Large-scale validation of a latex agglutination test for diagnosis of tuberculosis

Large-scale validation of a simple latex agglutination test for the diagnosis of tuberculosis is described. Soluble antigens extracted from a non-pathogenic saprophytic mycobacterium, Mycobacterium w, which shares antigenic determinants with Mycobacterium tuberculosis, were covalently linked to carboxylated polystyrene latex beads. Batch to batch reproducibility of coated latex was ensured. Latex reagents were standardized to overcome non-specific agglutination. Reagents of the test are stable for 1 year at 4 degrees C. A total of 1,058 serum samples of pulmonary and extrapulmonary tuberculosis patients or patients with other pulmonary diseases and healthy controls living in endemic areas were tested. Sensitivity of 94% for pulmonary tuberculosis and 87% for extrapulmonary tuberculosis was obtained. Specificity is 92.2% for healthy controls and patients with other respiratory diseases. We conclude that the latex agglutination test can be utilized for mass screening for both pulmonary and extrapulmonary tuberculosis where diagnosis by existing methods is much more difficult.     

Detection of anti-Leptospira antibodies in sera of patients in the latex agglutination test

The results of the preliminary evaluation of the sensitivity and specificity of the newly developed diagnostic test based on the determination of genus-specific antibodies to leptospires in the latex agglutination test, are presented. This test makes it possible to detect anti-Leptospira antibodies of any serogroup. The advantages of the developed test have been determined.     

Commercially available histoplasmin sensitized latex particles in an agglutination test for histoplasmosis

Summary A commercial preparation of histoplasmin sensitized latex particles was tested in an agglutination test with sera from 50 culturally confirmed cases of histoplasmosis in varying stages of the infection. The reactions were superior to those obtained with collodion agglutination and complement fixation tests in which the antigen histoplasmin was also used. The latex agglutination test with the commercially available antigen is easy to do, and can be done in any laboratory equipped to carry out agglutination tests with the common bacterial antigens. It warrants more extensive trial in the general hospital laboratory as a screening test for histoplasmosis, especially the primary, pulmonary type.     

The latex agglutination test versus counterimmunoelectrophoresis for rapid diagnosis of bacterial meningitis

A modified latex agglutination (LA) test was compared with Gram-staining and counterimmunoelectrophoresis (CIE) for the rapid detection in the cerebrospinal fluid (CSF) of antigen to Haemophilus influenzae type b, Neisseria meningitidis groups A, B and C, Escherichia coli K1, Streptococcus pneumoniae and group B streptococci, seven frequent causes of bacterial meningitis in children. Of 50 CSF samples from patients with culture-proven bacterial meningitis 90% were correctly shown by the LA test to contain antigen of the responsible organism. Gram-staining revealed organisms in 80% of 45 of these samples. In 75% of the 40 samples that were of sufficient volume for CIE, positive results for the appropriate antigen were obtained. The concentration of antigen detected in the CSF by the LA test varied from undetectable to 800 000 ng/ml. Patients with a high concentration (more than 2000 ng/ml or a positive result at dilutions of CSF over 1/8) were significantly more likely to have a poor response to therapy (two died and two had persistent pleocytosis or bacteria in the CSF) than patients with a lower concentration (4/16 v. 0/18, P < 0.05). After appropriate therapy was begun the concentration of antigen fell dramatically, but measurable amounts of antigen persisted in the CSF for up to 6 days. The LA test detected bacterial antigen at concentrations 2 to 70 times below the lower limit detected by CIE. In seven additional patients who had received antibiotics before lumbar puncture was performed the LA test detected antigen from meningitis-causing bacteria even though cultures of the CSF were sterile. In another 145 patients who did not have meningitis the results of the LA test were negative. The LA test, done as described in this article, is easier to perform than CIE and should be a useful addition to the diagnostic tests carried out on the CSF of any patient suspected of having meningitis.     

Rapid latex agglutination test for detection of staphylococcal enterotoxins A to E that uses high-density latex particles

A rapid reversed passive latex agglutination method that uses high-density latex particles for the detection of staphylococcal enterotoxins (SE) A to E was developed. It took 3 h for incubation, much less than the 16 h needed with a customary latex agglutination test for SE detection such as a commercial test kit (SET-RPLA; Denka Seiken Co. Ltd., Tokyo, Japan). The rapid test was shown to be highly specific and sensitive for SE detection (detection limit, about 0.5 ng of SE per ml), comparable to the SET-RPLA test. The rapid test was also efficient in SE detection in foods and culture supernatants of staphylococcal strains, similar to the SET-RPLA test. This showed that a rapid test with high-density latex particles is fully reliable for use.     

Evaluation of a latex agglutination test for rapid detection of rotavirus in faecal specimens

Two methods for detecting rotaviruses (latex agglutination, electron microscopy) have been compared on 80 faecal samples. These samples were obtained from infants between the age of four and 30 months hospitalized for acute gastroenteritis in the Children's Hospital, Karl Marx University at Leipzig, in 1982. Complete agreement among the two techniques was found in 75 specimens. Sensitivity of latex agglutination could be estimated at 95%, the specificity also at 95%. Only one sample reacted nonspecifically. Performance of the latex agglutination proved quite simple. The results indicate that latex agglutination is suitable for rapid screening of rotavirus induced gastroenteritis in clinical practice thus enabling the rate of nosocomial rotavirus infections in children's hospitals to be reduced.     

Molecular cloning and characterisation of 23-kDa piroplasm surface proteins of Theileria sergenti and Theileria buffeli.

