DIAGN MICROBIOLINFECT DIS 1991;14:7-10
7
Application of DNA Probes to Antimicrobial Susceptibility Testing of Legionellapneumophila Mary Reznicek, Martha Bale, and Michael Pfaller
The commercial availability of a DNA probe assay for the detection of Legionella (Gen-Probe Incorporated, San Diego, CA) provides a unique opportunity to investigate the application of this technology to antibiotic susceptibility testing of L. pneumophila. We examined the ability of erythromycin, rifampin, and ciprofloxacin to kill L. pneumophila in buffered ACES-yeast extract broth (YEB). The test organism was incubated for a total of 96 hr in the presence of 10 i~g/ml erythromycin, 1 I~g/ml rifampin, or 1 i~g/ml ciprofloxacin. Growth was monitored at 24-hr intervals by quantitative plating and
the DNA probe assay. The correlation between organism concentration [colony-forming units (CFU) per roll and DNA probe activity (counts per rain) was excellent (r = 0.97). The percent decrease in CFU/ml at 96 hr relative to control counts was >99% for erythromycin, rifampin, and ciprofloxacin. The percent decrease in CPM at 96 hr versus control was 87% for erythromycin, 89% for rifampin, and 93% for ciprofloxacin. This data documents a novel application of DNA probe technology, which may be useful in future studies of in vitro susceptibility of Legionella to various agents.
INTRODUCTION
has been shown to be comparable to direct immunofluorescence for Legionella. The current literature continues to stress the need for simultaneous culture in addition to the use of rapid diagnostic techniques (Doebbeling et al., 1988; Edelstein et al., 1987; Pfaller MA, 1988). The emergence of DNA probe technology provides a unique opportunity to explore potential applications of probe technology to additional clinical questions. Up to now, antimicrobial susceptibility testing for LegioneUa has not proven to be clinically useful (Meyer et al., 1983), however, reports of erythromycin-resistant strains of Legionella may make susceptibility testing important in the future (Dowling et al., 1985). To our knowledge, no studies currently exist addressing (a) the in vitro response of DNA probe activity to antimicrobial therapy of Legionella, and (b) whether antibiotic inhibition of DNA probe activity correlates with conventional methods of susceptibility testing. In this study, we examined the DNA probe response to a strain of Legionella treated with three different antibiotics (erythromycin, rifampin, and ciprofloxacin) in a broth system and compared the results to simultaneous quantitative plating.
Legionella is most commonly known in clinical medicine as an opportunistic organism causing infections in the elderly, debilitated, and immunocompromised. Because of the length of time needed to culture the organism, combined with the need for prompt therapy, methods for rapidly diagnosing Legionella prior to culture confirmation have gained popularity. The newest technique, DNA probe detection, is being used increasingly due to its quick turnaround time and technical simplicity. The DNA probe test for Legionella is easily performed in 2 hr and has objective interpretation guidelines. The reliability of the DNA probe for Legionella has been established in the literature and
From the Department of Pathology, University of Iowa Hospitals and Clinics (M.R.), the Veterans Administration Medical Center (M.B.), and the Department of Pathology, University of Iowa College of Medicine (M.P.), Iowa City, IA. Address reprint requests to: Dr. M. Pfaller, Department of Pathology, 273 MRC, University of Iowa College of Medicine, Iowa City, IA 52242. ReceivedMay 10, 1990;revised and acceptedJuly 10, 1990. © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
8
MATERIALS AND METHODS Bacterial Strain
Legionella pneumophila serogroup 1, identified by direct immunofluorescence (CDC antisera), was obtained as a clinical isolate from a hospitalized patient at the University of Iowa Hospitals and Clinics. The organism was saved on a slant of buffered-charcoalyeast extract agar (BCYE) (Remel Laboratories) and frozen at -70°C. After thawing, the organism was plated on BCYE agar and grown at 35°C in a candle jar. The organism was transferred to fresh media twice before the experiment.
