An Easy and efficient method for native and immunoreactive <i>Echinococcus granulosus</i> antigen 5 enrichment from hydatid cyst fluid

Background:Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis. Methodology/Principal Findings: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results. Conclusions/Significance: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera

An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid

Background: Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis. Methodology/Principal Findings: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results. Conclusions/Significance: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.This work was supported by Regione Autonoma della Sardegna (http://www.regione.sardegna.it/)Pubblicat

Brucella abortus Infection Acquired in Microbiology Laboratories

We report an outbreak of laboratory-acquired Brucella abortus infection originating in the accidental breakage of a centrifuge tube. A total of 12 laboratory workers were infected (attack rate of 31%), with an incubation time ranging from 6 weeks to 5 months. Antibody titers were evaluated weekly in all personnel exposed, allowing the diagnosis of the infection in most cases before the onset of clinical symptoms, so that specific therapy could be administrated

A Retrospective study on burden of human echinococcosis based on hospital discharge records from 2001 to 2009 in Sardinia, Italy

Cystic Echinococcosis (CE) is an infective zoonosis that represents a worldwide important public health problem. In humans, its manifestations may range from asymptomatic infection to severe disease and possible death, and lead to economic losses from treatment costs and lost wages. Recent studies suggest that this disease has a large social impact in endemic areas, and estimates of burden in terms of monetary and no-monetary impact on human health are essential to allocate financial and technical resources. In Sardinia, the most affected Italian region per number of inhabitants, CE is still endemic, although three eradication campaigns have been carried out in 1962, 1978, and 1987, respectively. To date, the burden of human CE in Sardinia remains poorly defined. In this work, a retrospective study was carried out using public Hospital Discharge Records spanning from 2001 to 2009. During these years, a total of 1409 discharges were recorded: 1196 (84.88%) records corresponding to patients hospitalized for symptoms directly correlated to CE (primary diagnosis), and 213 (15.11%) records corresponding to patients hospitalized for symptoms not directly correlated to CE and with an afterwards or concurrent diagnosis of echinococcosis made during the hospitalization (secondary diagnosis). The annual regional average record (discharge rate) was 9.3/100,000 inhabitants. Direct cost associated with diagnosis, surgery or chemotherapy, medical care, and hospitalization in humans were evaluated in this work. Furthermore, burden of disease was also evaluated by using the disability-adjusted life years (DALYs), the preferred disease-burden measure of the World Health Organization. Knowing the burden of human CE in Sardinia is extremely important to enable the prioritization of control measures for this preventable neglected disease. This is the first study describing the measure of the overall disease burden in an Italian region endemic for this disease, performed by calculating the number of CE patients from Hospital Discharge Records

<i>Brucella abortus</i> infection acquired in microbiology laboratories

We report an outbreak of laboratory-acquired Brucella abortus infection originating in the accidental breakage of a centrifuge tube. A total of 12 laboratory workers were infected (attack rate of 31%), with an incubation time ranging from 6 weeks to 5 months. Antibody titers were evaluated weekly in all personnel exposed, allowing the diagnosis of the infection in most cases before the onset of clinical symptoms, so that specific therapy could be administrated

Serological and molecular detection of <i>Bartonella</i> spp. in humans, cats and dogs from Northern Sardinia, Italy

Bartonella spp. are aerobic, gram-negative pleomorphic bacteria, belonging to the α2 subgroup of the Proteobacteria. Many Bartonella spp. are pathogens of humans, dogs, and cats. Numerous domestic and wild animals can serve as chronically infected reservoir hosts for Bartonella spp. An increasing number of arthropod vectors have been implicated in the transmission of Bartonella spp. The main source of transmission to humans is cats by means of scratches, whereas infection is less likely to occur by cat bite. Most human cases of infection with Bartonella henselae, named cat scratch disease (CSD), present as an acute febrile lymphadenopathy. The presence of cat fleas is essential for the maintenance of the infection within the cat population. The role of dogs as reservoirs of Bartonella spp. is less clear, because they seem to be only accidental hosts. Nevertheless, dogs are excellent sentinels for human infections, because a similar disease spectrum develops in dogs

Comparison and evaluation of analytic and diagnostic performances of four commercial kits for the detection of antibodies against Echinococcus granulosus and multilocularis in human sera.

Cystic echinococcosis (CE) is a disease caused by Echinococcus granulosus sensu lato (s.l.), an ubiquitous worldwide zoonotic agent affecting humans and animals. Diagnosis of CE in humans is usually performed by imagine techniques along with immunoassays. The aim of our study was to evaluate and compare four commercial diagnostic kits, based on the detection of IgG antibodies against E. granulosus and E. multilocularis. The study was performed on a total of 259 sera: the positive (n = 74) and the negative (n = 185) group. The following analytic and diagnostic performances of the four kits were evaluated: operator skills, specificity, sensitivity, repeatability, reproducibility, accuracy, positive and negative predictive values. Based on the parameters evaluated, all four tests demonstrated excellent quality and proved to be reliable diagnostic tools to support the clinical evaluation of human patients suspected of having CE. The four commercial assays, in our hands, presented altogether, a range of performances from good to excellent, being immunoblotting (IB) the most reliable, used as gold standard, followed by the immunochromatographic test (ICT) and finally the two enzyme linked immunosorbent assay (ELISAs)

Summary of the ELISA results obtained with the Ag5 enriched antigen test and four commercial kits.

<p>Summary of the ELISA results obtained with the Ag5 enriched antigen test and four commercial kits.</p

Immunoreactive bands subjected to mass spectrometry analysis.

<p>HCF was separated by SDS-PAGE and the resulting gel was cut in two parts, one of them subjected to western blot experiment with serum 1, the other part stained and further processed for protein identification Left: Immunoreactive profile observed by western immunoblotting on non-reduced (N) and reduced (R) HCF samples. Right: reactive bands excised from the SDS-PAGE gel and subjected to LC-MS/MS identification. M: molecular weight markers.</p