Melioidosis in Patients with COVID-19 Exposed to Contaminated Tap Water, Thailand, 2021.

In September 2021, a total of 25 patients diagnosed with COVID-19 developed acute melioidosis after (median 7 days) admission to a COVID-19 field hospital in Thailand. Eight nonpotable tap water samples and 6 soil samples were culture-positive for Burkholderia pseudomallei. Genomic analysis suggested contaminated tap water as the likely cause of illness.

Effectiveness of Umonium38 against Burkholderia pseudomallei, Escherichia coli, Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus (MRSA).

AIMS: We investigated the antibacterial efficacy of Umonium38 and Virkon® against Burkholderia pseudomallei, Escherichia coli, Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus (MRSA) up to 14 days following treatment. METHODS AND RESULTS: Umonium38 was diluted to 0.5%, 1.0%, 1.5%, 2.0%, 2.5% and 3%, tested against the bacterial strains at various contact times (15 min to 24 h), and incubated for up to 14 days. A minimum concentration of 0.5% Umonium38 with a contact time of 15 min effectively killed approximately 108 CFU/ml of all four bacterial species. No growth was observed on agar plates from day 0 until day 14 for all six concentrations. The bacteria were also inactivated by a 30-minute treatment time using Virkon® 1% solution. CONCLUSIONS: Umonium38 effectively inactivates B. pseudomallei, E. coli, P. aeruginosa and MRSA at a concentration of ≥ 0.5% with a contact time of at least 15 min. The antimicrobial effect of Umonium38 remained for 14 days.

A multi-country study using MALDI-TOF mass spectrometry for rapid identification of Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei is the bacterial causative agent of melioidosis, a difficult disease to diagnose clinically with high mortality if not appropriately treated. Definitive diagnosis requires isolation and identification of the organism. With the increased adoption of MALDI-TOF MS for the identification of bacteria, we established a method for rapid identification of B. pseudomallei using the Vitek MS, a system that does not currently have B. pseudomallei in its in-vitro diagnostic database. RESULTS: A routine direct spotting method was employed to create spectra and SuperSpectra. An initial B. pseudomallei SuperSpectrum was created at Shoklo Malaria Research Unit (SMRU) from 17 reference isolates (46 spectra). When tested, this initial SMRU SuperSpectrum was able to identify 98.2 % (54/55) of Asian isolates, but just 46.7 % (35/75) of Australian isolates. Using spectra (430) from different reference and clinical isolates, two additional SMRU SuperSpectra were created. Using the combination of all SMRU SuperSpectra with seven existing SuperSpectra from Townsville, Australia 119 (100 %) Asian isolates and 31 (100 %) Australian isolates were correctly identified. In addition, no misidentifications were obtained when using these 11 SuperSpectra when tested with 34 isolates of other bacteria including the closely related species Burkholderia thailandensis and Burkholderia cepacia. CONCLUSIONS: This study has established a method for identification of B. pseudomallei using Vitek MS, and highlights the impact of geographical differences between strains for identification using this technique.

Soil Nutrient Depletion Is Associated with the Presence of Burkholderia pseudomallei.

Burkholderia pseudomallei is a soil-dwelling bacterium and the cause of melioidosis, which kills an estimated 89,000 people per year worldwide. Agricultural workers are at high risk of infection due to repeated exposure to the bacterium. Little is known about the soil physicochemical properties associated with the presence or absence of the organism. Here, we evaluated the soil physicochemical properties and presence of B. pseudomallei in 6,100 soil samples collected from 61 rice fields in Thailand. The presence of B. pseudomallei was negatively associated with the proportion of clay, proportion of moisture, level of salinity, percentage of organic matter, presence of cadmium, and nutrient levels (phosphorus, potassium, calcium, magnesium, and iron). The presence of B. pseudomallei was not associated with the level of soil acidity (P = 0.54). In a multivariable logistic regression model, the presence of B. pseudomallei was negatively associated with the percentage of organic matter (odds ratio [OR], 0.06; 95% confidence interval [CI], 0.01 to 0.47; P = 0.007), level of salinity (OR, 0.06; 95% CI, 0.01 to 0.74; P = 0.03), and percentage of soil moisture (OR, 0.81; 95% CI, 0.66 to 1.00; P = 0.05). Our study suggests that B. pseudomallei thrives in rice fields that are nutrient depleted. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected areas. IMPORTANCE: Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis. Humans acquire the disease following skin inoculation, inhalation, or ingestion of the bacterium in the environment. The presence of B. pseudomallei in soil defines geographic regions where humans and livestock are at risk of melioidosis, yet little is known about the soil properties associated with the presence of the organism. We evaluated the soil properties and presence of B. pseudomallei in 61 rice fields in East, Central, and Northeast Thailand. We demonstrated that the organism was more commonly found in soils with lower levels of organic matter and nutrients, including phosphorus, potassium, calcium, magnesium, and iron. We also demonstrated that crop residue burning after harvest, which can reduce soil nutrients, was not uncommon. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected areas.

