Rapid identification of lineage and drug resistance in clinical samples of Mycobacterium Tuberculosis

Background: Mycobacterium tuberculosis is a slow-growing bacterium, which could delay its diagnosis and, therefore, promote the spread of the disease. Whole-genome sequencing allows us to obtain the complete drug-resistance profile of the strain; however, bacterial cultivation of clinical samples, along with complex processing, is required. Methods: In this work, we explore AmpliSeq, an amplicon-based enrichment method for preparing libraries for targeted next-generation sequencing, to identify lineage and drug resistance directly from clinical samples. Results: In our study, 111 clinical samples were tested. The lineage was identified in 100% of the culture-derived samples (52/52), in 95% of the smear (BK)-positive clinical samples (38/40) and in 42.1% of the BK-negative clinical samples (8/19). The drug-resistance profile was accurately identified in all but 11 samples, in which some phenotypic and genotypic discrepancies were found. In this respect, our panels were not exact in the detection of streptomycin resistance for isolates derived from clinical samples, as an extremely high number of SNPs in the rrs and rrl genes were detected due to cross-contamination. Conclusion: This technique has demonstrated high sensitivity in obtaining the drug-resistance profile of the isolates, as even those samples with DNA concentrations below the detection limit of Qubit produced a result. AmpliSeq technology is cheaper than whole-genome sequencing, easy to perform by laboratory technicians and applicable to any microorganism using the Ion Torrent platform

Estimation of the mutation rate of Mycobacterium tuberculosis in cases with recurrent tuberculosis using whole genome sequencing

The study of tuberculosis latency is problematic due to the difficulty of isolating the bacteria in the dormancy state. Despite this, several in vivo approaches have been taken to mimic the latency process. Our group has studied the evolution of the bacteria in 18 cases of recurrent tuberculosis. We found that HIV positive patients develop recurrent tuberculosis earlier, generally in the first two years (p value = 0.041). The genome of the 36 Mycobacterium tuberculosis paired isolates (first and relapsed isolates) showed that none of the SNPs found within each pair was observed more than once, indicating that they were not directly related to the recurrence process. Moreover, some IS6110 movements were found in the paired isolates, indicating the presence of different clones within the patient. Finally, our results suggest that the mutation rate remains constant during all the period as no correlation was found between the number of SNPs and the time to relapse

Multidrug-resistant Mycobacterium tuberculosis, Bangui, Central African Republic

We investigated multidrug-resistant (MDR) Mycobacterium tuberculosis strains in Bangui, Central African Republic. We found 39.6% with the same spoligotype and synonymous single nucleotide polymorphism in the mutT1 gene. However, strains had different rpoB mutations responsible for rifampin resistance. MDR strains in Bangui may emerge preferentially from a single, MDR-prone family

Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela

BACKGROUND: Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD). RESULTS: Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised. Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis. CONCLUSION: This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies

Aging Influences Hepatic Microvascular Biology and Liver Fibrosis in Advanced Chronic Liver Disease.

Advanced chronic liver disease (aCLD) represents a major public health concern. aCLD is more prevalent and severe in the elderly, carrying a higher risk of decompensation. We aimed at understanding how aging may impact on the pathophysiology of aCLD in aged rats and humans and secondly, at evaluating simvastatin as a therapeutic option in aged animals. aCLD was induced in young (1 month) and old (16 months) rats. A subgroup of aCLD-old animals received simvastatin (5 mg/kg) or vehicle (PBS) for 15 days. Hepatic and systemic hemodynamic, liver cells phenotype and hepatic fibrosis were evaluated. Additionally, the gene expression signature of cirrhosis was evaluated in a cohort of young and aged cirrhotic patients. Aged animals developed a more severe form of aCLD. Portal hypertension and liver fibrosis were exacerbated as a consequence of profound deregulations in the phenotype of the main hepatic cells: hepatocytes presented more extensive cell-death and poorer function, LSEC were further capillarized, HSC over-activated and macrophage infiltration was significantly increased. The gene expression signature of cirrhosis significantly differed comparing young and aged patients, indicating alterations in sinusoidal-protective pathways and confirming the pre-clinical observations. Simvastatin administration for 15-day to aged cirrhotic rats improved the hepatic sinusoidal milieu, leading to significant amelioration in portal hypertension. This study provides evidence that aCLD pathobiology is different in aged individuals. As the median age of patients with aCLD is increasing, we propose a real-life pre-clinical model to develop more reliable therapeutic strategies. Simvastatin effects in this model further demonstrate its translational potential

Molecular Characterization of Mycobacterium tuberculosis Strains with TB-SPRINT

We evaluated Tuberculosis-Spoligo-Rifampicin-Isoniazid Typing (TB-SPRINT), a microbead-based method for spoligotyping and detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis. For that, 67 M. tuberculosis complex strains were retrospectively selected. Membrane-based spoligotyping, restriction fragment length polymorphism, DNA sequencing/pyrosequencing of rpoB, katG, and inhA promoter, TB-SPRINT, and SNP typing were performed. Concordance between spoligotyping methods was 99.6% (2,785/2,795 spoligotype data points). For most of the discordant cases, the same lineage was assigned with both methods. Concordance between phenotypic drug susceptibility testing and TB-SPRINT for detecting rifampicin and isoniazid resistance was 98.4% (63/64) and 93.8% (60/64), respectively. Concordance between DNA sequencing/pyrosequencing and TB-SPRINT for detecting mutations in rpoB, katG, and inhA were 98.4% (60/61), 100% (64/64), and 96.9% (62/64), respectively. In conclusion, TB-SPRINT is a rapid and easy-to-perform assay for genotyping and detecting drug resistance in a single tube; therefore, it may be a useful tool to improve epidemiological surveillance

Effect of spray-dried porcine plasma in peripartum sow feed on subsequent litter size

Nutritional strategies for sows designed to reduce peripartum stress are suggested to support postpartum recovery and productivity. Spray-dried plasma (SDP) in sow feed has been reported to benefit sow and litter performance. Stressed animals fed diets with SDP have a more efficient immune response supporting animal recovery and health. The objectives of the present study using 452 sows (147 parity 1 sows, 148 parity 2 sows) were to determine if 0, 0.5 or 2.5% spray-dried porcine plasma (SDPP) in peripartum feed provided from entry in maternity through day 5 of lactation affects sow productivity and serological immune and oxidation status markers around parturition. Post-weaning sow productivity parameters including litter size at the next parturition was evaluated, but peripartum diets were only provided during the first parturition. In the first parturition, total born litter size was lower (P < 0.05) especially for sows allotted to the peripartum diet with 2.5% SDPP. Percentage of stillborn pigs decreased quadratically (P < 0.05) for sows fed 0.5% or 2.5% SDPP compared to 0% SDPP in peripartum feed and this result was not affected by total born litter size. Serum glutathione peroxidase activity linearly increased (P < 0.01) with increased dietary SDPP for both prepartum and postpartum sampling periods. In the next parturition, total born pigs from combined data of parity 1 and 2 sows linearly increased (P < 0.05) and live born pigs tended (P = 0.09) to linearly increase as level of SDPP increased and this result was not affected by total born litter size in the first parturition. The change in total and live born pigs from the first to the next parturition linearly (P < 0.01) increased as dietary SDPP increased for parity 1 and 2 sows. The reduced percentage of stillborn pigs and increased litter size of parity 1 and 2 sows in the next parturition was independent of total born litter size in the first parturition suggesting SDPP in peripartum sow feed may have merit for reducing stillborn pigs and benefit litter size in the next parturition for parity 1 and parity 2 sows