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Molecular approaches to the identification of mycobacteria
Clinical Microbiology Newsletter October 1, 1992
Vol. 14, No. 19
Molecular Approaches to the Identification of Mycobacteria Edward P. Desmond, Ph.D. Research Scientist Microbial Diseases Laboratory California Department of Health Services Berkeley, CA 94704 A renewed interest in mycobacterial infections has been kindled by the occurrence of Mycobacterium avium infections in patients with acquired immunodeficiency syndrome (AIDS) and by an ominous reversal in the late 1980s of the trend of steady annual decreases in the incidence of tuberculosis (TB) in the USA. During the years 1984 to 1988 there were nearly 15,000 cases of TB above and beyond the number expected based on past experience (1). These excess cases are believed to be a function of the susceptibility of AIDS patients to TB and the spread of the infection by TB-infected AIDS patients, of immigration, and of overcrowding in prisons and homeless shelters. Fortunately, this era has coincided with technological developments improving the turnaround time for detection, identification, and susceptibility testing of mycobacteria. Conventional techniques using Middlebrook agar and egg-based media usually take 3 to 5 wk for growth of M. tuberculosis, and biochemical identification and susceptibility testing then require another similar incubation period. This 6-10-wk tumaround time was fast reduced by two to several weeks by the radiometric BACTEC 460 instrument from Becton Dickinson Diagnostic Instrument Systems (Sparks, MD). Further reductions in the time required to identify mycobacteria have been achieved by using commercial CMNEE/14(19)145-152,1992
(Gen-Probe and Syngene) DNA probes or high performance liquid chromatography (HPLC). These rapid, accurate techniques have contributed to improved patient care (2). Rapid detection and identification ofmycobacteria allow early implementation of control measures such as respiratory isolation of patients and an early start to presumptive chemotherapy regimens. Rapid susceptibility testing may allow early administration of effective therapy and/or prophylaxis for those exposed, even in cases of drug-resistant tuberculosis. A further dramatic improvement in the rapidity and accuracy of laboratory testing may be expected in the near future from application o f p e w e r molecular biology techniques to the mycobacteriology laboratory. The polymerase chain reaction (PCR) has the potential to detect, amplify, and identify very small quantities of mycobacterial DNA directly in a clinical specimen, possibly the same day it is collected (3, 4). Other techniques, including DNA hybridization (5), restriction fragment length polymorphism (6), and DNA amplification and sequencing (7), offer an opportunity for larger research and reference laboratories to improve the accuracy of mycobacterial identifications and trace outbreaks or epidemics of mycobacterial infections. BACTEC The BACTEC method, which began the move toward quicker mycobacteriolElsevier
ogy laboratory results, is a radiometric method in which specimens decontaminated by conventional techniques or blood specimens are inoculated into a broth medium containing 14C-labeled nutrients. If mycobacteria are present, they may be detected by production of radioactive CO2. The BACTEC system also identifies the TB complex using NAP (para-nitro-alphaacetyl-aminobetahydroxypropiophenone) and allows rapid susceptibility testing by the presence or absence of growth (characterized by 14CO2production) in broths containing various levels of antimycobacterial drugs. Problems with some BACTEC instruments have occurred as a result of lransfer of organisms by the needles that penetrate the rubber septum to sample the head-space gas mixture for the presence of 14CO2. In order to mitigate this problem, the manufacturer has re-
In This Issue Therapeutic Drug Monitoring Methods for the Antimicrobial Agents . . . . . . . . . . . . . . . . . . . . . . . 153 A discussion of the various methods and approaches for assaying antibiotic levels in body fluids Cholera in the Americas: Crisis or Catalyst . . . . . . . . . . . . . . . . . . . . A century-old pathogen is now threatening the population of the new world
156
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cently modified the instrument to increase the needle heating time and has recommended an accelerated schedule of routine replacement of the needleheating units. Users should be aware, however, that cross-contaminafion of bottles can occur, and that the bottle that becomes contaminated may not necessarily be the adjacent bottle in the run. The next step in speeding up mycobacteriology laboratory results came when both BACTEC and non-BACTEC laboratories began to use the molecular techniques that are the primary focus of this article.
AccuProbe TM
SNAP® probe probes
////// A T A G C A T A T C G T
//////
Nucleic acid from lysate of test organism
fives c a n o c c u r
(8).
DNA prohes have worked well for identifying cultural isolates. The organisms to be identified are lysed, and their nucleic acids extracted and reacted with the probe(s). The probes consist of labeled DNA segments with a sequence of nucleotides unique to mycobacteria. They hybridize with specific complementary sequences in the ribosomal RNA of the organism being identified, or in the DNA that codes for ribosomal RNA. The probes are linked with chemical labels, either acridinium es-
T A T C G T
//////
bound to porous nylon membrane acridinium ester
////I//
hybrid
A T A G C A DNA Probes Beginning in 1987, DNA probes for identifying a fimited number of species of mycobacteria became commercially available. Currently, two manufacturers, Gen-Probe and Syngene, market DNA probes for mycobacteria. The currently available probes identify M. tuberculosis and M. bovis (TB complex), M. avium-intracellulare complex (MAC), M. kansasii, and M. gordonae. Probes are used to identify mycobacteria following culture, since their sensitivity in detecting mycobacteria directly in specimens is approximately 100-fold less than culture (threshold of detection approximately 104 per ml versus approximately 102 per ml), and false posi-
////// A T A G C A
÷
:: :" "" ";: 7: :: T A T C G T
//////
1
Selective inactivation of acridinium esters on unhybridized single-stranded probe by selection reagent.
