Performance characteristics and costs of serological tests for brucellosis in a pastoralist community of northern Tanzania.

The control of brucellosis across sub-Saharan Africa is hampered by the lack of standardized testing and the use of tests with poor performance. This study evaluated the performance and costs of serological assays for human brucellosis in a pastoralist community in northern Tanzania. Serum collected from 218 febrile hospital patients was used to evaluate the performance of seven index tests, selected based on international recommendation or current use. We evaluated the Rose Bengal test (RBT) using two protocols, four commercial agglutination tests and a competitive enzyme-linked immunosorbent assay (cELISA). The sensitivity, specificity, positive predictive value, negative predictive value, Youden's index, diagnostic accuracy, and per-sample cost of each index test were estimated. The diagnostic accuracy estimates ranged from 95.9 to 97.7% for the RBT, 55.0 to 72.0% for the commercial plate tests, and 89.4% for the cELISA. The per-sample cost range was $0.69-$0.79 for the RBT, $1.03-$1.14 for the commercial plate tests, and $2.51 for the cELISA. The widely used commercial plate tests performed poorly and cost more than the RBT. These findings provide evidence for the public health value of discontinuing the use of commercial agglutination tests for human brucellosis in Tanzania.

Investigating the use of protein saver cards for storage and subsequent detection of bovine anti-Brucella abortus smooth lipopolysaccharide antibodies and gamma interferon.

Brucella abortus, a smooth strain of the genus Brucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples from Brucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucella smooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-γ) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-γ ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-γ. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-γ recovery over 10 days when stored at room temperature.

Evaluation of competitive ELISA for detection of antibodies to Brucella infection in domestic animals.

AIM: To evaluate competitive enzyme-linked immunosorbent assay (cELISA) for its suitability as an additional serological test for the diagnosis of animal brucellosis. METHODS: cELISA, which was developed at the Veterinary Laboratories Agency, has been evaluated for its accuracy and suitability as an additional serological test for the diagnosis of animal brucellosis. Samples from naturally and experimentally infected animals and those from Brucella-free flocks and herds were tested. RESULTS: Data obtained since 1991 were analyzed from routine surveillance, animals experimentally infected with Brucella, and stored sera to validate cELISA for the detection of antibodies to Brucella in cows, small ruminants, and pigs. The sensitivity of the test ranged from 92.31% to 100%, in comparison with 77.14% to 100% for the complement fixation test (CFT). Specificities for cELISA, indirect enzyme-linked immunosorbent assay, and CFT were greater than 90%. CONCLUSION: cELISA can be used on a variety of animal species, and an added advantage is its suitability for use on poor-quality samples such as those affected by hemolysis.

Time-resolved fluorescent resonance energy transfer assay for simple and rapid detection of anti-Brucella antibodies in ruminant serum samples.

Brucellosis is a globally significant zoonosis, the control of which is difficult and resource intensive. Serological tests form a vital part of a multifactorial approach to control and are often performed in large numbers. The aim of the present study was to develop a new assay to improve the efficiency, ease, and effectiveness of serological testing. An existing competitive enzyme-linked immunosorbent assay (cELISA) was adapted to a completely homogeneous time-resolved fluorescent resonance energy transfer (TR-FRET) assay. This was achieved by labeling an anti-Brucella monoclonal antibody with a long-lifetime donor fluorophore and Brucella smooth lipopolysaccharide with a compatible acceptor and optimizing the reading conditions. The assay was performed in a 96-well plate with a single 30-min incubation period and no separation (wash) steps and was concluded by a single plate-reading step. The performance of the assay was evaluated with a panel of serum samples from infected (n = 73) and uninfected (n = 480) sources and compared to the performance of the parent cELISA, an indirect ELISA (iELISA), and fluorescence polarization assay (FPA). The performance of the TR-FRET assay matched the performance of the iELISA, which had 100% diagnostic sensitivity and specificity, and surpassed the performance of the cELISA and the FPA. The results also demonstrated that the TR-FRET technique is effective with poor-quality serum samples from the field. To the knowledge of the authors, this is the first homogeneous TR-FRET assay to detect antibodies raised against an infectious disease. The technique appears to be sufficiently adaptable to meet the needs of many other similar testing requirements to identify infectious diseases.

A new homogeneous assay for high throughput serological diagnosis of brucellosis in ruminants.

The control and eradication of brucellosis is highly desirable but heavily resource intensive as high throughput serological testing is required. The aim of this study was to meet the needs of high throughput screening laboratories involved in this process through the development of a new assay. An existing cELISA used for the serodiagnosis of brucellosis in ruminants was converted to an AlphaLISA homogenous proximity based assay. This assay requires no separation steps and can be performed in low volume microtitre format. The Brucella AlphaLISA was validated on a panel of bovine, ovine and caprine sera from infected and uninfected animals. The diagnostic sensitivities (>96%) and specificities (>98%) obtained compared well to those from cELISA, iELISA and FPA performed on the same samples. The AlphaLISA met the testing criteria set for ELISAs as defined by the OIEELISA standards and had an analytical sensitivity similar to that of the parent cELISA. The method was also used on a small panel of serum samples from cattle that were experimentally infected with Yersinia enterocolitica O:9. Some false positive reactions were obtained as was also the case with results from FPA, iELISA, cELISA, CFT and SAT. Despite this, the methodological advantages of the AlphaLISA mean that this assay is well suited to high throughput serodiagnosis. This report is the first description of the use of AlphaLISA to detect pathogen specific antibodies. Furthermore, the relative ease with which the cELISA was converted to this platform indicates that this technology is ready to meet the high throughput testing requirements for the diagnosis of many other diseases.