1 Role of the laboratory in diagnosingmycobacterial diseases, 161

2 Amplification techniques for directdetection, 1622.1 The polymerase chain reaction,1622.2 Targets for detection, 1632.3 Technical aspects ofamplification assays, 1642.4 Commercial amplification tests,1652.5 Performance of in-housepolymerase chain reaction assaysand commercial amplification tests,167

2.6 Diagnosing extrapulmonarytuberculosis with amplificationmethods, 1672.7 Polymerase chain reaction formonitoring response to tuberculosistreatment, 168

3 Species identification of culturedmycobacteria, 1683.1 Nucleic acid probeidentification, 1683.2 Polymerase chain reactioncombined with restriction enzymeanalysis, 1693.3 Nucleic acid sequencedetermination, 170

161

Chapter 9 / Molecular diagnostics

KATHLEEN D. EISENACH

3.4 Methods for distinguishingspecies of the Mycobacteriumtuberculosis complex, 170

4 Molecular methods for drugsusceptibility testing, 1714.1 Analysis of mutational hotspotsin genes associated with drugresistance, 1714.2 Metabolic assays for assessingviability in the presence of drugs,1744.3 The future of moleculardiagnostics, 174

5 Acknowledgements, 1756 References, 175

1 Role of the laboratory in diagnosingmycobacterial diseases

The timely identification of persons infected withMycobacterium tuberculosis and rapid laboratoryconfirmation of tuberculosis are two key factors for the treatment and prevention of the disease. With the increasing incidence of drug-resistant M. tuberculosis strains, early detection of drug resistanceis an important task in the proper management ofpatients with tuberculosis. Other mycobacterialinfections can cause significant morbidity and mor-tality, especially in immunocompromised hosts; thus,a rapid and specific diagnosis of these infections isimportant for the implementation of appropriatedrug therapy.

A definitive diagnosis of tuberculosis and othermycobacterial infections requires identification of the causative organism in clinical specimens. Con-ventional procedures start with microscopic ex-

amination of smears for the presence of acid-fastbacilli, and continue with culture, followed by biochemical tests of the cultured organisms to identify the specific Mycobacterium species. The entireprocess often requires 4–6 weeks from the time of specimen collection, primarily because of the slow growth of mycobacteria. Determination of drug susceptibility of an isolate by culturing can add 3–6 weeks to this already long process. The radiometric BACTEC TB system (Becton DickinsonMicrobiology Systems, Sparks, MD) and the newautomated liquid culture systems shorten the time todetection and increase recovery rates. However,these culture systems require an average of 13–15 days to detect positive specimens. The BACTECsystem also offers the NAP (p-nitro-a-acetylamino-b-hydroxypropiophenone) test for identification ofM. tuberculosis complex isolates. Chromatographicmethods for identification of cell-wall mycolic acidsare used by some reference laboratories to provide a

Mycobacteria: Molecular Biology and Virulence Edited by colin Ratledge and Jeremy Dale

© 1999 Blackwell Science Ltd. ISBN: 978-0-632-05304-9

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more rapid and definitive species identification. Toexpedite the detection of drug resistance, drug-containing media can be directly inoculated with thepatient’s smear-positive specimen. With the directmethod, drug susceptibility results can be anticipatedwithin 2–4 weeks of arrival of the specimen to thelaboratory.

Novel molecular assays for diagnosis and drug sus-ceptibility testing offer several potential advantagesover the above methods including faster turnaroundtimes, very sensitive and specific detection of nucleicacids, and minimal, or possibly no, prior culture. The need for new technologies for rapid diagnosis oftuberculosis is clear. Great enthusiasm aroused bymolecular technologies has been evident in the fieldof mycobacterial research. The goals have been todevelop reliable procedures that can detect and identify mycobacteria directly in clinical specimens,methods for testing antimycobacterial drug suscepti-bility, and methods for assessing bacillary loads intuberculosis patients to determine the efficacy ofchemotherapy. The significant advances that havebeen made in the last decade towards these goals aredescribed in this chapter.

2 Amplification techniques for direct detection

The advent of nucleic acid probe technology offeredpromise of rapid, specific and direct microbial detec-tion in clinical samples. However, laboratory experi-ence demonstrated that if the number of targetmolecules in a clinical sample is low, the sensitivity ofnucleic acid probes was unacceptably low. With thedescription of the polymerase chain reaction (PCR)for amplification of nucleic acids in 1987, researchersin the field quickly recognized the technology’spotential to provide more sensitive tuberculosis diag-nostics and possibly obviate the need for mycobacter-ial culture. By 1990, several PCR assays designed to amplify mycobacterial nucleotide sequences hadbeen described. Subsequently other amplificationtechnologies were developed and applied to thedetection of mycobacteria.

2.1 The polymerase chain reaction

PCR is the most prominent gene amplification technology, being the most thoroughly investigated,widely adopted and extensively published method.PCR and other target amplification methods allow exponential multiplication of DNA or RNAsequences, beginning with as few as one copy andproducing as many as one billion copies within a fewhours. Amplified copies (PCR products or amplicons)can be detected and characterized with specificoligonucleotide probes by using a variety of for-mats. PCR involves exponential amplification usingtwo oligonucleotide primers that anneal to oppo-site strands of the target DNA, and are extended with a thermostable DNA polymerase; the extendedDNA product becomes the target for further am-plification through multiple cycles of denaturation,annealing, and extension. Modifications of the basicprocedure include the reverse transcriptase (RT)PCR, multiplex PCR, quantitative PCR and nestedPCR.

When the target is RNA, the RNA is transcribed in an RT reaction and the cDNA products are thenamplified in a traditional PCR, hence the designationRT-PCR. Ribosomal and messenger RNA sequences of mycobacteria are amplified in this manner.Applications of these assays are discussed later in thechapter.

Several independent amplifications carried outsimultaneously in one tube with a mixture of primersis referred to as multiplex PCR. To establish a specificmultiplex assay in which each template is amplifiedefficiently can be challenging since reaction con-ditions must be appropriate for each primer set.Multiplex PCR assays commonly consist of oneprimer set for amplification of the target sequenceand a separate set for an internal control DNAsequence. Other multiplex formats include primersfor multiple target sequences. For example, Kox et al. (1997) designed a multiplex PCR assay for co-amplification of the M. tuberculosis complex-specific IS6110 and a highly conserved stretch of the 16S rDNA. PCR products of this multiplex assay

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were analysed in a reverse crossblot hybridizationwith species-specific probes and a Mycobacterium-specific probe. This multiplex PCR enabled identifi-cation of M. tuberculosis and the most importantopportunistic mycobacteria in clinical specimens. An added advantage was the ability to detect simulta-neous infections by more than one mycobacterialspecies.