////// A T A G C A T A T C G T
//////
nylon membrane
Wash away uni 'bridized probe.
/ detection reagent
I
added
chemiluminescence
(If complementary sequence is~not present in the test organism, probe will remain single-stranded and acridinium ester label will be inactivated
Addition of enz' rme substrate and color develo )ment (If complementary sequence is not present in the test organism, probe will remain single-stranded and will not bind to solid phase.)
by selection reagent. Wash step not necessary.)
Figure 1. Schematic of DNA probes.
ters (C-en-Probe AccuProbe) or alkaline phosphatase (Syngene SNAP probe). The label is incoqxxated into a doublestranded hybrid only if the mycobacterium has the specific unique sequence complementary to the probe. Doublestranded hybrids are then separated from unhybridized single-stranded probe by binding to a nylon membrane
filter followed by a wash step (SNAP probe), or the (acridinium ester) singlestranded label is inactivated using a selection reagent (AccuProbe) (Fig. 1). Presence of the hybrid is then detected, either by chemiluminescence (AccuProbe) or by adding alkaline phosphatase substrate to produce a color reaction (SNAP probe).
N o ' r E : No tespemibility i= sssmned by the Publisher for any injury and/or damage to persons or propelty as a nmtter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas onntained in the material herein. No suggested test or procedure should be carried out unless, in the reader's judgment, its risk is justified. Because of rapid advances in medical sciences, we reonmmend that the independent verification of diagncees and drag dosages should he made. Discussions, views and r e c e m d a t i o n s as to medical procedures, choice of drags and drag dosages are the responsibility of the authors.
CiinicalMicrobiology Newsletter (ISSN 01964399) is issued twice monthly in one indexed volume per year by Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010. Subscliption price per year:. $135.00 including postage and handling in the United States, Canada, and Mexico. Add $52.00 for postage in the test of the world. Second-class postage paid at New York, NY and at additional mailing offices. Postmaster. Send address changes to Cliaical Microbiology Newsletter, Elsevier Science Publishing Co., Inc., 655 Avenue of the AmericaJ, Hew York, NY 10010.
146
0196-4399/92250.00 + 03.00
© 1992 Elsevier Science Publishing Co., Inc.
Clinical Microbiology Newsletter 14:19,1992
For identifying cultural isolates, DNA probes have shown very good sensitivity and specificity. M. tuberculosis complex probes have shown sensitivity close to 100% with clinical isolates when biochemical identification confirmed by HPLC is used as the "gold standard" of organism identification (8, 9). The specificity has also been quite good, with only a few strains of M. terrae complex giving false positive resuits with the M. tuberculosis complex probe. M. terrae complex isolates are infrequent in the clinical laboratory, so the specificity of the M. tuberculosis probe exceeds 99% (9). Probes for the M. avium-intracellulare complex have shown a high degree of specificity and satisfactory sensitivity. Specificity of both commercially available probes has been 97% or better (9, 10) when compared with the biochemical reaction pattern as the "gold standard." The sensitivities of commercial DNA probes for identification of MAC have been less favorable than specificities, probably because of genotypic diversity among the organisms that fit biochemically into the M. avium complex. The frequency of MAC isohates that do not react with the probes probably varies from one geographic region to another and from one patient population to another. The Syngene SNAP probe offers a third probe, the MAC "X" probe, along with probes for M. avium and M. intracellulare. When all three probes or a pool of probes is used, the SNAP probe has been reported to have a sensitivity of 97% (11) for MAC isolates. The Gen-Probe radioactive MAC probe has been reported to have a different geographic regions (8, 10). A nonradioactive probe (Accu- Probe) has succeeded this product, and studies have shown that it is somewhat less sensitive than the SNAP probe for identifying MAC isolates (9, 12). However, Gen-Probe has developed and evaluated a new version of the AccuProbe for MAC, which it plans to market in the near future. This product has improved sensitivity and may approach the sensitivity of the SNAP probe for MAC. Users of the AccuProbe system should be aware of a problem that has
Clinical Micmbinlogy Newsletter 14:19,1992
A Mycobacterial mycollc acid - 60 to 90 carbon branched fatty acids
C R ~ --c00H R1 = variable with keto, methoxy, carboxyl, epoxy, and other oxygen-containing structures, and/or double bonds R2 = aliphatic straight chain hydrocarbons of varying lengths B HPLC system Sample Injector
Solvents
Chromatogram
/
Pumps Mixer
Column Detector
C
"Reversed phase" column packing material
- Si-O-Si-{CH2)17CH 3
Figure 2. Highperformance liquid chromatography.
recently been reported with the sonicating waterbaths that are used for lysis of the mycobacteria (13). The quality control steps recommended by the manufacturer may indicate adequate function of the sonicating waterbath even when one or more of the transducers that produce the vibrations has failed or mineral buildup in the sonicator tank has occurred. In this situation, anomalous results may be obtained, depending on the position of the sample or control in the water bath. If probe results do not agree with clinical or cultural observations, the probe test should be repeated. DNA probes can be used to identify organisms directly from BACTEC or other broth media, or from solid media. Results are obtained within hours.
