Microbiological Laboratory Diagnosis of Human Brucellosis: An Overview

Brucella spp. are Gram-negative, non-motile, non-spore-forming, slow-growing, facultative intracellular bacteria causing brucellosis. Brucellosis is an endemic of specific geographic areas and, although underreported, represents the most common zoonotic infection, with an annual global incidence of 500,000 cases among humans. Humans represent an occasional host where the infection is mainly caused by B. melitensis, which is the most virulent; B. abortus; B. suis; and B. canis. A microbiological analysis is crucial to identifying human cases because clinical symptoms of human brucellosis are variable and aspecific. The laboratory diagnosis is based on three different microbiological approaches: (i) direct diagnosis by culture, (ii) indirect diagnosis by serological tests, and (iii) direct rapid diagnosis by molecular PCR-based methods. Despite the established experience with serological tests and highly sensitive nucleic acid amplification tests (NAATs), a culture is still considered the “gold standard” in the laboratory diagnosis of brucellosis due to its clinical and epidemiological relevance. Moreover, the automated BC systems now available have increased the sensitivity of BCs and shortened the time to detection of Brucella species. The main limitations of serological tests are the lack of common interpretative criteria, the suboptimal specificity due to interspecies cross-reactivity, and the low sensitivity during the early stage of disease. Despite that, serological tests remain the main diagnostic tool, especially in endemic areas because they are inexpensive, user friendly, and have high negative predictive value. Promising serological tests based on new synthetic antigens have been recently developed together with novel point-of-care tests without the need for dedicated equipment and expertise. NAATs are rapid tests that can help diagnose brucellosis in a few hours with high sensitivity and specificity. Nevertheless, the interpretation of NAAT-positive results requires attention because it may not necessarily indicate an active infection but rather a low bacterial inoculum, DNA from dead bacteria, or a patient that has recovered. Refined NAATs should be developed, and their performances should be compared with those of commercial and home-made molecular tests before being commercialized for the diagnosis of brucellosis. Here, we review and report the most common and updated microbiological diagnostic methods currently available for the laboratory diagnosis of brucellosis.


Laboratory Diagnosis of Brucella Infection
Brucella spp. are small (0.5-0.7 by 0.6-1.5 µm), non-motile, non-spore-forming, slowgrowing, facultative intracellular Gram-negative coccobacilli belonging to the Brucellaceae family along with the Mycoplana and Ochrobactrum spp. [1,2]. Brucellae can infect several animal species and currently comprise 12 well-classified species, 4 of which causing almost BC samples should be collected as soon as possible when BSI is suspected, even in the absence of fever. Drawing two or three separate BC sets due to a continuous low-grade bacteremia.
The positivity provides definitive diagnosis. Confirm the diagnosis of brucellosis in its early stages, when the serology is still negative or shows low/borderline antibody titers.
Screening tool (positive result to be confirmed with SAT). Good accuracy in acute cases.
Fast (10 min) and simple. No drawback due to the prozone phenomenon.
Low sensitivity in complicated and chronic cases. False-positive results due to cross-reaction. [ Not commonly used in human infection due to its technical complexity and problems in its standardization. [39] serum A, C Measures the difference in rotational velocity between a small antigen molecule in solution, labeled with a fluorochrome, and the same antigen molecule conjugated with its antibody.
Diagnosis of the zoonosis in domestic and feral animals as well as for brucellosis screening in the dairy industry.
Successfully used in animals. Only one study in human infections with high sensitivity and specificity. Rapid.
Easy to perform. Portable equipment. [45] serum A, C

Culture
A culture is considered the "gold standard" for the laboratory diagnosis of brucellosis. As human brucellosis pathogenesis is always characterized by an initial bacteremic phase, peripheral blood cultures (BC) should always be performed as soon as brucellosis is suspected. This represents an important method to confirm the disease, although it shows a sensitivity ranging from 10 and 90% [9]. The variables that could affect the positivity rate of BC are microbial (i.e., Brucella species involved), patient-related (e.g., age, duration of symptoms, acute or chronic and focal disease, first infection or relapse, and antibiotics use), and method-associated (e.g., blood volume, number of BC vials collected, BC system used, incubation time, and performance of blinded subcultures) [10]. BC isolation of Brucella spp. with accurate identification to the species level allows for defining the source of infection and the initial stage of the disease when serological tests are still negative or inconclusive and for differentiating between wild-type and vaccine Brucella strains [11,12]. During the initial stage of brucellosis, patients present a low bacterial load in the blood that can be only detected by following the recommended guidelines of BC collection, i.e., drawing at least two or three separate BC sets and avoiding the collection of only a single BC set ("solitary" BC) [13]. As the infection progresses, the organism is removed from the blood and enters the macrophages, with a reduction in the concentration of circulating bacteria, which makes their isolation difficult [14].
