Case Report: Soft tissue infection with Burkholderia thailandensis capsular variant: case report from the Lao PDR

Background Burkholderia thailandensis is an environmental bacteria closely related to Burkholderia pseudomallei that rarely causes infection in humans. Some environmental isolates have shown to express a capsular polysaccharide known as B. thailandensis capsular variant (BTCV), but human infection has not previously been reported. Although B. thailandednisis has been identified in environmental samples in Laos before, there have not been any human cases reported. Case A 44-year-old man presented to a district hospital in Laos with a short history of fever and pain in his left foot. Physical examination identified a deep soft-tissue abscess in his left foot and an elevated white blood count. A deep pus sample was taken and melioidosis was suspected from preliminary laboratory tests. The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines. Laboratory methods A deep pus sample was sent to Mahosot Hospital microbiology laboratory where a mixed infection was identified including Burkholderia sp. Conventional identification tests and API 20NE were inconclusive, and the B. pseudomallei-specific latex agglutination was positive. The isolate then underwent a Burkholderia species specific PCR which identified the isolate as B. thailandensis. The isolate was sent for sequencing on the Illumina NovaSeq 6000 system and multi-locus sequence typing analysis identified the isolate had the same sequence type (ST696) as B. thailandensis E555, a strain which expresses a B. pseudomallei-like capsular polysaccharide. Conclusion This is the first report of human infection with B. thailandensis in Laos, and the first report of any human infection with the B. thailandensis capsular variant. Due to the potential for laboratory tests to incorrectly identify this bacteria, staff in endemic areas for B. thailandensis and B. pseudomallei should be aware and ensure that appropriate confirmatory methods are used to differentiate between the species.


Background
Burkholderia thailandensis is an environmental bacteria closely related to Burkholderia pseudomallei that rarely causes infection in humans.Some environmental isolates have shown to express a capsular polysaccharide known as B. thailandensis capsular variant (BTCV), but human infection has not previously been reported.Although B. thailandednisis has been identified in environmental samples in Laos before, there have not been any human cases reported.

Case
A 44-year-old man presented to a district hospital in Laos with a short history of fever and pain in his left foot.Physical examination identified a deep soft-tissue abscess in his left foot and an elevated white blood count.A deep pus sample was taken and melioidosis was suspected from preliminary laboratory tests.The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines.), but as of yet infection with such a strain has not been reported 6 .In Laos, there have been more than 1000 culture confirmed melioidosis cases in the past two decades since the first report in 1999 7 but there have been no reports of B. thailandensis infection.We report here the first case of soft tissue infection caused by a mixture of bacteria including BTCV that occurred in Vientiane Province, Laos.

Case report
On 9 th January 2019, a 44 year old man was admitted to a local district hospital with a short history of fever and pain in his left foot.The patient was a farmer from Vientiane Province, Laos, and lived approximately 60 kilometers from Vientiane Capital.He was previously considered fit and healthy.Two days prior to presenting to hospital, he suffered a puncture wound to his left foot while cleaning out a fish pond on his property.He started having pain that afternoon and the area started to swell.The next day he went to work in a rice field and self-treated with antibiotics bought from a local pharmacy, however his symptoms did not improve.On admission to hospital, his physical examination identified a deep soft-tissue abscess in his left foot.The patient's white blood count was elevated at 16.3 × 10 9 cells/L, with a markedly elevated granulocyte count (98.1%) and blood glucose was normal (125 mg/dL).
The patient was treated with intravenous (IV) cloxacillin 1 g four times per day.On day three of admission (11 th Jan), the left foot abscess was incised and drained; a deep pus sample was sent to the Microbiology Laboratory at Mahosot Hospital, Vientiane.The same day, the patient developed a high-grade fever and worsening of the abscess on the left ankle with severe pain on the left foot and loss of ability to walk.Ceftriaxone (2 g/day) and metronidazole (500 g 8 hourly) were added and cloxacillin discontinued, along with appropriate wound debridement, dressing and cleaning with normal saline, although without improvement.On day eight, haemoculture, urine and throat swab cultures were collected and sent to the Mahosot Hospital Microbiology Laboratory, as melioidosis was suspected, based on preliminary pus culture results.B. pseudomallei was subsequently not isolated from any sample, but treatment was changed to ceftazidime (2 g three times per day) as per local melioidosis treatment guidelines.On 22 nd January IV treatment was discontinued, the patient was discharged well and with one week of co-trimoxazole oral treatment to complete at home after discharge.and B. cepacia targeting a Tat domain protein, 70-kDa protein and a conserved 12-kDa protein respectively using an adapted previously published method 8 and was identified as B. thailandensis.The isolate was then referred to the Mahidol Oxford Research Unit (MORU) in Bangkok where the latex agglutination test was confirmed as positive and the isolate was then identified by MALDI-TOF (Bruker Daltonik GmbH) as B. thailandensis using a recently constructed library 9 .The latex agglutination test (latex beads coated with 4B11 monoclonal antibody specific for the 200kDa exo-polysaccharide of B. pseudomallei) is used to rapidly identify suspect B. pseudomallei colonies in the Mahosot Hospital Microbiology Laboratory.
DNA was extracted from the isolate using QIAamp DNA Mini Kit (Catalogue number 56304, Qiagen, Germany), then processed for the 150-base-read library preparation and sequenced by Illumina NovaSeq 6000 system with paired-end runs at the Center for Medical Genomics, Faculty of Medicine, Ramathibodi Hospital, Bangkok, Thailand.FastQC v.0.11.9 (https://github.com/s-andrews/FastQC)was used to pre-process

