Metabolic requirements of CD160 expressing memory-like NK cells in Gram-negative bacterial infection.

Objective: Unique metabolic requirements accompany the development and functional fates of immune cells. How cellular metabolism is important in natural killer (NK) cells and their memory-like differentiation in bacterial infections remains elusive. Methods: Here, we utilise our established NK cell memory assay to investigate the metabolic requirement for memory-like NK cell formation and function in response to the Gram-negative intracellular bacteria Burkholderia pseudomallei (BP), the causative agent of melioidosis. Results: We demonstrate that CD160+ memory-like NK cells upon BP stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients which is maintained at least 3-month post-hospital admission. Using an in vitro assay, human BP-specific CD160+ memory-like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake, increased mTOR activation and mitochondrial membrane potential upon BP re-stimulation. Antigen-specific and cytokine-induced IFN-γ production of this memory-like NK cell subset are highly dependent on oxidative phosphorylation (OXPHOS) with some dependency on glycolysis, whereas the formation of CD160+ memory-like NK cells in vitro is dependent on fatty acid oxidation and OXPHOS and further increased by metformin. Conclusion: This study reveals the link between metabolism and cellular function of memory-like NK cells, which can be exploited for vaccine design and for monitoring protection against Gram-negative bacterial infection.

Clinical and microbiological features of positive blood culture episodes caused by non-fermenting gram-negative bacilli other than Pseudomonas and Acinetobacter species (2020-2023).

INTRODUCTION: Non-fermenting Gram-negative bacilli (NFGNB) other than Pseudomonas aeruginosa and Acinetobacter baumannii complex are pathogens of interest due to their ability to cause health-care associated infections and display complex drug resistance phenotypes. However, their clinical and microbiological landscape is still poorly characterized. METHODS: Observational retrospective study including all hospitalized patients presenting with a positive positive blood culture (BC) episode caused by less common NFGNB over a four-year period (January 2020-December 2023). Clinical-microbiological features and factors associated with mortality were investigated. RESULTS: Sixty-six less common NFGNB isolates other than Pseudomonas and Acinetobacter species causing 63 positive BC episodes were recovered from 60 patients. Positive BC episodes were predominantly sustained by Stenotrophomonas maltophilia (49.2%) followed by Achromobacter species (15.9%) that exhibited the most complex resistance phenotype. Positive BC episodes had bloodstream infection criteria in 95.2% of cases (60 out 63), being intravascular device (30.2%) and respiratory tract (19.1%) the main sources of infection. Fourteen-day, 30-day, and in-hospital mortality rates were 6.4%, 9.5%, and 15.9%, respectively. The longer time from admission to the positive BC episode, older age, diabetes, admission due to sepsis, and higher Charlson Comorbidity Index were identified as the main predictors of in-hospital mortality. CONCLUSIONS: Positive BC episodes sustained by NFGNB other than Pseudomonas and Acinetobacter species were predominantly sustained by Stenotrophomonas maltophilia and Achromobacter species, having bloodstream infection criteria in the vast majority of cases. Factors that have emerged to be associated with mortality highlighted how these species may have more room in prolonged hospitalisation and at the end of life for patients with chronic organ diseases.

Latent melioidosis activation presenting with urinary tract infection and bacteremia.

We report a rare case of latent melioidosis activation in a patient with a distant travel history to an endemic region. Melioidosis is an infection caused by Burkholderia pseudomallei which is highly endemic in Southeast Asia and northern Australia. The patient exhibited common clinical risk factors, presenting with urinary tract infection and bacteremia. The treatment course was complicated by the adverse effect of trimethoprim/sulfamethoxazole. This case underscores the importance of early detection and appropriate treatment of melioidosis, particularly given its expanding global distribution.

Medical imaging in melioidosis - 20-year experience in a non-endemic Australian city.