© 1992 Elsevier Science Publishing Co., Inc.
Based on experience from this laboratory and based on an assumed test batch size of eight isolates, reportable result using SNAP probe costs $16 for materials and approximately 20 min of personnel time (labor cost of $5 to $9), giving a total cost per reportable result of $21 to $25. AccuProbe, based on the same assumptions, costs about $15 per reportable result for materials and requires about 12 min labor (labor cost of $3 to $6), for a total cost per reportable result of $18 to $21. DNA probes are limited, of course, to identifying the three to five species for which commercial probes are available. These few species, especially M. tuberculosis and-MAC, make up the vast majority of isolates in clinical iabo-
0196--4399/92/$0.00 + 03.00
147
TABLE 1. Capabilities of rapid and molecular techniques for mycobacteria Range of Yields Isolate for Identificationof Mycobacteria Susceptibility Usual Turnaround CulturalIsolates? Detected/identified Testing Time Yes Wide range Yes 4-10 wk
Use Directlyon Method Conventional culture and biochemicalI.D. BACTEC
Specimen? Yes
Yes
TB complexonly
Widerange
Commercial Availability Yes
Yes
7-15 d
Yes
1 d after growth 1 d after growth
Yes
detected
DNA probes
No
HPLC a
No
Yes
Yes Yes Yes
Limitedrange Wide range Limitedrange
No No No
PCRb
MicrotiterDNA hybridization RFLPe and DNA f'mgerprinting
No
Yes
Wide range
No
7-10 d after growth
No
No
Strain I.D. for epidemiology
StrainI.D. only
No
7-10 d after growth
Some reagents
Same day specimen is collected
Yes
1-2 y?
available
a High performance liquid chromatography. b Polymerase chain reaction. c Restriction fragment length polymorphism.
ratories. Probes do not differentiate M. tuberculosis from M. bovis, which could be clinically significant because of the difference between these two species in susceptibility to pyrazinamide.
HPLC HPLC of mycobacterial mycolic acids has been developed as a method for identifying a broad range of mycobacterial species (14, 15). Mycobacterial species vary in the composition of the mycolic acids in their outer envelope (Fig. 2A). The mycolic acids from about two Ioopsful of growth can be extracted with alcoholic KOH and chloroform, then converted to a bromophenacyl ester derivative so that they will absorb UV light. The derivatized mycolic acids are then injected into an HPLC system through the sample injector (see Fig. 2B), then separated to yield a characteristic chromatogram. HPLC columns for this application are packed with a Cls straight-chain hydrocarbon bonded to a silicate backbone (Fig. 2C). Mycolic acids bind with varying affinities to this nonpolar solid phase and are eluted using a methanol-methylenechloride gradient. Different strains within a species
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may yield different chromatograms, making it necessary to construct a library of chromatograms for comparison. Computer programs are being written to aid in interpretation of the chromatogmms for identification. Initially, weeks to months of lead time and are required for gaining experience with the method and building a library of chromatograms. HPLC instruments generally cost $30,000 to $40,000, a high initial cost, except for those laboratories that have bought instruments for additional uses. The pluses of HPLC are that it identifies a much broader range of mycobacteria than commercial probes and costs much less for reagents and supplies (about $3) per reportable resul0. In our laboratory, by botching isolates for identification in groups of 16, labor time for HPLC is approximately 45 min per reportable result, including time required for interpretation of the chromatogram. This results in a labor cost of $11 to $20 per reportable result, for a total cost of $13 to $22 per result. HPLC is rapid, versatile, and cost effective for large laboratories and reference laboratories.
PCR The polymerase chain reaction will
© 1992 ElsevierSciencePublishingCo., Inc.