Amendments from Version 1
The text has been updated with the suggested grammatical changes.
The isolate was identified using assembled genome sequence data.When submitting the genome sequences to the database through fIDBAC 10 , the whole genome Average Nucleotide Identity (ANI) of the isolate was 98.97% compared to B. thailandensis E264.The species was then confirmed by incorporating B. pseudomallei K96243, B. thailandensis FDAARGOS_238 and B. thailandensis E555.The isolate shared only 92.95% compared to B. pseudomallei K96243, while the ANI was 99.72% and 99.78% when compared to B. thailandensis FDAARGOS_238 and B. thailandensis E555, respectively.B. thailandensis E555 is a strain which expresses a B. pseudomallei-like capsular polysaccharide (BTCV).The MLST analysis revealed that the patient strain (LPD1900722) shared the same sequence type (ST696) as B. thailandensis strains E555, differing from E264 (ST80).While the capsule gene cluster was absent in B. thailandensis E264, it was present in LPD1900722, similar to the configuration found in B. pseudomallei K96243 and B. thailandensis E555 (Figure 1A).A maximum-likelihood phylogeny tree was constructed based on complete 16S ribosomal RNA (BTH_RS17800 of B. thailandensis E264) which showed the clinical isolate grouping within the B. thailandensis clade (Figure 1B).

Discussion
B. thailandensis is a Gram negative bacterium that is closely related to B. pseudomallei, the causative agent of melioidosis.B. thailandensis is widely present in Southeast Asia and Northern Australia but has also been detected in North America and West Africa 3,12 .B. thailandensis is usually considered non-pathogenic and is commonly found in the environment (surface water and soil) in tropical or subtropical climates 13 .B. thailandensis was first discovered in Thailand in 1996 and is differentiated from B. pseudomallei phenotypically by its ability to assimilate arabinose and it was classified as a new species in 1998 14,15 .BTCV produce a capsular polysaccharide that cross-reacts with that of B. pseudomallei, giving rise to the potential for confusion in the laboratory 6 .Occasional cases of infection with B. thailandensis have been reported in the United States, China, Thailand and Malaysia 4,5,[16][17][18][19] .As B. thailandensis is found in the environment, and the patient pus sample yielded a very mixed culture result, it is difficult to comment on the clinical significance of the BTCV isolate.However, this is the first documented isolation of B. thailandensis from a clinical specimen in Laos, and the first report of the isolation of BTCV from a clinical sample of which we are aware.The potential for confusion with B. pseudomallei is significant and is something of which laboratory staff should be aware of to avoid incorrectly labelling a patient as having melioidosis.There have been no further B. thailandensis isolates identified from the Mahosot Hospital Microbiology laboratory since this isolate (up to May 2024).The isolate was initially mis-identified by latex agglutination, but subsequently confirmed to be B. thailandensis by PCR and MALDI-TOF having failed to identify as B. pseudomallei by API 20NE.Although the pathogenicity of the BTCV within this mixed infection was questionable, this undoubtedly had the potential to lead to misdiagnosis meaning that the patient could have ended up being treated unnecessarily for melioidosis, involving antibiotic treatment for more than 3 months.

Conclusion
This is the first case of B. thailandensis reported in Laos and the first report of infection with BTCV of which we are aware.Laboratory staff in melioidosis-endemic areas should be aware of the possibility of B. thailandensis and should ensure that appropriate confirmatory methods are used to differentiate between B. pseudomallei and B. thailandensis rather than relying on latex agglutination or other serological methods alone.

