Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis

14 CLINICAL I M M U N O L O G Y Newsletter

patients. J Infect Dis 175:1087-1092, 1997. 7. Field AK, Biron KK: "The end of innocence" revisited: resistance of herpesviruses to antiviral drugs. Clin Microbiol Rev: 7:1-13, 1994. 8. Gerna G, Sarasini A, Percivalle E et al.: Rapid screening for resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovirus from culture positive blood samples. J Clin Microbiol 33:738-741, 1995. 9. Jabs DA, Enger C, Forman Met al.: Incidence of foscarnet resistance and cidofovir resistance in patients treated for cytomegalovirus retinitis. Antimicrob Agents Chemother 42:2240-2244, 1998. 10. Jokela J, Erice A, Stanat Set al.: A standardized plaque reduction assay for CMV antiviral susceptibility. Abs 2nd National Conference of Human Retroviruses and Related Infections, Washington DC, Jan. 29 - Feb. 2, 1995, Abstract #283. 11. Lipson SM, Soni M, Biondo FX et al.: Antiviral susceptibility testing-flow cytometric analysis (AST-FCA) for the detection of cytomegalovirus drug resistance. Diagn Microbiol Infect Dis 28:123-129, 1997. 12. Lurain NS, Ammons, HC, Kapel KS et al.: Molecular analysis of human cytomegalovirus strains from two lung transplant recipients with the same donor. Transplantation 62:497-502, 1996. 13. Lurain NS, Spafford LE, Thompson KD:

Vol. 19, No. 1/2, 1999

Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir. J Viro168:4427-4431, 1994. 14. Mazeron MC, Jahn G, Plachter B: Monoclonal antibody E-13 (M-810) to human cytomegalovirus recognizes an epitope encoded by exon 2 of the major immediate early gene. J Gen Virol 73:2699-2703, 1992. 15. McSharry JJ: Uses of flow cytometry in virology. Clin Microbiol Rev 7:576-604, 1994. 16. McSharry JJ: Flow cytometry based antiviral resistance assays. Clin Immunol Newslett 9:113119, 1995. 17. McSharry JJ: Flow cytometric analysis of virally infected cells: in vitro and in vivo studies. In Rapid Detection of Infectious Agents, Steven Specter, Mauro Bendinelli, and Herman Friedman, eds., Plenum Press, New York, 1998, p. 39-56. 18. McSharry JJ, Lurain NS, Drusano GL et al.: Flow cytometric determination of ganciclovir susceptibilities of human cytomegalovirus clinical isolates. J Clin Microbiol 36:958-964, 1998. 19. McSharry JJ, Lurain NS, Drusano GL et al.: Rapid ganciclovir susceptibility assay using flow cytometry for human cytomegalovirus clinical isolates. Antimicrob Agents Chemother 42:2326-2331, 1998. 20. McSharry JJ, Luarain NS, McDonough AC et al.: Drug susceptibilities of human cytomegalovirus (HCMV) clinical isolates as determined by flow

cytometry. Abs of 38th ICAAC, San Diego, CA, Sept. 24-27, 1998, Abstract # H-106. 21. McSharry JJ, Talarico C, Davis M et al.: Comparison of phenotypic assays for determining susceptibility of human cytomegalovirus (HCMV) to 1263W94 and ganciclovir. Abs 38th ICAAC, San Diego, CA Sept. 24-27, 1998, Abstract #H- 107. 22. Palestine AG, Polis MA, DeSmet MD et al.: A randomized controlled trial of foscarnet in the treatment of cytomegalovirus retinitis in patients with AIDS, Ann Intern Med 115:665-673, 1991. 23. Pavic I, Hartmen A, Zimmermann Aet al.: Flow cytometric analysis of herpes simplex virus type 1 susceptibility to acyclovir, ganciclovir, and foscarnet. Antimicrob Agents Chemother 41:2686-2692, 1997. 24. Pepin J-M, Simon E Dussault Aet al.: Rapid determination of human cytomegalovirus susceptibility to ganciclovir directly from clinical specimen primocultures. J Clin Microbiol 30:2917-2920, 1992. 25. Polis MA, Spooner KM, Baird BF et al.: Anticytomegalovirai activity and safety of cidofovir in patients with human immunodeficiency virus infection and cytomegalovirus viruria. Antimicrob Agents Chemother 39:882-886, 1995. 26. Stanat SC, Reardon JE, Erice EJ et al.: Ganciclovir-resistant cytomegalovirus clinical isolates: Mode of resistance to ganciclovir. Antimicrob Agents Chemother 35:129 l-1297, 1981.

