IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS: USE OF SIX DIFFERENT ANTIGENIC FRACTIONS FROM Strongyloides venezuelensis PARASITIC FEMALES

Imunodiagnóstico da estrongiloidíase humana: o uso de seis diferentes frações antigênicas de fêmeas parasitas de Strongyloides venezuelensis

SUMMARY

The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis.

Strongyloidiasis; Serodiagnosis; Heterologous antigens; Strongyloides venezuelensis; Parasitic females

RESUMO

O objetivo deste estudo foi avaliar seis frações antigênicas diferentes de fêmeas parasitas de Strongyloides venezuelensis para o diagnóstico sorológico da estrongiloidíase humana. As frações solúveis e de membrana de fêmeas parasitas de S. venezuelensis foram preparadas em solução salina tamponada (SSF e SMF, respectivamente), Tris-HCl (TSF e TMF, respectivamente) e tampão alcalino (ASF e AMF, respectivamente). As amostras de soro obtidas de pacientes com estrongiloidíase, com outras parasitoses e indivíduos saudáveis, foram analisadas pelo ensaio imunoenzimático (ELISA). As frações solúveis SSF, TSF e ASF apresentaram sensibilidade de 85,0%, 75,0%, e 80,0% e especificidade de 93,1%, 93,1% e 87,5%, respectivamente. As frações de membrana, SMF, TMF e AMF demonstraram sensibilidade de 80,0%, 75,0%, e 85,0%, e especificidade de 95,8%, 90,3% e 91,7%, respectivamente. Em conclusão os presentes resultados sugerem que as frações obtidas a partir de fêmeas parasitas, especialmente o SSF e SMF, podem ser utilizadas como fonte alternativa de antígenos no imunodiagnóstico da estrongiloidiase humana.

Human strongyloidiasis is often caused by the nematode Strongyloides stercoralis. This parasite is found worldwide but is mainly located in tropical and subtropical regions¹⁷. In humans, S. stercoralis can cause an asymptomatic chronic gastrointestinal infection. However, in immunocompromised individuals this parasite can cause a fatal hyperinfection syndrome or disseminated strongyloidiasis¹⁴.

A definitive diagnosis of strongyloidiasis is made through the detection of larvae in feces; however, parasitological methods have low sensitivity because of the irregular and intermittent release of larvae into feces¹⁰. Immunological methods have been widely used in the diagnosis of human strongyloidiasis because of their high sensitivity³. A major limitation of these methods is that it can be difficult to obtain sufficient quantities of S. stercoralis larvae for fractionation and analysis. If alternative antigens were available, including those from heterologous species such as Strongyloides venezuelensis, more satisfactory results for the immunodiagnosis of human strongyloidiasis might be possible^2,4,7.

Antigenic preparations from S. venezuelensis filariform larvae are most often used in the standardization and application of immunological techniques^4,7. Considering the life cycle of Strongyloides spp., it is necessary to study other antigenic sources, such as female parasites, as they are found in the intestinal mucosa of the host and might elicit an immune response^5,6. The aim of our study was to evaluate different antigenic fractions from S. venezuelensis parasitic females for their application in the immunodiagnosis of human strongyloidiasis.

Antigenic fractions from S. venezuelensis parasitic females were evaluated by samples of immunocompetent individuals. Serum samples were obtained from individuals at the Hospital das Clínicas of the Faculdade de Medicina at the Universidade de São Paulo (HCFMUSP). Of these, 20 patients were harboring S. stercoralis larvae. Thirty-two patients were infected with other parasites: hookworm (n = 4); Ascaris lumbricoides (n = 2); Blastocystis spp. (n = 3); Enterobius vermicularis (n = 1); Endolimax nana (n = 3); Giardia intestinalis (n = 3); Hymenolepis nana (n = 1); Schistosoma mansoni (n = 9); hookworm and H. nana (n = 1); S. mansoni, A. lumbricoides, Entamoeba coli, Blastocystis spp., and E. nana (n = 1); G. intestinalis and E. nana (n = 1); A. lumbricoides and Blastocystis spp (n = 1); E. nana, S. mansoni, and Blastocystis spp (n = 1); hookworm, Escherichia coli, Entamoeba dispar/histolytica, and Schistosoma mansoni (n = 1). The remaining 40 samples were from seemingly healthy volunteers based on their clinical observation, without evidence of contact with S. stercoralis or previous history of strongyloidiasis, and negative in all parasitological diagnostic methods. For each group, one stool sample was analyzed by the techniques described by LUTZ¹²and RUGAI et al.¹⁹and an agar plate culture method¹⁸. The study received approval (protocol 266.046) from the Research Ethics Committee of the Universidade de São Paulo (Sao Paulo, Brazil).

