Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

BACKGROUND & OBJECTIVES: El Tor Vibrio cholerae O1 carrying ctxB C trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. METHODS: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpA C , tcpA E, hlyA C and hlyA E were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. RESULTS: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. INTERPRETATION & CONCLUSIONS: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxB C).

Circumsporozoite proteins of human malaria parasites Plasmodium falciparum and Plasmodium vivax.

Monoclonal antibodies were raised against sporozoites of two species of malaria parasites, Plasmodium falciparum and Plasmodium vivax. The antibodies reacted with polypeptides (circumsporozoite proteins) that are uniformly distributed over the entire surface of sporozoites, as shown by indirect immunofluorescence and by the circumsporozoite precipitin reaction. The epitopes recognized by the monoclonal antibodies were expressed on sporozoites from different geographical isolates of the homologous species but were not detected on sporozoites of heterologous species nor on blood forms of the parasite. The monoclonal antibody to P. falciparum specifically immunoprecipitated two polypeptides of apparent 67,000 mol wt (Pf67) and 58,000 mol wt (Pf58) from extracts of [35S]methionine-labeled P. falciparum sporozoites. Similarly, the anti-P. vivax monoclonal immunoprecipitated two proteins of 51,000 mol wt (Pv51) and 45,000 mol wt (Pv45) from extracts of metabolically labeled P. vivax sporozoites. The extracts were also reacted with the serum of human volunteers successfully vaccinated with sporozoites of either P. vivax or P. falciparum. The patterns of immunoprecipitation were almost identical to those obtained with the corresponding monoclonal antibodies. The circumsporozoite proteins of P. falciparum and P. vivax play a role in immune protection. Incubation of the appropriate monoclonal antibody with viable sporozoites of the homologous species significantly reduced parasite infectivity, as determined by sporozoite neutralization assays carried out in splenectomized chimpanzees.

Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis.

Sera from four patients with parasitologically confirmed gnathostomiasis, 15 patients with presumptive gnathostomiasis, 64 patients with various parasitic infections and 19 healthy adults were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for their reactivities against somatic extract of Gnathostoma spinigerum third-stage larvae (L3). It was found that the L3 extract was highly complex consisting of more than 20 antigenic components, a few of which gave reactions with sera from the healthy controls. Extensive cross-reactions of the parasite's antigen with sera from patients with other parasitic infections occurred. A specific antigen of G. spinigerum with a mol. wt of 24,000 (24k) was found to react with all parasitologically proven patients, five of the presumptive patients, one of the patients with other parasitic infections and none of the healthy individuals. This 24k component of G. spinigerum is a potential diagnostic antigen for use in the immunodiagnosis of human gnathostomiasis.

Purification of Gnathostoma spinigerum specific antigen and immunodiagnosis of human gnathostomiasis.

Specific antigen of G. spinigerum which has been shown to be a protein with a relative mol. wt of 24,000 (24K) was prepared from the advanced third-stage larvae (L3) obtained from the livers of naturally infected eels. The L3 were ground and extracted with water. Purification procedures involved gel filtration, chromatofocussing and anion exchange column chromatographies, while characterization of the specific antigen was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining, Western blot analysis and isoelectric focussing. The specific antigen which has a pI of 8.5 was used as antigen in the indirect enzyme-linked immunosorbent assay (ELISA) to detect specific antibody in four groups of individuals, namely five parasitologically diagnosed gnathostomiasis patients (group 1); 15 clinically diagnosed gnathostomiasis patients (group 2); 136 patients with other parasitic infections (group 3); and 25 normal healthy parasite-free controls. Sensitivity, specificity and predictive values (positive and negative) of the assay were 100%.

Immunodiagnosis of human trichinellosis and identification of specific antigen for Trichinella spiralis.

Crude antigens obtained from the infective stage larvae of Trichinella spiralis were used in an ELISA for detecting IgG antibodies to T. spiralis in serum samples collected from three groups of individuals. The individuals of the first group were parasitologically confirmed trichinellosis patients, while those of group 2 were patients with other helminthiasis and group 3 were healthy, parasite-free individuals. The specificity of the assay was 96.8% when performed on sera of groups 2 and 3. Cross-reaction was observed with the sera of patients with capillariasis, gnathostomiasis, opisthorchiasis, and strongyloidiasis and opisthorchiasis with hookworm infection. The sensitivity of the test was 100% when performed on sera of group 1, which were collected 57 days after infection. Western blot analysis revealed that a specific antigen for T. spiralis was a component of M(r) 109.

Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection.

