Serology using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant women

Toxoplasma gondii causes severe fetal disease during acute infection in pregnant women, thus demanding early diagnosis for effective treatment and fetus preservation. Fetal tests are inefficient and risky, and diagnosis is based on maternal IgM serology, which had weak screening ability due to increased sensitivity, with alternative IgG avidity tests. Here, we performed ELISA and avidity assays using a recombinant T. gondii antigen, rROP2, in samples from 160 pregnant women screened from a large public hospital who were referred due to positive IgM assays. IgG serology and avidity assays were compared using whole T. gondii extract or rROP2. ELISA IgG detection with rROP2 showed good agreement with assays performed with T. gondii extract, but rROP2 IgG avidity assays were unrelated to whole extract antigen IgG avidity, regardless of the chaotrope used. These data show that avidity maturation is specific to individual antigen prevalence and immune response during infection. ELISA rROP2 IgG assays may be an alternative serological test for the diagnosis of toxoplasmosis during pregnancy, although our data do not support their use in avidity assays.

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

THE SCHISTOSOMULA TEGUMENT ANTIGEN AS A POTENTIAL CANDIDATE FOR THE EARLY SEROLOGICAL DIAGNOSIS OF SCHISTOSOMIASIS MANSONI

If Schistosoma mansoni infection could be detected in its early stages, especially before the egg deposition in the host tissues, the development of severe pathologic lesions could be efficiently prevented. We therefore developed an indirect enzyme-linked immunosorbent assay based on the detection of specific IgG against schistosomula antigens (ELISA-SmTeg). The assay was applied in sera samples from non-infected and infected mice collected seven and 15 days post-infection. The results were compared to the number of adult worms obtained by perfusion of the murine hepatic system 50 days post-infection. The sensitivity and specificity of the ELISA-SmTeg were 100% (p = 0.0032 and 0.0048 respectively for seven and 15 days of infection) with a cutoff value of 0.15 (p = 0.0002). Our findings show a novel low-cost serological assay using antigens which are easy to obtain, which was able to detect all the infected mice as early as seven days post-infection.

DETECTION OF Leptospira spp. AND Brucella abortus ANTIBODIES IN FREE-LIVING JAGUARS (Panthera onca) IN TWO PROTECTED AREAS OF NORTHERN PANTANAL, BRAZIL

 This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection.

LATERAL FLOW ASSAY FOR CRYPTOCOCCAL ANTIGEN: AN IMPORTANT ADVANCE TO IMPROVE THE CONTINUUM OF HIV CARE AND REDUCE CRYPTOCOCCAL MENINGITIS-RELATED MORTALITY

SUMMARYAIDS-related cryptococcal meningitis continues to cause a substantial burden of death in low and middle income countries. The diagnostic use for detection of cryptococcal capsular polysaccharide antigen (CrAg) in serum and cerebrospinal fluid by latex agglutination test (CrAg-latex) or enzyme-linked immunoassay (EIA) has been available for over decades. Better diagnostics in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce mortality. Recently, the cryptococcal antigen lateral flow assay (CrAg LFA) was included in the armamentarium for diagnosis. Unlike the other tests, the CrAg LFA is a dipstick immunochromatographic assay, in a format similar to the home pregnancy test, and requires little or no lab infrastructure. This test meets all of the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User friendly, Rapid/robust, Equipment-free, and Delivered). CrAg LFA in serum, plasma, whole blood, or cerebrospinal fluid is useful for the diagnosis of disease caused by Cryptococcusspecies. The CrAg LFA has better analytical sensitivity for C. gattii than CrAg-latex or EIA. Prevention of cryptococcal disease is new application of CrAg LFA via screening of blood for subclinical infection in asymptomatic HIV-infected persons with CD4 counts < 100 cells/mL who are not receiving effective antiretroviral therapy. CrAg screening of leftover plasma specimens after CD4 testing can identify persons with asymptomatic infection who urgently require pre-emptive fluconazole, who will otherwise progress to symptomatic infection and/or die.

Detection of viable and viable nonculturable Vibrio cholerae O1 through cultures and immunofluorescence in the Tucumán rivers, Argentina

Vibrio cholerae has been sporadically isolated from rivers in Tucumán, Argentina, since the outbreak in 1991. The aim of this study was to determine the environmental reservoir of the bacterium in these rivers, assessing the presence of Vibrio cholerae non-O1 and O1 (the latter both in its viable culturable and non culturable state) and its relationship to environmental physicochemical variables. 18 water samplings were collected in the Salí River (in Canal Norte and Banda) and the Lules River between 2003 and 2005. Physical-chemical measurements (pH, water temperature, electrical conductivity and dissolved oxygen) were examined. Vibrio cholerae was investigated with conventional culture methods and with Direct Immunofluorescence (DFA-VNC) in order to detect viable non culturable organisms. All isolated microorganisms corresponded to Vibrio cholerae non-O1 and non-O139 (Lules 26%, Canal Norte 33% and Banda 41%). The majority was found during spring and summer and correlated with temperature and pH. Non culturable Vibrio cholerae O1 was detected year round in 38 of the 54 water samples analyzed. Application of the Pearson correlation coefficient revealed that there was no relationship between positive immunofluorescence results and environmental physicochemical parameters. Genes coding for somatic antigen O1 were confirmed in all DFA-VNC-positive samples, whereas the virulence-associated ctxA and tcpA genes were confirmed in 24 samples.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

Use of limitations of radiolabeled anti-CEA antibodies and their fragments for photoscanning detection of human colorectal carcinomas.

Fifty-three patients with histologically proven carcinoma were injected with highly purified [131I]-labeled goat antibodies or fragments of antibodies against carcinoembryonic antigen (CEA). Each patient was tested by external photoscanning 4, 24, 36 and 48 h after injection. In 22 patients (16 of 38 injected with intact antibodies, 5 of 13 with F(ab')2 fragments and 1 of 2 with Fab' fragments), an increased concentration of 131I radioactivity corresponding to the previously known tumor location was detected by photoscanning 36-48 h after injection. Blood pool and secreted radioactivity was determined in all patients by injecting 15 min before scanning, [99mTc]-labeled normal serum albumin and free 99mTc04-. The computerized subtraction of 99mTc from 131I radioactivity enhanced the definition of tumor localization in the 22 positive patients. However, in spite of the computerized subtraction, interpretation of the scans remained doubtful for 12 patients and was entirely negative for 19 additional patients. In order to provide a more objective evaluation for the specificity of the tumor localization of antibodies, 14 patients scheduled for tumor resection were injected simultaneously with [131I]-labeled antibodies or fragments and with [125I]-labeled normal goat IgG or fragments. After surgery, the radioactivity of the two isotopes present either in tumor or adjacent normal tissues was measured in a dual channel scintillation counter. The results showed that the antibodies or their fragments were 2-4 times more concentrated in the tumor than in the normal tissues. In addition, it was shown that the injected antibodies formed immune complexes with circulating CEA and that the amount of immune complexes detectable in serum was roughly proportional to the level of circulating CEA.

Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathology study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tisue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.

The use of mannan antigen and anti-mannan antibodies in the diagnosis of invasive candidiasis: recommendations from the Third European Conference on Infections in Leukemia.

INTRODUCTION: Timely diagnosis of invasive candidiasis (IC) remains difficult as the clinical presentation is not specific and blood cultures lack sensitivity and need a long incubation time. Thus, non-culture-based methods for diagnosing IC have been developed. Mannan antigen (Mn) and anti-mannan antibodies (A-Mn) are present in patients with IC. On behalf of the Third European Conference on Infections in Leukemia, the performance of these tests was analysed and reviewed. METHODS: The literature was searched for studies using the commercially available sandwich enzyme-linked immunosorbent assays (Platelia™, Bio-Rad Laboratories, Marnes-la-Coquette, France) for detecting Mn and A-Mn in serum. The target condition of this review was IC defined according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Sensitivity, specificity and diagnostic odds ratios (DOR) were calculated for Mn, A-Mn and combined Mn/A-Mn testing. RESULTS: Overall, 14 studies that comprised 453 patients and 767 controls were reviewed. The patient populations included in the studies were mainly haematological and cancer cases in seven studies and mainly intensive care unit and surgery cases in the other seven studies. All studies but one were retrospective in design. Mn sensitivity was 58% (95% confidence interval [CI], 53-62); specificity, 93% (95% CI, 91-94) and DOR, 18 (95% CI 12-28). A-Mn sensitivity was 59% (95% CI, 54-65); specificity, 83% (95% CI, 79-97) and DOR, 12 (95% CI 7-21). Combined Mn/A-Mn sensitivity was 83% (95% CI, 79-87); specificity, 86% (95% CI, 82-90) and DOR, 58 (95% CI 27-122). Significant heterogeneity of the studies was detected. The sensitivity of both Mn and A-Mn varied for different Candida species, and it was the highest for C. albicans, followed by C. glabrata and C. tropicalis. In 73% of 45 patients with candidemia, at least one of the serological tests was positive before the culture results, with mean time advantage being 6 days for Mn and 7 days for A-Mn. In 21 patients with hepatosplenic IC, 18 (86%) had Mn or A-Mn positive test results at a median of 16 days before radiological detection of liver or spleen lesions. CONCLUSIONS: Mn and A-Mn are useful for diagnosis of IC. The performance of combined Mn/A-Mn testing is superior to either Mn or A-Mn testing.

