Toxicon 58 (2011) 664671

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Toxicon
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Clinical picture and laboratorial evaluation in human loxoscelism

Ceila M.S. Malaque a, *, Marcelo L. Santoro b, Joo Luiz C. Cardoso a, Mayra R. Conde a, Christina T.G. Novaes a, Jos Y. Risk a, Francisco O.S. Frana a, Carlos R. de Medeiros a, Hui W. Fan a
a b

Hospital Vital Brazil, Instituto Butantan, Av. Dr. Vital Brasil 1500, 05503-900, So Paulo-SP, Brazil Laboratory of Pathophysiology, Instituto Butantan, Av. Dr. Vital Brasil 1500, 05503-900, So Paulo-SP, Brazil

a r t i c l e i n f o
Article history: Received 1 April 2011 Received in revised form 2 August 2011 Accepted 21 September 2011 Available online 2 October 2011 Keywords: Loxosceles Hemolysis Clinical chemistry Necrosis Jaundice

a b s t r a c t
Loxosceles spiders are found globally, especially in South and North America. In Brazil, approximately 10,000 cases of Loxosceles spp. spider bites are reported annually. Herein we analyzed 81 patients diagnosed as either cutaneous or cutaneous-hemolytic loxoscelism, in a geographical area where most accidents are caused by Loxosceles gaucho, and we report their clinical and laboratory data obtained during week 1 and 2 after the bite. Massive hemolysis was noticed in only 2 cases, but high serum bilirubin and LDH levels, suggestive of hemolysis, were noticed in 25 cases on admission. Anemia was not frequent (14.7%), and reticulocytosis was particularly noticed during week 2 (in 56% of patients). High D-dimer levels were suggestive of endothelial cell activation and intravascular thrombin generation, but thrombocytopenia was noticed in only 17.6% of patients in week 1. Acute kidney injury (AKI) only occurred in patients with massive hemolysis. The denitive diagnosis of overt disseminated intravascular coagulation (DIC) could not be established on admission. Fever was associated with the presence of hemolysis (p 0.03). Altogether, these ndings provide evidence that mild hemolysis is frequent in loxoscelism and suggest that AKI is uncommon, exclusively occurring in patients with massive hemolysis. 2011 Elsevier Ltd. All rights reserved.

1. Introduction The brown recluse spiders, Loxosceles spp, are found globally, especially in South and North America (Gertsch, 1961; Southcott, 1976; Gertsch and Ennik, 1983; Platnick, 2010). In Brazil, approximately 20,000 cases of spider bites are reported annually, and Loxosceles bites account for almost 50% of bites involving all venomous species. Three species have been mostly implicated in human envenomation in Brazil: Loxosceles gaucho, Loxosceles intermedia and Loxosceles laeta (Brasil, 2001). Loxosceles spp venoms have been demonstrated to produce cutaneous necrosis and, occasionally, hemolysis by multiple pathways. Sphingomyelinase D, a key component of Loxosceles venom (Forrester et al.,
* Corresponding author. Tel./fax: 55 11 3726 7962. E-mail address: cmalaque@butantan.gov.br (C.M.S. Malaque). 0041-0101/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2011.09.011

1978) activates complement, endothelial and epithelial cells, as well as endogenous metalloproteinases (Patel et al., 1994; Gomez et al., 1999; Tambourgi et al., 2000; Veiga et al., 2001; Tambourgi et al., 2005; Paixo-Cavalcante et al., 2006; Tambourgi et al., 2007). Clinical manifestations evoked by Loxosceles envenomation vary depending on the amount of venom injected, anatomic location of the bite, and host susceptibility, as well as on spider species, sex and age (Futrell, 1992; Gonalves de Andrade et al., 1999; de Oliveira et al., 2005; McGlasson et al., 2007). Classically, loxoscelism have been classied into two forms: cutaneous and cutaneous-hemolytic (also known as systemic or viscerocutaneous loxoscelism). In cutaneous loxoscelism, patients manifest a slow-progression cutaneous lesion that may evolve into necrosis; this form can be associated with nonspecic systemic signs, such as scarlatiniform or morbilliform rash, headache, malaise, nausea, vomiting and low-