S. venezuelensis parasitic females were obtained from Wistar rats (Rattus norvegicus) that were experimentally infected using protocol CPE-IMT 2011/126. To recover parasitic females, rats were euthanized on day 14 post-infection. The small intestines were then removed as described by NAKAI & AMARANTE¹⁵. Briefly, the intestine was longitudinally sectioned and placed on a sedimentation chalice in contact with saline solution for four h at 37 ºC; the females were then counted and stored at -20 ºC until use.

For antigenic extraction, approximately 5,000 parasitic females f were incubated with three different extraction buffers: 10 mM phosphate-buffered saline (PBS, pH 7.2), 25 mM Tris-HCl (pH 7.5), or 0.15 M NaOH. All the extraction buffers were supplemented with protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Each extraction buffer was used to generate soluble (S) and membrane (M) antigenic fractions. For the soluble fractions, parasitic females were added to PBS (S), Tris-HCl (T), or NaOH a and disrupted in an ice bath using a tissue homogenizer. The suspensions were centrifuged at 12,400 × g for 30 min at 4 ºC and supernatants collected. The soluble fractions in PBS, Tris-HCl, and NaOH were designated SSF, TSF, and ASF, respectively. For membrane fractions, SSF and ASF pellets were resuspended in 1% sodium dodecyl sulfate (SDS), boiled for five min at 100 ºC, and centrifuged (12,400 × g, 30 min, 4 ºC); subsequently, the supernatants were collected. These membrane fractions were designated SMF and AMF. The TSF pellets were resuspended in 7 M urea, 2 M thiourea, and 2% CHAPS ([(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate); then, they were disrupted in an ice bath using a tissue homogenizer. The samples were centrifuged (12,400 × g, 30 min, 4 ºC) and supernatants were collected; these fractions were designated TMF. All fractions were analyzed for protein content according to the method of LOWRY et al.¹¹, subdivided into aliquots, and stored at -20 ºC until use. Fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by LAEMMLI⁹under reducing conditions. Each of the antigenic fractions (SSF, TSF, ASF, SMF, TMF, and AMF) was electrophoresed using a 12% acrylamide separation gel. The gels were then analyzed using PlusOne Coomassie table PhastGel Blue R-350 (GE HealthCare Bio-Sciences AB, Piscataway, NJ, USA).

Enzyme-linked immunosorbent assays (ELISAs) were performed according to CORRAL et al.², with some modifications. Briefly, the wells of polystyrene microplates (Corning-Costar, New York, NY, USA) were coated with SSF, TSF, ASF, SMF, TMF, or AMF (5 µg/mL) in carbonate-bicarbonate buffer (0.06 mM, pH 9.6) and incubated overnight at 4 °C. After incubation, plates were washed (3 × 5 min) with PBS containing 0.05% (v/v) Tween 20 (PBS-T), and blocked with PBS-T supplemented with 3% (w/v) nonfat milk (PBS-TM) for 45 min at 37 °C. Serum samples were diluted 1:400 and 1:200 in PBS-TM for the soluble and membrane fractions, respectively. Fc-specific goat anti-human IgG conjugated to peroxidase (Sigma-Aldrich, St. Louis, MO, USA) was diluted 1:30,000 in PBS-TM. Color was developed by adding enzyme substrate (4 mM orthophenylenediamine, 0.03% H₂O₂, 0.1 M citrate phosphate buffer, pH 5.5) and incubated at room temperature for 15 min in the dark. The reactions were stopped by adding 2 N H₂SO₄. The optical density (OD) was determined at 492 nm using an ELISA reader (Thermo Fischer Scientific, Waltham, MA, USA).

Statistical analyses were performed using GraphPad Prism version 5.0 (Graph Pad Software Inc. San Diego, USA). The ELISA index (EI) was calculated according to the following formula: EI = OD/cutoff. An EI value greater than one for a sample was considered positive. Tests were evaluated by calculating sensitivity, specificity, accuracy, kappa index (k), and likelihood ratio (LR). Any p-value below 0.05 was considered statistically significant.

To our knowledge, the present study is the first to use different antigenic fractions from S. venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. We used six antigenic fractions from parasitic females; three fractions were soluble and three were membrane-derived. The concentration of proteins from SSF, TSF, and ASF were 0.174, 0.174 and 0.708 mg/mL, respectively. Membrane fractions showed protein concentration of 0.631 (SMF), 0.300 (TMF), and 0.261 mg/mL (AMF). Although differences were observed in protein concentration, there was no change in the pattern of electrophoretic migration for the six fractions investigated, presenting bands of 25 and 150 kDa, and almost all samples presenting a band of approximately 23 kDa (Fig. 1).

