Susceptibility Testing Of Mycobacteria Research Articles

Luciferase Reporter Mycobacteriophages for Detection, Identification, and Antibiotic Susceptibility Testing of Mycobacterium tuberculosis in Mexico

The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.

Evaluation of the MB/BacT Mycobacterium Detection System for Susceptibility Testing of Mycobacterium tuberculosis

The MB/BacT mycobacterium detection system was evaluated for its performance in the susceptibility testing of Mycobacterium tuberculosis. Eighty-three M. tuberculosis isolates were processed. Results for all isoniazid-, rifampin- and streptomycin-susceptible, isoniazid-resistant, and rifampin-resistant M. tuberculosis isolates with the MB/BacT system agreed 100% with those obtained by the agar proportion method. The agreements between the two methods for streptomycin- and ethambutol-resistant isolates were 96.4 and 90.4%, respectively. The susceptibility test results were obtained in 7 days, on average. These data demonstrate that the MB/BacT system is an accurate, nonradiometric method for rapid susceptibility testing of M. tuberculosis.

Quantitative analysis of mRNA as a marker for viability of Mycobacterium tuberculosis.

Numerous assays which use conserved DNA or rRNA sequences as targets for amplification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of therapeutic efficacy owing to the persistence of amplifiable nucleic acid beyond the point at which smears and cultures become negative. Semiquantitative analysis of rRNA has been used to reduce the time required for antimicrobial susceptibility testing of Mycobacterium tuberculosis, although growth for up to 5 days in the presence of some drugs is still required to discriminate resistant strains. The purpose of the present study was to determine whether quantitative analysis of M. tuberculosis mRNA could be used to assess bacterial viability and to illustrate the application of this technique to rapid determination of drug susceptibility. Levels of mRNA encoding the 85B protein (alpha-antigen), IS6110 DNA, and 16S rRNA were compared in parallel cultures of M. tuberculosis that were treated with either no drug, 0. 2 microg of isoniazid per ml, or 1 microg of rifampin per ml. Exposure of sensitive strains to isoniazid or rifampin for 24 h reduced the levels of 85B mRNA to <4 and <0.01%, respectively, of those present in control cultures without drug. In contrast, the levels of IS6110 DNA and 16S rRNA did not diminish over the same period. Strains which were resistant to either isoniazid or rifampin demonstrated no reduction in 85B mRNA in the presence of the drug to which they were nonresponsive. Quantitative analysis of 85B mRNA offers a potentially useful tool for the rapid determination of M. tuberculosis drug susceptibility and for the monitoring of therapeutic efficacy.

Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis

There is still an urgent requirement for more sensitive, cost-effective methods for detection and susceptibility testing of mycobacteria in clinical samples. We have been investigating a simple bacteriophage-based system which could be used for both purposes. As this depends upon the detection of phages which have successfully infected cells, a key step is the efficient removal or inactivation of phages remaining free in the culture medium. We demonstrate here the use of ferrous ammonium sulphate as an effective agent for the inactivation of mycobacteriophage D29 without impairing phage replication in previously infected host bacteria. Using this property, we report the detection of viable Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis using simple low-cost technology. The method is highly sensitive, since it is able to detect 10 colony-forming units of M. smegmatis. It is also rapid, with the detection of M. tuberculosis in sputum specimens within 48 h.

Evaluation of a new method for rapid drug susceptibility testing of Mycobacterium avium complex isolates by using the mycobacteria growth indicator tube.

The reliability of the Mycobacteria Growth Indicator Tube (MGIT [BBL]) for rapid drug susceptibility testing of Mycobacterium avium complex (MAC) isolates was evaluated. MICs of amikacin, clarithromycin, clofazimine, ethambutol, and rifabutin were determined by the MGIT system for 16 MAC strains. The results were compared with those obtained by the BACTEC broth macrodilution method. The turnaround times were 6 to 8 days (median, 7 days) for the MGIT and 5 to 7 days (median, 6 days) for the BACTEC system. Agreements with BACTEC system-determined MICs, within +/-1 log2 dilution, were 100, 100, 88, 63, and 44% for amikacin, clofazimine, rifabutin, clarithromycin, and ethambutol, respectively. Within +/-2 log2 dilutions, agreement with BACTEC system-determined MICs increased to 100% for all the tested drugs. In addition, if MGIT-determined MICs were evaluated according to the thresholds adopted for the interpretation of BACTEC system-determined ones, ethambutol was the only drug for which susceptible strains were frequently misclassified as resistant. It is concluded that the MGIT system is a promising, nonradiometric alternative to the BACTEC method for rapid susceptibility testing of MAC isolates; however, additional studies are required to confirm our results and to determine the optimal criteria for the interpretation of ethambutol MICs.