Standard PCR amplification does not providequantitative information regarding the absoluteamount of nucleic acid in a sample. Quantificationcan be accomplished using several different PCRformats. The endpoint or limiting dilution methodinvolves serially diluting the target sample and com-paring the endpoint signal to a standard curve. Moreprecise results can be obtained with a competitivePCR, although this type of assay is more difficult toestablish. A control must be constructed so that it isamplified with the same primers, contains similar G+C content, and is of similar size as the target. Bothtemplates must amplify with equal efficiency. A com-petitive PCR based on IS6110 has been developed by DesJardin et al. (1998) for the purpose of quantify-ing M. tuberculosis DNA in sputum samples. Endpointdilution and competitive methods are labour inten-sive and require multiple reactions for each sample.To simplify this, DesJardin et al. (1998) developedanother IS6110 PCR using an automated, real-timePCR system. The basis for this assay is the ABI Prism7700 Sequence Detection system (TaqMan; AppliedBiosystems, Inc./Perkin Elmer, Foster City, CA)which uses a fluorogenic probe with the amount offluorescence detected being proportional to theamount of accumulated PCR product. The amount oftarget DNA in a sample is interpolated from a stan-dard curve that is generated with each run.Quantification of PCR products occurs real timeduring the exponential phase of amplification. Sinceno postamplification handling is necessary, this elimi-nates potential sources of carryover contaminationand reduces handling time. Comparable results havebeen observed with the IS6110 competitive andTaqMan PCRs.

In a nested PCR, a second round of amplification is

performed, using the amplicon of the first reaction asa target and a pair of primers complementary tosequences within this amplicon. Nested PCR providesincreased sensitivity, but this is achieved at the risk ofcross-contamination, since the tubes containingamplicons have to be opened to add new reagents or transfer amplicons to a second reaction tube.Alternatively, a reaction can be run with two sets ofprimers in one tube, providing the primer pairs canbe designed with different annealing temperatures.Nested PCR can increase the specificity of the reac-tion, since the internal primers anneal only if theamplicon has the corresponding expected sequence.Increased sensitivity has been achieved with nestedPCR assays that target single-copy sequences, such asthe 65-kDa and 38-kDa (Pab) genes (Hance et al.1989; Miyazaki et al. 1993).

2.2 Targets for detection

A suitable target for amplification may be a single-copy gene in the mycobacterial genome or one that ispresent as a repeated sequence. The choice of targetand design of primers within the gene target areequally important in terms of assay sensitivity andspecificity. Both genus-specific and species-specificgene targets have been utilized. Some of the targetsinclude the genes for insertion elements (IS6110,IS1081), immunodominant antigens (38-kDa antigen(Pab), 65-kDa protein, MPB70 (18kDa), 85 proteincomplex (30/32kDa), MPB64) and ribosomalsequences (16S rRNA, 23S rRNA). Predominantamong these is the insertion sequence IS6110/IS986(McAdam et al. 1990; Thierry et al. 1990), which istypically present in multiple copies in M. tuberculosis.The high copy number of IS6110 is thought to resultin increased sensitivity, although given the scale of amplification involved in PCR, this is unlikely to be a significant factor. Within IS6110, the choice ofprimers can affect the PCR results. The primers ofEisenach et al. (1990) are widely used and demon-strate high sensitivity and specificity. There havebeen reports of false positives with the IS6110 PCRwhich suggest some primers may lack specificity.

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Since IS6110 is a member of the widely distributedIS3 family of insertion sequences (see Chapter 2), it isplausible that some primers will detect other IS3-likeelements. A potentially more serious problem is theexistence of strains that lack IS6110. However, therehave been few reports of such strains and it isunlikely that false-negative PCR results are attribut-able to strains lacking this element.

Of the single-copy targets, the 65-kDa protein geneand the 16S rRNA gene have been frequently used.These highly conserved genes serve as Mycobacterium-specific targets. Careful design of primers and PCRconditions can provide an assay capable of detectingany mycobacterial species, with identification tospecies level provided by a second set of primers or byhybridization with species-specific probes (Brisson-Noël et al. 1989; Hance et al. 1989; Böddinghaus et al.1990). Investigators have also resorted to the use ofrRNA sequences as targets that can be amplified viaRT-PCR. The advantages are that the 16S rRNAsequences are found in high copy numbers (ª2000molecules/cell) with stretches of sequence that arehighly variable among species and serve as targets for species-specific amplification, whereas otherstretches are conserved and serve as a genus-specifictarget.

2.3 Technical aspects of amplificationassays

Mycobacterial cell lysis methods

The main objectives of sample preparation areefficient release of mycobacterial nucleic acid andremoval of any substances in the sample that may beinhibitory to the PCR; at the same time, it is desirableto avoid introducing chemicals that may themselvesmay be inhibitory. The challenge has been to developa practical method combining these objectives withsimplicity of operation. The methods that are suitablein the research laboratory have not proven suitablefor the clinical setting. Lysis of mycobacteria can be difficult because of the thick lipid-rich cell wall, components of which can also contaminate thenucleic acid preparation. The methods used com-

monly involve a combination of physical disruption (boiling, sonication, glass bead beating, or cycles offreezing–thawing), chemical degradation (guani-dinium salts, sodium hydroxide, sodium dodecyl sul-phate (SDS), chelex agents), and enzymatic digestion(lysozyme, proteinase K). For simplicity, a crudelysate is frequently used in the amplification reaction;however, where possible it is preferable to use apurified sample in which the DNA is concentratedand interfering substances have been eliminated. Thelatter can be accomplished by the traditional methodof phenol–chloroform extraction and ethanol pre-cipitation. As a rapid and simple alternative, Eisenachet al. (1991) have used the GeneClean reagents(Bio101, La Jolla, CA) for purifying and con-centrating DNA.

Controls and elimination of inhibitors

Heparin, haemoglobin, phenol, SDS, and otherundefined substances in clinical specimens are potentinhibitors of Taq polymerase activity. Inhibition of Taqpolymerase during the PCR can cause false-negativeresults, thus decreasing assay sensitivity. Inhibitionrates have been reported as high as 23%. Inhibitionoccurs most often when crude lysates are used and can often be rectified by diluting the sample orpurifying the DNA. Nested PCR formats, whichenable dilution of the sample in the second round ofPCR, have been applied with the explicit purpose of overcoming PCR inhibitors.

