QuantiFERON®-CMV assay for the assessment of cytomegalovirus cell-mediated immunity.

Cytomegalovirus (CMV) infection has historically been a major complication among immunocompromised patients, such as solid-organ and stem-cell transplant recipients and patients with advanced HIV infection. While the introduction of antiretroviral therapy has almost eradicated CMV infection in HIV-infected patients, CMV disease remains a significant problem in transplant recipients once antiviral prophylaxis is discontinued. QuantiFERON(®)-CMV allows the assessment of cellular immunity against CMV by detecting the production of IFN-γ following in vitro stimulation with CMV antigens. Preliminary studies have shown a correlation between a lack of detectable cell-mediated immunity measured by the QuantiFERON-CMV assay and a higher incidence of CMV infection and disease in immunocompromised patients. Measurement of cell-mediated immunity against CMV appears to be a promising strategy to identify patients at highest risk for the development of CMV disease and, therefore, to individualize preventive strategies for CMV in transplant recipients.

A recyclable assay to analyze the NH(2)-terminal trimming of antigenic peptide precursors.

The proteasome plays an essential role in the production of MHC class I-restricted antigenic peptides. Recent results have indicated that several peptidases, including tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, could act downstream of the proteasome by trimming NH(2)-terminal extensions of antigenic peptide precursors liberated by the proteasome. In this study, we have developed a solid-phase peptidase assay that allowed us to efficiently purify and immobilize proteasome, tripeptidyl peptidase II, and puromycin-sensitive aminopeptidase. Whereas the first peptidase was active against small fluorogenic peptides, the latter two could also digest antigenic peptide precursors and could be used repeatedly with different precursors. Using three distinct antigenic peptide precursors, we found that tripeptidyl peptidase II never cleaved within the antigenic peptide sequence, suggesting that, aside from its proteolytic activities, it may also play a role in protecting antigenic peptides from complete hydrolysis in the cytosol. This method should be valuable for high throughput screenings of substrate specificity and potential inhibitors.

Comparison of a multiplexed bead-based assay with an immunofluorescence and an enzyme-immuno assay for the assessment of Epstein-Barr virus serologic status

Dans cette étude, nous avons testé la performance diagnostique d'une nouvelle technique d'analyse multiplexée qui permet la détection d'anticorps de différentes spécificités dans la même réaction. En l'absence de gold standard, nous avons choisi de comparer la performance diagnostique de l'analyse avec deux méthodes de référance que sont l'IF et EIA, et avec un consensus déterminé selon une règle de majorité entre les trois méthodes.¦393 sérums analysés par IF, conservés par congélation, ont été décongelés pour être analysés par EIA et BBA. Pour chaque sérum, les anticorps recherchés ont été les anti-VCA (Viral Capsid Antigen) IgM, anti-VCA IgG et anti-EBNA (Epstein-Barr Nuclear Associated) IgG. Les échantillons ont été classés en cinq groupes selon les résultats de l'IF : séronégatifs, infections aiguës, infections anciennes et deux types d'indéterminés.¦Pour chaque méthode, le résultat numérique (index ou titre) des analyses est converti en termes de positif, négatif ou douteux. Pour le résultat de chaque type d'anticorps, un consensus est établi selon une règle de majorité entre les trois méthodes, permettant une interprétation du stade de l'infection. Puis l'interprétation de chacune des méthodes a été comparée au consensus. Nous avons également comparé les trois méthodes les unes aux autres concernant la détection des anticorps.¦Globalement, nous observons une bonne corrélation qualitative entre les trois approches pour détecter les anti-VCA IgG et IgM. Pour pour les anti-EBNA IgG, il y a une divergence notable entre l'IF et les deux autres méthodes, l'IF apparaissant moins sensible que les autres méthodes, résultant en un nombre accru d'interprétations indéterminées du stade de l'infection.¦L'origine de cette divergence ne peut être due à une perte d'anticorps liée au stockage de longue durée des échantillons. En effet, EIA et BBA restent plus sensibles que IF, dont l'analyse a été faite sur des sérums frais.¦Cette divergence ne semble pas non plus être due aux différents antigènes utilisés par les trois méthodes. EIA et BBA utilisent le même antigène recombinant EBNA-1, alors que l'IF utilise des "cellules lymphoïdes choisies pour leur production sélective d'antigènes EBNA". Ces cellules sont probablement des cellules infectées par EBV qui devraient exprimer plus d'antigènes de latence que seul EBNA-1. Cette différence devrait donc plutôt en principe résulter en une meilleure sensibilité de l'IF par rapport aux deux autres méthodes.¦Les anti-EBNA IgG peuvent disparaître chez les patients immunocompromis chez qui se produit une réactivation d'EBV. Nous avons donc recherché le status immunitaire des patients du groupe dont les sérums étaient négatifs pour anti-EBNA IgG en IF et positifs par les autres méthodes: seulement 28 des 70 patients étaient immunocompromis.¦Par conséquent, il est probable que dans la majorité de ces résultats discordants, les anticorps anti-EBNA IgG détectés par BBA et EIA sont de vrais positifs non décelés par l'IF.¦En conclusion, BBA est meilleur que la méthode de référance qu'est l'IF, et est égal à EIA en ce qui concerne la performance diagnostique. En outre, ces deux nouvelles méthodes offrent une économie de temps en raison de manipulations moindres, et ne requièrent aucune formation en microscopie à fluorescence. Elles sont également plus économes en échantillons que IF. BBA a l'avantage de n'avoir besoin que de deux analyses pour donner un diagnostique, alors que IF et EIA ont en besoin d'une par anticorps. Enfin, BBA dispose de contrôles internes permettant de reconnaître les liaisons non antigène-spécifiques des anticorps. Par contre, BBA nécessite l'achat d'un lecteur par cytométrie de flux assez coûteux.

