Biology subjects

Thwaites, G. E.

Publications and source records attributed to Thwaites, G. E..

13 recordsLinked to original sources

bioRxiv2025

Single-cell profiling of blood and cerebrospinal fluid in tuberculous meningitis

Tuberculous meningitis (TBM) is the most severe form of tuberculosis, with a fatality rate of 20-50% in treated individuals. Although corticosteroid therapy can increase survival in HIV-negative people with TBM, better antimicrobial and host-directed therapies are required to improve outcome. There is, therefore, a need to better understand local immunopathologic pathways. Despite its power in identifying disease-specific cellular profiles, single-cell RNA-sequencing (scRNA-seq) has been underutilized in cerebral samples in brain infection. We employed scRNA-seq to analyze fresh pretreatment cerebrospinal fluid (CSF) from four TBM patients, along with paired peripheral blood mononuclear cells (PBMCs). While 29 cell subtypes were present in both tissues, their relative abundance varied significantly. In particular, CSF was enriched with highly inflammatory microglia-like macrophages, GZMK-expressing CD8+ T cells, and CD56bright NK cells. The latter two subsets exhibited features associated with dysfunctional cytotoxicity. Across multiple cell types, inflammatory signaling pathways were increased and oxidative phosphorylation was decreased in CSF compared to PBMCs. This study highlights the value of scRNA-seq for exploring CSF immunopathogenesis in TBM patients and offers a resource for future studies investigating the pathophysiology of TBM and other brain infections, including potentially targetable cell populations linked with immune-mediated pathology.

immunology↗

bioRxiv2023

Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study

Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. Therefore, we investigated the rifampicin tolerance of M. tuberculosis isolates, with or without pre-existing isoniazid-resistance. We determined the in-vitro rifampicin survival fraction by minimum duration of killing assay in isoniazid susceptible (IS, n=119) and resistant (IR, n=84) M. tuberculosis isolates. Then we correlated the rifampicin tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. The longitudinal IR isolates collected from patients were analyzed for changes in rifampicin tolerance and associated emergence of genetic variants. The median duration of rifampicin exposure reducing the M. tuberculosis surviving fraction by 90% (minimum duration of killing-MDK90) increased from 1.23 (95%CI 1.11; 1.37) and 1.31 (95%CI 1.14; 1.48) to 2.55 (95%CI 2.04; 2.97) and 1.98 (95%CI 1.69; 2.56) days, for IS and IR respectively, during 15 to 60 days of incubation. This indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log10-fold survival fraction enabled classification of tolerance as low, medium or high and revealed IR association with increased tolerance with faster growth (OR=2.68 for low vs. medium, OR=4.42 for low vs. high, P-trend=0.0003). The high tolerance in IR isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Furthermore, the high rifampicin tolerant IR isolates have survival potential similar to multi-drug resistant isolates. These findings suggest that IR tuberculosis needs to be evaluated for high rifampicin tolerance to improve treatment regimen and prevent the risk of MDR-TB emergence.

microbiology↗

bioRxiv2023

Whole blood transcriptional profiles and the pathogenesis of tuberculous meningitis

BackgroundMortality and morbidity from tuberculous meningitis (TBM) are frequent and strongly associated with the inflammatory response to Mycobacterium tuberculosis infection. However, the mechanisms driving the associations are uncertain. We sought to identify the gene modules, hubs and pathways associated with the pathogenesis and mortality from TBM, and to identify which best-predicted death. MethodsWe used whole blood RNA sequencing to obtain transcriptional profiles from 281 Vietnamese adults with TBM (207 HIV-negative; 74 HIV-positive), 295 with pulmonary TB (PTB), and 30 healthy controls. The TBM cohort was divided randomly into a discovery cohort (n=142) and a validation cohort (n=139). Weighted gene co-expression network analysis identified clusters of genes (or modules) and hub genes associated with death or disease severity. An overrepresentation analysis identified pathways associated with TBM mortality, with a consensus analysis identifying consensual patterns between HIV-positive and HIV-negative individuals. A multivariate elastic-net Cox regression model selected the candidate predictors of TBM mortality, then model prediction performance using logistic regression and internal bootstrap validation to choose best predictors. ResultsOverall, TBM mortality was associated with increased neutrophil activation and decreased T and B cell activation pathways. Death from TBM was associated with increased angiogenesis in HIV-positive adults, and with activated TNF signaling and down-regulated extracellular matrix organization in HIV-negative adults. PTB and TBM have similar transcriptional profiles compared to healthy controls, although inflammatory genes were more activated in HIV-positive than HIV-negative TBM. The expression of four hub genes - MCEMP1, NELL2, ZNF354C and CD4 - were strongly predictive of death from TBM (AUC 0.80 and 0.86 for HIV-negative and HIV-positive, respectively). ConclusionsWhole blood transcriptional profiling revealed that TBM is associated with a characteristic systemic inflammatory response, similar to that invoked by pulmonary tuberculosis, but with key gene modules, hubs and pathways strongly associated with death. Our analysis suggests a novel 4-gene biomarker for predicting death from TBM, but also opens a new window into TBM pathogenesis that may reveal novel therapeutic targets for this lethal disease.