Although the precise nature of such inhibitors isnot known, their presence may be monitored withcontrol templates. Purified M. tuberculosis DNA maybe spiked in duplicate test samples or back-spikedinto PCR-negative samples, an endogenous gene may be co-amplified along with the target DNA, orgenetically engineered or plasmid target DNA may be used as internal controls. The fastest and leastexpensive procedure is co-amplification with aninternal control. Eisenach et al. (1991) were the firstto describe adding an internal control which wasamplified with the IS6110 primers to the PCR reactionmixture. The control DNA was a plasmid containingthe 3¢ and 5¢ ends of the 123-bp IS6110 target and a

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large insert of DNA, resulting in a large 600-bp PCRproduct. The control product could be easily distin-guished from the 123-bp sample product on ethidiumbromide-stained gels.

Kolk et al. (1994) developed a novel strain of M.smegmatis with a modified IS6110 sequence integratedinto its genome. The efficacy of each step in the assayincluding the sample preparation method can bemonitored by adding the modified M. smegmatis strainto the clinical sample. The most common control forcell lysis and DNA extraction is a standardized aliquotof a broth culture containing a known number of M. tuberculosis organisms which is included in eachrun.

Contamination

The risk of false-positive results due to the carryoverof target DNA from a positive to a negative sample is amajor concern in the clinical application of PCR diag-nostics. Contamination is a severe problem in thecontext of the diagnosis of tuberculosis, wherebyamplification of one to 100 template molecules is usually sought. Contaminating DNA may comefrom clinical specimens containing large numbers of M. tuberculosis organisms, from M. tuberculosis culturesused as cell lysis controls, or target DNA used as positive PCR controls. Most frequently, the problemarises from the accumulation of PCR amplicons in thelaboratory. Amplification systems have been adaptedto include use of dUTP and uracil DNA glycosylase asa strategy to eliminate amplicon carryover.

2.4 Commercial amplification tests

The commercial PCR test for the detection of M. tuber-culosis complex is marketed by Roche DiagnosticSystems (Branchburg, NJ). The Roche Amplicor MTBamplifies a region of the 16S rDNA sequence that isgenus specific and detects PCR products by hybridiza-tion with a M. tuberculosis complex-specific probe.Another version of this test, available outside theUnited States, employs additional species-specificprobes that allow detection and identification of M. avium and M. intracellulare (F. Hoffmann-La Roche

Ltd, Basel, Switzerland). The Amplicor MTB systemcan be automated with the Roche Cobas instru-ment which consists of a Thermocycler TC9600, ahybridization system based on magnetic particle separation, and a microwell plate reader. Results with the Cobas Amplicor MTB system are com-parable to those of its manual version (Rajalahti et al.1998).

The Gen-Probe Amplified Mycobacterium tuberculosisDirect (MTD) Test (Gen-Probe, Inc., San Diego, CA) is a transcription-mediated amplification (TMA)system. The basis of TMA is conversion of the target(in this case 16S rRNA) into cDNA by reverse transcriptase, using a primer containing an RNApolymerase promoter. The product can therefore betranscribed by RNA polymerase to produce largenumbers of RNA transcripts, which become tem-plates for reverse transcription and further trans-cription in a cyclic geometric amplification. RNAproducts are detected by a hybridization protectionassay that uses an acridinium ester-labelled DNAprobe complementary to the rRNA target. The MTD test can detect <103 copies of rRNA, equivalentto one bacillus, and <5 bacilli even in the presence of a high number of unrelated organisms, thus being M. tuberculosis complex specific (Jonas et al.1993).

A modification of TMA uses the RNA self-sustainedsequence replication reaction (termed 3SR) in whichRNaseH degrades the RNA–DNA duplexes and allowsconversion to dsDNA that has an RNA polymerasepromoter site at each end (Compton 1991). NucleicAcid Sequence-Based Amplification (NASBA) is acommercial development of the 3SR (OrganonTeknika, Amsterdam, the Netherlands) (Van der Vlietet al. 1993).

Strand Displacement Amplification (SDA; BDMicrobiology Systems) which was first described byWalker et al. (1992) is based upon the annealing todenatured target DNA of oligonucleotide primerswhich possess 5¢ tails containing restriction sites for the enzyme HincII. An exonuclease-deficient DNA polymerase extends the 3¢ end of the annealedprimers and incorporates thiolated nucleotide dATPas in the newly synthesized strand. Within

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the primer sequence, HincII nicks the unmodifiedstrand of the hemiphosphorothiolated duplex pro-viding a free 3¢ end from which the polymerase can extend and displace the downstream DNAstrand. Exponential amplification is achievedthrough continuous polymerization and displace-ment of both sense and antisense DNA templates. Aprototype system, the BDProbeTec-SDA, has beenevaluated in a few studies (Ichiyama et al. 1997).Newer SDA systems are under development whichemploy thermostable enzymes. Such assays offer the potential to enhanced specificity and are capableof achieving >109-fold amplification in as little as15–20min (Spargo et al. 1996). Recent developmentsinclude amplification of long targets up to 2kb in sizeand linkage of SDA to an RT reaction. The use of SDAfor quantitative detection of DNA and RNA is alsobeing explored.

Increased sensitivity of mycobacterial detectioncan also be achieved with probe amplification tech-nologies, one such method being the ligase chainreaction (LCR). LCR involves the joining, catalysedsequentially by polymerase and a thermostableligase, of two oligonucleotide probes specific for adjacent sequences in the target DNA, once such segments are hybridized with the complementarysequences. The products of each cycle step serve astemplates for the next cycle, resulting in exponentialamplification. LCR is the basis of the Abbott LCx M. tuberculosis (MTB) assay (Abbott Laboratories,Abbott Park, IL). The LCx MTB assay employs fouroligonucleotide probes, designed in pairs, that arecomplementary to the M. tuberculosis complex-specific gene encoding the protein antigen b (Pab).The paired probes are labelled with different haptens,one for capture and the other for detection, so thatonly joined products have both haptens and aredetected in the microparticle enzyme immunoassay.LCR products are detected using the automated LCx Analyser, in which a sample of amplified productis automatically transferred to an incubation well and microparticles coated with anticapture haptenbind the amplification product as well as any unli-gated probes with capture hapten. Clinical evalua-

tions of the LCx MTB test have demonstrated sensi-tivities and specificities similar to PCR and otheramplification methods (Ausina et al. 1997; Tortoli et al. 1997).

