i REVIEW ARTICLE

Nucleic Acid Amplification for Mycobacterial Diagnosis

Gaby E. Pfyffer

Introduction Within the past few years nucleic acid amplifi- cation(NAA)-based techniques have become accessible to the clinical mycobacteriology laboratory, providing it with a large array of exciting tools for direct detection of acid- fast bacilli (AFB) in clinical specimens, species identifica- tion, detection of resistance to antimycobacterial agents, and molecular epidemiology. Direct detection represents indeed the most dramatic improvement in mycobacterial diagnostics inasmuch as tuberculosis (TB) or mycobacte- rioses can, theoretically, be diagnosed on the day of arrival of a specimen in the laboratory. In their pioneering work during the early 1990s Kirschner ut al.’ had pro- posed an elegant algorithm appIying polymerase chain reaction (PCR) prior to 14s rRNA gene sequencing for direct detection of mycobacteria in clinical specimens. Since then, several other molecular techniques have been introduced for rapid detection of AFB, mainly of Mycobncterium t~herc~his complex, in clinical speci- mens. Even though NAA-based detection is a most appealing approach present-day drawbacks which still limit their wider application in routine clinical diagnosis have to be overcome.

Home-brew NAA Protocols and Commercially Available, Kit-based Test Formats and their

Diagnostic Performance on Respiratory Specimens

Home-brew PCR protocols amplifying a large variety of chromosomal DNA elements have disclosed new ways to diagnose TB and, to a lesser extent. non-tuberculous mycobacteria (NTM). The rapidly growing number of published reports on direct detection of mycobacteria have concentrated on both genus-specific and M. tubercu- losis complex-specific PCR assays. Genus-specific proto- cols are targeting genes such as the ones coding for the 16s rRNA or the 65-kDa heat shock protein (hsp). Subsequent mycobacterial identification is done with

highly discriminating probes or gene sequencing’, or with restriction enzyme analysis’. Among the M. tukrcdosis complex-specific PCR assays the DNA element amplified most frequently is the IS61 70. Other targets are genes encoding the 3%kDa protein (protein antigen b), the MPBh4 protein, the mtp40, and pMTb4 (cf. Soini et aI. 3).

To guarantee a high degree of reproducibility and to facilitate the application of NAA-based techniques in the clinical mycobacteriology laboratory, several important methods have been developed and are now marketed in user-friendly, kit-based formats, targeting either DNA or RNA. Although some of these, e.g., PCR and strand dis- placement amplification (SDA). have the potential to detect mycobacteria other than ~M~ycobactrrium tukrdo- sis complex current experience is almost exclusively restricted to the diagnosis of TB. Molecular strategies include either target amplification as done by PCR’j, transcription-mediated amplification (TMA)“.;, ligase chain reaction (ICR)S,“. and SDA”‘,’ I, or signal amplifi- cation e.g., 0-p amplification’?. While PCR and TMA have recently been approved by the Food and Drug Administration in the IJnited States for use on smear-pos- itive, respiratory specimens. other techniques are, at pres- ent, either clinically being evaluated or still under development.

Applying home-brew PCR protocols for direct detection of M. tuhmdosis complex overall sensitivities between 77 and 100% and specificities between 88 and 100% were achievedi.’ i.‘i. Regarding commercially available, kit- based test formats, seven European laboratories have tested more than 7100 respiratory specimens with the manual version of Amplicor PCR (Roche, Somerville, NJ). When compared with culture and the patients’ clinical data a sensitivity of 87.9% and a specificity of 99.6% was obtained’. With the automated version of Amplicor TB PCR (referred to as the Cobas Amplicor MTB) the values are in the same range. For more than 8000 respiratory specimens, the ‘Amplified M. tubrrc~dosis Direct Test’ (MTD: &n-Probe. San Diego, CA) yielded overall sensitivities between X2 and 97%. and specificities between 97 and

22 G.E. Pfyffer

100% for pulmonary specimens (cf. Pfyffer et nl.‘). Data generated by utilizing LCK (LCx; Abbott, Abbott Park, IL) look very much alike. For SDA, finally, established on a fully automated system (BDProbeTec: Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) which simul- taneously amplifies target sequences of the IS67 10 and the 16s rKNA gene limited information is available yet.