probably revolutionize the way in which laboratory mycobacteriology is performed. Small amounts of mycobacterial DNA in a clinical specimen can serve as a template and be amplified by PCR using a polymerase enzyme and nucleotides (3, 4). Primers are used to select the area of the genome to be amplified, and after amplification a specific DNA probe is used to detect the amplified DNA. This can be done the same day that the specimen is collected, and the sensitivity of the method may enable it to detect one or a few organisms in a specimen. Commercial applications of PCR for mycobacteria are under development. Several problems and limitations of this method are worth noting: (i) Crosscontamination can be a problem with PCR. During 25 to 30 cycles of amplification, a single segment of DNA is amplified 33 million- to one billionfold. If a single piece of the amplified product contaminates a subsequent specimen, a false positive result could be obtained. However, the amplified DNA can be chemically modified to prevent it from serving as a template in later tests. (ii) As with DNA probes, the range of mycobacteria detected by PCR is limited to the species probed for. Cul-
Clinical MicrobiologyNewsletter14:19,1992
ture will be needed to detect other species. (iii) Although PCR detects and identifies mycobacteria in clinical specimens quickly, it does not detect their susceptibility or resistance to antimicrobial drugs. Culture may be necessary for susceptibility testing. It is also possible to use PCR to look for resistance to antibiotics by amplifying and probing for genes that code for resistance (16). (iv) Potential interference with the amplification process by inhibitorspresent in clinical specimens has yet to be evaluated. (v) At present, closelyrelated mycobacteria such as M. tuberculosis and M. bovis cannot be differentiated by the probes that are used to detect the amplified DNA product. DNA Hybridization Fu.aki and co-workers have recently published a microdilution plate DNAhybridization technique for species identification of mycobacterial isolates (5). Wells of a microdilution plate were coated with DNA from reference strains of mycobacteria. Unknown mycobacteria could then be identified by growing them in broth, exlracting their DNA, labeling it with photobiotin, hybridizing to the DNA coated on the wells of the microdilution plate, then detecting hybridization using an avidin-enzyme conjugate. The method could be commercially developed and serves to identify a broad range of mycobacteria. RFLP/DNA Fingerprinting Restriction fragment length polymorphism has proven useful for strain identification (to detect cross-contamination
References 1. Fox, J. 1990. TB: A grim disease of numbers. ASM News 56:363-365. 2. Good, R. C., and T. D. Masta'o. 1989. The modem mycobacteriology laboratory: how it can help the clinician. Clin Chest Med 10:315-322. 3. Eisenstein, B. 1990. The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis. N. Engl. J. Med. 322:178-183. 4. Peter, J. 1991. The polymerase chain reaction: amplifying our options. Rev. Inf. Dis. 13:166-171. 5. Kusunoki, S. et al. 1991. Application of colorimetric microdilution plate hybridization for rapid genetic identification of 22 Mycobacterium species. J. Clin. Microbiol. 29:1596-1603. 6. Clark-Curtiss,J. 1990. Genome structure of mycobacteria,p.77-96. In L McFadden (ed.), Molecular biology of the mycobacteria. Academic Press, San Diego.
7. Rogall, T. et al. 1990. Towards a phylogeny and defmition of species at the molecular level within the genus Mycobacterium. Int. J. Syst. Bacteriol. 40:323-330. 8. McFadden, L, Z. Kunze, and P. Seechurn. 1990. DNA probes for detection and identification, p. 139-172. In Molecular biology of the mycobacteria. Academic Press, San Diego. 9. Lira, S. et al. 1991. Genotypic identification of pathogenic Mycobacteriwn species by using a nonradioactive oligonucleotide probe. J. Clin. Microbiol. 29:1276--1278. 10. Musial, C. E. et al. 1988. Identification of mycobacteria from culture by using the Gen-probe Rapid Diagnostic System for Mycobacterium aviwn complex and Mycobacterium tuberculosis complex. J. Clin Microbiol. 26:2120-2123. 11. Cregan, P. et al. Abstr. Annu. Meet. Am. Soc. Microbiol. 1991, U14, p. 144. 12. Pratt-Rippin, K., and G. S. Hall. Abstr. Annu. Meet. Am. Soc. Microbiol. 1991, U12, p. 144. 13. Walton, D. T., and M. Valesco~ 1991. Identification Mycobacterium gordonae from culture by the Gen-Probe rapid diagnostic system: evaluation of 218 isolates and potential sources of false-negative results. J. Clin. Microbiol. 29: 1850-1854. 14. Butler, W. R., D. G. Aheam, and J. O. Kilburn. 1986. High-performance liquid chromatography of mycolic acids as a tool in the identification of Corynebacterium, Nocardia, Rhodococcus, and Mycobacterium species. J. Clin. Microbiol. 23:182-185. 15. Josk K., D. Dunbar, and L. Elliott.Abstr. Annu. Meet. Am Soc. Microbiol.1990, U31, p. 146. 16. Arthur, M. et al. 1990. Detection of ¢rythromycin resistance by the polymerase chain reaction using primers in conserved regions of erm rRNA methylase genes. Antimicrob. Agents Chemother. 34:2024-2026.
able to this organism occurs in approximately 0.2 to 0.4% of all live births and generally develops within the first month of life (2). Estimates of genital GBS colonization during pregnancy r a n ~ from 5 to 28% of all pregnant women (1). Of the infants born to colonized mothers, 50 to 75% will become colonized themselves during birth and 1 to 2% will develop GBS disease (2).
G B S Disease The two major syndromes associated with GBS are early onset and late onset disease. Early onset disease occurs within hours of birth and is due to transmission of the organism from a colonized mother to her infant prior to or during the birth process (1). Manifestations of early onset GBS infection in newborns include pneumonia, sepsis,
© 1992 Elsevier Science PublishingCo., Inc.
0196-4399/92/$0.00 + 03.OO
or trace epidemics) (6). In this technique, DNA is extracted from swains of mycobactetia and then subjected to cleavage by restriction endonuclease(s). Using small, labeled DNA probes, variants among restriction fragments can be detected by the method of Southern blotting. Conclusion Table 1 summarizes the uses and limitations of the methods mentioned in this article. In addition to improving the turnaround time and accuracy of mycobacterial laboratory work, these methods have made life interesting for those of us who work in these laboratoties. With PCR, this exciting developmental phase will likely continue for some time into the future.