Signal amplification formats that have beenapplied to mycobacterial detection include the Q-Beta replicase amplification system (formerly Gene-Trak, Framingham, MA) and the branched-chain DNA Signal amplification assay (Chiron Corp., Emeryville, CA). Q-Beta replicase amplifica-tion is based on the use of ‘detector’ probes that are geometrically amplified by Q-Beta replicase(RNA-directed RNA polymerase) following hy-bridization to specific RNA targets (Lizardi et al.1988). In this system, amplification occurs aftersample matrix and unhybridized detector probe areremoved from the reaction mixture. This is accomplished by using multiple rounds of reversibletarget capture on paramagnetic particles. The Q-Beta replicase assay, designed to target M. tuberculosis23S rRNA, was found to be sensitive and specific for direct detection in sputum samples (Shah et al. 1995). The Galileo was developed as a prototypeinstrument (Vysis Inc., Downers Grove, IL) forautomating the assay in a closed disposable cartridge,thereby simplifying the assay and preventing con-tamination of the assay from external sources (Smith et al. 1997). In the branched-DNA assay,signal amplification is achieved via hybridization of multiple alkaline phosphatase-labelled probes tobranched-chain oligonucleotide probes with multiplebinding sites, followed by incubation with a chemilu-minescent substrate. The chemiluminescent output is directly proportional to the concentration of DNAtarget present in the specimen. Shen et al. (1994)described a branched-DNA assay, which uses multi-ple probes complementary to the IS6110 sequence,for detection and semiquantification of M. tuberculosisin sputum specimens. When luminescent signalswere compared to semiquantitative AFB readingsand colony counts on solid media, a correlation wasobserved. Currently, there are no apparent plans tocommercialize mycobacterial assays based on thesetwo signal amplification methods.

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2.5 Performance of in-house polymerasechain reaction assays and commercialamplification tests

Experience with in-house-developed PCR tests hasdemonstrated overall sensitivities and specificities inthe range of 70–100%. This variability is not surpris-ing given the fact that laboratories differ in terms ofextraction procedures, target and primer sequences,sample input, PCR conditions and detection methods.For an extensive review on published results oneshould consult Richeldi et al. (1995), Herold et al.(1996), Sandin (1996) and Forbes (1997). In moststudies, sensitivity and specificity have been calcu-lated as a function of the culture technique, since thisis the reference method and corresponding clinicalinformation has often not been available. When dis-crepant results have been revised on the basis of apositive history for culture or the clinical diagnosis oftuberculosis, the specificity and positive predictivevalue of the PCR tests have increased. Studies inwhich sensitivities approach 100% were carried outwith larger proportions of smear-positive specimensthan commonly found in clinical populations.Separate analyses of smear-negative, culture-positivespecimens have shown that the sensitivities aresignificantly lower than those of smear-positive,culture-positive specimens. The diagnostic yield issignificantly increased if more than one specimen perpatient is analysed. The commercial tests give resultscomparable to those obtained with in-house PCRassays. Generally, among the commercial tests thesensitivities and specificities have been equivalent.Occasionally, an in-house IS6110 PCR has appearedto be more sensitive than the commercial tests, andthe Gen-Probe MTD to be more sensitive than theRoche Amplicor MTB; however, these differenceshave not been statistically significant (Vuorinen et al. 1995; Dalovisio et al. 1996; Huang et al. 1996;Ichiyama et al. 1996; Piersimoni et al. 1997; Cohen et al. 1998).

Although several technical factors affect the per-formance of amplification tests on clinical samples,the key factor, as for microscopy and cultures, is the

density of M. tuberculosis organisms in the specimen.A clear relationship between PCR performance andthe number of M. tuberculosis organisms in sputumspecimens has been found by Clarridge et al. (1993).Only 52% of the specimens with <50 cfu/mL werepositive in the IS6110 PCR. Of those with >100cfu/mL, 98% were positive. In a detailed analysis ofthe sensitivity and specificity of the 16S rRNAamplification test (Gen-Probe MTD test), Jonas et al.(1993) found that, like DNA amplification, the sensitivity is dependent on the bacterial load in thespecimen. The assay was positive on only 53% ofthose samples containing <100 cfu/mL. However,positivity was 100% on samples with greater than1000 cfu/mL. These data indicate that the perfor-mance of the amplification tests may be insufficientto diagnose tuberculosis in patients with paucibacil-lary disease. To improve sensitivity, Gen-Probe hasdeveloped a second generation test MTD2 which usesa 10-fold increase in volume of pretreated specimen.In a recent comparison of the MTD1 and MTD2,Gamboa et al. (1998) observed increased sensitivitywith both respiratory and non-respiratory speci-mens; however, the differences in sensitivitiesbetween the two methods was significant for onlyrespiratory specimens.

2.6 Diagnosing extrapulmonarytuberculosis with amplification methods

The real value of PCR diagnosis is in situations wherethe clinical picture is less clear or where smear and culture are less reliable. It is extrapulmonarytuberculosis (meningitis, pleuritis, peritonitis, peri-carditis, lymph-node tuberculosis, skin tuberculosis,etc.) for which a rapid and accurate laboratory diag-nosis would be most beneficial. Limitations of smearand culture are due to the lower number of organ-isms normally present in these types of specimens.There have been many investigations of PCRamplification of cerebrospinal fluid for the diagnosisof tuberculous meningitis. These were limited by thesmall numbers of patients studied, lack of corre-sponding culture results, and inadequate clinical

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diagnoses. A wide range of sensitivities (32–100%)has been reported. More clinical studies are needed toevaluate the diagnostic yield of PCR in extrapul-monary tuberculosis.

2.7 Polymerase chain reaction formonitoring response to tuberculosistreatment

Sputum microscopy and culture have traditionallybeen used for monitoring treatment response in pul-monary tuberculosis. Both techniques have obviouslimitations. PCR, which combines sensitivity withspeed, has been investigated as a means for assessingbacterial clearance. Yuen et al. (1997) reported thatpersistence of M. tuberculosis DNA in sputum was associated with more underlying illness, high radi-ographic scores on the extent of involvement, previ-ous drug treatment, high degree of sputum smearpositivity, and multidrug resistance. In this study,60% of the patients who were PCR positive after 6months of treatment were clinical relapses whereasnone of those who became PCR negative before 6months relapsed. Similarly, Kennedy et al. (1994)observed that their relapsed cases had PCR positivesputa beyond 6 months suggesting that PCR may beuseful for detecting relapses. In these studies, PCRconversion to negativity was seen at 1–2 months fol-lowing smear and culture conversion, and it was pro-posed that PCR is a suitable method for assessingtreatment response. Contrary to these observations,Hellyer et al. (1996) demonstrated the persistence ofM. tuberculosis DNA in sputum >12 months after startof treatment and >6 months after conversion in somepatients. DNA persistence was not associated withradiographic extent of disease or relapse. Althoughthe differences observed in these studies cannot be readily explained, the presence of amplifiable DNA over such long periods in culture-negativepatients is not surprising considering the exquisitesensitivity of PCR methods. It seems logical that theinability to distinguish live and dead organisms wouldpreclude DNA amplification from use in therapeuticmonitoring.