Quantitative–Competitive Polymerase Chain Reaction for Rapid Susceptibility Testing ofMycobacterium tuberculosisto Isoniazid

The objective of this study was to determine whether quantitative–competitive polymerase chain reaction (QC-PCR) can be used for rapid susceptibility testing ofMycobacterium tuberculosis(MTB). QC-PCR was used to determine relative amounts of mycobacterial DNA inoculated at different isoniazid (INH) concentrations. A total of six different INH-sensitive (INH-S) and five INH-resistant (INH-R) strains were inoculated in the presence of 0.0, 0.2, 1.0, and 10.0 μg/ml of INH. DNA was quantified using QC-PCR at Week 0 and weekly thereafter for 3 weeks. For the QC-PCR, 10-fold dilutions of control (240 bp) DNA having the same primer set as the target DNA (123 bp) were used. The amount of target DNA was estimated by using known amounts of the internal standard. For INH-S isolates there was ≥1 log difference in DNA concentration in the presence of each INH concentration compared to that of the control within 1 to 3 weeks. In contrast, for INH-R isolates there were no apparent differences in DNA concentration between the control suspensions and those containing 0.2 and 1.0 μg/ml INH during the 3-week incubation period. The highest INH concentration (10 μg/ml), however, did abolish the DNA increase seen in the other MTB suspensions. This preliminary study suggests that by using concentrations of 0.2 or 1.0 μg/ml of INH, QC-PCR may differentiate INH-R and INH-S MTB isolates within 1 week. This method may be of particular value when applied directly to clinical specimens with varying numbers of bacilli.

Impact of technological developments and organizational strategies on clinical laboratory cost reduction

Health care reform efforts, largely under the aegis of managed health care initiatives, have prompted clinical laboratories to increase efficiency and reduce both expenditures and test turn-around times. The adoption of newer technologies is viewed as a mechanism of achieving the latter objectives, but direct and indirect costs and outcomes are often difficult to project. Issues explored in this article include the impact on a large university hospital-based clinical microbiology laboratory following the application of various technological approaches to organism recognition and susceptibility testing and the consolidation of certain testing services. Included are applications of an automated blood culture system; radiometric detection, identification, and susceptibility testing of mycobacteria; and the use of molecular probes to identify various microorganisms. Assessment was made through retrospective review of direct costs, estimates of average test report turnaround times, work flow changes, and real or perceived outcomes. Both the application of technology per se and consolidation of an independent virology service into the general microbiology laboratory enabled improvement in test report times and led to direct or indirect cost reduction. Managerial strategies to bring about organization changes throughout all clinical laboratories in response to a major hospital-wide cost reduction program are also presented together with financial outcomes achieved.

Application of new technology to the detection, identification, and antimicrobial susceptibility testing of mycobacteria.

Mycobacteria are reemerging as important causes of human disease. The increase in mycobacterial infections has prompted the development of more rapid and efficient ways of detecting and characterizing mycobacteria in the clinical microbiology laboratory. Methods currently in use or under development include more sensitive methods of direct detection, improved techniques for culture, identification, and susceptibility testing, and the use of nucleic acid probes for identification and epidemiologic typing. Broad application of rapid and sensitive methods for detection and characterization of mycobacteria are essential if we are to limit the spread of Mycobacterium tuberculosis (TB) and provide optimal care for patients infected with TB or other Mycobacterium species.

THE LABORATORY DIAGNOSIS OF MYCOBACTERIAL DISEASES

Recent developments in diagnostic mycobacteriology are surveyed with emphasis on laboratory capabilities relevant to the more rapid and accurate detection and identification of mycobacterial pathogens. Some terminologic problems are reviewed, and some newly recognized mycobacterial pathogens are discussed. Newer methodologies presented in some detail include the radiometric technique for detection, identification, and susceptibility testing of mycobacteria, and the use of DNA probes for identification of mycobacterial species and species complexes. The utility of other developing methodologies, such as polymerase chain reaction technology, analysis of body fluids for tuberculostearic acid, and the use of ELISA are also assessed.

