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Microbiological Laboratory Diagnosis of Human Brucellosis: History
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Subjects: Health Care Sciences & Services
Contributor: Giovanni Gherardi

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. 

  • brucellosis
  • laboratory diagnosis
  • culture method
  • serological test
  • nucleic acid amplification test

1. Laboratory Diagnosis of Brucella Infection

Brucella spp. are small (0.5–0.7 by 0.6–1.5 µm), non-motile, non-spore-forming, slow-growing, 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 the totality of human infections: B. melitensis, B. abortus, B. suis, and B. canis, with B. melitensis being the most virulent [3]. DNA-DNA hybridization studies have demonstrated that the different Brucella species are very closely related, with a percentage of similarity close to 100%, and they can be considered different subspecies belonging to a single species [4]. Nevertheless, the sequencing of several specific genes indicated that specific genotypes are strongly correlated with the different species, supporting the usefulness of retaining the traditional classification. Moreover, the conventional nomenclature has been maintained because of clinical, practical, and epidemiological reasons, with different biovars being species specific, such as B. abortus being closely associated with cattle, B. melitensis being closely associated with small ruminants, B. suis being closely associated with swine, and B. canis being closely associated with canids.
Brucellosis is a zoonotic infection due to the ability of these species to infect non-preferential hosts, including humans [3]. It can affect any organ and body site, occurring in animals and humans, as an occasional host, with very rare cases of human-to-human transmission [5]. Endemic in some geographic areas, such as the eastern Mediterranean basin, the Middle East, Arabian Peninsula, Latin America, Southern Europe, Central Asia, the Indian subcontinent, and many African countries, brucellosis represents the most common zoonotic infection, with a total of about 500,000 new human cases per year. However, a high discrepancy between the reported rate and the actual incidence has been reported largely due to misdiagnosis and underdiagnosis, especially in endemic areas [6]. The One Health approach, based on the integration of human and animal health, plants, and ecosystems, which involves local, regional, national, and global multidisciplinary cooperation and efforts, is useful [7].
The treatment of brucellosis is a challenge for many physicians since it requires prolonged therapy with a combination of antimicrobial drugs rarely used for other types of bacterial infections [8][9]. The fast and precise diagnosis of human brucellosis is essential for the delivery of a prompt and adequate antimicrobial therapy. It also supports public health services by identifying exposure to sick animals and avoiding the consumption of contaminated food.
Due to the variable and nonspecific clinical symptoms in humans, the microbiological laboratory is crucial for the identification of human cases and their subsequent management. A laboratory diagnosis can be carried out using three different approaches and microbiological procedures: direct diagnosis by culture, indirect diagnosis by serological tests, and rapid diagnosis by molecular PCR-based methods. 

2. 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) [10][11][12]. 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 [13]. 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 [14]. 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) [15]. 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 [16]; 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][17][13][18]; the 2-ME test, which uses 2-mercaptoethanol to eliminate the IgM type, leaving only the IgG isotype [19]; the Coombs antiglobulin agglutination test [20] and the Brucella Coombs gel test [21][22]; the complement fixation test (CF) [23]; the immunocapture agglutination test, such as BrucellaCap [24][25]; the IgG avidity ELISA [26]; 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 [27] 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 [28].
The major test targeting cytosolic proteins is ELISA, also used directly on cerebrospinal fluid (CSF) samples for neurobrucellosis diagnosis [6][29]. Recently, rapid detection of brucellosis using a handheld quantum dot (QD) immunochromatographic test strip can be employed as preliminary screening of brucellosis [30]. 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 [31][32][33][34][35].

3. 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 [36][37][38]. 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 [39][40]. Moreover, Formalin-Fixed Paraffin-Embedded (FFPE) tissue acquired from surgical biopsy samples can be used following validated DNA extraction procedures [41].
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 [42][43][44][45]. Other gene targets used for molecular diagnosis of Brucella infection are [46][47][48][49] (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.
Amplification strategies in NAATs include conventional PCR methods, in-house PCR, nested PCR, PCR-enzyme immunoassay (PCR-EIA) in a microplate format, real-time PCR, quantitative RT-PCR, and multiplex real-time PCR (M-RT-PCR) [14][50][51][52][53][54][55]. Recently, a new method based on loop-mediated isothermal amplification (LAMP) has been developed for the detection of brucellae [56].
Routine performance of antibiotic susceptibility tests is not recommended for Brucella isolates due to their susceptibilities to first-line antimicrobial agents [57]. 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 [14][58].
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 [59][60][61][62][63][64].
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 [65][66]. 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.

This entry is adapted from the peer-reviewed paper 10.3390/pathogens10121623

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