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Is there an association between positive Strongyloides stercoralis serology and diabetes mellitus?
Acta Tropica 99 (2006) 102–105
Short communication
Is there an association between positive Strongyloides stercoralis serology and diabetes mellitus? Suzan C.L. Mendonc¸a, Maria do Ros´ario F. Gonc¸alves-Pires, ´ Rosˆangela M. Rodrigues, Alvaro Ferreira Jr., Julia M. Costa-Cruz ∗ Departamento de Imunologia, Microbiologia e Parasitologia, Instituto de Ciˆencias Biom´edicas, Universidade Federal de Uberlˆandia, Av. Par´a 1720, 38400-902 Uberlˆandia, MG, Brazil Received 6 September 2005; received in revised form 1 June 2006; accepted 20 June 2006 Available online 26 July 2006
Abstract The purpose of this study was to determine the frequency of association between positive Strongyloides stercoralis serology and diabetes mellitus. A total of 78 diabetic patients and 42 controls were evaluated. For a parasitological diagnosis, Baermann and Hoffman et al.’s methods were applied. The immunological diagnosis involved the indirect fluorescence antibody test, ELISA and Western blotting to detect IgG antibodies. The frequency of positive S. stercoralis serology in diabetics was 23% versus 7.1% in the control group (P < 0.05). The odds ratio for diabetics was 3.9 (CI, 1.6–15.9, P < 0.05). Diabetic patients with HbA1c ≤ 7 had a greater chance of testing negatively for S. stercoralis infection (OR: 1.5, P > 0.05). Provided there are related cases of disseminated strongyloidiasis in diabetics and there is a higher frequency of asymptomaticity of the infection in this group, the immunological screening of these patients at risk could prevent severe and fatal outcomes of the disease. © 2006 Elsevier B.V. All rights reserved. Keywords: Strongyloidiasis; Diagnosis; Diabetic; Diabetes mellitus; Strongyloides stercoralis
Strongyloides stercoralis infection is distributed worldwide, especially in tropical and subtropical regions. Although the disease is manifested in most cases as a chronic asymptomatic disease, potentially fatal outcomes may occasionally occur (Genta, 1989; Rahim et al., 2005; Lam et al., 2006). Severe manifestations of S. stercoralis infection are frequently correlated with predisposing factors such as immunosuppression caused by other diseases or corticoid treatment (Keiser and Nutman, 2004). Although the medical literature contains some case reports of disseminated strongyloidiasis in diabetic ∗ Corresponding author. Tel.: +55 34 32182187; fax: +55 34 32182333. E-mail address: [email protected] (J.M. Costa-Cruz).
0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2006.06.006
patients (Venturi and Viliotti, 1984; Debussche et al., 1988; Coovadia et al., 1993; Higashiyama et al., 1997; Ho et al., 1997; Al Samman et al., 1999; Emad, 1999; Linder et al., 2000), no controlled study was found that attempted to determine whether diabetics are predisposed to chronic strongyloidiasis. It is important to understand that the transmission of S. stercoralis is totally independent of the acquisition of diabetes mellitus. Because of the risk of fatality in these severe forms of infection, and because diabetes mellitus is highly prevalent worldwide, the frequency of association between positive S. stercoralis serology and this metabolic disorder is considered extremely important. This study involved a total of 120 ambulatory patients of the Federal University of Uberlˆandia School of Medicine Endocrinology Unit, state of Minas Gerais,
S.C.L. Mendon¸ca et al. / Acta Tropica 99 (2006) 102–105
Brazil. Of these, 78 type 2 diabetics were selected consecutively, 32 males and 46 females with a mean age of 54.1 ± 11.9 years (ranging from 28 to 75) and 42 controls, 17 males and 25 females with a mean age of 53.9 ± 10.7 years (ages from 30 to 77). The controls were ambulatory patients with other endocrinal diseases included: hypothyroidism (33.3%), obesity (28.5%), treated hyperthyroidism (16.7%), atoxic multinodular goiter (9.5%), dyslipidemia (4.8%) and one case of prolactinoma, acromegaly and thyroid nodular disease (7.2%), undergoing treatment by the same physician during the same period as the diabetics. All subjects seen at this public hospital located in a hyperendemic area for strongyloidiasis had similar background and current socio-economic conditions. This study was approved by the Federal University of Uberlˆandia’s Ethical Committee. Blood samples were collected from all the patients to determine their