Composition Vendor Newgen KnowledgeWorks (P) Ltd. PLoS Negl Trop Dis plos plosntds PLOS Neglected Tropical Diseases 1935-2735 Public Library of Science San Francisco, CA USA 10.1371/journal.pntd.0013962 PNTD-D-25-01265 Research Article Biology and life sciencesOrganismsBacteriaBurkholderiaBurkholderia pseudomallei Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensBacterial pathogensBurkholderiaBurkholderia pseudomallei Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensBacterial pathogensBurkholderiaBurkholderia pseudomallei Medicine and health sciencesPharmacologyDrugsAntimicrobialsAntibiotics Biology and life sciencesMicrobiologyMicrobial controlAntimicrobialsAntibiotics Medicine and health sciencesMedical conditionsInfectious diseasesBacterial diseasesMelioidosis Medicine and health sciencesSurgical and invasive medical procedures Research and analysis methodsSpecimen preparation and treatment Medicine and health sciencesMedical conditionsSkin diseasesSkin infections Medicine and health sciencesDermatologySkin diseasesSkin infections Medicine and health sciencesMedical conditionsInfectious diseasesSkin infections Medicine and health sciencesClinical medicineSigns and symptomsAbscesses Research and analysis methodsSpecimen preparation and treatmentStainingMembrane stainingGram staining Rapid diagnosis of skin and soft tissue melioidosis in children Diagnosis of skin and soft tissue melioidosis in children https://orcid.org/0009-0005-0398-5030 Suy Keang Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Visualization Writing – original draft Writing – review & editing 1 2 * Bott Seda Data curation Writing – review & editing 1 Leng Nara Resources Writing – review & editing 3 Sar Vuthy Resources Writing – review & editing 3 Soeng Sona Investigation Resources Writing – review & editing 1 Real Sophanith Data curation Writing – review & editing 1 Dance David AB Resources Writing – review & editing 4 5 Lee Sue J. Formal analysis Methodology Resources Visualization Writing – review & editing 6 7 8 Turner Paul Conceptualization Methodology Resources Supervision Writing – review & editing 1 6 Ling Clare L. Conceptualization Investigation Methodology Project administration Resources Supervision Visualization Writing – review & editing 1 6 Chandna Arjun Conceptualization Methodology Project administration Resources Supervision Writing – review & editing 1 6 9 Cambodia Oxford Medical Research Unit, Angkor Hospital for Children, Siem Reap, Cambodia Medical Department, Angkor Hospital for Children, Siem Reap, Cambodia Surgical Unit, Angkor Hospital for Children, Siem Reap, Cambodia Lao Oxford Mahosot Hospital Wellcome Trust Research Unit, Mahosot Hospital, Vientiane, Lao PDR, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom Centre for Tropical Medicine & Global Health, University of Oxford, Oxford, United Kingdom Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand Department of Infectious Diseases, Alfred Hospital and School of Translational Medicine, Monash University, Melbourne, Australia Clinical Research Department, London School of Hygiene & Tropical Medicine, London, United Kingdom Poonawala Husain Editor Tufts Medical Center, UNITED STATES OF AMERICA * E-mail: suykeang@angkorhospital.org

I have read the journal’s policy and the authors of this manuscript have the following competing interests: DABD acts as a paid independent consultant for InBios. All other authors have declared that no competing interests exist.

☯ These authors contributed equally to this work.

 322026 22026 20 2 e0013962 2972025 2012026 2026 Suy et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Melioidosis is an endemic infection caused by Burkholderia pseudomallei, found in tropical and subtropical regions. In resource-limited settings, culture-based diagnostics are often slow, delaying appropriate treatment, or unavailable. We conducted an interventional ambispective cohort study to assess the impact and diagnostic accuracy of the Active Melioidosis Detect Plus rapid diagnostic test (AMD-RDT) in Cambodian children with suspected skin and soft tissue melioidosis (SST-M). The retrospective cohort (July 2022–July 2023) received standard diagnostics; the prospective cohort (July 2023–December 2024) included AMD-RDT testing. Twenty-five retrospective culture-confirmed participants and 107 participants (31 culture-confirmed) in the prospective arm were analysed. Median time from pus collection to appropriate antibiotic initiation was 118.4 hours in the retrospective arm and 14.4 hours in the prospective arm (p = 0.057). Disseminated melioidosis workups were completed for 24% (6/25) and 80.6% (25/31) of retrospective and prospective participants respectively (p < 0.001), and detected two children with bacteraemia and three with intra-abdominal abscesses in the prospective arm. The AMD-RDT achieved a sensitivity of 90.3% (95% CI: 74.2–98.0%), specificity of 100% (95% CI: 95.3–100%), and an area under the receiver operating characteristic curve (AUC) of 0.95 (95% CI 0.89-1.00). Incorporating the AMD-RDT into the routine diagnostic pathway was associated with a reduction in time to effective antibiotic therapy and an increase in the proportion of participants completing a comprehensive diagnostic workup for systemic involvement. The high accuracy and rapid turnaround time support its use in resource-limited settings.

