Association between Strongyloides stercoralis infection and cortisol secretion in alcoholic patients

Acta Tropica 154 (2016) 133–138

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Association between Strongyloides stercoralis infection and cortisol secretion in alcoholic patients Mônica L.S. Silva a , Elizabete de J. Inês a , Alex Bruno da S. Souza a , Victória Maria dos S. Dias a , Cléa M. Guimarães b , Edimacia R. Menezes b , Larissa G. Barbosa b , Maria Del Carmen M. Alves b , Márcia Cristina A. Teixeira a , Neci M. Soares a,∗ a b

Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal da Bahia, Salvador, Bahia, Brazil Obras Sociais Irmã Dulce, Salvador, Bahia, Brazil

a r t i c l e

i n f o

Article history: Received 14 May 2015 Received in revised form 13 October 2015 Accepted 14 November 2015 Available online 22 November 2015 Keywords: Strongyloides stercoralis Cortisol Alcoholism Diagnosis

a b s t r a c t A higher prevalence of Strongyloides stercoralis infections has been reported in alcoholic patients compared to nonalcoholic patients living in the same area. Excessive alcohol consumption increases the levels of endogenous corticosteroids that subsequently enhance the fecundity of S. stercoralis parthenogenetic females. These corticosteroids also enhance the transformation of rhabditiform larvae into infective filariform larvae by mimicking the effect of the ecdysteroid hormones produced by the parasite, thus leading to autoinfection. In addition, alterations in the intestinal barrier and host immune response contribute to the development of hyperinfection and severe strongyloidiasis in alcoholic patients. The aim of this study was to evaluate the frequency of S. stercoralis infections in alcoholic patients and to determine the association between S. stercoralis infection and endogenous cortisol levels. The frequency of infection was evaluated in 332 alcoholic and 92 nonalcoholic patients. The parasitological diagnosis was carried out by agar plate culture, the modified Baermann–Moraes method and spontaneous sedimentation. The immunological diagnosis was performed using an ELISA with anti-S. stercoralis IgG. The cortisol levels were measured in serum samples by ELISA. The frequency of S. stercoralis infection in alcoholic patients was 23.5% (78/332), while in nonalcoholic patients, it was 5.4% (5/92) (p < 0.05). The cortisol levels were higher in alcoholic than in nonalcoholic patients (p < 0.05). However, among the alcoholic patients, the cortisol levels did not differ between S. stercoralis-infected and uninfected patients (p > 0.05). As demonstrated in this work, 81.3% (26/32) of patients with a high parasite load, considered as more than 11 larvae per gram of feces, presented serum cortisol levels above the normal reference value (24 mg/dL). High endogenous cortisol levels in alcoholic patients were not associated to susceptibility to S. stercoralis infection, however once infected, this may lead to a high parasite load. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Strongyloides stercoralis infection is widely distributed in tropical and subtropical regions and is considered one of the most neglected among the soil-transmitted helminthes (Schär et al., 2013). It is estimated that approximately 100 million people are infected worldwide (Olsen et al., 2009), with high prevalence in areas with low socioeconomic and sanitation conditions (Borda et al., 1996; Gamboa et al., 2009). The prevalence of infection in Sal-

∗ Correspondence to: Rua Barão de Jeremoabo, s/n Campus Universitário de Ondina, Ondina, Salvador, Bahia 40170 115, Brazil. Fax: +55 71 32836919. E-mail addresses: [email protected], [email protected] (N.M. Soares). http://dx.doi.org/10.1016/j.actatropica.2015.11.010 0001-706X/© 2015 Elsevier B.V. All rights reserved.