Antimicrobial Agents Antibiotic solutions were prepared from standard powders (erythromycin, Abbott Laboratories, North Chicago, IL: rifampin, Merrell Dow, Cincinnati, OH; ciprofloxacin, Miles Pharmaceuticals, West Haven, CT) to a stock concentration of 1000 p,g/ml in sterile distilled water and frozen at -70°C for storage. On day 1 of the experiment, the antibiotic stock solutions were diluted with sterile water to 10 times the final concentration. The final dilution was made with ACES-buffered-yeast extract broth (YEB) (Barbaree et al., 1983) to concentrations of 10 ~g/ml erythromycin, 1 ~g/ml ciprofloxacin and 1 ~g/ml rifampin. These concentrations were selected to approximate in vivo serum concentrations of the antibiotics and are not reflective of minimal inhibitory concentration data.
Time-Kill Study
Legionella pneumophila was grown in YEB for 72 hr at 35°C in a candle jar. The resulting suspension was used to inoculate tubes of YEB (total volume: 5 ml) containing no antibiotic (growth control), 10 ~g/ml erythromycin, 1 ~g/ml rifampin, and 1 ~g/ml ciprofloxacin, respectively. The final inoculum concentration in each tube was 2.5 x 105 colony-forming units (CFU)/ml as determined by quantitative plate counts. Following inoculation, the tubes were incubated at 35°C in a candle jar. The time course of growth and/or antibacterial activity in the controland antibiotic-containing tubes was assessed by sampling each tube and determining the number of viable CFU/ml by quantitative plating methods and number of counts per minute (CPM) of 125I-activity measured by the DNA probe assay at 0, 24, 72, and 96 h of incubation. Quantitative plate counts were performed by serially diluting an aliquot of well-mixed broth from each antibiotic tube and control tube, and by plating 10 ~I onto BCYE agar at 0, 24, 72, and 96 hr of
M. Reznicek et al.
incubation. The plates were incubated in a candle jar at 35°C and examined daily for 12 days. The colonies were counted and the results expressed as log CFU/ml. The Gen-Probe assay (Gen-Probe Legionella Rapid Diagnostic System, GenProbe, San Diego, CA) was performed exactly as recommended by the manufacturer. Briefly, a 100 ~gl aliquot of well-mixed broth was aspirated from each tube and transferred to a reaction tube containing a lysing solution and glass beads, centrifuged (500-3000 g) for 15 sec and sonicated for 15 min at 60°-70°C to release the ribosomal RNA from the bacterial cells. After sonication, 2.0 ml of a solution containing 125IDNA probe specific for Legionella species was added to each reaction tube. The tubes were vortexed and incubated at 72°C for 60 rain to allow hybridization to take place. The separation of the nucleic acid hybrids from the nonhybridized probe was accomplished by addition of 2.0 ml of a separation suspension containing a solid adsorbent, hydroxyapatite, to adsorb the probe-ribosomal RNA hybrids. Unreacted probe was not adsorbed and was separated from the bound probe by low-speed (500-3000 g) centrifugation. Following a single wash, the hydroxyapatite pellet containing the bound hybrid was counted in a gamma counter. The results (CPM) were calculated by subtracting the background CPM from the sample CPM.
RESULTS AND DISCUSSION The relationship between viable colony counts (CFU/ml) and DNA probe activity (cpm) for L. pneumophila is shown in Figure 1. Excellent correlation was noted between the probe activity (CPM) and the number of viable CFU/ml (r = 0.97). A significant decrease in the probe activity (CPM) was noted with each of the antibiotics. Compared to the control at 96 hr, exposure to erythromycin showed an 87% decrease, rifampin showed an 89% decrease, and ciprofloxacin showed a 93% decrease in cpm (Fig. 2). The decrease in probe activity relative to control was progressive over the 96-hr time period for each of the antibiotics. Similar to the DNA probe activity, the quantitative plating showed a significant decrease in CFU/ml. Compared to the control at 96 h, each of the antibiotics showed a >99.9% inhibition (Fig. 3). The decrease in CFU/ml relative to control was progressive over the 96-hr time period for each of the antibiotics, The most practical application of DNA probe technology to clinical medicine is in the rapid detection of an infectious agent that is difficult to culture. The literature has shown that the DNA
Legionella Susceptibility
Testing DNA Probes
9
8.0" 7.0
6.0" 5.0. U.
o
g' 4.0
.J
3.0 2.0'
f
1.0
310
2.0
410
510
Log CPM FIGURE 1. The relationship between viable colony counts (CFU/ml) and DNA probe activity (CPM) for Legionella pneumophila (r = 0.97).