eosinophil count. Glycated hemoglobin (HbA1c ) was analyzed in diabetics using the Bayer DCA2000 analyzer. Three fecal samples were collected from each subject on consecutive days and stored in plastic receptacles without preservatives at 4 ◦ C. Parasitological diagnoses were carried out using Baermann’s (1917) and Hoffmann et al.’s (1934) method. Three slides of each of the 360 samples were prepared for the Baermann analysis and three for the Hoffmann et al. analysis, making a total of 2160 slides examined by two investigators using a Nikon (Japan) microscope at 100 and 400× magnification. The IFAT was carried out according to Costa-Cruz et al. (1997) using cryo-microtome section of S. stercoralis larvae. The results were expressed as antibody titers, which were considered positive when ≥20. Reactive serum samples were retested in two-fold serial dilutions up to the end-point titer. ELISA and WB were performed as described by Silva et al. (2003) using saline extracts of S. stercoralis, obtained from 420,000 filariform larvae. For ELISA the results were expressed as antibody titers which were considered positive when ≥80. For WB sera samples were considered positive if at least two immunodominant proteins (molecular weight of 17, 20, 23, 25, 26, 28, 30, 31, 40, 41, 55, 66, 90, 97 or 100 kDa) of S. stercoralis fractions were recognized (Genta et al., 1988; Sato et al., 1990; Conway et al., 1993b; Lindo et al., 1994). The association between positive S. stercoralis serology and diabetes was calculated as an odds ratio and expressed as an estimated relative risk. The confidence interval for the odds ratio was calculated by Fleiss’s method (1979). The statistical significance of associations was calculated using the χ2 statistic with the Yates
103
correction. The mean age between groups was compared by Student’s t-test. Differences were considered statistically significant when P < 0.05. The frequency of positive stool examination in diabetic patients was 3.8% (three cases) being all control individuals negative. In addition to S. stercoralis larvae, the coprological examination revealed one diabetic woman with hookworm eggs and another with Giardia lamblia cysts. The frequency of positive S. stercoralis serology in diabetics was 23% (18 cases) versus 7.1% (3 cases) in the control group (P < 0.05). The odds ratio for diabetics was 3.9 (CI, 1.6–15.9, P < 0.05). The three patients whose stool examination detected S. stercoralis larvae also tested positive in the IFAT, ELISA and WB tests. The efficacy of the stool examination in detecting Strongyloides infection was very low: 18 out of 21 patients with positive serology had negative coprologic examination results. Curiously, one patient whose third stool sample contained a large number of larvae had two previous samples which tested negative for S. stercoralis infection. One patient with a normal white cell count had 16% eosinophils, another patient had a history of intermittent diarrhea and 5% eosinophilia and a third patient whose stool examination tested positive was asymptomatic, without eosinophilia. No other patient with positive serology presented eosinophilia, indicating that the majority of the cases did only have signs of a recent, but not present infection. Table 1 shows the characteristics of patients with positive tests, glycated hemoglobin (for diabetics only), IFAT and ELISA results expressed as titers and the immunodominant antigenic component identified by WB. Positive S. stercoralis serology was detected in 9 male (18.3%) and 12 female patients (16.9%). The mean age for positive patients was 59.2 years for females and 54.4 years for males. There was congruence between the serological tests for 79.5% of the diabetics and for 71.4% of the control group. Patients were considered negative, even when IFAT and/or ELISA were positive, if at least two immunodominant antigenic components were undetected in the WB test. A correlation was drawn between the frequency of S. stercoralis infection and the glycated hemoglobin level (HbA1c ) in order to estimate the extent to which the diabetic whose disease is inadequately controlled is predisposed to remain chronically infection. Today, the HbA1c measurement is the best way to demonstrate chronic metabolic control, given the information about the mean serum glucose level over the previous 120 days. Among the 39 cases with good metabolic control (HbA1c < 7), only 9% tested positive for S. stercoralis compared with 14% of those with poor metabolic control.
