Middle mountains forests of Nepal

Publication no. 3201

High mountains and high Himal forests of Nepal

Publication no. 4201

Multiple Introductions of Mycobacterium tuberculosis Lineage 2–Beijing Into Africa Over Centuries

The Lineage 2–Beijing (L2–Beijing) sub-lineage of Mycobacterium tuberculosis has received much attention due to its high virulence, fast disease progression, and association with antibiotic resistance. Despite several reports of the recent emergence of L2–Beijing in Africa, no study has investigated the evolutionary history of this sub-lineage on the continent. In this study, we used whole genome sequences of 781 L2 clinical strains from 14 geographical regions globally distributed to investigate the origins and onward spread of this lineage in Africa. Our results reveal multiple introductions of L2–Beijing into Africa linked to independent bacterial populations from East- and Southeast Asia. Bayesian analyses further indicate that these introductions occurred during the past 300 years, with most of these events pre-dating the antibiotic era. Hence, the success of L2–Beijing in Africa is most likely due to its hypervirulence and high transmissibility rather than drug resistance

First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal

BACKGROUND: Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. METHODS AND FINDINGS: We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42-4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43-5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. CONCLUSIONS: We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian reg

Back-to-Africa introductions of Mycobacterium tuberculosis as the main cause of tuberculosis in Dar es Salaam, Tanzania

In settings with high tuberculosis (TB) endemicity, distinct genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple MTBC genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in this setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.We thank S. Lecher, S. Li and J. Zallet for technical support. Calculations were performed at the sciCORE scientific computing core facility at the University of Basel. This work was supported by the Swiss National Science Foundation (grants 310030_166687 (S.G.) and 320030_153442 (M.E.) and Swiss HIV Cohort Study grant 740 to L.F.), the European Research Council (309540-EVODRTB to S.G.), TB-PAN-NET (FP7-223681 to S.N.), PathoNgenTrace projects (FP7-278864-2 to S.N.), SystemsX.ch (S.G.), the German Center for Infection Research (DZIF; S.N.), the Novartis Foundation (S.G.), the Natural Science Foundation of China (91631301 to Q.G.), and the National Institute of Allergy and Infectious Diseases (5U01-AI069924-05) of the US National Institutes of Health (M.E.)

Molecular epidemiology of "mycobacterium tuberculosis" in Nepal

Tuberculosis (TB) is a global health problem. One reason of conducting molecular epidemiology studies is to understand the uneven distribution of the disease in different parts of the world. The global population structure of MTBC can be studied by genotyping strains from different geographic regions, which describes the evolutionary relatedness of MTBC. The choice of appropriate genetic tools is fundamental that can elucidate local as well as global spread of disease. Genetic markers like large sequence polymorphisms (LSPs) and single nucleotide polymorphisms (SNPs) have been used to construct phylogenies of MTBC lineages that are informative for understanding the global distribution of MTBC. Additionally, markers such as direct repeats can differentiate strains within smaller geographical settings or cohort of patients. Studies have shown that the lineages diversity itself could be associated with differences in the pathogenesis and epidemiology of TB. Most importantly, the emergence of drug resistance, which results mostly among treatment failures, is a serious threat to TB control programs. Our aim was to use those markers to explore the phylogenetic diversity and distribution of MTBC in Nepal and compare it to the global phylogeography of MTBC. Furthermore, to identify the mutational hotspots conferring drug resistance. Understanding the molecular mechanisms of drug resistance will allow us to develop rapid molecular drug resistance detection tools and management of TB cases with improved and more rational drug therapies. We used SNPs based genotyping tool for 506 M. tuberculosis strains from Nepal. This revealed four major lineages of MTBC. This allowed us to map the MTBC structure in Nepal compared to the global diversity. Additionally, the use of spoligotyping and MIRU-VNTR (used for XDR strains only) provided data within particular geographical settings and within human populations. A total of 69 different spoligotypes with unique SIT numbers were identified. We found Beijing and Central Asian Strain (CAS) family as the predominant genotypes as was expected owing to geo-position of Nepal in Asia. Molecular analysis of drug resistance for most common anti-TB drugs (i.e. isoniazid, rifampicin) from our sample set confirmed that the polymorphisms were more or less similar as previously documented globally, although we found some additional non-synonymous mutations which need validation. In general, our findings showed that the rapid molecular tools currently developed will detect most of the drug resistance isolates in Nepal. Among drug resistance strains, the katG S315T was proportionally more represented by multi-drug resistance strains. However, the patterns of rpoB mutation were unrelated to multi-drug "resistance or MTBC genotypes. By performing 24 MIRU-VNTR loci plus additional 4 hyper- variable region intended to use for Beijing spoligotypes, we provide evidence of primary transmission of XDR strains. On the other hand, the aim was to identify risk factors, risk groups, and co-morbidities that may relate to the susceptibility to TB. The number of male patients constituted two-third of the total sample population and most of them were at the age of 15-24 years. However, female TB patients in Nepal seem to be associated with “virulent” strains of TB (Beijing genotype) and drug resistance. We identified four XDR cases; the younger age (median age 21 yrs.) of XDR-TB is a serious matter that requires immediate attention from NTP, Nepal.

Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy

Abstract Background Circulating exosomes from prostate cancer (PCa) patients undergoing radiotherapy are attractive candidate biomarkers for monitoring treatment response. Multiple workflows for isolation and content characterization of exosomes in biofluids have been attempted. We report a protocol to isolate and characterize exosomal miRNAs content and assess radiation-induced changes. Methods In this pilot study, we performed targeted exosomal miRNA profiling of 25 serum samples obtained from PCa patients with intermediate- and high-risk disease treated with curative radiotherapy (RT), and controls. Post-treatment blood samples were collected at least 28 days after radiation therapy as a paired follow-up sample. The complete workflow consisted of two phases: I) filtration and polyethylene glycol salt precipitation phase which enriched particles below 200 nm in size followed by characterization using electron microscopy, and II) flow cytometry. Finally, miRNA expression analysis between untreated and treated patient samples was performed using RNA extraction kit, and qRT-PCR. Results In our preliminary data, 1 ml of serum from PCa patients showed higher exosomal concentration (3.68E+10) compared to controls (6.07E+08). The overall expression of exosomes after RT was found to be higher compared to untreated samples; the median value changed from 3.68E+10 to 5.40E+10; p = 0.52. Using electron microscopy, we were able to visualize cup-shaped vesicles with morphology and size compatible with exosomes. The bead-based flow cytometry showed positivity for exosomal tetraspanins surface markers CD63 and CD9. All five miRNAs (hsa-let-7a-5p, hsa-miR-141-3p, hsa-miR-145-5p, hsa-miR-21-5p, hsa-miR-99b-5p) have been identified in exosomes. Despite overall changes in hsa-let-7a-5p expression after radiation, the difference was significant only in the high-risk group (p = 0.037). In addition, the radiation response to hsa-miR-21-5p was elevated in the high-risk group compared to the intermediate group (p = 0.036). Conclusions Herewith, we demonstrated a protocol for isolation of serum exosomes and exosomal miRNA amplification. The recovery of exosomal miRNAs and their differential expression after radiation treatment suggests promising biomarker potential that requires further investigation in larger patient cohorts