient 2, did not meet the criteria for a suspicious culture. All anaerobic bottles showed stable or decreasing GI readings over the 7-day period. Nevertheless, eight of the n i n e bactermic patients were detected by the BACTEC 460 system. Gram stains were performed on all bottles but no microorganisms were demonstrated. However, b l i n d subculture was performed on to blood and chocolate agar (Remel Inc., Leneka, KS); the latter is s u p p l e m e n t e d with cystine. After overnight i n c u b a t i o n at 35°C in a 5% CO2 atmosphere, the chocolate agar plates showed small grayish colonies, and the blood agar plates demonstrated faint growth. Gram stains revealed small gram-negative coccobacilli that resembled Haemophilus species microscopically. Organisms were tested to rule out more c o m m o n l y encountered species. The isolates were forwarded to the state laboratory for confirmation as F. tularensis by fluorescent antibody tests. All patients had clinical syndromes compatible with tularemia but only Patient 2 demonstrated serological evidence of the disease (a convalescent tularemia agglut i n i n titer of >1:320). Patients 1 and 3 had no serological evidence of tularemia. Furthermore, Patient 1 died from his illness, which is consistent with previous observations that patients with positive blood cultures for F. tularensis have a more severe manifestation of the disease and poorer prognosis (Centers for Disease Control, 1983; Evans et al., 1985; Fulkerson et al., 1979; Kaiser et al., 1985; Klotz et al., 1987; Ludmerer and Kissane, 1984; Provenza et al., 1986). Radiometric blood cultures containing F. tularensis and showing only small incremental changes in the GI readings at days 5, 6, and 7 may be a more widespread p h e n o m e n o n than presently recognized. It is interesting to note that growth indices for Coccidioides immitis were demonstrated to be nondiagnostic for 6B BACTEC bottles, which, however, contained visible mycelial elements (Ampel and Weiden, 1988). Based u p o n our experience, we believe that blood cultures from patients believed to have tularemia should be b l i n d l y subcultured to chocolate agar on day 7 regardless of the GI reading. This is particularly true of those aerobic bottles i n
Notes
119
w h i c h GI readings show a rise at the end of 7 days of culture. It may also be advisable to count the bottles at 10 and 14 days of culture, w h i c h m a y further enhance the n u m b e r of positive bottles. In nonradiometric blood culture systems such as the BD blood culture bottle (Becton-Dickinson, Cockeysville, MD) F. tularensis is occasionally isolated from blood (J.H. Riley, Ft. Smith, AK, personal communication). Perhaps these blood culture systems should also be b l i n d l y subcultured onto agar m e d i u m s if they are taken from patients in w h o m the suspicion of tularemia is high. This paper was presented in part at the Annual Meeting of the American Society for Microbiology, Las Vegas, Nevada, 1985. Fluorescent antibody staining of F. tularensis was performed by the Missouri Department of Health, Jefferson City, Missouri.
REFERENCES Ampel NM, Weiden MA (1988) Discrepancy between growth of Coccidioides immitis in bacterial blood culture media and a radiometric growth index. Diagn Microbio| Infect Dis 9:7. Centers for Disease Control (1983) Tularemic pneumonia Tennessee. Morbid Mortal Weekly Rep 32:363-369. Evans ME, Gregory DW, Schaffner W, McGee ZA (1985) Tularemia: A 30-year experience with 88 cases. Medicine 64:251-269. Fulkerson W, Spiekard A, Davis BW (1979) Opportunistic infections in chronic lymphocytic leukemia. South Med J 72:1487-1488. Kaiser AB, Rieves D, Price AH, Gelfand MR, Parrish RE, Decker MD, Evans ME (1985) Tularemia and rhabdomyolysis. JAMA 253:241-243. Klotz SA, Penn RL, Provenza JM (1987) The unusual presentations of tularemia. Bacteremia, pneumonia and rhabdomyotysis. Arch Int Med 147:214. Ludmerer KM, Kissane JM (eds) (1984) Fever, leukopenia, acute renal failure, and death in a 65 year old man. Am J IVied 77:117-124. Provenza JM, Klotz SA, Penn RL (1986) Isolation of Francisella tularensis from blood. J C/in Microbiol 24:453-455. Weaver RE, Hollis DG, Bottone EJ (1985) Gram negative fermentative bacteria and Frencisella tularensis. Manual of Clinical Microbiology. Eds. EH Lennette, A Balows, WJ Hausler, Jr., and HJ Shadomy, Washington D.C.: American Society for Microbiology, pp 316-319.