T-Cell Responses Are Associated with Survival in Acute Melioidosis Patients

Background

Melioidosis is an increasingly recognised cause of sepsis and death across South East Asia and Northern Australia, caused by the bacterium Burkholderia pseudomallei. Risk factors include diabetes, alcoholism and renal disease, and a vaccine targeting at-risk populations is urgently required. A better understanding of the protective immune response in naturally infected patients is essential for vaccine design.

Methods

We conducted a longitudinal clinical and immunological study of 200 patients with melioidosis on admission, 12 weeks (n = 113) and 52 weeks (n = 65) later. Responses to whole killed B. pseudomallei were measured in peripheral blood mononuclear cells (PBMC) by interferon-gamma (IFN-γ) ELIspot assay and flow cytometry and compared to those of control subjects in the region with diabetes (n = 45) and without diabetes (n = 43).

Results

We demonstrated strong CD4+ and CD8+ responses to B. pseudomallei during acute disease, 12 weeks and 52 weeks later. 28-day mortality was 26% for melioidosis patients, and B. pseudomallei-specific cellular responses in fatal cases (mean 98 IFN-γ cells per million PBMC) were significantly lower than those in the survivors (mean 142 IFN-γ cells per million PBMC) in a multivariable logistic regression model (P = 0.01). A J-shaped curve association between circulating neutrophil count and mortality was seen with an optimal count of 4000 to 8000 neutrophils/μl.Melioidosis patients with known diabetes had poor diabetic control (median glycated haemoglobin HbA_1c 10.2%, interquartile range 9.2–13.1) and showed a stunted B. pseudomallei-specific cellular response during acute illness compared to those without diabetes.

Conclusions

The results demonstrate the role of both CD4+ and CD8+ T-cells in protection against melioidosis, and an interaction between diabetes and cellular responses. This supports development of vaccine strategies that induce strong T-cell responses for the control of intracellular pathogens such as B. pseudomallei.     

Geographical distribution of Burkholderia pseudomallei in soil in Myanmar

BackgroundBurkholderia pseudomallei is a Gram-negative bacterium found in soil and water in many tropical countries. It causes melioidosis, a potentially fatal infection first described in 1911 in Myanmar. Melioidosis is a common cause of sepsis and death in South and South-east Asia, but it is rarely diagnosed in Myanmar. We conducted a nationwide soil study to identify areas where B. pseudomallei is present.Methodology/Principal findingsWe collected soil samples from 387 locations in all 15 states and regions of Myanmar between September 2017 and June 2019. At each site, three samples were taken at each of three different depths (30, 60 and 90 cm) and were cultured for B. pseudomallei separately, along with a pooled sample from each site (i.e. 10 cultures per site). We used a negative binomial regression model to assess associations between isolation of B. pseudomallei and environmental factors (season, soil depth, soil type, land use and climate zones). B. pseudomallei was isolated in 7 of 15 states and regions. Of the 387 sites, 31 (8%) had one or more positive samples and of the 3,870 samples cultured, 103 (2.7%) tested positive for B. pseudomallei. B. pseudomallei was isolated more frequently during the monsoon season [RR-2.28 (95% CI: 0.70–7.38)] and less in the hot dry season [RR-0.70 (95% CI: 0.19–2.56)] compared to the cool dry season, and in the tropical monsoon climate zone [RR-2.26; 95% CI (0.21–6.21)] compared to the tropical dry winter climate zone. However, these associations were not statistically significant. B. pseudomallei was detected at all three depths and from various soil types (clay, silt and sand). Isolation was higher in agricultural land (2.2%), pasture land (8.5%) and disused land (5.8%) than in residential land (0.4%), but these differences were also not significant.Conclusion/SignificanceThis study confirms a widespread distribution of B. pseudomallei in Myanmar. Clinical studies should follow to obtain a better picture of the burden of melioidosis in Myanmar.     