Editorial
Cervical Screening Tests for Group B Streptococci Audrey R. Wanger, Ph.D. Department of Pathology University of Texas Health Science Center Houston, Texas 77025 Group B streptococci (GBS) are a leading cause of neonatal infections in the United States (1). Disease attribut-
Clinical MicrobiologyNewsletter 14:19,1992
149
Vol. 14, No. 19
Molecular Approaches to the Identification of Mycobacteria Edward P. Desmond, Ph.D. Research Scientist Microbial Diseases Laboratory California Department of Health Services Berkeley, CA 94704 A renewed interest in mycobacterial infections has been kindled by the occurrence of Mycobacterium avium infections in patients with acquired immunodeficiency syndrome (AIDS) and by an ominous reversal in the late 1980s of the trend of steady annual decreases in the incidence of tuberculosis (TB) in the USA. During the years 1984 to 1988 there were nearly 15,000 cases of TB above and beyond the number expected based on past experience (1). These excess cases are believed to be a function of the susceptibility of AIDS patients to TB and the spread of the infection by TB-infected AIDS patients, of immigration, and of overcrowding in prisons and homeless shelters. Fortunately, this era has coincided with technological developments improving the turnaround time for detection, identification, and susceptibility testing of mycobacteria. Conventional techniques using Middlebrook agar and egg-based media usually take 3 to 5 wk for growth of M. tuberculosis, and biochemical identification and susceptibility testing then require another similar incubation period. This 6-10-wk tumaround time was fast reduced by two to several weeks by the radiometric BACTEC 460 instrument from Becton Dickinson Diagnostic Instrument Systems (Sparks, MD). Further reductions in the time required to identify mycobacteria have been achieved by using commercial CMNEE/14(19)145-152,1992
(Gen-Probe and Syngene) DNA probes or high performance liquid chromatography (HPLC). These rapid, accurate techniques have contributed to improved patient care (2). Rapid detection and identification ofmycobacteria allow early implementation of control measures such as respiratory isolation of patients and an early start to presumptive chemotherapy regimens. Rapid susceptibility testing may allow early administration of effective therapy and/or prophylaxis for those exposed, even in cases of drug-resistant tuberculosis. A further dramatic improvement in the rapidity and accuracy of laboratory testing may be expected in the near future from application o f p e w e r molecular biology techniques to the mycobacteriology laboratory. The polymerase chain reaction (PCR) has the potential to detect, amplify, and identify very small quantities of mycobacterial DNA directly in a clinical specimen, possibly the same day it is collected (3, 4). Other techniques, including DNA hybridization (5), restriction fragment length polymorphism (6), and DNA amplification and sequencing (7), offer an opportunity for larger research and reference laboratories to improve the accuracy of mycobacterial identifications and trace outbreaks or epidemics of mycobacterial infections. BACTEC The BACTEC method, which began the move toward quicker mycobacteriolElsevier
ogy laboratory results, is a radiometric method in which specimens decontaminated by conventional techniques or blood specimens are inoculated into a broth medium containing 14C-labeled nutrients. If mycobacteria are present, they may be detected by production of radioactive CO2. The BACTEC system also identifies the TB complex using NAP (para-nitro-alphaacetyl-aminobetahydroxypropiophenone) and allows rapid susceptibility testing by the presence or absence of growth (characterized by 14CO2production) in broths containing various levels of antimycobacterial drugs. Problems with some BACTEC instruments have occurred as a result of lransfer of organisms by the needles that penetrate the rubber septum to sample the head-space gas mixture for the presence of 14CO2. In order to mitigate this problem, the manufacturer has re-
In This Issue Therapeutic Drug Monitoring Methods for the Antimicrobial Agents . . . . . . . . . . . . . . . . . . . . . . . 153 A discussion of the various methods and approaches for assaying antibiotic levels in body fluids Cholera in the Americas: Crisis or Catalyst . . . . . . . . . . . . . . . . . . . . A century-old pathogen is now threatening the population of the new world
156
0196-4399/92/$0.00 + 03.00
cently modified the instrument to increase the needle heating time and has recommended an accelerated schedule of routine replacement of the needleheating units. Users should be aware, however, that cross-contaminafion of bottles can occur, and that the bottle that becomes contaminated may not necessarily be the adjacent bottle in the run. The next step in speeding up mycobacteriology laboratory results came when both BACTEC and non-BACTEC laboratories began to use the molecular techniques that are the primary focus of this article.
AccuProbe TM
SNAP® probe probes
////// A T A G C A T A T C G T
//////
Nucleic acid from lysate of test organism
fives c a n o c c u r
(8).
DNA prohes have worked well for identifying cultural isolates. The organisms to be identified are lysed, and their nucleic acids extracted and reacted with the probe(s). The probes consist of labeled DNA segments with a sequence of nucleotides unique to mycobacteria. They hybridize with specific complementary sequences in the ribosomal RNA of the organism being identified, or in the DNA that codes for ribosomal RNA. The probes are linked with chemical labels, either acridinium es-
T A T C G T
//////
bound to porous nylon membrane acridinium ester
////I//
hybrid
A T A G C A DNA Probes Beginning in 1987, DNA probes for identifying a fimited number of species of mycobacteria became commercially available. Currently, two manufacturers, Gen-Probe and Syngene, market DNA probes for mycobacteria. The currently available probes identify M. tuberculosis and M. bovis (TB complex), M. avium-intracellulare complex (MAC), M. kansasii, and M. gordonae. Probes are used to identify mycobacteria following culture, since their sensitivity in detecting mycobacteria directly in specimens is approximately 100-fold less than culture (threshold of detection approximately 104 per ml versus approximately 102 per ml), and false posi-
////// A T A G C A
÷
:: :" "" ";: 7: :: T A T C G T
//////
1
Selective inactivation of acridinium esters on unhybridized single-stranded probe by selection reagent.