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.

Enzyme-linked immunosorbent assay for detection and quantification of Cryptococcus neoformans antigen

An enzyme-linked immunosorbent assay was standardized for the detection of cryptococcal antigen in serum and cerebrospinal fluid. The system was evaluated in clinical samples from patients infected by human immunodeficiency virus with and without previous cryptococcosis diagnosis. The evaluated system is highly sensitive and specific, and when it was compared with latex agglutination there were not significant differences. A standard curve with purified Cryptococcus neoformans antigen was settled down for the antigen quantification in positive samples.

Detection of the acute phase of abdominal angiostrongyliasis with a parasite-specific IgG enzyme linked immunosorbent assay

Angiostrongylus costaricensis may cause intestinal lesions of varied severity when it accidentally infects man in Central and South America. First-stage larvae have never been detected in stools. Therefore, a parasite-specific IgG ELISA was evaluated for the determination of the acute phase of infection. The specificity and the sensitivity of the immunoassay was shown to be 76.2% and 91.1%, respectively. Eight serum samples taken from patients with histopathological diagnosis, at different time points (3 to 15 months) after surgical treatment, showed a sharp and early decline in antibody reactivity. The titration of anti-A. costaricensis antibodies has proved to be a useful method for the diagnosis of acute abdominal angiostrongyliasis.

High proportion of wrongly identified methicillin-resistant Staphylococcus aureus carriers by use of a rapid commercial PCR assay due to presence of staphylococcal cassette chromosome element lacking the mecA gene.

During a 9-month period, 217 patients were newly diagnosed as methicillin-resistant Staphylococcus aureus (MRSA) carriers by using a commercial rapid PCR-based test (GeneXpert). However, no MRSA was recovered by culturing the second swab in 61 of these patients. Further analyses showed that 28 (12.9%) of the patients harbored S. aureus isolates with a staphylococcal cassette chromosome element lacking the mecA gene and were thus incorrectly determined to be MRSA carriers.

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by enzyme-linked immunosorbent assay

The present study was conducted to detected IgG antibodies using Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by the enzyme-linked immunosorbent assay (ELISA). Sera from 90 subjects were analyzed (30 with strongyloidiasis, 30 with other parasites and 30 healthy individuals). Results were expressed in antibody titers, which were considered as positive when titer was > 80. Sensibility and specificity of the assay were 100% and 96.7%, respectively. It can be concluded that the heterologous alkaline extract could be employed in ELISA as a diagnostic aid in human strongyloidiasis, due to its advantages as easiness of obtaining, practicability in preparing, and high indexes of sensitivity and specificity.