vador, Brazil ranges from 4.6 to 6.6% (Inês et al., 2011; Santos et al., 2007); this region is classified as a hyperendemic region (Pires and Dreyer, 1993). S. stercoralis infections generally present as subclinical or chronic forms and may persist for decades. Severe strongyloidiasis mainly affects patients with impaired immunity, such as those infected with human T-lymphotropic virus type 1 (HTLV-1), (Chieffi et al., 2000; Furtado et al., 2013; Porto et al., 2002), patients taking glucocorticoids (Fardet et al., 2007; Koticha et al., 2013; Valar et al., 2007) and alcoholics (Oude Lashof et al., 2007; Teixeira et al., 2010). The high prevalence of S. stercoralis infection, above 20%, in chronic alcoholic patients has been demonstrated in southeast Brazil (Gaburri et al., 1997; Marques et al., 2010; Oliveira et al., 2002; Zago-Gomes et al., 2002) and is likely due to dulled men-

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tal function, the breakdown of local protective barriers, exposure to pathogens and malnutrition (MacGregor, 1986). Ethanol intoxication can elevate human endogenous corticosteroids that in turn suppress T cell function and increase the fecundity and survival of the parasites. Increased cortisol levels stimulate the transformation of rhabditiform larvae into filariform larvae by mimicking the effect of worm ecdysteroids; this transformation then leads to autoinfection (Concha et al., 2005). In addition, the chronic consumption of alcohol has a toxic effect on the smooth muscle contractile proteins of the small intestine and on vagal function, the reducing gastrointestinal transit and enhancing the risk of autoinfection (Addolorato et al., 1997; Wegener et al., 1991). The diagnosis of S. stercoralis infection generally relies on the detection of larvae in stool samples. However, the majority of cases involve a chronic infection with an intermittent and small larvae load in the feces, decreasing the sensitivity of parasitological tests (Dreyer et al., 1996; Uparanukraw et al., 1999) and consequently resulting in the underestimation of the prevalence of infection. Some studies comparing the efficacy of parasitological methods for S. stercoralis detection have demonstrated that agar plate culture is the most suitable method due to its high sensitivity (Inês et al., 2011; Jongwutiwes et al., 1999; Kobayashi et al., 1996). The realtime PCR is more sensitive and specific than PCR conventional for detecting S. stercoralis in human stool (de Paula et al., 2015). However, in chronic infection with low larvae load it had less sensitivity (Sultana et al., 2013) and, it is not readily adaptable for use in the clinical settings where laboratory facilities are often limited. Serological methods, despite their limited use in clinical laboratories, may support the diagnosis of strongyloidiasis by providing high sensitivity and specificity (Inês et al., 2013; Van Doorn et al., 2007). Recent studies have tested a 31-kDa recombinant antigen of S. stercoralis (termed NIE) in both ELISA and in the luciferase immunoprecipitation system (LIPS). The results suggest that these tools are useful not only for diagnostic purposes and prevalence studies (Bisoffi et al., 2014; Krolewiecki et al., 2010), but also to follow-up patients who received S. stercoralis treatment, to exclude persistence of infection (Buonfrate et al., 2015). A reliable diagnosis is particularly important for risk groups to prevent hyperinfection and disseminated strongyloidiasis. In the present study, we showed that (1) chronic alcoholism itself is an important factor that predisposes people to S. stercoralis infection, corroborating with previous studies; (2) there is a strong correlation between S. stercoralis infection and circulating antiS. stercoralis IgG, and (3) there is a positive correlation between endogenous cortisol levels and parasite load in alcoholic patients with S. stercoralis infection. To our knowledge, this is the first report demonstrating the association between cortisol levels and parasite load in alcoholic patients.

