Prospective evaluation of BDProbeTec strand displacement amplification (SDA) system for diagnosis of tuberculosis in non-respiratory and respiratory samples.

Nucleic acid amplification techniques (NAATs) have been demonstrated to make significant improvements in the diagnosis of tuberculosis (TB), particularly in the time to diagnosis and the diagnosis of smear-negative TB. The BD ProbeTec strand displacement amplification (SDA) system for the diagnosis of pulmonary and non-pulmonary tuberculosis was evaluated. A total of 689 samples were analysed from patients with clinically suspected TB. Compared with culture, the sensitivity and specificity for pulmonary samples were 98 and 89 %, and against final clinical diagnosis 93 and 92 %, respectively. This assay has undergone limited evaluation for non-respiratory samples and so 331 non-respiratory samples were tested, identifying those specimens that were likely to yield a useful result. These were CSF (n = 104), fine needle aspirates (n = 64) and pus (n = 41). Pleural fluid (n = 47) was identified as a poor specimen. A concern in using the SDA assay was that low-positive samples were difficult to interpret; 7.8 % of specimens fell into this category. Indeed, 64 % of the discrepant results, when compared to final clinical diagnosis, could be assigned as low-positive samples. Specimen type did not predict likelihood of a sample being in the low-positive zone. Although the manufacturers do not describe the concept of a low-positive zone, we have found that it aids clinical diagnosis.

Estimation of mutation rates in antibiotic research.

Luria and Delbruck identified that the frequency of mutation was subject to considerable fluctuation. They argued that the large fluctuation in the number of organism surviving exposure to bacteriophage meant that resistance was acquired through mutation rather than a physiological adaptation to the bacteriophage. Mutations that arose early in the broth culture would give rise to a "Jackpot culture" (1). Thus the size of a lineage of mutant cells depends on when the mutation occurred. It is said that the original idea came to Luria while he was observing a slot machine in Bloomington, IN (2). Early mutations are rare (like jackpots with a slot machine) thus when a series of cultures are compared the numbers of mutants would have "a distribution with an abnormally high variance" (1).

Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing with Mycobacterium tuberculosis.

Mycobacterium tuberculosis converts pyrazinamide to its active form by using the enzyme pyrazinamidase. This enzyme is coded for on the pncA gene, and mutations in the pncA gene result in absence of active enzyme, conferring resistance to the drug pyrazinamide. We investigated 27 strains of Mycobacterium tuberculosis suspected of being multidrug resistant. Each isolate was tested for susceptibility to pyrazinamide by the BACTEC 460TB method, and 19 were pyrazinamide resistant. The presence of active pyrazinamidase enzyme was sought by using the Wayne assay, which was positive in all of the sensitive isolates and four of the resistant isolates. The pncA gene was amplified by PCR, and mutations were sought by single-strand conformation polymorphism (SSCP) analysis. We identified four isolates which were phenotypically resistant to pyrazinamide, but which had active pyrazinamide enzyme on the Wayne assay and normal pncA gene SSCP. MICs measured by BACTEC 460TB and susceptibility testing at a lower pH of 5.5 confirmed genuine resistance. The pncA gene was sequenced in these four isolates and found not to have any mutations. This implies that an alternative mechanism of resistance exists in these strains. We conclude that genotypic assessment of pyrazinamide resistance is unreliable, because it depends on the identification of a single resistance mechanism. Phenotypic methods such as the BACTEC 460TB technique remain the best methods for pyrazinamide susceptibility testing.

Comparison of fitness of two isolates of Mycobacterium tuberculosis, one of which had developed multi-drug resistance during the course of treatment.

OBJECTIVES: We report the cases of two patients, brother and sister, both with pulmonary tuberculosis. Both patients complied poorly with treatment. One developed multi-drug resistant disease, whilst the other did not. We aimed to show that the two infecting strains were the same, and then to compare the fitness of the resistant strain to that of the sensitive strain. METHODS: The isolates were typed by RFLP. The fitness of the multi-drug resistant tuberculosis strain was determined by calculating the ratio of generation produced by the drug-resistant and a drug-susceptible strain in a mixed culture. The number of bacteria present in this broth culture was estimated using the Miles and Misra technique. The number of drug-resistant bacteria present was determined by inoculating aliquots of broth onto Middlebrook 7H10 agar with 5mg/l rifampicin. RESULTS: The infecting strain of Mycobacterium tuberculosis was shown to be the same on RFLP typing in both cases. It was found that the multi-drug resistant organism had decreased fitness compared to the sensitive organism. CONCLUSION: The decreased relative fitness of the resistant strain implies a physiologic cal cost for the development of drug resistance.

Physiological cost of rifampin resistance induced in vitro in Mycobacterium tuberculosis.

Drug-resistant Mycobacterium tuberculosis is a major threat to public health. In clinical practice, a limited number of resistance mutations in a short sequence of the beta subunit of RNA polymerase (encoded by rpoB) have been described. Spontaneous resistance to rifampin was induced in vitro in M. tuberculosis H37Rv (ATCC 9360). Only three resistance patterns could be detected by PCR-single-strand conformation polymorphism analysis. Sequence analysis revealed that Ser(531)-->Leu arose most frequently, followed by His(526)-->Arg and then either His(526)-->Tyr or His(526)-->Asp. The relative Darwinian fitness of all but one of the mutant genotypes was less than that of the susceptible parent and, for these mutations, there was a significant correlation between fitness and clinical isolation rate (regression analysis P = 0.026). The fitness deficit in some mutants was small, suggesting that there is little likelihood of a spontaneous reversion to susceptibility.

Length of time to laboratory diagnosis of Mycobacterium tuberculosis infection: comparison of in-house methods with reference laboratory results.

OBJECTIVES: To audit the time taken to obtain laboratory confirmation of infection with Mycobacterium tuberculosis using in-house methods of polymerase chain reaction (PCR) and culture and referral to a reference laboratory. METHODS: Retrospective collection of data from laboratory records covering a period of 1 year. RESULTS: Median time to microbiological diagnosis of a new infection using the in-house services in addition to the reference laboratory was 22.0 days. Using reference laboratory results alone, median time to diagnosis would have been 61.5 days. CONCLUSIONS: Development of on-site laboratory facilities to identify Mycobacterium tuberculosis can reduce the time to its identification by almost two-thirds.