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S.C.L. Mendon¸ca et al. / Acta Tropica 99 (2006) 102–105
Table 1 Characteristics of patients testing positive for strongyloidiasis in terms of sex, age, endocrinal disease, glycated hemoglobin (for diabetics only), IFAT and ELISA test results expressed in titers and protein fractions detected by Western blotting Sex
Age
Diagnosis
Stool examination
HbA1c (%)
IFAT
ELISA
Protein fractions (kDa)
F M M F F M F F M F F M M F M M F F M F F
60 63 51 62 50 39 64 46 48 78 52 53 45 75 62 54 47 60 75 52 64
Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Hypothyroidism Hypothyroidism Hypothyroidism
Positive Positive Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
9.2 9.6 10 8 7 6 12.4 10 10.2 5.8 7 6 9.9 7.9 6.2 6.3 11.4 7.5 ND ND ND
40 20 40 40 20 Negative 20 40 20 40 40 20 20 20 40 Negative 20 Negative 20 40 20
160 1280 320 160 80 80 80 160 320 2560 2560 2560 640 80 320 160 160 80 320 2560 160
17, 20, 30, 87, 90 17, 20, 30, 40, 55 20, 23, 25, 30, 90 50, 55, 66, 78 55, 66, 90 23, 25, 40, 55, 66 40, 66 17, 20, 30, 78, 90 40, 78, 90 14, 17, 20, 25, 55, 66, 90 17, 20, 25, 55, 66, 90 17, 30, 40, 55, 90 31, 90 60, 66, 90 90, 78, 25, 20, 17, 13 90, 66, 55, 40, 25, 20, 17 90, 66, 55, 25, 20, 17 17, 20, 31, 85 31, 40, 41 17, 20, 25, 55, 66, 90 23, 25, 31, 87
ND: not done; M: male; F: female.
This study suggests an association between diabetes mellitus and the serodiagnosis of strongyloidiasis. Definitive diagnosis of strongyloidiasis is usually made on the basis of detection of larvae in the stool. However, in a majority of uncomplicated cases of strongyloidiasis, mainly in endemic regions, the intestinal worm load is often very low, leading to low larval densities in feces and fluctuations in larval excretion. It is important to note that a negative result does not necessarily indicate an unequivocal absence of the infection (Uparanukraw et al., 1999; Sudarshi et al., 2003; Keiser and Nutman, 2004). Hence, there is a great need for highly specific and efficient serodiagnostic tests for S. stercoralis. Currently, the most widely used method has been the ELISA using filariform larvae antigen. Most reports on the ELISA for strongyloidiasis have shown consistently high sensitivity and specificity, varying from 83.8 to 94.6 and 90.2 to 100%, respectively, and positive and negative predictive values of 97 and 95%, respectively (Genta, 1988; Conway et al., 1993a; Lindo et al., 1994; Sudarshi et al., 2003). Grove (1982) followed 43 patients for 6 months after thiabendazole treatment demonstrated a significant fall in anti-Strongyloides serum antibody level in many patients who were negative in the followup fecal examination. Genta and Weil (1982) also followed serologically, for 2–4 months, seven patients after specific treatment and demonstrated a significant
decrease in antibody titers to filariform larvae in all of them. There is no clear explanation for the high occurrence of positive test results for strongyloidiasis in diabetic patients. Despite the numerous studies that have evaluated the immune system in diabetes, it has become increasingly difficult to compare them. For this reason, even after decades of investigation, questions about whether diabetes itself results in specific immunological defects and how such defects might predispose the patient to infection are still being debated (Bessman and Sapico, 1992; Sentochnik and Eliopoulos, 1994). One controlled study in Turkey (Nazligul et al., 2001) found no predisposition for intestinal parasitosis in diabetic patients; the diagnosis was based on only one stool sample from each patient and no cases of S. stercoralis infection were found. Our results provide evidence supporting the hypothesis of a possible association between positive S. stercoralis serology and diabetes. Provided there are related cases of disseminated strongyloidiasis in diabetics and there is a higher frequency of asymptomaticity of the infection in this group, the immunological screening of these patients at risk could prevent severe and fatal outcomes of the disease, particularly if there is poor metabolic control of the disease. Considering the high efficacy and low toxicity of new drugs used in the treatment of strongyloidiasis, diabetic patients should be
