Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics.

Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens.

Development of a Rapid Live SARS-CoV-2 Neutralization Assay Based on a qPCR Readout.

Measuring SARS-CoV-2 neutralizing antibodies after vaccination or natural infection remains a priority in the ongoing COVID-19 pandemic to determine immunity, especially against newly emerging variants. The gold standard for assessing antibody-mediated immunity against SARS-CoV-2 are cell-based live virus neutralization assays. These assays usually take several days, thereby limiting test capacities and the availability of rapid results. In this study, therefore, we developed a faster live virus assay, which detects neutralizing antibodies through the early measurement of antibody-mediated intracellular virus reduction by SARS-CoV-2 qRT-PCR. In our assay, Vero E6 cells are infected with virus isolates preincubated with patient sera and controls. After 24 h, the intracellular viral load is determined by qRT-PCR using a standard curve to calculate percent neutralization. Utilizing COVID-19 convalescent-phase sera, we show that our novel assay generates results with high sensitivity and specificity as we detected antiviral activity for all tested convalescent-phase sera, but no antiviral activity in prepandemic sera. The assay showed a strong correlation with a conventional virus neutralization assay (rS = 0.8910), a receptor-binding domain ELISA (rS = 0.8485), and a surrogate neutralization assay (rS = 0.8373), proving that quantifying intracellular viral RNA can be used to measure seroneutralization. Our assay can be adapted easily to new variants, as demonstrated by our cross-neutralization experiments. This characteristic is key for rapidly determining immunity against newly emerging variants. Taken together, the novel assay presented here reduces turnaround time significantly while making use of a highly standardized and sensitive SARS-CoV-2 qRT-PCR method as a readout.

Development and Validation of a Burkholderia pseudomallei Core Genome Multilocus Sequence Typing Scheme To Facilitate Molecular Surveillance.

Burkholderia pseudomallei causes the severe disease melioidosis. Whole-genome sequencing (WGS)-based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills, and variable bioinformatic approaches, with the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,351 accessory targets. The scheme was validated with 320 WGS data sets. We compared our novel typing scheme with single nucleotide polymorphism-based approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigating not only closely related strains but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugarcane field as the presumable source of a recent melioidosis case. In summary, we developed a robust cgMLST scheme that integrates high resolution, maximized standardization, and fast analysis for the nonbioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

Rational design of an acidic erythritol (ACER) medium for the enhanced isolation of the environmental pathogen Burkholderia pseudomallei from soil samples.

The soil bacterium Burkholderia pseudomallei causes melioidosis, a potentially fatal and greatly underdiagnosed tropical disease. Detection of B. pseudomallei in the environment is important to trace the source of infections, define risk areas for melioidosis and increase the clinical awareness. Although B. pseudomallei polymerase chain reaction (PCR)-based environmental detection provides important information, the culture of the pathogen remains essential but is still a methodological challenge. B. pseudomallei can catabolize erythritol, a metabolic pathway, which is otherwise rarely encountered among bacteria. We recently demonstrated that replacing threonine with erythritol as a single carbon source in the pH-neutral threonine-basal salt solution (TBSS-C50) historically used improved the isolation of B. pseudomallei from rice paddy soils. However, further culture medium parameters for an optimized recovery of B. pseudomallei strains from soils are still ill-defined. We, therefore, aimed to design a new erythritol-based medium by systematically optimizing parameters such as pH, buffer capacity, salt and nutrient composition. A key finding of our study is the enhanced erythritol-based growth of B. pseudomallei under acidic medium conditions. Our experiments with B. pseudomallei strains from different geographical origin led to the development of a phosphate-buffered acidic erythritol (ACER) medium with a pH of 6.3, higher erythritol concentration of 1.2%, supplemented vitamins and nitrate. This highly selective medium composition shortened the lag phase of B. pseudomallei cultures and greatly increased growth densities compared to TBSS-C50 and TBSS-C50-based erythritol medium. The ACER medium led to the highest enrichments of B. pseudomallei as determined from culture supernatants by quantitative PCR in a comparative validation with soil samples from the central part of Vietnam. Consequently, the median recovery of B. pseudomallei colony forming units on Ashdown's agar from ACER subcultures was 5.4 times higher compared to TBSS-C50-based erythritol medium (p = 0.005) and 30.7 times higher than TBSS-C50 (p < 0.001). In conclusion, our newly developed ACER medium significantly improves the isolation of viable B. pseudomallei from soils and, thereby, has the potential to reduce the rate of false-negative environmental cultures in melioidosis risk areas.

