Melioidosis is an infectious condition caused by the bacterium Burkholderia pseudomallei.¹ It can affect almost every organ in your body and can lead to severe illness and high mortality rates if not diagnosed and treated promptly.^2,3 However, melioidosis is difficult to diagnose due to the limited awareness of the condition and because its symptoms often resemble a number of other conditions, leading to misdiagnosis.^4,5 Thus, enhancing patient outcomes and lowering mortality depend heavily on prompt and correct diagnosis. This article will provide you with an understanding of the various techniques available for the diagnosis of melioidosis.

Understanding Melioidosis

Burkholderia pseudomallei is found predominantly in soil and water in tropical and subtropical regions.¹ It is endemic in Southeast Asia and Northern Australia, with notable hotspots in Thailand, Malaysia, and Singapore.^6,7 This bacteria can enter your body through direct contact with contaminated soil or water, mainly through cuts or wounds, or through inhalation and ingestion.¹ The time between exposure and the onset of symptoms is from 1 to 21 days. However, dormant infections may become active several years later.¹ 

Risk factors

Individuals who engage in activities involving soil handling, like gardening, agriculture, or construction are more susceptible to developing melioidosis. If you have recently been in contact with contaminated water, e.g., through swimming, fishing, etc., you also have the chance to develop it.⁸

Other risk factors include: 

• Severe alcohol consumption
• Diabetes
• Liver and renal disease
• Cancer
• Thalassemia
• Chronic lung disease
• Other immunocompromising disorders^1,8,9

Symptoms of melioidosis

• Symptoms of a localised melioidosis infection include ulcers, nodules, or skin abscesses, fever, and muscle aches
• For a pulmonary infection, the symptoms range from mild bronchitis to severe pneumonia. Cough, high fever, chest pain, headache, anorexia, and muscle pain are the main symptoms. Lesions similar to tuberculosis can also be seen in a chest X-ray
• Disseminated meliodosis can lead to widespread infection and rapid-onset septicemia. It can result in lesions in internal organs such as the liver, spleen, kidney, and prostate

General symptoms of septicemia due to melioidosis include:

• Fever
• Headache
• Difficulty breathing
• Abdominal and/or chest discomfort
• Muscle pain
• Disorientation
• Seizures⁸

Diagnosis of Meliodosis

If you suspect melioidosis, it is crucial to accurately diagnose it, as misdiagnoses of acute melioidosis infections frequently occur for localised skin conditions, septicaemic shock, and community-acquired pneumonia. On the other hand, persistent lung infections due to melioidosis frequently mimic tuberculosis in the lungs.⁵

Laboratory diagnostic techniques

 Laboratory techniques frequently utilised for the diagnosis of melioidosis include:

Microbiological method

For identifying Burkholderia pseudomallei, your blood, urine, sputum, or pus are collected. The samples are then cultured on standard blood, MacConkey agar media, or selective media like Ashdown’s agar, and incubated at 37°C to encourage bacterial growth. Growth is monitored for about 48 hours or more.^10,11 If Burkholderia pseudomallei colonies are present in the culture, they tend to appear as dry, wrinkled structures with a metallic sheen. They may also exhibit a distinctive earthy odour.¹²

Identification of Burkholderia pseudomallei

Biochemical tests

These tests help identify B. pseudomallei by studying how it reacts to different chemicals. Common tests include checking for oxidase and catalase activity, motility, triple sugar iron agar, indole test, citrate usage, acid production from carbohydrates, and gelatin hydrolysis.¹³ 

Molecular methods 

Polymerase chain reaction (PCR) 

PCR uses a method to find specific parts of B. pseudomallei's genetic code. This technique is used in laboratories to produce numerous copies of a specific segment of the genetic material (DNA) of B. pseudomallei to quickly identify and distinguish it based on its unique genetic fingerprints. There are new PCR tests that are faster and can tell B. pseudomallei apart from similar bacteria like B. thailandensis.¹¹

Direct detection by specific primer PCR amplification

Scientists created and tested this special PCR test to find B. pseudomallei, which can be utilized to identify B. pseudomallei in samples from patients and the environment. It mainly helps in large studies to understand how melioidosis spreads in communities. However, success depends on how well the test is designed and the quality of the sample.¹¹

Direct detection from environmental samples

Detecting B. pseudomallei in soil is challenging due to diverse microbial communities and PCR inhibitors like humic acid. Methods involve selective enrichment cultures, DNA purification, and using specialised PCR techniques to overcome these obstacles and accurately identify B. pseudomallei from environmental samples.¹¹