 Author summary

Melioidosis is a serious infection caused by the soil-dwelling bacterium Burkholderia pseudomallei. Diagnosis requires culture of specimens taken from infected sites, which is the gold standard, but is often slow or unavailable in resource-limited settings. In our study, the Active Melioidosis Detect Plus rapid test was added into routine diagnostic processes. We found children received appropriate treatment faster, with the majority undergoing further testing to look for systemic infection. We also found that the rapid test had excellent accuracy when compared to culture.

 http://dx.doi.org/10.13039/100010269 Wellcome Trust 220211/Z/20/Z This research was conducted by the MORU Tropical Health Network, which is funded through a core grant block award from the Wellcome Trust (220211/Z/20/Z). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability De-identified, individual participant data from this study will be available to researchers whose proposed purpose of use is approved by the MORU data access committee. Enquiries or requests for data can be sent to datasharing@tropmedres.ac. PLOS Publication Stage vor-update-to-uncorrected-proof Publication Update 2026-02-06 Introduction

Melioidosis is an important endemic infection prevalent in tropical and subtropical regions, caused by Burkholderia pseudomallei, a Gram-negative, soil-dwelling saprophyte [1]. The diagnosis of melioidosis requires microbiological confirmation due to clinical features that overlap with those of other bacterial infections. Traditional culture-based diagnostic methods, which remain the gold standard, typically take 48 hours or longer to yield results [2–4]. This delay presents critical challenges, particularly because B. pseudomallei exhibits intrinsic resistance to many commonly used first-line antibiotics and therefore requires specific treatment. Delaying the initiation of appropriate antimicrobial therapy can compromise patient outcomes.

In resource-limited settings, patients with skin and soft tissue infections without systemic symptoms are frequently treated by surgical debridement and discharged with empirical antibiotics targeting common pathogens such as Staphylococcus aureus and Streptococcus spp., which are ineffective against B. pseudomallei. Microbiological culture is not always available and, even when performed, results are often delayed, by which time patients are often uncontactable (common reasons include living far from hospitals with capacity for microbiological culture, peripatetic lifestyles, and unreliable access to a functioning telephone). Additionally, such patients are less likely to be thoroughly screened (with blood culture and abdominal imaging) for disseminated disease, which can lead to serious complications if left undetected. Current guidelines for melioidosis treatment recommend an initial intensive phase of treatment with intravenous antibiotics (ceftazidime or meropenem) for at least 10–14 days [5], yet in low-resource healthcare settings, cutaneous melioidosis is often managed with oral antibiotics (co-trimoxazole) alone [2]. This makes exclusion of disseminated disease imperative to prevent sub-optimal treatment.

The Active Melioidosis Detect Plus rapid diagnostic test (AMD-RDT) is a qualitative, membrane-based lateral flow immunoassay point-of-care test that detects the capsular polysaccharide of B. pseudomallei, developed by InBios (Seattle, WA, USA) [6]. Previous studies of the AMD-RDT have shown promise, demonstrating high sensitivity and near-perfect specificity for pus specimens when compared to culture [3,7]. This rapid test significantly reduces diagnostic turnaround time, offering an advantage where timely diagnosis is critical, especially in resource-limited settings where culture may not be readily available. However, the impact of the test on clinical management decisions remains underexplored. This study assessed the impact of the AMD-RDT on the management of children presenting with skin and soft tissue infections to a paediatric hospital in northern Cambodia.

 Methods Ethics statement

Written informed consent was not sought because specimens were collected as part of routine care at the discretion of the surgical team. However, the study was explained to patients and/or their caregivers, including their right to opt out of the study if they wished to do so (see Intervention section). The study was approved by the AHC Institutional Review Board (IRB-0099/23AHC; March 2023), the Cambodian National Ethics Committee for Human Research (NECHR149; May 2023), and the Oxford Tropical Research Ethics Committee (OxTREC: 528:23; July 2023). The study is reported in line with the CONSORT guidelines for feasibility trials (S1 Text) [8].

 Study setting

The study was conducted at Angkor Hospital for Children (AHC), Siem Reap, Cambodia. AHC is a non-governmental paediatric healthcare organisation with a nationwide catchment area. The hospital provides services from primary to tertiary care and has 89 beds situated on two medical wards, a surgical ward, a special care baby unit, and neonatal and paediatric intensive care units. The 12-bed surgical unit has approximately 700 annual admissions and a minor procedure room which provides low-risk surgical care to approximately 10,000 patients with 1,600 procedures conducted annually. Most children presenting with skin and soft tissue infections are treated in the minor procedure room, receiving wound management and/or incision and drainage of abscesses, before being discharged home the same day.

 Study design

This was an interventional ambispective cohort study with a before-and-after comparison. The study included both a retrospective and a prospective arm; with recruitment for the prospective arm beginning after the intervention, i.e., introduction of the AMD-RDT. For the retrospective arm, clinical and laboratory data were analysed from children with pus or pus swab specimens sent to the microbiology laboratory between 14th July 2022 and 13th July 2023 that were culture-positive for B. pseudomallei.

For the prospective arm, consecutive pus and pus swab specimens sent to the microbiology laboratory with a request for melioidosis culture from 14th July 2023 until 31st December 2024 were screened by laboratory staff to identify potential participants. Participants were included if they were aged under 16 years and were presenting with a skin or soft tissue infection (including cellulitis, wound infections, skin abscesses, lymphadenitis, or parotitis). Pus or pus swab specimens from which the initial Gram stain showed Gram-positive cocci only were excluded (S1 Fig). Patients were not enrolled if they had already been included in the study for the same illness episode (still receiving antibiotics or under active follow up).

 Data collection

A structured paper case report form was used to capture data for both study arms. Data were subsequently entered into the study’s REDCap database hosted at the Cambodia Oxford Medical Research Unit, Angkor Hospital for Children [9]. Clinical and laboratory data were retrieved from the Laboratory Information Management System (LIMS), Hospital Information System (HIS), clinical records, and the hospital and laboratory logbooks. Data included basic demographics, clinical presentation, management plan (including diagnostic tests, disposition, and treatment), microbiological workup, and results of the AMD-RDT. All variables were prospectively defined in a data dictionary to ensure consistency of interpretation across the research team.

 Intervention

Prior to commencement of the prospective arm, a meeting with the surgical team was held to introduce the study and explain the eligibility criteria, AMD-RDT procedures, and the interpretation of negative and positive AMD-RDT results. During the meeting, the preliminary nature of the rapid test results was emphasised and that the final diagnosis should be based on the culture results. It was explained that the initial test results (Gram stain with or without AMD-RDT) would be available approximately one hour after the specimens were collected.

