Cryo-Microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis

Abstracts

Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p <FONT FACE="Symbol">£</font> 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended

Strongyloides stercoralis; Strongyloides ratti; Indirect immunofluorescence reaction; Diagnosis; Human strongyloidiasis

Comparou-se o emprego de cortes em congelação de larvas de S. stercoralis e de S. ratti como fonte de antígenos na reação de imunofluorescência indireta (RIF) para o diagnóstico da estrongiloidíase humana. Os antígenos foram obtidos de coproculturas de fezes humanas e de ratos, respectivamente. Soros de 123 indivíduos foram analisados sendo 54 de pacientes com estrongiloidíase e 69 controles. Empregou-se conjugado anti IgG humano marcado com fluoresceína nos títulos ideais de 10 para S. stercoralis e 100 para S. ratti. A sensibilidade da RIF foi de 94,4% e 92,5% e a especificidade de 94,2% e 97,1% respectivamente para os antígenos de S. stercoralis e S. ratti. Para os 2 antígenos os títulos dos soros variaram de 20 a 2560. Houve correlação linear significativa (r = 0,85 p <FONT FACE="Symbol">£</font> 0,001) entre os títulos de anticorpos para os 2 antígenos. Concluiu-se que os 2 antígenos podem ser empregados na RIF. Diante da disponibilidade de obtenção de grande quantidade de antígeno em laboratório, economia do conjugado, por não haver diferença significativa entre sensibilidades e especificidades e haver correlação linear entre os títulos de anticorpos, a utilização do S. ratti é recomendada como antígeno no diagnóstico da estrongiloidíase humana

CRYO-MICROTOME SECTIONS OF COPROCULTURE LARVAE OF Strongyloides stercoralis AND Strongyloides ratti AS ANTIGEN SOURCES FOR THE IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS

Julia Maria COSTA-CRUZ(¹), Carina Barbosa BULLAMAH(¹), Maria do Rosário F. GONÇALVES-PIRES(¹), Dulcinéa Maria B. CAMPOS(²) &

Miguel Alípio VIEIRA(²)

SUMMARY

Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p £ 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended.

KEYWORDS:Strongyloides stercoralis; Strongyloides ratti; Indirect immunofluorescence reaction; Diagnosis; Human strongyloidiasis.

INTRODUCTION

Human strongyloidiasis is a parasitic infection by the nematode Strongyloides stercoralis (Bavay, 1876) which commonly runs a symptomless course but may evolve towards an insidious and serious opportunistic invasion of tissues and organs which is often difficult to detect, so that the condition is frequently only diagnosed at autopsy^{6, 8, 9, 11, 18, 27}.

Infection of several hosts including man by Strongyloides species is characterized by poor reliability of the negative results even for the so called "specific" fecal techniques available for diagnosis^{13, 16, 26, 31}. In fact, only positive results are accepted as definitive, that is to say, when Strongyloides larvae are found in feces^{1, 3, 21, 23, 29, 30, 32, 34}, duodenal fluid^{5, 20} or occasionally in other fluids or tissues from infected hosts^{2, 25, 35}.

The development of immunologic tests for the diagnosis of strongyloidiasis may help to clarify clinical cases and to provide epidemiological information^{7, 31}. Important among them is the indirect immunofluorescence reaction (IIF) with some options not only with respect to standardization of the antigens to be employed but also with respect to the application of the technique, using slides^{4, 10} or tubes^{12, 15, 17}.

Technologic advances in the field of immunoparasitology and the modern application of highly specific and accurate diagnostic tests have resulted in the demand for the production of specific parasite antigens, not always available on the immunobiology market. The purpose of the present study was to compare antigens from filarioid larvae of S. stercoralis and S. ratti in larval sections obtained with a cryo-microtome and submitted to the indirect immunofluorescence reaction.

MATERIALS AND METHODS

Parasites: S. stercoralis was obtained from positive stools of patients from "Hospital das Clínicas, Universidade Federal de Uberlândia", State of Minas Gerais, Brazil. The fecal samples were obtained prior to patient treatment and were processed for culture free from any preservative drug. The samples were mixed with an equal part of finely ground wood charcoal, moistened with water, spread in an uniform layer on Petri dishes and incubated at 25°C for 5 days. The larvae were then harvested by the BAERMANN-MORAES^{3, 24} technique, concentrated by centrifugation for 5 minutes at 1000 g and stored at -18°C until the time of use.

S. ratti (Sandground, 1925) was obtained from the feces of experimentally infected rats (Rattus rattus) at the Parasitology Laboratory of" Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás" and processed as described for S. stercoralis.

Antigens: Filarioid S. stercoralis and S. ratti larvae were separately embedded in "Tissue Tek" (MILES) and cut into 4 µm thick sections with a cryo-microtome (CRYOCUT 1800 REICHERT-JUNG). The sections were added to extra-thin slides (76mm × 76mm) previously freed from fat material in an alcohol-ether solution, then dried at room temperature and preserved at -18°C up to the time of use. Only slides containing at least 5 larval sections were employed, as counted at 200X magnification.