Clinical and molecular epidemiology of Staphylococcus argenteus infections in Thailand.

Molecular typing of 246 Staphylococcus aureus isolates from unselected patients in Thailand showed that 10 (4.1%) were actually Staphylococcus argenteus. Contrary to the suggestion that S. argenteus is less virulent than S. aureus, we demonstrated comparable rates of morbidity, death, and health care-associated infection in patients infected with either of these two species.

Maintenance of leptospira species in leptospira Vanaporn Wuthiekanun agar.

The maintenance of Leptospira species in liquid or semisolid medium is time-consuming and at risk of contamination due to the needs of routine subculture and dark field microscopy. Using Leptospira Vanaporn Wuthiekanun (LVW) agar, we maintained 100 pathogenic Leptospira isolates for 12 months without the need for subculture and confirmed the viability of all isolates by the naked eye.

Microevolution of Burkholderia pseudomallei during an acute infection.

We used whole-genome sequencing to evaluate 69 independent colonies of Burkholderia pseudomallei isolated from seven body sites of a patient with acute disseminated melioidosis. Fourteen closely related genotypes were found, providing evidence for the rapid in vivo diversification of B. pseudomallei after inoculation and systemic spread.

Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei.

Known mechanisms of resistance to ß-lactam antibiotics include ß-lactamase expression, altered drug target, decreased bacterial permeability, and increased drug efflux. Here, we describe a unique mechanism of ß-lactam resistance in the biothreat organism Burkholderia pseudomallei (the cause of melioidosis), associated with treatment failure during prolonged ceftazidime therapy of natural infection. Detailed comparisons of the initial ceftazidime-susceptible infecting isolate and subsequent ceftazidime-resistant variants from six patients led us to identify a common, large-scale genomic loss involving a minimum of 49 genes in all six resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a penicillin-binding protein 3 (BPSS1219) present within the region of genomic loss. The clinical ceftazidime-resistant variants failed to grow using commonly used laboratory culture media, including commercial blood cultures, rendering the variants almost undetectable in the diagnostic laboratory. Melioidosis is notoriously difficult to cure and clinical treatment failure is common in patients treated with ceftazidime, the drug of first choice across most of Southeast Asia where the majority of cases are reported. The mechanism described here represents an explanation for ceftazidime treatment failure, and may be a frequent but undetected resistance event.

Rapid isolation and susceptibility testing of Leptospira spp. using a new solid medium, LVW agar.

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO(2) incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO(2) for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC(90) values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 µg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

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.

Sensitivity and specificity of DPP® Fever Panel II Asia in the diagnosis of malaria, dengue and melioidosis.

Introduction. Rapid diagnostic tests (RDTs) that can facilitate the diagnosis of a panel of tropical infectious diseases are critically needed. DPP® Fever Panel II Asia is a multiplex lateral flow immunoassay comprising antigen and IgM panels for the diagnosis of pathogens that commonly cause febrile illness in Southeast Asia.Hypothesis/Gap Statement. Accuracy of DPP® Fever Panel II Asia has not been evaluated in clinical studies.Aim. To evaluate the sensitivity and specificity of DPP® Fever Panel II Asia for malaria, dengue and melioidosis.Methodology. We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon sepsis). We included 143 and 98 patients diagnosed with malaria or dengue based on a positive PCR assay and 177 patients with melioidosis based on a culture positive for Burkholderia pseudomallei. Controls included 200 patients who were blood culture-positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae, and cases of the other diseases. Serum samples collected from all patients within 24 h of admission were stored and tested using the DPP® Fever Panel II Asia antigen and IgM multiplex assays. We selected cutoff values for each individual assay corresponding to a specificity of ≥95 %. When assessing diagnostic tests in combination, results were considered positive if either individual test was positive.Results. Within the DPP® Fever Panel II Asia antigen assay, a combination of pLDH and HRPII for malaria had a sensitivity of 91 % and a specificity of 97 %. The combination of dengue NS1 antigen and dengue antibody tests had a sensitivity of 61 % and a specificity of 91 %. The B. pseudomallei CPS antigen test had a sensitivity of 27 % and a specificity of 97 %. An odds ratio of 2.34 (95 % CI 1.16-4.72, P=0.02) was observed for the association between CPS positivity and mortality among melioidosis patients.Conclusion. The performance of the DPP® Fever Panel II Asia for diagnosis of malaria was high and that for dengue and melioidosis was relatively limited. For all three diseases, performance was comparable to that of other established RDTs. The potential operational advantages of a multiplex and quantitative point-of-care assay are substantial and warrant further investigation.