Comparative virulence of three different strains of Burkholderia pseudomallei in an aerosol non-human primate model

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. B. pseudomallei is a potential bioterrorism agent due to its high infectivity, especially via inhalation, and its inherent resistance to antimicrobials. There is currently no vaccine for melioidosis and antibiotic treatment can fail due to innate drug resistance, delayed diagnosis and treatment, or insufficient duration of treatment. A well-characterized animal model that mimics human melioidosis is needed for the development of new medical countermeasures. This study first characterized the disease progression of melioidosis in the African green monkey (AGM) and rhesus macaque (RM) for non-human primate model down-selection. All AGMs developed acute lethal disease similar to that described in human acute infection following exposure to aerosolized B. pseudomallei strain HBPUB10134a. Only 20% of RMs succumbed to acute disease. Disease progression, immune response and pathology of two other strains of B. pseudomallei, K96243 and MSHR5855, were also compared using AGMs. These three B. pseudomallei strains represent a highly virulent strain from Thailand (HBPUB101034a), a highly virulent strains from Australia (MSHR5855), and a commonly used laboratory strains originating from Thailand (K96243). Animals were observed for clinical signs of infection and blood samples were analyzed for cytokine responses, blood chemistry and leukocyte changes in order to characterize bacterial infection. AGMs experienced fever after exposure to aerosolized B. pseudomallei at the onset of acute disease. Inflammation, abscesses and/or pyogranulomas were observed in lung with all three strains of B. pseudomallei. Inflammation, abscesses and/or pyogranulomas were observed in lymph nodes, spleen, liver and/or kidney with B. pseudomallei, HBPUB10134a and K96243. Additionally, the Australian strain MSHR5855 induced brain lesions in one AGM similar to clinical cases of melioidosis seen in Australia. Elevated serum levels of IL-1β, IL-1 receptor antagonist, IL-6, MCP-1, G-CSF, HGF, IFNγ, MIG, I-TAC, and MIP-1β at terminal end points can be significantly correlated with non-survivors with B. pseudomallei infection in AGM. The AGM model represents an acute model of B. pseudomallei infection for all three strains from two geographical locations and will be useful for efficacy testing of vaccines and therapeutics against melioidosis. In summary, a dysregulated immune response leading to excessive persistent inflammation and inflammatory cell death is the key driver of acute melioidosis. Early intervention in these pathways will be necessary to counter B. pseudomallei and mitigate the pathological consequences of melioidosis.     

Activities of Daily Living Associated with Acquisition of Melioidosis in Northeast Thailand: A Matched Case-Control Study

Background

Melioidosis is a serious infectious disease caused by the Category B select agent and environmental saprophyte, Burkholderia pseudomallei. Most cases of naturally acquired infection are assumed to result from skin inoculation after exposure to soil or water. The aim of this study was to provide evidence for inoculation, inhalation and ingestion as routes of infection, and develop preventive guidelines based on this evidence.

Methods/Principal Findings

A prospective hospital-based 1∶2 matched case-control study was conducted in Northeast Thailand. Cases were patients with culture-confirmed melioidosis, and controls were patients admitted with non-infectious conditions during the same period, matched for gender, age, and diabetes mellitus. Activities of daily living were recorded for the 30-day period before onset of symptoms, and home visits were performed to obtain drinking water and culture this for B. pseudomallei. Multivariable conditional logistic regression analysis based on 286 cases and 512 controls showed that activities associated with a risk of melioidosis included working in a rice field (conditional odds ratio [cOR] = 2.1; 95% confidence interval [CI] 1.4–3.3), other activities associated with exposure to soil or water (cOR = 1.4; 95%CI 0.8–2.6), an open wound (cOR = 2.0; 95%CI 1.2–3.3), eating food contaminated with soil or dust (cOR = 1.5; 95%CI 1.0–2.2), drinking untreated water (cOR = 1.7; 95%CI 1.1–2.6), outdoor exposure to rain (cOR = 2.1; 95%CI 1.4–3.2), water inhalation (cOR = 2.4; 95%CI 1.5–3.9), current smoking (cOR = 1.5; 95%CI 1.0–2.3) and steroid intake (cOR = 3.1; 95%CI 1.4–6.9). B. pseudomallei was detected in water source(s) consumed by 7% of cases and 3% of controls (cOR = 2.2; 95%CI 0.8–5.8).

Conclusions/Significance

We used these findings to develop the first evidence-based guidelines for the prevention of melioidosis. These are suitable for people in melioidosis-endemic areas, travelers and military personnel. Public health campaigns based on our recommendations are under development in Thailand.     