INTRODUCTION: Melioidosis may occasionally be encountered in non-endemic areas and medical imaging is frequently used to identify and characterise sites of disease. The purpose of this study is to describe the spectrum of imaging findings encountered in melioidosis patients treated in the tertiary public hospitals of Perth, Western Australia, between 2002 and 2022. METHODS: A database search and electronic medical record review was used to identify cases. Cases were included if they had Burkholderia pseudomallei isolated on culture and if they had at least one diagnostic imaging study performed at a Perth public tertiary hospital. The relevant imaging studies were reviewed, and imaging findings were recorded. RESULTS: Thirty-six cases were identified. The most common disease manifestation was bacteraemia (72%, 26 cases), followed by pulmonary infection (58%, 21 cases), skin and soft tissue infection (22%, eight cases), prostate abscess (14%, five cases) and septic arthritis (6%, two cases). A previously unreported case of isolated melioid pleural effusion was identified, as was a case of reactivated chronic latent pulmonary melioidosis with an apparent delay of over 20 years between the onset of symptoms and the time of infection. In cases with pulmonary melioidosis, the major lung abnormalities on CT chest could be categorised into one of two distinct patterns: nodular-predominant (78%) or consolidation-predominant (22%). CONCLUSION: Further research is required to assess the utility of the pattern-based categorisation of lung abnormalities on CT chest seen in the pulmonary melioidosis cases of this series.

A new computed tomography score-based staging for melioidosis pneumonia to predict progression.

Background: Melioidosis pneumonia, caused by the bacterium Burkholderia pseudomallei, is a serious infectious disease prevalent in tropical regions. Chest computed tomography (CT) has emerged as a valuable tool for assessing the severity and progression of lung involvement in melioidosis pneumonia. However, there persists a need for the quantitative assessment of CT characteristics and staging methodologies to precisely anticipate disease progression. This study aimed to quantitatively extract CT features and evaluate a CT score-based staging system in predicting the progression of melioidosis pneumonia. Methods: This study included 97 patients with culture-confirmed melioidosis pneumonia who presented between January 2002 and December 2021. Lung segmentation and annotation of lesions (consolidation, nodules, and cavity) were used for feature extraction. The features, including the involved area, amount, and intensity, were extracted. The CT scores of the lesion features were defined by the feature importance weight and qualitative stage of melioidosis pneumonia. Gaussian process regression (GPR) was used to predict patients with severe or critical melioidosis pneumonia according to CT scores. Results: The melioidosis pneumonia stages included acute stage (0-7 days), subacute stage (8-28 days), and chronic stage (>28 days). In the acute stage, the CT scores of all patients ranged from 2.5 to 6.5. In the subacute stage, the CT scores for the severe and mild patients were 3.0-7.0 and 2.0-5.0, respectively. In the chronic stage, the CT score of the mild patients fluctuated approximately between 2.5 and 3.5 in a linear distribution. Consolidation was the most common type of lung lesion in those with melioidosis pneumonia. Between stages I and II, the percentage of severe scans with nodules dropped from 72.22% to 47.62% (P<0.05), and the percentage of severe scans with cavities significantly increased from 16.67% to 57.14% (P<0.05). The GPR optimization function yielded area under the receiver operating characteristic curves of 0.71 for stage I, 0.92 for stage II, and 0.87 for all stages. Conclusions: In patients with melioidosis pneumonia, it is reasonable to divide the period (the whole progression of melioidosis pneumonia) into three stages to determine the prognosis.

The hidden burden of melioidosis in Nepal: a paradigm for the urgent need to implement a simple laboratory algorithm to detect Burkholderia pseudomallei in low-resource endemic areas.