Detection of Cryptosporidium - specific coproantigen in human immunodeficiency virus/acquired immunodeficiency syndrome patients by using a commercially available immunoenzymatic assay

The aim of this study was to verify the occurrence of Cryptosporidium infection in 52 human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients (group 1) and 38 clinically healthy individuals (group 2) by using enzyme immunoassay (EIA). All fecal samples collected were submitted to the Baermann, Lutz, and Ritchie methods, the Safranin/Methylene Blue, and Weber's chromotrope modified Trichrome staining techniques, and EIA. In group 1, parasitological staining techniques and EIA were both positive for Cryptosporidium sp. infection in 3/52 (5.8%) samples and both negative in 45/52 (86.5%) samples, while 4/52 (7.7%) samples were positive in EIA and negative in parasitological staining techniques. Concerning group 2, all samples were negative by EIA and microscopy for Cryptosporidium infection. In conclusion, EIA may be an alternative method for detecting Cryptosporidium-specific coproantigen in HIV/AIDS patients.

Whole blood assay to access T cell-immune responses to Mycobacterium tuberculosis antigens in healthy Brazilian individuals

The production of interferon gamma (IFNgamma) guarantees effective T cell-mediated immunity against Mycobacterium tuberculosis infection. In the present study, we simply compare the in vitro immune responses to Mycobacterium antigens in terms of IFNg production in a total of 10 healthy Brazilian volunteers. Whole blood and mononuclear cells were cultivated in parallel with PPD, Ag85B, and M. bovis hsp65, and five-days supernatants were harvested for cytokine detection by ELISA. The inter-assay result was that the overall profile of agreement in response to antigens was highly correlated (r² = 0.9266; p = 0.0102). Potential analysis is in current progress to dictate the usefulness of this method to access the immune responses also in tuberculosis patients and its contacts.

Circulating filarial antigen in the hydrocele fluid from individuals living in a bancroftian filariasis area - Recife, Brazil: detected by the monoclonal antibody Og4C3-assay

The purpose of this study was to examine the circulating filarial antigen (CFA) detected by the monoclonal antibody (mAb) Og4C3-ELISA in paired samples of serum and hydrocele fluid from 104 men with hydrocele, living in an endemic area of Wuchereria bancrofti. Nocturnal blood specimens were filtered and examined for microfilariae (MF) and ultrasound was used in order to identify the presence of adult worms (the filaria dance sign - FDS) in the lymphatic vessels of the scrotal area. Four groups were selected according to their parasitological status: group I - 71 MF- and FDS-; group II - 21 MF+ and FDS+; group III - 10 MF- and FDS+ and group IV- 2 MF+ and FDS-. CFA was identified simultaneously (fluid and serum) in 11 (15.5%), 21 (100%), 3 (30%), and 1 (50%) in groups I, II, III, and IV, respectively. In despite of high CFA+ level (antigen Og4C3) units/ml, the Geometrical Mean (GM) = 2696) in the sera of these 36/104 paired samples, when compared to the hydrocele fluid, (GM = 1079), showed a very good correlation between the CFA level in the serum and CFA level in the fluid (r = 0.731). CFA level in the serum of the 23 microfilaremics (groups II and IV) was extremely high (GM = 4189) and was correlated with MF density (r = 0.442). These findings report for the first time the potential alternative use of the hydrocele fluid to investigate CFA using the mAb Og4C3-ELISA.

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

Detection of Entamoeba histolytica/Entamoeba dispar in stool specimens by using enzyme-linked immunosorbent assay

Entamoeba histolytica actually comprises two genetically distinct but morphologically indistinguishable species. E. histolytica can cause invasive intestinal and extra intestinal disease, while E. dispar cannot. Identification and differentiation of E. dispar and E. histolytica in stool sample by microscopy is imprecise. Several weeks of culture and isoenzyme analysis are required to differentiate E. histolytica from E. dispar. In this study, we have used an enzyme-linked immunosorbent assay (ELISA) for detection of E. histolytica/E.dispar and compared it with microscopy. Eighty-eight samples were evaluated, trichrome staining was positive in 20.4% (18) and ELISA was positive in 29.5% (26). Both tests were positive in 14 (15.9%) samples, 4 (4.5%) only with direct microscopy, and 12 (13.6%) only with ELISA. Both tests were negative in 58 (65.9%) samples. Microscopy has low sensitivity and high specificity, low negative predictive value and high positive predictive value in comparison with ELISA. E. histolytica/E. dispar antigen detection ELISA tests are inexpensive compared to the specific tests, yield objective results and do not require experienced microscopists and can therefore be recommended for screening of stools worldwide and the results can be taken for treatment that are fitting with its clinic.