While waiting for their results, patients and/or caregivers had the study explained to them, were given time to ask questions, and had the opportunity to opt out. The study team phoned the surgical team to inform them of the initial Gram stain result and, if performed, the AMD-RDT result. All patient management decisions were the responsibility of the surgical team who had access to local hospital and national treatment guidelines for melioidosis.

 Laboratory procedures

The AMD-RDT was performed on pus or pus swab specimens for participants in the prospective arm, with results reported back to the treating surgical team approximately 60 minutes after specimen collection. Following pre-test steps for pus or pus swab specimens, the test was performed following the manufacturer’s instructions which was described previously [3].

For pus specimens, a heating step, as described in a recent evaluation of the test (and intended for inclusion in the manufacturer’s FDA approval application) [3], was performed prior to conducting the AMD-RDT to enhance sensitivity. Briefly, 30 μL of pus was collected using a pipettor and transferred into a 2 mL sterile microtube. Four drops of 20x lysis buffer were added, and the microtube was vortexed thoroughly in three 10-second rounds. The microtube was then placed in a heating block set to 95°C for 15 minutes, with brief vortexing at 5 and 10 minutes. For pus swab specimens, prior to performing the AMD-RDT, the swabs were expressed in a microtube containing 500 μL of sterile water. A 50 μL aliquot of the swab eluate was then transferred into a new 2 mL sterile microtube. Four drops of 20x lysis buffer were added, and the microtube was vortexed for three 10-second intervals. Following the pre-test steps, 50 μL of the prepared pus or pus swab specimens was applied to the AMD-RDT sample port, followed by the addition of two drops of chase buffer. The results were read and interpreted after 15–20 minutes at room temperature.

Following Gram stain (with or without AMD-RDT), routine and B. pseudomallei culture was performed on all pus and pus swab specimens using blood agar, chocolate agar, MacConkey agar, Ashdown agar, and in-house selective broth (S2 Text) that was subcultured onto Ashdown agar (S1 Fig). B. pseudomallei isolates were identified using latex agglutination (Mahidol University, Thailand) and by MALDI-TOF mass-spectrometry (bioMerieux VITEK MS) using in-house SuperSpectra [10,11]. The microbiology laboratory obtained ISO accreditation under standards ISO 15189 and ISO 15190 in November 2023.

 Outcomes

There were two co-primary outcomes. The first was time to initiation of an antimicrobial with activity against B. pseudomallei for participants with culture-confirmed B. pseudomallei infection. This was defined as the time between collection of the pus specimen and the start of an antimicrobial agent with activity against B. pseudomallei (ceftazidime, meropenem, or co-trimoxazole). The second co-primary outcome was the proportion of participants with culture-confirmed B. pseudomallei infection in whom a blood culture and abdominal ultrasound were completed. The secondary outcome was the diagnostic accuracy of the AMD-RDT on pus/pus swab specimens, using microbiological culture as the reference standard.

 Sample size

Sample size calculation was pragmatic and based on the resources available for the study which permitted prospective recruitment for 12 months, which was extended to 15 months during the study due to a slower than expected recruitment rate. Using routinely collected baseline data from previous years at the same hospital, it was expected that 100 participants would be recruited over 12 months, of which approximately 40 were anticipated to have B. pseudomallei identified. Retrospective cases spanned one calendar year prior to the introduction of the AMD-RDT.

 Statistical analysis

All variables were summarised and reported using descriptive statistics. Continuous variables were reported using mean with standard deviation or median with 25th and 75th centiles. Categorical variables were summarised with frequency and percentage. Comparisons were made using the Mann-Whitney-U or Fisher’s Exact test.

Kaplan-Meier analysis and Cox regression were used to compare the time from pus collection to initiation of an antimicrobial with activity against B. pseudomallei between the prospective and retrospective arms. Participants who received antibiotics with activity against B. pseudomallei empirically (i.e., prior to pus collection) were excluded from the time to effective treatment analysis. Time 0 was set as the time when the pus specimen was collected. Children who did not receive effective treatment were censored at maximum follow up time. Hazard ratios with 95% confidence intervals are reported.

In the prospective arm, the accuracy of the AMD-RDT compared to culture was determined by the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). These metrics were calculated with 95% confidence intervals using Stata version 18 (Stata Corp LLC, College Station, TX, USA) and R version 4.3.2.

 Results Retrospective arm

Between 14th July 2022 and 13th July 2023, 476 pus and pus swab specimens were collected from 408 patients and sent to the microbiology laboratory. Of those, 171 specimens included a request for melioidosis culture. Twenty-five (14.6%; 25/171) specimens from 25 unique participants grew B. pseudomallei (Fig 1). Median age was 5.4 years (interquartile range [IQR] 2.5 to 8.2 years) and 68.0% (17/25) of the cohort were male (Table 1). The most common clinical presentation was acute lymphadenitis (52.0%; 13/25), followed by skin infections (36.0%; 9/25), and parotitis (12.0%; 3/25). Fifteen (60%;15/25) participants came from within Siem Reap province, while the other ten (40%; 10/25) came from four other provinces (S2 Fig). Sixty percent (15/25) of participants were confirmed as having recovered after receiving the recommended three-month course of antibiotics, while 32.0% (8/25) were lost to follow-up prior to completion of treatment; all were prescribed at least one month of oral co-trimoxazole after diagnosis. Two participants (2/25; 8.0%) did not receive antibiotics with efficacy against B. pseudomallei, one was treated with surgical drainage alone and the other discharged with antibiotics without activity against B. pseudomallei prior to culture results being available.