////// A T A G C A T A T C G T
//////
nylon membrane
Wash away uni 'bridized probe.
/ detection reagent
I
added
chemiluminescence
(If complementary sequence is~not present in the test organism, probe will remain single-stranded and acridinium ester label will be inactivated
Addition of enz' rme substrate and color develo )ment (If complementary sequence is not present in the test organism, probe will remain single-stranded and will not bind to solid phase.)
by selection reagent. Wash step not necessary.)
Figure 1. Schematic of DNA probes.
ters (C-en-Probe AccuProbe) or alkaline phosphatase (Syngene SNAP probe). The label is incoqxxated into a doublestranded hybrid only if the mycobacterium has the specific unique sequence complementary to the probe. Doublestranded hybrids are then separated from unhybridized single-stranded probe by binding to a nylon membrane
filter followed by a wash step (SNAP probe), or the (acridinium ester) singlestranded label is inactivated using a selection reagent (AccuProbe) (Fig. 1). Presence of the hybrid is then detected, either by chemiluminescence (AccuProbe) or by adding alkaline phosphatase substrate to produce a color reaction (SNAP probe).
N o ' r E : No tespemibility i= sssmned by the Publisher for any injury and/or damage to persons or propelty as a nmtter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas onntained in the material herein. No suggested test or procedure should be carried out unless, in the reader's judgment, its risk is justified. Because of rapid advances in medical sciences, we reonmmend that the independent verification of diagncees and drag dosages should he made. Discussions, views and r e c e m d a t i o n s as to medical procedures, choice of drags and drag dosages are the responsibility of the authors.
CiinicalMicrobiology Newsletter (ISSN 01964399) is issued twice monthly in one indexed volume per year by Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010. Subscliption price per year:. $135.00 including postage and handling in the United States, Canada, and Mexico. Add $52.00 for postage in the test of the world. Second-class postage paid at New York, NY and at additional mailing offices. Postmaster. Send address changes to Cliaical Microbiology Newsletter, Elsevier Science Publishing Co., Inc., 655 Avenue of the AmericaJ, Hew York, NY 10010.
146
0196-4399/92250.00 + 03.00
© 1992 Elsevier Science Publishing Co., Inc.
Clinical Microbiology Newsletter 14:19,1992
For identifying cultural isolates, DNA probes have shown very good sensitivity and specificity. M. tuberculosis complex probes have shown sensitivity close to 100% with clinical isolates when biochemical identification confirmed by HPLC is used as the "gold standard" of organism identification (8, 9). The specificity has also been quite good, with only a few strains of M. terrae complex giving false positive resuits with the M. tuberculosis complex probe. M. terrae complex isolates are infrequent in the clinical laboratory, so the specificity of the M. tuberculosis probe exceeds 99% (9). Probes for the M. avium-intracellulare complex have shown a high degree of specificity and satisfactory sensitivity. Specificity of both commercially available probes has been 97% or better (9, 10) when compared with the biochemical reaction pattern as the "gold standard." The sensitivities of commercial DNA probes for identification of MAC have been less favorable than specificities, probably because of genotypic diversity among the organisms that fit biochemically into the M. avium complex. The frequency of MAC isohates that do not react with the probes probably varies from one geographic region to another and from one patient population to another. The Syngene SNAP probe offers a third probe, the MAC "X" probe, along with probes for M. avium and M. intracellulare. When all three probes or a pool of probes is used, the SNAP probe has been reported to have a sensitivity of 97% (11) for MAC isolates. The Gen-Probe radioactive MAC probe has been reported to have a different geographic regions (8, 10). A nonradioactive probe (Accu- Probe) has succeeded this product, and studies have shown that it is somewhat less sensitive than the SNAP probe for identifying MAC isolates (9, 12). However, Gen-Probe has developed and evaluated a new version of the AccuProbe for MAC, which it plans to market in the near future. This product has improved sensitivity and may approach the sensitivity of the SNAP probe for MAC. Users of the AccuProbe system should be aware of a problem that has
Clinical Micmbinlogy Newsletter 14:19,1992
A Mycobacterial mycollc acid - 60 to 90 carbon branched fatty acids
C R ~ --c00H R1 = variable with keto, methoxy, carboxyl, epoxy, and other oxygen-containing structures, and/or double bonds R2 = aliphatic straight chain hydrocarbons of varying lengths B HPLC system Sample Injector
Solvents
Chromatogram
/
Pumps Mixer
Column Detector
C
"Reversed phase" column packing material
- Si-O-Si-{CH2)17CH 3
Figure 2. Highperformance liquid chromatography.
recently been reported with the sonicating waterbaths that are used for lysis of the mycobacteria (13). The quality control steps recommended by the manufacturer may indicate adequate function of the sonicating waterbath even when one or more of the transducers that produce the vibrations has failed or mineral buildup in the sonicator tank has occurred. In this situation, anomalous results may be obtained, depending on the position of the sample or control in the water bath. If probe results do not agree with clinical or cultural observations, the probe test should be repeated. DNA probes can be used to identify organisms directly from BACTEC or other broth media, or from solid media. Results are obtained within hours.
