Three indigenous Thai medicinal plants for control of Aedes aegypti and Culex quinquefasciatus.

The potential larvicidal activity and insect growth regulator (IGR) properties of three selected indigenous medicinal Thai plants were tested against two species of mosquito with special reference to the late 3rd and early 4th instar larvae (L3 and L4, respectively). In case of larvicidal activity, Thevetia peruviana was the most potent, followed by Pueraria mirifica, and Butea superba was the least effective. In all cases, the late 3rd instar was more susceptible than the early 4th instar larvae, and the 48-hours exposure yielded more potent larvicidal activity than 24-hours exposure. However, at sublethal dosages, both P. mirifica and B. superba showed some dispersed effects interfering with ecdysis. A variety of toxic effects were observed and recorded in eight categories according to the stage of metamorphosis when death occurred. P. mirifica rendered the main deleterious effects in the pupa-adult period in both instar of Ae. aegypti and Cx. quinquefasciatus, whereas B. superba showed highest effect in black-pupa period of the late 3rd instar larval stage. The results were reversed for the early 4th instar larvae of both species of mosquito as the main effect appeared in the pupa-adult category. The overall results indicated that T. peruviana did not show any IGR properties; whereas, P. mirifica and B. superba seemed to exhibit the juvenile hormone type activity which resulted in abnormal death at various stages of development. B. superba was more promising than P. mirifica, and Ae. aegypti was about 2 times more susceptible than Cx. quinquefasciatus. In addition, L3 was always more susceptible than L4 with both mosquito species.

IgG- and IgG4-detected antigens of Dirofilaria immitis adult worms for bancroftian filariasis by enzyme-linked immunoelectrotransfer blot.

In Thailand, Wuchereria bancrofti filariasis has persisted along the border between Thailand and Myanmar, its dynamic distribution caused by the infected transmigrants between neighboring countries, and the availability of susceptible mosquito vectors. Dirofilaria immitis adult worm was used as a source of antigens, excretory-secretory (ES) and partial surface extracts, to detect human filariasis. ES products showed several stained bands with Coomassie brilliant blue ranging from 14.5-93 kDa and mostly being glycoproteins as shown by concurrent reaction with Concanavalin A, except those at 18, 16 and 14.5 kDa which stained only with Coomassie brilliant blue. Surface proteins of 33.5-91.5 kDa were stained with Coomassie brilliant blue and showed smear bands with Concanavalin A. By enzyme-linked immunoelectrotransfer blot, Bancroftian filariasis sera gave specific reactions with glycoprotein ES antigens at MW 20.5 kDa against anti-human IgG. A prominent band of 18 kDa appeared consistently with the IgG4-ES antigen system. Surface extracts reacting with IgG and IgG4 were considered to be unsuitable as antibodies from all cases of filariasis could not detect any bands.

Neurocysticercosis: utilizing the cystic fluid antigen from Taenia solium metacestodes for diagnosis by IgG-ELISA.

Cystic fluid, which has antigenic properties of whole Taenia solium cysticerci, was used to discriminate neurocysticercosis cases and other parasitic infections, especially helminthiases. Twenty-one neurocysticercosis and several kinds of 22 different parasitic infections, including HIV cases (n=234) evaluated a 90.48% sensitivity and 86.32% specificity of indirect ELISA as follows: a low antigen concentration of 5 microg/ml. serum dilution of 1:400, conjugate dilution of 1:2,000 and a cut-off value of 0.349. Eight different helminthic infections (n = 25); echinococcosis (8/10), gnathostomiasis (6/8), strongyloidiasis (5/14), hookworm infection (1/18), angiostrongyliasis (2/25), opisthorchiasis (1/18), onchocercosis (1/3) and toxocariasis (1/6) were cross-reactive with this antigen. No serum antibody from other brain infections in the study gave a reaction with the antigen. In this study, the cystic fluid antigen gave high sensitivity of the test. However, the antigen contains various antigenic molecules able to bind with antibodies from several of the above helminthic sera, especially echinococcosis and gnathostomiasis. In Thailand, gnathostomiasis is one of the more famous tropical diseases but echinococcosis is quite rare. Cystic fluid antigen should be further investigated for its specific finding in diagnosis.

Angiostrongyliasis: analysis of antigens of Angiostrongylus costaricensis adult worms versus IgG from infected patients with Angiostrongylus cantonensis.

