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.

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.

Effectiveness of a simplified method for isolation of Burkholderia pseudomallei from soil.

Detection of environmental Burkholderia pseudomallei indicates a risk for melioidosis and is important for the development of a global risk map. We describe a simple method for detecting B. pseudomallei using direct culture of soil in enrichment broth. This gives a rate of positivity comparable to that obtained with a standard method but is cheaper and labor saving.

Multiple phylogenetically-diverse, differentially-virulent Burkholderia pseudomallei isolated from a single soil sample collected in Thailand.

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia that causes the disease, melioidosis. Although the global genomic diversity of clinical B. pseudomallei isolates has been investigated, there is limited understanding of its genomic diversity across small geographic scales, especially in soil. In this study, we obtained 288 B. pseudomallei isolates from a single soil sample (~100g; intensive site 2, INT2) collected at a depth of 30cm from a site in Ubon Ratchathani Province, Thailand. We sequenced the genomes of 169 of these isolates that represent 7 distinct sequence types (STs), including a new ST (ST1820), based on multi-locus sequence typing (MLST) analysis. A core genome SNP phylogeny demonstrated that all identified STs share a recent common ancestor that diverged an estimated 796-1260 years ago. A pan-genomics analysis demonstrated recombination between clades and intra-MLST phylogenetic and gene differences. To identify potential differential virulence between STs, groups of BALB/c mice (5 mice/isolate) were challenged via subcutaneous injection (500 CFUs) with 30 INT2 isolates representing 5 different STs; over the 21-day experiment, eight isolates killed all mice, 2 isolates killed an intermediate number of mice (1-2), and 20 isolates killed no mice. Although the virulence results were largely stratified by ST, one virulent isolate and six attenuated isolates were from the same ST (ST1005), suggesting that variably conserved genomic regions may contribute to virulence. Genomes from the animal-challenged isolates were subjected to a bacterial genome-wide association study to identify genomic regions associated with differential virulence. One associated region is a unique variant of Hcp1, a component of the type VI secretion system, which may result in attenuation. The results of this study have implications for comprehensive sampling strategies, environmental exposure risk assessment, and understanding recombination and differential virulence in B. pseudomallei.

Melioidosis in Thailand: Present and Future.

A recent modelling study estimated that there are 2800 deaths due to melioidosis in Thailand yearly. The Thailand Melioidosis Network (formed in 2012) has been working closely with the Ministry of Public Health (MoPH) to investigate and reduce the burden of this disease. Based on updated data, the incidence of melioidosis is still high in Northeast Thailand. More than 2000 culture-confirmed cases of melioidosis are diagnosed in general hospitals with microbiology laboratories in this region each year. The mortality rate is around 35%. Melioidosis is endemic throughout Thailand, but it is still not uncommon that microbiological facilities misidentify Burkholderia pseudomallei as a contaminant or another organism. Disease awareness is low, and people in rural areas neither wear boots nor boil water before drinking to protect themselves from acquiring B. pseudomallei. Previously, about 10 melioidosis deaths were formally reported to the National Notifiable Disease Surveillance System (Report 506) each year, thus limiting priority setting by the MoPH. In 2015, the formally reported number of melioidosis deaths rose to 112, solely because Sunpasithiprasong Hospital, Ubon Ratchathani province, reported its own data (n = 107). Melioidosis is truly an important cause of death in Thailand, and currently reported cases (Report 506) and cases diagnosed at research centers reflect the tip of the iceberg. Laboratory training and communication between clinicians and laboratory personnel are required to improve diagnosis and treatment of melioidosis countrywide. Implementation of rapid diagnostic tests, such as a lateral flow antigen detection assay, with high accuracy even in melioidosis-endemic countries such as Thailand, is critically needed. Reporting of all culture-confirmed melioidosis cases from every hospital with a microbiology laboratory, together with final outcome data, is mandated under the Communicable Diseases Act B.E.2558. By enforcing this legislation, the MoPH could raise the priority of this disease, and should consider implementing a campaign to raise awareness and melioidosis prevention countrywide.

Gastrointestinal tract involvement in melioidosis.

Background: Little is known about the involvement of the human gut in carriage and disease associated with Burkholderia pseudomallei, the cause of melioidosis. Methods: A hospital-based study was conducted in Northeast Thailand to culture stools or rectal swabs from patients with melioidosis, stools from controls with non-infectious diseases, and gastric biopsies from patients undergoing routine endoscopic investigation. Results and Conclusion: B. pseudomallei was isolated from 9/83 (11%) stools and 9/58 (16%) rectal swabs from 141 patients with melioidosis. All stools from 244 control patients and 799 gastric biopsies from 395 patients with no evidence of melioidosis were culture negative for B. pseudomallei. It is not uncommon for melioidosis patients to shed B. pseudomallei in stool. Colonization of the gut of individuals without signs and symptoms of melioidosis may be rare.

The Effects of Signal Erosion and Core Genome Reduction on the Identification of Diagnostic Markers.

UNLABELLED: Whole-genome sequence (WGS) data are commonly used to design diagnostic targets for the identification of bacterial pathogens. To do this effectively, genomics databases must be comprehensive to identify the strict core genome that is specific to the target pathogen. As additional genomes are analyzed, the core genome size is reduced and there is erosion of the target-specific regions due to commonality with related species, potentially resulting in the identification of false positives and/or false negatives. IMPORTANCE: A comparative analysis of 1,130 Burkholderia genomes identified unique markers for many named species, including the human pathogens B. pseudomallei and B. mallei Due to core genome reduction and signature erosion, only 38 targets specific to B. pseudomallei/mallei were identified. By using only public genomes, a larger number of markers were identified, due to undersampling, and this larger number represents the potential for false positives. This analysis has implications for the design of diagnostics for other species where the genomic space of the target and/or closely related species is not well defined.