10.1371/journal.pntd.0013962.t001Characteristics of participants with confirmed melioidosis in the retrospective and prospective arms.
Characteristics Retrospective armN (%), Total = 25 Prospective armN (%), Total = 31 p-value
Median age (IQR), years 5.4 (2.5-8.2) 3.9 (3.6-6.8) 0.85
Sex
 Female 8 (32.0) 18 (58.1) 0.064
 Male 17 (68.0) 13 (41.9)
Clinical presentation
 Parotitis 3 (12.0) 15 (48.4) 0.009
 Skin infection 9 (36.0) 4 (12.9)
 Lymphadenitis 13 (52.0) 12 (38.7)
Gram stain of pus/pus swab
 No bacteria seen 24 (96.0) 10 (32.3) < 0.001
 Gram-negative bacilli only 1 (4.0) 19 (61.3)
 Mixed 0 2 (6.5)
Diagnostic workups
Blood culture performed 7 (28.0) 25 (80.6) < 0.001
 B. pseudomallei isolated 0 2
Abdominal ultrasound performed 6 (24.0) 29 (93.5) < 0.001
 Splenic abscess 2 2
 Liver abscess 0 2
Antimicrobial management
No antimicrobials with activity against B. pseudomallei 2 (8.0) 0
Empirical anti-B. pseudomallei treatment 7 (28.0) 11 (35.5) 0.58
Median time to effective antimicrobial treatment (IQR), hours* 118.4 (21.3-143.3) 14.4 (3.3-70.7) 0.057
Outcome
 Recovered** 16 (64.0) 22 (71.0) 0.77
 Lost to follow-up*** 9 (36.0) 9 (29.0)

* Includes n = 18 from retrospective and n = 20 from prospective arm who did not receive effective treatment before pus collection. Time to treatment was set to maximum follow up time for the two children in the retrospective arm who did not receive antibiotics with efficacy against B. pseudomallei. ** One participant was treated with surgical debridement alone and considered recovered at follow-up. *** One participant was treated with antibiotics without activity against B. pseudomallei and was uncontactable after leaving the study site.

 10.1371/journal.pntd.0013962.g001Enrolment flowchart for retrospective (A) and prospective (B) arms.

Culture positive specimens were included in the time to effective treatment and diagnostic workup analyses. All prospective specimens (n = 107) were included in the AMD-RDT diagnostic accuracy analyses. GPC = Gram-positive cocci.

 Prospective arm

There were 208 pus and pus swab specimens sent with a request for melioidosis culture to the microbiology laboratory from 202 unique patients between 14th July 2023 and 31st December 2024. Gram stain of the specimens identified 100 specimens with Gram-positive cocci (GPC) only, which were excluded. One of two specimens that were sent from the same patient was excluded. The remaining 107 specimens from 107 unique patients were enrolled. No participants opted out of the study. Sixty-nine specimens (64.5%; 69/107) had no bacteria seen on Gram stain, Gram-negative bacilli (GNB) were seen in 23 specimens (21.5%; 23/107), and mixed GPC and GNB were seen in 15 specimens (14.0%; 15/107) (S1 Table).

Thirty-one of the 107 specimens (28.9%) included in the study were culture positive for B. pseudomallei (Fig 1). Median age of children who were positive for B. pseudomallei was 3.9 years (IQR 3.6 to 6.8 years) and 58.1% were female (18/31). Fifteen specimens (48.4%; 15/31) were sent from participants with parotitis, twelve (38.7%; 12/31) from participants with acute lymphadenitis, and four (12.9%; 4/31) from participants with skin infections (Table 1). Twenty-one participants (67.7%; 21/31) came from within Siem Reap province while the other ten (32.3%; 10/31) came from three other provinces (S2 Fig).

Twenty-two participants (71.0%; 22/31) were confirmed as having recovered after receiving the recommended three months of antibiotics. Nine participants (29.0%; 9/31) were lost to follow-up prior to completion of three months therapy; but all were prescribed at least one month of co-trimoxazole after diagnosis.

 Time to effective treatment

In the retrospective arm, all participants received empirical treatment for their infection. Seven (28%; 7/25) received empirical antibiotics with activity against B. pseudomallei prior to pus collection; 16 were switched to effective melioidosis treatment following specimen collection and two, both presenting with skin infections, did not receive antimicrobials with activity against B. pseudomallei (S3A Fig in S3 Fig). Of the participants who received treatment active against B. pseudomallei, seven (30.4.0%; 7/23) received intensive phase treatment (intravenous ceftazidime) followed by oral co-trimoxazole, while 16 (69.6%; 16/23) received oral co-trimoxazole monotherapy.

In the prospective arm, all B. pseudomallei culture-positive participants received empirical treatment for their infection episodes. Of those, eleven (35.5%; 11/31) received antimicrobials with activity against B. pseudomallei prior to pus collection and 20 (64.5%; 20/31) were switched to effective treatment following pus collection (S3B Fig in S3 Fig). Twenty-one participants (67.7%; 21/31) received intensive phase treatment prior to oral therapy, whilst 10 (32.3%; 10/31) received oral co-trimoxazole monotherapy.

There was no difference in the frequency of effective empiric treatment before pus collection between the prospective and retrospective arms (11/31 (35.5%) v. 7/25 (28.0%), p = 0.58). Among the 18 children in the retrospective arm and 20 in the prospective arm who did not receive effective empiric treatment before pus collection, median time from pus collection to initiation of treatment with an antimicrobial with activity against B. pseudomallei was shorter in the prospective arm but this was not statistically significant (118.4 hours [IQR 21.3 to 143.3 hours] in the retrospective arm and 14.4 hours [IQR 3.3 to 70.7 hours; p = 0.057] in the prospective arm; Hazard ratio 0.53; 95% CI 0.27 to 1.03; p = 0.061; Fig 2).