The possibility of cross-reactivity was previously investigated by indirect ELISA with sera from Angiostrongylus cantonensis infections, normal controls and A. costaricensis antigen. 5 microg/ml of crude antigen from both sexes of each species reacted with diluted serum samples (1:800) of each of 20 cases of angiostrongyliasis and normal controls, and further with anti-human IgG conjugate at 1:1,000. The mean absorbance values were evaluated as follows; normal controls showed a value of 0.033 using A. costaricensis antigen lower than (0.085) A. costaricensis antigen. Both mean values of angiostrongyliasis cases were rather close (0.491) using A. costaricensis antigen and the other antigen (0.518). The present study continued with a crude antigen of 13 A. costaricensis females and males. Serum samples were analyzed; 27 sera of angiostrongyliasis, 30 negative controls and 193 cases of other parasitic infections (91 cases of nematodiasis; 45 cases of cestodiasis; 47 cases of trematodiasis and 10 cases of HIV) and 7 cases of other brain infections. This antigen was evaluated for ELISA with a concentration of 5 microg/ml, serum dilution 1:400 and anti-human IgG conjugate at 1:2,000. The test gave sensitivity and specificity at cut-off value 0.261; 92.59% and 73% respectively. The antigen was cross-reactive with 30 cases from 9 out of 10 different kinds of nematodiasis (gnathostomiasis, strongyloidiasis, ascariasis, hookworm infections, trichinosis, toxocariasis, trichuriasis, onchocercosis and Wuchereria bancrofti infections. Five cases from 3 of 6 kinds of cestodiasis (neurocysticercosis, echinococcosis and Hymenolepis nana infections) and 18 cases of 4 out of 5 kinds of trematodiasis (Paragonimus heterotremus infections, opisthorchiasis, schistosomiasis and fascioliasis). One case of other brain infections was observed. The crude antigen of A. costaricensis showed a high percentage sensitivity with serum antibodies of angiostrongyliasis cases. Low specificity of the test was observed by reactions of those serum antibodies with various kinds of antigenic molecules. This study provides baseline data for further immunodiagnosis of human angiostrongyliasis.

Comparative studies on protein, isoenzyme and DNA restriction endonuclease profiles of Thai Paragonimus metacercariae.

General proteins and 14 enzymes from metacercariae of Paragonimus heterotremus, P. siamensis and P. westermani were determined by vertical polyacrylamide gel electrophoresis. The isoenzyme profiles showed considerable interspecific polymorphism for general protein (PT), malate dehydrogenase (MDH), malic enzyme (ME) and tetrazolium oxidase (TO) while those of glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) showed similarity. The Pt-6 and To-I loci can be used as identification markers for these three species. The preliminary study of the molecular biology of Paragonimus heterotremus P. siamensis and P. westermani was based on analysis of metacercarial genomic DNA with restriction endonuclease Pst I. Agarose gel electrophoresis revealed restriction fragment length differences among the three species studied. The DNA restriction fragments were approximately 4-6 fragments, ranging from 5.35 to 14.67 kb. Among these. P westermani shared two homologous fragments with P. siamensis, ie, 5.35 and 7.22 kh, none with P. heterotremus, while P. heterotremus shared only one with P. siamensis, ie, 8.16 kb. Thus, the DNA restriction fragment length differences can be used to differentiate among these three species.

Immunoblotting and enzyme linked-immunosorbent assay for diagnosis of toxoplasma infection in HIV Thai patients.

Toxoplasma gondii has been recognized as an important cause of morbidity and mortality among immunocompromised persons. The diagnosis of T. gondii infection is most often based on serological tests results. Serological diagnosis can be limited in AIDS patients because of depressed antibody responses. Fifty serum samples were used in this study to investigate serological evidence of toxoplasmosis in HIV positive Thai patients by Platelia kit, the commercial enzyme-linked immunosorbent assay (ELISA) in which the membrane protein p-30 is the predominant antigen and immunoblot technique (IB). Sera of HIV positive Thai patients with Toxoplasma infection recognized the same antigenic component, the 32 kDa antigenic band, as is recognized by Toxoplasma positive sera from immunocompetent patients and it may represent a specific marker for diagnosis of Toxoplasma infection in HIV positive Thai patients.