Flow Cytometric Testing of Susceptibilities of Mycobacterium tuberculosis Ronald F. Schell, 1'2'3 Andrea V. M o o r e , 1'2 Renee M. Vena, 1'2 Scott M. Kirk, 4 and Steven M. Callister s'6 Wisconsin State Laboratory of HygieneI and Departments of Medical Microbiology and Immunology2 and Bacteriology, S University of Wisconsin, Madison, Wisconsin 53706; Microbiology Research Laboratory5 and Department of lnfectious Diseases,6 Gundersen Lutheran Medical Center, LaCrosse, Wisconsin 54601; and Bio-Rad Laboratories, Hercules, California 945474 Introduction A f t e r steadily d e c r e a s i n g f r o m 1952 to 1985, the n u m b e r o f cases o f tuberculosis increased by 2 0 % b e t w e e n 1985 and 1992. In r e s p o n s e to the r e s u r g e n c e o f tuberculosis and increases in resistance to antim y c o b a c t e r i a l agents t5 the Centers for D i s e a s e C o n t r o l and P r e v e n t i o n ( C D C ) stated that rapid and accurate susceptibility testing o f Mycobacterium tuberculosis is essential and should be p e r f o r m e d for control o f the disease. 2 R e d u c i n g the t i m e r e q u i r e d for susceptibility testing w o u l d greatly i m p r o v e the care o f patients and

0197-1859/99 (see frontmatter)

the control o f the disease. Classically, susceptibility testing o f M. tuberculosis has been p e r f o r m e d by g r o w i n g the tubercle bacillus on m e d i u m in the presence or absence of antituberculosis agents for two or three w e e k s o f incubation before obtaining results. 6 This is called the proportion m e t h o d and it is the " g o l d standard" for susceptibility testing o f M. tuberculosis. A n u m b e r o f methods, however, are practiced or h a v e been p r o p o s e d that greatly decrease the time r e q u i r e d to o b t a i n s u s c e p t i b i l i t y test results. The m o s t frequently used method, B A C T E C - 4 6 0 , requires four to 12 days o f

© 1999 Elsevier Science Inc.

incubation before results are available. 71° A p p r o x i m a t e l y nine days after initiation o f testing p r o c e d u r e s B A C T E C results are reported f r o m our laboratory. To decrease further the time required for susceptibility testing, n e w e r m e t h o d s h a v e u t i l i z e d m e t a b o l i c activity detected by fluorescent dyes, tl'12 q u a n t i f i c a t i o n o f total m y c o bacterial - R N A , 1 3 h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y m y c o l i c acid analysis TMand a b i o l u m i n e s c e n c e assay for d e t e c t i o n o f m y c o b a c t e r i a l A T E 15 M o r e recently, Jacobs et al. 16 described a rapid m e t h o d for drug susceptibility test-

