Coinfections with Schistosoma haematobium, Necator americanus, and Entamoeba histolytica/Entamoeba dispar in children: chemokine and cytokine responses and changes after antiparasite treatment.

The effect of polyparasite infections on cytokine and chemokine responses as well as the effect of antiparasite treatment was studied in children without parasite infection (the G0 group), in children singly infected with Schistosoma haematobium (the G1 group), and in children multiply infected with S. haematobium/Schistosoma mansoni, Entamoeba histolytica/Entamoeba dispar, and Necator americanus (the G3+ group). Linear regression analysis disclosed a significant risk for coinfection with hookworm and Schistosoma species. Polyparasite infections detected in 23% of children before treatment were present in 5% at 15 months after treatment. Chemokine responses to S. mansoni adult worm antigen (SmAg) diminished after treatment for macrophage inflammatory chemokine (MIP)-1alpha/chemokine (C-C motif) ligand (CCL)-3 (among G3+ children, by a factor of 200 [95% confidence interval {CI}, 33-1111]) and for MIP-1beta/CCL-4 (among G3+ children, by a factor of 26 [95% CI, 6-117]) but were enhanced for thymus- and activation-regulated chemokine/CCL-17 (among G3+ children, by a factor of 10 [95% CI, 3-32]) (P < .001 for all). In response to E. histolytica antigen, interleukin (IL)-13 levels increased after treatment among G1 children by a factor of 138 (95% CI, 12-1569) and among G3+ children by a factor of 21 (95% CI, 7-64) (P < .001 for both). Cellular production of interferon (IFN)-gamma in response to SmAg decreased 4 weeks after treatment among G3+ children, whereas T helper cell type 2 (Th2) IL-13 production was enhanced among G1 and G3+ children. In summary, polyparasite infections with S. haematobium/S. mansoni, E. histolytica/E. dispar, and N. americanus generated prominent proinflammatory cytokine and chemokine responses, and, after antihelminth treatment, the inflammatory chemokine response lessened as the Th2 responsiveness in coinfected children increased.

Basophil competence during hookworm (Necator americanus) infection.

A popular hypothesis to explain parasite survival in the presence of a pronounced T helper 2 phenotype in helminth-parasitized populations has been Fc epsilonRI blockade by parasite-induced polyclonal IgE. To begin to test the hypothesis that Fc epsilonRI-bearing cells would be refractory to activation in parasitized populations, we investigated basophil function in 43 individuals from a hookworm endemic area. Study individuals had high levels of total IgE and eosinophilia and a mean hookworm burden of 2,257 epg. Basophils from all members of this parasitized population were shown to release histamine to a number of agonists, including anti IgE and a hookworm allergen, calreticulin. These data would indicate that Fc epsilonRI blockade at the level of the basophil did not occur in this parasitized population despite the presence of possible immunologic blocking agents. This would suggest that this effector arm of the T helper 2 phenotype remains operative in infected populations.

Necator americanus: optimization of the golden hamster model for testing anthelmintic drugs.

Laboratory golden hamsters (Mesocricetus auratus) were infected with Necator americanus under several different parasite and host conditions to optimize the model for testing anthelminthic drugs. The results confirmed that male hamsters were more susceptible to infection than females. Host age in the range of 5-15 weeks was not a factor that impacted on adult worm burden, and similar worm burdens were achieved using doses of 150, 250 or 500 N. americanus L3 (NaL3). The largest numbers of adult hookworms were recovered on days 21-28 post-infection, with a significant decrease at days 40-50 post-infection. Therefore adult worm recovery is maximal approximately 11-18 days prior to patency and host blood loss. From these studies a drug evaluation protocol was developed using 150 NaL3 as the infectious dose and then evaluating the anthelminthic effects of the drugs albendazole, tribendimidine, and pyrantel pamoate on days 21-28 post-infection. The model confirms the anthelminthic activity of albendazole, tribendimidine, and pyrantel pamoate and has the potential as a laboratory animal model to detect emerging drug resistance.

Immune responses following experimental human hookworm infection.