Glyburide Reduces Bacterial Dissemination in a Mouse Model of Melioidosis

Background

Burkholderia pseudomallei infection (melioidosis) is an important cause of community-acquired Gram-negative sepsis in Northeast Thailand, where it is associated with a ∼40% mortality rate despite antimicrobial chemotherapy. We showed in a previous cohort study that patients taking glyburide ( = glibenclamide) prior to admission have lower mortality and attenuated inflammatory responses compared to patients not taking glyburide. We sought to define the mechanism underlying this observation in a murine model of melioidosis.

Methods

Mice (C57BL/6) with streptozocin-induced diabetes were inoculated with ∼6×10² cfu B. pseudomallei intranasally, then treated with therapeutic ceftazidime (600 mg/kg intraperitoneally twice daily starting 24 h after inoculation) in order to mimic the clinical scenario. Glyburide (50 mg/kg) or vehicle was started 7 d before inoculation and continued until sacrifice. The minimum inhibitory concentration of glyburide for B. pseudomallei was determined by broth microdilution. We also examined the effect of glyburide on interleukin (IL) 1β by bone-marrow-derived macrophages (BMDM).

Results

Diabetic mice had increased susceptibility to melioidosis, with increased bacterial dissemination but no effect was seen of diabetes on inflammation compared to non-diabetic controls. Glyburide treatment did not affect glucose levels but was associated with reduced pulmonary cellular influx, reduced bacterial dissemination to both liver and spleen and reduced IL1β production when compared to untreated controls. Other cytokines were not different in glyburide-treated animals. There was no direct effect of glyburide on B. pseudomallei growth in vitro or in vivo. Glyburide directly reduced the secretion of IL1β by BMDMs in a dose-dependent fashion.

Conclusions

Diabetes increases the susceptibility to melioidosis. We further show, for the first time in any model of sepsis, that glyburide acts as an anti-inflammatory agent by reducing IL1β secretion accompanied by diminished cellular influx and reduced bacterial dissemination to distant organs. We found no evidence for a direct effect of glyburide on the bacterium.     

Expression and Function of Macrophage Migration Inhibitory Factor (MIF) in Melioidosis

Background

Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast-Asia. We aimed to characterize the expression and function of MIF in melioidosis.

Methodology and Principal Findings

MIF expression was determined in leukocytes and plasma from 34 melioidosis patients and 32 controls, and in mice infected with B. pseudomallei. MIF function was investigated in experimental murine melioidosis using anti-MIF antibodies and recombinant MIF. Patients demonstrated markedly increased MIF mRNA leukocyte and MIF plasma concentrations. Elevated MIF concentrations were associated with mortality. Mice inoculated intranasally with B. pseudomallei displayed a robust increase in pulmonary and systemic MIF expression. Anti-MIF treated mice showed lower bacterial loads in their lungs upon infection with a low inoculum. Conversely, mice treated with recombinant MIF displayed a modestly impaired clearance of B. pseudomallei. MIF exerted no direct effects on bacterial outgrowth or phagocytosis of B. pseudomallei.

Conclusions

MIF concentrations are markedly elevated during clinical melioidosis and correlate with patients'' outcomes. In experimental melioidosis MIF impaired antibacterial defense.     

Landscape Changes Influence the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Soil in Northern Australia

Background

The soil-dwelling saprophyte bacterium Burkholderia pseudomallei is the cause of melioidosis, a severe disease of humans and animals in southeast Asia and northern Australia. Despite the detection of B. pseudomallei in various soil and water samples from endemic areas, the environmental habitat of B. pseudomallei remains unclear.

Methodology/Principal Findings

We performed a large survey in the Darwin area in tropical Australia and screened 809 soil samples for the presence of these bacteria. B. pseudomallei were detected by using a recently developed and validated protocol involving soil DNA extraction and real-time PCR targeting the B. pseudomallei–specific Type III Secretion System TTS1 gene cluster. Statistical analyses such as multivariable cluster logistic regression and principal component analysis were performed to assess the association of B. pseudomallei with environmental factors. The combination of factors describing the habitat of B. pseudomallei differed between undisturbed sites and environmentally manipulated areas. At undisturbed sites, the occurrence of B. pseudomallei was found to be significantly associated with areas rich in grasses, whereas at environmentally disturbed sites, B. pseudomallei was associated with the presence of livestock animals, lower soil pH and different combinations of soil texture and colour.

Conclusions/Significance

This study contributes to the elucidation of environmental factors influencing the occurrence of B. pseudomallei and raises concerns that B. pseudomallei may spread due to changes in land use.     

