Host-pathogen relationship in retreated tuberculosis with major rifampicin resistance-conferring mutations.

Introduction: It is assumed that host defense systems eliminating the pathogen and regulating tissue damage make a strong impact on the outcome of tuberculosis (TB) disease and that these processes are affected by rifampicin (RIF) resistance-conferring mutations of Mycobacterium tuberculosis (Mtb). However, the host responses to the pathogen harboring different mutations have not been studied comprehensively in clinical settings. We analyzed clinico-epidemiological factors and blood transcriptomic signatures associated with major rpoB mutations conferring RIF resistance in a cohort study. Methods: Demographic data were collected from 295 active pulmonary TB patients with treatment history in Hanoi, Vietnam. When recruited, drug resistance-conferring mutations and lineage-specific variations were identified using whole-genome sequencing of clinical Mtb isolates. Before starting retreatment, total RNA was extracted from the whole blood of HIV-negative patients infected with Mtb that carried either the rpoB H445Y or rpoB S450L mutation, and the total RNA was subjected to RNA sequencing after age-gender matching. The individual RNA expression levels in the blood sample set were also measured using real-time RT-PCR. Logistic and linear regression models were used to assess possible associations. Results: In our cohort, rpoB S450L and rpoB H445Y were major RIF resistance-conferring mutations [32/87 (36.8%) and 15/87 (17.2%), respectively]. H445Y was enriched in the ancient Beijing genotype and was associated with nonsynonymous mutations of Rv1830 that has been reported to regulate antibiotic resilience. H445Y was also more frequently observed in genetically clustered strains and in samples from patients who had received more than one TB treatment episode. According to the RNA sequencing, gene sets involved in the interferon-γ and-α pathways were downregulated in H445Y compared with S450L. The qRT-PCR analysis also confirmed the low expression levels of interferon-inducible genes, including BATF2 and SERPING1, in the H445Y group, particularly in patients with extensive lesions on chest X-ray. Discussion: Our study results showed that rpoB mutations as well as Mtb sublineage with additional genetic variants may have significant effects on host response. These findings strengthen the rationale for investigation of host-pathogen interactions to develop countermeasures against epidemics of drug-resistant TB.

NY-ESO-1-specific redirected T cells with endogenous TCR knockdown mediate tumor response and cytokine release syndrome.

BACKGROUND: Because of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive. METHODS: We conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide. RESULTS: In vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels. CONCLUSIONS: The trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS. TRIAL REGISTRATION NUMBER: NCT02366546.

Phenotypic and genotypic features of the Mycobacterium tuberculosis lineage 1 subgroup in central Vietnam.

Mycobacterium tuberculosis (Mtb) has different features depending on different geographic areas. We collected Mtb strains from patients with smear-positive pulmonary tuberculosis in Da Nang, central Vietnam. Using a whole genome sequencing platform, including genome assembly complemented by long-read-sequencing data, genomic characteristics were studied. Of 181 Mtb isolates, predominant Vietnamese EAI4_VNM and EAI4-like spoligotypes (31.5%), ZERO strains (5.0%), and part of EAI5 (11.1%) were included in a lineage-1 (L1) sublineage, i.e., L1.1.1.1. These strains were found less often in younger people, and they genetically clustered less frequently than other modern strains. Patients infected with ZERO strains demonstrated less lung infiltration. A region in RD2bcg spanning six loci, i.e., PE_PGRS35, cfp21, Rv1985c, Rv1986, Rv1987, and erm(37), was deleted in EAI4_VNM, EAI4-like, and ZERO strains, whereas another 118 bp deletion in furA was specific only to ZERO strains. L1.1.1.1-sublineage-specific deletions in PE_PGRS4 and PE_PGRS22 were also identified. RD900, seen in ancestral lineages, was present in majority of the L1 members. All strains without IS6110 (5.0%) had the ZERO spoligo-pattern. Distinctive features of the ancestral L1 strains provide a basis for investigation of the modern versus ancestral Mtb lineages and allow consideration of countermeasures against this heterogeneous pathogen.

Genotyping of Mycobacterium tuberculosis spreading in Hanoi, Vietnam using conventional and whole genome sequencing methods.

Hanoi is the capital of Vietnam, one of the 30 countries with a high tuberculosis (TB) burden. Fundamental data on the molecular epidemiology of the disease is required for future TB management. To identify lineages and genotypes of Mycobacterium tuberculosis (Mtb), conventional genotyping data from clinical isolates of the Hanoi area was compared with whole genome sequencing (WGS) analysis from 332 of 470 samples. It was obtained from lineage-specific single nucleotide variants (SNVs), large sequence polymorphisms, spoligotyping, and variable number of tandem repeats (VNTR) analysis using mycobacterial interspersed repetitive unit (MIRU) and Japan anti-tuberculosis association (JATA) locus sets. This information was directly compared with results obtained from WGS. Mini-satellite repeat unit variants were identified using BLAST search against concatenated short read sequences, the RepUnitTyping tool. WGS analysis revealed that the Mtb strains tested are diverse and classified into lineage (L) 1, 2 and 4 (24.7, 57.2 and 18.1% respectively). The majority of the L2 strains were further divided into ancient and modern Beijing genotypes, and most of the L1 group were EAI4_VNM strains. Although conventional PCR-based genotyping results were mostly consistent with information obtained through WGS analysis, in-depth analysis identified aberrant deletions and spacers that may cause discordance. JATA-VNTR sets, including hypervariable loci, separated large Beijing genotypic clusters generated by MIRU15 into smaller groups. The distribution of repeat unit variants observed within 33 VNTR loci showed clear variation depending on the three lineages. WGS-based pairwise-SNV differences within VNTR-defined genotypic clusters were greater in L1 than in L2 and L4 (P = .001). Direct comparisons between results of PCR-based genotyping and in silico analysis of WGS data would bridge a gap between classical and modern technologies during this transition period, and provide further information on Mtb genotypes in specific geographical areas.