S.C.L. Mendon¸ca et al. / Acta Tropica 99 (2006) 102–105
treated in the presence of eosinophilia if they are to undergo corticosteroid therapy or in situations of severe metabolic distress such as ketoacidosis. In most parasitic infections, exposure to the organism plays the major role in determining infection. However, because of its unusual life cycle, S. stercoralis may be different and host factors are associated with long-term S. stercoralis infection. Although it is not clear that the association is actually a case of cause and effect, these factors should be taken into account, together with determinants of exposure, when studying the causes of chronic S. stercoralis infection and estimating the likelihood of infection in clinical practice. Acknowledgement To Maria das Grac¸as Marc¸al for parasitological diagnosis supports. References Al Samman, M., Haque, S., Long, J.D., 1999. Strongyloidiasis colitis: a case report and review of the literature. J. Clin. Gastroenterol. 28, 77–80. Baermann, G., 1917. Eine einfache Methode zur Auffinfung von Ankylostomun (Nematoden) Larven in Erdproben. Mededeel. mith.H Geneesk. Batavia, Lab. Weltevreden Feestbundel, pp. 41–47. Bessman, A.N., Sapico, F.L., 1992. Infections in the diabetic patient: the role of immune dysfunction and pathogen virulence factors. J. Diabetes Complications 6, 258–262. Conway, D.J., Atkins, N.S., Lillywhite, J.E., Bailey, J.W., Robinson, R.D., Lindo, J.F., Bundy, D.A., Bianco, A.E., 1993a. Immunodiagnosis of Strongyloides stercoralis infection: a method for increasing the specificity of the indirect ELISA. Trans. R. Soc. Trop. Med. Hyg. 87, 173–176. Conway, D.J., Bailey, J.W., Lindo, J.F., Robinson, R.D., Bundy, D.A.P., Bianco, A.E., 1993b. Serum IgG reactivity with 41-, 31-, and 28kDa larval proteins of Strongyloides stercoralis in individuals with strongyloidiasis. J. Infect. Dis. 168, 784–787. Coovadia, Y.M., Rajput, M.C., Bhana, R.H., 1993. Disseminated strongyloidiasis in a diabetic patient. Trop. Geogr. Med. 45, 179–180. Costa-Cruz, J.M., Bullamah, C.B., Gonc¸alves-Pires, M.R.F., Campos, D.M.B., Vieira, M.A., 1997. 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˜ao Paulo 39, 313–317. Debussche, X., Toublanc, M., Camillieri, J.P., Assan, R., 1988. Overwhelming strongyloidiasis in a diabetic patient following ACTH treatment and Keto-acidosis. Diab. Metab. 14, 294–298. Emad, A., 1999. Exsudative eosinophilic pleural effusion due to Strongyloides stercoralis in a diabetic man. South. Med. J. 92, 58–60. Fleiss, J.L., 1979. Confidence intervals for the odds ratio in case-control studies: the state of the art. J. Chronic Dis. 32, 69–77. Genta, R.M., 1988. Predictive value of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of strongyloidiasis. Am. J. Clin. Pathol. 89, 391–394.
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Genta, R.M., 1989. Global prevalence of strongyloidiasis: critical review with epidemiologic insights into the prevention of disseminated disease. Rev. Infect. Dis. 11, 755–767. Genta, R.M., Weil, G.J., 1982. Antibodies to Strongyloides stercoralis larval surface antigens in chronic strongyloidiasis. Lab. Invest. 47, 87–90. Genta, R.M., Gatti, S., Linke, M.J., Cevini, C., Scaglia, M., 1988. Endemic strongyloidiasis in northern Italy: clinical and immunological aspects. Q. J. Med. 68, 679–690. Grove, D.I., 1982. Treatment of strongyloidiasis with thiabendaloze: an analysis of toxicity and effectiveness. Trans. R. Soc. Trop. Med. Hyg. 76, 114–118. Higashiyama, Y., Sakata, H., Obase, Y., Sakata, S., Shimafuji, T., Mori, M., Ishiro, T., Kohno, S., Saito, A., Hara, K., 1997. A case of bacterial meningitis induced by strongyloidiasis. Kansenshogaku Zasshi 71, 680–683. Ho, P.L., Luk, W.K., Chant, A.C., Yuen, K.Y., 1997. Two cases of fatal strongyloidiasis in Hong Kong. Pathology 29, 324–326. Hoffmann, W.A., Pons, J.A., Janer, J.L., 1934. The sedimentation concentration method in schistosomiasis mansoni. P. R. J. Public Health Trop. Med. 9, 283–291. Keiser, P.B., Nutman, T.B., 2004. Strongyloides stercoralis in the immunocompromised population. Clin. Microbiol. Rev. 17, 208–217. Lam, C.S., Tong, M.K., Chan, K.M., Siu, Y.P., 2006. Disseminated strongyloidiasis: a retrospective study of clinical course and outcome. Eur. J. Clin. Microbiol. Infect. Dis. 25, 14–18. Linder, J.D., Monkemuller, K.E., Lazenby, A.J., Wilcox, C.M., 2000. Streptococcus bovis bacteremia associated with Strongyloides stercoralis colitis. Gastrointest. Endosc. 