Can We Use Antibodies to Chlamydia trachomatis as a Surveillance Tool for National Trachoma Control Programs? Results from a District Survey

Background

Trachoma is targeted for elimination by 2020. World Health Organization advises districts to undertake surveillance when follicular trachoma (TF) <5% in children 1–9 years and mass antibiotic administration has ceased. There is a question if other tools could be used for surveillance as well. We report data from a test for antibodies to C. trachomatis antigen pgp3 as a possible tool.

Methodology

We randomly sampled 30 hamlets in Kilosa district, Tanzania, and randomly selected 50 children ages 1–9 per hamlet. The tarsal conjunctivae were graded for trachoma (TF), tested for C. trachomatis infection (Aptima Combo2 assay: Hologic, San Diego, CA), and a dried blood spot processed for antibodies to C. trachomatis pgp3 using a multiplex bead assay on a Luminex 100 platform.

Principal findings

The prevalence of trachoma (TF) was 0.4%, well below the <5% indicator for re-starting a program. Infection was also low, 1.1%. Of the 30 hamlets, 22 had neither infection nor TF. Antibody positivity overall was low, 7.5% and increased with age from 5.2% in 1–3 year olds, to 9.3% in 7–9 year olds (p = 0.015). In 16 of the 30 hamlets, no children ages 1–3 years had antibodies to pgp3.

Conclusions

The antibody status of the 1–3 year olds indicates low cumulative exposure to infection during the surveillance period. Four years post MDA, there is no evidence for re-emergence of follicular trachoma.     

Malaria Prevalence in Endemic Districts of Bangladesh

Background

Following the 1971 ban of DDT in Bangladesh, malaria cases have increased steadily. Malaria persists as a major health problem in the thirteen south-eastern and north-eastern districts of Bangladesh. At present the national malaria control program, largely supported by the Global Fund for AIDS, Tuberculosis and Malaria (GFATM), provides interventions including advocacy at community level, Insecticide Treated Net (ITN) distribution, introduction of Rapid Diagnostic Tests (RDT) and combination therapy with Coartem. It is imperative, therefore, that baseline data on malaria prevalence and other malaria indicators are collected to assess the effectiveness of the interventions and rationalize the prevention and control efforts. The objective of this study was to obtain this baseline on the prevalence of malaria and bed net use in the thirteen malaria endemic districts of Bangladesh.

Methods and Principal Findings

In 2007, BRAC and ICDDR,B carried out a malaria prevalence survey in thirteen malaria endemic districts of Bangladesh. A multi-stage cluster sampling technique was used and 9750 blood samples were collected. Rapid Diagnostic Tests (RDT) were used for the diagnosis of malaria. The weighted average malaria prevalence in the thirteen endemic districts was 3.97%. In five south-eastern districts weighted average malaria prevalence rate was 6.00% and in the eight north-eastern districts weighted average malaria prevalence rate was (0.40%). The highest malaria prevalence was observed in Khagrachari district. The majority of the cases (90.18%) were P. falciparum infections. Malaria morbidity rates in five south-eastern districts was 2.94%. In eight north-eastern districts, morbidity was 0.07%.

Conclusion and Significance

Bangladesh has hypoendemic malaria with P. falciparum the dominant parasite species. The malaria situation in the five north-eastern districts of Bangladesh in particular warrants urgent attention. Detailed maps of the baseline malaria prevalence and summaries of the data collected are provided along with the survey results in full, in a supplemental information     

Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei

Background

Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling.

Methods/Principal Findings

An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP)) was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011) was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected to occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provision of affordable and ‘low-tech’ methodology that is applicable in both developed and developing countries.

Conclusions/Significance

The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global map of environmental B. pseudomallei.     

Public Awareness of Melioidosis in Thailand and Potential Use of Video Clips as Educational Tools

Background

Melioidosis causes more than 1,000 deaths in Thailand each year. Infection occurs via inoculation, ingestion or inhalation of the causative organism (Burkholderia pseuodmallei) present in soil and water. Here, we evaluated public awareness of melioidosis using a combination of population-based questionnaire, a public engagement campaign to obtain video clips made by the public, and viewpoints on these video clips as potential educational tools about the disease and its prevention.

