Print version ISSN 0036-4665
Rev. Inst. Med. trop. S. Paulo vol.42 n.1 São Paulo Feb. 2000
PARASITOLOGICAL AND IMMUNOLOGICAL DIAGNOSES OF STRONGYLOIDIASIS IN IMMUNOCOMPROMISED AND NON-IMMUNOCOMPROMISED CHILDREN AT UBERLÂNDIA, STATE OF MINAS GERAIS, BRAZIL
Parasitological and immunological diagnoses were part of a study conducted among 151 children, 83 immunocompromised (IC) and 68 non-immunocompromised (non-IC) aged from zero to 12, seen at the University Hospital, Universidade Federal de Uberlândia, State of Minas Gerais, Brazil, from February, 1996, to June, 1998. Three fecal samples from each child were analyzed for the parasitological diagnosis by Baermann-Moraes and Lutz methods. The immunological diagnosis to detect IgG and IgM antibodies was carried out by the indirect immunofluorescence antibody test (IFAT) with cryo-microtome sections of Strongyloides stercoralis and Strongyloides ratti larvae as antigens and by the ELISA test with an alkaline extract of S. ratti as the antigens. Of the 151 children 5 (3.31%) were infected with larvae of S. stercoralis (2 cases IC, 2.41%, and 3 cases non-IC, 4.41%). The IFAT-IgG detected 7 (8.43%) serum samples positive among IC, and 2 (2.94%) cases among non-IC. The ELISA-IgG test detected 10 (12.05%) serum samples positive among IC, and 1 (1.47%) case among non-IC. The IFAT-IgM detected 6 (7.22%) positive cases among IC, and 3 (4.41%) cases among non-IC. ELISA-IgM test detected 10 (12.05%) positive cases among IC, and 3 (4.41%) cases among non-IC. It was concluded that the immunological tests can help in the diagnosis of strongyloidiasis in immunocompromised children.
KEYWORDS: Strongyloides stercoralis; Strongyloidiasis; Diagnosis; Children; Immunocompromised.
Strongyloides stercoralis is one of the most difficult parasitic infections to diagnose. In normal hosts, it causes chronic or subclinical infections that can persist for decades without any adverse effect. However, in immunocompromised hosts S. stercoralis may cause a frequently fatal hyperinfection or disseminated disease11. Many cases of strongyloidiasis were described among a variety of conditions of immunosuppression, especially impairment of cell-mediated immunity, such as hematologic malignancies10,19, lymphoma21, renal transplantation9 and HIV+ (human immunodeficiency virus infection) 8.
The parasitological methods have low sensitivity even when repeated several times and have to be performed on fresh stools, rarely a standard procedure in hospital clinical laboratories7,11. Several studies support the view that detection of parasite-specific antibodies may be a useful complement to the traditional parasitological diagnosis of strongyloidiasis using the indirect immunofluorescence antibody test (IFAT)4,12 and enzyme-linked immunosorbent assay (ELISA)3,13,22.
The purpose of the present study was to detect S. stercoralis infection in immunocompromised (IC) and non-immunocompromised (non-IC) children aged from zero to 12 years of age seen at the University Hospital, Universidade Federal de Uberlândia, State of Minas Gerais, Brazil, from February, 1996, to June, 1998.
The study was conducted among 151 children, 83 IC (46 malignancies, 8 protein-calorie malnutrition degree II and III, 6 HIV+, 3 acquired immune deficiency syndrome - AIDS, 3 renal insufficiency, 3 rheumatic fever, 3 falciform anemia, 2 nephrotic syndrome, 2 politraumatism, 1 renal transplantation, 1 cardiac insuficiency, 1 mellitus diabetes, 1 mucoviscidosis, 1 hepatic insufficiency, 1 Gauchers disease, 1 necrotic enterocolite) that were clinically confirmed18,23 and were being treated with immunosuppressive agents; and 68 non-IC hospitalized children with other non-immunosupressive infections. Fecal and serum samples were collected for both groups after previous written authorization from those responsible for the children. None of the patients of the two groups presented clinical or parasitological diagnosis of strongyloidiasis.