Vol. 19, No. 1/2, 1999

ing by means of luciferase reporter phages. Collectively, these approaches have reduced the time required for obtaining susceptibility results, from weeks to days. Unfortunately, none of these rapid methods, except BACTEC, is used routinely in clinical microbiology laboratories. We demonstrated recently that susceptibility testing of M. tuberculosis can be accomplished rapidly by using flow cytometry. Results of tests are available within 24 hours after M. tuberculosis organisms were incubated with ethambutol, isoniazid, rifampin, or streptomycin. 17 The method is based on the ability of mycobacteria to hydrolyze fluorescein diacetate (FDA) to free fluorescein by nonspecific cellular esterases. Accumulation of fluorescein in metabolically active mycobacterial cells can then be easily. detected by using a flow cytometer. By contrast, mycobacterial organisms that are killed or inhibited by antimycobacterial agents hydrolyze significantly less FDA and therefore have less fluorescence. Fluorescein-labeled bacteria can be easily detected by the flow cytometer (even if free fluorescein is present in the medium containing M. tuberculosis organisms), in the presence or absence of antituberculosis agents. Background fluorescein in the medium is not detected by the flow cytometer. The flow cytometric susceptibility test is performed as follows, t8 Clinical isolates of M. tuberculosis are cultured in 5.0 ml of 7H9 broth at 37"C in the presence of 5% CO2 until the turbidity of the suspension is equivalent to a McFarland 1.0 standard. Once sufficient growth is obtained, two-fold dilutions (volume 0.5 ml) of the antimycobacterial agents or the concentrations recommended by the National Committee for Clinical Laboratory Standards 6 are inoculated with 0.5 ml of lxl06 M. tuberculosis organisms. Drug-free suspensions of M. tuberculosis organisms are also included as controls. The suspensions are then incubated for 24 hours at 37°C in the presence of 5% CO2. Although lesser numbers of M. tuberculosis cells (>103 <106) can be used, the large size of the inoculum has a distinct advantage. The larger the inoculum, the greater the chance of detecting resistant organisms by the flow cytometric assay. After incubation, 0.2 ml of each assay

C L I N I C A L I M M U N O L O G Y Newsletter 15

suspension is removed and placed in a sterile screwcap microtube containing 0.2 ml of FDA (Sigma Chemicals, St. Louis, MO) prepared at 500 ng/ml in phosphate buffered saline at pH 7.4. Samples are then incubated at 370C for 30 min before being analyzed by a flow cytometer. Instruments manufactured by Becton Dickinson and BioRad can be used for detection of fluorescein-labeled M. tuberculosis cells. Initially, unstained viable M. tuberculosis cells are detected and differentiated from nonM. tuberculosis particles in 7H9 medium by forward and side angle light scatter. TMBackground events (particles) in the 7H9 medium and electronic noise are eliminated by thresholding. Subsequently, viable M. tuberculosis cells incubated in the presence or absence of antimycobacterial agents for 24 hours and stained with FDA are processed by the flow cytometer. The susceptibility or resistance of an isolate of M. tuberculosis to antituberculosis agents is determined by use of a susceptibility index. Calculation of the susceptibility index eliminates variability among isolates of M. tuberculosis to hydrolyze FDA in the absence of antituberculosis agents. The susceptibility index is calculated as follows. The mean channel fluorescence values obtained for an isolate of FDA-stained M. tuberculosis cells in the presence or absence of antimycobacterial agents are divided by the number of channels per log decade. The antilog of these values is then used to obtain the relative linear fluorescence value for each sample analyzed. Finally, the relative fluorescence value of each drug-containing sample is divided by the relative fluorescence value of the drugfree control to obtain the susceptibility index. Although the susceptibility index appears to be complicated, it is relatively easy to calculate. We conservatively set the cutoff value at 0.75. An isolate was considered susceptible to an antimycobacterial agent if the susceptibility index was 0.75 or less. We next determined the susceptibilities of 35 clinical isolates of M. tuberculosis to various concentrations of isoniazid (0.2, 1.0 and 5.0 lag/ml) using both the proportion method and the flow cytometry susceptibility index. Overall, there was agreement between the two methods for © 1999 ElsevierScienceInc.