used corticosteroids or any other immunosuppression drugs, neither were infected with immunosuppressive agents, such as HIV or HTLV-1. The great majority of patients (75.2%) in both groups came from upcoming neighborhoods, presented similar socioeconomic characteristics and, probably were submitted to the same risks of parasite infection. This study was approved by the Committee of Ethics in Research of the Nursing School, Federal University of Bahia, Brazil, and a written informed consent for participation was assigned from each patient when the clinical specimens were acquired. All laboratory results were send to patients or to the respective physician. 2.2. Strongyloidiasis diagnosis Three fresh fecal samples from each subject were examined by three different parasitological methods, spontaneous sedimentation, the modified Baermann–Moraes method and agar plate culture (APC), on alternate days. S. stercoralis larvae from one gram of feces obtained by the modified Baermann–Moraes method were quantified under a microscope (400× magnification). Detection of anti-S. stercoralis IgG was performed by ELISA (Inês et al., 2013). 2.3. Serum cortisol levels Serum samples from 78 alcoholic patients infected with S. stercoralis, 80 alcoholic and 76 nonalcoholic patients without S. stercoralis infection, as confirmed by three negative parasitological tests, were collected in the morning between 7:00 and 9:30 a.m. to measure the cortisol concentration by ELISA, following the manufacturer’s instructions (Cortisol AccuBindTM EIA, Monobind Inc., USA). 2.4. Strongyloides stercoralis soluble antigen S. stercoralis third-stage infective larvae (L3) were obtained from the feces of a hyperinfected patient. The larvae were cultured in animal charcoal for five days at 28 ◦ C, collected and concentrated by Rugai’s method, and washed 5 times in 0.15 mol/L of phosphate buffered saline (PBS, pH 7.2) by centrifuging for 7 min at 1.8 × g. Parasites were suspended for 5 min in 0.25% sodium hypochlorite and then washed 5 times in PBS as described above. The larvae were suspended in PBS with protease inhibitors (5 mmol/L EDTA, 1 mmol/L phenyl-methyl sulfonylfluoride [Sigma], 0.05 mmol/L TPCK/TLCK, 1 ␮g/mL leupeptin) and sonicated in an ice bath for 9 cycles of 80 s each at 40 kHz (Branson Sonifier Cell Disruptor, Branson Instruments, Danbury, CT, USA). The larvae homogenate was then centrifuged at 11,000 × g for 30 min at 4 ◦ C. The supernatant was analyzed for protein content according to Lowry et al. (1951), divided into aliquots, and stored at −70 ◦ C until use.

2. Materials and methods

2.5. Enzyme-linked immunosorbent assay

2.1. Patients and samples details

Indirect ELISAs for specific IgG were conducted as previously described by Inês et al. (2013). Briefly, the microtiter plates (Corning Costar polystyrene EIA/RIA plates, Corning) were coated with 10 ␮g/mL of S. stercoralis soluble antigen in 0.06 mol/L carbonate–bicarbonate buffer (pH 9.6), incubated overnight at 4 ◦ C, and washed 3 times with PBS containing 0.05% Tween-20 (PBS-T). The plates were blocked with 100 ␮L PBS-T containing 8% w/v skim milk (PBS-T-Milk) for 1 h at 37 ◦ C. After blocking, the wells were washed as described previously. Sera samples diluted at 1:100 were incubated in duplicate at a volume of 100 ␮L of sera per well for 1 h at 37 ◦ C. Then, 100 ␮L of conjugated anti-human IgG linked to horseradish peroxidase (Sigma–Aldrich, St. Louis, MO, USA) diluted at 1:4000 was added to the plate and incubated under the same conditions. The reaction was visualized by the addition of 100 ␮L of

The present study was conducted from September 2012 to March 2014 with 424 subjects attended by the National Health System in Salvador, Bahia, Brazil, that has the main constitutional guideline based on the integration of public actions and services in a regionalized and hierarchical network. From these, 332 were chronic alcoholics voluntarily hospitalized for alcoholism treatment (mean age of 43.9 ± 9.7), diagnosed according to the WHO criteria (F10.2, ICD 10, 2002) and 92 nonalcoholic, apparently healthy individuals attended at an outpatient service (mean age of 47.0 ± 13.7). The inclusion criteria were the following: adult males with fecal examination results and sure information on presence or absence of daily ethanol intake. None of the subjects had

M.L.S. Silva et al. / Acta Tropica 154 (2016) 133–138 Table 1 Strongyloides stercoralis and other intestinal parasite infections in alcoholic (n = 332) and nonalcoholic (n = 92) patients diagnosed by three different methods (APC, spontaneous sedimentation and modified Baermann–Moraes). Parasite

Strongyloides stercoralis Hookworm Ascaris lumbricoides Schistosoma mansoni Trichuris trichiura Hymenolepis nana Taenia sp. Giardia duodenalis Entamoeba histolytica/dispar Entamoeba coli Endolimax nana Iodamoeba butschlii Total

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Table 2 Strongyloides stercoralis infection diagnosed by parasitological methods and specific IgG detected by ELISA in alcoholic and nonalcoholic patients. Parasitological methodsa

Number of positive samples (%)

Alcoholics

Alcoholics

Nonalcoholics

p Value

78 (23.5%)* 39 (11.7%) 10 (3.0%) 29 (8.7%) 8 (2.4%) 2 (0.6%) 1 (0.3%) 5 (1.5%) 3 (0.9%) 14 (4.2%) 31 (9.3%) 0 (0%) 332

5 (5.4%) 1 (1.1%) 0 (0%) 4 (4.3%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 12 (13.0%) 29 (31.5%) 5 (5.4%) 92

<0.0001** 0.0009** 0.13 0.19 0.21 1.00 1.00 0.59 1.00 0.005 <0.0001 0.0004

ELISA

Positive Negative Total

Nonalcoholics

Positive

Negative

Total

Positive

Negative

Total

63 15 78

10 244 254

73 259 332

4 0 4

2 73 75

6 73 79

Agreement between ELISA and parasitological methods (Kappa index: 0.786). a Spontaneous sedimentation, modified Baermann–Moraes and agar plate culture. Table 3 Cortisol levels in alcoholic and nonalcoholic S. stercoralis-infected and uninfected patients. Alcoholics*

Nonalcoholics

*

Frequency of S. stercoralis in comparison with other parasites in alcoholic patients, p < 0.05. ** Frequency of S. stercoralis and hookworm in alcoholic and nonalcoholic patients, p < 0.05.

substrate (100 ␮L of 0.051 mol/L citrate–phosphate buffer [pH 5.0] containing 0.0037 mol/L p-phenylenediamine and 0.04% hydrogen peroxide), and incubating the plate for 20 min in the absence of light, followed by the addition of 20 ␮L of 8N sulfuric acid to stop the reaction. The absorbance (Abs) was measured at 450–630 nm with a microplate reader (Awareness Technology, USA). 2.6. Statistical analysis Statistical analyses were performed using the statistical software GraphPad 5.0 (San Diego, USA). The ELISA cut-off value, sensitivity and specificity were established by the ROC curve (receiver operating characteristic) using 40 sera samples from healthy adult individuals who were members of the laboratory staff and 23 samples from patients with other intestinal parasites as negative controls and 35 sera samples from S. stercoralis monoinfected patients as positive controls. Fisher’s exact test was performed for comparisons between frequencies of parasites among alcoholics and nonalcoholics and for cortisol levels and S. stercoralis infection. Frequencies by parasitological and immunological methods were calculated with 95% confidence intervals. The Spearman correlation test was used to correlate the cortisol levels with the parasite load in infected alcoholic patients. Agreement between ELISA and parasitological diagnosis of strongyloidiasis was achieved by Kappa index. Differences were considered as statistically significant when p < 0.05. 3. Results 3.1. S. stercoralis infection in alcoholic patients The mean ages of the alcoholic and nonalcoholic patients were 43.9 ± 9.7 and 47.0 ± 13.7 years, respectively. Table 1 shows that the frequency of S. stercoralis (23.5%; 95% CI: 19.2–28.3; 78/332) and hookworm (11.7%; 95% CI: 8.7–15.7; 39/332) in alcoholics was significantly higher than in nonalcoholic patients to S. stercoralis (5.4%; 95% CI: 2.0–12.4; 5/92) and hookworm (1.1%; 95% CI: 0.01–6.5; 1/92) infections (p < 0.05). Other intestinal parasites were identified in both groups as shown in Table 1. Polyparasitism was found in 13.3% (44/332) and 14.1% (13/92) of alcoholic and nonalcoholic patients, respectively. Among the 78 S. stercoralis infected patients, nine (11.5%) were infected with hookworm. The coinfections were confirmed by the finding

S. stercoralis infection Cortisol levels

Positive n (%)

Negative n (%)

Positive n (%)

Negative n (%)