Advanced third-stage larvae of G. spinigerum were obtained from two separate sources, namely from cysts in the livers of naturally infected eels (L3E) and from experimentally infected mice (L3M). Morphology of the L3E was studied microscopically. The larvae were homogenized in distilled water, 1% Triton X-100 or 1% sodium deoxycholate containing protease inhibitors. Protein compositions of the three crude extracts were compared, on the same weight basis, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining while their antigenicities were studied by Western blot analysis using serum of a patient with parasitologically confirmed gnathostomiasis. Distilled water was found to be the best extraction solution in solubilizing proteins especially the diagnostic antigen, namely the 24,000 (24 kDa) mol. wt component from the larvae. The L3E and L3M contained relatively equal amounts of the 24 kDa antigen. This diagnostic component was anatomically located in the body fluid, oesophagus and intestine of the larva.

Monoclonal antibody to a diagnostic Mr 24,000 antigen of Gnathostoma spinigerum.

Crude water extract (CA) was prepared from the advanced third-stage larvae of Gnathostoma spinigerum collected from livers of naturally infected eels. The extract was partially purified by chromatofocussing column chromatography and the fraction which contained specific antigen of G. spinigerum which was an Mr 24,000 glycoprotein was used to immunize five Balb/c mice for preparing immune splenocytes. Spleen cells were collected from one mouse which showed high serum titre by indirect enzyme-linked immunosorbent assay and contained specific antibody to the Mr 24,000 antigen as checked by Western blot analysis. The spleen cells were fused with myeloma Sp2/0 cells at a ratio of 10 spleen cells per one myeloma cell using polyethylene glycol 3350 as a fusogen. Thirteen out of 174 growing polyclones (7.5%) produced antibodies to the partially purified CA fraction. Among them, two polyclones produced antibody directed to the Mr 24,000 protein. These two polyclones were subjected to monocloning by limiting dilution and a monoclone GN6/24 which produced monoclonal antibody to the specific Mr 24,000 protein of G. spinigerum was obtained.

Antibodies against malaria sporozoites in patients with acute uncomplicated malaria and patients with cerebral malaria.

Serum samples from 95 patients with acute uncomplicated falciparum malaria (AM) and 95 patients with cerebral malaria (CM) were tested by the indirect immunofluorescent assay (IFA) for IgG and IgM antibodies against Plasmodium falciparum and P. vivax sporozoites. Forty-six (48%) CM patients were positive for antibodies against P. falciparum sporozoites whereas only 23 (24%) were positive for antibodies against P. vivax sporozoites (P less than 0.002). A similar result was obtained in AM patients. However, CM patients had significantly lower mean IgG anti-sporozoite titer for P. falciparum than did AM patients (P less than 0.05), especially when only anti-sporozoite antibody-positive CM and AM patients were compared (P less than 0.0005), suggesting that CM patients had relatively less exposure and were probably less immune to malaria than were AM patients. The persistence of anti-sporozoite antibodies also was investigated in paired sera taken 63 days apart from 108 patients with acute falciparum malaria. There were significant decreases in the mean antibody titers in the follow-up sera during the period of stay in the malaria-free area. It was proposed that determination of anti-sporozoite antibody be made as a substitute for, or in addition to, anti-blood stage antibody for seroepidemiological study of malaria, especially in the monitoring of the success of the malaria control program.

Monoclonal antibodies to Paragonimus heterotremus and their potential for diagnosis of paragonimiasis.

Monoclonal antibodies (MAbs) specific to the lung fluke (Paragonimus heterotremus) were produced against the soluble metabolic products (excretory-secretory antigen). Three hybrids secreting MAbs specific for P. heterotremus antigens were identified by an indirect enzyme-linked immunosorbent assay (ELISA) against a panel of homologous and 24 heterologous parasite antigens and Mycobacterium tuberculosis. Of the three specific clones, clone 10F2, which was IgG1 producing and which gave immune complex bands with 31.5-kD and 22-kD polypeptides by gel electrophoresis and immunoblotting, was selected for further characterization and evaluation of its possible diagnostic potential. The result obtained from an indirect immunofluorescent antibody test suggested that MAb 10F2 reacted with mucosa and contents of the worm's intestine. The antibody could be readily used to prepare an affinity-purified antigen for use in an indirect ELISA that was highly sensitive and specific for the detection of circulating antibody in sera of paragonimiasis patients.

Anti-sporozoite antibodies induced by natural infection.