Environmental Factors Associated With Soil Prevalence of the Melioidosis Pathogen Burkholderia pseudomallei: A Longitudinal Seasonal Study From South West India.

Melioidosis is a seasonal infectious disease in tropical and subtropical areas caused by the soil bacterium Burkholderia pseudomallei. In many parts of the world, including South West India, most cases of human infections are reported during times of heavy rainfall, but the underlying causes of this phenomenon are not fully understood. India is among the countries with the highest predicted melioidosis burden globally, but there is very little information on the environmental distribution of B. pseudomallei and its determining factors. The present study aimed (i) to investigate the prevalence of B. pseudomallei in soil in South West India, (ii) determine geochemical factors associated with B. pseudomallei presence and (iii) look for potential seasonal patterns of B. pseudomallei soil abundance. Environmental samplings were performed in two regions during the monsoon and post-monsoon season and summer from July 2016 to November 2018. We applied direct quantitative real time PCR (qPCR) together with culture protocols to overcome the insufficient sensitivity of solely culture-based B. pseudomallei detection from soil. A total of 1,704 soil samples from 20 different agricultural sites were screened for the presence of B. pseudomallei. Direct qPCR detected B. pseudomallei in all 20 sites and in 30.2% (517/1,704) of all soil samples, whereas only two samples from two sites were culture-positive. B. pseudomallei DNA-positive samples were negatively associated with the concentration of iron, manganese and nitrogen in a binomial logistic regression model. The highest number of B. pseudomallei-positive samples (42.6%, p < 0.0001) and the highest B. pseudomallei loads in positive samples [median 4.45 × 103 genome equivalents (GE)/g, p < 0.0001] were observed during the monsoon season and eventually declined to 18.9% and a median of 1.47 × 103 GE/g in summer. In conclusion, our study from South West India shows a wide environmental distribution of B. pseudomallei, but also considerable differences in the abundance between sites and within single sites. Our results support the hypothesis that nutrient-depleted habitats promote the presence of B. pseudomallei. Most importantly, the highest B. pseudomallei abundance in soil is seen during the rainy season, when melioidosis cases occur.

Melioidosis DS rapid test: A standardized serological dipstick assay with increased sensitivity and reliability due to multiplex detection.

BACKGROUND: Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection. METHODS AND PRINCIPAL FINDINGS: The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97-100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens. CONCLUSIONS: Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts.

Erythritol as a single carbon source improves cultural isolation of Burkholderia pseudomallei from rice paddy soils.

BACKGROUND: Isolation of the soil bacterium Burkholderia pseudomallei from tropical environments is important to generate a global risk map for man and animals to acquire the infectious disease melioidosis. There is increasing evidence, that the currently recommended soil culture protocol using threonine-basal salt solution with colistin (TBSS-C50) for enrichment of B. pseudomallei and Ashdown agar for subsequent subculture lacks sensitivity. We therefore investigated, if the otherwise rarely encountered erythritol catabolism of B. pseudomallei might be exploited to improve isolation of this bacterium from soil. METHODOLOGY/PRINCIPAL FINDINGS: Based on TBSS-C50, we designed a new colistin-containing medium with erythritol as the single carbon source (EM). This medium was validated in various culture protocols by analyzing 80 soil samples from 16 different rice fields in Vietnam. B. pseudomallei enrichment was determined in all culture supernatants by a specific quantitative PCR (qPCR) targeting the type three secretion system 1. 51 out of 80 (63.8%) soil samples gave a positive qPCR signal in at least one of the culture conditions. We observed a significantly higher enrichment shown by lower median cycle threshold values for B. pseudomallei in a two-step culture with TBSS-C50 for 48 h followed by EM for 96h compared to single cultures in TBSS-C50 for either 48h or 144h (p<0.0001, respectively). Accordingly, B. pseudomallei could be isolated on Ashdown agar in 58.8% (30/51) of samples after subcultures from our novel two-step enrichment culture compared to only 9.8% (5/51) after standard enrichment with TBSS-C50 for 48h (p<0.0001) or 25.5% (13/51; p<0.01) after TBSS-C50 for 144h. CONCLUSIONS/SIGNIFICANCE: In the present study, we show that specific exploitation of B. pseudomallei metabolic capabilities in enrichment protocols leads to a significantly improved isolation rate of this pathogen from soil compared to established standard procedures. Our new culture method might help to facilitate the creation of environmental risk maps for melioidosis in the future.