It has been suggested that quantitative rather

than qualitative assessment of DNA levels mightreflect bacterial load; such approach has been investi-gated by DesJardin et al. (1998). Competitive andreal-time PCR assays for IS6110 were used to quantifyM. tuberculosis DNA in sputum samples serially col-lected during the course of therapy. As anticipated,the amount of DNA corresponded to the numbers ofAFB on microscopy, however, neither the DNA levelnor AFB count correlated with the number of cul-tivable bacilli after initiation of therapy. Thus, thesetests were not considered appropriate markers oftreatment efficacy. Similar data have been observedin mice, where quantitative estimates of DNA did notcorrespond to the numbers of bacilli cultured fromthe drug-treated animals (de Wit et al. 1995).

RNA is less stable than DNA and would appear tobe a more suitable target for this purpose. Moore et al.(1996) used the Gen-Probe MTD to monitor rRNA insputum from patients receiving therapy and observeda poor correlation between smear and culture resultsand the presence of rRNA. In contrast to DNA andrRNA, prokaryotic mRNA has a very short half-lifeand should be a reliable target for indicating the pres-ence of viable organisms. DesJardin et al. (1996) havedeveloped a quantitative RT-PCR which amplifiesalpha antigen mRNA and shown this assay to be areliable marker of bacterial viability (Hellyer et al.1999). In a study of culture-positive patients receiv-ing standard antituberculosis therapy, a precipitousdrop in alpha antigen levels was observed in as littleas four days after the start of therapy (DesJardin et al.1999). The data suggest that ratios of DNA to mRNAlevels may provide the most meaningful assessmentof the efficacy of drug treatment. Further studiesinvolving more patients are needed to determinewhether this approach will be useful for identifyingpatients with a high risk of relapse.

3 Species identification of culturedmycobacteria

3.1 Nucleic acid probe identification

Nucleic acid probes for the identification of M. tuberculosis complex and M. avium complex were

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introduced by Gen-Probe in 1987. Use of theseradioisotopic DNA probes for rapid identification ofcultures at the species level was the first applicationof molecular biology techniques in the clinicalmycobacteriology laboratory. By 1990, chemilumi-nescent probes (AccuProbes) were available, andprobes for speciating M. kansasii and M. gordonaehad been developed. The AccuProbes are single-stranded oligomers complementary to the rRNA of these particular species of mycobacteria. Lysis ofthe mycobacterial cells releases RNA, and the acri-dinium ester-labelled probe binds with the rRNA ofthe target organism to form a stable DNA–RNAhybrid. Detection of the hybrid is accomplished by ahybridization protection assay, which is the samemethod used to detect transcription products in theMTD test.

Initially, AccuProbes were developed for cultureconfirmation of organisms grown on solid mycobac-terial media, however, they are now widely used toidentify mycobacteria in liquid culture, e.g. BACTEC12B medium (Evans et al. 1992; Telenti et al. 1994;Metchock & Diem 1995). The test requires about 106

organisms to produce clear-cut results. Overall sensi-tivity and specificity of the AccuProbes are close to100%. Discrepant results are rare, but have beenreported in a number of cases. Misidentification of M.terrae, M. avium complex, and M. celatum as M. tubercu-losis complex has been reported (Martin et al. 1993;Stockman et al. 1993; Butler et al. 1994). Other minorlimitations include the inability to distinguishmembers of the M. tuberculosis complex and with theMAC (M. avium complex) probe there is no distinc-tion between M. avium and M. intracellulare.

With emphasis on the rapid detection of M. tuberculosis, probes combined with the BACTECsystem offer the most easily available and reliablemethod for most clinical laboratories. DNA probeshave been widely adopted in industrialized countriesbut are not used in developing countries because ofcost. Alternative probe-based methods underresearch and development, such as the cultureconfirmation test based on the direct repeat locus andthe cycling probe technology (ID Biomedical,Vancouver, Canada), may ultimately be more afford-

able and amenable to low technology settings (Beggset al. 1996).

3.2 Polymerase chain reaction combinedwith restriction enzyme analysis

Amplification of a highly conserved gene combinedwith restriction enzyme analysis of PCR products hasbeen applied to the identification of several com-monly encountered mycobacterial species. Plikaytiset al. (1992) were the first to describe such an assay inwhich a portion of the highly conserved heat-shockprotein 65 (hsp65) gene was amplified using primerscommon to all mycobacteria and the PCR productdigested separately with two restriction enzymes.Telenti et al. (1993a) developed a similar methodwhich differed in the 65-kDa primers and restrictionenzymes used. A third method described byVaneechoutte et al. (1993) was based on the 16SrDNA target. With these methods the restriction frag-ment patterns were distinctive for M. tuberculosis, M.bovis, M. avium, M. intracellulare, M. kansasii and M.gordonae; however, the patterns occasionally variedwithin a species. Members of the M. tuberculosiscomplex consistently displayed the same patternsand could not be differentiated on a species level.Strains of M. avium were tightly clustered, whereas M.kansasii, M. intracellulare and M. gordonae each showedgreater variability within their clusters.

PCR-restriction enzyme pattern analysis can beperformed on isolates from solid and BACTEC media.Lysis of mycobacteria is usually accomplished bymechanical means and crude lysates used in the PCR.Most patterns can be recognized visually, however,computer-assisted analysis facilitates pattern compar-isons and storage of a large database. The methodrequires high-resolution gels and internal standards,since some fragments differ in size only by a fewnucleotides. If the laboratory is also performing diag-nostic PCR, one should consider using primersdirected to an unrelated genomic region to the onetargeted in the PCR-restriction assay. This is impor-tant for avoiding carryover of amplified productsfrom the PCR-restriction assay to a sensitive diagnos-tic PCR.

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3.3 Nucleic acid sequence determination

Direct sequencing of mycobacterial genes has becomean increasingly important method for identifyingmycobacterial species. This approach is also useful forthe detection of growth-deficient mycobacterialspecies directly in clinical specimens and in taxo-nomic characterization of mycobacterial strains.Genes that have been examined include those for the16S rRNA, dnaJ, superoxide dismutase, hsp65, and32-kDa protein. In general, the gene chosen for PCR- and sequencing-based identification should befound in all relevant mycobacterial species and not inother bacteria, it should contain enough sequencediversity between different species to allow for easy identification, and there should be very littlevariation among the strains belonging to one species.The 16S rRNA gene, with its conserved and variableregions, has become the preferred target (Rogall et al.1990a).