10.1371/journal.pntd.0013962.g002Kaplan-Meier plot of the time to effective treatment for both study arms.

Included retrospective arm (n = 18) and prospective arm (n = 20). Time to effective treatment was defined from time of pus collection to initiation of antibiotics with activity against B. pseudomallei.

Among participants who received anti-B. pseudomallei antibiotics after pus collection, 10 (50.0%; 10/20) in the prospective arm and four (25.0%; 4/16, p = 0.17) in the retrospective arm received them on the day of pus collection, while four (20.0%; 4/20) in the prospective arm and eight (50%; 8/16, p = 0.081) in the retrospective arm received them more than three days after pus collection.

 Diagnostic workups

Both blood culture and abdominal ultrasound were performed in 24.0% (6/25) of participants in the retrospective arm and 80.6% (25/31) in the prospective arm (p < 0.001).

In the retrospective arm, blood cultures were performed in seven out of 25 participants (28.0%), all of which were negative, while abdominal ultrasound was conducted in six participants (24.0%; 6/25), identifying splenic microabscesses in two cases (Table 1). In the prospective arm, blood cultures were collected in 25 out of 31 participants (80.6%; p < 0.001), with B. pseudomallei isolated in two cases. Abdominal ultrasound was performed in 29 participants (93.5%; 29/31; p < 0.001), revealing intra-abdominal abscesses in three participants: one with a splenic abscess, one with a liver abscess, and one with both liver and splenic abscesses.

 Accuracy of the AMD-RDT

The AMD-RDT was performed on 107 specimens from the prospective arm. Among 31 culture positive specimens (four pus specimens and 27 pus swab specimens), 28 had a positive AMD-RDT result. All three negative AMD-RDT results occurred in pus swabs. None of the culture negative specimens had positive AMD-RDT results (Table 2).

10.1371/journal.pntd.0013962.t002Results of culture and AMD-RDTs for specimens enrolled in the prospective arm.
AMD RDT Total
Positive Negative
B. pseudomallei culture Positive 28 3 31
Negative 0 76 76
Total 28 79 107

Receiver operating characteristic curve analysis showed an AUC of 0.95 (95% CI 0.89-1.00), with a sensitivity of 90.3% (95% CI 74.2 to 98.0%), a specificity of 100% (95% CI 95.3 to 100%), a PPV of 100% (95% CI 87.7 to 100%), and a NPV of 96.3% (95% CI 89.4 to 99.2%).

The three pus swab specimens that were culture positive for B. pseudomallei but tested negative on the AMD-RDT were sent from two participants with acute parotitis and one participant with subacute cervical lymphadenitis. Information regarding antibiotic use prior to presentation was not available. Gram stain of the specimens did not identify presence of bacteria in two while Gram-negative bacilli were seen on one. Two specimens yielded a single colony of B. pseudomallei on chocolate agar at 24 hours, with growth observed on all other culture media at 48 hours of incubation. The third swab grew B. pseudomallei on Ashdown agar after subculture from selective broth at 48 hours.

 Discussion

Melioidosis remains a significant diagnostic challenge in many endemic tropical regions due to limitations in laboratory capacity and prolonged turnaround time of culture-based diagnosis. To our knowledge, this is the first study assessing the impact of the AMD-RDT on the care of children presenting with suspected skin and soft tissue melioidosis. Using a pragmatic before-and-after study design, we found a reduction in time from pus collection to initiation of effective antibiotics after the AMD-RDT was incorporated into the routine diagnostic workflow. The proportion of children receiving effective antibiotics on the day of presentation increased by more than 50% (21/31; 67.7% vs. 11/25; 44.0%), with the proportion of children who never received an effective antibiotic dropping from close to one in ten to zero after the rapid test was introduced.

Nevertheless, even though the AMD-RDT took less than one hour to perform, only two-thirds of the participants (21/31) received effective antibiotics on the day of sample collection, and 10% (4/31) received effective antibiotics more than three days later. Delays were often observed in participants whose specimens were collected towards the end of a day, with results only being reviewed the next working day, which could be up to three days for specimens collected prior to a weekend. This illustrates the importance of considering the existing patient care pathway when introducing a new diagnostic test, including ensuring that there is a mechanism for results to be acted upon, otherwise the potential for impact will be limited. This observation is not unique to melioidosis. Similar findings have been reported in a meta-analysis of studies investigating interventions aimed at improving the time to effective antibiotics in bacteraemic patients [12].

After the training session and the introduction of the AMD-RDT, we saw a three-fold increase in the proportion of participants who underwent workup for disseminated disease. Two participants with bacteraemia and three with intra-abdominal abscesses were identified in the prospective arm, whose disseminated disease may have gone unrecognised leading potentially to unfavourable outcomes. In addition, the proportion of participants who received the recommended two-phase therapy doubled. These findings highlight the potential benefit of using the AMD-RDT, which provides same day diagnosis, together with appropriate training to enable timely and targeted treatment of melioidosis, including for patients with systemic involvement.

We found that the test had excellent sensitivity and specificity and demonstrated high diagnostic accuracy as shown by the AUC. These findings are consistent with those reported by Currie and colleagues who refined the sample preparation steps (heating for pus and lysis buffer for swabs to enhance test sensitivity) that we subsequently used in our study [3]. Our results are also aligned with previous studies in term of test specificity [4,7,13,14]. Taken together, the evidence to date supports the use of the AMD-RDT on pus specimens collected from both adults and children. The three false negative AMD-RDT results were observed in specimens where bacterial load was likely low, as evidenced by their specimen culture result. We also observed that despite recommended by the microbiology laboratory to collect pus samples, pus swabs are often sent by the treating clinical teams.