100 of the 105 total tests (95%). Three of the discrepancies were obtained at lower concentrations of isoniazid by flow cytometry. However, identical results were obtained at the next higher concentration of isoniazid. Another isolate was resistant to 0.2 lag/ml of isoniazid by the proportion method but susceptible by the flow cytometric procedure. When higher concentrations (0.1 and 5.0 lag/ml) were tested, identical results were obtained by the two methods. Another isolate was resistant to 5.0 lag of isoniazid by flow cytometry, but it was susceptible by the proportion method. This isolate, however, was resistant to the lower concentrations of isoniazid by both methods. In other studies, 26 isolates of M. tuberculosis were tested for susceptibility to 5 lag of ethambutol/ml. Agreement (92%) between the proportion method and the flow cytometric procedure was reached for 24 of the 26 isolates. When 24 isolates of M. tuberculosis were tested for susceptibility to 1 lag of rifampin/ml, four discrepancies were detected. Three isolates were resistant to 1.0 lag of rifampin/ml by the flow cytometric procedure but susceptible by the proportion method. By contrast, the fourth isolate was resistant by the proportion method but susceptible by flow cytometry. The overall agreement was 83%. A possible explanation for the discrepancies is the metabolic activity of the M. tuberculosis cells. If the majority of the population of M. tuberculosis cells used in the proportion method or the flow cytometric susceptibility test was not in the exponential growth phase, discrepancies can easily occur. With the proportion method, inactive M. tuberculosis cells would not be readily affected by the various concentrations of the antituberculosis agents. Likewise, hydrolysis of FDA would be inhibited. Therefore, similar levels of hydrolysis of FDA would be detected by the flow cytometer in the drug-treated suspensions of M. tuberculosis cells and the drug-free controls. We have detected discrepancies if nonlog-phase mycobacteria are used for testing by flow cytometry. Generally, these isolates are reported as resistant. Fortunately, results obtained by the flow cytometric method can be verified rapidly. Cultures of M. tuberculosis cells with the 0197-1859/99 (see frontmatter)

16 C L I N I C A L I M M U N O L O G Y

antimycobacterial agent are incubated for an additional 24 hours, exposed to FDA, and analyzed by flow cytometry. This replication practice cannot be performed by any other presently used tuberculosis susceptibility testing method. Another explanation for the discrepancies is the selection of the susceptibility index cutoff value. We conservatively set the cutoff value at 0.75. The value could have been raised to 0.90, 0.85, 0.80, 0.76 or any numerical value within these numbers. By increasing the cutoff value to 0.90, most of the discrepancies would not have been detected. Finally, it is assumed that the proportion method is correct because it is the gold standard. 7 However, usage of non-log-phase cultures of M. tuberculosis cells or selection of a subpopulation of resistant or susceptible organisms within the population of M. tuberculosis cells being tested can yield conflicting results. Although the flow cytometric susceptibility test for M. tuberculosis is rapid (24 hours), a major concern has been biosafety. It is possible that aerolization of droplet nuclei containing viable M. tuberculosis could occur at several points in the procedure, including sample aspiration, ejection of the sample from the nozzle inside the flow chamber, and during decontamination. Therefore, the test procedure is suitable only for public health laboratories or large reference laboratories with a Biosafety Level 3 tuberculosis facility and experience with Biosafety Level 3 precautions. Although this excludes many laboratories from performing the test, this is not bad. Generally, large reference and public health laboratories have the most experience working with M. tuberculosis. Centralization of isolation of the organism, identification, and susceptibility testing of M. tuberculosis does improve the quality of patient care and public health measures to control the disease. Recently, Moore et al. 19developed a biologically safer flow cytometric susceptibility test for M. tuberculosis. The test depends on detection and enumeration of actively growing M. tuberculosis organisms in drug-free and antimycobacterial agent containing medium. The susceptibilities of 17 clinical isolates of M. tuber-

0197-1859/99 (see frontmatter)

