Biomedical subjects
Publications and source records attributed to Robin Warren.
Concern has been raised about the efficacy of the heat killing of mycobacteria during the isolation of DNA. We demonstrate a method that allows for the efficient killing of Mycobacterium tuberculosis without compromising DNA integrity for subsequent molecular investigation. This method reduces the risk of infection to the research scientist.
In order to fully understand the global tuberculosis (TB) epidemic it is important to investigate the population structure and dissemination of the causative agent that drives the epidemic. Mycobacterium tuberculosis strain family 11 (F11) genotype isolates (found in 21.4% of all infected patients) are at least as successful as the Beijing genotype family isolates (16.5%) in contributing to the TB problem in some Western Cape communities of South Africa. This study describes key molecular characteristics that define the F11 genotype. A data-mining approach coupled with additional molecular analysis showed that members of F11 can easily and uniquely be identified by PCR-based techniques such as spoligotyping and dot blot screening for a specific rrs491 polymorphism. Isolates of F11 not only are a major contributor to the TB epidemic in South Africa but also are present in four different continents and at least 25 other countries in the world. Careful study of dominant compared to rare strains should provide clues to their success and possibly provide new ideas for combating TB.
The worldwide recrudescence of tuberculosis and the widespread appearance of antibiotic resistance have strengthened the need for rapid and specific diagnostic tools. The prevailing microbiological identification of Mycobacterium tuberculosis, the causative agent of tuberculosis, which implies the use of in vitro cultures and acid-fast staining microscopy, is time-consuming. Detection of M. tuberculosis directly in clinical samples through PCR amplification of mycobacterium-specific genes, designed to shorten diagnostic delay, demonstrated reliability and high sensitivity. However, the quality of the diagnosis depends on the specificity of the target sequence for M. tuberculosis complex strains. In the present study, we demonstrated the specificity of recA and pps1 inteins for this complex and thus the feasibility of using intein-coding sequences as a new target for PCR diagnosis. Indeed, the recA and pps1 genes of 36 clinical isolates of M. tuberculosis and 10 field strains of M. bovis were found to be interrupted by an intein sequence at the RecA-a and Pps1-b sites, respectively, while a large number of nontuberculous mycobacterial species failed to demonstrate these insertions. Besides, the MtuPps1, which was cloned and expressed in Escherichia coli, was shown to possess an endonuclease activity. The intein cleaves the 40-bp sequence spanning the intein insertion site Pps1-b in the inteinless pps1 gene. In addition to the PCR amplification of recA and pps1 intein genes as a tool for diagnosis, the specific endonuclease activity could represent a new molecular approach to identify M. tuberculosis.
During the early development of drug resistance in Mycobacterium tuberculosis (M. tuberculosis) infection only a small proportion of resistant bacteria are present within a milieu of sensitive bacteria. This complicates the use of molecular methods to predict the presence of a resistant phenotype and has been largely ignored in many of the newly developed molecular methods. In this study, mixtures of DNA from M. tuberculosis strains with known wild-type and mutant sequences were used to evaluate the sensitivity of three different molecular methods for detection of drug resistance. The dot-blot and amplification refractory mutation system (ARMS) methods showed sensitivities that approach those of routine phenotypic methods and are able to detect the presence of mutant sequences at a ratio of 1 in 50 (corresponding to 2% mutant sequences). This is 10-fold more sensitive than the commercial kit. The ARMS method was also used to investigate the use of molecular methods to identify mixed infections, and both drug-resistant and susceptible strain populations were identified in a single clinical isolate. These findings highlight the applicability of molecular methods to the rapid detection of drug resistance in tuberculosis patients, particularly in those who are non-compliant and in contacts of known drug-resistant tuberculosis patients, and assistance in limiting the spread of drug-resistant strains.
Microsatellite typing is frequently used in disease diagnosis and in genetic association studies. Inter-study consistency and comparability is essential in both applications. In this study, we show that the interlaboratory comparison of microsatellite sizes is often discrepant and misleading. This is a matter of great concern in the recent literature. However, accurate allele designation is easily attainable by the simple procedures we report, which are applicable to all gel-based genotyping methods. These involve: 1) the creation of dedicated standards for a specific microsatellite by PCR-amplifying representative alleles to generate an allelic ladder with comparable electrophoretic characteristics; 2) including both internal and external standards during electrophoresis to facilitate alignment. In addition, we recommend procedures that will improve inter-study comparability of all microsatellite analyses regardless of genotyping method. These involve: 1) cloning and sequencing representative microsatellite alleles to obtain accurate size designation; 2) sharing alleles of known sequence between laboratories to use as standards. We report on the typing of natural resistance-associated macrophage protein (NRAMP2) and interferon-y (IFNG) gene microsatellites as examples, the latter of which is crucial to many pathogenic processes. We describe in detail the varying allele sizes obtained by different methods, which prevent meaningful inter-study comparisons.
Management of Tuberculosis is complicated by the emergence of drug resistant strains of Mycobacterium tuberculosis and this poses a threat to the success of Tuberculosis control programmes. Drug susceptibility testing by culture is time-consuming and technically difficult. It is known that resistance to drugs is due to a number of genomic mutations in specific genes of M. tuberculosis. These mutations in combination with molecular techniques can be used as markers for drug resistance, since drug susceptible isolates lack the corresponding gene mutations. This review focuses on molecular mechanisms, methods and applications as a possible new diagnostic tool for the early molecular detection of drug resistance in M. tuberculosis.