Evaluation of loop mediated isothermal amplification for diagnosis of Mycobacterium tuberculosis complex in clinical samples

Tuberculosis (TB) remains an important global public health problem. The lack of rapid and accurate diagnostic testing is an important impediment to global tuberculosis control. Loop mediated isothermal amplification (LAMP) is a rapid method for nucleic acid amplification. In this study, we assessed the performance of an in-house LAMP assay for the detection of tuberculosis. Six oligonucleotide primers specific for Mycobacterium tuberculosis complex were designed corresponding to IS6110 gene sequence. Optimization of LAMP reaction was performed. A total of 133 clinical sputum samples and 80 bacterial cultures were studied by LAMP method. Sensitivity of this assay for detection of genomic DNA was 5 fg. This assay successfully detected M. tuberculosis complex not only in the bacterial cultures but also in the clinical sputum samples from patients with TB. The sensitivity of LAMP in culturepositive samples was 100% (60/60) and the specificity in culture-negative samples was 95.9% (70/73, 95% confidence interval 91.3 to 98.7%). Thus, LAMP is a rapid, highly sensitive and specific DNA amplification technique for early diagnosis of TB.Key words: Loop mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), IS6110 gene, Mycobacterium tuberculosis, diagnosis

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

Experiencing visual impairment in a lifetime home: an interpretative phenomenological inquiry

Lifetime home standards (LTHS) are a set of standards aimed at making homes more accessible. Previous research, however, indicates that LTHS do not adequately meet the needs of those with sensory impairments. Now, with visual impairment set to increase globally and acknowledging the recognised link between quality of dwelling and wellbeing, this article aims to examine the experiences of visually impaired people living in lifetime homes. The objectives are to investigate existing lifetime homes and to identify whether LTHS meet occupants’ needs. Qualitative semi-structured interviews were carried out with six visually impaired people living in homes designed to LTHS in Northern Ireland. Collected data was analysed using interpretative phenomenological analysis identifying three super-ordinate themes: (1) living with visual impairment; (2) design considerations and (3) coping strategies. A core theme of balance between psychological and physical needs emerged through interconnection of super-ordinate themes. Although there are benefits to living in lifetime homes, negative aspects are also apparent with occupants employing several coping strategies to overcome difficulties. Whilst residents experience negative emotions following visual impairment diagnoses, results suggest that occupants still regard their homes as key places of security and comfort in addition to then highlighting the need for greater consideration of specific individual needs within general guidelines

Changes in Community Mobility in Older Men and Women. A 13-Year Prospective Study

Community mobility, defined as “moving [ones] self in the community and using public or private transportation”, has a unique ability to promote older peoples’ wellbeing by enabling independence and access to activity arenas for interaction with others. Early predictors of decreased community mobility among older men and women are useful in developing health promoting strategies. However, long-term prediction is rare, especially when it comes to including both public and private transportation. The present study describes factors associated with community mobility and decreased community mobility over time among older men and women. In total, 119 men and 147 women responded to a questionnaire in 1994 and 2007. Respondents were between 82 and 96 years old at follow-up. After 13 years, 40% of men and 43% of women had decreased community mobility, but 47% of men and 45% of women still experienced some independent community mobility. Cross-sectional independent community mobility among men was associated with higher ratings of subjective health, reporting no depression and more involvement in sport activities. Among women, cross-sectional independent community mobility was associated with better subjective health and doing more instrumental activities of daily living outside the home. Lower subjective health predicted decreased community mobility for both men and women, whereas self-reported health conditions did not. Consequently, general policies and individual interventions aiming to improve community mobility should consider older persons’ subjective health

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid

Objectives To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. Methods The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. Results We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. Conclusions In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST

The non-clonality of drug resistance in Beijing-genotype isolates of Mycobacterium tuberculosis from the Western Cape of South Africa

Background. The Beijing genotype of M. tuberculosis is a virulent strain that is disseminating worldwide and has a strong association with drug resistance. In the Western Cape of South Africa, epidemiological studies have identified the R220 cluster of the Beijing genotype as a major contributor to a recent outbreak of drug-resistant tuberculosis. Although the outbreak is considered to be due to clonal transmission, the relationship among drug resistant isolates has not yet been established. Results. To better understand the evolution of drug resistance among these strains, 14 drug-resistant clinical isolates of the Beijing genotype were sequenced by whole-genome sequencing, including eight from R220 and six from a more ancestral Beijing cluster, R86, for comparison. While each cluster shares a distinct resistance mutation for isoniazid, mapping of other drug-resistance mutations onto a phylogenetic tree constructed from single nucleotide polymorphisms shows that resistance mutations to many drugs have arisen multiple times independently within each cluster of isolates. Thus, drug resistance among these isolates appears to be acquired, not clonally derived. This observation suggests that, although the Beijing genotype as a whole might have selective advantages enabling its rapid dissemination, the XDR isolates are relatively less fit and do not propagate well. Although it has been hypothesized that the increased frequency of drug resistance in some Beijing lineages might be caused by a mutator phenotype, no significant shift in synonymous substitution patterns is observed in the genomes. Conclusion. While MDR-TB is spreading by transmission in the Western Cape, our data suggests that further drug resistance (i.e. XDR-TB) at this stage is acquired.Peer Reviewe

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

Updating the approaches to define susceptibility and resistance to anti-tuberculosis agents: implications for diagnosis and treatment

11 páginas, 2 figuras, 1 tablaInappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance.I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038)Peer reviewe