Sera: Sera from 123 persons were employed (after previous written authorization) for the tests; 54 of them were from patients with a positive parasitological diagnosis of strongyloidiasis; 39 from patients with other enteroparasitoses (hookworm, 11 cases; Enterobius vermicularis, 5 cases; Ascaris lumbricoides, 5 cases; Hymenolepis nana, 5 cases; Trichuris trichiura, 4 cases; Taenia sp, 3 cases; Giardia lamblia, 3 cases; Entamoeba histolytica, 3 cases), all from the Hospital of "Universidade Federal de Uberlândia". Sera from 30 symptomless students from the same University and whose stools yielded negative results when submitted to 3 consecutive examinations by both the BAERMANN-MORAES^{3, 24} and HOFFMANN, PONS & JANER¹⁹ techniques were used as control. Serum from a patient who was passing S. stercoralis larvae in the feces was used as "positive control" and serum from a healthy person among the 30 noninfected controls was used as "negative control".

Indirect Immunofluorescence Reaction: Both negative and positive control sera diluted 1:20 in saline buffered at pH 7.2 with 0.01 M phosphate (PBS), plus the serum to be tested similarly diluted and buffered were added to slides containing larval antigens of S. stercoralis and S. ratti. The positive sera were further tested at increasing 2-fold dilutions. After incubation for 30 minutes at 37°C the slides were washed three times in PBS for 5 minutes and fluoresceinated anti-human IgG conjugate (Biolab) was added at the ideal titers of 10 for S. stercoralis and 100 for S. ratti, both diluted in PBS containing 1% EVANS blue as contrast stain. After an additional 30 minute incubation at 37°C the slides were washed again as described above, covered with a few drops of glycerine, pH 8.5 and a coverslip and examined with an Olympus BH 2 RFC immunofluorescence microscope at 200 and 400X. The results are expressed as titers, which were considered positive when ³ 20. The tests were performed simultaneously for each serum against each of the 2 antigens.

Statistical analysis: The normality of distribution was tested for two proportions at the significance levels of 5% and 1% to compare the reliability and specificity of the tests with the two Strongyloides antigens. Linear correlation, linear regression and confidence intervals of the titers were employed to compare the results obtained with S. ratti and S. stercoralis antigens.^22,23.

RESULTS

The reliability of the immunofluorescence-IgG tests for S. stercoralis and S. ratti antigens was 94.4% and 92.5%, respectively, and specificity was 94.2% and 97.1% with no significant difference at the 1% and 5% levels.

The IIF reaction was negative for 3 patients with a positive fecal parasitological diagnosis of strongyloidiasis; 2 of them were 4 and 5 year old girls and the remaining one was a 50 year old man also infected with Schistosoma mansoni. An 18 year old female patient had a negative result to the IIF reaction only when the S. ratti antigen was employed.

Of the 69 control people, 4 reacted positively to the test. One of these with an ancylostomid infection, was positive to both antigens, S. stercoralis and S. ratti; 2 other cases of ancylostomid-infected patients reacted only to the S. stercoralis antigen, and a patient with E. vermicularis infection reacted positively to both antigens.

Table 1 shows the distributions of antibody titers against S. stercoralis and S. ratti in the IIF reaction for the strongyloidiasis-infected patients and for the controls. It can be observed that the titers ranged from 20 to 2560.

Table 1

Sera	Titer – S. stercoralis										Total positive
	n	< 20	20	40	80	160	320	640	1280	2560	n	%
S. stercoralis	54	3	5	7	13	12	5	2	4	3	51	94.4
Other parasites controls	39	35	1		2	1					4	5.8
Uninfected controls	30	30									0	0
Sera	Titer – S. ratti										Total positive
	n	< 20	20	40	80	160	320	640	1280	2560	n	%
S. stercoralis	54	4	7	5	12	15	3	4	2	2	50	92.5
Other parasites controls	39	37		1	1						2	5.1
Uninfected controls	30	30									0	0

Distribution of antibody indirect immunofluorescence titers either against S. stercoralis or S. ratti in sera from infected patients of strongyloidiasis and in control ones.

The linear correlation between antibody titers for each of the antigens was significant (r = 0.85 p £ 0.001). Figure 1 illustrates this correlation between the titers of each antigen, the linear regression equation Y = 27.39 + 0.71 X, and the confidence intervals of the titers.

Fig. 1 - Correlation between titers of IgG antibodies anti S. ratti and S. stercoralis detected by the indirect immunofluorescence reaction in the 54 cases of strongyloidiasis. Regresion line is given by the equation Y = 27.39 + 0.71 X, with r = 0.85.