probe for the detection of Legionella fulfills that task (Doebbeling et al., 1988; Edelstein et al., 1987; Pfaller MA, 1988). Other potential uses for probe technology are currently under investigation. The possible application of this technology to antimicrobial susceptibility testing is an area of growing
14000" ERY'rHROMYClN(10pig/ml) 12000'
CIPROFLOXACIN(1~g/ml)
RIFAMPIN(114gJrni) Control
//'/"t Cont'ol
Cot~trol
....~""/
I0000"
.///./.t/'''/'/
8000" a. o
interest. Although antimicrobial susceptibility testing for Legionella has not proven to be clinically useful in the past (Meyer et al., 1983), the emergence of erythromycin-resistant laboratory strains of Legionella may make susceptibility testing a future necessity in the clinical laboratory (Dowling et al., 1985). In this paper, we have investigated (a) the response of the DNA probe to antibiotic treated Legionella, and (b) the correlation of this probe data to quantitative culture. Our results show that DNA probe activity decreases in response to antibiotic treatment of Legionella organisms over a 96-hr time period and that the DNA probe data correlates well with conventional quantitative culture methods. These data represent the first example of a novel application of DNA probe technology to the area of antimicrobial susceptibility testing. This technology could potentially be applied to other organisms for which DNA probes have been developed. The lack of correlation between in vitro susceptibility testing for Legionella and clinical results may be due to the intracellular location of the Legionella organism within macrophages in the infected host. Although organisms in broth may be fully susceptible to a number of antibiotics, certain agents may fail to penetrate into the macrophage allowing intracellular organisms to survive. Studying the effects of antimicrobial agents on intracellular Legionella organisms using the macrophage tissue culture system may be more clinically valid than conventional broth
6000" 4000 2000
Eryrnromy=n 24
48
72
96
" 0
24
48
] ~ L 72
Ritampin
96
~ 0
Ciprolloxmcm 24
48
72
96
Hours FIGURE 2. The change in probe activity (CPM) is shown at 24-hr intervals over a 96-hr time period for each antibiotic against Legionellapneumophila. The control activity is shown for comparison. Results are the mean _+ SEM of two separate experiments.
10
M. Reznicek et al.
ClPROFLOXACIN
i-°. .,,
]--
(1 ~l/ml)
,:~ Control
/:/t C.~lrol
/
/:
i
/
/:
~..............d
~3fl~n
0
0
. . 24
. . 48
.
72
96
0
24
72
48
96
24
48
72
96
Hours
FIGURE 3. The inhibition of Legionella pneumophila by quantitative plating (CFU/ml) is shown at 24-hr intervals over a 96-hr time period for each of the antibiotics. A control culture is shown for comparison. Results are the mean - SEM of two separate experiments. susceptibility testing. In s t u d y i n g the effects of antimicrobials on Legionella organisms in cell culture, we feel that the D N A probe for Legionella m a y provide a single objective m e a n s to assess bactericidal activity.
This study was supported in part by Gen-Probe Incorporated. The excellent secretarial skills of Ruth Kjaer are greatly appreciated.
REFERENCES Barbaree JM, Sanchez A, Sandens GN (1983) Tolerance of Legionella species to sodium chloride. Curr Microbiol 9:15. Doebbeling BN, Bale MJ, Koontz FP, Helms CM, Wenzel RP, Pfaller MA (1988) Prospective evaluation of the Gen-Probe assay for the detection of Legionellae in respiratory specimens. Eur J Clin Microbiol Infect Dis 7:748752. Dowling JN, McDevitt DA, Pasculle AW (1985) Isolation and preliminary characterization of erythromycin-resistant variants of Legionella micdadei and Legionella pneumophila. Antimicrob Agents Chemother 27:272-274.
Edelstein PH, Bryan RN, Enns RK, Kohne DE, Kacian DL (1987) Retrospective study of Gen-Probe rapid diagnostic system for the detection of Legionellae in frozen clinical respiratory tract samples. J Clin Microbio125:10221026. Meyer RD (1983) Legionella infections: A review of five years of research. Rev Infect Dis 5:258-278. Pfaller MA (1988) Laboratory diagnosis of infections due to Legionella species: practical application of DNA probes in the clinical microbiology laboratory. Lab Med 19:301304.
