Visual typing detection of brucellosis with a lateral flow immunoassay based on coloured latex microspheres.

AIMS: Treatment and preventive control strategies for Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) infection differ. A lateral flow immunoassay (LFIA) for the rapid typing and detection of brucellosis by using polychromatic dye-doped latex microspheres (LMs) as a labelling material was developed. METHODS AND RESULTS: This LFIA utilizes a double-antigen sandwich method in which the BP26 protein is used as the diagnostic antigen to detect brucellosis infection and the OMP31 protein is used as the identified antigen to distinguish between bovine and sheep brucellosis. Thus, people and animals infected with brucellosis can be diagnosed according to the different colours of the signals displayed on the detection lines. The results indicated that the accuracy of this assay was found to reach 98%, and the immunochromatographic test strip is highly accurate, shows good sensitivity and can facilitate typing diagnosis, among other features. CONCLUSIONS: The established LFIA can distinguish B. melitensis infection from B. abortus infection and produces results in a short period of time while retaining the advantages of LFIAs. SIGNIFICANCE AND IMPACT OF THE STUDY: This technology lays a foundation for the development of multi-disease test strips and the establishment of methods for rapid, multi-specimen quantitative detection and is thus of great importance for the development of medical diagnostic technologies.

Brucella Case That Led to Atlantoaxial Spinal Instability in Childhood.

BACKGROUND: Atlantoaxial subluxation caused by Brucella infection is rarely seen in children. CASE DESCRIPTION: C1-C2 dislocation, erosion in the odontoid bone, and compression to the brainstem were detected on cervical computed tomography and magnetic resonance imaging in a 6-year-old patient who suffered neck pain, deviation to the right in the neck, fever, and pain in his extremities. He was hospitalized in the Clinic of Pediatric Infectious Diseases. Positive Brucella agglutination tests were obtained, so C1-C2 stabilization and fusion were performed. Antibrucellosis antibiotic treatment was administered for 6 months. It was suggested that the dislocation was related to odontoid erosion and laxity of the atlantoaxial ligamentous structures during Brucella infection. CONCLUSIONS: Atlantoaxial dislocation and instability develops secondarily to paravertebral abscesses, only rarely. Like brucellosis of childhood, granulomatous infectious diseases rarely cause atlantoaxial subluxation or dislocation. Torticollis and neck pains should be taken seriously for the purpose of early diagnosis of patients at risk in endemic regions. Stabilization and fusion should be performed when instability is detected, and these patients should be assessed with both pediatric infectious diseases and neurosurgery clinics. Our case is the first one in the literature in which atlantoaxial instability developed due to Brucella infection and stabilization was performed.

The detection of brucellosis antibody in whole serum based on the low-fouling electrochemical immunosensor fabricated with magnetic Fe3O4@Au@PEG@HA nanoparticles.

It is a novel competitive challenge for electrochemical biosensor to directly, rapidly and ultrasensitively detect the disease markers in the whole serum due to biofouling caused by the complexity of actual samples. In this paper, poly (ethylene glycol) (PEG) and hyaluronic acid (HA) were utilized to modify Fe3O4 @Au nanoparticles (NPs). Based on the successfully preparation and characterization of Fe3O4 @Au@PEG@HA NPs with TEM, SEM, XRD, FTIR and EDS, respectively, a novel immunosensor of brucellosis with high selectivity, sensitivity and almost perfect protein-resistant properties in various external environments, especially, in complex biological systems was fabricated. More importantly, this immunosensor is capable of assaying brucellosis antibody in 100% serum without suffering from any significant biological interference. In addition, a wide linear response range from 10-15 g mL-1 to 10-11 g mL-1 towards antibody in 100% serum and a low limit of detection (LOD) of 0.36 fg mL-1 (3σ, n = 13) are demonstrated, which indicates that this immunosensor has a promising potential in clinical diagnosis.

Latex agglutination using the periplasmic proteins antigen of Brucella melitensis is a successful, rapid, and specific serodiagnostic test for ovine brucellosis.

Brucellosis, especially caused by Brucella melitensis, is considered the most-widespread zoonosis in the world, particularly in developing countries. This study was planned to develop an accurate test for diagnosis of ovine brucellosis using a specific hot saline extracted soluble Brucella melitensis periplasmic proteins (SBPPs). The efficacy of the latex agglutination test (LAT) using SBPPs compared to the Rose Bengal test (RBT), buffered plate agglutination test (BPAT), serum agglutination test (SAT), and an indirect enzyme-linked immunosorbent assay (i-ELISA) was evaluated in the field diagnosis of ovine brucellosis. The test performance was evaluated by estimating sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), disease prevalence (DP), positive likelihood ratio (PLR), and negative likelihood ratio (NLR) using test agreement and bacteriological culture in 1777 samples. The false-positive result was significantly (P ⩽0.05) lower in LAT than RBT, BPAT, SAT, and i-ELISA. With reference to test agreement, the Se, Sp, PPV, and PLR were highest (P ⩽0.05) in LAT 99.33%, 99.88%, 98.68%, and 827.25%, respectively. With reference to bacteriological culture, the LAT and i-ELISA tests showed a significant difference in Se with SAT. However, no significant difference in specificity was detected. The DP was 8.44% in the five tests. In conclusion, LAT using SBPPs of B. melitensis could be a suitable serodiagnostic field test for ovine brucellosis, with high sensitivity and specificity.

