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.     

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.     

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.     

A dot-blot ELISA comparable to immunoblot for the specific diagnosis of human parastrongyliasis

A dot-blot enzyme-linked immunosorbent assay (dot-blot ELISA) using an electroeluted 31-kDa glycoprotein from adult worms of Parastrongylus cantonensis as the specific antigen was evaluated for the immunological diagnosis of patients infected with P. cantonensis. The sensitivity and specificity for the detection of serum antibody to P. cantonensis in dot-blot ELISA were both 100%, as determined with serum samples of ten P. cantonensis-infected patients, 60 patients with other related parasitic infections, and 20 uninfected controls. The test was as sensitive and specific as the immunoblot test which revealed a reactive band of 31 kDa. Both the dot-blot ELISA and immunoblot detected all sera from ten P. cantonensis-infected individuals, but not with those of other heterologous parasitoses (gnathostomiasis, toxocariasis, filariasis, paragonimiasis, cysticercosis and malaria) or sera from healthy controls. The dot-blot ELISA is much simpler to perform than the immunoblot technique, and the test can be applied under field conditions where sophisticated facilities are lacking.     

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%.     

Multi-immunodot for rapid differential diagnosis of eosinophilic meningitis due to parasitic infections

A multi-dot enzyme-linked immunosorbent assay (ELISA) was developed for the rapid and simple differential diagnosis of eosinophilic meningitis due to helminth infections. Ultrafiltered, purified antigens of Parastrongylus (=Angiostrongylus) cantonensis, Gnathostoma spinigerum and Taenia solium metacestodes, the most common parasites that invade the central nervous system and cause eosinophilic pleocytosis, were dotted onto a single nitrocellulose membrane strip. Antigen-coated strips, when blocked with 5% skimmed milk and dried, were stable for at least 6 months at 4 degrees C. With peroxidase conjugated anti-human immunoglobulins and 4-chloro-1-naphthol as a substrate, antibodies in the corresponding patients' sera were clearly detected on the membrane strip as well-defined blue dots. Although cross-reactions between P. cantonensis and G. spinigerum antigens were observed with the use of partially purified antigens, the darkest dot correlated well with the infecting parasites in all cases. This fast, easy and economical multiple dot-blot ELISA method is useful for the differential diagnosis of eosinophilic meningitis caused by parasitic helminths, as semi-purified antigens can be easily obtained by ultrafiltration and used. Further improvements using highly specific parasite antigens may make this multi-immunodot test more suitable for wide-scale use in field studies and diagnostic laboratories.     

Recognition of deglycosylated larval proteins of Gnathostoma spinigerum by a monoclonal antibody and human gnathostomiasis antiserum.

The study on the recognition of 35S-labelled somatic antigens of Gnathostoma spinigerum advanced third-stage larva (aL3) has revealed that the mAb GN6/24 immunoprecipitated 26- and 24-kDa proteins from the undigested and N-glycosidase F-digested larval extracts, respectively. The recognition of the deglycosylated form of the glycoprotein indicated that the mAb reacted with the peptide epitope on the 26-kDa protein. Human gnathostomiasis antiserum immunoprecipitated most of the N-glycosidase F-digested larval proteins including the deglycosylated 26-kDa protein.     

Use of heterologous antigens for the immunodiagnosis of abdominal angiostrongyliasis by an enzyme-linked immunosorbent assay

Angiostrongylus costaricensis has a broad geographic distribution spanning from North to South America and the infections of vertebrates with this nematode can result in abdominal complications. Human infections are diagnosed by histological or serological methods because the isolation of larvae from feces is not feasible, as most parasites become trapped in intestinal tissues due to intense eosinophilic inflammation. Because A. costaricensis is difficult to maintain in the laboratory, an immunodiagnostic IgG enzyme-linked immunosorbent assay (ELISA) using antigens from the congeneric Angiostrongylus cantonensis species was evaluated against a panel of serum samples from patients who were histologically diagnosed with A. costaricensis infections. Sera from uninfected individuals and individuals infected with other parasites were used as controls. The sensitivity and specificity of the assay were estimated at 88.4% and 78.7%, respectively. Because the use of purified or cloned antigens has not been established as a reliable diagnostic tool, the use of heterologous antigens may provide a viable alternative for the development of an ELISA-based immunodetection system for the diagnosis of abdominal angiostrongyliasis.     

Development of a double antibody sandwich ELISA assay for the diagnosis of angiostrongyliasis

With the use of 2 monoclonal antibodies (MAbs) against excretory/secretory (ES) antigens of adult Angiostrongylus cantonensis, a new method was developed for double antibody sandwich ELISA for the detection of circulating antigens (CAg). To evaluate the sensitivity of the new procedure, the CAg in sera of rats (80) and mice (15) infected with A. cantonensis, as well as CAg in sera of clinically confirmed angiostrongyliasis patients (70), were evaluated. Cross-reaction testing was used to determine the specificity of serum from patients infected with Ascaris lumbricoides, Trichinella spiralis , Toxoplasma gondii , Schistosoma japonicum, Paragonimus westermani, Clonorchis sinensis, Echinococcus granulosus, Spirometra, and Taenia solium, as well as normal healthy people. The results proved that the sensitivity and the specificity of the new method were totally effective for the detection of A. cantonensis CAg. The assay is highly sensitive, specific, and reproducible, with easy handling and excellent cost effectiveness, and thereby provides a new method for the accurate diagnosis of angiostrongyliasis.     

Immunoelectrotransfer blot assay in acute and chronic human trichinellosis

An immunoelectrotransfer blot assay (IETB) using excretory secretory products of muscle larvae of Trichinella spiralis (ML-ESP) and the avidin biotin system was developed in order to characterize reactivity against ML-ESP in sera from patients with acute and chronic trichinellosis. A complete pattern of up to 13 bands was developed by sera from individuals with trichinellosis where doublets, triplets, or single bands were shown to have molecular weights of roughly 66, 55, 45, 36, 29, 24, and 14 kDa. The bands at approximately 55, 36, 29, and 14 kDa proved specific for T. spiralis. The band at approximately 55 kDa was present in all trichinellosis sera, whereas the approximately 14-kDa band was present in only a small percentage of sera. The development of approximately 36- and 29-kDa bands suggests a modulation of the reactivity against ML-ESP over time. IETB proved more sensitive for the population of chronic trichinellosis under study than a conventional diagnostic enzyme-linked immunosorbent assay, allowing negative or borderline serum samples to be determined. Thus, this technique, when applied for human trichinellosis surveillance, should provide a useful tool in endemic areas.     

Serodiagnosis of Toxocariasis by ELISA Using Crude Antigen of Toxocara canis Larvae

Toxocariasis is a worldwide zoonosis caused by larvae of ascarid nematodes of dogs or cats, Toxocara canis or T. cati. Diagnosis of human toxocariasis currently relies on serology that uses T. canis excretory-secretory antigen to detect specific IgG antibodies by ELISA. We investigated the serodiagnostic efficacy of ELISA using crude antigen of T. canis larvae (TCLA). Serum specimens of 64 clinically confirmed toxocariasis, 115 healthy controls, and 119 other tissue-invading helminthiases were screened by ELISA using TCLA. The ELISA using TCLA showed 92.2% (59/64 patient samples) sensitivity and 86.6% (103/119) specificity. Its positive diagnostic predictivity was 78.7% and negative predictivity was 97.8%. No serum of healthy controls reacted but that of anisakiasis (45.5%), gnathostomiasis (19.2%), clonorchiasis (15.8%), sparganosis (11.1%), and cysticercosis (6.3%) cross-reacted. Immunoblot analysis on TCLA recognized antigenic proteins of 28- and 30-kDa bands in their dominant protein quantity and strong blotting reactivity. The present results indicate that the ELISA using our TCLA antigen is acceptable by the sensitivity and specificity for serodiagnosis of human toxocariasis. ELISA with TCLA is recommended to make differential diagnosis for patients with any sign of organ infiltration and eosinophilia.     

Studies on suspected clinical and experimental angiostrongyliasis: serological responses

Serum antibodies in suspected angiostrongyliasis patient were detected by ELISA. The antibody titre was 1:51,200 in the serum and 1:6,400 in CSF with preadult A. cantonensis antigen. Other tests like AGD and CIEP failed to show any positive reaction with both preadult and adult worm antigens. Experimental infection with 100 A. cantonensis larvae in albino rats indicated positive CIEP reaction in serum from the day 5 to 375 after infection. No precipitin line was seen on the other hand, in AGD during observation period. Different rat groups infected with larval doses of 100, 500, 2,000, and 5,000 showed positive CIEP reaction, on the 21st day of infection when preadult worms were seen in CNS. There was no CIEP reaction when a low dose of 15 larvae was used. Cerebral fluid of rats infected with heavy dose of 5,000 larvae showed positive CIEP reaction on the 21st day.     

Characterization of Angiostrongylus cantonensis excretory-secretory proteins as potential diagnostic targets

Angiostrongyliasis results from infections with intra-arterial nematodes that accidentally infect humans. Specifically, infections with Angiostrongylus cantonensis cause eosinophilic meningitis and Angiostrongylus costaricensis infections result in eosinophilic enteritis. Immunological tests are the primary means of diagnosing infections with either pathogen since these parasites are usually not recoverable in fecal or cerebrospinal fluid. However, well-defined, purified antigens are not currently available in sufficient quantities from either pathogen for use in routine immunodiagnostic assays. Since A. costaricensis and A. cantonensis share common antigens, sera from infected persons will recognize antigens from either species. In addition to their potential use in angiostrongyliasis diagnosis, characterization of these proteins that establish the host-parasite interphase would improve our understanding of the biology of these parasites. The main objective of the present work was to characterize A. cantonensis excretory-secretory (ES) products by analyzing ES preparations by two-dimensional gel electrophoresis coupled with immunoblotting using pools of positive sera (PS) and sera from healthy individuals (SC). Protein spots recognized by PS were excised and analyzed by electrospray ionization (ESI) mass spectrometry. MASCOT analysis of mass spectrometry data identified 17 proteins: aldolase; CBR-PYP-1 protein; beta-amylase; heat shock protein 70; proteosome subunit beta type-1; actin A3; peroxiredoxin; serine carboxypeptidase; protein disulfide isomerase 1; fructose-bisphosphate aldolase 2; aspartyl protease inhibitor; lectin-5; hypothetical protein F01F1.12; cathepsin B-like cysteine proteinase 1; hemoglobinase-type cysteine proteinase; putative ferritin protein 2; and a hypothetical protein. Molecular cloning of these respective targets will next be carried out to develop a panel of Angiostrongylus antigens that can be used for diagnostic purposes and to further study host-Angiostrongylus interactions.     

Qualitative characterization of antibody responses to single and multiple Brugia pahangi infections in jirds

Antibody responses of jirds, singly and multiply inoculated with Brugia pahangi infective larvae (L3), to soluble somatic extracts of adult parasites were characterized by western blot analysis. Forty-two protein bands ranging in molecular weight from 12 to 160 kDa were recognized by sera from infected jirds. Antibody recognition of individual B. pahangi antigen bands in this assay appears to be independent of antibody enzyme-linked immunosorbent assay (ELISA) titers to crude parasite extract, severity of lymphatic lesions, levels of microfilaremia, numbers of L3 inoculated, or numbers of adult parasites in individual jirds. Antibody recognition of protein bands with molecular weights of 37 kDa, 21 kDa, and 17 kDa, however, did temporally correspond with certain parasitological and pathologic events. Antibody against the 37-kDa protein band first was identified at the onset of patency, reaching a 90% prevalence rate by 90 days postinfection (DPI). The prevalence of this antibody remained high. Antibody recognition of the 21-kDa protein band first occurred at 90 DPI and gradually increased in prevalence during the course of infection temporally similar to the increase in microfilaremia. Recognition of the 17-kDa protein band first occurred at 48 DPI, reached a maximum prevalence of 80% at 90 DPI, and decreased to a minimal prevalence by 160 DPI. Prevalence of antibody responses to the 17-kDa protein band corresponded temporally with the kinetics of the rise and fall of numbers of intralymphatic thrombi. The patterns of antibody response to these 3 bands were similar in both singly and multiply inoculated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

A note on the correlation between rainfall and the prevalence of Gnathostoma spp. infective stage larvae in swamp eels in Thailand

Gnathostoma spinigerum is the major causative agent of human gnathostomiasis, a parasitic zoonosis with a great variety of clinical manifestations. Generally, humans are infected by consumption of third-stage larvae (L3) of G. spinigerum in infected hosts in the form of partially cooked or uncooked food. Surveys of the contamination of Gnathostoma spp. L3 in swamp eels are useful for prevention and control of diseases and have been continuously performed in Thailand. The author performed a retrospective study on 33 previous cross-sectional surveys with geographical data and the prevalence of Gnathostoma spp. L3 that covered 12 provinces in Thailand. The relation between rainfall (derived from the geographical data) and the prevalence of Gnathostomo spp. L3 in swamp eels (derived from the overall infection rate of Gnathostoma spp. L3) was investigated. The least-square equation plot rainfall (y) versus prevalence (x) is y= 9.68x + 1,035.12 (r = 0.83; p < 0.01). A significant correlation was discerned between rainfall and the prevalence of eel infection but not for the season of the survey. Similar to the previous study, the prevalence of eel infection may depend on rainfall rather than season. However, this study focused on only 33 cross-sectional surveys in Thailand; further similar study in other countries to assess the correlation between rainfall and the prevalence of infection is required to substantiate this conclusion.     

Eosinophilic meningitis outbreak related to religious practice

Three patients with eosinophilic meningitis (EoM) were investigated in two hospitals in Porto Alegre, Southern Brazil. These patients had a common exposure after the ingestion of raw mollusks in a religious ritual. Two of them had an uncommon presentation with intense lower distal extremities pain and small fiber neuropathy as defined by an electroneuromyography (ENMG) study. All three patients were positive for Angiostrongylus cantonensis serology and recovered after antihelminthic and anti-inflammatory treatment. Increased awareness of A. cantonensis infection is important to avoid new infections and to improved recognition and handling of cerebral angiostrongyliasis.     

On the molecular weight and subunit composition of calf thymus ribonuclease H1

We have reinvestigated the molecular weight and subunit composition of calf thymus ribonuclease H1. Earlier studies suggested a variety of molecular weights for the enzyme in the range of 64K-84K and reported that the enzyme either was a single polypeptide of 74 kDa or consisted of from two to four subunits in the range of 21-34 kDa. Although we too find bands in this lower molecular weight range in our highly purified preparations following SDS-PAGE, our data suggest that the native structure of RNase H1 is a dimer of 68-kDa subunits. The evidence includes the following: (1) Western blot analysis of fractions taken at various stages of the purification indicates that the predominant antigenic form of the enzyme in crude extracts has a molecular weight of 68K but that during purification in the absence of sufficient protease inhibitors a variety of lower molecular weight forms appear concomitant with the disappearance of the 68-kDa band. (2) Activity gel analysis of the highly purified enzyme prepared in the presence of a battery of protease inhibitors reveals that the 68-kDa band (as well as several bands of lower molecular weight) possesses RNase H activity. (3) The 68-kDa band recognized by Western blotting with anti-RNase H immune sera is not detected by using preimmune sera. Furthermore, when immune sera are used, a trace of a 140-150-kDa antigenic form can sometimes be detected, consistent with the existence of a dimeric form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunodiagnosis of nocardiosis.

A specific immunodominant 54-kDa antigen was purified from a culture filtrate of Nocardia asteroides by immunoaffinity chromatography. The chromatography column was prepared with immunoglobulin G obtained from sera from patients with lepromatous leprosy. Unbound solutes consisted of specific, partially purified N. asteroides antigens, primarily a 54-kDa band, accompanied by two others of 31 and 62 kDa. The Western blot (immunoblot) technique was applied to detecting the immunologic response to nocardiae in the serum of nocardiosis patients. Each of the serum samples from immunosuppressed or immunocompetent patients infected with N. asteroides reacted with the 54-kDa band, and two reacted with the 31- and 62-kDa bands. There was no reaction to either the 54- or the 31-kDa antigen with all serum samples obtained from patients with tuberculosis, except for one, with all serum samples obtained from patients with leprosy, or with all sera obtained from healthy controls. The partially purified 54-kDa antigen, specific for N. asteroides, was used as the immunogen to generate monoclonal antibodies (mAbs) and two mAbs were selected. As determined by Western blot, both mAbs reacted with the 54-kDa band. Using indirect immunofluorescence or enzyme immunoassay with whole N. asteroides micro-organisms, the mAbs did not react with N. asteroides cells. No cross-reactivity with mycobacterial antigens, either culture-filtrate antigens or tuberculin, was exhibited with any of the two mAbs. These mAbs are candidates to be used for the development of a sensitive and specific diagnostic test for nocardiosis.     

Efficacy of albendazole combined with a marine fungal extract (m2-9) against Angiostrongylus cantonensis-induced meningitis in mice

The pathogenesis of angiostrongyliasis, resulting from Angiostrongylus cantonensis invasion of the human central nervous system, remains elusive. Anthelmintics are usually used to kill worms, although dead worms in the brain may cause severe inflammation which will lead to central nervous system damage. Therefore, combination therapy with anthelmintics and anti-inflammatory drugs in the treatment of human angiostrongyliasis needs further study. To evaluate the efficacy of albendazole combined with a marine fungal extract (m2-9) in A. cantonensis infection, BALB/c mice infected by the third-stage larvae of A. cantonensis were divided into three groups: mice treated with albendazole or m2-9 alone or in combination from day 5 post-inoculation (PI). Several efficacy parameters were recorded, including weight change, worm recovery, neurological function, behavioural analysis, eosinophil and leucocyte counts. The results showed that combination therapy increased body weight, reduced worm burden, improved learning ability, memory and action, decreased neurological dysfunction and leucocyte response in these mice. The combination of albendazole and m2-9 treatment significantly decreased leucocyte response and increased the frequency of rearing, compared to infected mice treated with either drug alone. Therefore, m2-9 is a natural product with potentially significant therapeutic value for angiostrongyliasis and is worthy of further study.     

Seasonal variation in the prevalence and intensity of canine Gnathostoma spinigerum infection in northeastern Thailand.

Gnathostoma spinigerum was found in gastric nodules in 4.1% of 2940 dogs surveyed in northeastern Thailand. The prevalence and worm burden of G. spinigerum exhibited a seasonal fluctuation. The parasites were more abundant in the rainy season and the early winter (August-December) than in the summer (April-March). Most parasites were sexually mature between August and December while immature worms were observed during March and April. The distribution of gnathostomes within the sampled dogs was highly dispersed and few animals were found to harbour more than five worms.