Melioidosis, an emerging infectious disease caused by the Gram-negative bacillus Burkholderia pseudomallei, is massively underdiagnosed in many low- and middle-income countries. The disease is clinically extremely variable, has a high case fatality rate, and is assumed to be highly endemic in South Asian countries, including Nepal. The reasons for underdiagnosis include the lack of awareness among clinicians and laboratory staff and limited microbiological capacities. Because costly laboratory equipment and consumables are likely to remain a significant challenge in many melioidosis-endemic countries in the near future, it will be necessary to make optimum use of available tools and promote their stringent implementation. Therefore, we suggest that health facilities in resource-poor countries, such as Nepal, introduce a simple and low-cost diagnostic laboratory algorithm for the identification of B. pseudomallei cultures. This screening algorithm should be applied specifically to samples from patients with fever of unknown origin and risk factors for melioidosis, such as diabetes. In addition, there could also be a role of low-cost, novel, promising serological point-of-care tests, which are currently under research and development.

Tracking melioidosis in Iran: Utilizing abattoir-based surveillance as a One Health approach.

BACKGROUND: Burkholderia pseudomallei, an environmental saprophyte bacterium, causes melioidosis in humans and animals. It was first discovered in Iran between 1967 and 1976 in small ruminants, equines, environments and humans. No subsequent studies have been conducted to determine the existence and prevalence of this pathogen in the country. OBJECTIVES: The present study aims to monitor the presence of B. pseudomallei in the ruminant population of the Golestan province of Iran, which largely depends on pastures. The ruminants can serve as sentinels to indicate the presence of the bacteria in the environment and its potential impact on human health in the One Health triad. METHODS: Liver and lung abscesses from domestic sheep, cattle and goats in three industrial and three conventional slaughterhouses were sampled and analysed using 23S ribosomal DNA polymerase chain reaction (rDNA PCR) with primers CVMP 23-1 and CVP-23-2 for B. pseudomallei, Burkholderia cepacia and Burkholderia vietnamiensis, as well as B. pseudomallei-specific TTS1 real-time PCR, along with microbiological and biochemical assays. RESULTS: Out of the 97 animals sampled, only 14 (15%) tested positive for 23S rDNA PCR. However, the follow-up evaluation using TTS1 real-time PCR and microbiological and biochemical assays did not confirm the presence of B. pseudomallei in the samples. CONCLUSIONS: Although B. pseudomallei was not detected in the current survey, conducting abattoir-based surveillance of ruminants is a cost-effective One Health approach to monitor pathogenic Burkholderia. Developing standards of clinical and laboratory good practices for Burkholderia infections is crucial for One Health surveillance.

Enhancing Diagnosis and Treatment Adjustments for Travelers Infected with Burkholderia pseudomallei: A Case Report - Beijing Municipality, China, June 2023.

What is already known about this topic?: Burkholderia pseudomallei (BP) infection leads to melioidosis, a tropical disease endemic to coastal provinces of southern China. Physicians in non-endemic areas, do not commonly consider this disease as a primary differential diagnosis for febrile patients. What is added by this report?: This article discusses a case of melioidosis in Northern China. The patient, who had recently visited a Southeast Asian region with high melioidosis prevalence, fell ill after exposure to contaminated water. The disease progressed quickly and with severity. What are the implications for public health practice?: Healthcare workers need to remain vigilant regarding travel-related diseases for accurate differential diagnosis and to provide timely and effective treatment, especially for patients with recent travel history or symptoms during travel.

First Case Report of Cystic Echinococcosis Caused by G7 Genotype Echinococcus intermedius Confirmed by Genetic Sequencing - Southern China, December 2023.

What is already known about this topic?: Echinococcosis exhibits a global distribution. In China, the primary endemic area is the northwest region. In December 2023, we documented a case of echinococcosis in an individual lacking any travel or residential history in endemic regions. What is added by this report?: This is the first laboratory-confirmed case of hepatic echinococcosis reported in Guangdong Province, associated with the G7 genotype of Echinococcus granulosus (E. granulosus). The most probable mode of transmission is a local infection resulting from E. granulosus introduced from endemic regions. What are the implications for public health practice?: As the circulation of agricultural products increases, it is essential to enhance the quarantine and management of livestock from epidemic areas to prevent and control the spread of echinococcosis to non-epidemic regions.

