Benchmarking CRISPR-BP34 for point-of-care melioidosis detection in low-income and middle-income countries: a molecular diagnostics study.

BACKGROUND: Melioidosis is a neglected but often fatal tropical disease. The disease has broad clinical manifestations, which makes diagnosis challenging and time consuming. To improve diagnosis, we aimed to evaluate the performance of the CRISPR-Cas12a system (CRISPR-BP34) to detect Burkholderia pseudomallei DNA across clinical specimens from patients suspected to have melioidosis. METHODS: We conducted a prospective, observational cohort study of adult patients (aged ≥18 years) with melioidosis at Sunpasitthiprasong Hospital, a tertiary care hospital in Thailand. Participants were eligible for inclusion if they had culture-confirmed B pseudomallei infection from any clinical samples. Data were collected from patient clinical records and follow-up telephone calls. Routine clinical samples (blood, urine, respiratory secretion, pus, and other body fluids) were collected for culture. We documented time taken for diagnosis, and mortality at day 28 of follow-up. We also performed CRISPR-BP34 detection on clinical specimens collected from 330 patients with suspected melioidosis and compared its performance with the current gold-standard culture-based method. Discordant results were validated by three independent qualitative PCR tests. This study is registered with the Thai Clinical Trial Registry, TCTR20190322003. FINDINGS: Between Oct 1, 2019, and Dec 31, 2022, 876 patients with culture-confirmed melioidosis were admitted or referred to Sunpasitthiprasong Hospital, 433 of whom were alive at diagnosis and were enrolled in this study. Median time from sample collection to diagnosis by culture was 4·0 days (IQR 3·0-5·0) among all patients with known survival status at day 28, which resulted in delayed treatment. 199 (23%) of 876 patients died before diagnosis and 114 (26%) of 433 patients in follow-up were treated, but died within 28 days of admission. To test the CRISPR-BP34 assay, we enrolled and collected clinical samples from 114 patients with melioidosis and 216 patients without melioidosis between May 26 and Dec 31, 2022. Application of CRISPR-BP34 reduced the median sample-to-diagnosis time to 1·1 days (IQR 0·7-1·5) for blood samples, 2·3 h (IQR 2·3-2·4) for urine, and 3·3 h (3·1-3·4) for respiratory secretion, pus, and other body fluids. The overall sensitivity of CRISPR-BP34 was 93·0% (106 of 114 samples [95% CI 86·6-96·9]) compared with 66·7% (76 of 114 samples [57·2-75·2]) for culture. The overall specificity of CRISPR-BP34 was 96·8% (209 of 216 samples [95% CI 93·4-98·7]), compared with 100% (216 of 216 samples [98·3-100·0]) for culture. INTERPRETATION: The sensitivity, specificity, speed, and window of clinical intervention offered by CRISPR-BP34 support its prospective use as a point-of-care diagnostic tool for melioidosis. Future development should be focused on scalability and cost reduction. FUNDING: Chiang Mai University Thailand and Wellcome Trust UK.

Genetic diversity, determinants, and dissemination of Burkholderia pseudomallei lineages implicated in melioidosis in northeast Thailand.

Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conducted a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identified three dominant lineages with unique gene sets enhancing bacterial fitness, indicating lineage-specific adaptation strategies. Crucially, recombination was found to drive lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage revealed heightened expression of lineage-specific genes in environmental versus infection conditions, notably under nutrient depletion, highlighting environmental persistence as a key factor in the success of dominant lineages. The study also revealed the role of environmental factors - slope of terrain, altitude, direction of rivers, and the northeast monsoons - in shaping B. pseudomallei geographical dispersal. Collectively, our findings highlight persistence in the environment as a pivotal element facilitating B. pseudomallei spread, and as a prelude to exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies.

BurkHostGEN: a study protocol for evaluating variations in the Burkholderia pseudomallei and host genomes associated with melioidosis infection.

Background: Melioidosis is a frequently fatal disease caused by an environmental bacterium Burkholderia pseudomallei. The disease is prevalent in northeast Thailand, particularly among rice field farmers who are at risk of bacterial exposure through contact with contaminated soil and water. However, not all exposure results in disease, and infection can manifest diverse outcomes. We postulate that genetic factors, whether from the bacterium, the host or the combination of both, may influence disease outcomes. To address this hypothesis, we aim to collect, sequence, and analyse genetic data from melioidosis patients and controls, along with isolates of B. pseudomallei obtained from patients. Additionally, we will study the metagenomics of the household water supply for both patients and controls, including the presence of B. pseudomallei. Methods: BurkHostGEN is an ongoing observational study being conducted at Sunpasitthiprasong Hospital, Ubon Ratchathani, Thailand. We are obtaining consent from 600 melioidosis patients and 700 controls, spanning both sexes, to collect 1 mL of blood for host DNA analysis, 3 mL of blood for RNA analysis, as well as 5 L of household water supply for metagenomic analysis. Additionally, we are isolating B. pseudomallei from the melioidosis patients to obtain bacterial DNA. This comprehensive approach will allow us to identify B. pseudomallei and their paired host genetic factors associated with disease acquisition and severity. Ethical approvals have been obtained for BurkHostGEN. Host and bacterial genetic data will be uploaded to European Genome-Phenome Archive (EGA) and European Nucleotide Archive (ENA), respectively. Conclusions: BurkHostGEN holds the potential to discover bacterial and host genetic factors associated with melioidosis infection and severity of illness. It can also support various study designs, including biomarker validation, disease pathogenesis, and epidemiological analysis not only for melioidosis but also for other infectious diseases.