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grade fever. The cutaneous-hemolytic form of loxoscelism in which in addition to the cutaneous lesion, hemolysis is present is much less common and seldom result in death; this type of loxoscelism is associated with acute kidney injury and disseminated intravascular coagulation (DIC) due to intravascular hemolysis (Futrell, 1992; Hogan et al., 2004; Isbister and Fan, 2011). Although the development of massive intravascular hemolysis is well recognized in cutaneoushemolytic loxoscelism, no report has demonstrated the occurrence of mild hemolysis in cutaneous loxoscelism. In fact, few clinical reports have reported laboratory data from patients bitten by Loxosceles spiders, mostly of patients with cutaneous-hemolytic loxoscelism (Taylor and Denny, 1966; Hostetler et al., 2003; Zambrano et al., 2005; Dyachenko et al., 2006; de Souza et al., 2008; McDade et al., 2010). Herein, we report a prospective clinical and laboratory data survey of patients with denitive or presumptive diagnosis of either cutaneous or cutaneous-hemolytic loxoscelism admitted to Hospital Vital Brazil, Butantan Institute. Our ndings suggest that mild hemolysis is frequent, and that it occurs in one-third of those patients. 2. Patients and methods Patients (n 81) with denite or presumptive diagnosis of either cutaneous or cutaneous-hemolytic loxoscelism who had been admitted to Hospital Vital Brazil, Butantan Institute, So Paulo, Brazil, during 20042006 were included in this study. Epidemiological and clinical information such as sex, age, time interval between bite and admission, clinical signs and symptomsadministered treatment, occurrence of complications, and laboratory data were collected and evaluated. Denite diagnosis was accomplished based on identication of the agent associated with a skin lesion showing features compatible with loxoscelism evolution. Presumptive diagnosis of cutaneous loxoscelism was carried out in patients who exhibited (a) a characteristic lesion associated with (b) compatible epidemiological history, (c) a characteristic time-course of loxoscelism lesion and, sometimes, (d) non-specic systemic symptoms and signs. The characteristic lesion of cutaneous loxoscelism, usually present in the rst 23 days after the bite, is manifested by a painful macula, whose color is a mingling of violaceous (which does not blanch upon diascopy) and pale areas, sometimes indurated, and which is frequently surrounded by an erythematous area. Serous and/ or hemorrhagic blisters can be observed. Patients admitted later may manifest dry necrosis or an ulcer with sharply dened rim and tissue of granulation (Futrell, 1992; Isbister and Fan, 2011). Thus, that clinical presentation could not be attributable to any other etiology. Cutaneous-hemolytic loxoscelism was dened when intravascular hemolysis was suspected, irrespective of local reaction severity. One case report of a patient included here with massive hemolysis has been published elsewhere (de Souza et al., 2008). 2.1. Treatment Polyvalent antivenom for Loxosceles sp, Phoneutria sp and Tityus sp (soro antiaracndico, SAA, Instituto Butantan) was administered to patients in the early stage of