To characterize the immune response following primary human hookworm infection, an adult volunteer was infected with 50 L3 larvae of Necator americanus, reinfected 27 months later and followed for a further 6 months. Clinical signs, blood picture, ex-vivo peripheral blood cytokine production (IFN-gamma, IL-5, IL-13, IL-10 to mitogen and hookworm antigen), acute phase proteins (APP) (C-reactive protein, CRP and alpha1-antitrypsin, alpha1-AT) and antibody levels were determined. Dermatitis, oedema, mild nausea and abdominal discomfort followed the primary infection. Eosinophil counts peaked early during both infections but remained elevated ( approximately 18%) throughout. Transient production of IL-5, IL-13 and APP also followed infection but there were negligible levels of IFN-gamma or IL-10. The onset of nausea, oedema and the initial rise in CRP, alpha1-AT, eosinophilia and IL-5 coincided (days 13-27) with the late larval migration and early establishment of the preadult worms in the intestine. Apart from the eosinophilia these responses declined to baseline levels within 4 months and were less pronounced on re-infection.

Platelet parameters in subjects infected with hookworm.

Hookworm is an important intestinal parasite infection with high prevalence in tropical countries. In addition to iron deficiency anaemia, the human hookworm appears to have evolved a number of complementary strategies to overcome the host's haemostatic processes. These include the inhibition of blood coagulation, platelet aggregation and mediator release, and the secretion of fibrinogenolytic enzymes. In this work, we studied the platelets in the subjects with detection of hookworm infection from screening stool examination comparing to those without. One hundred subjects were included, studied for stool parasite and platelet parameters. Of our 100 subjects, hookworms were identified in stools of six cases, giving an infection rate equal to 6%. The platelet parameters of the subjects showed a statistically significant lowering of mean platelet volume (MPV) in the subjects with hookworm infection. Also, we detected a non-significant lower platelet count in subjects with hookworm infections. Of interest, the alteration in platelet size, identified by platelet parameters, has never been documented in the literature. This observation might be a pathological process or might be an accidental finding in our study.

Albendazole therapy and reduced decline in haemoglobin concentration during pregnancy (Sierra Leone).

WHO recommends that anthelmintic treatment be included in strategies to improve maternal nutrition in areas where hookworms are endemic and anaemia is prevalent. At present, few countries have adopted this recommendation, partly owing to the lack of data to support the adverse effects of hookworms on maternal health. A longitudinal study was conducted on 125 women in Sierra Leone (in 1995/96) to measure the impact of single-dose albendazole (400 mg) and daily iron-folate supplements (36 mg iron and 5 mg folate) on haemoglobin and serum ferritin concentration during pregnancy. Women who received both albendazole and iron-folate supplements experienced no significant change (P > 0.05) in the prevalence of anaemia and iron-deficiency anaemia between the first and third trimesters. These prevalence levels significantly increased (P < 0.05) in women who received either albendazole or iron-folate supplements or neither. After controlling for baseline haemoglobin concentration and season, the mean decline in haemoglobin concentration between the first and third trimester in women who received albendazole was 6.6 g/L less than in women who received the control (P = 0.0034). The corresponding value for iron-folate supplements was 13.7 g/L haemoglobin (P < 0.001). The effects of albendazole and iron-folate supplements were additive. These findings lend support to WHO's recommendation for anthelmintic treatment during pregnancy.

T-lymphocyte subsets in patients with hookworm infection in Zaria, Nigeria.

Peripheral lymphocyte subsets CD3, CD4, CD8 were studied using monoclonal antibodies to determine the mechanism of immunosuppression observed in an earlier study with total T-cells using the sheep erythrocyte rosetting technique. The study was carried out in 37 Nigerian patients with hookworm infection (Necator americanus and Ancylostoma duodenale and 17 healthy Nigerians as controls. The leucocyte migration inhibition (LMI) test was also carried out to assess the functional integrity of the lymphocytes, while the hookworm status of the patients was assessed by the Stoll technique for measuring egg counts. The results of the T-cell studied showed that CD3 and CD4 cell percentages were significantly depressed in hookworm patients compared to controls (P<0.05). The difference between mean CD8 percentages and absolutes in hookworms patients and controls were not significant. Absolute CD3 and CD4 numbers were not significantly lowered compared to controls in the study and CD4/CD8 ratios were insignificantly reduced. Patients with polyparasitism including hookworm infection showed significant reduction in CD3 and CD4 cells when compared with controls and with patients with hookworm infection alone. The leucocyte migration inhibition response to purified protein derivative of M. tuberculosis (PPD) was significantly decreased in hookworm patients compared to controls, confirming that functionally cell mediated immunity is depressed in hookworm infection.