γδ T Cells Mediate Protection Against Neutrophil-associated Lung Inflammation in Pulmonary Melioidosis.

Pulmonary melioidosis is a severe tropical infection caused by Burkholderia pseudomallei and is associated with high mortality despite early antibiotic treatment. γδ T cells have been increasingly implicated as drivers of the host neutrophil response during bacterial pneumonia, but their role in pulmonary melioidosis is unknown. Here, we report that in patients with melioidosis, a lower peripheral blood γδ T cell concentration is associated with higher mortality even when adjusting for severity of illness. γδ T cells were also enriched in the lung and protected against mortality in a mouse model of pulmonary melioidosis. γδ T cell deficiency in infected mice induced an early recruitment of neutrophils to the lung, independent of bacterial burden. Subsequently, γδ T cell deficiency resulted in increased neutrophil-associated inflammation in the lung as well as impaired bacterial clearance. Additionally, γδ T cells influenced neutrophil function and subset diversity in the lung after infection. Our results indicate that γδ T cells serve a novel protective role in the lung during a severe bacterial pneumonia by regulating excessive neutrophil-associated inflammation.

EBUS-TBNA diagnosis of mediastinal melioidosis in patient presented with lymphadenopathy and SVC thrombosis: A rare manifestation in non-diabetic patient.

Melioidosis is a tropical infectious disease that ranks as northeastern Thailand's third most common infectious cause of death. The manifestations of melioidosis vary depending on the organs involved and often resemble malignancy and tuberculosis. We present a case of an atypical melioidosis presentation in a patient with low-grade fever and facial swelling without any risk factors. Chest CT revealed a 3.3-cm heterogeneous enhancing right lower paratracheal lymph nodes with thrombosis of the superior vena cava and azygos vein. Endobronchial ultrasound-guided transbronchial needle aspiration of lymph node was performed, and Burkholderia pseudomallei was identified through lymph node culture. The patient underwent a three-week intravenous course of ceftazidime and a 12-week oral course of trimethoprim-sulfamethoxazole. Oral anticoagulation was also administered. Follow-up computed tomography of the thorax after completion of treatment revealed no residual lymphadenopathy and thrombosis.

Characteristic of pancreatic involvement in melioidosis: Case report and literature review.

Background: Intra-abdominal abscesses are a frequent manifestations of melioidosis whereas pancreas is barely affected by this condition. Herein, by delving into the clinical manifestations, diagnostic processes, and the ultimate clinical outcome, we report a case of an unusual presentation of pancreatic melioidosis in a Chinese patient, aiming to shed light on a diagnosis that is not commonly associated with the pancreas. Case presentation: The patient, a 32-year-old male farmer, suffered from persistent burning pain in his upper abdomen, accompanied by nausea, vomiting, fever and other symptoms, presented to the clinic. His body temperature spiked to 38.5 °C without apparent reason for this fever. A thorough examination, including the blood culture and the imaging examination, led to a diagnosis of pancreatic melioidosis. The patient was promptly treated with intravenous meropenem and ceftazidime. As a consequence, his symptoms eased and discharged in stable condition. The patient continued his treatment with oral trimethoprim-sulfamethoxazole (co-trimoxazole) for three months to control the infection. Following 6 months of regular follow-up, the patient fully recovered. Conclusions: In tropical regions such as Hainan, it is crucial to consider atypical infection like B. pseudomallei in the differential diagnosis, even when they present in atypical locations such as a pancreatic pseudocyst. Detecting pancreatic involvement in melioidosis relies heavily on sensitive bacterial culture and imaging examination. This retrospective study of patients' infection diagnosis aims to shed light on the clinical treatment, and prognosis associated with pancreatic melioidosis, thereby raising awareness about the risk of pancreatic affection in melioidosis cases.

Genome sequence of two novel virulent clinical strains of Burkholderia pseudomallei isolated from acute melioidosis cases imported to Israel from India and Thailand.