Vol. 19, No. 1/2, 1999

Newsletter

culosis to ethambutol, isoniazid, and rifampin were tested by the agar proportion and flow cytometric methods. Subsequently, all flow cytometric susceptibility test samples were inactivated by exposure to paraformaldehyde before analysis by a flow cytometer. Agreement between the results from the two methods was 98%. Results are available 72 hours after initiation of testing procedures. However, the authors still do not recommend processing samples outside a Biosafety Level 3 tuberculosis facility. In conclusion, flow cytometric susceptibility assays (FDA dependent or dependent on enumeration of bacteria) will greatly improve susceptibility testing for M. tuberculosis. The assays are extremely simple to perform and can be completed in 72 hours or less after initiation of testing. The actual test components cost less than $3.00. Although the cost of a flow cytometer is high, when the high cost of supplies for performing susceptibility testing by the BACTEC method is considered, the cost of a flow cytometer can be justified. References 1. Bitch AB, Cauthen GM, Onorato IM et al.: Nationwide survey of drug-resistant tuberculosis in the United States. JAMA 271:665-671, 1994. 2. Centers for Disease Control and Prevention. Tuberculosis morbidity - - United States, 1995. Morbid Mortal Weekly Rep, 45:365-370, 1996. 3. Cohn DL, Bustreo F, Raviglione MC: Drugresistant tuberculosis: review of the worldwide situation and the WHO/IUATLD global surveillance project. Clin Infect Dis 24 (Suppl. 1): S121-S!30, 1997. 4. Daniel TM, Debanne SM: Estimation of the annual risk of tuberculosis infection for white men in the United States. J Infect Dis 175:15351537, 1997.

radiometric and conventional methods. Antimicrob Agents Chemother 27:11-15, 1985. 8. Lee C, Heifets LB: Determination of minimal concentrations of antituberculosis drugs by radiometric and conventional methods. Am Rev Respir Dis 136:349-352, 1987. 9. Roberts GD, Goodman NL, Heifets LB et al.: Evaluation of the BACTEC radiometric method for recovery of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis from acid-fast smear positive specimens. J Clin Microbiol 18:689-696, 1983. 10. Siddiqi SH, Hawkins JE, Laszio Aet al.: lntedaboratory drug susceptibility testing of Mycobacterium tuberculosis by a radiometric procedure and two conventional methods. J Clin Microbiol 22:919-923, 1985. 11. Jarnagin JL, Luchsinger DW: The use of fluorescein diacetate and ethidium bromide as a stain for evaluating viability of mycobacteria. Stain Technol 55:253-257, 1980. 12. Kaprelyants S, Kell DB: Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry. J App Bacteriol 72:410-422,

1992. 13. Kawa DE, Pennell DR, Kubista LN et al.: Development of a rapid method for determining the susceptibility of Mycobacterium tuberculosis to isoniazid by using the Gen-Probe DNA Hybridization System. Antimicrob Agents Chemother 33:1000-1005, 1989. 14. Garza-Gonzales E, Guerrero-Olazaran M, Tijerina-Menchaca R et al.: Determination of drug susceptibility of Mycobacterium tuberculosis through mycolic acid analysis. J Clin Microbiol 35:1287-1289, 1997, 15. Arain TM, Resconi AE, Hickey MJ et al.: Bioluminescence screening in vitro (Bio-Siv) assays for high-volume antimycobacterial drug discovery. Antimicrob Agents Chemother 40:1536-1541, 1996. 16. Jacobs WR Jr, Barlena RG, Udani R et al.: Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 260:819-822, 1993.

5. Raviglione MC, Snider DE Jr., Kochi A: Global epidemiology of tuberculosis: morbidity and mortality of a worldwide epidemic. JAMA 273:220-226, 1995.

17. Norden MA, Kurzynski TA, Bownds SE et al.: Rapid susceptibility testing of Mycobacterium tuberculosis (H37Ra) by flow cytometry. J Clin Microbiol 33:1231-1237, 1995.

6. National Committee for Clinical Laboratory Standards: Antimycobacterial susceptibility testing for Mycobacterium tuberculosis. Proposed standard M24-T. National Committee for Clinical Laboratory Standards, Villanova, PA, 1995.

18. Kirk SM, Schell RE Moore AV et al.: Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid and rifampin in 24 hours. J Clin Microbiol 36:1568-1573, 1998.

7. Heifets LB, Iseman MD, Cook JLet al.: Determination of in vitro susceptibility of Mycobacterium tuberculosis to cephalosporins by

© 1999 Elsevier Science Inc.

19. Moore AV, Kirk SM, Callister SM et al.: Safe determination of susceptibility of Mycobacterium tuberculosis to antimycobacterial agents by flow cytometry. J Clin Microbiol, in press.