5–23 ␮g/dL 24–30 ␮g/dL >30 ␮g/dL Total Mean ± SD

21 (26.9) 29 (37.2) 28 (35.9) 78 28.2 ± 6.5

21 (26.3) 42 (52.5) 17 (21.2) 80 26.3 ± 5.2

4 (100) 0 (0) 0 (0) 4 14.2 ± 6.4

60 (80) 14 (18.4) 2 (2.6) 76 17.5 ± 6.9

*

Cortisol levels in alcoholic and nonalcoholic patients (p < 0.05).

of hookworm eggs in the spontaneous sedimentation technique and by the examination of larva morphology diagnosed by Baermann–Moraes and APC. The ELISA cut off value obtained in this study was 0.135. Sensitivity and specificity by ROC curve were 88.6% and 98.4%, respectively. The overall frequency of anti-S. stercoralis IgG in alcoholic patients was 22% (95% CI: 17.9–26.8; 73/332), significantly higher than in nonalcoholic patients 7.6% (95% CI: 3.2–15.9; 6/79). Although the ELISA for specific IgG showed high level of agreement when compared with parasitological methods (k = 0.786), it was unable to detect specific IgG in 15 sera samples from S. stercoralis infected alcoholic patients. However, 10 sera samples from alcoholic patients with negative parasitological findings for S. stercoralis and other infections by parasites were positive for anti-S. stercoralis IgG by ELISA (Table 2). 3.2. Evaluation of endogenous cortisol levels in S. stercoralis-infected and uninfected alcoholic patients Serum cortisol levels were higher (73.4%; 116/158) among the alcoholic patients, ranging from 6.5 to 41.3 ␮g/dL, than in the nonalcoholic patients (20%; 16/80), ranging from 3.8 to 36.3 ␮g/dL (p < 0.05). However, as shown in Table 3, there were no significant differences between cortisol levels in the serum from S. stercoralis-infected (mean = 28.2 ± 6.5 ␮g/dL) and uninfected (mean = 26.3 ± 5.2 ␮g/dL) alcoholic patients (p > 0.05). There was a positive correlation between high cortisol levels and a parasite load ranging from 11 to 100 larvae per gram of feces (Table 4, r = 0.2; p < 0.05). 4. Discussion Chronic alcoholism is an important factor that predisposes individuals to S. stercoralis infection. Marques et al. (2010) and Zago-Gomes et al. (2002) in Vitória, ES, Brazil, found infection frequencies of 20.5% and 21.7% in alcoholic patients, respectively. In alcoholic patients with liver cirrhosis, the frequencies ranged from 40.2% to 44.4% (Gaburri et al., 1997; Oliveira et al., 2002). Fur-

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Table 4 Cortisol levels (mg/dL) and parasite load (larvae per gram of feces) in S. stercoralisinfected alcoholic patients (n = 78). Serum cortisol levels (␮g/dL) Parasite load

5–23

>24

Number of patients

1–10 11–50 51–100 >100

15 (32.6%) 2 (11.8%) 2 (50.0%) 2 (18.2%)

31 (67.4%) 15 (88.2%)* 2 (50.0%) 9 (81.8%)

46 17 4 11

*

Positive correlation between cortisol levels and parasite load (p < 0.05; r = 0.2).