52, 796–798. Lindo, J.F., Conway, D.J., Atkins, N.S., Bianco, A.E., Robinson, R.D., Bundy, D.A., 1994. Prospective evaluation of enzyme-linked immunosorbent assay and immunoblot methods for the diagnosis of endemic Strongyloides stercoralis infection. Am. J. Trop. Med. Hyg. 51, 175–179. Nazligul, Y., Sabuncu, T., Ozbilge, H., 2001. Is there a predisposition to intestinal parasitosis in diabetic patients? Diab. Care 24, 1503–1504. Rahim, S., Drabu, Y., Jarvis, K., Melville, D., 2005. Strongyloidiasis: a mistaken diagnosis and a fatal outcome in a patient with diarrhoea. Trans. R. Soc. Trop. Med. Hyg. 99, 215–217. Sato, Y., Inoue, F., Matsuyama, R., Shiroma, Y., 1990. Immunoblot analysis of antibodies in human strongyloidiasis. Trans. R. Soc. Trop. Med. Hyg. 84, 403–406. Sentochnik, D.E., Eliopoulos, G.M., 1994. Infection and diabetes. In: Joslin’s Diabetes Mellitus, 13th ed. Lea & Febiger, Malvern, pp. 867–888. Silva, L.P., Barcelos, I.S.C., Passos-Lima, A.B., Espindola, F.S., Campos, D.M.B., Costa-Cruz, J.M., 2003. Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis. Mem. Inst. Oswaldo Cruz 98, 687–691. Sudarshi, S., Stumpfle, R., Armstrong, M., Ellman, T., Parton, S., Krishnan, P., Chiodini, P.L., Whitty, C.J., 2003. Clinical presentation and diagnostic sensitivity of laboratory tests for Strongyloides stercoralis in travellers compared with immigrants in a non-endemic country. Trop. Med. Int. Health 8, 728–732. Uparanukraw, P., Phongsri, S., Morakote, N., 1999. Fluctuations of larval excretion in Strongyloides stercoralis infection. Am. J. Trop. Med. Hyg. 60, 967–973. Venturi, M., Viliotti, W.M., 1984. Disseminated strongyloidiasis in a diabetic patient. Rev. Paul. Med. 102, 283.
Short communication
Is there an association between positive Strongyloides stercoralis serology and diabetes mellitus? Suzan C.L. Mendonc¸a, Maria do Ros´ario F. Gonc¸alves-Pires, ´ Rosˆangela M. Rodrigues, Alvaro Ferreira Jr., Julia M. Costa-Cruz ∗ Departamento de Imunologia, Microbiologia e Parasitologia, Instituto de Ciˆencias Biom´edicas, Universidade Federal de Uberlˆandia, Av. Par´a 1720, 38400-902 Uberlˆandia, MG, Brazil Received 6 September 2005; received in revised form 1 June 2006; accepted 20 June 2006 Available online 26 July 2006
Abstract The purpose of this study was to determine the frequency of association between positive Strongyloides stercoralis serology and diabetes mellitus. A total of 78 diabetic patients and 42 controls were evaluated. For a parasitological diagnosis, Baermann and Hoffman et al.’s methods were applied. The immunological diagnosis involved the indirect fluorescence antibody test, ELISA and Western blotting to detect IgG antibodies. The frequency of positive S. stercoralis serology in diabetics was 23% versus 7.1% in the control group (P < 0.05). The odds ratio for diabetics was 3.9 (CI, 1.6–15.9, P < 0.05). Diabetic patients with HbA1c ≤ 7 had a greater chance of testing negatively for S. stercoralis infection (OR: 1.5, P > 0.05). Provided there are related cases of disseminated strongyloidiasis in diabetics and there is a higher frequency of asymptomaticity of the infection in this group, the immunological screening of these patients at risk could prevent severe and fatal outcomes of the disease. © 2006 Elsevier B.V. All rights reserved. Keywords: Strongyloidiasis; Diagnosis; Diabetic; Diabetes mellitus; Strongyloides stercoralis
Strongyloides stercoralis infection is distributed worldwide, especially in tropical and subtropical regions. Although the disease is manifested in most cases as a chronic asymptomatic disease, potentially fatal outcomes may occasionally occur (Genta, 1989; Rahim et al., 2005; Lam et al., 2006). Severe manifestations of S. stercoralis infection are frequently correlated with predisposing factors such as immunosuppression caused by other diseases or corticoid treatment (Keiser and Nutman, 2004). Although the medical literature contains some case reports of disseminated strongyloidiasis in diabetic ∗ Corresponding author. Tel.: +55 34 32182187; fax: +55 34 32182333. E-mail address: [email protected] (J.M. Costa-Cruz).