OBJECTIVE: Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations. DATA DESCRIPTION: We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii.

Development of an Antigen Capture Lateral Flow Immunoassay for the Detection of Burkholderia pseudomallei.

Early diagnosis is essential for the successful management of Burkholderia pseudomallei infection, but it cannot be achieved by the current gold standard culture technique. Therefore, this study aimed to develop a lateral flow immunoassay (LFIA) targeting B. pseudomallei capsular polysaccharide. The development was performed by varying nitrocellulose membrane reaction pads and chase buffers. The prototype LFIA is composed of Unisart CN95 and chase buffer containing tris-base, casein, and Surfactant 10G. The assay showed no cross-reactivity with E. coli, S. aureus, P. aeruginosa, and P. acne. The limit of detections (LODs) of the prototype LFIA was 107 and 106 CFU/mL B. pseudomallei in hemoculture medium and artificial urine, respectively. These LODs suggest that this prototype can detect melioidosis from positive hemoculture bottles but not straight from urine. Additionally, these LODs are still inferior compared to Active Melioidosis Detect (AMDTM). Overall, this prototype holds the potential to be used clinically with hemoculture bottles. However, further improvements should be considered, especially for use with urine samples.

Hemophagocytic lymphohistiocytosis complicating septicemic melioidosis: A case report.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. People infected with B. pseudomallei experience fever and skin changes, pneumonia, abscesses, and septic shock that could cause death. Hemophagocytic lymphohistiocytosis is a severe inflammatory syndrome due to the excess activation of macrophages and T cells. We report a 50-year-old hypertensive and diabetic male patient presented with high-grade intermittent fever with loss of appetite and weight loss for two months and a history of jaundice, backache and swelling of both feet for 15 days. Blood and bone marrow culture grew Burkholderia pseudomallei. A liver biopsy revealed Kupffer cell hyperplasia and hemophagocytosis. The patient was treated with an injection of dexamethasone 4mg intravenous three times a day for five days and tapered over 15 days with ceftazidime 2 gm intravenous three times a day for six weeks. Early suspicion in the diagnosis of hemophagocytic lymphohistiocytosis in septicemia can prevent severe complications, even death.

Disseminated Melioidosis Presenting as Sepsis, Neurological Melioidosis, Liver and Prostate Abscess in United Arab Emirates.

Background: Melioidosis is an infection caused by Burkholderia pseudomallei, a Gram-negative bacterium. It is a disease endemic to Southeast Asia and northern Australia although its global incidence has been rising. It most commonly infects people with certain identified risk factors such as diabetes, alcoholism, thalassemia, and underlying chronic disease involving lungs, kidney and liver. This bacterium is capable of producing a wide array of clinical manifestations ranging from asymptomatic disease to localised infections such as in the lung, bone or skin to disseminated infection. Case description: This is a case, from United Arab Emirates, of a 40-year-old male recently diagnosed with diabetes who presented with multiple abscesses and was eventually diagnosed with disseminated melioidosis. He was treated successfully with antibiotics and drainage of abscesses. Conclusion: In non-endemic regions, melioidosis can be easily missed in common diagnostic approaches. This gap of awareness could delay the diagnosis and allow further deterioration of the patient due to complications. Thus, case reports like this can enlighten internists about changing incidences and complexity of clinical presentations, thus preparing them to better handle such patients in the future. LEARNING POINTS: Owing to its considerably rare incidence in non-endemic regions including the United Arab Emirates, melioidosis can easily be overlooked or misdiagnosed.Moreover, due to similarity with multiple other diseases and infections as well as significant absence from standard medical curricula, melioidosis is rarely on the differential list of an internist.This report aims to enhance awareness and alertness to aid earlier detection and avoid severe complications.

Diagnostic accuracy of automation and non-automation techniques for identifying Burkholderia pseudomallei: A systematic review and meta-analysis.