cutaneous loxoscelism, who manifested neither necrosis nor ulcer on admission (Pauli et al., 2009); on the other hand, antivenom treatment was prescribed to all patients with cutaneous-hemolytic loxoscelism. Antivenom was given intravenously and the number of vials varied according to envenomation severity: 05 vials for cutaneous loxoscelism, and 10 vials for cutaneous-hemolytic loxoscelism (Brasil, 2001). The classication of severity was based on clinical manifestations on admission. Prednisone (4060 mg/day, or 1 mg/kg/day for children) was administered for 57 days. 2.2. Laboratory analyses A previous study showed that patients seldom arrive prior to 24 h at the hospital, but in different time periods thereafter (Malaque et al., 2002). Thus, in order to standardize the analysis of results, blood samples collected in different time periods after arrival were grouped according to the time period they were obtained: week 1 (days 17) or 2 (days 814) after Loxosceles sp bite. Blood samples were obtained at Hospital Vital Brazil, and sent to Hospital Universitrio (University of So Paulo, So Paulo-SP) to be analyzed in automated equipments. Hematological (complete blood count), hemostatic [brinogen assay, activated partial thromboplastin time (aPTT), prothrombin time (PT), D-dimer assay] and biochemical [serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total and direct bilirubin, urea, creatinine, C-reactive protein (CRP), lactate dehydrogenase (LDH), creatine kinase (CK), Na and K)] tests were carried out, regardless in which day the blood samples were collected. If several laboratory results from blood samples collected over the two-week period were available, the most altered ones were included in the analysis. Presumptive diagnosis of intravascular hemolysis was suspected when jaundice was present and/or serum levels of total bilirubin (TB) were higher than 1.0 mg/dL and indirect bilirubin (IB) higher than >0.7 mg/dL. Serum creatinine levels were considered abnormal when values were higher than 1.3 mg/dL. 2.3. Statistical analyses A database of this pooled information was constructed and statistical calculations were performed using the softwares Epi-Info 6.04c (CDC, Atlanta, GA, USA) and Stata 8.0. Comparisons between proportions (c2 test or Fishers exact test), means or medians (Students t-test or MannWhitneys rank sum), whenever was appropriate, or determination of correlation among variables were performed using Stata 8.0 and Sigmastat 3.5. Whenever necessary, data transformation was undertaken to obtain homocedasticity and normal distribution. Differences with p < 0.05 were considered statistically signicant. 3. Results A total of 81 patients with loxoscelism were included in this study. The median age was 36 years (range: 375 years; 7.4% less than 14 years old), and no gender difference was observed (female/male: 49.4%/50.6%). In 14 cases (17.3%),

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the spider was captured and identied as L. gaucho (12) or Loxosceles sp. (2). Diagnosis was based solely on clinical ndings in 67 cases (82.3%). The time interval between the bite and admission to hospital ranged from 1 h to 9 days (median: 48 h; only 12.3% were admitted less than 12 h). Based on clinical signs and symptoms on admission, 90% of cases were classied as cutaneous loxoscelism, and 10% as cutaneous-hemolytic loxoscelism. Systemic symptoms were noticed in 87.7% (71/81) of patients (Table 1). Forty-four patients (54.3%, 44/81) received antivenom (SAA) (mean: 2.4 vials) and 31.8% (14/44) of them manifested some type of early anaphylactic reaction. Necrosis was observed in 57.9% (44/76) of patients, and mild local infection was observed exclusively in patients who developed scares (5.2%, 4/76). Two patients developed massive hemolysis and acute kidney injury (AKI), and were transferred to the Intensive Care Unit in Emlio Ribas Institute of Infectology. Both were submitted to dialysis. No death occurred. As shown Fig. 1, in week 1, a high proportion of patients had high serum levels of total (40.9%, 27/66) and indirect (34.4%, 22/64) bilirubin, as well as of LDH (50.0%, 28/56); however, serum levels of bilirubin and LDH diminished in week 2. Generally, hemoglobin levels were within the normal reference range in most cases (85.3%, 64/75) during week 1, and a drastic fall in hemoglobin levels was solely noticed in two patients who developed massive hemolysis and AKI. The proportion of patients who exhibited elevated reticulocyte counts increased from week 1 (30.3%, 10/33) to week 2 (64.0%, 16/25) (Fig. 1). As shown in Fig. 2, CK levels were raised in 19 patients on week 1 (30.6%, 19/62), and such proportion decreased to