Use of delipidized antigens of Taenia solium metacestodes in IgG-ELISA for detection of neurocysticercosis.

The development of IgG-ELISA for detecting neurocysticercosis is aimed at the routine laboratory, and requires a particular antigen preparation, an acceptable number of serum samples to be tested (both homologous and heterologous) and patients with a diversity of helminthic infections to rule out cross-reactions. This study characterizes IgG-antibodies from cases of neurocysticercosis by assaying the sera against ether-delipidized antigens (5 microg/ml) prepared from metacestodes of Taenia solium. The test had a sensitivity of 90% and a specificity of 83%. IgG-antibodies from heterologous serum samples elicited a number of false positives (25/147) from six different helminthic infections, ie paragonimiasis, echinococcosis, opisthorchiasis, ascariasis, taeniasis and fascioliasis. In additional tests to detect antibody levels to these stage-related antigens, one of three serum samples from T. solium-infected cases gave negative at OD value of 0.187 while the others yielded 0.472 and 0.576. Conversely, assays of all serum samples from neurocysticercosis cases reacted against antigens from Echinococcus granulosus cystic fluid, Paragonimus heterotremus and Opisthorchis viverrini adult worms. In comparison, the antigens from these three species yielded higher mean OD values when assayed against the corresponding infected serum samples. Furthermore, neurocysticercosis cases yielded OD values that are separate and distinct from those of paragonimiasis cases.

Evaluation of Bithynia funiculata snail antigens by ELISA-serodiagnosis of human opisthorchiasis.

Four batches of crude somatic antigens from: (1) Opisthorchis viverrini adult worms, (2) Bithynia funiculata-whole body, (3) B. funiculata-head-foot, and (4) B. funiculata-visceral mass were assayed against sera from 81 opisthorchiasis patients, 30 parasite-free healthy individuals, and 50 individuals infected with other helminthic infections, and their antibody levels determined. By IgG-ELISA, the antigenic reactive proteins were found in both the head-foot and the visceral mass of B. funiculata snails, but the whole snail antigens gave the best results. Furthermore, it was as good as when O. viverini antigens were used. Antibody levels of sera from patients with opisthorchiasis assayed against antigens from whole B. funiculata snails were significantly higher than those of the other two groups. The cut-off value for positivity at 0.228 gave 80.2% sensitivity and 81.2% specificity. Cross reactions were observed with sera from patients with paragonimiasis and strongyloidiasis. No cross reactions were found to occur with sera from healthy individuals.

The natural first intermediate host of Paragonimus siamensis (Miyazaki and Wykoff, 1965) in Thailand.

The first intermediate host of six-known the Paragonimus species in Thailand had not been found until the Filopaludina (Siamopaludina) martensi martensi snail was discovered to maintain the cercariae of a Paragonimus species. An extensive study examined cercarial development through to adult worms by infecting 3 genera of 7 crab species with penetration of cercariae and feeding of snails containing such cercariae. These crabs provided many metacercariae which were fed to cats and bandicoots. The animals gave many Paragonimus adult worms which were characterized as Paragonimus siamensis by the following criteria: 6-lobed ovary and cuticular spines in groups. It is concluded that the Filopaludina martensi martensi snail is a susceptable natural first intermediate host of P. siamensis. Second intermediate hosts Somanniathelphusa brandti, S. sexpunctatum and S. bangkokensis were experimentally infected; prior to this study only S. germaini and S. dugasti had ever been naturally infected with metacercariae of this species.

Gnathostomiasis in Thailand: a survey on intermediate hosts of Gnathostoma spp. with special reference to a new type of larvae found in Fluta alba.

To clarify current status of gnathostomiasis in Thailand, a survey on intermediate hosts has been carried out at various localities since 1987. It was found that Fluta alba (Fresh water eel) as well as Channa striata (snake-headed fish) might be important in playing a role of transmitting the infection either among humans or reservoir animals. During the three years from 1987 to 1989, larvae of Gnathostoma spinigerum were found in 80-100% of F. alba obtained from markets in Nakhon Nayok, with a maximum recovery of 2,582 larvae per eel. Among larvae found in these eels, five were peculiar in possessing four rows of hooklets with complicated branches at the base. Epithelial cells of the intestine of these larvae contained 1-2 nuclei. These observations indicate that the larvae are different from those of reported species of Gnathostoma from Thailand including G. spinigerum, suggesting a possibility of the advanced third-stage larvae of G. malaysiae.