thermore, a meta-analysis of three case-control studies in Brazil, showed a significantly increased risk of S. stercoralis infection in the alcoholic patients (OR: 6.69, CI: 1.47–33.8) (Schär et al., 2013). In the present study, the frequency of helminthes in alcoholic and nonalcoholic patients groups showed significant difference for both S. stercoralis and hookworm (p < 0.0001 and p = 0.0009, respectively). The poor hygienic habits of alcoholic patients related with soil-transmitted helminthes may have increased the risk of infection to these parasites. However, the occurrence of S. stercoralis in alcoholics was two times higher (p < 0.05) than the frequency of hookworm observed. The autoinfection process associated to altered defense mechanisms against S. stercoralis could be involved in rendering alcoholic patients more susceptible to the parasite. Serological tests for the detection of parasite-specific antibodies may be a useful alternative method to support the strongyloidiasis diagnosis due to the difficulty of detecting larvae by traditional parasitological methods. The enzyme-linked immunosorbent assay (ELISA) is the most widely used serological test and presents high sensitivity and specificity that can reach 100%, depending on the antigen, specimen type and antibody isotype (Feliciano et al., 2010; Gonc¸alves et al., 2012; Inês et al., 2013; Koosha et al., 2004; Rodrigues et al., 2007; Van Doorn et al., 2007). In this study, the ELISA sensitivity (88.6%) and specificity (98.4%) were higher than those obtained previously in our laboratory (Inês et al., 2013) that reach 76.0% and 92.9%, respectively. The improvement in the performance of ELISA can be explained by the antigen preparation using filariform larva cultivated in animal charcoal, followed by Rugai’s method concentration, cleaning fecal debris so often observed in larva obtained directly from agar plate cultures. Although serology is a complementary diagnostic tool, it might overestimate the prevalence of disease due to cross-reactivity with other nematode infections and its difficulty in distinguishing between recent and past infections (Requena-Méndez et al., 2013). In this study, anti-S. stercoralis IgG was detected in the sera of 80.8% (n = 63) of alcoholic patients infected with S. stercoralis. Generally, IgG titers are detected two weeks after infection and can persist for up to 20 weeks post infection (Levenhagen and Costa-Cruz, 2014). However, we showed here that 19.2% of S. stercoralis-infected alcoholic patients did not have circulating S. stercoralis-specific IgG detected by ELISA (n = 15). It is possible that alterations in the immune response induced by corticosteroid metabolites modified antibody production, as previously demonstrated by others studies of immunocompromised patients (Abdul-Fattah et al., 1995; Huaman et al., 2003; Schaffel et al., 2001). Moreover, as the sensitivity of our in-house ELISA was not reached 100%, we should not exclude a few false-negative results, especially with patients producing low antibody levels to S. stercoralis. Alcohol induces modulation of both the innate and adaptive immune response. The effects may vary depending on acute or chronic exposure, the time of alcohol consumption and the blood alcohol level (Crews et al., 2006). In vitro and in vivo studies have demonstrated a decreased ability of antigen presentation by monocytes and dendritic cells after alcohol exposure (Mandrekar et al., 2004; Szabo et al., 1993, 1995, 2001). Chang et al. (2002) argued

