Immune complex detection in saliva samples: an innovative proposal for the diagnosis of human strongyloidiasis.

Human strongyloidiasis is caused by helminth Strongyloides stercoralis. It has a worldwide distribution, often neglected and cause of severe morbidity. The parasitological diagnosis is hindered by the low and irregular amount of larvae in feces. The goal of the present study was to detect IgG and IgG immune complex using conventional serum samples and saliva as alternative samples. We collected samples from 60 individuals, namely: group I composed of 30 healthy individuals; and group II composed of 30 individuals eliminating S. stercoralis larvae in feces. We calculated the area under the curve, general index of diagnostic accuracy, Kappa index and determined the correlations between different diagnostic tests. The detection of IgG levels was performed by an immunoenzymatic assay with alkaline extract of S. venezuelensis larvae as antigen. Positivity of anti-S. stercoralis IgG in serum samples from group I was 3·3%, and from group II 93·3%. The detection of immune complex indicated that group I exhibited 3·3% and group II 56·7%. In the saliva samples, IgG detection was 26·7% for group I and 43·3% for group II. Immune complex was detected in 20% of group I, and 30% of group II. IgG immune complex in conventional serum samples and saliva as alternative samples can be considered biomarkers for the diagnosis of active strongyloidiasis.

Specific IgG and immune complex responses to parthenogenetic females and eggs of nematode Strongyloides venezuelensis for the diagnosis of immunosuppression in infected rats.

In the present study, antigens from parthenogenetic females and eggs of Strongyloides venezuelensis, or anti-parthenogenetic-female and anti-egg antigens were used to detect specific IgG and immune complex responses, respectively. Serum samples from experimentally infected immunocompetent and immunosuppressed rats were analysed on days 5, 8, 13 and 21 post-infection (dpi). An enzyme-linked immunosorbent assay (ELISA) was performed using alkaline parasite extract for specific IgG detection, and anti-parthenogenetic-female or anti-egg antigens for immune complex detection. The data were analysed using analysis of variance (ANOVA), followed by a Bonferroni test. When parthenogenetic female or egg extracts were used as antigens, specific IgGs were not detected in either immunocompetent or immunosuppressed rats. When anti-parthenogenetic-female or anti-S. venezuelensis-eggs were used, immune complexes were detected for the duration of the infection in immunosuppressed animals and were only detected between 5 and 13 dpi in immunocompetent animals. The duration of infection was not significantly different between the immunocompetent and immunosuppressed groups when anti-parthenogenetic-female or anti-S. venezuelensis-eggs were used. Parthenogenetic female extracts yielded significant differences between antibody and immune complex responses in immunocompetent rats from 5 to 13 dpi, but only on day 5 dpi in immunosuppressed rats. Exposure to S. venezuelensis egg extract yielded significant differences in both antibody and immune complex detection between immunocompetent and immunosuppressed rats for the duration of the infection. In conclusion, ELISA using alternative antigens may be a successful strategy for identifying immune complexes in serum samples and diagnosing active strongyloidiasis, particularly under conditions of immunosuppression.

F-actin accumulates in the vulva of female Strongyloides venezuelensis.

Little is known about the actin cytoskeleton architecture in female Strongyloides venezuelensis and thus to investigate the distribution and concentration of actin, female worms were labelled with phalloidin-rhodamine and visualized under confocal microscopy. Our results demonstrate that filamentous actin accumulates in the vulva and the concentration of F-actin at this site suggests its important role, especially during oviposition, in the life cycle of S. venezuelensis.

Use of larval, parasitic female and egg antigens from Strongyloides venezuelensis to detect parasite-specific IgG and immune complexes in immunodiagnosis of human strongyloidiasis.

The aim of this study was to use larval, parasitic female and egg antigens from Strongyloides venezuelensis to detect parasite-specific IgG and immune complexes in human serum samples by enzyme-linked immunosorbent assay (ELISA). In total, 95 serum samples were analysed, consisting of 30 patients harbouring S. stercoralis larvae, 30 healthy subjects and 35 patients with other parasites. Sensitivity, specificity and diagnostic efficiency were calculated. A significant statistical difference was found in the detection of immune complexes and antibodies in patients harbouring S. stercoralis larvae from larval and eggs antigens, with higher positivity using larval antigen. The larval antigen showed the highest values for sensitivity, specificity and diagnostic efficiency in ELISA from detection of immune complexes. For the first time we used IgG anti-larvae, IgG anti-parasitic females or IgG anti-eggs for immune complex detection. We concluded that the association of antibody and immune complex detection could be used in the diagnosis of human strongyloidiasis.

Immunolocalization and pathological alterations following Strongyloides venezuelensis infection in the lungs and the intestine of MHC class I or II deficient mice.