Table 1 Frequency of systemic signs and symptoms found in patients with loxoscelism on admission at Hospital Vital Brazil, So Paulo, Brazil, 2004 2006. Patients were also grouped according to serum indirect bilirubin (IB) levels i.e., those with normal levels (IB 0.7 mg/dL) and those with raised levels (IB > 0.7 mg/dL) and compared statistically. Clinical signs and symptoms Morbiliform or scarlatiniform rash Total IB  0.7 (n 47) IB > 0.7 (n 25) Fever Total IB  0.7 (n 47) IB > 0.7 (n 25) Malaise Total IB  0.7 (n 47) IB > 0.7 (n 25) Headache Total IB  0.7 (n 47) IB > 0.7 (n 25) Nausea Total IB  0.7 (n 47) IB > 0.7 (n 25) Jaundice Total IB  0.7 (n 47) IB > 0.7 (n 25) N (%) 52 (64.2) 28 (59.5) 18 (72.0) 37 (46.3) 19 (40.4) 17 (68.0) 30 (37) 15 (31.9) 12 (48.0) 29 (35.8) 17 (36.2) 9 (36.0) 20 (24.7) 10 (21.3) 8 (32.0) 8 (10) 0 (0) 8 (32.0) p

0.5021

0.0297

0.2200

0.8805

0.4143

0.0003

6.2% (2/32) in week 2. Serum levels of ALT (17.2%, 11/64) and AST (9.2%, 6/65) were altered in week 1. In regard to ALT levels, the highest values were noticed in three patients who developed hemolysis. A mild increase in ALT levels, close to the upper reference level, was noticed in most other patients, and four of them developed hemolysis. Alterations in serum AST levels were noticed in 10.8% (7/ 65) of patients in week 1. Most of these patients (85.7%, 6/7) also exhibited levels of indirect bilirubin between 0.9 and 3.8 mg/dL. In those two patients who manifested massive hemolysis, the increase in AST levels was ve to ten times higher than the upper reference level. Most serum levels of creatinine, urea, Na and K were within the normal range of reference. However, elevated levels of creatinine and urea were noticed in those two patients who developed intravascular hemolysis, and such levels were raised as soon as 24 after the bite. One of these patients showed mild hyponatremia and moderate hyperkalemia. Leukocytosis was present in 50.8% (38/75) and 48.6% (17/35) of patients admitted in week 1 and 2, respectively (Fig. 3). Neutrophilia was noticed in 37.8% (28/74) and 62.8% (22/35) of patients admitted in week 1 and 2, respectively. CRP levels were raised in 85.3% (29/34) of patients in week 1, reaching values as high as 152 mg/L; in week 2, 41.2% (7/ 17) of CRP values were high (Fig. 3). Plasma brinogen levels were elevated in 34% (17/34) and 90.9% (20/22) of patients in week 1 and 2, respectively. The highest values observed in patients were 817 mg/dL and 558 mg/dL in week 1 and 2, respectively. Platelet counts of most patients were within the normal reference, and thrombocytopenia was found in 17.6% (13/74) and 2.8% (8/35) of patients admitted in week 1 and 2, respectively. In addition, 7.5% (4/53) of patients admitted in week 1 showed INR ratios >1.3, and 13.2% (7/53) of them showed values lower than 1.0. For aPTT values, 21.6% (11/51) of patients had an aPTT ratio greater than 1.25 in week 1. D-dimer levels were increased in 53.5% (23/43) of patients in week 1, and 25% (3/12) in week 2. Two patients who manifested massive hemolysis could not be included in the criteria adopted to diagnose overt DIC (Taylor et al., 2001), since few laboratory hemostatic tests were available on admission. Inasmuch as some patients showed high levels of indirect bilirubin and LDH, which were suggestive of intravascular hemolysis, patients were grouped on the basis of indirect bilirubin levels on admission (IB  0.7 and IB > 0.7). Of 72 patients who had bilirubin levels analyzed on admission (week 1 or 2), 25 (34.7%) had increased indirect bilirubin levels (>0.7 mg/dL). No group differences were observed on account of age, creatinine levels (Table 2), leukocyte and platelet counts or hemoglobin levels. However, the presence of fever (Table 1) and increased levels of CRP in week 1 were more frequent in patients with increased indirect bilirubin (IB) levels. Surprisingly, high bilirubin levels were more frequent in men (p 0.002). Reticulocyte percentage in week 2 was statistically higher in the group with increased indirect bilirubin levels. Despite the apparent higher D-dimer levels in patients with increased indirect bilirubin levels, they were not statistically different from the values observed in the other group (Table 2).

