INTRODUCTION

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Since their introduction to the clinical mycobacteriology laboratory, amplification techniques have been welcomed as being able to have a strong impact on the speed and accuracy of diagnostic results. However, the promise of timely and sensitive detection of Mycobacterium tuberculosis complex (MTB) directly from clinical specimens is still unfulfilled because of the unsatisfactory sensitivity of current amplification assays. A number of amplification systems have been described; besides in-house assays, commercial systems have been developed with the aim of providing standardized, easy-to-use kits having the potential of "walk-away" automation. Moreover, recent evidence of inhibitory samples has brought companies to develop kits containing a second target to be used as an internal amplification control (IAC). The IAC monitors amplification and detection steps, thereby making negative test results truly reliable.

To date, a few commercial systems for the detection of MTB in clinical samples are available in Italy. Of these, the Amplified Mycobacterium tuberculosis Direct Test (Gen-Probe, Inc., San Diego, Calif.) has recently been upgraded, featuring a larger amount of sediment sample combined with a shorter assay time, and is marketed as the enhanced AMTD (AMTDII), whereas the COBAS AMPLICOR MTB System (Roche Diagnostic Systems, Inc., Branchburg, N.J.) exhibits an internal control for monitoring of amplification inhibitors coupled with a high degree of automation.

The purpose of this study was to carry out a comparative evaluation of these assays.

	MATERIALS AND METHODS

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Study design. Four hundred eighty-six clinical specimens, consecutively received for culture of acid-fast bacilli (AFB) by the Regional Mycobacteria Reference Centre in Vicenza, Italy, were used in this study. The specimens, almost entirely collected from inpatients for whom tuberculosis (TB) was strongly suspected, were submitted to the reference laboratory from different hospitals within the whole region.

Specimen collection and processing. The specimens investigated were collected from 323 patients and included 257 sputum samples, 2 bronchoalveolar lavages, 37 bronchial washings, 4 gastric aspirates, 70 urine samples, 37 normally sterile body fluid (pleural, pericardial, synovial, cerebrospinal fluid [CSF], and ascites fluid) samples, and 79 miscellaneous samples, such as pus and biopsy specimens.

Pretreatment of selected clinical specimens for amplification. (i) Pretreatment of CSF. CSF was treated with NALC-NaOH and centrifuged at 12,000 × g for 10 min. The pellet was resuspended in PBS and frozen in aliquots until the amplification techniques were performed.

(ii) Pretreatment of pleural and other sterile fluids. After decontamination with NALC-NaOH, the sediment was washed twice with sterile distilled water before being stocked for amplification assays.

Culture. A 0.5-ml portion of the processed sediment was cultivated by the radiometric BACTEC technique (Becton-Dickinson Diagnostic Instrument Systems) and with Löwenstein-Jensen (LJ) solid medium. All media were incubated for 6 weeks at 36 ± 1°C. BACTEC 12B culture vials were tested for growth twice a week for the first 3 weeks and weekly thereafter. The radiometric growth index (GI) was recorded by the BACTEC instrument; a GI of >50 was considered suspect, and smears were made daily to confirm the presence of AFB.

Microscopy. To detect AFB, smears were stained with auramine-rhodamine fluorescent stain.

Identification of mycobacteria. Isolates were identified by specific DNA probes (Accuprobe; Gen-Probe, Inc., San Diego, Calif.), by standard biochemical tests, and by the high-performance liquid chromatography method (6).

Amplification procedures. Amplification assays were run in three separate areas which had been set up in two different rooms.

Gen-Probe AMTDII. The Gen-Probe AMTD assay (Gen-Probe, Inc., San Diego, Calif.) was performed according to the instructions supplied by the manufacturer. Each run included positive and negative amplification controls: the former was prepared from a 10⁴ to 10⁵ dilution of a 1 McFarland nephelometric standard suspension of M. tuberculosis ATCC 27294, while the latter was made from a similarly prepared suspension of Mycobacterium gordonae ATCC 14470. A cutoff value of 30,000 relative light units was used for positive specimens. To tentatively detect inhibitory substances, frozen aliquots of discrepant samples were tested retrospectively after 1:5 and 1:10 dilution with PBS.

COBAS AMPLICOR. The COBAS AMPLICOR test was done by following the instructions supplied by the manufacturer. The procedure, starting from a 100-µl sediment sample portion, consisted of two steps: specimen preparation and combined, fully automated amplification and detection. The IAC DNA sequence contained primer-binding regions identical to those of the MTB target sequence. A unique probe-binding region differentiated the IAC from the target amplicon. The IAC was introduced into each amplification reaction and was coamplified with the possible target DNA from the clinical specimen. In addition, each run included positive and negative amplification controls. A colorimetric reading exhibiting absorbance values greater than 0.350 optical density units was considered as positive. Specimens showing IAC inhibition were repeated after 1:5 and 1:10 dilutions of the sample with a mixture of 50% Respiratory Lysys Reagent and 50% Neutralizing Reagent.

Patients' clinical evaluation. Clinical assessment included the patients' medical history, signs, symptoms, chest X-ray, pathology, and microbiology results, as well as follow-up observations. All of the records were carefully reviewed, with the aim of setting up the combination of culture and clinical diagnosis as the "gold standard." After this analysis, amplification results were reclassified as appropriate.

Statistical analysis. Statistical comparisons were calculated by using the chi-square test; P < 0.05 was considered significant.

	RESULTS

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Respiratory specimens. A total of 296 respiratory specimens collected from 194 patients were tested. Altogether, 133 specimens yielded a culture positive for AFB; 114 isolates were found to belong to the MTB, while the remaining 19 strains were identified as nontuberculous species. Amplification results with smears, cultures, and clinical data are summarized in Table 1. A total of 126 specimens were from patients with a diagnosis of tuberculosis, and 170 were from patients with nontuberculous pulmonary disease, based on clinical and microbiological findings.

Extrapulmonary specimens. A total of 190 extrapulmonary specimens collected from 129 patients were tested. Altogether, 44 specimens yielded a culture positive for AFB; 33 isolates were found to belong to the MTB, while the remaining 11 strains were identified as nontuberculous species. The amplification results with smears, cultures, and clinical data are summarized in Table 3. A total of 41 specimens were from patients with a diagnosis of extrapulmonary tuberculosis, and 149 were from nontuberculous patients, based on clinical and microbiological findings.

Specimen inhibition. On the basis of our retrospective study, the overall inhibition rate for AMTDII was 2.9%, ranging from 2.1 to 3.4% for extrapulmonary and respiratory specimens, respectively. The inhibition rate for AMPLICOR was slightly higher (3.9%), ranging from 2.6 to 4.7% for extrapulmonary and respiratory specimens, respectively. Proper dilution according to the manufacturer's protocol was able to overcome inhibition in 12 of 19 (63.1%) samples. AMTDII-inhibiting samples (except one) were different from those showing inhibition by the AMPLICOR assay. Almost entirely, they included sputa, biopsy specimens, and sterile fluids. Moreover, inhibitory samples were not found in all sputum specimens collected from the same patient.

	DISCUSSION

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The laboratory diagnosis of MTB infection by culture techniques usually requires 1 to 8 weeks. The present study demonstrates that amplification tests can detect MTB in clinical samples within a few hours. The AMTDII turnaround time is shorter than that of AMPLICOR (2.5 versus 7.5 h). However, the latter assay offers a considerable advantage in reducing hands-on time and gives the opportunity to run the system overnight. The kits contain all of the reagents needed for sample amplification and detection and appeared to fit well in the workflow of a reference laboratory performing amplification twice a week. From the analytical point of view, differences among cutoff values, positive and negative controls, and samples were broad enough to allow easy discrimination by both assays.

In comparison with the gold standard, the sensitivity and specificity of AMTDII were 85.7 and 100% for respiratory specimens and 82.9 and 100% for extrapulmonary specimens, respectively. No significant differences in sensitivity between respiratory and extrapulmonary specimens were observed. Negative results obtained from smear-positive or smear-negative MTB-yielding samples were shown to depend almost entirely on the presence of inhibitors of enzymatic amplification. In fact, most of these samples (13 of 16), which in a routine setting were likely to be misdiagnosed to contain NTM or considered as negative, turned out positive by simple dilution. A considerable increase in the specimen volume (from 50 to 450 µl) in comparison with the former version has been claimed as the most likely explanation of this previously undescribed inhibition (5).

In our view, an overall inhibition rate for MTB-yielding samples of 8.8% stresses the importance of monitoring inhibitory substances in clinical specimens. Systematic inclusion of the IAC would greatly contribute to the accuracy of the assay, also providing important information when testing nonapproved types of samples.

In this context, removal of inhibitory substances may be an alternative approach to improve sensitivity (P. Singer and F. Vlaspolder, Abstr. 20th Annu. Conf. Eur. Soc. Mycobacteriol., p. 76, 1999). However, because the nature of the inhibition is still unclear and probably affects amplification techniques unevenly, it seems unlikely we will be able to find a routine-fitting procedure able to remove all inhibitory substances (3, 4).

The sensitivity and specificity of AMPLICOR were 94.2 and 100% for respiratory specimens and 85.0 and 100% for extrapulmonary specimens, respectively. No significant differences in sensitivity between respiratory and extrapulmonary specimens were observed. Data from the literature about AMPLICOR sensitivity and specificity are in agreement with our findings (Bodmer et al. [1], 92.6 and 99.6%; Rajalahti et al. [8], 83 and 99%; Wang and Tay [10], 96.1 and 100%; and Reischl et al. [9], 83.5 and 98.8%, respectively) and document that the automated AMPLICOR assay exhibits higher sensitivity and specificity than those obtained by the manual version (7).

Using different kinds of clinical samples for amplification, we observed an overall inhibition rate of 3.9%. No significant difference between respiratory (4.7%) and extrapulmonary specimens (2.6%) was found. False-negative inhibitory samples were easily detected and soon reclassified as true positive or, when repeatedly inhibitory, as uninterpretable. Moreover, a positive IAC strengthens the predictive value of negative tests.

Negative results obtained by the AMPLICOR assay for two smear-positive, MTB-yielding noninhibitory samples remain unexplained. The same results were obtained upon a repeat assay, when a frozen aliquot of both samples was tested retrospectively. For smear-negative, culture-positive noninhibitory samples, a low number of mycobacteria, unequally distributed in the test suspension, is perhaps the most likely explanation.

Of the 20 samples (12 respiratory and 8 extrapulmonary) that were both smear and culture negative, obtained from patients strongly suspected of having TB, 11 were AMTDII positive and nine were AMPLICOR positive.

None of the specimens from patients found negative for TB by culture and clinical criteria or yielding NTM was positive by any assay.

We can conclude that, although at present, amplification assays cannot replace culture techniques, AMTDII and AMPLICOR were shown to be rapid and specific for the detection of MTB in clinical samples. Their protocols were easy to perform and suitable for a routine microbiology laboratory's workflow. On the basis of our data, the difference between AMPLICOR and AMTDII sensitivities was found to depend on the use of the IAC, which was lacking in the latter assay. Evidence supporting the IAC as an essential feature of commercial amplification assays is thriving, and it is likely to represent the landmark of the "second generation" kits. Moreover, the testing of three consecutive specimens per patient has to be considered as the minimum requirement, especially if IAC is not available.

In our opinion, transcription-mediated amplification (TMA) is potentially an excellent diagnostic technique for both respiratory and extrapulmonary specimens, provided that an IAC is included with the assay. In this context, the recent development of the VIDAS Probe MTB System (bioMerieux, Inc., Rockland, Mass.) featuring TMA automation coupled with IAC highlights a new technological challenge to current assays (2).

Finally, companies should make every effort to improve the sensitivity of amplification assays for smear-negative, culture-positive noninhibitory samples.