7
Application of DNA Probes to Antimicrobial Susceptibility Testing of Legionellapneumophila Mary Reznicek, Martha Bale, and Michael Pfaller
The commercial availability of a DNA probe assay for the detection of Legionella (Gen-Probe Incorporated, San Diego, CA) provides a unique opportunity to investigate the application of this technology to antibiotic susceptibility testing of L. pneumophila. We examined the ability of erythromycin, rifampin, and ciprofloxacin to kill L. pneumophila in buffered ACES-yeast extract broth (YEB). The test organism was incubated for a total of 96 hr in the presence of 10 i~g/ml erythromycin, 1 I~g/ml rifampin, or 1 i~g/ml ciprofloxacin. Growth was monitored at 24-hr intervals by quantitative plating and
the DNA probe assay. The correlation between organism concentration [colony-forming units (CFU) per roll and DNA probe activity (counts per rain) was excellent (r = 0.97). The percent decrease in CFU/ml at 96 hr relative to control counts was >99% for erythromycin, rifampin, and ciprofloxacin. The percent decrease in CPM at 96 hr versus control was 87% for erythromycin, 89% for rifampin, and 93% for ciprofloxacin. This data documents a novel application of DNA probe technology, which may be useful in future studies of in vitro susceptibility of Legionella to various agents.
INTRODUCTION
has been shown to be comparable to direct immunofluorescence for Legionella. The current literature continues to stress the need for simultaneous culture in addition to the use of rapid diagnostic techniques (Doebbeling et al., 1988; Edelstein et al., 1987; Pfaller MA, 1988). The emergence of DNA probe technology provides a unique opportunity to explore potential applications of probe technology to additional clinical questions. Up to now, antimicrobial susceptibility testing for LegioneUa has not proven to be clinically useful (Meyer et al., 1983), however, reports of erythromycin-resistant strains of Legionella may make susceptibility testing important in the future (Dowling et al., 1985). To our knowledge, no studies currently exist addressing (a) the in vitro response of DNA probe activity to antimicrobial therapy of Legionella, and (b) whether antibiotic inhibition of DNA probe activity correlates with conventional methods of susceptibility testing. In this study, we examined the DNA probe response to a strain of Legionella treated with three different antibiotics (erythromycin, rifampin, and ciprofloxacin) in a broth system and compared the results to simultaneous quantitative plating.
Legionella is most commonly known in clinical medicine as an opportunistic organism causing infections in the elderly, debilitated, and immunocompromised. Because of the length of time needed to culture the organism, combined with the need for prompt therapy, methods for rapidly diagnosing Legionella prior to culture confirmation have gained popularity. The newest technique, DNA probe detection, is being used increasingly due to its quick turnaround time and technical simplicity. The DNA probe test for Legionella is easily performed in 2 hr and has objective interpretation guidelines. The reliability of the DNA probe for Legionella has been established in the literature and
From the Department of Pathology, University of Iowa Hospitals and Clinics (M.R.), the Veterans Administration Medical Center (M.B.), and the Department of Pathology, University of Iowa College of Medicine (M.P.), Iowa City, IA. Address reprint requests to: Dr. M. Pfaller, Department of Pathology, 273 MRC, University of Iowa College of Medicine, Iowa City, IA 52242. ReceivedMay 10, 1990;revised and acceptedJuly 10, 1990. © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
8
MATERIALS AND METHODS Bacterial Strain
Legionella pneumophila serogroup 1, identified by direct immunofluorescence (CDC antisera), was obtained as a clinical isolate from a hospitalized patient at the University of Iowa Hospitals and Clinics. The organism was saved on a slant of buffered-charcoalyeast extract agar (BCYE) (Remel Laboratories) and frozen at -70°C. After thawing, the organism was plated on BCYE agar and grown at 35°C in a candle jar. The organism was transferred to fresh media twice before the experiment.