molecular biology↗

bioRxiv2022

Distribution and origins of Mycobacterium tuberculosis L4 in Southeast Asia

Molecular and genomic studies have revealed that Mycobacterium tuberculosis Lineage 4 (L4, Euro-American lineage) emerged in Europe before becoming distributed around the globe by trade routes, colonial migration, and other historical connections. Although L4 accounts for tens or hundreds of thousands of TB cases in multiple Southeast Asian countries, phylogeographic studies have either focussed on a single country or just included Southeast Asia as part of a global analysis. Therefore, we interrogated public genomic data to investigate the historical patterns underlying the distribution of L4 in Southeast Asia and surrounding countries. We downloaded 6037 genomes associated with 23 published studies, focusing on global analyses of L4 and Asian studies of M. tuberculosis. We identified 2256 L4 genomes including 968 from Asia. We show that 81% of L4 in Thailand, 51% of L4 in Vietnam and 9% of L4 in Indonesia belong to sub-clades of L4 that are rarely seen outside East and Southeast Asia. These sub-clades have been transmitted between East and Southeast Asian countries, with no recent European ancestor. Although there is considerable uncertainty about the exact direction and order of intra-Asian transmissions, due to differing sampling frames between countries, our analysis suggests that China may be the intermediate between Europe and Southeast Asia for two of the three predominantly East and Southeast Asian L4 sub-lineages. This new perspective on L4 in Southeast Asia raises the possibility of investigating host population specific evolution and highlights the need for more structured sampling from Southeast Asian countries to provide more certainty of the historical and current routes of transmission.

microbiology↗

bioRxiv2020

The expression of virulence by the Cryptococcus neoformans VNIa-5 lineage is plastic and associated with host immune background.

Cryptococcus neoformans most frequently causes disease in immunocompromised patients. However, in Vietnam and east Asia, disease is frequently reported in apparently immunocompetent patients. We have previously shown that almost all such disease is due to a specific lineage of C. neoformans - VNIa-5. However, in HIV-infected patients, infections due to this lineage are not associated with worse outcomes. Here, we demonstrate that the VNIa-5 lineage presents different virulence phenotypes depending on its source. Isolates derived from immunocompetent patients are more virulent than those from HIV-infected patients or the environment. Moreover, the virulence phenotype is plastic - sterile culture filtrate from highly virulent VNIa-5 strains can induce increased virulence in less virulent VNIa-5 isolates, which in turn can then induce increased virulence in their low virulence states. We present evidence that this phenomenon is driven by secreted proteins associated with extra-cellular vesicles.

microbiology↗

bioRxiv2019

Ciprofloxacin facilitates the transfer of XDR plasmids from commensal E. coli into epidemic fluoroquinolone-resistant Shigella sonnei

The global dissemination of a ciprofloxacin-resistant (cipR) S. sonnei clone outlines the mobility of this important agent of diarrheal disease, and threatens the utility of ciprofloxacin as a first-line antimicrobial for shigellosis. Here, we aimed to track the emergence of cipR S. sonnei in Vietnam to understand how novel antimicrobial resistant (AMR) Shigella clones become established in new locations. From 2014 to 2016, we isolated and genome sequenced 79 S. sonnei from children hospitalized with dysenteric diarrhea in southern Vietnam. The novel cipR S. sonnei clone displaced the resident ciprofloxacin-susceptible lineage while acquiring resistance against third-generation cephalosporins, macrolides, and aminoglycosides. This process was not the result of a single clonal expansion, as we identified at least thirteen independent acquisitions of ESBL-encoding plasmids. The frequency and diversity of the variable AMR repertoire in an expanding clonal background of S. sonnei is unprecedented and we speculated that it was facilitated by horizontal gene transfer from commensal organisms in the human gut. Consequently, we characterized non-Shigella Enterobacteriaceae from Shigella-infected and healthy children by shotgun metagenomics. We identified a wide array of AMR genes and plasmids in the commensal Enterobacteriaceae, including an E. coli isolated from a Shigella-infected child with an identical ESBL plasmid to that characterized in the infecting S. sonnei. We confirmed that these AMR plasmids could be exchanged between commensal E. coli and S. sonnei and found that supplementation of ciprofloxacin into the conjugation media significantly increased the conjugation frequency of IncI/blaCTX-M-15, IncB/O/blaCTX-M-27 and IncF/blaCTX-M-27 plasmids. In a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines. Our work highlights the role of the gut microbiota in transferring resistance plasmids into enteric pathogens and provides essential data to restrict the use of ciprofloxacin globally.