Fig. 1
- Electrophoretic profiles of soluble (SSF, TSF, and ASF) and membrane (SMF, TMF, and AMF) fractions from Strongyloides venezuelensis parasitic females, obtained using 12% SDS-PAGE and Coomassie blue staining. MW, molecular weight standard in kilodaltons (kDa).

The diagnostic parameters and efficiency of ELISA for the six antigenic fractions are shown inFig. 2. Among the soluble fractions, SSF had the highest value of sensitivity and specificity (85% and 93.1%, respectively). The value of sensitivity and specificity for the membrane fraction SMF were also high, at 80% and 95.8%, respectively. The k values ranged from 0.619 to 0.753 for the soluble fractions, and 0.630 to 0.772 for the membrane fractions. Of the six fractions tested, those obtained using PBS provided optimal diagnostic parameters. An LR greater than 10 indicates high performance with respect to the detection of specific IgG antibodies in patients with strongyloidiasis⁷. In this study, the antigenic fractions obtained with PBS had high LRs (12.2-19.2), indicating that these preparations had high ability to discriminate between strongyloidiasis patients and controls.

Fig. 2
- ROC curve indicating the optimum point of reaction (cut-off), sensitivity (Se), specificity (Sp), diagnostic efficiency (DE), kappa index (k), likelihood ratio (LR), and area under curve (AUC) for IgG detection in serum samples using soluble (SSF, TSF, and ASF) and membrane (SMF, TMF, and AMF) fractions from Strongyloides venezuelensis parasitic females.

Differences among values for diagnosis were observed; it may be due to changes in the action of buffers used for the production of antigenic fractions, which can facilitate the antigen-antibody interaction. In general, there are major concerns regarding the antigenic preparations used in serological tests, especially regarding the evaluation of buffers used for the extraction of proteins, which is fundamental to obtaining antigens⁴. Cross-reactivity was observed for the six antigenic fractions with the following parasites: hookworms (1/4 in SSF and TMF), Schistosoma mansoni (2/9 in TSF and TMF), A. lumbricoides (1/2 in ASF), G. intestinalis (1/3 in AMF), Blastocystis spp (1/3 in ASF), and poly-infection with hookworms and H. nana (1/1 in SSF, ASF, and AMF).

The various forms of the S. venezuelensis filarial larvae during the parasite life cycle can be easily obtained in murine models¹³. The use of parasitic females has not been widely explored, possibly because of the difficulties involved in obtaining sufficient numbers for antigen production. However, specific proteins are present in parasitic females, and these proteins present new possibilities for antigen production¹⁶. Recently, GONÇALVES et al.^5,6showed that antigens from S. venezuelensis parasitic females could be used in the immunodiagnosis of human strongyloidiasis.

This study is the first to report the use of membrane antigens from S. venezuelensis parasites females. Our results showed high specificity of the membrane antigens compared with those in soluble preparations. The use of membrane fractions from S. venezuelensis infective larvae has shown promising results for the immunodiagnosis of human strongyloidiasis².

It is difficult to obtain specific antigenic fractions for the immune diagnosis of strongyloidiasis, and a reliable diagnostic test is urgently required. To produce antigens for the diagnosis of human strongyloidiasis, the use of heterologous species maintained in experimental models has been proposed. Therefore, female parasites represent a viable antigenic source; however, its use as a source of antigens remains uncommon. GONÇALVES et al.^5,6, who used an alkaline extract similar to ASF from S. venezuelensis parasitic females, reported similar results with respect to sensitivity and specificity as seen in the present study. Other antigenic fractions from the female parasite were not investigated by these authors. In our current study, we have shown that different antigenic extracts from female parasites can be used in the diagnosis of human strongyloidiasis.

The increasing use of recombinant antigens for the serological diagnosis of human strongyloidiasis has shown some promise^1,8. However, there are some limitations regarding resources in areas that are endemic for this helminthiasis. This lack of resources makes the application of these methodologies problematic in most regions. If a laboratory has adequate infrastructure and reagents for the production of recombinant antigens, then this technique could be viable. Nevertheless, in countries where the conditions are limited, the antigenic extracts preparations should be based on protocol, lower cost, and with good sensitivity and specificity results. Thus, the use of S. venezuelensis parasitic females, especially soluble and membrane fractions extracted with PBS, could provide an alternative source of antigens for the immunodiagnosis of human strongyloidiasis.

ACKNOWLEDGMENTS

This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2013/04236-9), Brazil.