Serum samples from 120 individuals living in a malaria-endemic area, 31 patients with Plasmodium falciparum infection, and 58 healthy blood donors were tested for antibodies against P. falciparum and P. vivax sporozoites. Specific antibodies were determined by the circumsporozoite precipitation (CSP) reaction and indirect immunofluorescent (IFA) tests for IgG and IgM antibodies. It was found that a high proportion of adults living in the endemic area had IFA anti-sporozoite antibodies, usually IgG. Children and healthy donors were either negative or had low antibody titers. A positive correlation was found between IgG antibody titers against P. falciparum sporozoites and those against P. vivax sporozoites. CSP reactivity was demonstrated in 5 of 31 sera from patients with falciparum malaria, and was always associated with a high level of IFA antibodies. The anti-sporozoite antibodies were found to be stage- and species-specific.

Plasmodium falciparum strain-specific human antibody inhibits merozoite invasion of erythrocytes.

The extent to which human antibodies involved in functional immunity react with antigenic determinants varying between different isolates or strains of the human malaria parasite Plasmodium falciparum will influence the design of vaccines against malaria. We identified nine immune sera from Cambodian refugees which blocked in vitro invasion of erythrocytes by merozoites of the Camp strain of P. falciparum and agglutinated Camp strain merozoites. However, none of these sera blocked invasion of erythrocytes by merozoites of the FCR-3 strain. We conclude that antibodies in these human sera recognized antigenic determinants present on the surface of viable merozoites of the Camp strain but not the FCR-3 strain. These parasite strains and in vitro assays can be used to analyze strain-specific functional immunity in humans.

Factors contributing to the development of cerebral malaria. I. Humoral immune responses.

Humoral immune responses to malaria were studied in 100 patients with cerebral malaria of whom 53 had added complications, 108 patients with acute malaria, and 100 blood donors. The methods employed were indirect hemagglutination (IHA), indirect fluorescent antibody (IFA), enzyme-linked immunosorbent assay (ELISA), and parasite growth inhibition (PGI) tests. Patients with cerebral malaria, especially those with complications, had histories of fewer attacks of malaria in the previous 5 years than did those with acute malaria, suggesting that the cerebral malaria patients were less immune. The combined cerebral malaria group (complicated and uncomplicated) did not show defective humoral immune responses, since the initial seronegative rate and the mean initial IHA and IFA antibody titers were not significantly different from those of acute malaria patients and the mean initial ELISA titer was even higher than that of the acute malaria group. Reduced humoral responses were found only in complicated cerebral malaria patients, as their mean initial IHA titer was lower and their IHA seronegative rate was higher than those in acute malaria patients and in the uncomplicated cerebral malaria group. The combined cerebral malaria group had greater PGI activity than that of acute malaria patients, but this increased activity was entirely due to the higher results obtained in the complicated cerebral malaria group. The increased PGI activity returned to normal after recovery. An IgG preparation from seven of eight of these sera failed to exert the growth inhibition effect. Factors other than IgG were therefore responsible for the inhibition of parasite growth.

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.

Diagnosis of Plasmodium vivax malaria using a specific deoxyribonucleic acid probe.

A deoxyribonucleic acid (DNA) probe which specifically distinguishes Plasmodium vivax from P. falciparum malaria has been derived from a P. vivax genomic DNA library. This probe, VPL101, consists of 3.2 kilobase pairs and does not hybridize with up to 6 micrograms of human or P. falciparum DNA. VPL101 contains at least two copies of a 205 base pair repeat sequence. The subcloned repeat probe, VPL101/5, reacted with 73 of 76 microscopically diagnosed P. vivax samples but not with any of 17 human DNA samples or any of 8 P. falciparum DNA samples from cultured parasites. It was possible to detect P. vivax in mixed infections in which only P. falciparum parasites were identifiable by microscopy. This P. vivax DNA probe provides a useful epidemiological tool for malaria control programmes.

Genotypes and phenotypes of Shiga toxin producing-Escherichia coli isolated from healthy cattle in Thailand.

Shiga toxin producing-Escherichia coli (STEC) has not yet been identified as an important aetiologic agent of human disease in Thailand. To evaluate the potential for STEC to contribute to human disease in Thailand, 139 fecal samples were collected from healthy cattle from five different provinces and analysed by genotypic and phenotypic methods for STEC. Of 139 samples, 27 (19.4%) were positive for stx1 and/or stx2 by multiplex polymerase chain reaction, or for O157 lipopolysaccharide (LPS) by immunoassay. Isolates positive for stx and/or O157 were subdivided into 49 strains that varied in the presence of the virulence determinants stx1+/stx2+ (22 strains), stx2+ (22 strains), stx1+ (4 strains), and O157 LPS (1 strain). Within these 49 distinguishable strains, other virulence determinants varied as follows: hlyA+ (77.6%), eae+ and tir+ (4.1%), and katP+ (6.12%). The most predominant profile (22 isolates) was stx1+/stx2+, eae-, tir-, etpD-, hlyA+, katP-. For further characterization of the isolated strains by two molecular typing assays, plasmid profiles and ERIC PCR were performed. The results suggest that the genetic and phenotypic profiles of STEC associated with human disease are not prevalent at this time in cattle in Thailand.