Effect of single-dose albendazole and single-dose mebendazole on Necator americanus.

One hundred two children (43 males and 59 females) aged 6 to 14 years with positive stool examination by Kato-Katz and/or Harada-Mori culture techniques for N. americanus, were randomly divided into two groups. Group I with 48 children were treated with a single dose albendazole, 400 mg. Group II, 54 children, received a single dose mebendazole, 600 mg. After treatment, repeated stool examination was performed on Day 14, Day 21 and Day 28. The children were considered cured when stool examination was negative on all three occasions by both methods. The cure rate was 64% in Group I and 11% in Group II. The difference was statistically significant (p less than 0.05). The eggs reduction rate was 98% in Group I and 95% in Group II. Mild and transient side effects such as nausea, dizziness and headache were observed in both groups. Albendazole, 400 mg, as a single dose treatment was shown to be superior to mebendazole, 600 mg, single dose for the mass treatment of hookworm infection, especially that of Necator americanus, in an endemic area.

Antibody responses in human gnathostomiasis.

Sera from 4 patients with parasitologically confirmed gnathostomiasis and from 18 healthy individuals were studied by SDS-PAGE and Western blot analysis using radioiodinated protein A to detect antibody responses against crude aqueous somatic extract of advanced third stage larvae of Gnathostoma spinigerum (L3G). It was found that the L3G extract was highly complex, comprising of more than 40 polypeptides among which more than 20 components were antigenic in human. The relative M.W. of the proteins ranged from 13 kd to 150 kd with the major antigenic bands at 150, 135, 120, 94, 84, 82, 72, 55, 54, 49, 43, 38, 35, 32 and 28 kd. All 4 sera from gnathostomiasis patients gave almost an identical pattern of reactivities against the L3G antigens whereas sera from the normal individuals gave much lower reactivities against the L3G antigen of M.W. 38 kd and, in certain individuals, those of 49 and 43 kd. The present findings suggest that the serum antibody response against the parasite is specific and may be useful in a specific or a confirmed immunodiagnosis of human gnathostomiasis.

Occurrence of a common epitope in circumsporozoite proteins of Plasmodium falciparum isolated from different areas in Thailand.

Fifteen isolates of P. falciparum sporozoites obtained from patients with acute falciparum malaria from various malaria endemic areas in Thailand were tested for the presence of a common antigenic determinant in their CS protein molecules. SDS-PAGE and Western blot analysis using MAB or human serum antibodies specific to the CS proteins of the parasites revealed a common epitope shared in the CS proteins of all strains of P. falciparum tested. However, the CS proteins exhibited M.W. variation when different strains of the parasites were compared. A similar result was obtained when the human serum antibodies were used. The present study clearly indicated the occurrence of the common epitope in phenotypically different CS proteins among isolates of P. falciparum sporozoites and supported the notion that antigens containing these repetitive epitopes could be used as the candidates for the sporozoite vaccine against P. falciparum infection.

Antibodies against circumsporozoite proteins of Plasmodium falciparum induced by natural infection.

Sera from 10 individuals who lived in a malaria endemic area, 10 patients with acute uncomplicated falciparum malaria and 10 patients with cerebral malaria and hyperimmune mouse serum were tested for their reactivities against Plasmodium falciparum sporozoite antigens by Western blot analysis using 125I-labeled staphylococcal protein A as the detecting reagent. These sera were shown by indirect immunofluorescence and/or circumsporozoite precipitation test to have antibodies reacting against the parasites. It was found that all serum antibodies from the three groups of individuals and the mouse serum reacted in a similar pattern with circumsporozoite (CS) proteins of P. falciparum. Ten sera from normal individuals were negative in all reactions. Monoclonal antibody (MAB) specific against CS proteins of the parasites showed that the proteins exhibited as four different molecular weight (MW) polypeptides, i.e., 67,000, 65,000, 60,000, and 58,000 daltons. These CS proteins of P. falciparum were found to be species and stage specific. Radioimmunoprecipitation using 35S-methionine-labeled parasites and sera of individuals from the various categories or MABs gave a similar result. Another protein antigen of P. falciparum sporozoites had a MW of 80,000 daltons. This antigen was not species specific, probably not membrane associated and was present in a minute quantity in the parasite's extract.