A hookworm allergen which strongly resembles calreticulin.

Immmoglobulin E-rich plasma from patients from Papua New Guinea infected with Necator americanus has been used to probe an adult N. americanus cDNA library for the presence of hookworm allergens. Using this approach, one hookworm allergen has been identified as calreticulin, which was subsequently expressed in Escherichia coli. Little serological cross reactivity was seen between the recombinant calreticulins of this hookworm and its host. Prospective roles for hookworm calreticulin in the host-parasite relationship are discussed in depth.

Necator americanus in inbred mice: evidence in support of genetically determined differences in the cellular immune response to a primary infection.

The blood and broncho-alveolar leucocyte (BAL) responses, to a primary Necator americanus infection, were studied in male BALB/c and NIH mice. Following percutaneous infection, a significant blood leucocytosis occurred in both the BALB/c and NIH strains. The peak response occurred, in both strains, on day 10 p.i. and this was reflected in the eosinophil response which peaked at the same time-point. The change in blood eosinophil numbers, as a result of infection, was found to be the greatest recorded for any cell type. In BALB/c mice, however, infection elicited a significantly more intense total leucocyte, lymphocyte and eosinophil response than in NIH mice. In contrast, the BAL response was more intense in the NIH strain. Peak BAL responses were observed between days 12 and 16 p.i., in both strains, and the change in broncho-alveolar eosinophil numbers, as a result of infection, was found to be the greatest recorded for any cell type. The relationship between the observed leucocyte responses and resistance to the migrating larvae of a primary infection is discussed.

The specificity of the human IgE response to Necator americanus.

Human IgE responses to helminth infections have been described as both problematic and beneficial, in that type 1 hypersensitivity to parasite infections can reputedly cause pathology and/or parasite expulsion, (Pritchard, Quinnell & Walsh 1995). In the present communication, we can report that the IgE response to helminth infections may be beneficial in a diagnostic sense, in that antibodies of this isotype showed minimal cross-reactivity against antigens from the parasites tested. This information may be of benefit to workers attempting to establish ELISA assays for the specific diagnosis of zoonotic hookworm infections, for example, Ancylostoma caninum (Croese 1988), particularly in regions where human hookworm infection is endemic.

Immunosuppression in Nigerians with hookworm infection.

T rosette cells results in patients with (Necator americanus and Ancylostoma duodenale) hookworm infection, divided into groups on the basis of the degree of anaemia (Group I had marked anaemia Hb 1-7, hypochromia ), Group II had moderate anaemia (Hb 8-11, hypochromia ++) and Group III had no signs of anaemia despite the underlying hookworm infection), were compared with results in 28 healthy controls who had no signs of other infection and excreting no ova of hookworm or other parasites. The level of sheep erythrocyte rosette-forming T-lymphocytes (SE-TL) in the patients (Group I:42 +/- 8%; Group II:45 +/- 11%; Group III:47 +/- 6%) was markedly reduced compared to controls (58 +/- 13%) (P < 0.001). The leucocyte migratory responses to purified protein derivative (P.P.D); were significantly (P < 0.05) reduced in all the subgroups as compared with the controls. The results support the hypothesis of the existence of immunosuppression in patients with hookworm infection.

[Differential diagnostic value of occult blood in stool in hookworm infection in the Third World. Study from Papua New Guinea]. / Differentialdiagnostische Wertigkeit von okkultem Blut im Stuhl bei Hakenwurmbefall in der Dritten Welt. Studie aus Papua Neu Guinea.

