Introduction
Diagnosing brucellosis in nonendemic regions is challenging because it is a nonspecific febrile disease, and appropriate testing is critical [
1,
2]. Although serologic tests and PCR can be used to diagnose brucellosis, the standard test method is to detect
Brucella spp. via blood culture. However, it remains challenging to accurately identify
Brucella spp. in clinical microbiology laboratories (CMLs), especially in nonendemic countries.
Brucella, classified as a Category B biological warfare pathogen by the Centers for Disease Control and Prevention (CDC), illustrates the importance of proper suspicion and testing protocols to avoid diagnostic delays and potential laboratory-acquired infections (LAIs) [
2]. Laboratories lacking experience in
Brucella diagnosis may encounter misidentification, particularly with automated identification instruments. While human brucellosis is predominantly caused by
Brucella melitensis,
Brucella abortus, and
Brucella suis [
1], most misidentification reports are limited to
B. melitensis and
B. suis [
3‐
11]. We present a case involving the delayed diagnosis of
B. abortus bacteremia, which was initially misidentified, and a review of the relevant literature on the misidentification of brucellosis, laboratory safety, and nomenclature issues.
Discussion and conclusions
Diagnosing brucellosis, especially in nonendemic areas or from returning travelers, is challenging [
1,
2]. In this case, insufficient experience in conducting appropriate biochemical testing delayed accurate diagnosis, thereby increasing the risk of LAI [
21]. However, brucellosis was subsequently confirmed during hospitalization through additional MALDI–TOF MS database and 16S rRNA sequencing. In South Korea,
B. abortus is the main pathogen of human and bovine brucellosis, and brucellosis was designated a notifiable infectious disease in 2000. The first human case was reported in 2002, with reports increasing to more than 250 cases in 2006 [
22]. Since then, human brucellosis incidence has been on the decline due to active eradication policies, with fewer than 10 cases per year since 2014 [
23]. However, imported cases of
B. melitensis have been reported [
24,
25], requiring vigilance by CMLs.
Reports on the misidentification of
Brucella spp. were most often with
B. melitensis or
B. suis being misidentified as
Ochrobactrum anthropi (Table
1). Misidentification not only delays correct diagnosis but also increases the risk of LAI [
2]. Manipulating unknown
Brucella isolates on an open bench rather than in a biosafety cabinet (BSC) exposes many workers through aerosolization and increases the risk of LAI. In a recent assessment of the risk of exposure to brucellosis in laboratory workers in New York from 2015 to 2017,
Brucella exposure incidents occurred in 10 of 11 confirmed brucellosis cases [
26]. In the present case, brucellosis was clinically suspected, and the worker wore a mask and conducted all work in a Class II BSC, so there was no exposure. The worker was monitored for fever but did not develop symptoms. The use of MALDI–TOF MS is increasing, and safe work practices, including working with slow-growing organisms in a BSC and not using MALDI–TOF MS unless a biothreat agent is excluded, are recommended [
26].
Table 1
Misidentified cases related to the genus Brucella
Elsaghir et al., 2003 [ 3] | API 20NE | Ochrobactrum anthropi | Brucella melitensis | Positive |
| RapID NF Plus | O. anthropi | Brucella suis | NT |
Carrington et al., 2012 [ 5] | RapID NF Plus | O. anthropi | B. suis | Positive |
| VITEK 2 | O. anthropi | B. suis | Positive |
| VITEK MS | O. anthropi | B. melitensis | NT |
Poonawala et al., 2018 [ 7] | VITEK MS | O. anthropi | B. melitensis | NT |
Khaliulina Ushakova et al., 2020 [ 9] | Bruker MALDI Biotyper | O. anthropi | B. melitensis | Positive |
| VITEK 2 | B. melitensis | Haematobacter massiliensis | Negative |
VITEK 2 | B. melitensis | Herbaspirillum frisingense | NT |
Gopalsamy et al., 2021 [ 10] | VITEK 2 | O. anthropi | B. suis | Positive |
| Bruker MALDI Biotyper | Ochrobactrum deajoenense | B. melitensis | Positive |
Current case | VITEK 2 | Pseudomonas fluorescens | Brucella abortus | Positive |
MALDI–TOF MS is commonly used for rapid and accurate identification of microorganisms. The Bruker SR database has been reported to be able to rapidly and accurately identify biothreat agents, including
Brucella spp., while the in vitro diagnostics (IVD) and research use only (RUO) databases cannot [
28]. In the identification of
Brucella using VITEK MS, the IVD database failed, but the RUO database was reported to identify 56.9% of strains at the genus level [
28]. In this case, the CDC MicrobeNet database identified the isolate as
Brucella spp. and the Bruker SR database identified it as
B. melitensis, both of which were successful in differentiating the genus
Brucella. However, due to export restrictions, SR databases are not readily available for CMLs, especially those outside of Europe [
29]. The use of MALDI-TOF MS to differentiate
Brucella is not limited to the development of in-house databases; it also extends to reports that have been integrated into primary or public databases [
30,
31]. Therefore, if MALDI–TOF MS is performed in a situation where brucellosis is suspected, it would be helpful to use publicly available CDC MicrobeNet database.
The nomenclature of the genus
Brucella has long been controversial [
32]. Recently, a reclassification of
Ochrobactrum spp. to the genus
Brucella was proposed due to genomic similarities [
33], and both classifications are currently "validly published" nomenclature [
34]. As the new classification has been applied to some microbial identification systems, guidelines have been published to reduce clinical confusion [
35‐
37]. Given the known limitations of automated identification methods, including MALDI–TOF MS, in differentiating
Brucella spp. and
Ochrobactrum spp., it is important to distinguish them by morphologic and phenotypic characteristics [
36]. This clinical isolate did not grow on MacConkey agar, suggesting
Brucella spp.
Traditionally, subtyping of
Brucella spp. has been based on multilocus variable-number tandem-repeat analysis (MLVA) [
38]. With the increase in the amount of available WGS data, cgMLST for
B. melitensis, which can be used to accurately perform epidemiological studies and outbreak analyses, has been developed [
38]. In this study, a new
Brucella-wide cgMLST scheme was used to perform phylogenetic analysis [
20]. This clinical isolate is different from a previously reported
B. abortus strain in South Korea. Although there is a lack of WGS data on
B. abortus strains in South Korea, cgMLST may allow for more accurate analysis of transmission.
The limitations of automated identification systems for identifying Brucella spp. are well-recognized. Although MALDI–TOF MS is widely used in CMLs, it has limitations in identifying Brucella spp. without additional analysis. CMLs in nonendemic areas also require attention regarding the diagnosis of brucellosis because of diagnostic delays and the risk of LAI. It is important that clinicians' suspicions are well communicated and that CMLs perform appropriate testing with precaution to biosafety.
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