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.

Mouse IgE response to an allergen from Entamoeba histolytica.

Attempts were made to demonstrate the presence of allergenic components in an extract of axenically cultured Entamoeba histolytica (HK 9). Various strains of mice were immunized i.p. with a low dose of the extract mixed with Al(OH)3 and boosted with the same mixture 4 wk later. A high titer of IgE antibody response was demonstrated in BALB/c, DBA/2 (H-2d), CBA (H-2k), and SWM strains but not in C3H/He (H-2k) strain. The extract was fractionated by gel filtration with Sephadex G-200 and then by DEAE-cellulose chromatography with a gradient buffer with progressive increase in molarity. Fractions having the strongest activity to stimulate the IgE antibody response in the mouse were subjected to SDS polyacrylamide gel electrophoresis. From the results of gel filtration and polyacrylamide gel electrophoresis, the molecular weight on the partially purified allergen was estimated to be 25,000 to 27,000 daltons.

Detection of Paragonimus heterotremus in experimentally infected cat feces by antigen capture-ELISA and by DNA hybridization.

An antigen capture enzyme-linked immunosorbent assay (antigen capture-ELISA) and DNA hybridization technique were developed and evaluated for their application in the detection of Paragonimus heterotremus infection in experimentally infected cats. An IgG fraction prepared from serum of a rabbit immunized with P. heterotremus excretory-secretory (ES) products was used as the capture antibody. An IgG1 monoclonal antibody specific to the 22- and 31.5-kDa ES products of P. heterotremus was used as the antigen probe. As little as 0.24 ng of the ES products could be detected by this technique. A specific P. heterotremus DNA probe derived from the P. heterotremus genomic DNA library containing 1,500 base pairs was used in a dot-blot hybridization assay for the detection of parasite DNA. The radioactively labeled probe could detect DNA released from as few as 2 P. heterotremus eggs. Both ELISA and DNA hybridization were found to have 100% specificity, with sensitivities of 73.7% and 100%, respectively.

Cell-bound haemagglutinin (CHA) of V. cholerae 01 as protective antigen.

The study of immunogenicity of cell-bound haemagglutinin (CHA) of Vibrio cholerae E1 Tor in mice revealed that the CHA was a good antigen when it was adsorbed onto the surface of sheep red blood cells and given orally to mice. The antigen not only induced high levels of various class antibodies which sustained in the intestinal tracts for a long period of time (longer than 6 months) but also the antibodies were protective against homologous cholera challenge. The degree of protection seems to correlate with the level of IgA in the intestinal washing. The protective ability was conferred mainly by anti-CHA.

Antigenic diversity of Plasmodium vivax and their geographic distribution in Thailand.

Fifty-eight monoclonal antibodies (MAbs) raised against the erythrocytic stages of Plasmodium vivax were selected for typing of 501 P. vivax isolates from different geographic locations throughout Thailand. Based on their reactivities in the indirect fluorescent antibody test, these MAbs were classified into five groups: group I MAbs showing generalized staining of all blood stages; group II MAbs reacting with merozoites and their organelles; group III MAbs reacting with the surface membrane of merozoites; Group V MAbs reacting with the surface membrane of trophozoites and schizonts; and group VII MAbs reacting with internal components of the parasites. Sixteen MAbs reacted with more than 95% of the isolates; the epitopes recognized by these MAbs were considered as being invariant. The remaining MAbs reacted with 30-90% of the isolates, and the epitopes recognized by these MAbs were regarded as being variable. The variant epitopes were associated with > 200-, 135-, and 100-kilodalton (kDa) molecules of all blood stages, the 95-kDa molecule on merozoite organelles, the 200-kDa molecule on the surface of trophozoites and schizonts, and the 85-kDa molecule of the parasite internal components. Antigenic diversity occurred among the P. vivax population in the endemic areas of Thailand and was shown to vary from place to place and was highest in the area with the highest rate of transmission along the Myanmar border in western Thailand and along the Cambodian border in eastern Thailand, including Trat (48.4%), Tak (41.7%), Chantaburi (36.5%), and Mae Hong Son (36.4%). Demonstration of antigenic diversity of P. vivax parasites signals a note of caution in the development of vaccines for vivax malaria. The vaccines should be directed against protective, conserved and not against variant epitopes.