REFERENCES

• ¹ Bisoffi Z, Buonfrate D, Sequi M, Mejia R, Cimino RO, Krolewiecki AJ, et al. Diagnostic accuracy of five serologic tests for Strongyloides stercoralis infection. PLoS Negl Trop Dis. 2014;8:e2640.
• ² Corral MA, Paula FM, Gottardi M, Meisel DMCL, Chieffi PP, Gryschek RC. Membrane fractions from Strongyloides venezuelensis for using in the immunodiagnosis of human strongyloidiasis. Rev Inst Med Trop Sao Paulo. 2015;57:77-80.
• ³ Costa-Cruz JM, Bullamah CB, Gonçalves-Pires MR, Campos DM, Vieira MA. Cryo-microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis. Rev Inst Med Trop Sao Paulo. 1997;39:313-7.
• ⁴ Feliciano ND, Gonzaga HT, Fatima Goncalves-Pires MR, Ribeiro Gonçalves AL, Rodrigues RM, Ueta MT, et al. Hydrophobic fractions from Strongyloides venezuelensis for use in the human immunodiagnosis of strongyloidiasis. Diagn Microbiol Infect Dis. 2010;67:153-61.
• ⁵ Gonçalves AL, Nunes DS, Gonçalves-Pires MR, Ueta MT, Costa-Cruz JM. Use of larval, parasitic female and egg antigens from Strongyloides venezuelensis to detect parasite-specific IgG and immune complexes in immunodiagnosis of human strongyloidiasis. Parasitology. 2012;139:956-61.
• ⁶ Gonçalves AL, Silva CV, Ueta MT, Costa-Cruz JM. Antigen, antibody and immune complex detection in serum samples from rats experimentally infected with Strongyloides venezuelensis. Exp Parasitol. 2012;130:205-8.
• ⁷ Gonzaga HT, Vila-Verde C, Nunes DS, Ribeiro VS, Cunha-Júnior JP, Costa-Cruz JM. Ion-exchange protocol to obtain antigenic fractions with potential for serodiagnosis of strongyloidiasis. Parasitology. 2013;140:69-75.
• ⁸ Krolewiecki AJ, Ramanathan R, Fink V, McAuliffe I, Cajal SP, Won K, et al. Improved diagnosis of Strongyloides stercoralis using recombinant antigen-based serologies in a community-wide study in northern Argentina. Clin Vaccine Immunol. 2010;17:1624-30.
• ⁹ Laemmli UK. Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature. 1970;227:680-5.
• ¹⁰ Levenhagen MA, Costa-Cruz JM. Update on immunologic and molecular diagnosis of human strongyloidiasis. Acta Trop. 2014;135:33-43.
• ¹¹ Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-75.
• ¹² Lutz A. O Schistosoma mansoni e a schistosomose segundo observações feitas no Brasil. Mem Inst Oswaldo Cruz. 1919;11:121-5.
• ¹³ Machado ER, Ueta MT, Gonçalves-Pires MR, De Oliveira JB, Faccioli LH, Costa-Cruz JM. Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by enzyme linked immunosorbent assay. Mem Inst Oswaldo Cruz. 2003;98:849-51.
• ¹⁴ Marcos LA, Terashima A, Canales M, Gotuzzo E. Update on strongyloidiasis in the immunocompromised host. Curr Infect Dis Rep. 2011;13:35-46.
• ¹⁵ Nakai ES, Amarante AFT. Infecção experimental de camundongos (Mus musculus) e ratos (Rattus norvegicus) com Strongyloides venezuelensis. Rev Bras Parasitol Vet. 2001;10:1-6.
• ¹⁶ Northern C, Grove DI. Strongyloides-stercoralis: antigenic analysis of infective larvae and adult worms. Int J Parasitol. 1990;20:381-7.
• ¹⁷ Paula FM, Costa-Cruz JM. Epidemiological aspects of strongyloidiasis in Brazil. Parasitology. 2011;138:1331-40.
• ¹⁸ Paula FM, Gottardi M, Corral MA, Chieffi PP, Gryschek RC. Is the agar plate culture a good tool for the diagnosis of Strongyloides stercoralis in candidates for transplantation? Rev Inst Med Trop Sao Paulo. 2013;55:291.
• ¹⁹ Rugai E, Mattos T, Brisola AP. Nova técnica para isolar larvas de nematóides das fezes: modificações do método de Baermann. Rev Inst Adolfo Lutz. 1954;14:5-8.

Publication Dates

• Publication in this collection
  Sep-Oct 2015

History

• Received
  16 Mar 2015
• Accepted
  19 June 2015