The characteristics of slow growth (i.e., several hours), low bacterial load in blood, and CO 2 production of brucellae, require a modification of the common procedures used in the automated BC systems [15,16]. Although the normal time of incubation in the automatic BC systems is up to 5 days, the incubation needs to be prolonged up to 7 days to detect severe cases of brucellosis [17,18]. In some cases, the American Society for Microbiology and the WHO recommend prolonging the incubation of BC bottles up to 4 weeks along with blinded subcultures, although this protocol presents several disadvantages, such as expensive and labor-intensive work, organizational problems, and delay in the diagnosis from automatic BC systems [10,19].
Different BC methods have been used for the detection of Brucella spp., such as the manual monophasic and biphasic methods [20], lysis-based BC (white blood cells are lysed by saponin to release Brucella prior to inoculate them onto culture media), blood clot cultures (blood clots in which leukocytes containing phagocytosed organisms are cultured), and the automated new generation BC systems, with the most common being the Bactec 9000 or Bactec FX series from Becton Dickinson and the BacT/Alert BC system from bioMerieux. The use of new BC instruments has increased the sensitivity of blood cultures and shortened the time to detection of Brucella species [21,22].
After the initial hematogenous spread, in 25-35% of patients, brucellae can move towards remote organs with the development of focal infections. This allows for the isolation of brucellae from specimens in other sites other than blood, such as bone marrow, urines, bone tissue, pleural and synovial fluid aspirates, liver biopsy specimens, lymph nodes, cerebrospinal fluid, and abscesses after an incubation up to 14 days in a 5% CO 2enriched atmosphere at 35 • C or by inoculation of specimens into broth media (usually those used for blood cultures) [23][24][25][26][27][28][29][30].
The rapid identification of Brucella spp. recovered from culture is essential (i) to establish a timely diagnosis; (ii) to avoid biological risk for laboratory personnel; (iii) to confirm the presence of the disease in its early stages, when antibody titers are still absent, low, or borderline; (iv) to distinguish between wild and vaccine Brucella strains; and (v) to track the source, since the individual species and their naturally occurring hosts are strongly associated.
Clinical microbiology laboratories should identify suspected colonies based on few morphologic, biochemical, and serological tests. Brucella spp. can be isolated mostly from blood agar and chocolate agar media after 24-72 h of incubation. Colonies are small (1-2 mm), convex, nonpigmented, nonhemolytic with an entire edge, and frequently smooth. Rough variants can occur with B. canis [31]. The presumptive identification of brucellae is based on the Gram staining appearance (faintly stained Gram-negative very small coccobacilli resembling fine sand, often clustered in microcolonies); specific traditional phenotypic reactions, such as positive oxidase, catalase, and urease activity; no fermentation of sugars; H 2 S production; growth in the presence of thionine and basic fuchsin; oxidative metabolic tests; and lack of motility.
Several commercial systems may misidentify brucellae for closely related bacterial species, mostly belonging to the related genus Ochrobactrum [75]. Besides conventional phenotypic methods, the identification of brucellae to the species level can be also performed by Matrix-Assisted Laser Desorption Ionization-Time Of Flight mass spectrometry (MALDI-TOF) technology, although its accuracy is sometimes contradictory [76][77][78][79]. The presumptive identification of brucellae should be confirmed by a molecular method.
Brucellae represent the most common etiology of laboratory-acquired infections [80] because of the high transmission of these microorganisms within confined environments. Indeed, a very low infectious dose (10-100 cells by aerosol or subcutaneous routes) is required, and several entry routes to access the host are exploited by these bacteria (e.g., respiratory mucosa, conjunctivae, abraded skin, and gastrointestinal tract) [80]. In addition, viable brucellae can persist in the environment for weeks and even months [80].
Routine bacteriological manual procedures such as homogenization of tissues, centrifugation and vortexing of bacterial suspensions, performance of subcultures, and biochemical testing may create dangerous aerosols and spillage of living bacteria, increasing the risk for unintentional transmission to working personnel [81].
The Centers for Disease Control recommended BSL-2 practices, containment equipment, and facilities for routine handling of clinical specimens of human or animal origins, whereas BSL-3 practices, containment equipment, and facilities are recommended for all manipulations of cultures of pathogenic Brucella spp. [82]. Furthermore, in areas of endemicity of brucellosis, all positive blood culture vials should be initially processed in safety cabinets, pending final identification of the organism. Bacterial isolates presumptively identified as Bordetella bronchiseptica, Ochrobactrum spp., Bergeyella zoohelcum, or Psychrobacter phenylpyruvicus should be managed in a similar manner until the presence of a Brucella spp. has been firmly ruled out.