Methods

A questionnaire was developed to evaluate public awareness of melioidosis, and knowledge about its prevention. From 1 March to 31 April 2012, the questionnaire was delivered to five randomly selected adults in each of 928 districts in Thailand. A video clip contest entitled “Melioidosis, an infectious disease that Thais must know” was run between May and October 2012. The best 12 video clips judged by a contest committee were shown to 71 people at risk from melioidosis (diabetics). Focus group interviews were used to evaluate their perceptions of the video clips.

Results

Of 4,203 Thais who completed our study questionnaire, 74% had never heard of melioidosis, and 19% had heard of the disease but had no further knowledge. Most participants in all focus group sessions felt that video clips were beneficial and could positively influence them to increase adherence to recommended preventive behaviours, including drinking boiled water and wearing protective gear if in contact with soil or environmental water. Participants suggested that video clips should be presented in the local dialect with simple words rather than medical terms, in a serious manner, with a doctor as the one presenting the facts, and having detailed pictures of each recommended prevention method.

Conclusions

In summary, public awareness of melioidosis in Thailand is very low, and video clips could serve as a useful medium to educate people and promote disease prevention.

Presented in Part

World Melioidosis Congress 2013, Bangkok, Thailand, 18–20 September 2013 (abstract OS VII-04).     

Variation in Bordetella pertussis Susceptibility to Erythromycin and Virulence-Related Genotype Changes in China (1970-2014)

Objectives

To investigate changes in virulence-related genotypes and in the antimicrobial susceptibility of Bordetella pertussis isolates collected from the 1970s to 2014 in the northern part of China.

Methods

A total of 124 B. pertussis isolates from three periods, the 1970s, 2000–2008, and May 2013–Sept 2014, were typed by multilocus sequence typing (MLST) and tested for antimicrobial susceptibility and virulence-related genes. A fragment of the 23S rRNA gene from each of the 99 isolates from 2013–2014 was amplified and sequenced.

Results

All isolates from 2000–2008 and 2013–2014 were identified as ST2, whereas isolates from the 1970s were ST1. PtxA2/ptxC1/ptxP1/prn1/fim2-1/fim3-1/tcfA2, which was the same as the vaccine strain, was the only type in the 1970s. During the 2000s and 2013–2014, the virulence type ptxA1/ptxC1/ptxP1/prn1/fim2-1/fim3-1/tcfA2 was dominant, with frequencies of 68.4% and 91.9%, respectively. Nine ptxP3 strains, which were more virulent, were detected after 2000. All 124 isolates were susceptible to levofloxacin, sulphamethoxazole/trimethoprim and tetracycline. The isolates from the 1970s and 2000–2008 were susceptible to all tested macrolides, whereas 91.9% of the 2013–2014 isolates were highly resistant (minimal inhibitory concentration, MIC >256 μg/ml). No ptxP3 strain was resistant to macrolides. All erythromycin-resistant strains except for one had the A2047G mutation in the 23S rRNA gene.

Conclusions

Macrolide resistance of the B. pertussis population has been a serious problem in the northern part of China. Because most of the epidemic clone of the pathogen expresses the same antigen profiles as the vaccine strain, except ptxA, improvements in immunization strategies may prevent the spread of infection and drug resistance.     

Induction of Mouse Melioidosis with Meningitis by CD11b+ Phagocytic Cells Harboring Intracellular B. pseudomallei as a Trojan Horse

Background

Approximately 3–5% of patients with melioidosis manifest CNS symptoms; however, the clinical data regarding neurological melioidosis are limited.

Methods and Findings

We established a mouse model of melioidosis with meningitis characterized by neutrophil infiltration into the meninges histologically and B. pseudomallei in the cerebrospinal fluid (CSF) by bacteriological culturing methods. As the disease progresses, the bacteria successively colonize the spleen, liver, bone marrow (BM) and brain and invade splenic and BM cells by days 2 and 6 post-infection, respectively. The predominant cell types intracellularly infected with B. pseudomallei were splenic and BM CD11b⁺ populations. The CD11b⁺Ly6C^high inflamed monocytes, CD11b⁺Ly6C^low resident monocytes, CD11b⁺Ly6G⁺ neutrophils, CD11b⁺F4/80⁺ macrophages and CD11b⁺CD19⁺ B cells were expanded in the spleen and BM during the progression of melioidosis. After adoptive transfer of CD11b populations harboring B. pseudomallei, the infected CD11b⁺ cells induced bacterial colonization in the brain, whereas CD11b⁻ cells only partially induced colonization; extracellular (free) B. pseudomallei were unable to colonize the brain. CD62L (selectin) was absent on splenic CD11b⁺ cells on day 4 but was expressed on day 10 post-infection. Adoptive transfer of CD11b⁺ cells expressing CD62L (harvested on day 10 post-infection) resulted in meningitis in the recipients, but transfer of CD11b⁺ CD62L-negative cells did not.