microbiology↗

bioRxiv2019

Pro-Resolving Mediator Profiles And 5-Lipoxygenase Activity In Cerebrospinal Fluid Correlate with Disease Severity and Outcome in Adults with Tuberculous Meningitis

Tuberculous meningitis (TBM) is the most lethal form of tuberculosis infection, characterized by a dysregulated immune response that frequently leads to neurological injury and death despite the best available treatment. The mechanisms driving the inflammatory response in TBM are not well understood. To gain insights into these mechanisms we used a lipid mediator profiling approach to investigate the regulation of a novel group of host protective mediators, termed specialized pro-resolving mediators (SPM), in the cerebrospinal fluid (CSF) of adults with TBM enrolled into a randomised placebo-controlled trial of adjunctive aspirin treatment. We found distinct lipid mediator profiles with increasing disease severity, changes that were linked with an upregulation of inflammatory eicosanoids in patients with severe TBM and a decrease in the production of a number of 5-lipoxygenase (ALOX5)-derived SPM. CSF pro-resolving mediator concentrations were also associated with 80-day survival. In survivors, we found a significant increase in pro-resolving mediator concentrations, including the ALOX5-derived resolvin (Rv)T2, RvT4 and 15-epi-Lipoxin (LX)B4, compared to those who died. Aspirin administration increased the ratio of pro-resolving to pro-inflammatory mediators decreasing the concentrations of the prothrombic mediator TxA2, changes that were linked with early reductions in brain infarcts and deaths. Together, these findings identify a CSF SPM signature that is associated with disease severity and 80-day mortality in TBM. Furthermore, the therapeutic manipulation of the ratio between pro-resolving mediators and pro-inflammatory/thrombogenic mediators in the CSF, by aspirin for example, offers a novel treatment strategy to reduce the morbidity and mortality caused by TBM.\n\nAuthors SummaryInfections of the brain and the meninges by Mycobacterium tuberculosis (M. tb) lead to severe inflammation and are associated with poor outcomes. The mechanisms leading to this disease remain poorly defined. Herein, we investigated how M. tb infection regulates the concentrations of specialized pro-resolving mediators that are central in controlling the bodys ability to clear infections. In these investigations, we found that disease survival was linked with increased concentrations of a number of these protective molecules including resolvins and lipoxins. Treatment of M. tb-infected patients with aspirin decreased the production of the immunosuppressive and thrombogenic mediator thromboxane A2 improving the balance between protective and inflammatory molecules. Of note, these changes were linked with reduced disease severity and improved survival. Therefore, the present findings suggest a previously unappreciated role for pro-resolving mediators in TBM pathogenesis.

immunology↗

bioRxiv2018

Genomics of Cryptococcus neoformans

C. neoformans var. grubii (C. neoformans) is an environmentally acquired pathogen causing 181 000 HIV-associated deaths each year. We used whole genome sequencing (WGS) to characterise 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. We found that 91% of our clinical isolates belonged to one of three highly clonal sub-clades of VNIa, which we have termed VNIa-4, VNIa-5 and VNIa-93. Parsimony analysis revealed frequent, long distance transmissions of C. neoformans; international transmissions took place on 13% of VNIa-4 branches, and intercontinental transmissions on 7% of VNIa-93 branches. The median length of within sub-clade internal branches was 3-6 SNPs, while terminal branches were 44.5-77.5 SNPs. The short median internal branches were partly driven by the large number (12-15% of internal branches) of polytomies in the within-sub-clade trees. To simultaneously explain our observation of no apparent molecular clock, short internal branches and frequent polytomies we hypothesise that C. neoformans VNIa spends much of its time in the environment in a quiescent state, while, when it is sampled, it has almost always undergone an extended period of growth. Infections with VNIa-93 were associated with a significantly reduced risk of death by 10 weeks compared with infections with VNIa-4 (Hazard Ratio = 0.45, p = 0.003). We detected a recombination in the mitochondrial sequence of VNIa-5, suggesting that mitochondria could be involved in the propensity of this sub-clade to infect HIV-uninfected patients. These data highlight the insight into the biology and epidemiology of pathogenic fungi which can be gained from WGS data.