Identification by sequencing conserved gene targets

Sequencing technology uses a method to read genetic information to identify B. pseudomallei bacteria quickly.¹¹ 16S rRNA gene sequencing is commonly used. However, it cannot always distinguish between B. pseudomallei and similar bacteria like B. thailandensis because their genes are very similar. Nevertheless, an alternative gene target known as groEL gene sequencing can better tell apart B. pseudomallei from B. thailandensis.¹¹

Mass spectrometry 

MALDI-TOF mass spectrometry is used to rapidly identify bacterial proteins, in order to confirm the presence of B. pseudomallei. This revolutionary technique requires only a small amount of bacteria, gives results in minutes, is cheaper than other methods, and is becoming more common in labs. However, sometimes it misidentifies closely related bacteria due to database limitations.^11,14

Serological methods

Serological methods involve testing blood serum in order to detect antibodies that are produced by your immune system against B. pseudomallei. These tests help identify whether you have been exposed to the bacterium by looking for antibodies that recognise and bind to antigens on the bacterial surface.¹⁵

Indirect Hemagglutination Assay (IHA)

The indirect hemagglutination assay is a common test for melioidosis in areas where the disease is common. This test measures if certain antibodies in the blood can clump together red blood cells in the presence of B. pseudomallei.¹⁶ It uses sheep blood cells mixed with patient blood to see if they stick together, indicating infection. However, sometimes, the test misses cases, especially in very sick patients. It can also take a long time for the test to show a clear result. ¹¹

ELISA

ELISA is a serological test that checks if the patient has antibodies against B. pseudomallei in their blood.¹⁴ This test uses bacterial components like lipopolysaccharide (LPS), whole cells, or recombinant proteins to detect antibodies. They are quick and reduce human error compared to traditional tests like IHA. Different antigens show varying sensitivity and specificity, however, LPS-based ELISAs may be more specific. Nevertheless, variability in B. pseudomallei strains affects test accuracy, especially with LPS-based assays.¹¹

Rapid diagnostic tests

Lateral flow tests

 Similar to home pregnancy tests, these provide a simple and rapid method for detecting B. pseudomallei with visual results. Lateral flow tests provide quick results by detecting B. pseudomallei antigens or antibodies in clinical samples. These tests can find B. pseudomallei by looking for specific parts of the bacteria or the body's response to it.¹¹

Immunochromatographic test (ICT) 

 ICTs are point-of-care test strips and cassettes designed to detect IgG and IgM antibodies against B. pseudomallei. These tests provide rapid results, potentially aiding in early diagnosis and treatment initiation. Limited evaluations show ICTs have similar sensitivity to other serological methods like IHA, and ELISA. Despite promising results, ICTs are not widely accessible for routine clinical use yet.¹¹

Emerging technologies

New methods are being developed that could make it even faster and easier to find B. pseudomallei, like using special tools that can quickly read genetic information. Advances in diagnostic technologies, such as CRISPR-based assays and portable sequencing devices, are being developed to offer faster and more accurate detection of B. pseudomallei.¹⁷

Imaging techniques

Imaging is a useful tool for diagnosing melioidosis since it may reveal whether or not the body has pus-filled formations or abscesses. Understanding the location of these abscesses is crucial for treatment planning. Imaging can help physicians decide where to obtain bodily samples for examination, such as those from infected organs or abscesses. This supports the melioidosis diagnosis.¹⁸

Common imaging modalities

 X-ray: Radiation is used in X-rays to generate images of the body's internal structures. They're often used first to look for lung infections in melioidosis.¹⁹

Ultrasound: This method employs sound waves to generate images of organs. It's good for finding abscesses in organs like the liver, spleen, and prostate.²⁰

Computed tomography (CT): CT scans employ X-rays to produce precise cross-sectional pictures of the body. For the diagnosis of periarticular abscesses and osseous erosions (degradation or loss of bone tissue), a CT scan is preferable. It is used to provide information about the disease's severity and extent. They can detect lung diseases and infections in the lungs and other deep body regions and are excellent at displaying structures. They are also used for the diagnosis of neurological symptoms associated with melioidosis.¹⁸

Magnetic resonance imaging (MRI): MRI employs magnets and radio waves for creating detailed images of soft tissues. MRI is useful for finding infections in the brain and nervous system/neurological symptoms, which can happen in severe cases of melioidosis.¹⁸

Summary

Melioidosis is a severe infectious disease caused by the bacteria Burkholderia pseudomallei. Due to the frequent misdiagnosis of this condition and its severity, an accurate and timely diagnosis is critical. Laboratory diagnostic techniques include microbiological methods, molecular methods like PCR, and serological methods such as ELISA and ICTs for identifying the bacterium. Imaging techniques include X-ray, MRI, CT scan, and ultrasound which play a significant role in detecting internal abscesses and guiding appropriate treatment. However, enhancing diagnostic strategies and advancing research are vital to improving patient outcomes and ensuring timely treatment of melioidosis.