To parasitological diagnosis, three fecal samples from each child were collected in plastic vials without preservatives with intervals of 4 to 15 days and analyzed by the BAERMANN1, MORAES17 and LUTZ15 methods. Six slides were prepared for each method for each of the 453 samples. The total number of slides examined was thus 5,436. All the families of the children received the results of the laboratory diagnosis. The positive cases were given specific treatment.
To immunological diagnosis, S. stercoralis larvae were obtained from the feces of patients seen at the same University Hospital and S. ratti was obtained from the feces of experimentally infected rats (Rattus rattus). The larvae of each antigen were mixed with an equal part of finely ground charcoal, moistened with water, spread in an uniform layer on Petri dishes and incubated at 25 °C for 5 days. The filariform larvae were then harvested by the BAERMANN1, MORAES17 technique, concentrated by centrifugation at 1000g for 5 minutes and stored at 20 °C until the time of use.
For IFAT, S. stercoralis and S. ratti antigens were prepared according to COSTA-CRUZ et al.4. For ELISA, the alkaline extract was prepared with the addition of 1 ml of 0.15 M NaOH at 4 °C for 6 hours of agitation. Subsequently, adding 0.5 ml of 0.3 M of HCl until the pH reached 7.0. This preparation was centrifuged at 3000g for 15 minutes at 4 °C and the supernatant was submitted to LOWRY et al.14 method for protein detection.
The immunofluorescence - IgG tests for S. stercoralis and S. ratti antigens utilized in the present work was done according to COSTA-CRUZ et al.4. The authors described 94.4% and 94.2% respectively of sensitivity and specificity to S. stercoralis and respectively 92.5% and 97.1% to S. ratti for IgG antibodies, considered titers positive when ³ 20. For IgM antibodies, IFAT was standardized previously by PEIXOTO20 with 37.0% and 29.6% positive serum samples detected in patients with strongyloidiasis, by S. stercoralis and S. ratti antigens respectively; considered titers positive when ³ 40. The tests, in the present study, were performed simultaneously for each serum using each of the two antigens. The fluoresceinated anti-human IgG and IgM conjugate (Biolab, Brazil) were added at the ideal titers of 100 (for both antigens) and 20 (for both antigens), respectively.
ELISA was previously described by COSTA-CRUZ et al.5 with a sensitivity of 93.4% and specificity of 96.9% for IgG antibodies. For IgM antibodies, ELISA was standardized previously by COSTA-CRUZ et al.6 and 44.26% positive serum samples were detected in patients with confirmed strongyloidiasis. The cut off was determined as BASSI et al.2 with two standard deviations. The results were expressed as titers, which were considered positive when ³ 80. The anti-human IgG and IgM labeled with peroxidase (Sigma) was added respectively at titers 4000 and 2000.
All positive serum sample IgM antibodies were tested using the "Kit Reumatex â" (Doles, Brazil) and FTA sorbent (Laborclin, Brazil).
The normality of distribution was tested for two proportions and X2 at the significance level of 5%16 to compare the results obtained with two groups.
From the 151 children studied, 139 (92.05%) were from the State of Minas Gerais (86, 56.95%, from Uberlândia) 11 (7.28%) from the State of Goiás and 1 (0.66%) from the State of Mato Grosso do Sul.
The distribution of intestinal parasites and comensal in IC and non-IC children is showed in Table 1. Of the 151 children studied 5 (3.31%) were infected with larvae of S. stercoralis. Two of these cases (2.41%) were IC, both were girls in the category of two to four years and 3 (4.41%) were non-IC, all were boys, one case being in the category of two years of age or under and two cases in the category of between ten and twelve. No statistical difference was observed by test for the two proportions (Z= 0.69, p>0.05) in the occurrence of S. stercoralis between the two groups.
Frequency of intestinal parasites and comensal among immunocompromised and non-immunocompromised children seen at the University Hospital of Uberlândia, MG, Brazil, from February, 1996 to June, 1998
The IFAT-IgG detected 9 (5.96%) positive serum samples, 7 (8.43%) IC and 2 (2.94%) non-IC. The 7 (8.43%) cases of IC were detected by the S. stercoralis antigen, 4 (4.82%) of these were also S. ratti antigen positives. The S. stercoralis antigen detected 2 (2.94%) positive cases among non-IC and the S. ratti antigen only 1 (1.47%) case. The IFAT-IgM detected 9 (5.96%) positive cases, 6 (7.22%) cases among IC and 3 (4.41%) among non-IC. Of the 6 positive cases among IC 5 (6.02%) cases were detected by S. stercoralis and 6 (7.23%) cases by S. ratti antigens. The S. stercoralis antigen detected 3 (4.41%) cases among non-IC and the S. ratti antigen only 1 (1.47%) case. No statistical difference was observed using the test for the two proportions between the two antigens or the two groups (p>0.05).
The ELISA-IgG test detected 11 (7.28%) positive serum samples, 10 (12.05%) among IC and 1 (1.47%) among non-IC. The ELISA-IgM test detected 13 (8.61%) positive cases, 10 (12.05%) among IC and 3 (4.41%) among non-IC. Statistical difference was observed between the two groups in ELISA-IgG (Z=3.84, p<0.05) and ELISA-IgM (Z=2.75, p<0.05).
All IgM positive serum samples were negative in rheumatic factor research.
From the 151 children 19 (12.58%) were positive for S. stercoralis, considering the parasitological and immunological diagnoses, 16 (10.60%) were from the State of Minas Gerais [9 (5.96%) from Uberlândia] and 3 (1.99%) from the State of Goiás, aged from five months to 12 years, and of both sexes.
Identification of S. stercoralis positive cases from parasitological and immunological diagnoses among immunocompromised and non-immunocompromised children seen at the University Hospital of Uberlândia, MG, Brazil, from February, 1996 to June, 1998
|Fig. 1 - Comparison of parasitological (Baermann-Moraes-BM and Lutz-L) and immunological (IFAT-G and IFAT-M, S.stercoralis-S.s. and S. ratti-S.r., ELISA-G and ELISA-M) methods among 83 immunocompromised and 68 non-immunocompromised children seen at the University Hospital of Uberlândia, MG, Brazil, from February, 1996 to June, 1998. *Z=2.41; #Z=3.84 **Z=2.75 (p<0.05)|
Considering immunosuppression as a risk factor of strongyloidiasis and the high positive rate of immunological tests among IC, the present study shows the value of IFAT and ELISA in the detection of IgG and IgM antibodies in diagnosis of human strongyloidiasis among immunocompromised children, contributing to the establishment of the diagnosis of this parasitosis and the possibility of early treatment of these children.
Diagnóstico parasitológico e imunológico da estrongiloidíase em crianças imunodeprimidas e imunocompetentes na cidade de Uberlândia, MG, Brasil
O diagnóstico parasitológico e imunológico da estrongiloidíase foi realizado em 151 crianças (83 imunodeprimidas -ID e 68 imunocompetentes -IC) de zero a 12 anos de idade internadas no Hospital de Clínicas da Universidade Federal de Uberlândia, Minas Gerais, Brasil, no período de fevereiro de 1996 a junho de 1998. Para o diagnóstico parasitológico três amostras de fezes de cada indivíduo foram processadas pelos métodos de Baermann-Moraes e de Lutz. O diagnóstico imunológico para a detecção de anticorpos IgG e IgM foi realizado através das reações de imunofluorescência indireta (RIFI) utilizando-se como antígeno cortes de 4 micra de larvas filarióides de Strongyloides stercoralis e Strongyloides ratti e do teste ELISA utilizando-se como antígeno extrato alcalino de larvas de S. ratti. Das 151 crianças, 5 (3,31%) estavam infectadas com S. stercoralis (2 casos ID, 2,41% e 3 casos IC, 4,41%). A RIFI- IgG detectou 7 (8,43%) amostras de soros positivas nas ID, e 2 (2,94%) nas IC. O teste ELISA-IgG detectou 10 (12,05%) amostras de soros positivas nas ID, e 1 (1,47%) nas IC. A RIFI-IgM detectou 6 (7,22%) casos positivos nas ID, e 3 (4,41%) nas IC. O teste ELISA-IgM detectou 10 (12,05%) casos positivos nas ID e 3 (4,41%) nas IC. Concluiu-se que os testes imunológicos podem contribuir para o diagnóstico da estrongiloidíase em crianças imunodeprimidas.
This work was supported by FAPEMIG and CAPES. We thank Prof. Vanderli A. de Campos for the statistical analyses and Dr. David G. Francis for a critical reading of the manuscript.
1. BAERMANN, G. - Eine einfache methode zur auffindung von Ankylostomum (Nematoden) larven in Erdproben. Mededeel mit H. Geneesk. Batavia, Lab. Weltevreden. Feestbundel, 1917. p. 41-47. [ Links ]
2. BASSI, G.E.; ISHIRI, D.K.; FERREIRA, A.W. & CAMARGO, M.E. - A reação imunoenzimática para cisticercose no líquido cefalorraquiano. Considerações sobre o limiar de reatividade. Rev. bras. Pat. clín., 27: 49-52, 1991. [ Links ]
3. BRASIL, R.; BADARÓ, R.; ANDRADE, J.A.F. et al. - Diagnóstico sorológico da estrongiloidíase humana através do método imunoenzimático. Rev. Soc. bras. Med. trop., 21: 139-143, 1988. [ Links ]
4. COSTA-CRUZ, J.M.; BULLAMAH, C.B.; GONÇALVES-PIRES, M.R.F. et al. - 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. S. Paulo, 39: 313-317, 1997. [ Links ]
5. COSTA-CRUZ, J.M.; SILVA, L.P.; GONÇALVES-PIRES, M.R.F. & CAMPOS, D.M.B. - Detecção de IgG pelo teste ELISA, utilizando extratos antigênicos salino e alcalino de Strongyloides ratti, no diagnóstico da estrongiloidíase humana. In: CONGRESSO BRASILEIRO DE PARASITOLOGIA, 16., Poços de Caldas, 1999. p. 129. [ Links ]
6. COSTA-CRUZ, J.M.; GONÇALVES-PIRES, M.R.F.; PAULA, F.M. & CAMPOS, D.M.B. - Padronização do teste ELISA para detecção de anticorpos IgM, utilizando extratos salino e alcalino de Strongyloides ratti no diagnóstico da estrongiloidíase humana. Rev. Soc. bras. Med. trop., 32 (supl. 1): 299, 1999. [ Links ]
7. DREYER, G.; FERNANDES-SILVA, E.; ALVES, S. et al. - Patterns of detection of Strongyloides stercoralis in stool specimens: implications for diagnosis and clinical trials. J. clin. Microbiol., 34: 2569-2571, 1996. [ Links ]
8. FERREIRA, M.S.; NISHIOKA, A.S.; BORGES, A.S. et al. - Strongyloidiasis and infection due to human immunodeficiency virus: 25 cases at a Brazilian teaching hospital, including seven cases of hyperinfection syndrome. Clin. infect. Dis., 28:154-155, 1999. [ Links ]
9. GERMAN, J.C.; FLORES, J.H.; CHIESURA, G. et al. - Estrongiloidiasis fatal en paciente inmunodeprimido pos-transplante renal. Rev. Hosp. Clín. Fac. Med. S. Paulo, 47: 31-33, 1992. [ Links ]
10. GRAEFF-TEIXEIRA, C.; LEITE, C.S.M.; SPERHACKE, C.L. et al. - Prospective study of strongyloidosis in patients with hematologic malignancies. Rev. Soc. bras. Med. trop., 30: 355-357, 1997. [ Links ]
11. GROVE, D.I. - Human strongyloidiasis. Advanc. Parasit., 38: 251-309, 1996. [ Links ]
12. GROVE, D.I. & BLAIR, A.J. - Diagnosis of human strongyloidiasis by immunofluorescence, using Strongyloides ratti and S. stercoralis larvae. [ Links ]Amer. J. trop. Med. Hyg., 30: 344-349, 1981.
