Validation of salmonellosis and shigellosis diagnostic test kits at a provincial hospital in Thailand.

Rapid Diagnosis of salmonellosis and shigellosis was performed using six different diagnostic test kits which recently have been made available commercially. They were Salmo-Dot, Typhi-Dot, Shigel Dot A, B, C, and D test kits for detection of Salmonella spp., group D salmonellae, and groups A, B, C, and D Shigella spp., respectively. The principle of all test kits is a membrane (dot) ELISA using specific monoclonal antibodies to the respective pathogens as the detection reagents. The present study was designed to validate the accuracy of the test kits, at a laboratory in a provincial hospital in Thailand, in comparison with the conventional bacterial culture method alone or with the combined results of the culture and the Western blot analysis (WB) for detecting the respective bacterial lipopolysacchharides (LPS) in specimens. Five hundred rectal swab samples of patients with diarrhea who seeked treatment at the hospital, were evaluated. The diagnostic accuracy of the Salmo-Dot was 91.0% when compared with the conventional bacterial culture method alone but was 100.0% in comparison with the combined results of the culture and the WB. The Typhi-Dot and the Shigel-Dot A, B, C, and D showed 100%, 99.2%, 95.0%, 94.0% and 96.4%, respectively when compared with the culture alone and all were 100% in comparison with the combination of the results of the bacterial culture and the WB. The Shigel-Dot A revealed antigen of type 1 Shigella dysenteriae in several specimens in which the bacteria could not be recovered by the culture method. This difference is important as type 1 Shigella dysenteriae have high epidemic potential and often cause severe morbidity. Unawareness of their presence by the conventional culture may have great impact on disease surveillance for public health. The pathogen detection using the six diagnostic test kits is sensitive, specific, rapid, and relatively simple and less expensive. Several specimens can be tested at the same time without much increase in turn around time. Moreover, these kits produce no contaminated waste as compared with the bacterial culture method. The test kits should be used for rapid screening of specimens of patients with diarrhea especially in areas where culture facilities are inadequate.

Differential diagnosis of schistosomiasis mekongi and trichinellosis in human.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.

Comparative studies on schistosomulicidal activity of mouse and rat eosinophils.

Eosinophils from interleukin (IL)-5 transgenic mice were shown to have antibody-dependent killing activity against the larvae of Schistosoma japonicum. However, in comparison with rat eosinophils, the schistosomulicidal activity of mouse eosinophils was lower. Flow cytometric analysis of the cells binding to mouse immunoglobulins demonstrated that rat cells were superior to mouse cells in the binding of mouse IgG. However, the adherence and schistosomulicidal activity of mouse cells were inhibited by rat anti-mouse Fcgamma receptor monoclonal antibody. These results suggest that the mechanism of killing by mouse eosinophils is mediated by IgG antibodies.

The B cell epitope of paramyosin recognized by a protective monoclonal IgE antibody to Schistosoma japonicum.

Passive immunization of mice with a monoclonal IgE antibody to Schistosoma japonicum (SJ18 epsilon. 1) induces significant protection to a challenge infection and the target molecule of SJ18 epsilon. 1 is paramyosin. In the present study, we demonstrate the B cell epitope of paramyosin recognized by SJ18 epsilon. 1 by using a series of deletion mutants expressed in Escherichia coli. SJ18 epsilon 1 reacted with the recombinant paramyosin containing 113 amino acids (Glu301. Ala413) but not with a shorter peptide (Glu301-Asp343). Further epitope mapping carried out by a multi-pin system using heptameric peptides synthesized sequentially from 71 amino acids of paramyosin (Asp343-Ala413) demonstrated significant binding of SJ18 epsilon. 1 to the sequence, 359Ile-Arg-Arg-Ala362. Replacement set analysis of the pentameric peptide, 358Leu-Ile-Arg-Arg-Ala362, revealed that replacement of each residue with a hydrophobic or hydrophilic amino acid did not inhibit binding of SJ18 epsilon. 1, whereas replacement of positively charged Arg. or hydrophobic Ala with a negatively charged amino acid, Glu, showed reduction in binding of the antibody. Moreover, replacement of each amino acid including Arg with a positively charged amino acid, Lys, resulted in a drastic loss of the binding, indicating that binding of the antibody was markedly affected by the change of charges of the peptide as well as by the conformational alteration. The target epitope of SJ18 epsilon. 1 was common among paramyosins of S. mansoni, Taenia solium and Echinococcus granulosus but not among nematode paramyosins, suggesting that the epitope is specific for platyhelminthes.

Assessment of putative tests for protective immunity to falciparum malaria.

Sera from clinically immune individuals comprising 10 hospitalised patients (Group I), 30 persons residing in a malaria endemic area in Thailand (Group II) and 8 persons from a hyperendemic area in Ivory Coast (Group III) were tested by the parasite growth inhibition (PGI), indirect fluorescent antibody test of ring-infected erythrocyte surface antigen (RESA-IFA), urease-ELISA and Western blot. Paired sera from patients recovering from malaria (Group IV) as well as sera from blood donors were also tested. In the PGI test, sera were tested against three uncloned isolates of P. falciparum comprising SO, I4 and AE9 (PGI-SO, PGI-I4 and PGI-AE9 respectively). When growth inhibition of greater than or equal to 30% against any one of the three isolates was considered positive, the positive rate for the combined Groups I, II and III was 78.7%. Further analysis showed that the positive rates for PGI-SO, PGI-I4 and PGI-AE9 were 63.8%, 59.5% and 59.5% respectively and were not significantly different (p greater than 0.05). Comparison between PGI-SO, PGI-I4 and PGI-AE9 activities of Groups I, II and III sera showed no significant differences in any comparison groups except with PGI-AE9 in which Group III sera were more frequently positive than Group II sera (p = 0.004). Follow-up of PGI-SO and PGI-AE9 activities in Group IV patients showed mostly a decrease or no change in the activities of the convalescent sera taken 63 days later. RESA-IFA positive rate in the combined Groups I, II and III sera was 91.7%. There were no significant differences either in the seropositive rates or in the geometric mean antibody titers (GMT) between Groups I, II and III sera. Follow-up in Group IV patients showed no change in antibody titers in 64% of cases, decrease and increase in titers in 29% and 7% of cases respectively. The urease-ELISA seropositive rate in the combined Groups (I, II and III) was 89.5% which is not significantly different from that of RESA-IFA (p greater than 0.05). Comparison between individual Groups (I, II and III) likewise showed no significant differences in both GMT and seropositive rates. Follow-up in Group IV sera showed either no change or a decrease in antibody titers in 55.6% and 44.4% of cases respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibody against a ring-infected erythrocyte surface antigen in cerebral and non-cerebral malaria patients.

An indirect fluorescent antibody test for glutaraldehyde-fixed, ring-infected erythrocyte surface antigen was performed on admission sera from 45 patients with complicated cerebral Plasmodium falciparum malaria, 33 with uncomplicated cerebral malaria, 91 non-cerebral malaria patients, and 53 blood donors from a non-malarious area. 14 (31%), 28 (85%), 64 (70%), and 1 (2%), respectively, had titres greater than or equal to 1/25, considered as positive. The seropositive rate and the geometric mean reciprocal titre of patients with complicated cerebral malaria were significantly lower than those of uncomplicated and non-cerebral patients, particularly in the 6-14 and 15-29 year age groups. Compared with non-cerebral patients, lower seropositive rates for patients with complicated cerebral malaria were demonstrated only in those who had been ill 4 or more days before admission; whereas lower rates for patients with complications, when compared with rates in those with uncomplicated cerebral malaria, occurred irrespective of the duration of illness.