Nail discoloration induced by doxycycline.

All tetracyclines are deposited in calcifying areas of the bones and teeth and may cause discoloration. Although hyperpigmentation of the skin, teeth and nails have been reported and well documented due to other tetracycline intake, it has been rarely reported that discolored nails induced by doxycycline in pediatric patients. Here we report an 11-year-old-boy with nail discoloration caused by doxycycline intake.

Pancytopenia As the Initial Manifestation of Brucellosis in Children.

Presenting with severe thrombocytopenia and pancytopenia is rare in children with brucellosis, and at the beginning it can be misdiagnosed as a hematological or a viral hemorrhagic disease. The follow-ups of 52 patients diagnosed with brucellosis from January, 2008, to December, 2013, in our clinic have shown the following results. Eleven out of these 52 patients revealed the fact that they had pancytopenia at the admission phase. Anemia and leukopenia were defined as hemoglobin levels and leukocyte counts below the standard values in terms of ages, thrombocytopenia as thrombocyte counts below 150,000/mm(3), and severe thrombocytopenia as thrombocyte counts below 20,000/mm(3). The most frequent admission symptoms and findings of the patients with pancytopenia were fever (75%), fatigue (50%), splenomegaly (75%), and hepatomegaly (41%). Laboratory results were hemoglobin 9.3±0.96 gram/dL, white blood cell count 2226±735.9/mm(3), and thrombocyte count 70,090±47,961/mm(3). The standard tube agglutination test was positive for all patients, and Brucellosis spp. were isolated in the blood cultures of six (54%) patients. Three of the 11 patients had severe thrombocytopenia, and they were admitted with complaints of epistaxis, gingival bleeding, petechiae, and purpura. At the beginning, two of three cases were misdiagnosed as Crimean-Congo hemorrhagic fever (CCHF), another zoonotic endemic disease in Turkey. Pancytopenia improved with treatment of brucellosis on all patients. In conclusion, brucellosis can show great similarity with hematologic and zoonotic diseases like CCHF. Brucellosis should be considered in the differential diagnosis of pancytopenia, treatment-resistant immune thrombocytopenia, and viral hemorrhagic disease, especially in countries where brucellosis is endemic.

Polycarbonate membranes: a novel surface for solid-phase determinations with utility in field format serological assays.

A dipstick suitable for immunoassay procedures has been developed, and its utility demonstrated by the development of tests for the determination of immunoglobulin levels in newborn bovine blood, and for the detection of bovine antibodies against Brucella abortus. The dipstick is of simple design, consisting of three components: a polycarbonate membrane, an adhesive, and a support material. The polycarbonate membrane is a porous filter material upon which the immunoassay is undertaken. Biomolecules bind to the membrane with sufficient affinity to permit the development of useful immunoassay procedures; however, this membrane exhibits low non-specific binding of reagents. Collectively, these properties make polycarbonate a useful material for solid-phase immunoassay procedures.

[Antibody neutralization test in brucellosis]. / Reaktsiia neitralizatsii antitel pri brutselleze

The authors present the results of study of the antibody neutralization test in brucellosis with the use of erythrocytes sensitized with lipopolysaccharide. This test was shown to be highly specific and sensitive in detection of brucellae and brucellosis antigen in pathological material, food and objects of external environment.

Epidemiological and clinical aspects of human brucellosis in eastern Anatolia.

BACKGROUND: We investigated the seroprevalence of human brucellosis and risk factors in a village in Eastern Anatolia, Turkey, where a brucellosis outbreak among cattle had recently occurred. PATIENTS AND METHODS: All 501 inhabitants enrolled were screened with the Rose Bengal Test and were asked to fill out a questionnaire to determine the signs and symptoms of brucellosis and the risk factors. Patients' laboratory findings and clinical responses were also evaluated. RESULTS: The Rose Bengal Test was positive in 44 persons. In comparison of age groups, fever (20.2%), arthralgia (19.2%) and weight loss (8.4%) were frequently seen in children (p<0.05). Hepatomegaly (17.9%) was also prevalent in the age group of 0 to 14 years (p<0.05). The consumption of dairy products, a family history of brucellosis, and raising livestock were significantly related to seropositivity (p<0.05). Rifampicin plus doxycyline was the most common regimen administered in these cases (54.5%) and also had the highest relapse rate (p<0.05). CONCLUSION: Brucellosis remains an important public-health problem in the rural areas of Turkey. Appropriate public health measures and education must be pursued to eradicate human brucellosis.

Serological diagnosis of Brucella infections in odontocetes.