that the decreased B cell proliferation in ethanol-consuming mice was due in part to excessive IL-4 production and an inability of the B cell to interact properly with IL-4. On the other hand, 3.9% of sera samples from alcoholic patients without infection by intestinal parasites (n = 10) were reactive to S. stercoralis antigens by ELISA, which suggests either immunological memory of past infections and/or current infection with low parasite discharge, despite the use of the highly sensitive APC method. Moreover, cross reactions with antigens from other intestinal parasites could result in false positive reactions. In spite of high sensitivity and specificity of our in-house ELISA would be interesting to compare it with other accurate methods, such as PCR and NIE-LIPS. Different mechanisms may be responsible for the stimulation of hyperinfection in susceptible hosts, although corticosteroid therapy remains the most frequent risk factor. Acute or chronic ethanol administration in rats increases the plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone and thus interferes with the infective filariform larvae and exacerbates the autoinfection (Concha et al., 2005). High cortisol levels have been demonstrated in alcoholic patients (Gianoulakis et al., 2003; Thayer et al., 2006). Marques et al. (2010) demonstrated a positive correlation between the frequency of S. stercoralis infection with the daily ethanol ingestion. In this work, the cortisol levels in alcoholic patients were 1.7 times higher than in nonalcoholic patients, and a positive correlation between serum cortisol concentration and the parasite load of S. stercoralis infection was demonstrated for the first time, to our knowledge. The high endogenous cortisol levels were not a predisposing factor for S. stercoralis infection, but, once infected, high levels of metabolic cortisol may increase the differentiation of rhabditiform larvae into infective filariform larvae, thus favoring autoinfection. In this way, the amount of females may be increased in the duodenum, and the number of rhabditiform larvae in the stools may be enhanced, thus increasing the chance of identifying larvae during a fecal examination. Furthermore, chronic alcohol ingestion can directly change the morphology of the intestinal villi, thereby altering mucosal permeability (Worthington et al., 1978), decreasing intestinal peristalsis, and favoring the delay of rhabditiform larvae in the intestinal lumen. The alteration of the host immune response is a factor that contributes to parasite survival (Oliveira et al., 2002; Zago-Gomes et al., 2002). Strongyloidiasis is primarily controlled by the Th2 subset of CD4 lymphocytes. Eosinophils act as antigen-presenting cells and increase IL-4, IL-5, IL-13 production, consequently inducing specific IgE, IgM and IgG antibodies, favoring the elimination of the parasite (Galioto et al., 2006; Padigel et al., 2006, 2007). A modified immunological response against parasite antigens was observed in individuals coinfected with S. stercoralis and human T-lymphotropic virus type 1 (HTLV-1); these individuals had increased production of IFN-␥ and decreased IL-4, IL-5, and IL-13 levels and IgE response (Carvalho and Porto, 2004; Machado et al., 2004; Porto et al., 2002). This deviation of the immune response toward a Th1 response also results in lower serum antibody levels and decreased eosinophil production, thus impairing the host defense against helminth infection (Porto et al., 2002). However, studies in humans have shown a reduction in Th1 cytokines and an increase in the Th2 cytokine profile following alcohol exposure (Dominguez-Santalla et al., 2001; Szabo, 1999). Furthermore, mice maintained chronically on an ethanolcontaining diet had some alterations in their ability to produce Th1 and Th2 immune responses yet were capable of generating a protective Th2 immune response against S. stercoralis infection (Krolewiecki et al., 2001). Teixeira et al. (2010) reported a clinical case of S. stercoralis hyperinfection in a chronic alcoholic patient with preserved Th2 immune response. In this study, the majority of S. stercoralis-infected alcoholic patients had an increased par-