0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2006.06.006
patients (Venturi and Viliotti, 1984; Debussche et al., 1988; Coovadia et al., 1993; Higashiyama et al., 1997; Ho et al., 1997; Al Samman et al., 1999; Emad, 1999; Linder et al., 2000), no controlled study was found that attempted to determine whether diabetics are predisposed to chronic strongyloidiasis. It is important to understand that the transmission of S. stercoralis is totally independent of the acquisition of diabetes mellitus. Because of the risk of fatality in these severe forms of infection, and because diabetes mellitus is highly prevalent worldwide, the frequency of association between positive S. stercoralis serology and this metabolic disorder is considered extremely important. This study involved a total of 120 ambulatory patients of the Federal University of Uberlˆandia School of Medicine Endocrinology Unit, state of Minas Gerais,
S.C.L. Mendon¸ca et al. / Acta Tropica 99 (2006) 102–105
Brazil. Of these, 78 type 2 diabetics were selected consecutively, 32 males and 46 females with a mean age of 54.1 ± 11.9 years (ranging from 28 to 75) and 42 controls, 17 males and 25 females with a mean age of 53.9 ± 10.7 years (ages from 30 to 77). The controls were ambulatory patients with other endocrinal diseases included: hypothyroidism (33.3%), obesity (28.5%), treated hyperthyroidism (16.7%), atoxic multinodular goiter (9.5%), dyslipidemia (4.8%) and one case of prolactinoma, acromegaly and thyroid nodular disease (7.2%), undergoing treatment by the same physician during the same period as the diabetics. All subjects seen at this public hospital located in a hyperendemic area for strongyloidiasis had similar background and current socio-economic conditions. This study was approved by the Federal University of Uberlˆandia’s Ethical Committee. Blood samples were collected from all the patients to determine their eosinophil count. Glycated hemoglobin (HbA1c ) was analyzed in diabetics using the Bayer DCA2000 analyzer. Three fecal samples were collected from each subject on consecutive days and stored in plastic receptacles without preservatives at 4 ◦ C. Parasitological diagnoses were carried out using Baermann’s (1917) and Hoffmann et al.’s (1934) method. Three slides of each of the 360 samples were prepared for the Baermann analysis and three for the Hoffmann et al. analysis, making a total of 2160 slides examined by two investigators using a Nikon (Japan) microscope at 100 and 400× magnification. The IFAT was carried out according to Costa-Cruz et al. (1997) using cryo-microtome section of S. stercoralis larvae. The results were expressed as antibody titers, which were considered positive when ≥20. Reactive serum samples were retested in two-fold serial dilutions up to the end-point titer. ELISA and WB were performed as described by Silva et al. (2003) using saline extracts of S. stercoralis, obtained from 420,000 filariform larvae. For ELISA the results were expressed as antibody titers which were considered positive when ≥80. For WB sera samples were considered positive if at least two immunodominant proteins (molecular weight of 17, 20, 23, 25, 26, 28, 30, 31, 40, 41, 55, 66, 90, 97 or 100 kDa) of S. stercoralis fractions were recognized (Genta et al., 1988; Sato et al., 1990; Conway et al., 1993b; Lindo et al., 1994). The association between positive S. stercoralis serology and diabetes was calculated as an odds ratio and expressed as an estimated relative risk. The confidence interval for the odds ratio was calculated by Fleiss’s method (1979). The statistical significance of associations was calculated using the χ2 statistic with the Yates
103
