Ability of PCR assay to identify Mycobacterium tuberculosis in BACTEC 12B vials

Abstract

Introduction of PCR to directly detect Mycobacterium tuberculosis in clinical specimens has shown promise; however, interfering substances in clinical material have contributed to lowered assay sensitivities. We evaluated the ability of a PCR assay to detect M. tuberculosis in BACTEC 12B broth cultures. Clinical specimens were processed and inoculated into BACTEC 12B vials. Evaluation was approached in two phases, starting with an initial evaluation in which an aliquot of 12B broth was removed when the growth index (GI) was .10 and stored at 4°C until assayed by PCR. Of the 290 specimens initially assayed, 129 were culture negative for mycobacteria as well as PCR negative for M. tuberculosis. Except for one, cultures (n = 102) which grew mycobacteria other than M. tuberculosis were all PCR negative. The remaining 59 broths were all culture and PCR positive for M. tuberculosis; 39%o (n = 23) of these cultures when assayed by PCR had GIs of s50. Following initial evaluation, 200 12B BACTEC vials with GIs of :10 were assayed in a similar manner except that specimens were amplified twice weekly to determine PCR's impact on the length of time to identification of M. tuberculosis as compared with standard laboratory practices. Utilization of PCR resulted in a mean time to detection of M. tuberculosis of 14 days, compared with 29 days by using commercially available nucleic acid probes to identify M. tuberculosis complex from growth of BACTEC 12B subcultures on solid media. In light of an overall sensitivity and specificity of 100 and 99.7%, respectively, coupled with the ability to identify M. tuberculosis days or weeks before other methods can be applied, we conclude that PCR might prove to be a rapid alternative for identification of M. tuberculosis in culture and allow for earlier setup of susceptibility testing.

The application of molecular techniques to the diagnosis and epidemiology of mycobacterial diseases

“…tuberculosis DNA in specimens that were smear-positive (and therefore contained large numbers of mycobacteria) whereas detection was less reliable (SO-SS%) for specimens that were smear-negative but culture-positive (containing lower numbers). Sensitivity of detection of MTB can be increased for smear-negative, culture-positive specimens by culture in BACTEC medium for 7-10 d prior to PCR giving sensitivities and specificities of 100°/o and 99.7% with a mean detection time of 14 d (Forbes and Hicks 1994). Many studies have shown apparently genuine PCR positive results with specimens from which no mycobacteria could be grown ( Table 2).…”

“…(1992) found that after sonication as few as 10 organisms could be detected, while with boiling, freezing and thawing and treatment with nonionic detergents and proteinase K the limit of detection was 10' bacteria; Sritharan and Barker (1991), Kocagiiz rt al. (1993) and Forbes and Hicks (1994) found that boiling allowed detection of as few as 10 organisms. Wilson et nl.…”

“…Although IS6110 has been shown in a number of studies to be specific for the Myco. tuberculosis complex Eisenach et al 1991 ;Kolk et al 1992), there have also been several reports of false positives using IS6llO-based PCR (Clarridge et al 1993 ;Forbes and Hicks 1994). In most cases, these are believed to be due to contamination or to a lack of specificity of the primers.…”

Evaluation of a rapid air thermal cycler for detection of Mycobacterium tuberculosis

“…Due to the resurgence of tuberculosis, the emergence of multiple-drug-resistant Mycobacterium tuberculosis strains, and a need to differentiate between M. tuberculosis and other mycobacteria for isolation and treatment purposes, rapid and accurate detection of M. tuberculosis in clinical specimens is critical. PCR using IS6110 primers has been shown to be a rapid, sensitive, and specific procedure for the detection of M. tuberculosis complex in clinical samples (1,4,6,12,14,15).…”

Confirmation of the presence of Mycobacterium tuberculosis and other mycobacteria in mycobacterial growth indicator tubes (MGIT) by multiplex strand displacement amplification

“…Commercially available amplification systems (1,2,14,22) are being developed and evaluated for the detection and identification of AFB in clinical specimens and in culture. The application of these assays for culture identification is gaining popularity because of the observed sensitivity, specificity, and timeliness of testing (9). It should be possible to design a panel of primers to identify commonly isolated AFB to the species level, thereby eliminating time-consuming biochemical identification.…”