Survey of antimicrobial resistance in clinical Burkholderia pseudomallei isolates over two decades in Northeast Thailand.

A 21-year survey conducted in northeast Thailand of antimicrobial resistance to parenteral antimicrobial drugs used to treat melioidosis identified 24/4,021 (0.6%) patients with one or more isolates resistant to ceftazidime (n = 8), amoxicillin-clavulanic acid (n = 4), or both drugs (n = 12). Two cases were identified at admission, and the remainder were detected a median of 15 days after starting antimicrobial therapy. Resistance to carbapenem drugs was not detected. These findings support the current prescribing recommendations for melioidosis.

Randomized soil survey of the distribution of Burkholderia pseudomallei in rice fields in Laos.

Melioidosis is a major cause of morbidity and mortality in Southeast Asia, where the causative organism (Burkholderia pseudomallei) is present in the soil. In the Lao People's Democratic Republic (Laos), B. pseudomallei is a significant cause of sepsis around the capital, Vientiane, and has been isolated in soil near the city, adjacent to the Mekong River. We explored whether B. pseudomallei occurs in Lao soil distant from the Mekong River, drawing three axes across northwest, northeast, and southern Laos to create nine sampling areas in six provinces. Within each sampling area, a random rice field site containing a grid of 100 sampling points each 5 m apart was selected. Soil was obtained from a depth of 30 cm and cultured for B. pseudomallei. Four of nine sites (44%) were positive for B. pseudomallei, including all three sites in Saravane Province, southern Laos. The highest isolation frequency was in east Saravane, where 94% of soil samples were B. pseudomallei positive with a geometric mean concentration of 464 CFU/g soil (95% confidence interval, 372 to 579 CFU/g soil; range, 25 to 10,850 CFU/g soil). At one site in northwest Laos (Luangnamtha), only one sample (1%) was positive for B. pseudomallei, at a concentration of 80 CFU/g soil. Therefore, B. pseudomallei occurs in Lao soils beyond the immediate vicinity of the Mekong River, alerting physicians to the likelihood of melioidosis in these areas. Further studies are needed to investigate potential climatic, soil, and biological determinants of this heterogeneity.

Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis.

BACKGROUND: Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium. METHODS: We evaluated our hypothesis during a prospective study of 418 consecutive patients presenting to a hospital in northeast Thailand with an acute febrile illness. Admission blood samples were taken for Leptospira culture and PCR. A single tube nested PCR that amplified a region of the rrs gene was developed and applied, amplicons sequenced and a phylogenetic tree reconstructed. RESULTS: 39/418 (9%) patients were culture-positive for Leptospira spp., and 81/418 (19%) patients were culture-negative but rrs PCR-positive. The species associated with culture-positive leptospirosis (37 L. interrogans and 2 L. borgpetersenii) were comparable to those associated with culture-negative, PCR-positive leptospirosis (76 L. interrogans, 4 L. borgpetersenii, 1 unidentified, possibly new species). CONCLUSION: Molecular speciation failed to identify a unique bacterial subset in patients with culture-negative, PCR-positive leptospirosis. The rate of false-negative culture was high, and we speculate that antibiotic pre-treatment is the most likely explanation for this.

Detection and differentiation of Burkholderia species with pathogenic potential in environmental soil samples.

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil.

Evaluation of antigen-detecting and antibody-detecting diagnostic test combinations for diagnosing melioidosis.