Serological Tests
Indirect diagnosis is based on serological tests aimed at detecting specific antibodies in the serum of patients. Interpretive criteria are uniformly defined, such as a highly specific titer in agglutination assays, a cutoff value in enzyme-linked immunosorbent assay (ELISA), or the presence of a clear specific band in the lateral flow immunoassay. Nevertheless, these criteria are often controversial depending on laboratory-dependent differences and on the clinical and epidemiological characteristics (e.g., age, duration of illness, occupational risks, history of the disease, endemicity, and repeated exposition) [83][84][85]. The main disadvantages of serological tests are their low specificity; the difficulty in interpretating results, especially in patients with repeated exposure to Brucella; and distinguishing between active and past infection [33]. Serology showed also low sensitivity during the early stage of the disease and suboptimal specificity due to cross-reaction with other bacterial species. Other major causes of false-negative results of serological tests are prozone effect, low-affinity antibodies, and infections due to B. canis [10]. Despite these limitations, serological tests represent the main diagnostic methods for the diagnosis of brucellosis in endemic and low-to-middle-income countries because they are low cost and user friendly and have a high negative predictive value.
A wide variety of serological tests for the diagnosis of human brucellosis are available, mostly applied in the veterinary field. The major challenge for the development of serological tests is the complexity of antigenic structures, such as outer membrane proteins, cytosolic proteins, and immunodominant LPS. Several antigens are used for serological tests, mostly obtained from B. melitensis and B. abortus, whereas whole cell preparations are used in the indirect fluorescent-antibody (IFA) [31]. However, most serological tests used for the laboratory diagnosis of brucellosis are divided into methods that target brucellar smooth LPS (S-LPS) and those targeting cytosolic proteins.
The methods directed at S-LPS comprise the Rose Bengal test (RBT), based on a slide agglutination test [32]; the standard agglutination test (SAT) in tubes, the most common serological assay used for diagnosing B. abortus, B. melitensis, and B. suis infections, with the new developed SAT miniaturized test [6,12,33,34]; the 2-ME test, which uses 2-mercaptoethanol to eliminate the IgM type, leaving only the IgG isotype [35]; the Coombs antiglobulin agglutination test [36] and the Brucella Coombs gel test [37,38]; the complement fixation test (CF) [39]; the immunocapture agglutination test, such as BrucellaCap [40,41]; the IgG avidity ELISA [42]; the fluorescent resonance energy transfer (FRET) assay, which labels a given antigen and its complementary antibody with adequate fluorophores and measures the amount of energy transferred after the excitation of the donor fluorophore [44] against the Brucella S-LPS; and the fluorescent polarization immunoassay (FPA), based on measuring the difference in rotational velocity between a small antigen molecule in solution, labeled with a fluorochrome, and the same antigen conjugated with its antibody, against brucellar S-LPS [45].
The major test targeting cytosolic proteins is ELISA, also used directly on cerebrospinal fluid (CSF) samples for neurobrucellosis diagnosis [6,43]. Recently, rapid detection of brucellosis using a handheld quantum dot (QD) immunochromatographic test strip can be employed as preliminary screening of brucellosis [46]. Contrary to enzyme immunoassay (EIA) and IFA, agglutination-based tests cannot differentiate the types of antibodies involved [1]. Novel experimental antigens and tests have been developed, i.e., synthetic oligosaccharides, recombinant Brucella proteins-providing higher sensitivity, rapid and reliable results, reduction in costs, and simplicity of technical execution [86][87][88][89][90].

Nucleic Acid Amplification Tests (NAATs)
Molecular methods, also called NAATs, allow for the diagnosis of brucellosis in a few hours with high sensitivity and specificity. NAATs remain positive for a long time in patients apparently asymptomatic and when clinical relevance is unclear. However, a positive test may not necessarily indicate an active infection but could be the result of a low bacterial inoculum in frequently exposed healthy individuals, DNA from dead organisms, or successfully treated patients. Thus, the interpretation of results from NAATs should be carefully conducted, always taking into consideration the clinical and epidemiological setting involved.
Used first on peripheral blood with good performance, serum samples represent the sample of choice for the molecular diagnosis of human brucellosis with better yield [47][48][49]. A molecular diagnosis of brucellosis can be also performed in other specimens (e.g., from the genitourinary, osteoarticular, cardiovascular, and central nervous systems), useful in the diagnosis of focal brucellosis affecting any organ and tissue where cultures are often negative [91,92]. Moreover, Formalin-Fixed Paraffin-Embedded (FFPE) tissue acquired from surgical biopsy samples can be used following validated DNA extraction procedures [50].