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Based on experience from this laboratory and based on an assumed test batch size of eight isolates, reportable result using SNAP probe costs $16 for materials and approximately 20 min of personnel time (labor cost of $5 to $9), giving a total cost per reportable result of $21 to $25. AccuProbe, based on the same assumptions, costs about $15 per reportable result for materials and requires about 12 min labor (labor cost of $3 to $6), for a total cost per reportable result of $18 to $21. DNA probes are limited, of course, to identifying the three to five species for which commercial probes are available. These few species, especially M. tuberculosis and-MAC, make up the vast majority of isolates in clinical iabo-
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TABLE 1. Capabilities of rapid and molecular techniques for mycobacteria Range of Yields Isolate for Identificationof Mycobacteria Susceptibility Usual Turnaround CulturalIsolates? Detected/identified Testing Time Yes Wide range Yes 4-10 wk
Use Directlyon Method Conventional culture and biochemicalI.D. BACTEC
Specimen? Yes
Yes
TB complexonly
Widerange
Commercial Availability Yes
Yes
7-15 d
Yes
1 d after growth 1 d after growth
Yes
detected
DNA probes
No
HPLC a
No
Yes
Yes Yes Yes
Limitedrange Wide range Limitedrange
No No No
PCRb
MicrotiterDNA hybridization RFLPe and DNA f'mgerprinting
No
Yes
Wide range
No
7-10 d after growth
No
No
Strain I.D. for epidemiology
StrainI.D. only
No
7-10 d after growth
Some reagents
Same day specimen is collected
Yes
1-2 y?
available
a High performance liquid chromatography. b Polymerase chain reaction. c Restriction fragment length polymorphism.
ratories. Probes do not differentiate M. tuberculosis from M. bovis, which could be clinically significant because of the difference between these two species in susceptibility to pyrazinamide.
HPLC HPLC of mycobacterial mycolic acids has been developed as a method for identifying a broad range of mycobacterial species (14, 15). Mycobacterial species vary in the composition of the mycolic acids in their outer envelope (Fig. 2A). The mycolic acids from about two Ioopsful of growth can be extracted with alcoholic KOH and chloroform, then converted to a bromophenacyl ester derivative so that they will absorb UV light. The derivatized mycolic acids are then injected into an HPLC system through the sample injector (see Fig. 2B), then separated to yield a characteristic chromatogram. HPLC columns for this application are packed with a Cls straight-chain hydrocarbon bonded to a silicate backbone (Fig. 2C). Mycolic acids bind with varying affinities to this nonpolar solid phase and are eluted using a methanol-methylenechloride gradient. Different strains within a species
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may yield different chromatograms, making it necessary to construct a library of chromatograms for comparison. Computer programs are being written to aid in interpretation of the chromatogmms for identification. Initially, weeks to months of lead time and are required for gaining experience with the method and building a library of chromatograms. HPLC instruments generally cost $30,000 to $40,000, a high initial cost, except for those laboratories that have bought instruments for additional uses. The pluses of HPLC are that it identifies a much broader range of mycobacteria than commercial probes and costs much less for reagents and supplies (about $3) per reportable resul0. In our laboratory, by botching isolates for identification in groups of 16, labor time for HPLC is approximately 45 min per reportable result, including time required for interpretation of the chromatogram. This results in a labor cost of $11 to $20 per reportable result, for a total cost of $13 to $22 per result. HPLC is rapid, versatile, and cost effective for large laboratories and reference laboratories.
PCR The polymerase chain reaction will
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probably revolutionize the way in which laboratory mycobacteriology is performed. Small amounts of mycobacterial DNA in a clinical specimen can serve as a template and be amplified by PCR using a polymerase enzyme and nucleotides (3, 4). Primers are used to select the area of the genome to be amplified, and after amplification a specific DNA probe is used to detect the amplified DNA. This can be done the same day that the specimen is collected, and the sensitivity of the method may enable it to detect one or a few organisms in a specimen. Commercial applications of PCR for mycobacteria are under development. Several problems and limitations of this method are worth noting: (i) Crosscontamination can be a problem with PCR. During 25 to 30 cycles of amplification, a single segment of DNA is amplified 33 million- to one billionfold. If a single piece of the amplified product contaminates a subsequent specimen, a false positive result could be obtained. However, the amplified DNA can be chemically modified to prevent it from serving as a template in later tests. (ii) As with DNA probes, the range of mycobacteria detected by PCR is limited to the species probed for. Cul-
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ture will be needed to detect other species. (iii) Although PCR detects and identifies mycobacteria in clinical specimens quickly, it does not detect their susceptibility or resistance to antimicrobial drugs. Culture may be necessary for susceptibility testing. It is also possible to use PCR to look for resistance to antibiotics by amplifying and probing for genes that code for resistance (16). (iv) Potential interference with the amplification process by inhibitorspresent in clinical specimens has yet to be evaluated. (v) At present, closelyrelated mycobacteria such as M. tuberculosis and M. bovis cannot be differentiated by the probes that are used to detect the amplified DNA product. DNA Hybridization Fu.aki and co-workers have recently published a microdilution plate DNAhybridization technique for species identification of mycobacterial isolates (5). Wells of a microdilution plate were coated with DNA from reference strains of mycobacteria. Unknown mycobacteria could then be identified by growing them in broth, exlracting their DNA, labeling it with photobiotin, hybridizing to the DNA coated on the wells of the microdilution plate, then detecting hybridization using an avidin-enzyme conjugate. The method could be commercially developed and serves to identify a broad range of mycobacteria. RFLP/DNA Fingerprinting Restriction fragment length polymorphism has proven useful for strain identification (to detect cross-contamination
References 1. Fox, J. 1990. TB: A grim disease of numbers. ASM News 56:363-365. 2. Good, R. C., and T. D. Masta'o. 1989. The modem mycobacteriology laboratory: how it can help the clinician. Clin Chest Med 10:315-322. 3. Eisenstein, B. 1990. The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis. N. Engl. J. Med. 322:178-183. 4. Peter, J. 1991. The polymerase chain reaction: amplifying our options. Rev. Inf. Dis. 13:166-171. 5. Kusunoki, S. et al. 1991. Application of colorimetric microdilution plate hybridization for rapid genetic identification of 22 Mycobacterium species. J. Clin. Microbiol. 29:1596-1603. 6. Clark-Curtiss,J. 1990. Genome structure of mycobacteria,p.77-96. In L McFadden (ed.), Molecular biology of the mycobacteria. Academic Press, San Diego.
7. Rogall, T. et al. 1990. Towards a phylogeny and defmition of species at the molecular level within the genus Mycobacterium. Int. J. Syst. Bacteriol. 40:323-330. 8. McFadden, L, Z. Kunze, and P. Seechurn. 1990. DNA probes for detection and identification, p. 139-172. In Molecular biology of the mycobacteria. Academic Press, San Diego. 9. Lira, S. et al. 1991. Genotypic identification of pathogenic Mycobacteriwn species by using a nonradioactive oligonucleotide probe. J. Clin. Microbiol. 29:1276--1278. 10. Musial, C. E. et al. 1988. Identification of mycobacteria from culture by using the Gen-probe Rapid Diagnostic System for Mycobacterium aviwn complex and Mycobacterium tuberculosis complex. J. Clin Microbiol. 26:2120-2123. 11. Cregan, P. et al. Abstr. Annu. Meet. Am. Soc. Microbiol. 1991, U14, p. 144. 12. Pratt-Rippin, K., and G. S. Hall. Abstr. Annu. Meet. Am. Soc. Microbiol. 1991, U12, p. 144. 13. Walton, D. T., and M. Valesco~ 1991. Identification Mycobacterium gordonae from culture by the Gen-Probe rapid diagnostic system: evaluation of 218 isolates and potential sources of false-negative results. J. Clin. Microbiol. 29: 1850-1854. 14. Butler, W. R., D. G. Aheam, and J. O. Kilburn. 1986. High-performance liquid chromatography of mycolic acids as a tool in the identification of Corynebacterium, Nocardia, Rhodococcus, and Mycobacterium species. J. Clin. Microbiol. 23:182-185. 15. Josk K., D. Dunbar, and L. Elliott.Abstr. Annu. Meet. Am Soc. Microbiol.1990, U31, p. 146. 16. Arthur, M. et al. 1990. Detection of ¢rythromycin resistance by the polymerase chain reaction using primers in conserved regions of erm rRNA methylase genes. Antimicrob. Agents Chemother. 34:2024-2026.
able to this organism occurs in approximately 0.2 to 0.4% of all live births and generally develops within the first month of life (2). Estimates of genital GBS colonization during pregnancy r a n ~ from 5 to 28% of all pregnant women (1). Of the infants born to colonized mothers, 50 to 75% will become colonized themselves during birth and 1 to 2% will develop GBS disease (2).
G B S Disease The two major syndromes associated with GBS are early onset and late onset disease. Early onset disease occurs within hours of birth and is due to transmission of the organism from a colonized mother to her infant prior to or during the birth process (1). Manifestations of early onset GBS infection in newborns include pneumonia, sepsis,
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or trace epidemics) (6). In this technique, DNA is extracted from swains of mycobactetia and then subjected to cleavage by restriction endonuclease(s). Using small, labeled DNA probes, variants among restriction fragments can be detected by the method of Southern blotting. Conclusion Table 1 summarizes the uses and limitations of the methods mentioned in this article. In addition to improving the turnaround time and accuracy of mycobacterial laboratory work, these methods have made life interesting for those of us who work in these laboratoties. With PCR, this exciting developmental phase will likely continue for some time into the future.
Editorial
Cervical Screening Tests for Group B Streptococci Audrey R. Wanger, Ph.D. Department of Pathology University of Texas Health Science Center Houston, Texas 77025 Group B streptococci (GBS) are a leading cause of neonatal infections in the United States (1). Disease attribut-
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