This study has limitations. First, the results may have been confounded by temporal changes in healthcare worker behaviour. Nevertheless, a before-and-after design is an appropriate approach for evaluating implementation of an intervention with proven accuracy into routine care [15]. Second, it is unclear how much the increase in systemic diagnostic work ups was influenced by the surgical team training provided during implementation, rather than early diagnosis of melioidosis by the AMD-RDT prompting clinicians to investigate for systemic disease. Although empirical prescribing practices did not appear to alter, it is important to ensure that the rollout of any new test is supported by appropriate diagnostic stewardship. Third, although the pre-test steps may have improved the accuracy of the test, they limit its use to a laboratory setting with appropriate safety measures. Fourth, a higher than anticipated empirical prescribing rate of anti-B. pseudomallei antibiotics resulted in the group available for the time to effective treatment analysis being smaller than expected, and whilst it did indicate a reduction in time to effective therapy (118 vs 14 hours), the estimate was imprecise and did not reach statistical significance. There were also a large number of patients lost to follow-up (29% in the prospective arm and 36% in the retrospective arm) which limits our ability to determine final clinical outcome for both groups, although this did not impact the study’s co-primary outcomes. Finally, differences in the baseline characteristics, in particular a higher proportion of samples with bacteria visible on Gram stain in the prospective cohort which may reflect higher bacterial loads, may have introduced bias, which is a limitation of this non-randomised study design. Another study design limitation was that the study was conducted in a single-centre and in a paediatric only population, which may limit generalisability of the findings.

In conclusion, introduction of the AMD-RDT was associated with a reduction in time to effective antibiotic therapy and, together with training of the clinical staff, an increase in the proportion of participants completing a comprehensive diagnostic workup for systemic involvement. With excellent accuracy, the test is applicable for both children and adults in resource-limited settings where access to culture diagnosis may not be feasible or delayed. Where possible, the test should be an adjunct to, rather than a replacement for, culture-based diagnosis, as this could lead to loss of critical information on antimicrobial susceptibility over time. To maximise impact, it is imperative that the test is embedded within functioning care pathways that allow timely action following the result. Future research should look to replicate our findings and explore the cost-effectiveness of incorporating the AMD-RDT into routine diagnostic protocols in resource-limited settings.

 Supporting information Diagnostic workflow for participants in the prospective arm.

Different media types and incubation conditions were used to allow detection of a broad range of pathogens with varying culture requirements. GPC: Gram-positive cocci; AMD-RDT: Active Melioidosis Detect Plus rapid diagnostic test; B. pseudomallei: Burkholderia pseudomallei; MALDI-TOF MS: Matrix-assisted laser desorption/ionisation mass spectrometry.

(TIF)

 Case Distribution in Cambodia by Provinces and District of their origin.

(source of the basemap shapefile: https://data.humdata.org/dataset/cod-ab-khm).

(TIF)

 Number of participants receiving empirical antibiotics with activity against <italic>Burkholderia pseudomallei.</italic>

A: Retrospective arm, number of participants = 25; B: Prospective arm, number of participants = 31.

(TIF)

 <p>Summary of information reported in accordance with CONSORT 2010 checklist (source of the checklist: <ext-link ext-link-type="uri" xlink:href="https://www.equator-network.org/reporting-guidelines/consort-2010-statement-extension-to-randomised-pilot-and-feasibility-trials/" xlink:type="simple">https://www.equator-network.org/reporting-guidelines/consort-2010-statement-extension-to-randomised-pilot-and-feasibility-trials/</ext-link>).</p> <p>(DOCX)</p> </caption> </supplementary-material> <supplementary-material id="pntd.0013962.s005" mimetype="application/vnd.openxmlformats-officedocument.wordprocessingml.document" position="float" xlink:href="info:doi/10.1371/journal.pntd.0013962.s005" xlink:type="simple"> <label>S2 Text</label> <caption> <title>In-house media preparation: A.

Selective Broth, B: Ashdown Agar.

(DOCX)

 Characteristics of participants in prospective arm.

(DOCX)

We are grateful to all the participants and their caregivers, the AHC surgical team, and the COMRU laboratory and research staff for their specimen processing, data management and study monitoring. We acknowledge the support of InBios, who provided the AMD-RDTs used in the study. After preparation of the initial draft, the corresponding author (KS) used the free version of OpenAI’s ChatGPT (GPT-4o, May 2024 version) in order to enhance readability and grammar. After using this tool, all authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