The specific targets used for molecular tests involve mainly genes encoding outer membrane proteins, such as omp2 and omp31, and omp28 genes, also named bp26 [51][52][53][54]. Other gene targets used for molecular diagnosis of Brucella infection are [55][56][57][58] (i) the gene 16S rRNA, although cross-reactions have been reported; (ii) the insertion sequence IS711, in which the performance has been questioned due to its sequence variation and its absence in some strains; and (iii) bcsp31, the most frequently used gene, encoding the synthesis of an immunogenic membrane protein.
Routine performance of antibiotic susceptibility tests is not recommended for Brucella isolates due to their susceptibilities to first-line antimicrobial agents [93]. However, it is recommended that all strains should be sent to a reference laboratory for accurate identification to the species level and for determination of its biovar, for several reasons, such as the identification of zoonotic source, epidemiological studies during outbreaks, the description of strains circulating and spreading in a particular geographic area, the differentiation between wild-type and vaccine strains, and veterinary control programs [10,94].
Molecular methods used to identify brucellae to species level and genotyping include (i) fluorescence in situ hybridization (FISH) test, targeting a partial region of the 16S rRNA gene with rapid and precise detection of all human pathogenic species; (ii) a novel recA gene-based PCR assay able to differentiate between Brucella spp. and the related species Ochrobactrum spp.; (iii) the "AMOS PCR" test used to differentiate four Brucella species, namely B. abortus, B. melitensis, B. ovis, and B. suis and vaccine strains; (iv) the Bruce ladder multiplex PCR assay, proving to identify and accurately differentiate between reference and vaccine isolates with high reproducibility; and (v) whole genome sequencing by the identification of specific SNPs with the differentiation of five different Brucella species [66][67][68][69][70][71].
Commercial NAATs available for the diagnosis of brucellosis are still limited, and published comparative studies for assessing the different performances of commercial and home-made molecular tests are scarce and, in several cases, only report a small sample size [72,73]. Rapid, reliable, and affordable detection of Brucella spp. via molecular methods remains a challenge. Thus far, there are not validated commercial or home-made NAATs that can guarantee a high reproducibility of results; thus, direct methods by culture and indirect methods by serological tests remain the main tools for the laboratory diagnosis of brucellosis and the methods of choice for the follow-up of infections by Brucella spp.

Conclusions
In this mini review, we reported and discussed the most updated and common methods currently available for the microbiological laboratory diagnosis of brucellosis. The fast and precise diagnosis of human brucellosis is essential for delivering a prompt and adequate antimicrobial therapy, for informing public health services, and for avoiding the spread of the disease through exposure to sick animals and the consumption of con-taminated food. Moreover, the clinical symptoms of human brucellosis are variable and nonspecific; therefore, a microbiological laboratory analysis is essential.
A laboratory diagnosis is based on three different microbiological approaches: direct diagnosis by culture, indirect diagnosis by serological tests, and rapid diagnosis by molecular NAATs.
Despite the availability of long-term experience (serological) and highly sensitive (NAATs) tests, a culture is still considered the "gold standard" in the laboratory diagnosis of brucellosis due to its clinical and epidemiological relevance. The recent development of automated BC systems has increased the sensitivity of BCs and shortened the detection time of Brucella species by monitoring CO 2 production, thus achieving a detection rate of more than 95% within a one week incubation period. Prolonged incubation is required in the case of focal complications along with blind subcultures. Rapid and accurate identification of Brucella species has been accomplished with the introduction of MALDI-TOF analysis, nucleic acid amplification assays, and hybridization tests.
The indirect diagnosis is based on serological tests detecting antibodies in the serum of patients. The main limitations of serological tests are the lack of common interpretative criteria (depending on laboratory variability, and clinical and epidemiological differences), the suboptimal specificity due to interspecies cross-reactivity, and the low sensitivity during the early stage of disease. Despite being imperfect, serological tests remain the main diagnostic tool in endemic areas thanks to their affordability, user friendliness, and high negative predictive value. Generally, a sensitive serological test method is used as an initial screening followed by a more specific confirmatory test such as the SAT. Despite the development of new serological tests being hampered by the complexity of antigenic structures, promising serological tests based on new synthetic antigens have been recently developed. In addition, a novel point-of-care test could warrant reliable results without the need for dedicated equipment and expertise, or the shipping of samples to distant laboratories.
NAATs are rapid tests that can help to diagnose brucellosis in a few hours with high sensitivity and specificity. Nevertheless, the interpretation of NAATs results requires attention because a positive test may not necessarily indicate an active infection but rather a low bacterial inoculum, DNA from dead organisms, or recovered patients. Commercial NAATs available for the diagnosis of brucellosis are still limited, and published comparative studies assessing the different performances of commercial and home-made molecular tests are still scarce.
The One Health approach, based on globally integrated multidisciplinary efforts on human and animal health, should be pursued for the optimal management of brucellosis.