As a whole, data derived from the many different studies (some selected examples are listed in Table I) demonstrate excellent sensitivities and specificities for smear-positive specimens (always compared with conven- tional diagnostics and patients’ clinical data). However, these parameters are considerably lower for smear- negatives precluding, thus, the general use of NAA-based techniques as a screen to safely rule out TB.

Terra incognita: Extrapulmonary Specimens Compared with respiratory specimens, much less infor- mation is available on the diagnostic performance charac- teristics of NAA-based techniques in the extrapulmonary situation, such as tuberculous pleuritis or TB meningitis, for which a rapid and accurate diagnosis is of utter impor- tance. The problems arising when dealing with extrapul- monary specimens are well-known, both to the clinician and the laboratorian. For the former, (i) any mycobacter- ial species may be responsible for disease: (ii) as far as TB is concerned, non-respiratory TB is far less frequently seen than the pulmonary form: and (iii) disease may manifest

quite frequently at inaccessible body sites requiring thus, invasive procedures for specimen collection. For the labo- ratorian, on the other hand, (i) a large array of different types of specimens are submitted which may require indi- vidual, labour-intensive pretreatment and DNA extrac- tion procedures: (ii) very often, disappointingly little amount of specimen is sent to the laboratory: and (iii) in most cases, a paucibacillary situation is encountered resulting in negative smears. Above all, the few studies dealing with extrapulmonary specimens comprise only a small number of positive samples, usually not exceeding 10% of the total number of specimens analysed (Table II). Under such low ‘prevalence’ conditions, performance parameters are of rather limited value.

Usually, most extrapulmonary specimens such as urine, gastric juice, lymph node, and biopsy specimens are analysed by NAA-based techniques without major prob- lems while others (e.g., pleural or ascitic fluid specimens) are known to be difficult’,’ j. Similarly, the diagnosis of TB meningitis appears to be a hot issue as well. As illustrated in Table III much has to be improved towards a more reli- able PCK-based diagnosis. Likewise, difficulties were also reported when utilizing MTD inasmuch as the test yields reliable results only after cerebrospinal fluid (CSF) speci- mens have been pretreated with sodium dodecyl sulfate (SDS)-NaOH prior to amplification’. Most recently, Lang and co-worl~ers*h have presented an interesting approach by lowering the cut-off value of the test for CSF speci- mens. More important, these authors also showed that freezing of CSF specimens prior to MTD is not advisable.

Table 1. Diagnostic performance of NAA-based techniques currently used for the direct detection of M. tubercufosis complex in respiratory specimens’

Home-brew yrotocols PCR

Kit-based test formats PCR (Amplicor: Roche)

PCR (Cobas Amplicor; Roche) 16s rRNA

TMA (MTD; Gen-Probe) rRNA

LCR (Xx: Abbott) 3%kDa protein

SDA (BDProbeTec; Becton Dickinson)

Qp (Galileo: Gene-Trak)

Target

IS6110 IS6170(ne)

1hS i-RNA

IS6 7 10 plus 1 hS rRNA 2 3s rRNA

Sensitivity (‘Xj)b Overall Smear-negatives

85.0 73.0 77.1 57.9

74.0 53.0 97.8 - 90.8 51.0 84.3 57.9 92.4 59.6 83.0 68.0 100 - 77.0 57.0 77.1 36.8 90.8 53.0 90.2 70.0 97.9 92.3

93.0 YX.9 100

- 100

99.0 99.3 99.0

100 98.4 96.5

14 27 2x 26 28 29 27

Y 26 30 31 11

84.0 69.2 Y7.0 12

Specificityh (‘Xl)

8X.0

References

14 26

%elected examples “after resolution of discrepant results

Direct Detection of Mycobacteria 23

Table II. Diagnostic performance characteristics of commercially available NAA-based techniques used for direct detection of Al. t~fheri~~k~sis com- plex in extrapulmonary specimens”

Technique Number 01‘ specimens Sensitivity I’!%)h analysed Overall Smear-negiltivcs

I’CK IAmplicorJ 241) x7.7 325” 7x.5

‘I’MA l~l’Tl)l 522 Y3.1 Y0.5 L/Z’ x(1.x Th.6 ‘07 Y4.4 Y’.;

IL’R (ILki I47 13.: 526 7X.5 il.1

~‘Values are given after resolution of discrepant results. “,~l~~c‘ohc~ct~riunl specific assay.

‘Enhanced LITD.

Table III. NAA-based diagnosis of TB meningitis

Technique No. 01 specimens analyscd Scnsitivitv I’%)

I’CR 34 64.i PCK 33 il.0 I’CR 40 X5.0 1’CR 9; 3 3.0 Amplicor I’C’K hY 60.0 Rill’ll 54 100 MTD x4 8 3.0

,‘cut-off value 2 1 1 000 RI.lIs

Spccil’icity” y!,

Y Y 5 Y 3.5 Yi.7 YX.4 Yh. 3 100 100

Specificity t’!%)

X8.1 Y4.1 93.1 100 100

9h.N 100

kfercncea

2 3;

34 I’Uyl’kr. (:.E.. unpublished data

35 $6

Kekrences

3; ;X 39

40 41

i 1 h

False-negative and ‘False-positive’ Results- How to Cope?

Difficulties in the interpretation of results generated by Conversely, not all NAA positive/culture negative NAA-based techniques become obvious when results results may necessarily be considered false-positive. diverge from the clinician’s view and from the results Again. the results generated by molecular methods obtained by classical microbiological methods, i.e.. smear should be interpreted in conjunction with the patients’ and culture. False-negative results (amplification nega- clinical data, i.e.. medical history, physical examination, tive/culture positive) may be due to several factors: (1) chest X-ray, tuberculin testing, and response to therapy. there may be an intrinsically low sensitivity of the ampli- In doing so, many of the NAA results will prove to be cor- fication procedure itself when dealing with clinical speci- rect (i.e.. true-positive), in particular, if specimens origi- mens. Although manufacturers of kit-based tests claim a nate fromTB patients still being under treatment or if the detection limit of I 1 AFB/ml of specimen, in prnsi the sit- clinical symptoms are highly consistent with TB. The lat- uation looks quite different: When analysing CSF which ter has to be emphasized since culture never attains a had been artificially spiked with cells of M. tubrrculosis, sensitivity of 100% - even when working with the ‘gold consistently positive results were obtained only as long as standard’, i.e.. using a culture consisting of a combina- the concentration of TB cells was not < 1 OL/ml of CSF:: tion of liquid and solid media. A lot more difficult is a sit- (2) in paucibacillary situations, false-negatives may result uation where specimens originate from symptomatic from sampling errors, in particular when taking into patients with a history of TB many years ago, or from account the well-known tendency of mycobacteria to patients who had admittedly close contacts to family aggregate: (3) missing the diagnosis may, furthermore. be members suffering from active TB;. As long as sufficient caused by the presence of exogenous DNA polymerase experience with NAA-based techniques is lacking such inhibitors such as anticoagulants and detergents as well results should be considered inconclusive even though as endogenous inhibitors (cf. Richeldi et nl.17). Inhibition they may indicate in these cases an early stage of TR rates being as high as 23% have been reported’“. reactivation or a new infection. respectively. There

Interestingly, inhibition of amplification is not only con- fined to DNA but occurs also in NAA-based techniques targeting RNA”.

24 G.E. Pfyffer

Table IV. Indications for direct detection of M. ttrbrrctkxis complex in clinical specimens’

Smear

Positive

Negative

Type of clinical specimen

Respiratory (sputum, bronchial and tracheal aspirate, bronchoalveolar lavage)

Non-respiratory (lymph node. cerebrospinal fluid, urine, gastric fluid. specimens, other body fluids, pus, biopsies, etc.) Respiratory

Non-respiratoty

Direct testing recommendedh -.