correction. The mean age between groups was compared by Student’s t-test. Differences were considered statistically significant when P < 0.05. The frequency of positive stool examination in diabetic patients was 3.8% (three cases) being all control individuals negative. In addition to S. stercoralis larvae, the coprological examination revealed one diabetic woman with hookworm eggs and another with Giardia lamblia cysts. The frequency of positive S. stercoralis serology in diabetics was 23% (18 cases) versus 7.1% (3 cases) in the control group (P < 0.05). The odds ratio for diabetics was 3.9 (CI, 1.6–15.9, P < 0.05). The three patients whose stool examination detected S. stercoralis larvae also tested positive in the IFAT, ELISA and WB tests. The efficacy of the stool examination in detecting Strongyloides infection was very low: 18 out of 21 patients with positive serology had negative coprologic examination results. Curiously, one patient whose third stool sample contained a large number of larvae had two previous samples which tested negative for S. stercoralis infection. One patient with a normal white cell count had 16% eosinophils, another patient had a history of intermittent diarrhea and 5% eosinophilia and a third patient whose stool examination tested positive was asymptomatic, without eosinophilia. No other patient with positive serology presented eosinophilia, indicating that the majority of the cases did only have signs of a recent, but not present infection. Table 1 shows the characteristics of patients with positive tests, glycated hemoglobin (for diabetics only), IFAT and ELISA results expressed as titers and the immunodominant antigenic component identified by WB. Positive S. stercoralis serology was detected in 9 male (18.3%) and 12 female patients (16.9%). The mean age for positive patients was 59.2 years for females and 54.4 years for males. There was congruence between the serological tests for 79.5% of the diabetics and for 71.4% of the control group. Patients were considered negative, even when IFAT and/or ELISA were positive, if at least two immunodominant antigenic components were undetected in the WB test. A correlation was drawn between the frequency of S. stercoralis infection and the glycated hemoglobin level (HbA1c ) in order to estimate the extent to which the diabetic whose disease is inadequately controlled is predisposed to remain chronically infection. Today, the HbA1c measurement is the best way to demonstrate chronic metabolic control, given the information about the mean serum glucose level over the previous 120 days. Among the 39 cases with good metabolic control (HbA1c < 7), only 9% tested positive for S. stercoralis compared with 14% of those with poor metabolic control.
104
S.C.L. Mendon¸ca et al. / Acta Tropica 99 (2006) 102–105
Table 1 Characteristics of patients testing positive for strongyloidiasis in terms of sex, age, endocrinal disease, glycated hemoglobin (for diabetics only), IFAT and ELISA test results expressed in titers and protein fractions detected by Western blotting Sex
Age
Diagnosis
Stool examination
HbA1c (%)
IFAT
ELISA
Protein fractions (kDa)
F M M F F M F F M F F M M F M M F F M F F
60 63 51 62 50 39 64 46 48 78 52 53 45 75 62 54 47 60 75 52 64
Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Diabetes Hypothyroidism Hypothyroidism Hypothyroidism
Positive Positive Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
9.2 9.6 10 8 7 6 12.4 10 10.2 5.8 7 6 9.9 7.9 6.2 6.3 11.4 7.5 ND ND ND
40 20 40 40 20 Negative 20 40 20 40 40 20 20 20 40 Negative 20 Negative 20 40 20
160 1280 320 160 80 80 80 160 320 2560 2560 2560 640 80 320 160 160 80 320 2560 160
17, 20, 30, 87, 90 17, 20, 30, 40, 55 20, 23, 25, 30, 90 50, 55, 66, 78 55, 66, 90 23, 25, 40, 55, 66 40, 66 17, 20, 30, 78, 90 40, 78, 90 14, 17, 20, 25, 55, 66, 90 17, 20, 25, 55, 66, 90 17, 30, 40, 55, 90 31, 90 60, 66, 90 90, 78, 25, 20, 17, 13 90, 66, 55, 40, 25, 20, 17 90, 66, 55, 25, 20, 17 17, 20, 31, 85 31, 40, 41 17, 20, 25, 55, 66, 90 23, 25, 31, 87
ND: not done; M: male; F: female.