In 633 persons of the Madang province in Papua-New Guinea hookworm-infection, blood hemoglobin and the presence of blood in stools were studied. 60% of the subjects were infected with hookworm (Necator americanus). There was a significant linear correlation between the intensity of hookworm infection and blood haemoglobin level. The Colo-Rectal test gave positive results in 17% of the cases without worm-infection. In infected persons, the Colo-Rectal test gave an increasing rate of positive results as the parasite infection increased: 24% for subjects with low levels of infection, and 57% and 67% for moderate and high levels, respectively. The mean blood-haemoglobin level was lower in persons with positive Colo-Rectal-Test and hookworm infection (11.8 g%) than among those with negative Colo-Rectal-Test and hookworm infection (15.0 g%, p < 0.01). In young populations with a known high prevalence of hookworm infection, Colo-Rectal test is therefore suitable for the detection of moderate or severe hookworm infections. It may serve as a simple and useful method to work up anaemia is such populations.

Hookworm (Necator americanus) infection and storage iron depletion.

The relationship between iron status and the intensity of infection with hookworm was investigated in a rural population on Karkar Island, Mandang Province, Papua New Guinea. There was a significant negative correlation between plasma ferritin level and hookworm burden, which was strongest in males. In contrast, there was no correlation between plasma ferritin and hookworm egg count, and no consistent correlation between haemoglobin level or haematocrit and either measure of hookworm intensity. The results suggest that the role of hookworm in the aetiology of anaemia may be difficult to assess without the accurate measurement of hookworm burden.

Necator americanus in the DSN hamster: density-dependent expulsion of adult worms during primary infection.

Neonatal hamsters were exposed to varying doses of Necator americanus larvae and changes in the stability of the resulting worm burdens were monitored over a period of 25 weeks. No change in worm burdens was evident for the first 5 weeks of infection, irrespective of the infection intensity, but the more heavily infected groups subsequently lost worms in a density-dependent manner. Male and female hamsters lost comparable proportions of their established parasite burdens indicating that there was no host sex-linked difference in this respect. By week 15 infections had stabilized and the residual worm burdens, usually a maximum of 30 worms survived for a considerably longer period of time. Initially the percentage of male worms varied from 45% to 50% but as infection progressed male worms comprised a significantly increasing proportion of the total worm burden. By week 25 the percentage of male worms was usually in excess of 60%. The growth of infected animals was not severely affected by N. americanus, even when heavy worm burdens established initially, but a significant effect was detected particularly in week 5, prior to worm loss, when the adult worms would have been feeding on intestinal tissues and causing blood loss for a period of about 2 weeks. The most severe depression in the packed cell volume was also recorded in week 5, indicating that anaemia had been initiated in infected hamsters. Whilst, the regulation of parasite burdens in weeks 5-10 post-infection may have resulted from host immunity, the persistence of the residual worm burdens, the marked density-dependent anaemia and the subtle effect on host weight, all reflected well-documented aspects of chronic human necatoriasis.

[Uncinariasis in younger infants]. / Uncinariasis en el lactante menor.

In order to determine the main clinical and laboratory manifestations associated with the infestation by an uncinaria during the first year of life, 42 children were studied from a group hospitalized between the years 1980 and 1985 in whom the main diagnosis at their time of leave from the hospital was parasitosis. Over 31 patients (74%) showed some degree of malnutrition, and severe in 24 of them (57% of the total). Clinical manifestations were mainly related to anemia (paleness, lack of air) and active intestinal bleeding (enterorrhagia, melena). Among the laboratory findings, 80% of the patients were shown to have anemia and in 24% of them, their hemoglobin levels were less than 5 g/dL, in more than half the anemia was normocytic normochromic. Also included are particularities concerning uncinariasis in the breast-feeding infant and some differences with the infection in the adult.

Epidemiology and immunology of Necator americanus infection in a community in Papua New Guinea: humoral responses to excretory-secretory and cuticular collagen antigens.