In the direct sequencing method described byKirschner et al. (1993), preparation of nucleic acidswas accomplished by simple mechanical disruption ofthe bacteria and a 1-kb fragment of 16S rRNA genewas PCR amplified. Because of the specificity of oneof the primers, mycobacterial DNA was preferentiallyamplified, permitting the correct identification ofmycobacteria in samples containing more than oneorganism. The other primer targeted a conservedregion in Escherichia coli and was biotinylated to allowfor the single-stranded solid-phase sequencing tech-nique. A third primer was used in the sequencingreactions which provided the nucleic acid sequenceof region A (Rogall et al. 1990a). Most mycobacterialspecies have a unique sequence in region A, there-fore precise identification is possible by comparingthe sequence of the unknown isolate with the knownsignature sequences (Rogall et al. 1990b). Althoughregion A can be used for routine identification, theadditional analysis of region B may be required forisolates that are indistinguishable on the basis ofregion A or for isolates that show unique sequencesin region A, possibly indicating previously unde-scribed taxa.

16S rRNA sequence determination represents a

highly accurate and rapid method for identifyingmycobacteria. More advanced instrumentation, i.e. a rapid-ramping thermal cycler, an automatedsequence detection system, and computer-assistedanalysis would allow final identification to be com-pleted within 1 day. This technique offers severaladvantages in terms of speed, accuracy and versatil-ity; however, its use is restricted to reference or clini-cal research laboratories because of the cost andtechnical expertise required.

3.4 Methods for distinguishing species ofthe Mycobacterium tuberculosis complex

The similarity of M. tuberculosis, M. bovis and M.africanum in clinical presentation and treatment ofthese infections has resulted in the laboratory notfully identifying these species. However, it is impor-tant to differentiate members of the M. tuberculosiscomplex on the species level so that the incidence ofM. bovis infections in humans and animals can be doc-umented. Traditionally, strains of M. bovis and M.tuberculosis have been distinguished by several bio-chemical properties; however, these results are oftennot available for 6–8 weeks and not always reliable.With the two species being virtually identical on thegenetic level it has been difficult to identify sequencediversity on which a molecular method could bebased.

Early descriptions of the insertion element IS6110indicated that M. tuberculosis and M. bovis could be distinguished on the basis of IS6110 copy number,with the M. bovis strains having one to two copies ofIS6110 and the M. tuberculosis strains having 10–15copies. Plikaytis et al. (1991) described an IS6110 PCRassay based on this assumption for differentiating thetwo species. Subsequent DNA fingerprint datademonstrated that this was an invalid approach sincesome M. bovis strains have high copy numbers ofIS6110 and vice versa. Del Portillo et al. (1996) proposed that PCR amplification of the mtp40 genecould be used as a diagnostic tool for detecting M. tuberculosis infections and for differentiating themfrom M. bovis infections. The basis for this was thatmtp40 appeared to be present in only M. tuberculosis

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strains (Parra et al. 1991). An extensive evaluation ofthe mtp40 PCR indicated that the mtp40 gene is foundin most, but not all, M. tuberculosis strains and isabsent in most, but not all, M. bovis strains castingdoubt on the reliability of this method (Weil et al.1996).

Recently the nucleotide sequences for the oryRgene and the pyrazinamidase (pncA) gene were determined with point mutations being observed in the two species (Scorpio & Zhang 1996; Sreevatsanet al. 1996). Subsequently, molecular methods basedon these differences were developed. One methoduses PCR and single-stranded conformation poly-morphism analysis to detect a single characteristicmutation in the pncA gene of M. bovis (Scorpio et al.1997). Another amplifies a region of the oryR genewith the PCR products being subject to restrictionanalysis following digestion with AluI (Sreevatsan et al. 1996). To provide a simpler method, De losMonteros et al. (1998) developed an allele-specificPCR method based on the oryR sequences which wasshown to be reliable for distinguishing M. bovis fromM. tuberculosis.

Another promising PCR method developed by M.Beggs (personal communication) takes advantage ofsequence differences in the direct repeat (DR) locus.In this assay one primer was complementary to theregion flanking the DR locus (a sequence which isconserved in all M. tuberculosis complex strains) andthe other to a spacer sequence which appeared to beunique to M. bovis (Beggs et al. 1996). M. bovis strainsconsistently yielded a 580-bp product whereas most M. tuberculosis strains yielded no product. The occa-sional product observed with the M. tuberculosisstrains could be easily distinguished on the basis of its larger size. The assay was developed in a multiplex format such that the primers for IS6110are included in the PCR, thus enabling the simultaneous confirmation of the presence of M. tuberculosis complex DNA. A large collection of M. bovis and M. tuberculosis strains from diverse host and geographical origin has been tested demonstrating the diagnostic utility of this assay.Advantages of this method are that the PCR assay isvery simple to perform with cultured cells being

placed directly in the PCR reaction (no DNA isolationrequired) and the results are straightforward.

Bacille Calmette–Guérin (BCG), which is used as avaccine against M. tuberculosis, a recombinant vehiclefor multivalent vaccines, and as cancer immunother-apy, can cause disease in humans, especially thosewith cellular immunodeficiencies. Therefore, theability to rapidly and specifically identify BCG can beclinically important. Several methods have beenreported to differentiate BCG from other members ofthe M. tuberculosis complex. These include DNAfingerprinting methods with the DR and IS1081probes (van Soolingen et al. 1992; see also Chapter 6)and amplification of a specific region containing the major polymorphic tandem repeat followed byrestriction enzyme analysis (Frothingham 1995).Recently, the RD1 region was found to be present inall virulent M. bovis and M. tuberculosis strains testedbut deleted from all BCG strains tested (Mahairas et al. 1996). With this information, Talbot et al. (1997)developed a multiplex PCR to detect the RD1 dele-tion. The assay included two primers complementaryto regions flanking the RD1 and one primer com-plementary to DNA within the RD1 sequence, withresults based on the size of the PCR products. In anevaluation of a large, representative collection ofBCG and other M. tuberculosis complex strains, theRD1 PCR gave consistent and easy to interpretresults, thus demonstrating that it is a promising toolfor the rapid and specific identification of BCG.

4 Molecular methods for drug susceptibility testing

4.1 Analysis of mutational hotspots ingenes associated with drug resistance

In the last few years there has been considerableprogress in our understanding of the mechanisms ofaction of antimycobacterial agents and the basis ofresistance to these compounds (Musser 1995; Heymet al. 1996; see also Chapter 15). To date, there isinformation about 12 genes involved in resistance in M. tuberculosis. Of greatest interest is the basis ofresistance to the two key drugs, isoniazid (INH) and

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rifampin (RMP), as resistance to these compounds is likely to influence patient care. This information has allowed the development of novel strategies fordetecting resistance at the genotype level. Thesestrategies have the potential to provide results morerapidly than the traditional methods that rely ongrowth or inhibition of growth in the presence of theindividual drugs.