This study suggests an association between diabetes mellitus and the serodiagnosis of strongyloidiasis. Definitive diagnosis of strongyloidiasis is usually made on the basis of detection of larvae in the stool. However, in a majority of uncomplicated cases of strongyloidiasis, mainly in endemic regions, the intestinal worm load is often very low, leading to low larval densities in feces and fluctuations in larval excretion. It is important to note that a negative result does not necessarily indicate an unequivocal absence of the infection (Uparanukraw et al., 1999; Sudarshi et al., 2003; Keiser and Nutman, 2004). Hence, there is a great need for highly specific and efficient serodiagnostic tests for S. stercoralis. Currently, the most widely used method has been the ELISA using filariform larvae antigen. Most reports on the ELISA for strongyloidiasis have shown consistently high sensitivity and specificity, varying from 83.8 to 94.6 and 90.2 to 100%, respectively, and positive and negative predictive values of 97 and 95%, respectively (Genta, 1988; Conway et al., 1993a; Lindo et al., 1994; Sudarshi et al., 2003). Grove (1982) followed 43 patients for 6 months after thiabendazole treatment demonstrated a significant fall in anti-Strongyloides serum antibody level in many patients who were negative in the followup fecal examination. Genta and Weil (1982) also followed serologically, for 2–4 months, seven patients after specific treatment and demonstrated a significant
decrease in antibody titers to filariform larvae in all of them. There is no clear explanation for the high occurrence of positive test results for strongyloidiasis in diabetic patients. Despite the numerous studies that have evaluated the immune system in diabetes, it has become increasingly difficult to compare them. For this reason, even after decades of investigation, questions about whether diabetes itself results in specific immunological defects and how such defects might predispose the patient to infection are still being debated (Bessman and Sapico, 1992; Sentochnik and Eliopoulos, 1994). One controlled study in Turkey (Nazligul et al., 2001) found no predisposition for intestinal parasitosis in diabetic patients; the diagnosis was based on only one stool sample from each patient and no cases of S. stercoralis infection were found. Our results provide evidence supporting the hypothesis of a possible association between positive S. stercoralis serology and diabetes. Provided there are related cases of disseminated strongyloidiasis in diabetics and there is a higher frequency of asymptomaticity of the infection in this group, the immunological screening of these patients at risk could prevent severe and fatal outcomes of the disease, particularly if there is poor metabolic control of the disease. Considering the high efficacy and low toxicity of new drugs used in the treatment of strongyloidiasis, diabetic patients should be
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treated in the presence of eosinophilia if they are to undergo corticosteroid therapy or in situations of severe metabolic distress such as ketoacidosis. In most parasitic infections, exposure to the organism plays the major role in determining infection. However, because of its unusual life cycle, S. stercoralis may be different and host factors are associated with long-term S. stercoralis infection. Although it is not clear that the association is actually a case of cause and effect, these factors should be taken into account, together with determinants of exposure, when studying the causes of chronic S. stercoralis infection and estimating the likelihood of infection in clinical practice. Acknowledgement To Maria das Grac¸as Marc¸al for parasitological diagnosis supports. References Al Samman, M., Haque, S., Long, J.D., 1999. Strongyloidiasis colitis: a case report and review of the literature. J. Clin. Gastroenterol. 28, 77–80. Baermann, G., 1917. Eine einfache Methode zur Auffinfung von Ankylostomun (Nematoden) Larven in Erdproben. Mededeel. mith.H Geneesk. Batavia, Lab. Weltevreden Feestbundel, pp. 41–47. Bessman, A.N., Sapico, F.L., 1992. Infections in the diabetic patient: the role of immune dysfunction and pathogen virulence factors. J. Diabetes Complications 6, 258–262. Conway, D.J., Atkins, N.S., Lillywhite, J.E., Bailey, J.W., Robinson, R.D., Lindo, J.F., Bundy, D.A., Bianco, A.E., 1993a. Immunodiagnosis of Strongyloides stercoralis infection: a method for increasing the specificity of the indirect ELISA. Trans. R. Soc. Trop. Med. Hyg. 87, 173–176. Conway, D.J., Bailey, J.W., Lindo, J.F., Robinson, R.D., Bundy, D.A.P., Bianco, A.E., 1993b. Serum IgG reactivity with 41-, 31-, and 28kDa larval proteins of Strongyloides stercoralis in individuals with strongyloidiasis. J. Infect. Dis. 168, 784–787. Coovadia, Y.M., Rajput, M.C., Bhana, R.H., 1993. Disseminated strongyloidiasis in a diabetic patient. Trop. Geogr. Med. 45, 179–180. Costa-Cruz, J.M., Bullamah, C.B., Gonc¸alves-Pires, M.R.F., Campos, D.M.B., Vieira, M.A., 1997. 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˜ao Paulo 39, 313–317. Debussche, X., Toublanc, M., Camillieri, J.P., Assan, R., 1988. Overwhelming strongyloidiasis in a diabetic patient following ACTH treatment and Keto-acidosis. Diab. Metab. 14, 294–298. Emad, A., 1999. Exsudative eosinophilic pleural effusion due to Strongyloides stercoralis in a diabetic man. South. Med. J. 92, 58–60. Fleiss, J.L., 1979. Confidence intervals for the odds ratio in case-control studies: the state of the art. J. Chronic Dis. 32, 69–77. Genta, R.M., 1988. Predictive value of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of strongyloidiasis. Am. J. Clin. Pathol. 89, 391–394.
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