Melioidosis molecular diagnostics: An update

Abstract

Melioidosis, a fatal tropical disease, presents a wide array of clinical manifestations, including abscesses, pneumonia, septic shock, bacteraemia, osteomyelitis, septic arthritis, and skin infection. The Centers for Disease Control and Prevention (CDC) has classified Burkholderia pseudomallei (B. pseudomallei), a gram-negative bacterium found in soil, as a Tier 1 select agent. Referred to as the "great mimicker," this organism can infect several organs imitating the symptoms of different illnesses. According to worldwide data, there are around 165,000 cases and 89,000 deaths annually. Current diagnostic procedures rely primarily on culturing B. pseudomallei, are slow and have low sensitivity, resulting in delayed treatment and higher fatality rates. This review examines the substantial difficulties related to diagnosing melioidosis in response to the urgent need for precise and prompt diagnosis. We have summarized the results of diagnostic kits that are currently sold in the market and assessed the market for melioidosis diagnostic kits.

Keywords: Melioidosis; artificial intelligence; diagnostics; early detection; liquid biopsy; point-of-care-test.

Figure 1.

The morphological characteristics of B. pseudomallei and its colony as cultured on blood agar plates. B. pseudomallei is a Gram-negative, rod-shaped bacterium: (a) The results of Gram staining for B. pseudomallei reveal pink-stained cells under the microscope, one of its distinctive features is the “safety pin” structure, which is due to bipolar staining; (b) The colonies are usually cream to white in colour, but over time they may develop a characteristic wrinkled appearance when incubated for longer duration. Used with permission (doi .Org/10 .1016/j.Heliyon.2024.e30299).

Figure 2.

Challenges of Melioidosis. Melioidosis caused by B. pseudomallei is highly underreported worldwide. The reasons for its underdiagnosis are multifactorial. Their resistance to common antibiotics and non-specific symptoms that mimic the symptoms of other diseases lead to misdiagnosis and delayed treatment. Moreover, the gold standard of disease involving bacterial culture is slow and requires specialized conditions, leading to high mortality rates. The limited availability of advanced molecular diagnostics in endemic regions further complicates timely and accurate detection.

Figure 3.

Antibiotic resistance mechanism in B. pseudomallei. B. pseudomallei exhibits intrinsic resistance to a range of antibiotics such as penicillin, gentamicin, streptomycin, tobramycin, ampicillin, and first- and second-generation cephalosporins. This resistance is mediated through several mechanisms: 1) Alteration of an antibiotic target site, thereby reducing drug binding efficacy; 2) Degradation of antibiotics by β-lactamase enzymes, hydrolysing the antibiotics containing β-lactam rings; 3) Inactivation of antibiotics by the antibiotic altering enzymes; 4) Efflux of antibiotics reduce the intracellular antibiotic concentration by RND (Resistance-nodulation-division) efflux pumps.

Figure 4.

Development of diagnostic kit for melioidosis detection. The current diagnostic kits for melioidosis are primarily based on the following technologies: Immunohistochemistry, real-time PCR, and lateral flow assays. These assays are tailored to detect patient samples (blood, urine, sputum, etc.) containing specific biomarkers associated with the disease. Rigorous validation procedures are conducted to optimize sensitivity and specificity, culminating in producing prototype kits designed for precise and reliable detection of melioidosis. The major companies involved in developing and producing these diagnostic kits are also highlighted for each category.

Figure 5.

Milestones in melioidosis detection techniques. Significant progress has been made in the detection techniques for melioidosis. This timeline highlights the chronological account of important milestones in the evolution of diagnostic methods for melioidosis. Over the decades, diagnostic approaches have progressed from basic culture-based methods and serological assays to more precise biochemical tests and PCR-based molecular techniques. The development of rapid diagnostic tools and the adoption of next-generation sequencing have further enhanced diagnostic accuracy and strain typing, significantly improving the management and control of melioidosis.

Figure 6.

Current and future diagnostic techniques used for melioidosis diagnosis. This cartoon representation presents a comprehensive overview highlighting the progression from traditional methods to cutting-edge technologies, aiming to achieve earlier detection, improved accuracy, and better patient outcomes in melioidosis management. Existing techniques include bacterial culture, which remains the gold standard but is time-consuming, along with imaging studies and serological assays such as ELISA, immunohistochemistry, and molecular methods like real-time PCR, which offer higher sensitivity and specificity.

Figure 7.

Future of melioidosis diagnostic techniques. Future diagnostic advancements include point-of-care diagnostics, including lateral flow assays, which provide rapid results and are particularly useful in resource-limited settings. CRISPR-Cas-based assays, diagnostic biomarkers, and AI models are also expected to enhance detection speed, accuracy, and accessibility, addressing current limitations and improving early diagnosis in endemic regions.