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

Using RT qPCR for quantifying Mycobacteria marinum from in vitro and in vivo samples

Mycobacterium marinum, the causative agent of fish tuberculosis, is rarely a human pathogen causing a chronic skin infection. It is now wildely used as a model system in animal models, especially in zebra fish model, to study the pathology of tuberculosis and as a means of screening new anti-tuberculosis agent. To facilitate such research, quantifying the viable count of M. marinum bacteria is a crucial step. The main approach used currently is still by counting the number of colony forming units (cfu), a method that has been in place for almost 100 years. Though this method well established, understood and relatively easy to perform, it is time-consuming and labor-intensive. The result can be compromised by failure to grow effectively and the relationship between count and actual numbers is confused by clumping of the bacteria where a single colony is made from multiple organisms. More importantly, this method is not able to detect live but not cultivable bacteria, and there is increasing evidence that mycobacteria readily enter a "dormant" state which confounds the relationship between bacterial number in the host and the number detected in a cfu assay. DNA based PCR methods detect both living and dead organisms but here we describe a method, which utilizes species specific Taq-Man assay and RT-qPCR technology for quantifying the viable M. marinum bacterial load by detecting 16S ribosomal RNA (16S rRNA).Postprin

Spontaneous Emergence of Multiple Drug Resistance in Tuberculosis before and during Therapy

The emergence of drug resistance in M. tuberculosis undermines the efficacy of tuberculosis (TB) treatment in individuals and of TB control programs in populations. Multiple drug resistance is often attributed to sequential functional monotherapy, and standard initial treatment regimens have therefore been designed to include simultaneous use of four different antibiotics. Despite the widespread use of combination therapy, highly resistant M. tb strains have emerged in many settings. Here we use a stochastic birth-death model to estimate the probability of the emergence of multidrug resistance during the growth of a population of initially drug sensitive TB bacilli within an infected host. We find that the probability of the emergence of resistance to the two principal anti-TB drugs before initiation of therapy ranges from 10−5 to 10−4; while rare, this is several orders of magnitude higher than previous estimates. This finding suggests that multidrug resistant M. tb may not be an entirely “man-made” phenomenon and may help explain how highly drug resistant forms of TB have independently emerged in many settings

Low Dose Aerosol Fitness at the Innate Phase of Murine Infection Better Predicts Virulence amongst Clinical Strains of Mycobacterium tuberculosis

Background: Evaluation of a quick and easy model to determine the intrinsic ability of clinical strains to generate active TB has been set by assuming that this is linked to the fitness of Mycobacterium tuberculosis strain at the innate phase of the infection. Thus, the higher the bacillary load, the greater the possibility of inducting liquefaction, and thus active TB, once the adaptive response is set. Methodology/Principal Findings: The virulence of seven clinical Mycobacterium tuberculosis strains isolated in Spain was tested by determining the bacillary concentration in the spleen and lung of mice at weeks 0, 1 and 2 after intravenous (IV) inoculation of 10 4 CFU, and by determining the growth in vitro until the stationary phase had been reached. Cord distribution automated analysis showed two clear patterns related to the high and low fitness in the lung after IV infection. This pattern was not seen in the in vitro fitness tests, which clearly favored the reference strain (H37Rv). Subsequent determination using a more physiological low-dose aerosol (AER) inoculation with 10 2 CFU showed a third pattern in which the three best values coincided with the highest dissemination capacity according to epidemiological data. Conclusions/Significance: The fitness obtained after low dose aerosol administration in the presence of the innate immune response is the most predictive factor for determining the virulence of clinical strains. This gives support to a mechanism o

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

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research

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 5 mg/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 physiological cost for the development of drug resistance, (C) 2000 The British Infection Society