BACKGROUND: Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in many tropical developing countries and has a high mortality. Here we evaluated combinations of a lateral flow immunoassay (LFI) detecting B. pseudomallei capsular polysaccharide (CPS) and enzyme-linked immunosorbent assays (ELISA) detecting antibodies against hemolysin co-regulated protein (Hcp1) or O-polysaccharide (OPS) for diagnosing melioidosis. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon-sepsis). Cases included 192 patients with a clinical specimen culture positive for B. pseudomallei. Controls included 502 patients who were blood culture positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae or were polymerase chain reaction assay positive for malaria or dengue. Serum samples collected within 24 hours of admission were stored and tested using a CPS-LFI, Hcp1-ELISA and OPS-ELISA. When assessing diagnostic tests in combination, results were considered positive if either test was positive. We selected ELISA cut-offs corresponding to a specificity of 95%. Using a positive cut-off OD of 2.912 for Hcp1-ELISA, the combination of the CPS-LFI and Hcp1-ELISA had a sensitivity of 67.7% (130/192 case patients) and a specificity of 95.0% (477/502 control patients). The sensitivity of the combination (67.7%) was higher than that of the CPS-LFI alone (31.3%, p<0.001) and that of Hcp1-ELISA alone (53.6%, p<0.001). A similar phenomenon was also observed for the combination of CPS-LFI and OPS-ELISA. In case patients, positivity of the CPS-LFI was associated with a short duration of symptoms, high modified Sequential (sepsis-related) Organ Failure Assessment (SOFA) score, bacteraemia and mortality outcome, while positivity of Hcp1-ELISA was associated with a longer duration of symptoms, low modified SOFA score, non-bacteraemia and survival outcome. CONCLUSIONS/SIGNIFICANCE: A combination of antigen-antibody diagnostic tests increased the sensitivity of melioidosis diagnosis over individual tests while preserving high specificity. Point-of-care tests for melioidosis based on the use of combination assays should be further developed and evaluated.

Survival of Burkholderia pseudomallei and Pathogenic Leptospira in Cola, Beer, Energy Drinks, and Sports Drinks.

Burkholderia pseudomallei and pathogenic Leptospira in contaminated drinking water can cause melioidosis and leptospirosis, respectively. Here, we evaluated their survival in beverages. We mixed six isolates (three isolates per organism) in four beverages (Coca-Cola®, Red Bull®, Singha® beer, and Gatorade®) and distilled water as the control at two final concentrations (1 × 107 colony-forming units [CFU]/mL and 1 × 103 CFU/mL). The solution was kept at two temperatures (37°C and 4°C). At 4°C and at the high concentration, pathogenic Leptospira survived in Coca-Cola® up to 3 minutes and in Singha, Red Bull®, and Gatorade up to 15 minutes, whereas B. pseudomallei survived in these beverages up to 8 hours, and 14, 14, and 28 days, respectively. The survival time of both organisms was shorter at 37°C (P = 0.01) and at the lower concentration (P = 0.001). In conclusion, Leptospira can survive in some beverages for up to 15 minutes, whereas B. pseudomallei can survive in some beverages for up to 4 weeks.

Dosing regimens of cotrimoxazole (trimethoprim-sulfamethoxazole) for melioidosis.

Melioidosis is an infectious disease with a propensity for relapse, despite prolonged antibiotic eradication therapy for 12 to 20 weeks. A pharmacokinetic (PK) simulation study was performed to determine the optimal dosing of cotrimoxazole (trimethoprim-sulfamethoxazole [TMP-SMX]) used in current eradication regimens in Thailand and Australia. Data for bioavailability, protein binding, and coefficients of absorption and elimination were taken from published literature. Apparent volumes of distribution were correlated with body mass and were estimated separately for Thai and Australian populations. In vitro experiments demonstrated concentration-dependent killing. In Australia, the currently used eradication regimen (320 [TMP]/1,600 [SMX] mg every 12 h [q12h]) was predicted to achieve the PK-pharmacodynamic (PD) target (an area under the concentration-time curve from 0 to 24 h/MIC ratio of >25 for both TMP and SMX) for strains with the MIC90 of Australian strains (< or = 1/19 mg/liter). In Thailand, the former regimen of 160/800 mg q12h would not be expected to attain the target for strains with an MIC of > or = 1/19 mg/liter, but the recently implemented weight-based regimen (<40 kg [body weight], 160/800 mg q12h; 40 to 60 kg, 240/1,200 mg q12h; >60 kg, 320/1,600 mg q12h) would be expected to achieve adequate concentrations for strains with an MIC of < or = 1/19 mg/liter. The results were sensitive to the variance of the PK parameters. Prospective PK-PD studies of Asian populations are needed to optimize TMP-SMX dosing in melioidosis.