 References Limmathurotsakul D, Golding N, Dance DA, Messina JP, Pigott DM, Moyes CL, et al. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat Microbiol. 2016;1(1):15008. doi: 10.1038/nmicrobiol.2015.8 26877885 Chandna A, Bonhoeffer M, Miliya T, Suy K, Sao S, Turner P. Improving Treatment and Outcomes for Melioidosis in Children, Northern Cambodia, 2009-2018. Emerg Infect Dis. 2021;27(4):1169–72. doi: 10.3201/eid2704.201683 33754990 Currie BJ, Woerle C, Mayo M, Meumann EM, Baird RW. What is the Role of Lateral Flow Immunoassay for the Diagnosis of Melioidosis?. Open Forum Infect Dis. 2022;9(5):ofac149. doi: 10.1093/ofid/ofac149 35493111 Radhakrishnan A, Behera B, Mishra B, Mohapatra PR, Kumar R, Singh AK. Clinico-microbiological description and evaluation of rapid lateral flow immunoassay and PCR for detection of Burkholderia pseudomallei from patients hospitalized with sepsis and pneumonia: A twenty-one months study from Odisha, India. Acta Trop. 2021;221:105994. doi: 10.1016/j.actatropica.2021.105994 34118206 Currie BJ, Mayo M, Ward LM, Kaestli M, Meumann EM, Webb JR, et al. The Darwin Prospective Melioidosis Study: a 30-year prospective, observational investigation. Lancet Infect Dis. 2021;21(12):1737–46. doi: 10.1016/S1473-3099(21)00022-0 34303419 Houghton RL, Reed DE, Hubbard MA, Dillon MJ, Chen H, Currie BJ, et al. Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis. PLoS Negl Trop Dis. 2014;8(3):e2727. doi: 10.1371/journal.pntd.0002727 24651568 Rizzi MC, Rattanavong S, Bouthasavong L, Seubsanith A, Vongsouvath M, Davong V, et al. Evaluation of the Active Melioidosis Detect™ test as a point-of-care tool for the early diagnosis of melioidosis: a comparison with culture in Laos. Trans R Soc Trop Med Hyg. 2019;113(12):757–63. doi: 10.1093/trstmh/trz092 31638152 Eldridge SM, Chan CL, Campbell MJ, Bond CM, Hopewell S, Thabane L, et al. CONSORT 2010 statement: extension to randomised pilot and feasibility trials. BMJ. 2016;355:i5239. doi: 10.1136/bmj.i5239 27777223 Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG. Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inform. 2009;42(2):377–81. doi: 10.1016/j.jbi.2008.08.010 18929686 Anuntagool N, Naigowit P, Petkanchanapong V, Aramsri P, Panichakul T, Sirisinha S. Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. J Med Microbiol. 2000;49(12):1075–8. doi: 10.1099/0022-1317-49-12-1075 11129718 Watthanaworawit W, Roberts T, Hopkins J, Gassiep I, Norton R, Robinson MT, et al. A multi-country study using MALDI-TOF mass spectrometry for rapid identification of Burkholderia pseudomallei. BMC Microbiol. 2021;21(1):213. doi: 10.1186/s12866-021-02276-1 34266382 Anton-Vazquez V, Hine P, Krishna S, Chaplin M, Planche T. Rapid versus standard antimicrobial susceptibility testing to guide treatment of bloodstream infection. Cochrane Database Syst Rev. 2021;5(5):CD013235. doi: 10.1002/14651858.CD013235.pub2 34097767 Woods KL, Boutthasavong L, NicFhogartaigh C, Lee SJ, Davong V, AuCoin DP, et al. Evaluation of a Rapid Diagnostic Test for Detection of Burkholderia pseudomallei in the Lao People’s Democratic Republic. J Clin Microbiol. 2018;56(7):e02002-17. doi: 10.1128/JCM.02002-17 29720430 Shaw T, Tellapragada C, Ke V, AuCoin DP, Mukhopadhyay C. Performance evaluation of Active Melioidosis Detect-Lateral Flow Assay (AMD-LFA) for diagnosis of melioidosis in endemic settings with limited resources. PLoS One. 2018;13(3):e0194595. doi: 10.1371/journal.pone.0194595 29579128 Lancaster GA, Thabane L. Guidelines for reporting non-randomised pilot and feasibility studies. Pilot Feasibility Stud. 2019;5:114. doi: 10.1186/s40814-019-0499-1 31608150 10.1371/journal.pntd.0013962.r001 Decision Letter 0 Poonawala Husain Academic Editor 2026 Husain Poonawala This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

28 Oct 2025

Rapid diagnosis of skin and soft tissue melioidosis in children

PLOS Neglected Tropical Diseases

Dear Dr. Suy,

Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases. After careful consideration, we feel that it has merit but does not fully meet PLOS Neglected Tropical Diseases's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Husain Poonawala

Academic Editor

PLOS Neglected Tropical Diseases

Ana LTO Nascimento

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

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Paul Brindley

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PLOS Neglected Tropical Diseases

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Additional Editor Comments :

The outcomes and analysis plan do not seem to align. Is the definition of "antimicrobials with activity against B. pseudomallei" not similar to "effective treatment following pus collection"? This is a distinction without a difference.

I would like to see more explanation of why time to effective treatment was chosen. I don't understand the rationale for separating out analysis by whether they got empiric treatment or not.

The sample sizes for the K-M curves are quite small - is there a reason that the analysis separates those who received empiric antibiotics from those who did not? Can you perform an analysis that uses the time from any antibiotic administration, perhaps only excluding those who received antibiotics prior to presentation at the hospitial? The effect sizes are quite wide

If a statistician is not already involved, I would recommended obtaining their help.

I am not sure how the blood culture and ultrasound workups are relevant to performance of the LDT - it seems out of place from the other two aims. It would be more relevant towards the role of diagnostic pathways in the workup of B. pseudomallei.

Lines 263 and 224 - I think it would be more accurate to say that all were prescribed a month of antibiotics. Received implies that they took the medication, but that cannot be confirmed since they were lost to follow-up.

The pus swabs were probably negative because swab is not a good sample collection device and there was likely very little specimen, highlighting the need for good sample collection as part of the diagnostic workup (and the larger issue that swabs should not be used at all)

I consider the 33% loss to follow-up a significant limitation and recommend including it in the discussion.

Is the real finding of this study that patients with B. pseudomellei require more thorough workup on initial presentation? I think the diagnostic performance of the test is a minor part of the improved outcomes, and what really made a difference was the training of the surgical team and improved diagnostic workup. My suggestion is to consider reframing the article on the role of all the changes that helped improve the diagnosis.

I would like to respectfully point out to the authors that submission of any material into ChatGPT results in a loss of confidentiality and to use AI tools for research with a full understanding of what happens to the information once submitted there.

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Note: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Reviewers' Comments:

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: 1.The manuscript mentions that “routine and B. pseudomallei culture was performed using blood agar, chocolate agar, MacConkey agar, Ashdown agar, and selective broth that was subcultured onto Ashdown agar.” To improve reproducibility and clarity, please specify the composition or source of the selective broth used in this study (e.g., enrichment broth type, manufacturer, or reference). Was it an in-house selective enrichment medium or a commercial formulation?