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asite load, although disseminated strongyloidiasis had not been observed in any of these patients. The cellular immune response in S. stercoralis-infected alcoholic patients may include immunoregulatory mechanisms that control disseminated strongyloidiasis, but this has not yet been studied. High frequency of S. stercoralis infection in ethanol abusers may be multifactorial involving exposure to S. stercoralis infection, malnutrition, break down of local imune responses, and/or alterations in intestinal barriers. Moreover, high levels of human endogenous corticosteroid may stimulate the autoinfection leading to a high parasite load, as observed herein. Acknowledgments This work was supported by Ministério da Saúde/Fundac¸ão de Amparo à Pesquisa do Estado da Bahia (PPSUS/FAPESB) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazilian agencies. References Abdul-Fattah, M.M., Nasr, M.E., Yousef, S.M., Ibraheem, M.I., Abdul-Wahhab, S.E., Soliman, H.M., 1995. Efficacy of ELISA in diagnosis of strongyloidiasis among the immune-compromised patients. J. Egypt. Soc. Parasitol. 25, 491–498. Addolorato, G., Montalto, M., Capristo, E., Certo, M., Fedeli, G., Gentiloni, N., Stefanini, G.F., Gasbarrini, G., 1997. Influence of alcohol on gastrointestinal motility: lactulose breath hydrogen testing in orocecal transit time in chronic alcoholics, social drinkers and teetotaler subjects. Hepatogastroenterology 44, 1076–1081. Bisoffi, Z., Buonfrate, D., Sequi, M., Mejia, R., Cimino, R.O., Krolewiecki, A.J., Albonico, M., Gobbo, M., Bonafini, S., Angheben, A., Requena-Mendez, A., ˜ Munoz, J., Nutman, T.B., 2014. Diagnostic accuracy of five serologic tests for Strongyloides stercoralis infection. PLoS Negl. Trop. Dis. 8, e2640. Borda, C.E., Rea, M.J., Rosa, J.R., Maidana, C., 1996. Intestinal parasitism in San Cayetano Corrientes Argentina. Bull. Pan Am. Health Org. 30, 227–233. Buonfrate, D., Sequi, M., Mejia, R., Cimino, R.O., Krolewiecki, A.J., Albonico, M., ˜ J., Nutman, T.B., Degani, M., Tais, S., Angheben, A., Requena-Mendez, A., Munoz, Bisoffi, Z., 2015. Accuracy of five serologic tests for the follow up of Strongyloides stercoralis infection. PLoS Negl. Trop. Dis. 9, e0003491. Carvalho, E.M., Porto, A.F., 2004. Epidemiological and clinical interaction between HTLV-1 and Strongyloides stercoralis. Parasite Immunol. 26, 487–497. Chang, M.P., Wang, Q., Norman, D.C., 2002. Diminished proliferation of B blast cell in response to cytokines in ethanol-consuming mice. Immunopharmacol. Immunotoxicol. 24, 69–82. Chieffi, P.P., Chiattone, C.S., Feltrim, E.N., Alves, R.C., Paschoalotti, M.A., 2000. Coinfection by Strongyloides stercoralis in blood donors infected with human T-cell leukemia/lymphoma virus type 1 in São Paulo City, Brazil. Mem. Inst. Oswaldo Cruz 95, 711–712. Concha, R., Harrington Jr., W., Rogers, A.I., 2005. Intestinal strongyloidiasis: recognition, management, and determinants of outcome. J. Clin. Gastroenterol. 39, 203–211. Crews, F.T., Bechara, R., Brown, L.A., Guidot, D.M., Mandrekar, P., Oak, S., Qin, L., Szabo, G., Wheeler, M., Zou, J., 2006. Cytokines and alcohol. Alcohol. Clin. Exp. Res. 30, 720–730. de Paula, F.M., Malta, F.deM., Marques, P.D., Sitta, R.B., Pinho, J.R., Gryschek, R.C., Chieffi, P.P., 2015. Molecular diagnosis of strongyloidiasis in tropical areas: a comparison of conventional and real-time polymerase chain reaction with parasitological methods. Mem. Inst. Oswaldo Cruz 110, 272–274. ˜ Dominguez-Santalla, M.J., Vidal, C., Vinuela, J., Pérez, L.F., González-Quintela, A., 2001. Increased serum IgE in alcoholics: relationship with Th1/Th2 cytokine production by stimulated blood mononuclear cells. Alcohol. Clin. Exp. Res. 25, 1198–1205. Dreyer, G., Fernandes-Silva, E., Alves, S., Rocha, A., Albuquerque, R., Addiss, D., 1996. Patterns of detection of Strongyloides stercoralis in stool specimens: implications for diagnosis and clinical trials. J. Clin. Microbiol. 34, 2569–2571. Fardet, L., Généreau, T., Poirot, J.L., Guidet, B., Kettaneh, A., Cabane, J., 2007. Severe strongyloidiasis in corticosteroid-treated patients: case series and literature review. J. Infect. 54, 18–27. Feliciano, N.D., Gonzaga, H.T., Gonc¸alves-Pires, Mdo R., Gonc¸alves, A.L., Rodrigues, R.M., Ueta, M.T., Costa-Cruz, J.M., 2010. Hydrophobic fractions from Strongyloides venezuelensis for use in the human immunodiagnosis of strongyloidiais. Diagn. Microbiol. Infect. Dis. 67, 153–161. Furtado, K.C.Y.O., Costa, C.A., de S. Ferreira, L., Martins, L.C., da C. Linhares, A., Ishikawa, E.A., de J. Batista, E., Sousa, M.S., 2013. Occurrence of strongyloidiasis among patients with HTLV-1/2 seen at the outpatient clinic of the Núcleo de Medicina Tropical, Belém, State of Pará, Brazil. Rev. Soc. Bras. Med. Trop. 46, 241–243. Gaburri, D., Gaburri, A.K., Hubner, E., Lopes, M.H., Ribeiro, A.M., de Paulo, G.A., Pace, F.H., Gaburri, P.D., Ornellas, A.T., Ferreira, J.O., Chebli, J.M., Ferreira, L.E., de

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