Conclusions/Significance

We suggest that B. pseudomallei-infected CD11b⁺ selectin-expressing cells act as a Trojan horse and are able to transmigrate across endothelial cells, resulting in melioidosis with meningitis.     

Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the “gold standard” for the diagnosis of melioidosis; results can take 3–7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.     

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.     

Overexpression of the Endothelial Protein C Receptor Is Detrimental during Pneumonia-Derived Gram-negative Sepsis (Melioidosis)

Background

The endothelial protein C receptor (EPCR) enhances anticoagulation by accelerating activation of protein C to activated protein C (APC) and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B.) pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia.

Methodology/Principal Findings

Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT) mice and mice with either EPCR-overexpression (Tie2-EPCR) or EPCR-deficiency (EPCR^−/−). Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR^−/− mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma.

Conclusion/Significance

Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumo)sepsis.     

A Novel Sample Processing Method for Rapid Detection of Tuberculosis in the Stool of Pediatric Patients Using the Xpert MTB/RIF Assay

Background

Tuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available.

Methods

We developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa.

Results

The assay’s analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6–0.9) and 84% (95% CI 0.6–0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77–1) and 94% (95% CI 0.7–0.99), respectively.

Conclusion

This novel approach may permit simple and rapid detection of TB using pediatric stool samples.     

Sensitivity and Specificity of a Urine Circulating Anodic Antigen Test for the Diagnosis of Schistosoma haematobium in Low Endemic Settings

Background

Elimination of schistosomiasis as a public health problem and interruption of transmission in selected areas are key goals of the World Health Organization for 2025. Conventional parasitological methods are insensitive for the detection of light-intensity infections. Techniques with high sensitivity and specificity are required for an accurate diagnosis in low-transmission settings and verification of elimination. We determined the accuracy of a urine-based up-converting phosphor-lateral flow circulating anodic antigen (UCP-LF CAA) assay for Schistosoma haematobium diagnosis in low-prevalence settings in Zanzibar, Tanzania.

Methodology

A total of 1,740 urine samples were collected in 2013 from children on Pemba Island, from schools where the S. haematobium prevalence was <2%, 2–5%, and 5–10%, based on a single urine filtration. On the day of collection, all samples were tested for microhematuria with reagent strips and for the presence of S. haematobium eggs with microscopy. Eight months later, 1.5 ml of urine from each of 1,200 samples stored at -20°C were analyzed by UCP-LF CAA assay, while urine filtration slides were subjected to quality control (QCUF). In the absence of a true ‘gold’ standard, the diagnostic performance was calculated using latent class analyses (LCA).

Principal Findings

The ‘empirical’ S. haematobium prevalence revealed by UCP-LF CAA, QCUF, and reagent strips was 14%, 5%, and 4%, respectively. LCA revealed a sensitivity of the UCP-LF CAA, QCUF, and reagent strips of 97% (95% confidence interval (CI): 91–100%), 86% (95% CI: 72–99%), and 67% (95% CI: 52–81%), respectively. Test specificities were consistently above 90%.

Conclusions/Significance

The UCP-LF CAA assay shows high sensitivity for the diagnosis of S. haematobium in low-endemicity settings. Empirically, it detects a considerably higher number of infections than microscopy. Hence, the UCP-LF CAA employed in combination with QCUF, is a promising tool for monitoring and surveillance of urogenital schistosomiasis in low-transmission settings targeted for elimination.     

The Influence of Malnutrition and Micronutrient Status on Anemic Risk in Children under 3 Years Old in Poor Areas in China

Background

Malnutrition and anemia affect large numbers of young children living in poor areas of China. Multi-micronutrient deficiencies may be related to the prevalence of anemia in different populations, and identifying the risk factors that render children susceptible to anemia is the first step in combating anemia effectively.

