Comparative study of the accuracy of different technique for the laboratory diagnosis of schistosomiasis mansoni in areas of low endemicity in Barra Mansa City, Rio de Janeiro State, Brazil

Schistosomiasis constitutes a major public health problem, with an estimated 200 million people infected worldwide. Many areas of Brazil show low endemicity of schistosomiasis, and the current standard parasitological techniques are not sufficiently sensitive to detect the low-level helminth infections common in areas of low endemicity (ALEs). This study compared the Kato-Katz (KK); Hoffman, Pons, and Janer (HH); enzyme-linked immunosorbent assay- (ELISA-) IgG and ELISA-IgM; indirect immunofluorescence technique (IFT-IgM); and qPCR techniques for schistosomiasis detection in serum and fecal samples, using the circumoval precipitin test (COPT) as reference. An epidemiological survey was conducted in a randomized sample of residents from five neighborhoods of Barra Mansa, RJ, with 610 fecal and 612 serum samples. ELISA-IgM (21.4%) showed the highest positivity and HH and KK techniques were the least sensitive (0.8%). All techniques except qPCR-serum showed high accuracy (82­95.5%), differed significantly from COPT in positivity , and showed poor agreement with COPT. Medium agreement was seen with ELISA-IgG (Kappa = 0.377) and IFA (Kappa = 0.347). Parasitological techniques showed much lower positivity rates than those by other techniques. We suggest the possibility of using a combination of laboratory tools for the diagnosis of schistosomiasis in ALEs.

Evaluation of ‘cattle’ and ‘Indian Bison’ type antigens of Mycobacterium avium subspecies paratuberculosis for diagnosis of Bovine Johne’s Disease using ‘indigenous ELISA’ and AGPT .

Two antigens (‘cattle’ type and ‘Indian Bison’ type) of Mycobacterium avium subspecies paratuberculosis were evaluated for diagnosis of Johne’s disease (JD) in a gaushala (cattle herd). Of the 160 cows of Sahiwal and Hariana breeds screened, 81 (50.6%) tested positive in ELISA and 66 (41.8%) in AGPT test. Using the two antigens, 33.5% tested positive in both the tests while 41.1% tested negative. Exclusively, only 8.2% tested positive in ELISA while 17.1% tested positive in AGPT. Two antigens together detected 58.9% prevalence of MAP in the gaushala. Individually, indigenous ELISA using antigen from native source of MAP proved superior to AGPT in the diagnosis of JD in cows.

Comparison between precipitin and ELISA tests in the bloodmeal detection of Aedes aegypti (Linnaeus) and Aedes fluviatilis (Lutz) mosquitoes experimentally fed on feline, canine and human hosts

The identification of arthropod bloodmeals is important in many epidemiological studies, as, the understanding of the life cycle of vectors and the patogens they transmit, as well as helping to define arthropods' control strategies. The precipitin test has been used for decades, but ELISA is slowly becoming more popular. To compare the two tests for sensitivity, specificity and accuracy to detect small insect bloodmeals, Aedes aegypti or Ae. fluviatilis mosquitoes were fed either on feline, canine or human hosts. Mosquitoes were frozen at 6, 12, 24, 48 or 72 h after feeding. Precipitin test showed better specificity and accuracy and ELISA test showed higher sensitivity. Better results with both tests were achieved when mosquitoes were frozen within 48 h from feeding

Combined immunoprecipitation and agglutination for the detection of the heterodimeric molecule: human chorionic gonadotropin as a study model.

Double-particle reversed passive hemagglutination (RPHA) was performed for the detection of an intact heterodimeric form of human chorionic gonadotropin (intact hCG) composed in Profasi hCG (P-hCG). This technique relied on two mixed types of human O RBC, which were individually coated with two distinct monoclonal antibodies that recognized alpha or beta subunit of hCG, i.e. ALC-1 and BEL-5, respectively. The positive hemagglutination result was achieved by this technique. However, in the BEL-5 coated single-particle control system, positive results for both P-hCG and beta subunit hCG solution were realized. The occurrence of beta-multimer hCG was a causative molecule revealed by the hemagglutination inhibition technique. Thereby, the novel method called "combined immunoprecipitation and agglutination" was developed to overcome this problem. The free beta subunit together with the betamultimer hCG were eliminated from other forms presented in P-hCG after using the ALC-1 coated particles. The precipitated particles, which captured the heterodimer hCG molecule, reacted further with soluble BEL-5 to subsequently form a trellis. A positive result was obtained only with P-hCG, but not with beta subunit hCG or hLH. This study is inferable as a model for the detection of heterodimeric molecule by an elementary method.