The cDNA encoding a 23-kDa piroplasm membrane protein (p23) of Theileria sergenti Chitose (C)-type was isolated and its nucleotide sequence was determined. The gene encodes a polypeptide of 223 aa with a 28 residue N-terminal signal sequence and a hydrophobic, valine-rich, C-terminal transmembrane domain, as deduced from its nucleotide sequence. Southern blot hybridisation analysis proved that p23 gene was a single copy gene and had allelic forms of the gene in the parasite population. By PCR, the open reading frames of T. sergenti Ikeda (I)-type and Theileria buffeli (B)-type p23 were amplified from genomic DNA and their nucleotide sequences were also determined. Comparison of C-type sequence with that of I-type and B-type revealed 90.5% and 93.5% sequence similarity, respectively, at the aa level. These results suggest that a conserved molecule in these benign Theileria spp. could be a candidate antigen for the development of an anti-piroplasm vaccine.     

Theileria sergenti proliferates in SCID mice with bovine erythrocyte transfusion.

The unavailability of in vitro or in vivo experimental systems has been the major factor hampering the progress of research studies on Theileria sergenti, causative agent of theileriosis, a major disease of cattle in Japan. We report the first successful propagation of T. sergenti in SCID mice into which uninfected bovine erythrocytes (Bo-RBC) were supplied periodically. The infectivity of T. sergenti proliferated in an SCID mouse was ascertained by successful transfer of infection into another SCID mouse into which uninfected Bo-RBC were supplied periodically.     

Rapid latex agglutination test for detection of staphylococcal enterotoxins A to E that uses high-density latex particles.

A rapid reversed passive latex agglutination method that uses high-density latex particles for the detection of staphylococcal enterotoxins (SE) A to E was developed. It took 3 h for incubation, much less than the 16 h needed with a customary latex agglutination test for SE detection such as a commercial test kit (SET-RPLA; Denka Seiken Co. Ltd., Tokyo, Japan). The rapid test was shown to be highly specific and sensitive for SE detection (detection limit, about 0.5 ng of SE per ml), comparable to the SET-RPLA test. The rapid test was also efficient in SE detection in foods and culture supernatants of staphylococcal strains, similar to the SET-RPLA test. This showed that a rapid test with high-density latex particles is fully reliable for use.     

Flow cytometry to evaluate Theileria sergenti parasitemia using the fluorescent nucleic acid stain, SYTO16

BACKGROUND: Although the infection of Theileria sergenti is demonstrated by intraerythrocytic localization of this parasite, much time and labor are necessary in order to determine this. We applied flow cytometry to evaluate T. sergenti parasitemia using the fluorescent nucleic acid stain method. METHODS: Peripheral blood samples from cattle infected with T. sergenti were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16, and hydroethidine (HE). Stained parasitized erythrocytes were measured by a flow cytometer equipped with a single argon laser operating at 488 nm. RESULTS: SYTO16-stained intraerythrocytic parasites were detected on the FL1 (525 nm) and parasitized cells were separated completely from unparasitized cells. However, HE-stained erythrocytes could not be divided clearly into parasitized and unparasitized cells. SYTO16-stained parasites were reproducibly detected at a percentage above 0.1%. Contaminating leukocytes, which were indicated by CD18-positive cells, were eliminated from the analysis by narrowing the light scatter gate of the erythrocyte fraction. A correlation (r = 0.983) between the percentage of SYTO16-positive cells and parasitemia in grazing cattle was observed. CONCLUSIONS: Flow cytometric detection using SYTO16 is a rapid and reliable method of monitoring parasitemia in T. sergenti-infected cattle.     

Diagnosis of disseminated candidiasis in hospitalized patients using the Cand-Tec latex agglutination assay

A total of 1,303 sera from 202 patients at risk for disseminated candidiasis were analyzed for the presence of Candida antigen using a commercially available latex agglutination test (Cand-Tec). Twentythree patients had disseminated candidiasis documented by positive blood cultures, deep biopsy culture and histopathology or autopsy. Six patients had transient candidemia, 15 patients had candiduria, 62 patients were not colonized yet treated empirically with amphotericin B, and 46 patients were not colonized and not treated with amphotericin B. The sensitivity and specificity of the Candida antigen test for the diagnosis of disseminated candidiasis was 87% and 36% (threshold titer of 12), 70% and 60% (14), and 30% and 85% (18), respectively. In contrast to previous studies we were unable to demonstrate a prognostic role for the Candida antigen test in patients with documented disseminated candidiasis. The lack of sensitivity and specificity of the Cand-Tec Candida antigen test precludes its use in the diagnosis of disseminated candidiasis.     

Prevalence of Theileria sergenti infection in Korean native cattle by polymerase chain reaction

This study was performed to investigate the prevalence of theileriosis and to compare the prevalence of this disease in Korean native cattle reared under different environmental conditions, namely, in a grazing area and a non-grazing area by polymerase chain reaction. Three hundred and one Korean native cattle (276 cows and 25 bulls) that had not received prior treatment or been vaccinated to prevent theileriosis were examined by PCR for Theileria sergenti infection from 2001 to 2002. In our study, the parasitemia range in T. sergenti-positive cattle by microscopy were from 0.1 to 3% (mean 0.8%). In terms of mean prevalence, 204 of the 301 Korean native cattle (67.8%) were positive reaction by PCR. Our results also revealed that the infection rate among cows (70.3%) was significantly higher than that among bulls (40.0%) (p < 0.01). T. sergenti infection among the over 3 year-old-group (75%) had a significant higher prevalence than that among the less than 3 year-old-group (61.8%) (p < 0.05). Our data also showed that grazing areas (76.1%) had the significant higher prevalence than non-grazing areas (51%) (p < 0.001). In conclusion, this study demonstrates that the prevalence of T. sergenti infection is high and that its prevalence in grazing cattle is higher than that in non-grazing cattle. Therefore, life-long treatment and the development of an optimal vaccine are needed to reduce the numbers of bovine theileriosis in both grazing and non-grazing areas.