13. LINDO, J.F.; CONWAY, D.J.; ATKINS, N.S. et al. - Prospective evaluation of enzyme-linked immunosorbent assay and immunoblot methods for the diagnosis of endemic Strongyloides stercoralis infection. Amer. J. trop. Med. Hyg., 51: 175-179, 1994. [ Links ]
14. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.J. - Protein measurement with the folin phenol reagent. J. biol. Chem., 93: 265-275, 1951. [ Links ]
15. LUTZ, A.V. - Schistosoma mansoni e a schistosomose, segundo observações feitas no Brasil. Mem. Inst. Oswaldo Cruz, 11: 121-125, 1919. [ Links ]
16. MALETA, C.H.M. - Bioestatística em Saúde Pública. 2. ed. Belo Horizonte, Coopmed, 1992. [ Links ]
17. MORAES, R.G. - Contribuição para o estudo do Strongyloides stercoralis e da estrongiloidíase no Brasil. Rev. Serv. esp. Saúde públ., 1: 507-624, 1948. [ Links ]
18. NUCCI, M. & PULCHERI, W. - Infecções no paciente imunodeprimido. In: SCHECHTER, M. Doenças infecciosas: conduta diagnostica e terapêutica. 2. ed. Rio de Janeiro, Guanabara-Koogan, 1999. p.543-556. [ Links ]
19. NUCCI, M.; PORTUGAL, R.; PULCHERI, W. et al. - Strongyloidiasis in patients with hematologic malignancies. Clin. infect. Dis., 21: 675-677, 1995. [ Links ]
20. PEIXOTO, V. - Detecção de imunoglobulina M (IgM) pelo teste de imunofluorescência indireta no diagnóstico da estrongiloidíase humana. Uberlândia, 1997. (Monografia - Curso de Ciências Biológicas/UFU). [ Links ]
21. SANDLUND, J.T.; KAUFFMAN, W. & FLYNN, P.M. - Strongyloides stercoralis infection mimicking relapse in a child with small noncleaved cell lymphoma. Amer. J. clin. Oncol., 20: 215-216, 1997. [ Links ]
22. SATO, Y.; KOBAYASHI, J. & SHIROMA, Y. - Serodiagnosis of strongyloidiasis. The application and significance. Rev. Inst. Med. trop. S. Paulo, 37: 35-41, 1995. [ Links ]
23. STILLWELL, M. & CAPLAN, E.S. - O paciente politraumatizado com septicemia. In: MOELLLERING, R.C. Clínicas de doenças infecciosas da América do Norte. Rio de Janeiro, Interlivros, 1989. v. 1/89, p.155-185. [ Links ]
Received: 13 September 1999
Accepted: 13 December 1999
(1) Laboratório de Parasitologia, Departamento de Patologia, Universidade Federal de Uberlândia, MG, Brasil.
(2) Enfermaria de Pediatria, Departamento de Pediatria, Universidade Federal de Uberlândia, MG, Brasil.
(3) Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, GO, Brasil
Correspondence to: Profª Dra. Julia Maria Costa Cruz, Laboratório de Parasitologia, Departamento de Patologia, Universidade Federal de Uberlândia, Av. Pará 1720, 38400-902 Uberlândia, Minas Gerais, Brasil. FAX: +55.34.2182333. E-mail: firstname.lastname@example.org