Public and patient involvement
There was no formal patient or public involvement in the design or conduct of this work.

accurate laboratory diagnosis clearly conveyed.
There are a few sentences that need to be revised to improve reading.Please see text below to be revised: The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines.
Can you include the identification of Burkholderia thailandensis somewhere in here, as this has been skipped completely in the abstract.
○ Some environmental isolates of B. thailandensis, including one from Lao PDR (Laos), have been shown to express a capsular polysaccharide that cross-reacts with that of B. pseudomallei, known as B. thailandensis capsular variants (BTCV), but as yet infection with such a strain has not been reported6.
○ do you mean "but as of yet"?

○
The potential for confusion with B. pseudomallei is significant and is something of which laboratory staff should be aware to avoid incorrectly labelling a patient as having melioidosis."should be aware of" ○ I would also like to ask the authors why they chose to provide a 16S phylogeny over a whole genome, cgMLST or MLST phylogeny.Given the authors performed typing on the isolate and further describe relatedness of strains using MLST, I expected to see a B. thailandensis MLST phylogeny.I think the paper would be more robust, provide greater insight into the relatedness of strains and align with the manuscript text better with a MLST phylogeny in Figure 1.

Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes
Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?Yes

Is the case presented with sufficient detail to be useful for other practitioners? Yes
Competing Interests: No competing interests were disclosed.
of performing a whole-genome or MLST phylogeny to gain a more comprehensive understanding of the relatedness between Burkholderia thailandensis strains.In response, we are planning a separate, more detailed study where we will expand our dataset to encompass all available B. thailandensis genomes.This will allow us to conduct an in-depth genomic analysis, which will provide a more robust comparison of strain relationships, including the identification of virulence genes and their geographic distribution.This study serves as a foundation for future analyses.We appreciate your understanding of the study's scope and look forward to exploring these important aspects in the future.
Competing Interests: No competing interests were disclosed.

Mohd Firdaus Raih
Universiti Kebangsaan Malaysia, Bangi, Malaysia Increasing reports of B. thailandensis infections is an important contribution to the field in order to highlight the threat also posed by B. thailandensis which was previously thought to be less or even non-infectious.
The manuscript is clearly written and all the elements required of a case report is presented.
Is the background of the case's history and progression described in sufficient detail?Yes

Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes
Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?Yes Is the case presented with sufficient detail to be useful for other practitioners?Yes Competing Interests: No competing interests were disclosed.

Abdel Rahman Zueter
The Hashemite University, Zarqa, Jordan The case description is concise and to the point.It provides essential clinical details, including the patient's symptoms, physical examination findings, and initial treatment.The progression from preliminary suspicion of melioidosis to the adjustment of treatment according to local guidelines is logical and well-explained.
Since the case is very rare, it is good to add a table to review all similar cases Is the background of the case's history and progression described in sufficient detail?Yes

Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes
Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?Yes Is the case presented with sufficient detail to be useful for other practitioners?Yes

Figure 1 .
Figure 1.(A) Read coverage of LPD1900722 mapped to capsular polysaccharide gene cluster of B. thailandensis E264 (top), B. pseudomallei K96243 (middle) and B. thailandensis E555 (bottom).(B) Phylogenetic tree analysis of full-length of 16s ribosomal rna of Burkholderia spp.and clinical isolate (LPD1900722) using a maximum-likelihood with 100 bootstraps.Number on the branches indicate branch length (black) and bootstrap values (red).
Burkholderia species specific PCR which identified the isolate as B. thailandensis.The isolate was sent for sequencing on the Illumina NovaSeq 6000 system and multilocus sequence typing analysis identified the isolate had the same sequence type (ST696) as B. thailandensis E555, a strain which expresses a B. pseudomallei-like capsular polysaccharide.
ConclusionThis is the first report of human infection with B. thailandensis in Laos, and the first report of any human infection with the B. thailandensis capsular variant.Due to the potential for laboratory tests to incorrectly identify this bacteria, staff in endemic areas for B. thailandensis and B. pseudomallei should be aware and ensure that appropriate confirmatory methods are used to differentiate between the species.Plain Language Summary >Burkholderia thailandensis is a bacteria that is found in the environment.Rarely, this bacteria can cause infection in humans.Here we report a B. thailandensis infection in a 44 year old male in Laos.The patient sustained a puncture wound in his left foot and when presenting at a district hospital was prescribed cloxacillin.The wound did not improve and on day three of admission, a pus sample was sent to Mahosot Hospital Microbiology Laboratory for investigation.A preliminary diagnosis of melioidosis, caused by the bacteria Burkholderia pseudomallei, was made and antibiotic treatment was changed.Additional laboratory investigation determined that the isolate was actually B. thailandensis and antibiotic treatment was further changed.Due to the inconclusive results of the against B. pseudomallei K96243, B. thailandensis E264, B. thailandensis FDAARGOS_238 and B. thailandensis E555.