Comprehensive Evaluation of Performance, Laboratory Application, and Clinical Usefulness of Two Direct Amplification Technologies for the Detection of<i>Mycobacterium tuberculosis</i>Complex

Della‐Latta, Phyllis; Whittier, Susan

doi:10.1093/ajcp/110.3.301

Cited by 40 publications

(26 citation statements)

References 13 publications

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“…These figures are similar to those reported elsewhere (1,4). One in six test results above this value were not followed by a positive culture for tuberculosis, and false-positive rates of 5% or below were only achieved at RLU readings of 2 million or above.…”

supporting

confidence: 90%

Interpreting the Results of the Amplified Mycobacterium tuberculosis Direct Test for Detection of M. tuberculosis rRNA

Middleton¹,

Cullinan

Wilson

et al. 2003

J Clin Microbiol

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The Amplified Mycobacterium tuberculosis Direct (AMTD) test detects M. tuberculosis rRNA. By using culture of M. tuberculosis as a gold standard, a number of different diagnostic indices were examined in an attempt to determine the diagnostic performance of the AMTD test and demonstrate how it might usefully be interpreted during the early management of disease.Recent developments in diagnostic methods have decreased the time needed for detection and identification of Mycobacterium tuberculosis, but the process still requires at least 2 weeks. The Amplified Mycobacterium tuberculosis Direct test (AMTD test; Gen-Probe, Inc., San Diego, Calif.) can be used to detect M. tuberculosis rRNA in respiratory specimens (sputum, broncho-alveolar lavage [BAL], and bronchial and tracheal secretions) and can be performed in approximately 3.5 h. With this test specific RNA amplification products are hybridized to complementary acridinium-ester-labeled DNA probes. Subsequent degradation of the acridinium-ester probes results in luminescence that is measured in relative light units (RLU).According to the manufacturers an RLU reading of Ͼ30,000 signifies a positive test, but they recommend repeating the tests at between 30,000 and 100,000 RLU. Sensitivities and specificities of the test at other RLU values, in comparison with a clinical diagnosis of tuberculosis, have been reported (1) with results of around 90% for each. Here we report the performance of the AMTD test with a consecutive series of hospital patients suspected of tuberculous infection and demonstrate how the results might usefully be interpreted during their early management.From 1994 to 2001 434 sputum and 339 BAL samples from separate patients were received for AMTD testing and mycobacterial culture. All were processed by a modified Petroff's method (3) prior to AMTD testing and culture on Löwenstein-Jensen slopes and in Middlebrook 7H12 radiometric broth (Becton Dickinson, Sparks, Md.). Cultured M. tuberculosis was identified by biochemical tests (5) and/or Accu-Probe culture confirmation tests (Gen-Probe, Inc.).Of the 773 respiratory samples, 178 were smear positive (146 sputum, 32 BAL). Ninety-two cultures grew M. tuberculosis, 66 from sputa (54 smear positive, 12 smear negative) and 26 from BAL (12 smear positive, 14 smear negative). Seventy-five cultures (10%) grew nontuberculous mycobacteria. The median RLU value for specimens with positive cultures for M. tuberculosis was 2,345,744 (range, 3,094 to 3,648,998 RLU), and for those with negative cultures (including nontuberculous mycobacteria) the RLU value was 7,031 (range, 448 to 3,609,521 RLU). The median RLU value for specimens with only positive cultures for nontuberculous mycobacteria was 12,870 (range, 1,205 to 3,424,688). Figure 1 shows the distribution of RLU readings for AMTD tests on samples with negative cultures or with cultures of either M. tuberculosis or nontuberculous mycobacteria.Using the culture results as the "gold standard," we calculated the sensitivities, false-positive rates (100 Ϫ % s...

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confidence: 90%

Interpreting the Results of the Amplified Mycobacterium tuberculosis Direct Test for Detection of M. tuberculosis rRNA

Middleton¹,

Cullinan

Wilson

et al. 2003

J Clin Microbiol

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“…Reducing the number of sputum specimens tested per patient from three to two would reasonably decrease the costs. However, multiple specimens are required to maximise the sensitivity of the PCR test in the detection of M. tuberculosis especially in smear-negative specimens, and to confirm the exclusion of M. tuberculosis in smear-positive specimens [6,7,10]. The fact that patients expectorate bacteria infrequently and specimens are of inconsistent quality is supported by the findings of NELSON et al [11] in which 13% of smear-positive and 7% of smear-negative TB cases were detected by conventional tests only from the third sputum specimen tested.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to rapid differentiation between this complex and nontuberculous mycobacteria in smear-positive sputum specimens, NAA assays detect a considerable proportion of smear-negative culture-positive TB cases [4,6,7]. However, the implementation and performance of these tests demands financial resources and experienced laboratory personnel; therefore the cost-effectiveness of NAA assays in diagnosing pulmonary TB should be carefully assessed.…”

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confidence: 99%

Economic evaluation of the use of PCR assay in diagnosing pulmonary TB in a low-incidence area

Rajalahti¹,

Ruokonen²,

Kotomäki³

et al. 2004

Eur Respir J

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Economic evaluation of the use of PCR assay in diagnosing pulmonary TB in a low-incidence area. I. Rajalahti, E-L. Ruokonen, T. Kotomäki, H. Sintonen, M.M. Nieminen. #ERS Journals Ltd 2004. ABSTRACT: To determine whether polymerase chain reaction (PCR) testing in the initial diagnosis of pulmonary tuberculosis (TB) is cost-effective in a low-prevalence population, an economic evaluation was carried out between the smear and culture (NOPCR) and smear, culture and PCR (zPCR) strategies.A decision tree model based on retrospective laboratory data was developed to assess the strategies of testing patients with suspicion of TB. Direct healthcare costs prior to confirmation of TB or nontuberculous mycobacteria by PCR or culture were included. Effectiveness was measured by the probability of correct treatment and isolation decisions.In the baseline situation NOPCR costs J29.50 less than the zPCR strategy per patient tested. According to sensitivity analyses, reducing PCR test price, shortening test performance time or increasing the proportion of smear-positive patients in the tested population would contribute to cost savings with the zPCR strategy.Routine polymerase chain reaction testing of all specimens from suspected tuberculosis patients in a low-prevalence population was not cost-saving. When the polymerase chain reaction assay was applied only to smear-positive sputum specimens, the smear and culture strategy was clearly dominated by it, i.e. the polymerase chain reaction smear-positive sputum strategy was less costly and more effective in producing correct treatment decisions and isolations. To date the global tuberculosis (TB) epidemic has shown no notable decline. In low-prevalence countries, factors contributing to the spread of the disease, especially in hospital settings, are delays in diagnosis and initiation of treatment [1]. In addition to lack of suspicion of TB, diagnosis is prolonged by the time-consuming identification of the Mycobacterium tuberculosis complex in sputum specimens [2]. The acid-fast smear rapidly identifies patients with infectious TB, but is neither sensitive nor specific for M. tuberculosis bacteria. In countries like Finland (494 TB cases and 505 cases with nontuberculous mycobacteria in 2001) where nontuberculous mycobacteria are frequently detected, smear-positive nontuberculous cases may be isolated and treated with inappropriate drug combinations unnecessarily until culture results are available. Culture is sensitive in detecting mycobacteria for species identification and susceptibility testing, but requires a mean time of 2-3 weeks [3]. In smear-negative TB cases this may lead to unnecessary diagnostic procedures that increase healthcare costs and cause distress to the patients.Commercial nucleic acid amplification (NAA) assays are rapid, sensitive and specific tools for the detection of the M. tuberculosis complex in sputum specimens [4][5][6]. In addition to rapid differentiation between this complex and nontuberculous mycobacteria in smear-positive sputum specimens, NAA ...

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“…The overall AMTD2 test specificity ranged from 92.1 to 100%. Some papers showed specificity close to 100% (26, 57) while others reported false-positive results, ranging from about 1 to 7.1% (1,20,34,45,49). In this context, most false-positive results exhibited low RLUs close to the traditional cutoff of 30,000 RLUs.…”

Section: Currently Available Commercial Direct Amplification Tests (Cmentioning

confidence: 96%

Relevance of Commercial Amplification Methods for Direct Detection of Mycobacterium tuberculosis Complex in Clinical Samples

2003

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Mycobacteria are a group of acid-fast, aerobic, slow-growing organisms whose genus includes more than 90 different species. The causative agents of tuberculosis (TB), which is currently considered a global emergency, with more than 2 million people dying every year and 8 million new cases, belong to the Mycobacterium tuberculosis complex (MTB). Moreover, although of lesser public health importance, many other species referred to as nontuberculous mycobacteria (NTM) have also been associated with human disease with increasing frequency worldwide (65). As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed as early as possible to stop the spread of the disease. Active TB is currently diagnosed by conventional laboratory procedures including specimen digestion and decontamination, microscopic examination for the presence of acid-fast bacilli (AFB), isolation by culture on solid and/or liquid media, and identification and drug susceptibility testing of the recovered isolate. Because of the slow growth of mycobacteria, the above-reported laboratory procedures may require turnaround times of 3 to 4 weeks or longer.During the last decade, several molecular methods have been developed for direct detection and identification of MTB in clinical specimens. These methods, being able to potentially reduce the diagnostic time from weeks to days, have been acquiring greater and greater relevance in the field of laboratory TB diagnosis. The basic principle of any molecular diagnostic test is the detection of a specific nucleic acid sequence by hybridization to a complementary sequence, a probe, followed by detection of the hybrid. However, the sensitivity of nucleic acid probe tests that do not involve amplification is much lower than that of amplified ones. Any portion of nucleic acid can be copied by using the specific polymerase, provided that some sequence data are known for the setup of appropriate primers. In general, amplification of target nucleic acid sequences is composed of three parts: denaturation, primer annealing, and primer extension. Discovery of PCRs in 1986 made this process reiterative, leading to an exponential increase in the production of the amplified target. Soon after, alternative amplification techniques were developed and patented by companies, which used different enzymes and strategies, but they are all based on reiterative reactions. Many different amplification targets including both DNA or RNA fragments have been proposed. The target most frequently amplified in MTB is the IS6110 (31) repetitive element, of which 10 to 16 copies are present in most clinical isolates. Numerous techniques for nucleic acid extraction have been proposed, as have different types of controls for monitoring the efficacy of nucleic acid extraction and amplification procedures. Currently, the U.S. Food and Drug Administration (FDA) requires that culture (still considered the "gold standard" for TB diagnosis) must be done in conjunction with the performance of each amplification-based t...

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Comprehensive Evaluation of Performance, Laboratory Application, and Clinical Usefulness of Two Direct Amplification Technologies for the Detection ofMycobacterium tuberculosisComplex

Cited by 40 publications

References 13 publications

Interpreting the Results of the Amplified Mycobacterium tuberculosis Direct Test for Detection of M. tuberculosis rRNA

Interpreting the Results of the Amplified Mycobacterium tuberculosis Direct Test for Detection of M. tuberculosis rRNA

Economic evaluation of the use of PCR assay in diagnosing pulmonary TB in a low-incidence area

Relevance of Commercial Amplification Methods for Direct Detection of Mycobacterium tuberculosis Complex in Clinical Samples

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