Study of two different enzyme immunoassays for the detection of Mayaro virus antibodies

This paper presents the evaluation of an enzyme immunoassay in which Mayaro virus-infected cultured cells ara used as antigen (EIA-ICC) and an IgM antibody capture ELISA (MAC-ELISA) for Mayaro serologic diagnosis using 114 human sera obtained during a Mayaro outbreak occurred in Bolivia, in 1987. Results were compared with those obtained by haemagglutination-inhibition test (HAI). MAC-ELISA was the most sensitive technique for anti-Mayaro IgM detection. MAC-ELISA was twice sensitive as IgM EIA-ICC. The data shows that MAC-ELISA is a practical and valid technique for diagnosis of recent mayaro infection. IgG-ICC showed hight sensitivity and high specificity compared to HAI. The combination of anti-Mayaro IgG and IgM EIA-ICC results presented the highest sensitivity of the study. Anti-Mayaro IgG and IgM simultaneous detection by ELISA-ICC can be used for recent infection diagnosis (in spite of a less sensitive IgM detection than by MAC-ELISA), for surveillance and sero-epidemiologic studies, and for studies of IgG and IgM responses to Mayaro infection.

Technique for the detection of Toxoplasma gondii antigens in mouse urine

A simple and rapid staphylococcal coagglutination test for the detection of Toxoplasma gondii antigens in mice urine is described. A suspension of protein-A containing Staphylococcus aureus coated with rabbit hyperimmune serum was used as reagent. The sensitivity of the antigen assay was found to be at least 118 ng of the antigen protein per ml. No coagglutination was observed when the reagent was challenged against antigenic solutions of other parasites. The suitability of the method for detecting antigens of T. gondii in urine samples was studied by experimental toxoplasma infection in mice. Before the staphylococcal test, the urine samples were double serially diluted in 0.1 M PBS. From the second day on all samples from infected mice were positive at 1/16 dilution. At this dilution, all samples from non infected mice were negative or did not produce coagglutination. This method might be used in the rapid etiological diagnosis also in human cases of acute toxoplasmosis.

Evaluation of three serological tests for the detection of human plague in northeast Brazil

The passive haemagglutination (PHA) test, enzyme-linked immunosorbent assay (ELISA) and the dot enzyme-immunosorbent assay (DOT-ELISA) were used to detect the levels of IgG antibodies against the Fraction 1 (F1) antigen of Yersinia pestis in sera of plague-infected patients from Northeast Brazil. Twenty three selected PHA-positive sera of subjects with bacteriological confirmation of plague were also positive in the DOT-ELISA but only 19 were detected by the conventional ELISA technique. Another group of 186 serum samples from subjects diagnosed as plague-infected by clinical and epidemiological parameters, but PHA-negative, were screened with DOT-ELISA and 11 gave positive results. The specificity of the assays on the serological detection of plague was confirmed in inhibition tests using purified F1 antigen. These results suggest that DOT-ELISA can be an useful, simple and more sensitive alternative for the serodiagnosis of plague in Northeast Brazil.

Detection of promoter activity by flow cytometric analysis of GFP reporter expression.

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.