Should not be universally applied. Recommended in the following situations: -X-ray abnormal but not highly suggestive of TB ~ special disposition of the patient (immunosuppressive therapy [cancer patient. transplant recipient]. HIV infection, chronic lung disease, old age etc.) - epidemiological concerns (inmate of shelter home, HIV ward. or prison: drug abuser) -clinical symptoms of TB but culture remains negative (untreated patient) Always if TB is part of the differential diagnosis

-Clinical symptoms highly suggestive of TB (night sweats, weight loss, cough, etc.) -X-ray highly suggestive of TB (cavity, infiltrate etc.) - haemoptysis ~ unsuccessful antibiotic treatment Always if TB is part of the differential diagnosis

“see also Bottger et al. (1995)“’ Yesting of multiple specimens is often necessary

remain, finally, cases which can safely be considered false-positives arising from cross-reaction with mycobac- teria other than M. tuberculosis complex’“, from contami- nation with other clinical samples, or from carry-over of amplicons in the laboratory. In an interlaboratory study involving 30 expert laboratories performing NAA-based techniques for the direct detection of M. tuberculosis com- plex (home-brew PCR, Amplicor PCR, MTD) Noordhoek et ~1.~” have beautifully illustrated that lack of specificity was the problem rather than lack of sensitivity. Most importantly, reliability of a test procedure was not associ- ated with the use of any particular method. Such studies clearly underline the need for good laboratory practice and for reference reagents to monitor the performance of the assays which have to consist of ample internal qual- ity controls including inhibitory controls as well as refined processing methods for a large variety of clinical specimens. No less important is a regular contamination monitoring of all separate areas in the laboratory where NAA-based techniques are to be performed, and the participation of the laboratory in external proficiency testing schemes.

More Questions Appearing on the Horizon Apart from the difficulties in explaining divergent results little sound information is currently available concerning the viability of cells detected with these molecular proce- dures. Applying single-tube, reverse transcriptase, nested PCR Jou et aLzl have clearly demonstrated that targeting mRNA appears to be a far better approach to judge mycobacterial viability than by amplification tests directed at DNA and RNA targets.

Although some authors have suggested that PCR may be useful in monitoring response to therapyZ2 a recent study has demonstrated that neither PCR nor SDA are able to do so, since as many as 22% of the clinically cured patients have still had positive results after more than 1 year13. Utilizing the ABI 7700 System (TaqMan) and competitive PCR for quantification of IS6 1 IO DNA in spu- tum during TB treatment pointed in the same direction inasmuch as the rate of disappearance of M. tuberculosis DNA did not correlate with the decline in cultivated bacilli either24. Analogous findings have been reported for MTD: In 23 patients examined (i) conversion with rRNA (as compared with smear and culture) had taken longest, and (ii) 56% of the patients had a period of shedding non-cul- tivable M. tuberculosis which lasted from 7 to 245 days”j.

Current Diagnostic Value of NAA-Based Test Systems

For both patient and clinician the most obvious advantage of NAA-based techniques is the rapidity by which mycobacterial disease can be diagnosed (PCR and LCR, 5-6 h; MTD, 3 h). Although all currently available meth- ods have, from a technical point of view, the potential to amplify and detect minute amounts of target nucleic acid, they have, however, to be improved to achieve higher reli- ability, in particular as far as smear-negative specimens are concerned.

As to laboratory strategies, ideally, molecular direct detection should, by and large, replace the conventional detection and identification methodology, i.e., free the clinical mycobacteriology laboratory from much of the cumbersome work. At present, this has, however, not yet

Direct Detection of Mycobacteria 25

been accomplished. Therefore. these assays can currently only be used as an adjunct to the general standard proce- dures. A laboratory offering today’s new molecular possi- bilities has to strive for carefully working out, together with the clinician, in which situation and with which types of specimens direct detection is indicated (e.g.. for M. tlrherculosis in Table IV). Finally, any integration of new molecular technologies in a laboratory has also to address personnel resources and cost-effectiveness. With respect to the latter all NAA-based technology, especially the commercial tests, suffer from high costs. Although convincing cost-benefit calculations are still unavailable, it is quite obvious that on a more balanced view. a straightforward application of NAA-based techniques will pav off, in particular when taking into account patient management and the high costs of hospitalization and therapy,

Thus, the role of molecular tests in direct detection of mycobacterial disease will eventually be determined once the answers to the plethora of questions become clearer. Aiming at this goal, more prospective work is needed to define the practical value of these new tools in the clinical mycobacteriology laboratory.

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