Methods

In this cross-sectional study, a total of 1370 children under 3 years old were selected based on probability proportional to size sampling principles from poor counties of China. Basic characteristics data were collected by questionnaire; then anthropometrics and hemoglobin were measured in the field and anemia prevalence evaluated. Venous blood was drawn from children aged 12–35 months (N = 553) to evaluate micronutrient status. Logistic regression was used to identify the risk factors for children’s anemia.

Results

Among children aged 0–35 months, the prevalence of stunting, low body weight and wasting was 17.5%, 8.6% and 5.1%, respectively, and 25.6% of the children were affected by anemia, with more anemic infants and younger children than older children (P <0.01). There were 26.5%, 12.8%, 14.1% and 20.0% of the children aged 12–35 months affected by iron deficiency, vitamin D deficiency, folic acid deficiency and vitamin B₁₂ deficiency, respectively. For children aged 0–11 months who were breastfed, the mothers’ anemic status was the only factor associated with the child’s anemia (OR = 2.6; 95% CI: 1.2–5.4, P < 0.05). For children aged 12–35 months, multivariate logistic regression indicated that anemia was significantly associated with iron and vitamin B₁₂ deficiency (OR = 5.3; 95% CI: 1.9–14.5, P < 0.01) and monotonous diet (OR = 2.3; 95% CI: 1.1–4.7, P < 0.05) after adjusting for age and gender.

Conclusion

The prevalence of anemia was higher in children under 2 years old and requires urgent intervention. An effective intervention strategy should include iron and vitamin B₁₂ supplements_, improving dietary diversity and controlling breastfeeding mothers'' anemia.     

Shifts in the Antibiotic Susceptibility,Serogroups, and Clonal Complexes of Neisseria meningitidis in Shanghai,China: A Time Trend Analysis of the Pre-Quinolone and Quinolone Eras

Background

Fluoroquinolones have been used broadly since the end of the 1980s and have been recommended for Neisseria meningitidis prophylaxis since 2005 in China. The aim of this study was to determine whether and how N. meningitidis antimicrobial susceptibility, serogroup prevalence, and clonal complex (CC) prevalence shifted in association with the introduction and expanding use of quinolones in Shanghai, a region with a traditionally high incidence of invasive disease due to N. meningitidis.

Methods and Findings

A total of 374 N. meningitidis isolates collected by the Shanghai Municipal Center for Disease Control and Prevention between 1965 and 2013 were studied. Shifts in the serogroups and CCs were observed, from predominantly serogroup A CC5 (84%) in 1965–1973 to serogroup A CC1 (58%) in 1974–1985, then to serogroup C or B CC4821 (62%) in 2005–2013. The rates of ciprofloxacin nonsusceptibility in N. meningitidis disease isolates increased from 0% in 1965–1985 to 84% (31/37) in 2005–2013 (p < 0.001). Among the ciprofloxacin-nonsusceptible isolates, 87% (27/31) were assigned to either CC4821 (n = 20) or CC5 (n = 7). The two predominant ciprofloxacin-resistant clones were designated China^{CC4821-R1-C/B} and China^CC5-R14-A. The China^{CC4821-R1-C/B} clone acquired ciprofloxacin resistance by a point mutation, and was present in 52% (16/31) of the ciprofloxacin-nonsusceptible disease isolates. The China^CC5-R14-A clone acquired ciprofloxacin resistance by horizontal gene transfer, and was found in 23% (7/31) of the ciprofloxacin-nonsusceptible disease isolates. The ciprofloxacin nonsusceptibility rate was 47% (7/15) among isolates from asymptomatic carriers, and nonsusceptibility was associated with diverse multi-locus sequence typing profiles and pulsed-field gel electrophoresis patterns. As detected after 2005, ciprofloxacin-nonsusceptible strains were shared between some of the patients and their close contacts. A limitation of this study is that isolates from 1986–2004 were not available and that only a small sample of convenience isolates from 1965–1985 were available.

Conclusions

The increasing prevalence of ciprofloxacin resistance since 2005 in Shanghai was associated with the spread of hypervirulent lineages CC4821 and CC5. Two resistant meningococcal clones China^{CC4821-R1-C/B} and China^CC5-R14-A have emerged in Shanghai during the quinolone era. Ciprofloxacin should be utilized with caution for the chemoprophylaxis of N. meningitidis in China.