Baseline data from an immuno-epidemiological study of hookworm infection in a rural village in Madang Province, Papua New Guinea are reported. Necator americanus was found to be the commonest helminth infection, with a prevalence of near 100% and intensity of 40 worms per host in adults. Enterobius vermicularis, Ascaris lumbricoides and Trichuris trichiura were also present, at prevalences of 53, 10 and 3% respectively; Ancylostoma duodenale was absent. The frequency distribution of N. americanus was highly over-dispersed, and was well described by a negative binomial distribution with aggregation parameter, k, of 0.370. Intensity of infection was significantly related to host age, but did not differ between the sexes. Haemoglobin levels and haematocrit values were indicative of anaemia in the community, but were unrelated to hookworm infection. Levels of antibodies (IgG, IgA and IgM combined) against adult Necator cuticular collagen and excretory-secretory (ES) products were determined. Serum concentrations of the two types of antibody were significantly correlated with each other. Significant positive correlations were found between anti-ES antibody levels and hookworm egg production, and between anti-collagen antibody levels and host age. It is suggested that the level of anti-collagen antibodies may reflect cumulative exposure to infection, whereas levels of anti-ES antibodies may be more dependent on current worm burden. No evidence was found to suggest that either antibody response is important in regulating parasite population growth. Similarly, the presence of a positive correlation between eosinophil concentration and infection intensity in adults indicates that eosinophilia reflects, rather than determines, the host's worm burden.

The clinical and immunologic responses of normal human volunteers to low dose hookworm (Necator americanus) infection.

Five normal human volunteers were exposed to approximately 50 infective larvae of Necator americanus and were observed for the development of clinical signs or symptoms and for changes in blood eosinophil levels, IgG antibody titers, total and parasite-specific IgE, and lymphocyte blastogenic responses for 6-10 weeks. Bronchoalveolar lavage was performed on four subjects prior to infection and at times when larval migration through the pulmonary tree was likely. Eggs were demonstrated in the stools of four volunteers who remained untreated for more than 6 weeks; one volunteer had to be treated at day 40 because of severe gastrointestinal symptoms. All others also complained of abdominal pain and flatulence between days 35-40. All volunteers developed marked blood eosinophilia which peaked between days 38-64 and ranged from 1,350-3,828 eosinophils/mm3. Small increases in total and parasite-specific IgE and IgG were noted in some volunteers. One volunteer showed a significant lymphocyte blastogenic response. With the exception of mucosal erythema, bronchoalveolar lavage results were unremarkable. Our data indicate that a single small inoculum of hookworm larvae is capable of producing significant transient gastrointestinal morbidity and marked blood eosinophilia but does not induce other prominent T cell- and B cell-dependent immune responses.

Antigen expression during development of the human hookworm, Necator americanus (Nematoda).

The accumulated and de novo synthesized antigens expressed by L3, L4 and adult Necator americanus, recognized by both the natural host, man, and the experimental host, the hamster, were identified by immunoblotting and immunoprecipitation analysis. Following infection of neonatal hamsters serum samples were taken on days 17, 35 and 117. Only serum taken 117 days after infection showed significant reactivity in immunoblotting experiments, recognizing adult epitopes of 30,000, 33,000, 48,000 and 69,000 mol. wt thereby suggesting that few accumulated antigens are shared between developmental stages. By contrast, immunoprecipitation analysis of metabolically labelled proteins suggested that L3 and in particular L4 larvae synthesize some antigens which comigrate with those synthesized and accumulated by adult worms. In addition, L4 larvae synthesize a 41,000 mol. wt excretory/secretory (ES) stage specific antigen. Parallel experiments using serum samples from infected humans, demonstrated that hamsters and man recognize many antigens of identical molecular weight. Notable in this respect are accumulated adult antigens of 30,000, 33,000, 48,000 and 69,000 and de novo synthesized antigens of 30,000, 33,000, 44,000, 46,000 and 69,000 mol. wt. Some individual human sera mainly recognized L3 antigens of 47,000-69,000 mol. wt in immunoblotting experiments whilst others simultaneously recognized adult epitopes. This differential recognition of developmental stages by individual human sera suggests that genetic or epidemiological factors are operative and warrants further study. Overall, these studies confirm the pronounced immunogenicity of Necator americanus in both man and an animal model and pave the way for analysis of the relevance of these antigens to field situations.