Proteins anchored via glycosylphosphatidylinositol and solubilizing phospholipases in Trypanosoma cruzi

The presence of GPI anchors and phospholipases capable of solubilizing them in Trypanosoma cruzi has been investigated in epimastigotes, metacyclic trypomastigotes from axenic cultures and tissue culture trypomastigotes. The GPI anchored proteins in epimastigote forms are scarce when compared to their abundance in the parasite forms which can infect mammals, and GPI-solubilizing phospholipases C have been found in all life cycles stages. In epimastigote and metacyclic forms, the activity is found in the soluble fraction upon cell lysis, whereas in tissue cultured trypomastigotes it is membrane bound and, being mostly sensitive to p-chloromercuriphenylsulfonate, resembles closely the GPI specific phospholipase of Trypanosoma brucei. Sequential immunoprecipitations with monoclonal antibodies and anti-CRD indicated the presence of several sub-populations among the surface proteins of metacyclic trypomastigotes, five of these belonging to the GPI-anchored 90 kD family. Among this family, the epitopes recognized by MAb-1G7 are present in three members, one of them also expressing the 3F6 epitope. There are 2 members recognized only by MAb-3F6 but not by MAb-1G7, one of them being probably galactosylated on the GPI since it can be immunoprecipitated by anti-CRD. Very strangely, the epitope recognized by the MAb-WIC29.26 was always present on the gp72, as originally described, but under certain circumstances appeared cryptic on one of the 90 kD species. During epimastigote transformation into metacyclic trypomastigotes in vitro, the ability of the GPI of the 1G7-antigen to be solubilized by phospholipase C and D varies depending on the age of the culture and presence or absence of fetal calf serum. Different patterns of solubilization were also obtained for 1G7-Ag, depending on whether the test is performed with parasite lysates or with antigen affinity purified from them. Our data indicate that the phospholipase C resistance observed does not arise from acylation on the inositol, as previously described for acetylcholinesterase from human erythrocytes, being rather due to factors which either modify the GPI or affect the action of the phospholipases...

Application of alcohol- or acetone-fixed Schistosoma eggs as an alternative to lyophilized eggs for the circumoval precipitin test.

Antigenicity of Schistosoma mansoni and S. japonicum eggs preserved in ethanol or acetone were assessed in a circumoval precipitin (COP) assay. The egg antigens were found to retain sufficiently their COP reactivity for the diagnosis of both schistosomiasis mansoni and japonica, although their reactivity became lower than that of lyophilized eggs. These alternative preparations for COP tests have advantages, such as keeping eggs directly in fixatives soon after the egg-purification process. Furthermore, evaporation-process may cause eggshell cleavages which facilitate the reaction. The possible usefulness of those eggs in COP assays in local endemic areas is discussed.

Simple serological tests for detecting classical heat labile enterotoxin (LT-I) of Escherichia coli.

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) remains a problem in Southeast Asia. At present, no routine laboratories as yet are available for ETEC detection. In this study, attempts were made to produce reagents for use in simple serological tests for detecting LT. The serological methods were the Biken, the staphylococcal coagglutination and the reverse passive hemagglutination tests. For the Biken test, medium was prepared locally by mixing constituents as described previously by Honda et al., (1981). Anti-CT-B subunit was prepared by immunizing a rabbit with commercial CT-B subunits (Sigma). Other chemical reagents e.g. colistin, lincomycin etc. were obtained from the local supplies. Using the locally made reagents to detect LT from 100 WHO reference strains of E. coli by the Biken test, it was found that the test had 100%, 92%, 96%, 100% and 92.5% of specificity, sensitivity, accuracy, positive predictive value and negative predictive value, respectively. Protein A rich Staphylococcus aureus from the stock culture of the Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University were grown in suitable medium i.e. blood agar containing lincomycin (BA-Lin). Suitable amount of the rabbit anti CT-B subunit (0.1 ml) was used to sensitize each ml of the formalinized, heat-fixed bacteria. The sensitized bacteria were used for detecting LT in the lysates of the 100 E. coli reference strains. The lysates were prepared by growing the E. coli strains on BA-Lin medium for 8 hours, then a loopful of each strain was inoculated into colistin solution (20,000 unit/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Cellulose acetate precipitation test in sero-diagnosis of amoebiasis.

Cellulose acetate precipitation (CAP) test for the detection of antibody against Entamoeba histolytica using the axenic antigen, was performed on the 127 serum samples obtained from patients with amoebic liver abscess (14), amoebic hepatitis (21), amoebic dysentry (11), amoebic colitis (31), other parasitic infestations (25) and normal individuals (25). The percent positivity was 100, 95.23, 90.9, 67.74 and 16 and 12 respectively whereas the corresponding figures for the indirect immunoflourescence (IFAT) test were 100, 100, 100, 74.19, 12 and 8 respectively. Although CAP is not as good a test as IFAT, yet it can be recommended for routine testing due to its sensitivity, speed of performance and applicability to a single serum sample.

Circumoval precipitin (COP) test using air-dried eggs of Schistosoma japonicum and S. mansoni.

In an attempt to establish a simplified circumoval precipitin (COP) test for the diagnostic purpose of schistosomiasis, air-dried eggs of both Schistosoma japonicum and S. mansoni were tested as antigens for this assay. Twenty-six sera from mice infected with S. japonicum showed positive COP reactions as assessed by air-dried eggs. Among 36 serum samples from patients with schistosomiasis japonica, five exhibited false negative reaction when assessed with air-dried eggs and showed a minimum level of COP reaction when assessed with lyophilized eggs. Similarly, all of 30 serum samples from jirds (Meriones unguiculatus) infected with S. mansoni gave positive COP reaction when assessed using air-dried egg. Diagnostic sensitivity of air-dried egg-system was comparable to those of fresh egg-or lyophilized egg-systems. A simple COP technique employing air-dried eggs, instead of lyophilized or fresh ones, would be thus useful for the serodiagnosis of schistosomiasis in local endemic areas, where sophisticated laboratory facilities are not available.