Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis

Morata, Pilar; Queipo-Ortuño, María Isabel; Reguera, J.M.; García-Ordóñez, Miguel Ángel; Cárdenas, Ana Marı́a; Colmenero, Juan

doi:10.1128/jcm.41.1.144-148.2003

Cited by 75 publications

(49 citation statements)

References 35 publications

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“…in blood culture and/or (ii) the presence of compatible clinical signs or symptoms, together with the presence of specific antibodies at significantly high titers or a seroconversion, or also a fourfold increase in titer between two sequential samples from the same patient (4,5,24). Significant titers were considered to be a SAT titer of Ն1/160, a CT titer of Ն1/320, or a CF titer of Ն1/16.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Different PCR Assays for Early Detection of Acute and Relapsing Brucellosis in Humans in Comparison with Conventional Methods

Mitka¹,

Anetakis²,

Souliou

et al. 2007

J Clin Microbiol

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Human brucellosis is a significant public health problem in many Mediterranean countries including Greece. The conventional serological methods, as well as blood cultures, have serious limitations, especially in chronic, relapsing, and focal forms of the disease. Four different PCR assays were applied in 4,926 buffy coat, whole-blood, and serum samples received from 200 patients admitted with brucellosis to the Infectious Diseases Hospital, Thessaloniki, Greece, for the rapid diagnosis of acute infection and relapses and compared to blood culture and serological tests (i.e., Wright's seroagglutination test, Coombs' antibrucella test, and the complement fixation test). The four PCR assays had excellent sensitivity and specificity and were able to detect all of the cases of acute disease. Buffy coat and whole blood were the optimal specimens. All four PCR assays were negative in all follow-up samples from 183 patients who had completed a successful treatment and were positive in all follow-up samples from 17 patients who had relapses in the first year after therapy, including the times of the relapses. In conclusion, PCR is a very useful tool for the rapid diagnosis of acute brucellosis and a good marker for the posttreatment follow-up and the early detection of relapses.

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Section: Methodsmentioning

confidence: 99%

Evaluation of Different PCR Assays for Early Detection of Acute and Relapsing Brucellosis in Humans in Comparison with Conventional Methods

Mitka¹,

Anetakis²,

Souliou

et al. 2007

J Clin Microbiol

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“…Clinical applications in human disease are reported (Matar et al, 1996;Queipo-Ortuno et al, 1997;Morata et al, 2003). We have described previously (Al-Nakkas et al, 2002) a single-tube nested PCR for diagnosis of brucellosis in venous blood samples using primers from the genus-specific IS711 sequence present in varying copy number across all members of the genus Brucella.…”

Section: Introductionmentioning

confidence: 99%

Large-scale evaluation of a single-tube nested PCR for the laboratory diagnosis of human brucellosis in Kuwait

Al-Nakkas

Mustafa

Wright

2005

Journal of Medical Microbiology

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A single-tube nested PCR assay identifying a 52 bp fragment from the genus-specific Brucella IS711 gene was used prospectively in clinical practice for the diagnosis of human brucellosis in Kuwait. Patients with suspected brucellosis and with other infections were investigated as clinically indicated but in all of them culture, serology and PCR for Brucella were carried out. Out of 263 suspected cases of brucellosis, diagnostic tests were positive in 199, serology was positive in 199 and culture in 89, while the Brucella PCR was positive in 193 (sensitivity 96 . 98 %, 95 % confidence interval 94 . 5-99 . 5 %). Chronic brucellosis, involving symptoms for more than 1 year, was diagnosed in 49 out of these 193 patients. Diagnoses in four out of the six patients with positive serology but negative PCR and culture were non-Brucella bacterial meningitis and viral meningitis. False-negative PCR results were possible in the remaining two; both had been on long-term antibiotics for previously diagnosed brucellosis but their adherence may have been questionable, allowing a relapse. The PCR was negative in 244 patients with other infections (specificity 100 %) and in 180 control subjects with negative Brucella culture and serology. PCR results were available within 24 h of sample receipt, providing diagnostic information rapidly. The PCR is expensive and technically demanding and may not be appropriate for all cases. Future studies will help define how it may best be used. For example Brucella and tuberculous meningitides can be very similar, Brucella PCR may allow prompt distinction between the two, avoiding the need for prolonged empirical treatment for both.

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“…The most frequently described PCR target for the diagnosis of human brucellosis is the bcsp31 gene encoding a 31-kDa antigen conserved among Brucella spp. (8,9,15). PCR identification of Brucella strains at the species or biovar level has been more challenging.…”

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confidence: 99%

Real-Time Multiplex PCR Assay for Detection of Brucella spp., B. abortus , and B. melitensis

et al. 2004

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The identification of Brucella can be a time-consuming and labor-intensive process that places personnel at risk for laboratory-acquired infection. Here, we describe a real-time PCR assay for confirmation of presumptive Brucella isolates. The assay was designed in a multiplex format that will allow the rapid identification of Brucella spp., B. abortus, and B. melitensis in a single test.Brucellosis is a zoonotic disease that causes abortion, fetal death, and genital infections in animals. In humans, this highly diverse illness initially presents as fever, malaise, and myalgia and may later develop into a chronic illness affecting various organs and tissues. The causative agents of brucellosis are facultative intracellular gram-negative bacteria belonging to the genus Brucella. This genus has been further classified into six species according to host preference and pathogenicity. Genetic studies, however, indicate that the six classical species of Brucella represent a single genomospecies based upon DNA-DNA reassociation (14). In spite of this evidence, the conventional classification scheme has been maintained. Human brucellosis has been attributed to B. abortus, B. melitensis, B. suis, and B. canis and more recently to strains resembling Brucella isolated from marine mammals (13). Laboratory detection of Brucella and species identification is based largely on culture isolation and phenotypic characterization. This process is lengthy and labor-intensive and has been associated with a heightened risk of laboratory-acquired infections (7). To surmount these problems, nucleic acid amplification has been explored for the rapid detection and confirmation of Brucella.A number of nucleic acid sequences have been targeted for the development of Brucella genus-specific PCR assays, including 16S rRNA, the 16S-23S intergenic spacer region, omp2, and bcsp31 (1, 9, 11, 12). The most frequently described PCR target for the diagnosis of human brucellosis is the bcsp31 gene encoding a 31-kDa antigen conserved among Brucella spp. (8,9,15). PCR identification of Brucella strains at the species or biovar level has been more challenging. Recently, Redkar et al.(10) described real-time PCR assays for the detection of B. abortus, B. melitensis, and B. suis biovar 1. These PCR assays target the specific integration of IS711 elements within the genome of the respective Brucella species or biovar. The assays, however, were designed to be tested in separate PCRs. Using similar PCR targets, but in a multiplex format, we have developed a real-time triplex assay that permits rapid confirmation of Brucella spp., B. abortus, and B. melitensis isolates in a single test.The primers and TaqMan probes (Qiagen, Alameda, Calif

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Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis

Cited by 75 publications

References 35 publications

Evaluation of Different PCR Assays for Early Detection of Acute and Relapsing Brucellosis in Humans in Comparison with Conventional Methods

Evaluation of Different PCR Assays for Early Detection of Acute and Relapsing Brucellosis in Humans in Comparison with Conventional Methods

Large-scale evaluation of a single-tube nested PCR for the laboratory diagnosis of human brucellosis in Kuwait

Real-Time Multiplex PCR Assay for Detection of Brucella spp., B. abortus , and B. melitensis

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