Melioidosis in Vietnam: Recently Improved Recognition but still an Uncertain Disease Burden after Almost a Century of Reporting.

The first cases of human melioidosis were described in Vietnam in the 1920s, almost a century ago. It was in Vietnam in the thirties that the saprophytic nature of B. pseudomallei was first recognized. Although a significant number of French and U.S. soldiers acquired the disease during the Vietnam wars, indigenous cases in the Vietnamese population were only sporadically reported over many decades. After reunification in 1975, only two retrospective studies reported relatively small numbers of indigenous cases from single tertiary care hospitals located in the biggest cities in the South and the North, respectively. Studies from provincial hospitals throughout the country were missing until the Research Network on Melioidosis and Burkholderia pseudomallei (RENOMAB) project started in 2014. From then on seminars, workshops, and national scientific conferences on melioidosis have been conducted to raise awareness among physicians and clinical laboratory staff. This led to the recognition of a significant number of cases in at least 36 hospitals in 26 provinces and cities throughout Vietnam. Although a widespread distribution of melioidosis has now been documented, there are still challenges to understand the true epidemiology of the disease. Establishment of national guidelines for diagnosis, management, and reporting of the disease together with more investigations on animal melioidosis, genomic diversity of B. pseudomallei and its environmental distribution are required.

Melioidosis in Africa: Time to Uncover the True Disease Load.

Melioidosis is an often fatal infectious disease with a protean clinical spectrum, caused by the environmental bacterial pathogen Burkholderia pseudomallei. Although the disease has been reported from some African countries in the past, the present epidemiology of melioidosis in Africa is almost entirely unknown. Therefore, the common view that melioidosis is rare in Africa is not evidence-based. A recent study concludes that large parts of Africa are environmentally suitable for B. pseudomallei. Twenty-four African countries and three countries in the Middle East were predicted to be endemic, but no cases of melioidosis have been reported yet. In this study, we summarize the present fragmentary knowledge on human and animal melioidosis and environmental B. pseudomallei in Africa and the Middle East. We propose that systematic serological studies in man and animals together with environmental investigations on potential B. pseudomallei habitats are needed to identify risk areas for melioidosis. This information can subsequently be used to target raising clinical awareness and the implementation of simple laboratory algorithms for the isolation of B. pseudomallei from clinical specimens. B. pseudomallei was most likely transferred from Asia to the Americas via Africa, which is shown by phylogenetic analyses. More data on the virulence and genomic characteristics of African B. pseudomallei isolates will contribute to a better understanding of the global evolution of the pathogen and will also help to assess potential differences in disease prevalence and outcome.

Nitrogen fixation in distinct microbial niches within a chemoautotrophy-driven cave ecosystem.

Microbial sulfur and carbon cycles in ecosystems driven by chemoautotrophy-present at deep-sea hydrothermal vents, cold seeps and sulfidic caves-have been studied to some extent, yet little is known about nitrogen fixation in these systems. Using a comprehensive approach comprising of (15)N2 isotope labeling, acetylene reduction assay and nitrogenase gene expression analyses, we investigated nitrogen fixation in the sulfide-rich, chemoautotrophy-based Frasassi cave ecosystem (Italy). Nitrogen fixation was examined in three different microbial niches within the cave waters: (1) symbiotic bacterial community of Niphargus amphipods, (2) Beggiatoa-dominated biofilms, which occur at the sulfide-oxygen interface, and (3) sulfidic sediment. We found evidence for nitrogen fixation in all the three niches, and the nitrogenase gene (homologs of nifH) expression data clearly show niche differentiation of diazotrophic Proteobacteria within the water streams. The nifH transcript originated from the symbiotic community of Niphargus amphipods might belong to the Thiothrix ectosymbionts. Two abundantly expressed nifH genes in the Beggiatoa-dominated biofilms are closely related to those from Beggiatoa- and Desulfovibrio-related bacteria. These two diazotrophs were consistently found in Beggiatoa-dominated biofilms collected at various time points, thus illustrating species-specific associations of the diazotrophs in biofilm formation, and micron-scale niche partitioning of sulfur-oxidizing and sulfate-reducing bacteria driven by steep redox gradients within the biofilm. Finally, putative heterotrophs (Geobacter, Azoarcus and Desulfovibrio related) were the active diazotrophs in the sulfidic sediment. Our study is the first to shed light on nitrogen fixation in permanently dark caves and suggests that diazotrophy may be widespread in chemosynthetic communities.