The present study, investigated the mechanisms involved in the immune responses of Major Histocompatibility Complex class I or class II knockout mice, following Strongyloides venezuelensis infection. Wild-type C57BL/6 (WT), MHC II(-/-) and MHC I(-/-) mice were individually inoculated with 3000 larvae (L3) of S. venezuelensis and sacrificed on days 1, 3, 5, 8, 13 and 21 post-infection (p.i.). Samples of blood, lungs and small intestines were collected. The tissue samples were stained with hematoxylin-eosin for the pathological analysis. The presence of the parasite was demonstrated by immunoperoxidase analysis. MHC II(-/-) mice presented a significantly higher number of adult worms recovered from the small intestine on day 5p.i. and presented elevated numbers of eggs in the feces. The infection by S. venezuelensis was completely eliminated 13 days after infection in WT as well as in MHC I(-/-) mice. In MHC II(-/-) mice, eggs and adult worms were still found on day 21 p.i., however, there was a significant reduction in their numbers. In the lung, the parasite was observed in MHC I(-/-) on day 1 p.i. and in MHC II(-/-) mice on days 1 and 5 p.i. In the small intestine of WT mice, a larger number of parasites were observed on day 8 p.i. and their absence was observed after day 13 p.i. Through immunohistochemistry analysis, the parasite was detected in the duodenum of WT on days 5 and 8 p.i., and in knockout mice on days 5, 8 and 13 p.i.; as well as in posterior portions of the small intestine in MHC I(-/-) and MHC II(-/-) on day 13 p.i., a finding which was not observed in WT mice. We concluded that immunohistochemistry analysis contributed to a more adequate understanding of the parasite localization in immunodeficient hosts and that the findings aid in the interpretation of immunopathogenesis in Strongyloides infection.

Evaluation of strongyloidiasis in kennel dogs and keepers by parasitological and serological assays.

Strongyloides stercoralis is an intestinal nematode with worldwide distribution, particularly in tropical and subtropical regions. Due to the low sensitivity of traditional parasitological methods, the detection of serum specific antibodies may serve as an alternative test for the diagnosis. The aims of the present study were to verify the occurrence of S. stercoralis and the presence of specific IgG antibodies to the parasite in kennel dogs and keepers, using parasitological and serological assays. A total of 181 dogs were examined from 7 breeding kennels in the city of Uberlândia, southeastern region of Brazil and distributed as follows: kennel A (n=41), kennel B (n=16), kennel C (n=11), kennel D (n=63), kennel E (n=11), kennel F (n=18) and kennel G (n=21). Fecal and serum samples from 11 keepers responsible for kennel cleaning and dog control were also collected in five of the seven kennels (two from kennel A, one from kennel B, four from kennel D, two from kennel E and two from kennel G). Overall, enteroparasites were detected by parasitological assays in 66, 36.5% (95% CI: 2.5-43.4%) of the 181 dogs tested. Only one (0.6%) dog was copropositive for S. stercoralis. Among the keepers only one fecal sample, 9.1% (95% CI: 8.6-9.4%) was positive for hookworm by the Lutz method. Serological assays showed that 44 (24.3%) of the 181 dogs were seropositive for S. stercoralis in at least one of the tests in the following kennels: 21 (11.6%) in kennel A; 1 (0.6%) in kennel B; 5 (2.7%) in kennel C; 6 (3.3%) in kennel D; 1 (0.6%) in kennel E; 9 (4.9%) in kennel F and 1 (0.6%) in kennel G. Among the keepers no S. stercoralis seropositive samples were identified using IFAT but 2 (18.2%) of the keepers from kennel D and 1 (9.1%) from kennel G were seropositive by ELISA. The present study demonstrated that the occurrence of S. stercoralis infection in kennel dogs and keepers is low in the city of Uberlândia and that serological assays can contribute to the diagnosis of canine as well as human strongyloidiasis.

Parasitological and serological diagnosis of Strongyloides stercoralis in domesticated dogs from southeastern Brazil.

Canine strongyloidiasis is a parasitic infection caused by the nematode Strongyloides stercoralis and presents a great zoonotic potential. Its confirmation, using coproparasitological methods, is difficult. The detection of serum specific antibodies, however, may facilitate the diagnosis. The aims of this study were to determine the presence of S. stercoralis through the use of parasitological methods and to detect specific antibodies to the parasite in serum samples from domestic dogs by using the indirect fluorescent antibody test (IFAT) on slides and the enzyme-linked immunosorbent assay (ELISA). A total of 215 dogs of various breeds, from the cities of Uberlândia, Araxá and Campo Belo in the State of Minas Gerais, were examined and distributed according to age into the following groups: (I) 19 males and 20 females of 1-2 months old; (II) 11 males and 20 females of 2-month- to 1-year-old and (III) 41 males and 104 females, from 1 to 7 years old. Coproparasitological results showed that 63/215 (29.3%) of the dogs presented some kind of parasite, with two (0.9%) dogs (one from Araxá and the other from Uberlândia) passing S. stercoralis larvae in the feces. Serological results revealed antibodies to S. stercoralis in 45/215 (20.9%) of the dogs, with seropositivity rates of 0% (0/39) in Group I, 22.6% (7/31) in Group II, and 26.2% (38/145) in Group III. No serological cross-reactivity between S. stercoralis and hookworms or Ascaridae was found. Hookworm infections were seen in 31 dogs, but only one of these dogs (infected with both hookworm and Cystoisospora spp.) was S. stercoralis seropositive by IFAT. The present study demonstrated, for the first time, natural S. stercoralis infections in dogs diagnosed by coproparasitological and serological methods. It was concluded that the detection of specific antibodies to S. stercoralis by IFAT and ELISA may contribute to the diagnosis of canine strongyloidiasis.