Antimicrobial Agents Antibiotic solutions were prepared from standard powders (erythromycin, Abbott Laboratories, North Chicago, IL: rifampin, Merrell Dow, Cincinnati, OH; ciprofloxacin, Miles Pharmaceuticals, West Haven, CT) to a stock concentration of 1000 p,g/ml in sterile distilled water and frozen at -70°C for storage. On day 1 of the experiment, the antibiotic stock solutions were diluted with sterile water to 10 times the final concentration. The final dilution was made with ACES-buffered-yeast extract broth (YEB) (Barbaree et al., 1983) to concentrations of 10 ~g/ml erythromycin, 1 ~g/ml ciprofloxacin and 1 ~g/ml rifampin. These concentrations were selected to approximate in vivo serum concentrations of the antibiotics and are not reflective of minimal inhibitory concentration data.
Time-Kill Study
Legionella pneumophila was grown in YEB for 72 hr at 35°C in a candle jar. The resulting suspension was used to inoculate tubes of YEB (total volume: 5 ml) containing no antibiotic (growth control), 10 ~g/ml erythromycin, 1 ~g/ml rifampin, and 1 ~g/ml ciprofloxacin, respectively. The final inoculum concentration in each tube was 2.5 x 105 colony-forming units (CFU)/ml as determined by quantitative plate counts. Following inoculation, the tubes were incubated at 35°C in a candle jar. The time course of growth and/or antibacterial activity in the controland antibiotic-containing tubes was assessed by sampling each tube and determining the number of viable CFU/ml by quantitative plating methods and number of counts per minute (CPM) of 125I-activity measured by the DNA probe assay at 0, 24, 72, and 96 h of incubation. Quantitative plate counts were performed by serially diluting an aliquot of well-mixed broth from each antibiotic tube and control tube, and by plating 10 ~I onto BCYE agar at 0, 24, 72, and 96 hr of
M. Reznicek et al.
incubation. The plates were incubated in a candle jar at 35°C and examined daily for 12 days. The colonies were counted and the results expressed as log CFU/ml. The Gen-Probe assay (Gen-Probe Legionella Rapid Diagnostic System, GenProbe, San Diego, CA) was performed exactly as recommended by the manufacturer. Briefly, a 100 ~gl aliquot of well-mixed broth was aspirated from each tube and transferred to a reaction tube containing a lysing solution and glass beads, centrifuged (500-3000 g) for 15 sec and sonicated for 15 min at 60°-70°C to release the ribosomal RNA from the bacterial cells. After sonication, 2.0 ml of a solution containing 125IDNA probe specific for Legionella species was added to each reaction tube. The tubes were vortexed and incubated at 72°C for 60 rain to allow hybridization to take place. The separation of the nucleic acid hybrids from the nonhybridized probe was accomplished by addition of 2.0 ml of a separation suspension containing a solid adsorbent, hydroxyapatite, to adsorb the probe-ribosomal RNA hybrids. Unreacted probe was not adsorbed and was separated from the bound probe by low-speed (500-3000 g) centrifugation. Following a single wash, the hydroxyapatite pellet containing the bound hybrid was counted in a gamma counter. The results (CPM) were calculated by subtracting the background CPM from the sample CPM.
RESULTS AND DISCUSSION The relationship between viable colony counts (CFU/ml) and DNA probe activity (cpm) for L. pneumophila is shown in Figure 1. Excellent correlation was noted between the probe activity (CPM) and the number of viable CFU/ml (r = 0.97). A significant decrease in the probe activity (CPM) was noted with each of the antibiotics. Compared to the control at 96 hr, exposure to erythromycin showed an 87% decrease, rifampin showed an 89% decrease, and ciprofloxacin showed a 93% decrease in cpm (Fig. 2). The decrease in probe activity relative to control was progressive over the 96-hr time period for each of the antibiotics. Similar to the DNA probe activity, the quantitative plating showed a significant decrease in CFU/ml. Compared to the control at 96 h, each of the antibiotics showed a >99.9% inhibition (Fig. 3). The decrease in CFU/ml relative to control was progressive over the 96-hr time period for each of the antibiotics, The most practical application of DNA probe technology to clinical medicine is in the rapid detection of an infectious agent that is difficult to culture. The literature has shown that the DNA
Legionella Susceptibility
Testing DNA Probes
9
8.0" 7.0
6.0" 5.0. U.
o
g' 4.0
.J
3.0 2.0'
f
1.0
310
2.0
410
510
Log CPM FIGURE 1. The relationship between viable colony counts (CFU/ml) and DNA probe activity (CPM) for Legionella pneumophila (r = 0.97).