Whole genome sequencing, analyses of drug resistance-conferring mutations, and correlation with transmission of Mycobacterium tuberculosis carrying katG-S315T in Hanoi, Vietnam.

Drug-resistant tuberculosis (TB) is a serious global problem, and pathogen factors involved in the transmission of isoniazid (INH)-resistant TB have not been fully investigated. We performed whole genome sequencing of 332 clinical Mycobacterium tuberculosis (Mtb) isolates collected from patients newly diagnosed with smear-positive pulmonary TB in Hanoi, Vietnam. Using a bacterial genome-wide approach based on linear mixed models, we investigated the associations between 31-bp k-mers and clustered strains harboring katG-S315T, a major INH-resistance mutation in the present cohort and in the second panel previously published in South Africa. Five statistically significant genes, namely, PPE18/19, gid, emrB, Rv1588c, and pncA, were shared by the two panels. We further identified variants of the genes responsible for these k-mers, which are relevant to the spread of INH-resistant strains. Phylogenetic convergence test showed that variants relevant to PPE46/47-like chimeric genes were significantly associated with the same phenotype in Hanoi. The associations were further confirmed after adjustment for the confounders. These findings suggest that genomic variations of the pathogen facilitate the expansion of INH-resistance TB, at least in part, and our study provides a new insight into the mechanisms by which drug-resistant Mtb maintains fitness and spreads in Asia and Africa.

Complete Genome Sequences of Three Representative Mycobacterium tuberculosis Beijing Family Strains Belonging to Distinct Genotype Clusters in Hanoi, Vietnam, during 2007 to 2009.

We present here three complete genome sequences of Mycobacterium tuberculosis Beijing family strains isolated in Hanoi, Vietnam. These three strains were selected from major genotypic clusters (15-MIRU-VNTR) identified in a previous population-based study. We emphasize their importance and potential as reference strains in this Asian region.

Complete Genome Sequence of a Mycobacterium tuberculosis Strain Belonging to the East African-Indian Family in the Indo-Oceanic Lineage, Isolated in Hanoi, Vietnam.

The East African-Indian (EAI) family of Mycobacterium tuberculosis is an endemic group mainly observed in Southeast Asia. Here, we report the complete genome sequence of an M. tuberculosis strain isolated as a member of the EAI family in Hanoi, Vietnam, a country with a high incidence of tuberculosis.

Primary drug-resistant tuberculosis in Hanoi, Viet Nam: present status and risk factors.

INTRODUCTION: Resistance of Mycobacterium tuberculosis (MTB) to anti-tuberculosis (TB) drugs presents a serious challenge to TB control worldwide. We investigated the status of drug resistance, including multidrug-resistant (MDR) TB, and possible risk factors among newly diagnosed TB patients in Hanoi, the capital of Viet Nam. METHODS: Clinical and epidemiological information was collected from 506 newly diagnosed patients with sputum smear- and culture-positive TB, and 489 (96.6%) MTB isolates were subjected to conventional drug susceptibility testing, spoligotyping, and 15-locus variable numbers of tandem repeats typing. Adjusted odds ratios (aORs) were calculated to analyze the risk factors for primary drug resistance. RESULTS: Of 489 isolates, 298 (60.9%) were sensitive to all drugs tested. Resistance to isoniazid, rifampicin, streptomycin, ethambutol, and MDR accounted for 28.2%, 4.9%, 28.2%, 2.9%, and 4.5%, respectively. Of 24 isolates with rifampicin resistance, 22 (91.7%) were MDR and also resistant to streptomycin, except one case. Factors associated with isoniazid resistance included living in old urban areas, presence of the Beijing genotype, and clustered strains [aOR = 2.23, 95% confidence interval (CI) 1.15-4.35; 1.91, 1.18-3.10; and 1.69, 1.06-2.69, respectively). The Beijing genotype was also associated with streptomycin resistance (aOR = 2.10, 95% CI 1.29-3.40). Human immunodeficiency virus (HIV) coinfection was associated with rifampicin resistance and MDR (aOR = 5.42, 95% CI 2.07-14.14; 6.23, 2.34-16.58, respectively). CONCLUSION: Isoniazid and streptomycin resistance was observed in more than a quarter of TB patients without treatment history in Hanoi. Transmission of isoniazid-resistant TB among younger people should be carefully monitored in urban areas, where Beijing strains and HIV coinfection are prevalent. Choosing an optimal treatment regimen on the basis of the results of drug susceptibility tests and monitoring of treatment adherence would minimize further development of drug resistance strains.