Richard M. Anthony

Combined Species Identification, Genotyping, and Drug Resistance Detection of Mycobacterium tuberculosis Cultures by MLPA on a Bead-Based Array

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

Development of Multiplex Assay for Rapid Characterization of Mycobacterium tuberculosis

ABSTRACT We have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in the Mycobacterium tuberculosis genome. The assay was validated on a reference panel of well-characterized strains, and the results show that M. tuberculosis can be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance (rpoB, katG, inhA, embB), members of the M. tuberculosis complex (16S rRNA, IS6110, TbD1), the principal genotypic group (katG, gyrA), and Haarlem and Beijing strains (ogt, mutT2, mutT4) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinical M. tuberculosis isolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmed M. tuberculosis clinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.

Resistant mutants of Mycobacterium tuberculosis selected in vitro do not reflect the in vivo mechanism of isoniazid resistance.

Objectives The high prevalence of isoniazid-resistant Mycobacterium tuberculosis is often explained by a high mutation rate for this trait, although detailed information to support this theory is absent. We studied the development of isoniazid resistance in vitro, making use of a laboratory strain of M. tuberculosis. Methods Spontaneous isoniazid-resistant mutants were characterized by molecular methods allowing identification of the most commonly encountered resistance-conferring mutations. Additionally, we determined the in vitro mutation rates for isoniazid and rifampicin resistance, and characterized the genome of a triple-resistant strain. Results Results confirm that the in vitro mutation rate for isoniazid resistance (3.2 × 10−7 mutations/cell division) is much higher than the rate for rifampicin resistance (9.8 × 10−9 mutations/cell division). However, in the majority of the in vitro mutants katG was partially or completely deleted and neither of the two most common in vivo mutations, katG-S315T or inhA-C(-)15T, were found in 120 isogenic mutants. This implies that clinically prevalent resistance mutations were present in <0.8% of isoniazid-resistant strains selected in vitro (95% CI 0%–2.5%). The triple-resistant strain had acquired isoniazid resistance via a 49 kbp deletion, which included katG. Apart from previously identified resistance-conferring mutations, three additional point mutations were acquired during sequential selection steps. Conclusions These outcomes demonstrate that the in vivo mechanism of isoniazid resistance is not reflected by in vitro experiments. We therefore conclude that the high in vitro mutation rate for isoniazid resistance is not a satisfactory explanation for the fact that isoniazid monoresistance is significantly more widespread than monoresistance to rifampicin.

DNA array technology and diagnostic microbiology.

Near instantaneous detection of pathogens from clinical material, combined with simultaneous prediction of their antimicrobial resistance profiles, would revolutionize the impact of microbiology on the management of infection. Array-based assays allow a range of characteristics to be rapidly and simultaneously determined. At present these systems have found their primary role as research tools for the monitoring of mRNA expression in the form of DNA microarrays or ‘chips’. As fabrication costs reduce and validated targeted arrays are developed, it is inevitable they will be used for more routine applications. Microfluidics offers the exciting possibility of combining purification, amplification and detection in a single disposable device; microarrays are particularly suitable for use within these systems. Arrays will become an important tool for clinical diagnostics.

Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing

ABSTRACT The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

Proton Transfer Reaction Mass Spectrometry detects rapid changes in volatile metabolite emission by Mycobacterium smegmatis after the addition of specific antimicrobial agents

The metabolic activity of plants, animals or microbes can be monitored by gas headspace analysis. This can be achieved using Proton Transfer Reaction Mass Spectrometry (PTR-MS), a highly sensitive detection method for trace gas analysis. PTR-MS is rapid and can detect metabolic responses on-line as they occur. Here, we study the headspace of actively growing cultures of paired ciprofloxacin sensitive and resistant bacterial strains (Mycobacterium smegmatis in Middlebrook M7H9 liquid media) after the addition of the antibiotics ciprofloxacin and gentamicin in real time. Following the emission patterns of the mycobacteria over time allowed volatile markers specific for the bacterial response to each antibiotic to be detected. A proportion of the measured responses were very rapid, occurring within three hours after the addition of the compounds and varied between isolates with different resistance phenotypes. Specifically, we observed a two fold increase of m73 (unidentified C4 compound) within 10h after the addition of ciprofloxacin and a threefold increase of m45 (acetaldehyde) within 4h after the addition of gentamicin as compared to values before the addition. Monitoring the emission of specific volatiles into the culture headspace thus has the potential for rapid drug susceptibility testing. Moreover, these and other differences in the measured responses to the two tested compounds provide evidence that monitoring multiple compounds may also give an indication of the mechanism of action of the compound added.

Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001). All tested strains showed similar mutation rates (1.33×10−8 to 2.49×10−7) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10−7, more than 10 times higher than that of the INH susceptible parental strain (5.46–7.44×10−8). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance mutations, especially in regions that are highly endemic for (multi)drug resistant tuberculosis.

Simplified Automated Image Analysis for Detection and Phenotyping of Mycobacterium tuberculosis on Porous Supports by Monitoring Growing Microcolonies

Background Even with the advent of nucleic acid (NA) amplification technologies the culture of mycobacteria for diagnostic and other applications remains of critical importance. Notably microscopic observed drug susceptibility testing (MODS), as opposed to traditional culture on solid media or automated liquid culture, has shown potential to both speed up and increase the provision of mycobacterial culture in high burden settings. Methods Here we explore the growth of Mycobacterial tuberculosis microcolonies, imaged by automated digital microscopy, cultured on a porous aluminium oxide (PAO) supports. Repeated imaging during colony growth greatly simplifies “computer vision” and presumptive identification of microcolonies was achieved here using existing publically available algorithms. Our system thus allows the growth of individual microcolonies to be monitored and critically, also to change the media during the growth phase without disrupting the microcolonies. Transfer of identified microcolonies onto selective media allowed us, within 1-2 bacterial generations, to rapidly detect the drug susceptibility of individual microcolonies, eliminating the need for time consuming subculturing or the inoculation of multiple parallel cultures. Significance Monitoring the phenotype of individual microcolonies as they grow has immense potential for research, screening, and ultimately M. tuberculosis diagnostic applications. The method described is particularly appealing with respect to speed and automation.

Retooling National TB Control Programmes (NTPs) with New Diagnostics: The NTP Perspective

Background A delay is evident between the development of new policies on TB diagnostics and their implementation at country level. The Stop TB Partnership would benefit from information from national TB program (NTP) managers on progress towards implementation of new recommendations as well as the opportunities and challenges encountered in the process. Methods and Findings To solicit information on the introduction of new TB diagnostics at country level, questionnaires were sent out to NTP managers of high-burden TB countries and a subset of managers was interviewed. The results indicate that about 50% of high-burden TB countries are using the TB diagnostic tools newly recommended by the World Health Organization (WHO). Most NTP managers reported that new diagnostics would only be implemented when officially endorsed by the WHO. All countries have plans to adopt newly endorsed diagnostics at reference laboratory level, while approaches to optimize smear microscopy at lower levels of the health service are given less attention. NTP managers reported diverse challenges to the implementation of new diagnostics. Conclusions More information on the obstacles and advantages of introducing new diagnostic tools should be provided to NTP managers to ensure the rational adoption of new diagnostics. A single recommendation covering the introduction of a package of diagnostic tools might be preferable to NTP managers and facilitate implementation in high-burden TB countries.

Current status and opportunities for therapeutic drug monitoring in the treatment of tuberculosis

ABSTRACT Introduction: Tuberculosis remains a global health problem and pharmacokinetic variability has been postulated as one of the causes of treatment failure and acquired drug resistance. New developments enable implementation of therapeutic drug monitoring, a strategy to evaluate drug exposure in order to tailor the dose to the individual patient, in tuberculosis treatment. Areas covered: Literature on pharmacokinetics and pharmacodynamics of anti-tuberculosis drugs was explored to evaluate the effect of drug exposure in relation to drug susceptibility, toxicity and efficacy. New, down-sized strategies, like dried blood spot analysis and limited sampling strategies are reviewed. In addition, molecular resistance testing of Mycobacteria tuberculosis, combining a short turn-around time with relevant information on drug susceptibility of the causative pathogen was explored. Newly emerging host biomarkers provide information on the response to treatment. Expert opinion: Therapeutic drug monitoring can minimize toxicity and increase efficacy of tuberculosis treatment and prevent the development of resistance. Dried blood spot analysis and limited sampling strategies, can be combined to provide us with a more patient friendly approach. Furthermore, rapid information on drug susceptibility by molecular testing, and information from host biomarkers on the bacteriological response, can be used to further optimize tuberculosis treatment.