probe for the detection of Legionella fulfills that task (Doebbeling et al., 1988; Edelstein et al., 1987; Pfaller MA, 1988). Other potential uses for probe technology are currently under investigation. The possible application of this technology to antimicrobial susceptibility testing is an area of growing
14000" ERY'rHROMYClN(10pig/ml) 12000'
CIPROFLOXACIN(1~g/ml)
RIFAMPIN(114gJrni) Control
//'/"t Cont'ol
Cot~trol
....~""/
I0000"
.///./.t/'''/'/
8000" a. o
interest. Although antimicrobial susceptibility testing for Legionella has not proven to be clinically useful in the past (Meyer et al., 1983), the emergence of erythromycin-resistant laboratory strains of Legionella may make susceptibility testing a future necessity in the clinical laboratory (Dowling et al., 1985). In this paper, we have investigated (a) the response of the DNA probe to antibiotic treated Legionella, and (b) the correlation of this probe data to quantitative culture. Our results show that DNA probe activity decreases in response to antibiotic treatment of Legionella organisms over a 96-hr time period and that the DNA probe data correlates well with conventional quantitative culture methods. These data represent the first example of a novel application of DNA probe technology to the area of antimicrobial susceptibility testing. This technology could potentially be applied to other organisms for which DNA probes have been developed. The lack of correlation between in vitro susceptibility testing for Legionella and clinical results may be due to the intracellular location of the Legionella organism within macrophages in the infected host. Although organisms in broth may be fully susceptible to a number of antibiotics, certain agents may fail to penetrate into the macrophage allowing intracellular organisms to survive. Studying the effects of antimicrobial agents on intracellular Legionella organisms using the macrophage tissue culture system may be more clinically valid than conventional broth
6000" 4000 2000
Eryrnromy=n 24
48
72
96
" 0
24
48
] ~ L 72
Ritampin
96
~ 0
Ciprolloxmcm 24
48
72
96
Hours FIGURE 2. The change in probe activity (CPM) is shown at 24-hr intervals over a 96-hr time period for each antibiotic against Legionellapneumophila. The control activity is shown for comparison. Results are the mean _+ SEM of two separate experiments.
10
M. Reznicek et al.
ClPROFLOXACIN
i-°. .,,
]--
(1 ~l/ml)
,:~ Control
/:/t C.~lrol
/
/:
i
/
/:
~..............d
~3fl~n
0
0
. . 24
. . 48
.
72
96
0
24
72
48
96
24
48
72
96
Hours
FIGURE 3. The inhibition of Legionella pneumophila by quantitative plating (CFU/ml) is shown at 24-hr intervals over a 96-hr time period for each of the antibiotics. A control culture is shown for comparison. Results are the mean - SEM of two separate experiments. susceptibility testing. In s t u d y i n g the effects of antimicrobials on Legionella organisms in cell culture, we feel that the D N A probe for Legionella m a y provide a single objective m e a n s to assess bactericidal activity.
This study was supported in part by Gen-Probe Incorporated. The excellent secretarial skills of Ruth Kjaer are greatly appreciated.
REFERENCES Barbaree JM, Sanchez A, Sandens GN (1983) Tolerance of Legionella species to sodium chloride. Curr Microbiol 9:15. Doebbeling BN, Bale MJ, Koontz FP, Helms CM, Wenzel RP, Pfaller MA (1988) Prospective evaluation of the Gen-Probe assay for the detection of Legionellae in respiratory specimens. Eur J Clin Microbiol Infect Dis 7:748752. Dowling JN, McDevitt DA, Pasculle AW (1985) Isolation and preliminary characterization of erythromycin-resistant variants of Legionella micdadei and Legionella pneumophila. Antimicrob Agents Chemother 27:272-274.
Edelstein PH, Bryan RN, Enns RK, Kohne DE, Kacian DL (1987) Retrospective study of Gen-Probe rapid diagnostic system for the detection of Legionellae in frozen clinical respiratory tract samples. J Clin Microbio125:10221026. Meyer RD (1983) Legionella infections: A review of five years of research. Rev Infect Dis 5:258-278. Pfaller MA (1988) Laboratory diagnosis of infections due to Legionella species: practical application of DNA probes in the clinical microbiology laboratory. Lab Med 19:301304.