2.In Supplementary Figure S1 (“Diagnostic workflow for participants in the prospective arm”), could the authors clarify the rationale for using a CO₂-enriched atmosphere for blood and chocolate agar, but aerobic (O₂) incubation for MacConkey agar?

Typically, blood and chocolate agars are incubated in 5% CO₂ to support the growth of fastidious organisms (e.g., Streptococcus, Haemophilus), whereas MacConkey agar is incubated aerobically for enteric Gram-negative bacteria. It would be helpful if the authors could confirm that this was the intended rationale or provide a brief justification in the Methods or figure legend for clarity and reproducibility.

Reviewer #2: The methods are clearly articulated and are appropriate for the research questions. A power calculation was not performed. While the reason for this is stated clearly, it is likely that the small size of study has contributed to a non-statistically significant result for the first co-primary outcome.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results are well presented and clearly demonstrate the clinical utility of the AMD-RDT in improving diagnostic timeliness and treatment initiation for suspected melioidosis.The findings convincingly support the test’s value as an adjunct to culture-based diagnosis in resource-limited settings.

Reviewer #2: The analysis is presented clearly and is easy to follow. For the first co-primary outcome, the difference in time to effective treatment was not statistically significant, and this could be stated more clearly in the results.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The discussion section is well-written, coherent, and effectively links the study findings with existing literature. The authors have clearly articulated the significance of introducing the AMD-RDT in clinical workflows and provided a balanced interpretation of their results. The linkage between diagnostic turnaround time, clinical outcomes, and system-level implementation challenges is thoughtfully addressed.

Reviewer #2: I would suggest some additional discussion of aspects of the data that warrant consideration, as well as expanded discussion on potential limitations of the study.

There are significant differences in the baseline characteristics of the two groups (retrospective and prospective). These differences are presented in Table 1, but are not discussed at all. Do the authors have any theories for why: (1) more females were affected in the prospective cohort, and more males in the retrospective cohort; (2) parotitis made up nearly half of the cases in the prospective cohort, but only 12% of the cases in the retrospective cohort; (3) skin infections made up more than a third of the cases in the retrospective cohort, but only 12.9% of the prospective cohort; (4) GNBs were more identified on microscopy in 61.3% cases in the prospective cohort but only 4% of cases in the retrospective cohort? Is it possible that a higher rate of parotitis and lower rate of skin infection may have contributed to seeing more positive microscopy, and that this might have inflated the sensitivity of the lateral flow assay?

Do the authors think it is possible that the sensitivity of the lateral flow assay (LFA) may be higher in samples that have a high enough bacterial load to be positive on Gram stain microscopy? If the LFA had been tested on the earlier cohort, where the vast majority of cases had no bacteria seen on microscopy, is it possible that the LFA may have performed less well, in terms of sensitivity? The authors should discuss this possibility, and acknowledge that the unbalanced cohorts introduces a risk of bias and that this is an important limitation.

The authors acknowledge the potential for the training to have contributed to higher rates of investigation for cases in the prospective arm (the second co-primary outcome). It would be good to see a little more discussion of this, as I suspect that training is more likely to lead to this kind of change in behaviour than a positive diagnostic test, especially in a cohort of patients who will already have a positive diagnostic test (albeit the culture result will come later). The training is probably also largely responsible for the higher proportion of patients getting intensive phase intravenous treatment. It would be interesting to see more discussion about whether or not this is always required.

While it does not appear to have been part of the analysis plan, a comparison of length of stay would be interesting to see. It may be that while the group who had the LFA test received a diagnosis and effective treatment sooner, they may also have had longer hospital stays, and the question of if the additional intravenous therapy is necessary or beneficial may not be clear.

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: This study is well designed and the results and discussion are well presented. The conclusions are sound, and appropriate based on the results of the analysis. However, further discussion about the limitations of the study design and the potential impacts of disparate before- and after- cohorts and training linked to introduction of the new test would be valuable.

**********

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Reviewer #1: No

Reviewer #2: Yes: Joshua R Francis

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Figure resubmission:

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 10.1371/journal.pntd.0013962.r002 Author response to Decision Letter 1 Submission Version 1

31 Dec 2025

Submitted filename: ROMEO_Response to Reviewers.docx

 10.1371/journal.pntd.0013962.r003 Decision Letter 1 Poonawala Husain Academic Editor 2026 Husain Poonawala This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

20 Jan 2026

Dear Dr. Suy,

We are pleased to inform you that your manuscript 'Rapid diagnosis of skin and soft tissue melioidosis in children' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Husain Poonawala

Academic Editor

PLOS Neglected Tropical Diseases

Ana LTO Nascimento

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

***********************************************************

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; line-height: 16.0px; font: 14.0px Arial; color: #323333; -webkit-text-stroke: #323333}span.s1 {font-kerning: none

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: No further revisions are required.

Reviewer #2: The methods are clearly articulated.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: No further revisions are required.

Reviewer #2: The changes to the results are good, and have strengthened the article.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -No further revisions are required.

Reviewer #2: The discussion and conclusions are appropriate.

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: No further revisions are required.

Reviewer #2: I have no suggestions for further modifications.

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: No further revisions are required.

Reviewer #2: Congratulations to the authors on a worthwhile study and high quality manuscript.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: Yes: Prof Joshua Francis

 10.1371/journal.pntd.0013962.r004 Acceptance letter Poonawala Husain Academic Editor 2026 Husain Poonawala This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Dear Doctor Suy,

We are delighted to inform you that your manuscript, "Rapid diagnosis of skin and soft tissue melioidosis in children," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases