Evaluation of Amplicor PCR for direct detection of Mycobacterium tuberculosis from sputum specimens

Abstract

We evaluated the Amplicor PCR assay (Roche Molecular Systems, Branchburg, N.J.) for direct detection of Mycobacterium tuberculosis in sputum. A total of 532 specimens from 270 patients were decontaminated and stored at 4 or ؊75؇C until assayed by PCR. This assay used three-step sample preparation, biotinylated primer pairs, AmpErase, and a microtiter format for amplicon capture and detection. Amplicor PCR results were compared with clinical history, culture from a Lowenstein-Jensen slant, and results from the BACTEC TB-460 system. Eighty-seven cultures from 15 patients grew M. tuberculosis; of these, 83 (95%) were positive with the Amplicor PCR test. The false negatives were most likely due to sample variation and inhibitors. Of the 445 specimens from which M. tuberculosis was not isolated, 428 (96%) were negative with the Amplicor PCR test. Of the 17 M. tuberculosis culture-negative, Amplicor-positive specimens, 15 were reclassified as true positives because previous cultures grew M. tuberculosis. Of the 445 specimens which did not grow M. tuberculosis, Mycobacterium spp. other than M. tuberculosis were isolated from 150 specimens. Three of these 150 specimens were Amplicor positive; two were from a patient with a history of tuberculosis, and one specimen gave a false-positive result. We do not feel that this represents cross-reactivity, because repeated Amplicor testing of the isolate gave negative results. The microtiter plate has 96 wells. Allowing for six controls, 90 decontaminated specimens can be tested by one technologist in 7.5 h. This PCR assay took 7.5 h to complete and is a sensitive and specific, rapid method for the direct detection of M. tuberculosis from sputum.

Evaluation of Amplicor‐ and IS6110‐PCR for direct detection of Mycobacterium tuberculosis complex in Singapore

“…These results were in concordance with those reported by others, where the sensitivity ranged from 95% to 99% in smear-positive and 55% to 66% in smear-negative samples (Damato et al 1995;Moore & Curry 1995). Beavis et al (1995) reported an overall higher sensitivity of Amplicor (95%) and AFstaining (93%). Hence, sensitivity of PCR in detecting Mtc is relative to Mycobacterium loads, where the AF-staining result may serve as a rough indicator.…”

“…In this study, we did not observe amplification inhibition-associated PCR failure in the culture-positive samples, as did other investigators (Beavis et al 1995;Schirm et al 1995;Moore & Curry 1995). In general practice, we used NaOH to decontaminate samples, NPV ϭ Negative predictive value c Positive ϭ 37 samples positive for Mtc, including 31 culture-positive, and 6 culture-negative samples (but PCR-positive and from patients under antimycobacterial treatment but with previous culture-positive results).…”

“…Twenty-five l of inoculum were used for PCR tests but a 20-fold inoculum of 500 l was used culture. Another reason could be the unequal distribution of bacteria in samples (Beavis et al 1995).…”

“…The introduction of the standard Amplicor test facilitated the possibility of PCR usage in routine clinical laboratories. Since then, a number of studies has been carried out in the United States (Beavis et al 1995;Vuorinen et al 1995) and Europe (Damato et al 1995;Schirm et al 1995). However, no evaluation has been done in South-East Asian countries, where tuberculosis is more prevalent.…”

Detection of Mycobacterium tuberculosis in BACTEC 12B broth cultures by the Roche Amplicor PCR assay

“…Theoretically, the PCR, by which nucleic acid is multiplied many times, could be more sensitive than the DNA probe method for identification of MTBC. Data from several studies evaluating a commercial PCR kit (Amplicor MTB Assay; Roche Molecular Systems, Inc., Somerville, N.J.) for direct detection of MTBC in clinical specimens have been published, but studies that have investigated the ability of the assay to detect MTBC in broth culture systems have been limited (2,3,5,9,12). The purpose of this study was to further evaluate the reliability of the PCR assay for detection of MTBC in BACTEC 12B broth cultures.…”