One hundred and eighteen clinical samples were collected and processed for culture during the study period. These samples were obtained from patients with relevant infections who were receiving care in various hospital settings, including inpatient rooms and intensive care units such as the ICU, PICU, and NICU. Sputum samples were predominant, reflecting that pneumonia is the most common manifestation of melioidosis.²⁰ Burkholderia pseudomallei was detected in a limited number of sputum samples. Other prevalent pathogenic bacteria in sputum included P. aeruginosa, observed in 32/59 (54.23%) of cases. P. aeruginosa showed similar predominance in BAL, pus, and sputum samples., with occurrences of 19/22 (86.36%), 10/17 (58.82%), and 6/10 (60%), respectively. Additional pathogenic bacteria grown on routine media (CBA and MCA) from these clinical samples included K. pneumoniae, E. coli, A. baumannii, B. cepacia, and S. maltophilia. On ASA media, P. aeruginosa was also observed, although its colony morphology distinctly differs from that of B. pseudomallei,²¹ demanding further identification to confirm its species.

This study utilized three different media types (selective: ASA; routine/non-selective: CBA and MCA) to cultivate and assess the selectivity for B. pseudomallei (Figure 2). The results indicated that all three media were suitable for the growth of B. pseudomallei. However, ASA demonstrated a faster colony growth within 24-48 hours. In contrast, on CBA and MCA media, the colonies appeared very small and were nearly invisible, potentially leading to false-negative results. On CBA (Figure 2A), the colonies were not clearly visible as they grew amidst other bacterial colonies, making them obscured due to their small and smooth nature, thereby necessitating a longer incubation period of over 48 hours for visible growth. The colonies eventually developed a metallic luster and could appear dry or wrinkled after extended incubation.²² Similarly, on MCA media (Figure 2B), the colonies that could not ferment lactose were almost colorless and could develop a metallic luster and pink rugose appearance after 48 hours.²³

ASA media displayed colonies with a clearer, larger size, showcasing a pink or pale color, and by 48 hours, the colonies exhibited a distinct purple, wrinkled morphology (Figure 2C). Observations from this study noted that ASA media was slightly oily and challenging to scratch due to its glycerol content. It is recommended to incubate sterile ASA media at 37 °C for at least 24 hours before use. This pre-incubation significantly improves the separation and purity of colony streaks in the 3rd and 4th quadrants.  The microscopic appearance of the B. pseudomallei colony showed rod-shaped, medium/fine in size, and gram-negative (red) (Figure 2D). The selective growth of Burkholderia colonies on ASA media stems from their inherent resistance to polymyxin and gentamicin,²³ with ASA media containing 4 mg/L of gentamicin, which suppresses the growth of non-Burkholderia bacteria. The TSI reaction indicates that the slant/butt is alkaline and non-fermented (K/K) (Figure 2E). Oxidase testing confirmed that all colonies of B. pseudomallei were oxidase-positive (Figure 2F). Antibiotic susceptibility testing demonstrated marked sensitivity to amoxicillin/clavulanic acid, limited susceptibility to amikacin, and complete resistance to gentamicin, as indicated by the absence of an inhibition zone (Figure 2G). These findings highlight the potential efficacy of amoxicillin/clavulanic acid against the tested isolates.

The diagnosis of melioidosis presents significant challenges in microbiology laboratories, often leading to underdiagnosis due to a lack of clinical awareness, which can contribute to the high mortality rates associated with the disease due to inappropriate or delayed initial treatment.²⁴ In hospitals, empirical therapy is generally based on clinical judgment and diagnosis,²⁵ administered to prevent severe infections while awaiting the results of microbiological cultures. In this study, patients received empirical therapies, including ceftriaxone, cefotaxime, ciprofloxacin, and levofloxacin. However, these therapies are often not effective as B. pseudomallei is intrinsically resistant to many common antimicrobial agents, including penicillins (except amoxicillin/clavulanic acid), third-generation cephalosporins (except ceftazidime), aminoglycosides, quinolones, and macrolides.²⁶ Antibiotic sensitivity testing revealed high resistance levels to the four antibiotics, with all isolates showing resistance to amikacin and gentamicin. Comparatively, dominant gram-negative bacteria found in clinical samples not suspected of melioidosis, such as E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii, remain over 80% sensitive to amikacin. This disparity highlights the difficulty in selecting effective antibiotic therapy for melioidosis, given the limited options available.

The percentage results for antimicrobial sensitivity utilized cutoff values established by the European Unit Reference Laboratory for Antimicrobial Resistance (EURL-AR) in 2013 to guide antimicrobial therapy in hospitals. These cutoff values are crucial for comparing antimicrobial sensitivity, aiding in the management of resistance, and assisting microbiologists in interpreting test results. The criteria are defined as follows: sensitivities below 60% are not recommended for therapy due to high resistance rates; sensitivities between 60-80% may be considered in consultation with the hospital’s antimicrobial resistance control program; sensitivities above 80% are generally recommended for therapeutic use. Antibiotics such as piperacillin-tazobactam, ceftazidime, cefepime, tigecycline, trimethoprim-sulfamethoxazole, amoxicillin-clavulanic acid, and meropenem, which demonstrated 100% sensitivity, are recommended as definitive therapy options for melioidosis patients.