BACKGROUND: Burkholderia pseudomallei, a Gram-negative pathogen, causes melioidosis. Although various clinical laboratory identification methods exist, culture-based techniques lack comprehensive evaluation. Thus, this systematic review and meta-analysis aimed to assess the diagnostic accuracy of culture-based automation and non-automation methods. METHODS: Data were collected via PubMed/MEDLINE, EMBASE, and Scopus using specific search strategies. Selected studies underwent bias assessment using QUADAS-2. Sensitivity and specificity were computed, generating pooled estimates. Heterogeneity was assessed using I2 statistics. RESULTS: The review encompassed 20 studies with 2988 B. pseudomallei samples and 753 non-B. pseudomallei samples. Automation-based methods, particularly with updating databases, exhibited high pooled sensitivity (82.79%; 95% CI 64.44-95.85%) and specificity (99.94%; 95% CI 98.93-100.00%). Subgroup analysis highlighted superior sensitivity for updating-database automation (96.42%, 95% CI 90.01-99.87%) compared to non-updating (3.31%, 95% CI 0.00-10.28%), while specificity remained high at 99.94% (95% CI 98.93-100%). Non-automation methods displayed varying sensitivity and specificity. In-house latex agglutination demonstrated the highest sensitivity (100%; 95% CI 98.49-100%), followed by commercial latex agglutination (99.24%; 95% CI 96.64-100%). However, API 20E had the lowest sensitivity (19.42%; 95% CI 12.94-28.10%). Overall, non-automation tools showed sensitivity of 88.34% (95% CI 77.30-96.25%) and specificity of 90.76% (95% CI 78.45-98.57%). CONCLUSION: The study underscores automation's crucial role in accurately identifying B. pseudomallei, supporting evidence-based melioidosis management decisions. Automation technologies, especially those with updating databases, provide reliable and efficient identification.

Electrochemical Immunoassay for Capturing Capsular Polysaccharide of Burkholderia pseudomallei: Early Onsite Detection of Melioidosis.

This study presented the detection and quantification of capsular polysaccharide (CPS) as a biomarker for the diagnosis of melioidosis. After successfully screening four monoclonal antibodies (mAbs) previously determined to bind CPS molecules, the team developed a portable electrochemical immunosensor based on antibody-antigen interactions. The biosensor was able to detect CPS with a wide detection range from 0.1pg/mL to 1 µg/mL. The developed biosensor achieved high sensitivity for the detection of CPS spiked into both urine and serum. The developed assay platform was successfully programmed into a Windows app, and the sensor performance was evaluated with different spiked concentrations. The rapid electro-analytical device (READ) sensor showed great unprecedented sensitivity for the detection of CPS molecules in both serum and urine, and results were cross-validated with ELISA methods.

Multi-systemic melioidosis in a patient with type 2 diabetes in non-endemic areas: A case report and review of literature.

BACKGROUND: Melioidosis, an infectious disease caused by Burkholderia pseudomallei (B. pseudomallei), occurs endemically in Southeast Asia and Northern Australia and is a serious opportunistic infection associated with a high mortality rate. CASE SUMMARY: A 58-year-old woman presented with scattered erythema on the skin of her limbs, followed by fever and seizures. B. pseudomallei was isolated successively from the patient's urine, blood, and pus. Magnetic resonance imaging showed abscess formation involving the right forehead and the right frontal region. Subsequently, abscess resection and drainage were performed. The patient showed no signs of relapse after 4 months of follow-up visits post-treatment. CONCLUSION: We present here a unique case of multi-systemic melioidosis that occurs in non-endemic regions in a patient who had no recent travel history. Hence, it is critical to enhance awareness of melioidosis in non-endemic regions.

Development of a novel sequence based real-time PCR assay for specific and sensitive detection of Burkholderia pseudomallei in clinical and environmental matrices.

BACKGROUND: Melioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence. METHODS: An extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls. RESULTS: A novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil. CONCLUSIONS: The findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices.