Strongyloides stercoralis excretory/secretory protein strongylastacin specifically recognized by IgE antibodies in infected human sera.

The infective, microscopic Strongyloides stercoralis larvae in contaminated soil can penetrate human skin with the help of excretory/secretory proteases. These proteases play a critical role in infection and transmigration of the parasite to the intestines. Strongylastacin is similar to astacin (from the digestive gland of the crayfish Astacus astacus), a multi-domain protein with a signal peptide, a pro-enzyme, a catalytic domain containing the zinc binding consensus astacin family signature sequence HEXXHXXGFXHEXXRXDR, and a second conserved zinc binding motif SIMHY at N- terminal region. An EGF-1 like domain and a CUB domain are located at the COOH- terminal. In this study, the excretory/secretory Strongylastacin gene from S. stercoralis infective larval stage was cloned and expressed as a 45 kDa in Escherichia coli. Immunoblot analysis showed the presence of natural IgG antibodies against strongylastacin in six infected and six non-endemic normal sera. These findings were confirmed in an ELISA of 32 S. stercoralis infected and 32 presumed normal human sera; all contained natural anti-strongylastacin IgG antibodies. By contrast, IgE antibodies specific to strongylastacin were present in sera from individuals infected with S. stercoralis but not in uninfected control sera. Moreover, recombinant strongylastacin did not cross-react with IgE antibodies either from patients infected with filaria or patients with tropical pulmonary eosinophilic (TPE) who had increased IgE antibodies. The present authors conclude that strongylastacin, an excretory/secretory antigen, elicits specific IgE antibodies in S. stercoralis infected humans. Non-specific IgG antibodies to strongylastacin are present in both infected and normal humans. Further investigation is needed to understand the role of the host protective response against strongylastacin.

A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection.

BACKGROUND: We investigated whether luciferase immunoprecipitation systems (LIPS) can be the basis for a more rapid, specific, and standardized assay for the diagnosis of Strongyloides stercoralis infection. METHODS: A LIPS assay was developed based on immunoglobulin (Ig) G or IgG4 antibody to a recombinant Strongyloides antigen (NIE) and was compared with an NIE enzyme-linked immunosorbent assay (ELISA). A second antigen, S. stercoralis immunoreactive antigen (SsIR), was tested alone and in combination with NIE. The assays were tested using serum samples from patients with parasitologically proven S. stercoralis or filarial infections and from healthy, uninfected control subjects. RESULTS: The NIE LIPS assay based on IgG antibody easily differentiated between S. stercoralis-infected and uninfected patients (P< .0001) and demonstrated improved specificity compared with the NIE ELISA (100% vs. 95%). Serum from filaria-infected patients did not cross-react when tested with the NIE LIPS assay. When SsIR was used in combination with NIE in the LIPS format, sensitivity and specificity improved to 100%, with a 7-fold difference between positive and negative values. No advantage was found in using a LIPS assay based on IgG4. At posttreatment follow-up, a significant decline in antibody titers was detected using the NIE ELISA (P< .0017) and the NIE LIPS assay (P< .0001). CONCLUSIONS: LIPS addresses several limitations of current ELISAs and represents a major advance in the diagnosis of S. stercoralis infection.

Strongyloides stercoralis recombinant NIE antigen shares epitope with recombinant Ves v 5 and Pol a 5 allergens of insects.

A new recombinant protein (NIE) for immunodiagnosis of human Strongyloides infection has 13% to 18% amino acid identity with antigen 5 insect venom allergen, but the C-terminal segment of NIE showed highest identity with Ves v 5 (yellow jacket) and Pol a 5 (paper wasp). A rabbit polyclonal anti-NIE antibody identified a single band of NIE antigen as well as bands of Pol a 5 and Ves v 5 antigens, and mouse anti-Pol a 5 and anti-Ves v 5 sera reacted with recombinant NIE antigen by Western blot. A cyanogen bromide-digested C-terminal fragment of NIE was reactive with mouse anti-Ves v 5 and Pol a 5 antibodies as well as with rabbit anti-NIE serum. Although IgE and IgG antibodies from pooled sera from Strongyloides-infected patients reacted with Pol a 5 and Ves v 5 recombinant antigens on immunoblots, neither antigen inhibited human IgG reaction with NIE antigen in a competitive enzyme-linked immunosorbent assay.

Identification of an astacin-like metallo-proteinase transcript from the infective larvae of Strongyloides stercoralis.

Strongyloides stercoralis, an important nematode pathogen of humans, is transmitted by contact with soil contaminated with the microscopic larvae of the parasite. We determined the cDNA sequence and deduced amino acid structure of a metallo-proteinase that is abundantly transcribed expressed by infective stage larvae of S. stercoralis. This deduced structure of the enzyme revealed a multi-domain protein that included an NH2-terminal peptidase. This peptidase consisted of a signal peptide, a pro-enzyme region, and a mature peptidase domain that included the metal ion co-ordinating motifs, HETSHALGVIH and SIMHY ("Met-turn"), characteristic of the catalytic active site of members of the metzincin superfamily of zinc metallo-endopeptidases. It was phylogenetically and structurally similar to astacin from the digestive gland of the crayfish Astacus astacus, to the HCH-1 peptidase of Caenorhabditis elegans required for hatching and migration of a post-embryonic neuroblast, and to the morphogenetically important peptidases, bone morphogenetic protein-1 (BMP-1) and Drosophila tolloid. In addition, the Strongyloides enzyme, designated strongylastacin, includes a central epidermal growth factor (EGF) domain followed by a carboxyl CUB (complement sub component C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein) domain. Inspection of the dbEST database revealed the presence of at least 9 transcript clusters that are related to greater or lesser extent to strongylastacin; based on these expressed sequence tags, strongylastacin was expressed only in the infective third stage larvae, whereas other transcript clusters were expressed both in filariform and rhabditiform stages or only in the rhabditiform stage. Based on the deduced sequence, structure, and expression profile, strongylastacin is the probable candidate for the zinc-dependent metalloprotease, Ss40, known to be deployed by larvae of S. stercoralis to penetrate human skin to initiate infection.

Evaluation of 7SL RNA gene sequences for the identification of Leishmania spp.

We evaluated the use of 7SL RNA gene sequences for the identification of Leishmania spp. A fragment (approximately 137 basepairs) of the 7SL RNA gene from 13 reference strains and 18 clinical isolates of 11 different Leishmania species was amplified and sequenced using conserved primers. Reference strains from each Leishmania spp. complex showed unique sequences. The nucleotide sequences were compared pairwise and a range of 81.0-99.3% intercomplex similarity was observed. Clinical isolates of the same species had sequences identical to the corresponding reference strains; thus, the intraspecies similarity was 100%. A phylogenetic tree derived from the 7SL RNA gene partial sequences was constructed and is in agreement with accepted phylogenetic schemes.

Helminthic infection down-regulates type 1 immune responses in human T cell lymphotropic virus type 1 (HTLV-1) carriers and is more prevalent in HTLV-1 carriers than in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis.

Human T cell lymphotropic virus type 1 (HTLV-1) infection is associated with an exacerbated type 1 immune response and secretion of high levels of proinflammatory cytokines. In contrast, helminthic infection induces a type 2 immune response. In the present study, the cytokine profile in HTLV-1 carriers coinfected with helminths (Strongyloides stercoralis and/or Schistosoma mansoni) was compared with that in HTLV-1 carriers not coinfected with helminths. Levels of interferon (IFN)- gamma were higher in HTLV-1 carriers not coinfected with helminths than in HTLV-1 carriers coinfected with helminths (P<.05). The overall frequency of IFN- gamma -expressing CD8+ and CD4+ cells was decreased in HTLV-1 carriers coinfected with helminths (P<.05). The percentage of interleukin (IL)-5- and IL-10-expressing T cells in HTLV-1 carriers coinfected with helminths was higher than that in HTLV-1 carriers not coinfected with helminths (P<.05). Moreover, we found that the prevalence of helminthic infection was 7-fold higher in HTLV-1 carriers than in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (P<.05). These data show that helminthic infection decreases activation of type 1 cells, which may influence the clinical outcome of HTLV-1 infection.

Majority of interferon-gamma-producing CD4+ cells in patients infected with human T cell lymphotrophic virus do not express tax protein.

The present study was conducted to determine whether interferon (IFN)-gamma production by CD4(+) cells in patients infected with human T cell lymphotropic virus (HTLV) is associated with expression of Tax, an HTLV type 1 (HTLV-1) transactivator. The frequency of IFN-gamma production from CD4(+) cells was greater in HTLV-1-infected patients (n=21) than in uninfected (n=3) and Strongyloides stercoralis-infected patients (n=4), and greater in patients with HTLV-1 with detectable Tax than in patients with HTLV-1 with undetectable Tax. In the patients with HTLV-1 with detectable Tax, the majority of CD4(+) cells making IFN-gamma did not express Tax.

Case report: Rectal adminstration of ivermectin to a patient with Strongyloides hyperinfection syndrome.

Strongyloides hyperinfection syndrome may be complicated by paralytic ileus that interferes with the absorption of oral anti-helminthics. We report on the administration of ivermectin as a rectal enema preparation to a renal transplant recipient with Strongyloides hyperinfection syndrome and progressive ileus. Attempts at treatment using nasogastric albendazole and ivermectin were unsuccessful despite clamping the nasogastric tube after drug administration. Ivermectin tablets were ground to a powder, resuspended in a commercially available suspending agent, and administered per rectum. The suspending agent was chosen for its near-physiologic osmolality to allow longer retention, in contrast to many enema preparations that have a laxative effect. The patient improved markedly within 72 hours of initiation of the therapy per rectum and recovered fully. Ivermectin administered as an enema may be beneficial in patients with severe strongyloidiasis who are unable to absorb or tolerate oral therapy.

Characterization of a recombinant immunodiagnostic antigen (NIE) from Strongyloides stercoralis L3-stage larvae.

Due to the process of internal autoinfection, even chronic asymptomatic infections with Strongyloides stercoralis have the potential to become severe disseminated disease with fatal outcome. Intermittent and scanty larval excretion makes parasitologic diagnosis difficult. Serodiagnosis is helpful, but antigen preparation from infective larvae requires access to patients or immunosuppressed experimental animals. For these reasons, attention has turned to recombinant antigens for immunodiagnosis. A 31-kDa candidate antigen (NIE) derived from an L3 cDNA library is described in this report. Multiple alignment of the deduced amino acid sequence of NIE showed approximately 12-18% identity with various other organisms, including 17.9% of Asp1 of Ancylostoma caninum, 12.6% of Hemonchus contortus, and 17.6% of insect venom allergen 5 of yellow jacket. By ELISA, antibodies to the purified recombinant NIE antigen were demonstrated in 87.5% of 48 sera from strongyloides-infected patients and in only 6.5% of sera from presumed normal controls. Immunoreactivity of purified NIE antigen with parasite-specific IgE from sera of strongyloides-infected patients indicated its potential use as an immediate sensitivity skin test antigen. This application of the NIE antigen was supported by its capacity to trigger release of histamine upon in vitro exposure to blood from strongyloides-infected patients and its failure to produce histamine release from blood of normal controls.

Age-prevalence and household clustering of Strongyloides stercoralis infection in Jamaica

The epidemiology of Strongyloides stercoralis was studied in families of clinical (reference) cases and their neighbours at endemic foci in Jamaica. Thirteen foci were studied based on the place of residence of a reference case. For each household of a reference case, the 4 most proximal neighbourhood households (spatial controls) were included in the study. Out of 312 persons contacted 244 were followed up using questionaires, stool examimation and serology. Prevalence of infection based on based on stool examination was 3.5 percent and on ELISA 24.2 percent. Prevalence increased with age but was not related to gender. Reference cases were significantly older than the general study population. The prevalence of infection based on both serology and stool examination was significantly higher in referecne than in neighbouring households (the reference cases, themselves, were not included in the analysis). Furthermore, prevalence of infection was highest among persons who shared a bedroom with a reference case and decreased significantly with increasing spatial separation. This is indicative of close contact transmission which has not been previously shown for a geohelminth, but which is common among microparasites.(AU)

Age-prevalence profile and household clustering of cases of strongyloides infection at endemic foci at Jamaica - abstract

Strongyloides sterocalis infections were examined in families of clinical cases and also in those of their most proximal neighbours. Thirteen clinical cases in Kingston, Jamaica led to the identification of thirteen endemic foci. In addition to the clinical cases, 299 persons were contacted using questionnaires, stool examination and serology. Two hundred and thirty-one persons were fully compliant. The stool prevalence of S.sterocalis was 3.5 percent, while that based on ELISA was 24.2 percent (not including the 13 clinical cases). Both estimates of infection prevalence were significantly higher in the households of the clinical cases compared with the neighbours. The clinical cases were significantly older than the general study population. Furthermore, prevalence was highest among persons who shared a bedroom with a clinical case and decreased with spatial separation. These data strongly suggest that human strongyloides is a close-contact infection. This is likely to be facilitated by the direct phase of the parasite's life cycle and has significant implications for control of infections in endemic areas (AU)_

Immunoepidemiologic studies of Strongyloides stercoralis and human T lymphoropic virus type I infection in Jamaica

Epidemilogic investigations of Strongyloides stercoralis and human T lymphotropic virus type I (HTLV-I) infections were conducted. Of 312 persons contacted, 209 (67 percent) provided blood and stool samples. Prevalences of S. stercoralis and HTLV-I antibodies were 26.8 percent and 8.1 percent (n = 198), respectively, and S. stercoralis larvae were detected in 4 percent. HTLV-I antibodies were significantly more common in persons positive for S. stercoralis larvae (10 [58.8 percent] of 17) compared with seropositive larvae-negative (4 [8.9 percent] of 45) or seronegative persons (9 [6.2 percent] of145) (P< .002). IgE levels increased with age in S. stercoralis-seropositive persons who were HTLV-I negative (P, .))2). However, there was an age-related depression of serum IgE in HTLV-I-positive persons (P < .003) that was sufficient to annul the IgE level-raising effect of S stercoralis seropositivity. The data provide evidence that HTLV-I infection is associated with increased frequency of larvae in the stool of S. stercoralis-infected persons and suggest that the mechanism may involve suppression of the IgE response (AU)

HTLV-1 infection - a risk factor for severe strongyloidiasis? - abstract

Epidemiological associations of Strongyloides stercoralis and Human T-Lymphotropic Virus Type-1 (HTLV-1) were investigated at eleven (11) foci endemic for strongyloidiasis in Jamaica. Each focus was identified on the basis of residency of a parasitologically-proved case of strongyloidiasis who presented at the University Hospital. Three hundred and twelve (312) persons were contacted, and blood and stool samples were collected for HTLV-1 and S. stercoralis serology and S. stercoralis coproculture, respectively. Overall compliance was 66.6 per cent. The prevalence of S. stercoralis in the pooled foci was 8.2 per cent including and 4.0 per cent excluding the hospital presenters, and for HTLV-1 it was 11.1 per cent and 8.1 per cent. Seroprevalence of S. stercoralis infection was 30 per cent and 26.8 per cent with and without the clinical cases, respectively. The prevalence of each infection was correlated with the age of the host (Spearman Rank Correlation, p<0.001). HTLV-1 was clustered in S. stercoralis larval shedders (58.8 per cent, n=17) compared with non-shedders (8.9 per cent, n=45) (Fisher's exact test, p<0.001), but particularly so among clinical cases 7 of whom (n=9) had HTLV-1 antibodies. The association was explained on the basis of age-related total serum IgE levels in HTLV-1/S. stercoralis infection cases. Individuals uninfected by S. stercoralis displayed a tendency for decreased IgE levels with age. This especially so in HTLV-1 carriers (ANCOVA: age * HTLV-1 interaction term t = 3.176, p<0.002). In marked contrast, S. stercoralis seropositive individuals had significantly elevated IgE titres in older persons compared with seronegative controls (ANCOVA: age * S. stercoralis interaction term t = 3.733, p<0.001). In persons seropositive for both HTLV-1 and S. stercoralis, however, the elevation of IgE levels previously observed in S. stercoralis positive individuals was completely subsumed by the negative influence of HTLV-1. It is suggested that impairment of host immunity by HTLV-1, and/or S. stercoralis in the presence of HTLV-1, exacerbates strongyloidiasis resulting in increased frequency of both larval shedding and parasite disease. Effect of S. stercoralis (S) and HTLV-1 (H) infection status on the host age/serum IgE association. This figure is designed to illustrate general trends; ungrouped data were used in statistical analyses (AU)

Gastrointestinal parasitic infection in healthy Jamaican carriers of HTLV-I

A subsample (1.6 percent; n = 13,260) of a healthy Jamaican population of food-handlers, studied by Murphy et al. (1991), who were serologically positive (n = 99) or negative (n = 113) for HTLV-I was investigated for intestinal parasitic infection using coprological methods. Helminth infection included Ascaris lumbricoides (2.8 percent), Trichuris trichiura (7.1 percent) and hookworms (6.1 percent). Entamoeba coli was found in 21.8 percent of samples, while E. hartmanni, Giardia lamblia, Endolimax nana, Iodamoeba butschlii and Chilomastrix mesnili each occurred in less than 10 percent of responders. T. trichiura displayed a higher prevalence (10.6 vs 3 percent (chi 2 = 4.623;p = 0.03) in the HTLV-I negative group. G. lamblia was detected more frequently among HTLV-I carriers compared to controls (9.1 and 3.5 percent respectively), but the association was not statistically significant (chi 2 = 2.825;p = 0.09). Infection with intestinal parasites is likely to occur independent of HTLV-I status: however, possible HTLV-I-induced immunosuppression may lead to higher intensity infections of certain organisms thus facilitating easier detection using parasitological methods. The immunomodulatory potential of HTLV-I infection in the aetiology of non-malignant diseases requires further investigation. (AU)

Interaction between HTLV-1 and Strongyloides stercoralis infection resulting in lowered IgE titres in Jamaica - abstract

In Japan, a positive association exists between the presence of serum antibodies to HTLV-1 and S. stercoralis. Also, it has been shown in Jamaica that coincidental HTLV-I infection may influence the outcome of treatment of S. stercoralis, or even underlie development of severe strongyloidiasis in some persons. However, the relationship between HTLV-I and S. stercoralis remains unclear. This paper highlights a hitherto unreported association between the occurrence of serum antibodies to HTLV-I, to S. stercoralis, and total serum IgE and strongyloidiasis in a Jamaican community. Blood and stool samples were collected from 67 persons from 6 geographical locations in Kingston. Sera were analysed for antibodies to S. stercoralis and HTLV-I, while stool samples were subjected to charcoal coproculture. As in Japan, individuals serologically positive for S. stercoralis tended to be infected more often with HTLV-I (33 per cent) than seronegative individuals (15 per cent), but the difference was not significant (two-tailed Fisher's exact test; P = 0.15). However, parasitologically-proved strongyloidiasis and HTLV-I seroconversion were strongly associated; while occurrence of HTLV-I was 67 per cent in individuals whose stool contained S. stercoralis larvae, it was only 15 per cent in their parasitologically negative counterparts (two-tailed Fisher's exact test; P = 0.01). Analyses strongly suggest that serological status for S. stercoralis HTLV-I affect IgE titres interaction term, F = 3.54; P = 0.06). IgE titres were lower in HTLV-I seropositive than seronegative individuals in both groups with and without S. stercoralis antibodies (HTLV-I main effect, F = 11.13; P = 0.002), but more strongly so in the former group (Table). S. stercoralis infection appeared to elevate reagin levels in some individuals in the HTLV-I negative group (n=13) (one-way ANOVA, F = 2.09; P - 0.15). In contrast, however, serum IgE titres were depressed in the group (n-5) with HTLV-I and concomitant S. stercoralis infection to levels perhaps even lower than those seen in HTLV-I individuals who did not have S. stercoralis (one-way ANOVA, F = 2.39; p = 0.15). Thus, it appears that HTLV-I and S. stercoralis operate synergistically to effect a significant reduction in IgE serum antibodies in infected individuals. If expressed at the level of the intestinal mucosa, this may permit increased rates of autoinfection of the parasite and result in correspondingly greater worm loads and patient morbidity. Lower levels of IgE antibodies against S. stercoralis are known to be associated with disseminated disease (AU)

Coincidental HTLV-I infection influences outcome of treatment of strongyloidiasis - abstract

It is uncertain whether HTLV-I infection and Strongyloidiasis are related other than by chance. A consecutive series of Jamaican patients and controls have been analysed retrospectively for anti-Strongyloides and HTLV-1 antibodies to determine whether either influences the outcome of anti-helminthic therapy. Twenty-seven Jamaicans (16 M, 11F) mean age 50.2 years (range 16-85), who were found to have Strongyloides stercoralis infection were studied at the University Hospital of the West Indies. At the same time, a parasite-negative group of 13 patients (6M, 7F) of mean age 37.6 years, (range 23-53), with minor or no gastrointestinal disease served as controls. Pretreatment blood samples were taken from the Strongyloides group and controls. Serum was subsequently tested for IgG antibodies to filariform Strongyloides stercoralis larval antigens by ELISA and to HTLV-1 by ELISA and Western Blot. Outcome of the treatment of Strongyloidiasis with thiabendazole (25 -mg/kg b.d. orally for 10 days was determined at 2 months. Strongyloides reciprocal antibody titre was considerably higher in patients than controls, mean 870 vs 167; median 1,024 vs 8 (p<0.001). The sensitivity of the antibody test was 93 percent, but the specificity was 69 percent at best. There was no correlation with the anti-Strongyloides antibody titre and outcome with anti-helminthic therapy. HTLV-1 antibodies were found only in the Strongyloides patients, 12 of 27 (44 percent); antibody titres were high and positive with both test used: only one patient was known beforehand to have a disease associated with HTLV-1 infection. Of those 12 with HTLV-1 antibodies, 3 (25 percent) were cured, 7 still had the infection at 2 months, a further 2 had died or defaulted from follow-up. Of the 15 patients without HTLV-1 antibodies, 9 (60 percent) were cured, 3 still had the infection and 3 had died or defaulted. By chi-square analysis, the difference is significant whether one includes all the deaths and defaulters on an intention-to-treat basis or just those who were available at 2 months post-therapy. However, since none of the deaths were related to Stongyloidiasis or HTLV-1 injection, it is probably justifiable to exclude the deaths from the computation. These results show that the association of Strongyloidiasis and HTLV-1 is more than chance clustering. Not only is the prevalence of HTLV-1 antibodies far higher in the patients with Strongyloidiasis than in the normal Jamaican population, but that concurrent asymptomatic HTLV-1 infection interferes with anti-helminthic treatment (AU)