Most B. pseudomallei isolates were identified in patients displaying clinical features of pulmonary tuberculosis and pleural effusion, aligning with typical manifestations of melioidosis. Melioidosis often presents as chronic pneumonia, which can closely mimic tuberculosis, usually regarded together in clinical assessments.^22,26 Among these patients, 5/9 (55.5%) were diagnosed with tuberculosis alongside other comorbidities. These patients were treated with antituberculosis drugs such as rifampicin, ethambutol, and isoniazid, which may contribute to increased antibiotic resistance. Concurrent infections of M. tuberculosis and B. pseudomallei were identified, although co-infection is rare. A similar case of concurrent tuberculosis and melioidosis was also documented in Singapore in 2014, marking the first reported instance of these conditions occurring simultaneously in the site of infection.²⁷ This study highlighted cases where both tuberculosis and melioidosis occurred simultaneously in the same patient. Although antibiotic therapy can alleviate the acute symptoms of melioidosis, the infection is known for its potential to relapse, with symptoms sometimes reappearing months or even years after apparent clinical resolution.²⁸

Melioidosis is infrequently reported in Indonesia, with many cases noted only by their clinical symptoms without confirmatory laboratory evidence. This study documents the occurrence of B. pseudomallei infection in North Sumatra Province and its surrounding areas, including a notable case of coinfection with pulmonary tuberculosis. Approximately half of the melioidosis cases present with pneumonia, which may progress to fatal septicemia, mimicking unilateral pneumonia or other chronic diseases. Chronic forms of melioidosis often manifest as persistent pneumonia or non-healing skin sores.^{11,13,15,16,20} In this study, we identified a pediatric case where a child with a burn wound was infected by B. pseudomallei.²⁹ A study in India noted melioidosis in 8% of children, primarily at local lesion sites.³⁰ Research in Southeast Asia indicates that children more frequently exhibit local manifestations such as skin lesions or lymphadenopathy, ranging from 46%-63%.³¹ Geographically, the distribution of melioidosis cases in this study extended beyond North Sumatra; one pediatric patient with a burn lesion infection originated from Aceh Province, specifically in Aceh Singkil District, and was referred to Universitas Sumatera Utara Hospital for surgical treatment. The other eight cases were from North Sumatra Province (Figure 3). Transmission of B. pseudomallei typically occurs through unprotected occupational exposure to soil or water, ingestion of contaminated water or food, and inhalation.³⁰ North Sumatra, with a predominant traditional farming community, poses a higher risk for melioidosis. Additionally, its proximity to Thailand and Malaysia, countries with high incidences of melioidosis, increases the risk of cross-border transmission, particularly for travelers.

A high index of suspicion is essential for diagnosing melioidosis in non-endemic regions. Clinicians should consider melioidosis in patients presenting with fever who have any of the following: history of residence or travel to endemic areas; occupational or recreational exposure to soil or water potentially harboring B. pseudomallei, including military personnel; or risk factors like diabetes mellitus or kidney disease.³¹ Early engagement with clinical microbiology laboratories is crucial when investigating potential cases to enhance the recognition of significant pathogens in mixed cultures. Moreover, B. pseudomallei is categorized as a hazard group 3 biological agent, requiring stringent containment measures during handling. Employing selective agar is recommended to improve culture sensitivity from non-sterile clinical samples.²¹

In Thailand, the management of melioidosis is informed by a series of clinical trials spanning the last 25 years and involves a dual-phase treatment approach: an acute phase aimed at mitigating life-threatening sepsis, followed by an eradication phase designed to eliminate residual bacteria and reduce recurrence risk.²⁵ The acute phase employs ceftazidime, with carbapenem reserved for severe cases or when initial treatment fails, and amoxicillin/clavulanic acid (co-amoxiclav) as a secondary option. The eradication phase typically involves trimethoprim/sulfamethoxazole or co-amoxiclav as alternatives, with treatment customization based on the patient’s clinical manifestations and response.^26,32

Diagnostic investigations for melioidosis depend on the resources available, which are often limited in regions where the disease is endemic. Supporting laboratory tests include those for acute renal failure, liver function abnormalities, anemia, and coagulation disorders, which are prevalent in severe cases of melioidosis.⁴ All patients should undergo chest X-ray imaging, which might show localized alveolar patch infiltrates, diffuse bilateral alveolar effusions, or multiple nodular lesions indicative of hematogenous spread. Differentiating upper lobe infections from pulmonary TB can be challenging due to similarities in clinical and radiological presentations. Conditions such as empyema or pulmonary abscess are well-documented, warranting repeated chest radiographs for patients with respiratory involvement. Arterial blood gases are recommended for those with pulmonary issues or sepsis. Comprehensive imaging such as ultrasound, CT scans, or other modalities should be used to check for abscesses in organs like the liver and spleen and to assess potential prostate involvement.⁹

Currently, there is no vaccine for melioidosis; thus, preventive measures are crucial. Even individuals who have previously had melioidosis can be reinfected if exposed again. The diverse clinical presentations make it challenging to differentiate melioidosis from other bacterial infections, acute or chronic. Confirming a diagnosis relies on proper specimen collection, culture, and isolation of B. pseudomallei, with culture remaining the diagnostic gold standard.