The majority of resistance to RMP involves mis-sense mutations in a well-characterized region of the rpoB gene (encoding the subunit of the RNA polymerase) (Telenti et al. 1993b); thus, investigationof RMP resistance is relatively straightforward. In contrast, resistance to INH is associated with avariety of mutations affecting one or more genes such as those encoding catalase–peroxidase (katG)(Zhang et al. 1992), the enoly acyl carrier proteinreductase involved in mycolic acid biosynthesis(inhA) (Banerjee et al. 1994), and the recentlydescribed alkyl-hydroperoxide reductase (ahpC),which is involved in cellular response to oxidativestress (Deretic et al. 1995; Wilson & Collins 1996).Investigation of INH resistance is more complex sincethe analysis of limited regions in all three genes isrequired.

New techniques undergoing clinical evaluationinvolve screening mutational hotspots in the genesencoding drug targets using PCR followed by analysiswith either automated DNA sequencing, single-strand conformation polymorphism (PCR-SSCP) orsolid-phase hybridization (Telenti & Persing 1996).

Sequencing remains the gold standard for thedetection of mutations. Automated sequencing hasbeen applied to the identification of mutations ingenes involved in resistance to INH, RMP, strepto-mycin and fluoroquinolones (Kapur et al. 1995).However, a complete surveillance of all known muta-tion sites for some of these genes (e.g. katG) requiresmultiple reactions per isolate. The requirement of anautomated sequencer and the technical demands ofthis multistep process limits this method to sophisti-cated reference laboratories.

The principle of PCR-SSCP is based on the fact thatthe two denatured strands of a PCR-amplified DNAmolecule adopt stable intramolecular conformations;

changes can be easily recognized by their altered elec-trophoretic mobility compared to the wild-typepattern. This analysis can be performed on an auto-mated sequencer to render results in 24h, or manu-ally using a modified silver-staining procedure orradioactive labelling during the amplification step.SSCP requires long electrophoresis steps underhighly controlled conditions and technical expertiseto ensure reproducibility. Furthermore, the elec-trophoresis patterns for some mutations can be verysimilar to wild-type patterns. As with automatedsequencing, SSCP would be best suited for referencelaboratories. Recently Telenti et al. (1997) set out to define the sensitivity and specificity of SSCP at the reference laboratory level. A blind assessment of the accuracy of targeted mutation analysis was conducted using selected regions of four genes (katG, inhA, ahpC and rpoB). PCR-SSCP successfullydetected >96% of the RMP-resistant strains and 87%of the INH-resistant strains and was 100% specific.

These methods generally require large amounts ofamplified product to achieve unambiguous results.Although this limitation does not apply to analysis ofisolated colonies or BACTEC cultures, direct detec-tion of drug resistance markers within a clinical spec-imen may be hindered by the presence of inhibitorsor by small numbers of mycobacteria. To overcomethese problems, Whelen et al. (1995) devised a single-tube heminested PCR that provided high sensitivityof detection of M. tuberculosis in sputum and sufficientproduct for subsequent analysis by sequencing orSSCP.

Hybridization of DNA to oligonucleotide probes is awell-established technique for detecting mutations.Successful hybridization under stringent conditions isdependent upon a perfect match between the targetand a short probe. This is the basis of a commercialtest designed to detect mutations within the 69-bphypervariable region of the rpoB (Inno-LiPA Rif.TB,Innogenetics N.V., Zwijndrecht, Belgium). The lineprobe kit consists of a membrane strip onto which 10 oligonucleotide probes are immobilized; onespecific for M. tuberculosis complex, five overlappingwild-type probes that encompass the entire hyper-variable region, and four for specific rpoB mutations.

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Biotinylated PCR products are hybridized with theprobes, and hybrids are determined by an immuno-enzymatic procedure that results in a visual colour.Evaluations of the line probe assay with RMP-resistant strains have shown >90% concordancewith phenotypic RMP susceptibility testing results(Cooksey et al. 1997; Telenti et al. 1997). In Cooksey’sstudy, five resistant isolates (two with codon inser-tions and three which had no mutations in the 69-bpregion) were identified as RMP sensitive by the lineprobe assay. Mutations in four isolates which demon-strated resistant subpopulations by phenotypic sus-ceptibility testing were correctly identified. However,in Telenti’s study the LiPA missed one isolate whichcontained a mixed population. De Beenhouwer et al.(1995) described using the LiPA for detecting RMPresistance directly in clinical specimens. Results fromthese studies indicate that the line probe assay mayserve an important role as a rapid and convenientscreen for rifampin resistance in M. tuberculosis.

Other novel strategies that have been described fordetecting rpoB mutations include dideoxy finger-printing (Felmlee et al. 1995), heteroduplex forma-tion analysis (Williams et al. 1994), RNA/RNAmismatch (Nash et al. 1997) and molecular beaconprobes (Piatek et al. 1998). Dideoxy fingerprintingwas recently used to enhance visibility of mobilityshifts of rpoB-specific amplification products. Thistechnique combines elements of dideoxy sequencingand SSCP, resulting in increased sensitivity for muta-tion detection. Heteroduplex formation involvesmixing denatured PCR product from the test strainwith product from a susceptible control strain.Hybridization results in formation of heteroduplexproducts which exhibit different electrophoreticmobility compared with homoduplex hybrids. TheRNA/RNA mismatch assay involves transcription ofsingle-stranded RNA from the test strain PCR prod-ucts and complementary single-stranded RNA fromPCR products from an RMP-susceptible strain. RNasecleaves the RNA/RNA duplex at any positions of basemismatch, and the RNase reactions are analysed byagarose gel electrophoresis. An advantage of thisassay over SSCP and heteroduplex analysis is that alarger region of the gene can be screened giving the

potential of detecting more mutations within a singleassay. The mismatch assay is simple to perform andinterpret and has the capability of detecting resistantsubpopulations. It has also been used to detectmacrolide resistance in M. avium (Nash & Inderlied1996). Another novel approach to mutation analysisis the use of fluorogenic reporter molecules calledmolecular beacons (Tyagi & Kramer 1996) for allelicdiscrimination in a real time PCR assay. Molecularbeacons are single-stranded probes that possess astem-and-loop structure with the loop portion beingcomplementary to the target sequence. A fluorescentmoiety is attached to one end of the stem and a non-fluorescein, quenching moiety to the other.When the molecular beacon hybridizes with thecomplementary target, the probe–target hybrid being stronger and more stable that the stem hybrid,the fluorophore is separated from the quencher, permitting the fluorophore to fluoresce. The power ofmolecular beacons is their ability to hybridize only totarget sequences that are perfectly complementary.Piatek et al. (1998) have successfully used molecularbeacon analysis to detect a broad range of pointmutations, as well as insertions and deletions, in the81-bp region of the M. tuberculosis rpoB. Molecularbeacons have also been designed to detect mutationsin the three genes associated with INH resistance (D.Alland, personal communication).

Cleavase fragment length polymorphism (CFLP) isa new alternative to SSCP and sequencing for muta-tional screening. CFLP has been applied to theidentification and positioning of katG mutations asso-ciated with INH resistance in M. tuberculosis (Brow etal. 1996). CFLP is based on the observation that dena-tured single strands of DNA can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as CleavaseI. The cleavage patterns are characteristic of thesequence analysed so that each DNA has its ownstructural fingerprint. Point mutations change thestructural conformation around the site of the muta-tion and are reflected as changes in the structuralfingerprint. By detecting conformational changeswith the use of enzymatic cleavage rather than elec-trophoretic mobility, sequence differences in much

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larger molecules are detectable. The Cleavase technology, under development by Third WaveTechnologies (Madison, WI), has potential utility fordifferentiating drug-sensitive and drug-resistantstrains and for distinguishing mycobacteria at thelevel of genus and species.

4.2 Metabolic assays for assessing viabilityin the presence of drugs

Methods for detecting specific genetic mutationshave limited practical value owing to our incompleteunderstanding of all the mutations associated withthe development of resistance. Molecular methodsthat provide a direct measurement of bacterialmetabolism can potentially circumvent this problem.One such approach has been the application of theGen-Probe MTD amplification assay to the BACTECsystem (Kawa et al. 1989; Miyamoto et al. 1996;Martin-Casabona et al. 1997). Detection of rRNA indrug containing BACTEC vials can shorten the turn-around time for susceptibility results. However, thestability of the rRNA requires incubation in the pres-ence of antimycobacterial agents for 3–5 days toobtain reliable discrimination of drug-sensitive and drug-resistant isolates. Cangelosi et al. (1996)recently described hybridization and RT-PCR assaysfor M. tuberculosis pre-16S rRNA. Results for RMP andciprofloxacin were obtained within 24 and 48h,respectively, of exposure to the drugs. This systemwas unable to detect any depletion of rRNA precursormolecules in the presence of INH or ethambutol.Quantitative analysis of mRNA as a marker of viabil-ity has been proposed by Hellyer et al. (1999) to be auseful method for rapid drug susceptibility testing.Using a quantitative RT-PCR assay, M. tuberculosisstrains that were susceptible to INH and RMP showedmarked reduction in alpha antigen mRNA expressionwithin 24h of exposure to these drugs. In contrast,alpha antigen mRNA levels in resistant strains werenot reduced.

An innovative method for examining metabolicactivity is the biological assay based on the luciferasereporter phage (LRP) technology (Jacobs et al. 1993;see also Chapter 3). The luciferase reporter phage

is an ingenious tool for evaluating viability and in-volves infecting mycobacterial cells with a phage carrying the firefly luciferase gene. In the presence ofadenosine triphosphate (ATP), found only in livingorganisms, luciferase produces light from its substrateluciferin. When mycobacteria are infected with the reporter phage and then treated with drugs, lightis produced only by viable or drug-resistant myco-bacteria. Recently, Riska et al. (1997) have shown that the LRP assay when coupled with BACTEC and the NAP compound can differentiate between M. tuberculosis complex and non-tuberculous myco-bacteria and characterize drug susceptibility patternswithin 24–48h. Recent achievements indicate thatthe development of an even more efficient LRP protocol is possible. First, new reporter mycobacte-riophages with higher degrees of sensitivity havebeen generated (Carrière et al. 1997). These phagescan detect mycobacteria in BACTEC vials at growthindices as low as 10. Second, it has been possible todetect M. tuberculosis in processed AFB smear-positivesputum samples within 24–48h, without the need forsubculturing in the BACTEC system. Third, a newformat for photographic detection of light output has been developed (Riska et al. 1999). The film-based LRP assay, when incorporated with the newmycobacteriophages that can induce prolonged lightproduction in infected host cells, should furtherenhance the efficiency of this diagnostic technology.

4.3 The future of molecular diagnostics

Recent technological advances have enabled the clin-ical mycobacteriology laboratory to detect M. tubercu-losis in clinical specimens and to screen for resistanceto the commonly used antituberculosis drugs within24–48h. Many clinical laboratories are routinelyusing either a commercial amplification system or anin-house PCR assay to test acid-fast, smear-positiverespiratory specimens for primary diagnosis.Unfortunately, these rapid diagnostic tests have notreplaced acid-fast smears or mycobacterial cultures.Smear microscopy provides an index of the degree of contagiousness, facilitating informed decisionsregarding public health measures. Mycobacterial

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cultures allow determination of complete drug sus-ceptibility profiles, which are recommended for allpatients to ensure optimal treatment. High sensitivityand specificity must be achieved with all sample typesbefore amplification techniques can replace classicdiagnostic methods. High specificity can be achievedif the laboratory staff is properly trained and complieswith the stringent quality control requirements. Lackof sensitivity most likely results from the use of smallsample volumes and irregular dispersion of theorganisms in the paucibacillary samples. These shortcomings suggest the need for improved samplepreparation methods and/or the performance ofmore than one test on each sample. Such issues con-tinue to be addressed, and we can expect that thesecond- and third-generation tests will provideimproved sensitivity and specificity. Among the mostpromising recent developments is the microarraytechnology (DNA chips) which, combined with DNA or RNA amplification, could provide rapididentification of a wide range of mycobacterialspecies and drug susceptibility results. The microbiol-ogy laboratory wish list for future amplificationsystems includes those that are speedy, automated,sensitive, specific, are not at risk of cross-contamina-tion, affordable and amenable to quantification.Thanks to the efforts of numerous academic scientistsand commercial firms, this potential is becoming areality. Hopefully, the ensuing competition betweenassays will eventually result in decreased costs for thisstate-of-the-art revolutionary technology.

5 Acknowledgements

The author would like to thank Donald Cave, TobinHellyer, Lucy DesJardin, Marjorie Beggs, VivianJonas-Taggart, Bill Keating, David Alland and JohnChan for helpful comments and providing informa-tion prior to publication.

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