Ascaris lumbricoides and Necator americanus are soil-transmitted parasites with global geographic distribution, and they represent some of the most common and neglected infections in the world. Periodic treatment with mass drug administration (MDA) in endemic areas is the recommended action put forth by the World Health Organization. However, MDA can cause the selection of subpopulations that possess the genetic ability to overcome the mechanism of drug action. In fact, beta-tubulin gene mutations (codons 167, 198 and 200) are correlated with benzimidazole resistance in nematodes of veterinary importance. It is possible that these SNPs also have strong correlation with treatment resistance in the human geohelminths A. lumbricoides, Trichuris trichiura and hookworms. Here, we aimed to investigate the presence of some of these canonical molecular markers associated with parasite resistance to benzimidazole in N. americanus and A. lumbricoides collected from six Brazilian states. Nested-PCR and PCR-RFLP were used to detect mutations at codons 167 and 198 in 601 individual eggs of A. lumbricoides collected from 62 human stool samples; however, no mutations were found. Codons 198 and 200 were tested in 552 N. americanus eggs collected from 48 patients using the same methodology, which presented a relative frequency of 1.4% and 1.1%, respectively. The presence of these SNPs in N. americanus eggs is an important finding, indicating that with high benzimidazole drug pressure there is potential for benzimidazole resistance to be selected in this hookworm. However, at these low frequencies it does not indicate that there is at present any benzimidazole resistance problem. This is the first systematic study performed in South America, and the study yielded a landscape of the genetic variants in the beta-tubulin gene and anthelmintic resistance to soil-transmitted parasites detected by a simple, rapid and affordable genotyping assay of individual eggs. The soil-transmitted helminths Ascaris lumbricoides and the hookworms (Necator americanus and Ancylostoma duodenale) are the most prevalent intestinal helminths of humans. The standard approach for geohelminth control is large-scale preventive chemotherapy predominantly using benzimidazoles through mass drug administration. Nevertheless, this inexpensive and highly effective strategy can potentially select subpopulations of parasites that become resistant to treatment. In veterinary parasites, intensive reliance on the same anthelmintics has led to the emergence of benzimidazole resistance, but this is not yet proven to helminth parasites of humans. Here we describe a PCR-RFLP approach for single nucleotide polymorphisms (SNPs) detection at codons 167 and 198 in the beta-tubulin gene of A. lumbricoides, and SNPs at codons 198 and 200 in the beta-tubulin gene of N. americanus of Brazilian populations. A. lumbricoides samples showed no mutations in any codons, and mutation at codons 198 and 200 were observed at low frequencies in N. americanus. The observed data posit important public health issues: the mutations are a substrate for selective pressure and a long-term mass drug administration could allow to the selection of parasites resistant to benzimidazole in Brazil.

Background: Blomia tropicalisis a dust mite and an important source of allergens in tropical regions. Up to now, the assays to diagnose atopy to this mite use whole body extract as antigens. However, anti-B. tropicalisIgE antibodies cross-react withAscaris lumbricoidesantigens, hindering the diagnosis of allergy to this mite. In this study,B. tropicalisrecombinant allergens were evaluated with the purpose of developing an immunodiagnostic assay for allergy to this mite with greater specificity than those commercially available.Methods: TwoB. tropicalisallergens (Blo t 5 and Blo t 21) were cloned into a plasmidial expression vector, expressed inEscherichia coliand purified by affinity chromatography. Sixty-three sera containing anti-B. tropicalisextract (BtE) IgE antibodies were used to investigate IgE reactivity to the recombinant Blot 5 and 21 allergens. Inhibition assays with 20 sera pre-adsorbed withA. lumbricoidesextract were performed using rBlo t 5, rBlo t 21, and BtE as antigens. All the assays were carried using indirect ELISA.Results: Eighty-two point nine percent and 80.0% of the sera with anti-BtE antibodies from 35 children reacted with rBlo t 5 and rBlo t 21, respectively, whereas 92.8% and 89.3% of the 28 sera with anti-BtE antibodies from adult asthma patients reacted with the same allergens, and 96.4% of these sera reacted with a mixture of rBlo t 5 and rBlo t 21. In an inhibition ELISA, the absorption of sera byA. lumbricoidesextract affected less the reaction with rBlo t 5 and rBlo t 21 than with BtE.Conclusions: The rBlo t 5 and rBlo t 21 allergens contain important epitopes recognized by IgE antibodies of individuals allergic toB. tropicalisantigens. Moreover, the assays using the recombinant allergens had lower IgE cross-reactivity withA. lumbricoidesantigens, a fact which would confers higher specificity to serodiagnostic assays than the crude mite extract. However, additional recombinant allergens should be evaluated in order to reach the same sensitivity of the commercially available assays based on mite extract.

Current schistosomiasis control strategies are mainly based on chemotherapy, but the development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission. When it comes to vaccine candidates, several genes encoding Schistosoma mansoni proteins expressed at the mammalian host–parasite interface have been tested. Among the most promising molecules are the proteins present on the tegument and digestive tract of the parasite. In this study, we evaluate the potential of SmKI-1, the first Kunitz-type protease inhibitor functionally characterized in S. mansoni, as a vaccine candidate. Bioinformatic analysis points to the C-terminal fragment as the main region of the molecule responsible for the development of a potential protective immune response induced by SmKI-1. Therefore, for the vaccine formulations, we produced the recombinant (r) SmKI-1 and two different fragments, its Kunitz (KI) domain and its C-terminal tail. First, we demonstrate that mice immunized with recombinant SmKI-1 (rSmKI-1) or its fragments, formulated with Freund’s adjuvant, induced the production of IgG-specific antibodies. Further, all vaccine formulations tested here also induced a Th1-type of immune response, as suggested by the production of IFN-γ and TNF-α by protein-stimulated cultured splenocytes. However, the protective effect conferred by vaccination was only observed in groups which received rSmKI-1 or C-terminal domain vaccines. Mice administered with rSmKI-1 demonstrated reduction of 47% in worm burden, 36% in egg number in mouse livers, and 33% in area of liver granulomas. Additionally, mice injected with C-terminal domain showed reduction of 28% in worm burden, 38% in egg number in liver, and 25% in area of liver granulomas. In contrast, KI domain immunization was unable to reduce worm burden and ameliorate liver pathology after challenge infection. Taken together, our data demonstrated that SmKI-1 is a potential candidate for use in a vaccine to control schistosomiasis, and its C-terminal tail seems to be the main region of the molecule responsible for protection conferred by this antigen.