Brucella ceti causes disease in Odontoceti. The absence of control serum collections and the diversity of cetaceans have hampered the standardization of serological tests for the diagnosis of cetacean brucellosis. Without a "gold" standard for sensitivity and specificity determination, an alternative approach was followed. We designed an indirect enzyme-linked immunosorbent assay (iELISA) that recognizes immunoglobulins G (IgGs) from 17 odontocete species as a single group. For the standardization, we used Brucella melitensis and Brucella abortus lipopolysaccharides, serum samples from seven resident odontocetes with no history of infectious disease displaying negative rose bengal test (RBT) reactions, and serum samples from seven dolphins infected with B. ceti. We compared the performance of the iELISA with those of the protein G ELISA (gELISA), the competitive ELISA (cELISA), and the immunofluorescence (IF) and dot blot (DB) tests, using 179 odontocete serum samples and RBT as the reference. The diagnostic potential based on sensitivity and specificity of the iELISA was superior to that of gELISA and cELISA. The correlation and agreement between the iELISA and the gELISA were relatively good (R(i/g)2 = 0.65 and kappa(i/g) = 0.66, respectively), while the correlation and agreement of these two ELISAs with cELISA were low (R(i/c)2 = 0.46, R(g/c)2 = 0.37 and kappa(i/c) = 0.62, kappa(g/c) = 0.42). In spite of using the same anti-odontocete IgG antibody, the iELISA was more specific than were the IF and DB tests. An association between high antibody titers and the presence of neurological symptoms in dolphins was observed. The prediction is that iELISA based on broadly cross-reacting anti-dolphin IgG antibody would be a reliable test for the diagnosis of brucellosis in odontocetes, including families not covered in this study.

A case of disseminated intravascular coagulation caused by Brucella melitensis.

OBJECTIVE: Although the haematological abnormalities were common accompaniments of brucellosis, overt disseminated intravascular coagulopathy was reported only in a case at the literature. We report here a case of Brucella melitensis sepsis which showed an acute onset with clinical and hematological findings disseminated intravascular coagulopathy METHODS: The patient had a physical examination, coagulation screening tests, tests of thrombin generation and fibrinolysis, bone marrow aspirate, serum Brucella agglutination test and blood culture. A case of Brucella infection presenting at the onset as a disseminated intravascular coagulation with gingival bleeding and echimotic lesions on abdomen is reported. A hemogram showed severe thrombocytopenia, anemia, and leukopenia. Anisocytozis, poikilocytozis, shift to the left of the granulocytic series, fragmented red blood cells, toksic granulation, were present in the peripheral smear. Bone marrow aspirate revealed a hypercellular marrow without granulomata or malignant precursors. Prothrombin time: 20 s, activated partial thromboplastin time:53.9 s were found longer than normal. D-dimer: 1056 mug/l (50-228 mug/l) was found. Fibrinogen was too low to detected in serum. Patient had Brucella melitensis isolated from blood cultures. RESULTS: After appropriate antimicrobial therapy, the clinical and hematological status of the patient improved, and 4 days later with disappearance of all hematological abnormalities. CONCLUSISON: Diagnosis of brucellosis may be delayed, particularly if uncommon features such as pancytopenia and disseminated intravascular coagulopathy are present. Hence, brucellosis must be considered in the differential diagnosis of all those conditions leading to pancytopenia and disseminated intravascular coagulopathy in areas endemic for brucellosis.

Factors affecting detection of Brucella melitensis by BACTEC NR730, a nonradiometric system for hemocultures.

The detection of Brucella bacteremia by subculture does not always correlate with a positive signal in the BACTEC NR730 nonradiometric system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.). The effect of the inoculum size, pH, sodium polyanetholesulfonate, carbon sources (i-erythritol, sodium pyruvate, monosodium glutamate, D-glucose, and L-alanine), and urea in the release of CO2 was evaluated by using the reference strain Brucella melitensis 16M. In standard NR6 vials with or without blood, inocula 5 to 10 times larger (at least 265 CFU per vial) than those usually found in the blood of patients with brucellosis were necessary to produce a positive growth value (GV) in 4 days or less, and similar results were obtained with vials supplemented with the substrates listed above. GVs were consistently lower in vials with sodium polyanetholesulfonate than in vials without this agent. Vials with no blood inoculated with 265 CFU per vial showed turbidity 1 day before GVs became positive, proving that the major limiting detection factor was the low level of release of CO2 and not an inadequate growth medium. In NR6 vials buffered to pH 6.2, GVs became positive faster and were higher than those in standard vials. NR6 vials at pH 6.2 with 0.3% sodium pyruvate yielded a positive GV in the first day of bacterial turbidity.

[Sacroiliitis caused by Brucella melitensis]. / Sakroiliitis durch Brucella melitensis.

Technetium scintigraphy established sacroiliitis in a 21-year-old man who had complained of hip pain for two weeks. Brucella melitensis was cultured from hip-joint fluid. He was treated with 160 mg trimethoprim and 800 mg sulphamethoxazole, three times daily for six weeks. He also had prophylactically 600 mg rifampicin daily by mouth for the last three weeks. He was symptom-free four weeks after onset of treatment. At follow-up one year later there was no sign of recurrence. Brucella titres and blood sedimentation rate were within normal limits.