Comparison of improved BACTEC and Lowenstein-Jensen media for culture of mycobacteria from clinical specimens

A 4-month trial involving 2,563 routine clinical specimens was conducted to compare the improved BACTEC TB system (12B medium) with the conventional Lowenstein-Jensen (LJ) media for the isolation, identification, and susceptibility testing of mycobacteria. One hundred sixty-two mycobacterial isolates were recovered, 147 (91%) with BACTEC and 118 (73%) with LJ media. Of these, 62 were Mycobacterium tuberculosis complex strains, 59 (95%) of which were isolated with BACTEC and 54 (87%) of which were isolated with LJ media. Of the remaining 100 isolates, which were mycobacteria other than tuberculosis (MOTT), BACTEC and LJ media detected 88 and 64%, respectively. The contamination rate was significantly higher in BACTEC (5%) than in LJ media (3.3%). The mean isolation time for M. tuberculosis complex with BACTEC was 15.5 days, compared with 25.6 days with LJ. For MOTT, the mean isolation times were 5.8 and 21.4 days, respectively. Identification of 32 M. tuberculosis complex isolates and 38 isolates of MOTT by the BACTEC NAP (p-nitro-alpha-acetylamino-beta-hydroxypropiophenone) inhibition test gave 100% agreement with conventional biochemical identifications. The results of susceptibility testing of 18 M. tuberculosis complex isolates with BACTEC agreed completely with those obtained by the resistance ratio method.

The Bactec-system in the diagnosis of tuberculosis comparison of a conventional and the radiometric method (Bactec) for culturing, differentiation and susceptibility testing of Mycobacteria

A method developed in the USA (2, 6, 8) for the culturing, identification and differentiation of mycobacteria by means of a radiometric method (Bactec) was compared with a conventional method by examining 802 specimens received. Obviously better results were obtained with the new method: The number of positive cultures was 102 (12.7%) for Bactec against 87 (10.8%) for the conventional method. When using the radiometric method, the contamination rate (5.1%) was higher than for the conventional method (3.1%). It would, however, seem that this disadvantage can be offset by an increase in the alkali concentration during pretreatment of the specimens. After elimination of all paired samples one or both specimens were found to be contaminated, 743 specimens remained for direct comparison. Of these, 101 (13.6%) were positive when the Bactec method, and 84 (11.3%), when the conventional method was used. The superiority of the new method was most obvious with sputum specimens: 14.5% were positive when Bactec, and 12.2%, when the conventional method was used. For the Bactec method, the mean period until positive results could be recognized by daily readings was 15 days against 28 days for the conventional method with weekly readings. Sensitivity testing can be completed within 8 days. Owing to the costs of the radiometric method, it is recommended to limit its use of defined situations.

Susceptibility Testing of Mycobacteria

Susceptibility Testing of Mycobacteria J. Kenneth McClatchy, Ph.D. J. Kenneth McClatchy, Ph.D. J. Kenneth McClatchy, Ph.D. is Chief of Clinical Laboratories, National Jewish Hospital and Research Center, Denver, CO 80206. This article is reprinted from the Technical Improvement Service, Volume 29. American Society of Clinical Pathologists. Search for other works by this author on: Oxford Academic PubMed Google Scholar Laboratory Medicine, Volume 9, Issue 3, 1 March 1978, Pages 47–52, https://doi.org/10.1093/labmed/9.3.47 Published: 01 March 1978

Cooperative field test of drug-impregnated discs for susceptibility testing of mycobacteria.

Cooperative field test of drug-impregnated discs for susceptibility testing of mycobacteria.

BACTERIOLOGICAL DIAGNOSIS OF TUBERCULOSIS

Today, microscopic examination and culture are still essential for active tuberculosis diagnosis and therapeutic decisions. Nucleic acid amplification and line probe assays speed up identification and susceptibility testing of mycobacteria in AFB smear positive specimens or in culture. They are also efficient for comparison of M. tuberculosis strains with each other (genotyping). On the other hand, today, molecular tests are not relevant for the diagnosis in smear negative specimens and even more for diagnosis of culture-negative tuberculosis.