microbiology↗

bioRxiv2017

MULTILOCUS SEQUENCE TYPING REVEALS A UNIQUE CO-DOMINANT POPULATION STRUCTURE OF CRYPTOCOCCUS NEOFORMANS VAR. GRUBII IN VIETNAM

Cryptococcosis is amongst the most important invasive fungal infections globally, with cryptococcal meningitis causing an estimated 180,000 deaths each year in HIV infected patients alone. Patients with other forms of immunosuppression are also at risk, and disease is increasingly recognized in apparently immunocompetent individuals. Cryptococcus neoformans var. grubii (serotype A, molecular type VNI) has a global distribution and is responsible for the majority of cases. Here, we used the consensus ISHAM Multilocus Sequence Typing (MLST) for C. neoformans to define the population structure of clinical isolates of Cryptococcus neoformans var. grubii from Vietnam (n=136) and Laos (n=81). We placed these isolates into the global context using published MLST data from 8 other countries (total N = 669). We observed a phylo-geographical relationship in which Laos was similar to its Southeast Asian neighbor Thailand in being dominated (83%) by Sequence Type (ST) 4 and its Single Locus Variant ST6. On the other hand, Vietnam was uniquely intermediate between Southeast Asia and East Asia having both ST4/ST6 (35%) and ST5 (48%) which causes the majority of cases in East Asia. Analysis of genetic distance (Fst) between different populations of Cryptococcus neoformans var. grubii supported the intermediate nature of the population from Vietnam. A strong association between ST5 and infection in apparently immunocompetent, HIV-uninfected patients was observed in Vietnam (OR: 7.97, [95%CI: 3.18-19.97], p < 0.0001). Our study emphasizes that Vietnam, with its intermediate Cryptococcus neoformans var. grubii population structure, provides the strongest epidemiological evidence of the relationship between ST5 and infection of HIV-uninfected patients. Human population genetic distances within the region suggest these differences in CNVG population across Southeast Asia are driven by ecological factors rather than host factors.\n\nAuthor summaryCryptococcus neoformans is a yeast that causes meningitis in people, usually with damaged immune systems. There are >180,000 deaths in HIV-infected patients each year, most occurring where there are the highest HIV/AIDS disease burdens. Vietnam and Laos have contributed significantly to clinical trials aiming to improve the treatment of cryptococcal meningitis, but the relationship of isolates from these countries to the global population is not yet described. Here, we address this knowledge gap by using Multilocus Sequence Typing to study the population of Cryptococcus neoformans var. grubii (CNVG) in Laos and Vietnam, with the specific aim of incorporating these populations into the wider global context. We found that, in most countries, a single lineage (family) of strains was responsible for most disease. The Vietnamese CNVG population was unusual in that 2 main lineages circulated at the same time. The Vietnamese CNVG population occupies a middle ground between Thailand/Laos in the west and China in the east. The differences in population structure moving from West to East are probably due to ecological differences. Disease in HIV uninfected patients was almost always due to members of a single family of strains (ST5).

molecular biology↗

bioRxiv2017

N VITRO AND IN VIVO CHARACTERISATION OF ISOLATES OF CRYPTOCOCCUS NEOFORMANS CAUSING MENINGITIS IN HIV-INFECTED AND UNINFECTED PATIENTS IN VIETNAM

We previously observed a substantial burden of cryptococcal meningitis in Vietnam atypically arising in HIV-uninfected individuals. This disease was associated with a single genotype of Cryptococcus neoformans (Sequence Type (ST)5), which was significantly less common in HIV-infected individuals. Aiming to compare the phenotypic characteristics of ST5 and non-ST5 C. neoformans we selected 30 representative Vietnamese isolates, compared their in vitro pathogenic potential and in vivo virulence. ST5 and non-ST5 organisms exhibited comparable characteristics with respect to in vitro virulence markers including melanin production, replication at 37{degrees}C, and growth in cerebrospinal fluid. However, the ST5 isolates had significantly increased variability in cellular and capsular sizing compared with non-ST5 organisms (p<0.001). Counter-intuitively, mice infected with ST5 isolates had significantly longer survival with lower fungal burdens at day 7 than non-ST5 isolates. Notably, ST5 isolates induced significantly greater initial inflammatory responses than non-ST5 strains, measured by TNF- concentrations (p<0.001). Despite being generally less virulent in the mouse model, we hypothesize that the significant within strain variation seen in ST5 isolates in the tested phenotypes may represent an evolutionary advantage enabling adaptation to novel niches including apparently immunocompetent human hosts.