DISCUSSION

The IIF reaction using larvae of S. stercoralis or S. ratti as antigens (cryo-microtome slices) can be employed as a subsidiary diagnosis of human strongyloidiasis because of its high accuracy, as shown by the present results.

The difficult mass production of S. stercoralis larvae^{14, 28}, with the involved risk of infectivity for laboratory technicians, can be avoided because of the feasibility of safe and easy mass production of S. ratti larvae, an adequate substitute antigen, as proven in the present study. In fact, by means of the larval cryo-microtome technique a new model of IIF is presented that permits besides the safer and richer antigen production – a considerable economy of conjugate. All of these relevant advantages seem to be gained with an apparently negligible or non-existing loss in the reliability or accuracy of that immunodiagnostic technique, since the present results revealed an almost perfect equivalence of the use of either S. stercoralis or S. ratti larvae as antigens. Live S. ratti larvae have been previously used as antigens¹⁷ in another model for the same reaction, using glass tubes, with the addition of 50 µl of human anti-globulin diluted at 1:20.

An economy of antigens, compared to the reactions performed in tubes that utilize 4¹² to 50¹⁷ larvae per tube or 1000 larvae/ml¹⁵, was proven in the present study.

The results obtained with the model presented in this paper were similar to those of COUDERT et al.¹⁰ who employed filarioid larvae of S. stercoralis included in striated guinea pig muscles, also cut with a cryo-microtome.

CAMPOS et al.⁴ obtained 82% accuracy in the reaction using Strongyloides cebus larvae within the heart cavities of mice challenged with anti-gammaglobulin human conjugate. In the present study, embedding the larvae in "Tissue Tek" without any other support represents a convenient practical simplification.

The lack of serum IgG positivity in 3 patients with strongyloidiasis might mean that they had the acute form of this infection with the presence of larvae in the feces.

S. ratti antigen was not reactive in 2 of 3 ancylostomid-infected patients who reacted to the S. stercoralis antigen. Cross reaction of sera from ancylostomid and Strongyloides infection, although not always perceptible (1 of 11 patients, 9.1%) had already been reported by CAMPOS et al.⁴ in 2 of 50 cases.

This new diagnostic indirect immunofluorescence test could be applied to "risk populations" such as children, pregnant women, immunodepressed patients (organ transplant surgery, chemotherapy, radiotherapy, AIDS) for the prompt indication of specific treatment of strongyloidiasis thus preventing serious outbreaks of this nematodiasis.

RESUMO

Cortes em congelação de larvas de Strongyloides stercoralis e de S. ratti como fonte de antígenos para o imunodiagnóstico da estrongiloidíase humana

Comparou-se o emprego de cortes em congelação de larvas de S. stercoralis e de S. ratti como fonte de antígenos na reação de imunofluorescência indireta (RIF) para o diagnóstico da estrongiloidíase humana. Os antígenos foram obtidos de coproculturas de fezes humanas e de ratos, respectivamente. Soros de 123 indivíduos foram analisados sendo 54 de pacientes com estrongiloidíase e 69 controles. Empregou-se conjugado anti IgG humano marcado com fluoresceína nos títulos ideais de 10 para S. stercoralis e 100 para S. ratti. A sensibilidade da RIF foi de 94,4% e 92,5% e a especificidade de 94,2% e 97,1% respectivamente para os antígenos de S. stercoralis e S. ratti. Para os 2 antígenos os títulos dos soros variaram de 20 a 2560. Houve correlação linear significativa (r = 0,85 p £ 0,001) entre os títulos de anticorpos para os 2 antígenos. Concluiu-se que os 2 antígenos podem ser empregados na RIF. Diante da disponibilidade de obtenção de grande quantidade de antígeno em laboratório, economia do conjugado, por não haver diferença significativa entre sensibilidades e especificidades e haver correlação linear entre os títulos de anticorpos, a utilização do S. ratti é recomendada como antígeno no diagnóstico da estrongiloidíase humana.

ACKNOWLEDGEMENTS

This work was supported by CNPq. We thank Prof. Vanderli A. de Campos for the statistical analyses and Prof. Dr. Uriel Franco Rocha for suggestions.

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Recebido para publicação em 16/06/1997

Aceito para publicação em 21/11/1997

(1

) Laboratório de Parasitologia, Departamento de Patologia, Universidade Federal de Uberlândia, Uberlândia, MG, Brasil.

(2

) Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, Goiânia, GO, Brasil.

Correspondence to: Julia Maria Costa-Cruz. Laboratório de Parasitologia, Departamento de Patologia, Universidade Federal de Uberlândia. Av. Pará 1720, 38400-902 Uberlândia, MG, Brasil. Fax: [phone].

Publication Dates

• Publication in this collection
  09 Oct 1998
• Date of issue
  Nov 1997

History

• Accepted
  21 Nov 1997
• Received
  16 June 1997