Detection of maternal transmission of resistant Gram-negative bacteria in a Cambodian hospital setting.

Infection with Extended-spectrum beta-lactamase -producing Enterobacterales (ESBL-E) is common in infants and leads to increased intensive care unit admission and mortality, but the role of maternal transmission in colonization of infants is unclear. Using paired isolates from 50 pairs of mothers and neonates admitted to a Cambodian hospital, we investigated antimicrobial resistance in Escherichia coli and Klebsiella pneumoniae using whole genome sequencing. We detected a wide variety of ESBL-E genes present in this population along with high levels of multidrug resistance. From 21 pairs where the same organism was present in both mother and neonate, we identified eight pairs with identical or near-identical isolates from both individuals suggestive of transmission at or around birth, including a pair with transmission of multiple strains. We found no evidence for transmission of plasmids only from mother to infant. This suggests vertical transmission outside hospitals as a common cause of ESBL-E colonization in neonates.

Raising awareness of antimicrobial resistance: development of an 'antibiotic footprint calculator'.

Non-academic partners can be vital in successful public engagement activities on antimicrobial resistance. With collaboration between academic and non-academic partners, we developed and launched an open-access web-based application, the 'antibiotic footprint calculator', in both Thai and English. The application focused on a good user experience, addressing antibiotic overuse and its impact, and encouraging immediate action. The application was unveiled in joint public engagement activities. From 1 Nov 2021 to 31 July 2022 (9 month period), 2554 players estimated their personal antibiotic footprint by using the application.

Abundance of ACVR1B transcript is elevated during septic conditions: Perspectives obtained from a hands-on reductionist investigation.

Sepsis is a complex heterogeneous condition, and the current lack of effective risk and outcome predictors hinders the improvement of its management. Using a reductionist approach leveraging publicly available transcriptomic data, we describe a knowledge gap for the role of ACVR1B (activin A receptor type 1B) in sepsis. ACVR1B, a member of the transforming growth factor-beta (TGF-beta) superfamily, was selected based on the following: 1) induction upon in vitro exposure of neutrophils from healthy subjects with the serum of septic patients (GSE49755), and 2) absence or minimal overlap between ACVR1B, sepsis, inflammation, or neutrophil in published literature. Moreover, ACVR1B expression is upregulated in septic melioidosis, a widespread cause of fatal sepsis in the tropics. Key biological concepts extracted from a series of PubMed queries established indirect links between ACVR1B and "cancer", "TGF-beta superfamily", "cell proliferation", "inhibitors of activin", and "apoptosis". We confirmed our observations by measuring ACVR1B transcript abundance in buffy coat samples obtained from healthy individuals (n=3) exposed to septic plasma (n = 26 melioidosis sepsis cases)ex vivo. Based on our re-investigation of publicly available transcriptomic data and newly generated ex vivo data, we provide perspective on the role of ACVR1B during sepsis. Additional experiments for addressing this knowledge gap are discussed.

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a.

Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.

Corrigendum: An Evolutionary Arms Race Between Burkholderia pseudomallei and Host Immune System: What Do We Know?

[This corrects the article DOI: 10.3389/fmicb.2020.612568.].

An Evolutionary Arms Race Between Burkholderia pseudomallei and Host Immune System: What Do We Know?

A better understanding of co-evolution between pathogens and hosts holds promise for better prevention and control strategies. This review will explore the interactions between Burkholderia pseudomallei, an environmental and opportunistic pathogen, and the human host immune system. B. pseudomallei causes "Melioidosis," a rapidly fatal tropical infectious disease predicted to affect 165,000 cases annually worldwide, of which 89,000 are fatal. Genetic heterogeneities were reported in both B. pseudomallei and human host population, some of which may, at least in part, contribute to inter-individual differences in disease susceptibility. Here, we review (i) a multi-host-pathogen characteristic of the interaction; (ii) selection pressures acting on B. pseudomallei and human genomes with the former being driven by bacterial adaptation across ranges of ecological niches while the latter are driven by human encounter of broad ranges of pathogens; (iii) the mechanisms that generate genetic diversity in bacterial and host population particularly in sequences encoding proteins functioning in host-pathogen interaction; (iv) reported genetic and structural variations of proteins or molecules observed in B. pseudomallei-human host interactions and their implications in infection outcomes. Together, these predict bacterial and host evolutionary trajectory which continues to generate genetic diversity in bacterium and operates host immune selection at the molecular level.