microbiology↗

bioRxiv2017

The decline of malaria in Vietnam, 1991-2014

A central component of malaria control initiatives throughout the world is the use of artemisinin-based combination therapies (ACTs) for treatment of uncomplicated P. falciparium malaria. Despite the well-documented clinical efficacy of ACTs, the population-level effects of ACT case management on malaria transmission have not been studied thoroughly until recently. An ideal case study for the population-level effects of artemisinin use can be found in Vietnam, where a major increase of malaria cases in the 1980s was followed by the gradual adoption of artemisinin-based clinical case management. We assembled annual data from Vietnams National Institutes for Malariology, Parasitology, and Entomology showing the degree to which artemisinin therapies were adopted in different provinces, the effort placed on vector control, and the funding available to provincial malaria control programs, from 1991 to 2014. Data on urbanization were also collected for this period. We found that a 10% increase in the artemisinin proportion of treatments procured by a provincial control program corresponded to a 32.8% (95% CI: 27.7 - 37.5%) decline in estimated malaria cases; the association persisted and the effect size was nearly unchanged if confirmed cases or suspected cases were used. There was no consistent effect of vector control on malaria cases in Vietnam as a whole, nor was any effect found when the data were broken up regionally. The association between urbanization and malaria was generally negative and sometimes statistically significant. This was most pronounced in the central region of Vietnam, where a 10% increase in urbanization corresponded to a 43.3% (95% CI: 21.6 - 58.9%) decrease in suspected malaria incidence; this association was not statistically significant if confirmed cases or estimated cases were used. The decline of malaria in Vietnam from 1991 to 2014 can largely be attributed to the rapid adoption of artemisinin-based drugs. Recent analyses of aggregated data from Africa have shown that insecticide-treated nets have had the greatest effect on lowering malaria prevalence over the past fifteen years, suggesting that the success of different types of malaria interventions is region specific. Continuing global efforts on malaria elimination should focus on both vector control measures and increased access to artemisinin-combination therapies.

epidemiology↗

bioRxiv2017

Non-annual seasonality of influenza-like illness in a tropical urban setting

In temperate countries, influenza and other viral respiratory diseases often have distinct seasonal peaks occurring during colder, wintertime months. However, little is known about the dynamics of influenza and viral respiratory disease dynamics in the tropics, despite high morbidity and a clear epidemiological link between tropical and temperate countries. In temperate countries, the dynamics of influenza and other respiratory diseases are often analyzed using syndromic surveillance data describing influenza-like illness (ILI) as ILI is highly correlated with virological surveillance for influenza. To obtain a detailed picture of respiratory disease incidence patterns in a large tropical city, we established an mHealth study in community outpatient clinics in Ho Chi Minh City, Vietnam (11N latitude). From August 2009 through December 2015, clinics reported daily case numbers of ILI using standard mobile-phone SMS messaging. A subset of these clinics performed molecular diagnostics for influenza A and B viruses. Unlike the annual patterns seen in temperate countries, ILI activity in Ho Chi Minh City exhibited strong non-annual periodicity and was not correlated with PCR-confirmed influenza. The dominant periodicity in the data was approximately 200 days. This was confirmed by a time series decomposition, a step-wise regression analysis on annual and non-annual covariates, and a forecasting exercise showing that forecasting was 30% to 40% more accurate when a 200-day non-annual cycle was included in the forecast. This suggests, for the first-time, that a non-annual cycle may be an essential driver of ILI dynamics in the tropics. This raises new questions about the seasonality and drivers of respiratory disease transmission in tropical countries.

epidemiology↗

bioRxiv2016

Structure of general-population antibody titer distributions to influenza A virus

Seroepidemiological studies aim to understand population-level exposure and immunity to infectious diseases. Results from serological assays are normally presented as binary outcomes describing the presence or absence of pathogen-specific antibody, despite the fact that many assays measure continuous quantities. A populations natural distribution of antibody titers to an endemic infectious disease may in fact include information on multiple serological states - e.g. naivete, recent infection, non-recent infection - depending on the disease in question and the acquisition and waning patterns of host immunity. In this study, we investigate a collection of 20,152 general-population serum samples from southern Vietnam collected between 2009 and 2013 from which we report antibody titers to the influenza virus HA1 protein using a continuous titer measurement from a protein microarray assay. We describe the distributions of antibody titers to subtypes 2009 H1N1 and H3N2. Using a model selection approach to fit mixture distributions, we show that 2009 H1N1 antibody titers fall into four titer subgroups and that H3N2 titers fall into three subgroups. For H1N1, our interpretation is that the two highest-titer subgroups correspond to recent infection and historical infection, which is consistent with 2009 pandemic attack rates. For H3N2, observations censored at the highest titer dilutions make similar interpretations difficult to validate.

epidemiology↗