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Fig. 1. Total bilirubin, indirect bilirubin, LDH and hemoglobin levels, and reticulocyte counts measured in patients with loxoscelism on week 1 (black points) and 2 (green points) after envenomation. Horizontal red lines represent median values, and yellow areas evidence the limits of normal reference range. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

Fig. 2. Creatine kinase (CK), ALT, AST, sodium, potassium, urea and creatinine levels measured in patients with loxoscelism on week 1 (black points) and 2 (green points) after envenomation. Horizontal red lines represent median values, and yellow areas evidence the limits of normal reference range. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

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Fig. 3. Counts of white blood cells (WBC), neutrophils and platelets, levels of CPR, brinogen and D-dimer, and values of INR and aPTT ratio measured in patients with loxoscelism on week 1 (black points) and 2 (green points) after envenomation. Horizontal red lines represent median values, and yellow areas evidence the limits of normal reference range. Blue horizontal line represent the normal upper limit for aPTT ratio. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

4. Discussion
Table 2 Comparison of epidemiological and laboratory parameters between 72 patients with loxoscelism, grouped on the basis of indirect bilirubin (IB) levels on admission (week 1 or 2), and compared statistically. Hospital Vital Brazil, So Paulo, Brazil, 20042006. Variable Age (years) IB > 0.7 mg/dL Mean S.D. 36.16 18.30 (n 25) Median 36 Male:female 19:6 (n 37) IB  0.7 mg/dL p 35.98 14.89 (n 47) 33.0 18:29 (n 35) 62.09 51.45 (n 44) 55 0.9639

Sex

0.002 0.4134

Interval Mean S.D. 51.9 45.03 between (n 24) bite and Median 30.5 hospital admission (h) Hemoglobin mean S.D. 13.27 2.23 (g/dL) (n 24) (week 1) Median 13.8 Creatinine (mg/dL) (week 1) LDH (U/L) (week 1) Mean S.D. 1.18 0.71 (n 24) Median 1.0

13.68 1.41 (n 43) 14 0.96 0.21 (n 40) 0.9

0.7189

0.1396

Mean S.D. 722.85 1607.42 181.74 41.22 0.0066 (n 20) (n 34) Median 211 174.5 1.9 0.78 (n 15) 1.8 0.0226

Reticulocytes Mean S.D. 4.01 3.99 (%) (n 10) (week 2) Median 2.4 D-Dimer (ng/mL) (week 1) CRP (mg/L) (week 1) Mean S.D. 2087.3 2913.9 (n 16) Median 827.5 Mean S.D. 61.7 39.50 (n 14) Median 60.5

940.7 1271.0 0.0884 (n 24) 497.5 30.8 40.9 (n 20) 11 0.0038

Loxosceles spp. occur in North America, Central America, South America, Europe, Africa, Australia and parts of Asia (Gertsch, 1961; Southcott, 1976; Gertsch and Ennik, 1983; Platnick, 2010). Most of the published reports of Loxosceles bites are of either individual cases or limited series of clinical cases. On the other hand, reports of laboratory tests on loxoscelism are of individual cases, mostly of patients with cutaneous-hemolytic loxoscelism (Nance, 1961; Gonzalez et al., 1986; Schenone et al., 1989; Murray and Seger, 1994; Williams et al., 1995; Dyachenko et al., 2006). Diagnosis of loxoscelism is usually presumptive and made through epidemiological information and evolution of the clinical picture, since few patients bring the biological agent for its correct identication. Besides the presence of a cutaneous lesion at the site of the bite, the presence of systemic manifestations is not infrequent in loxoscelism, and can be ancillary to the diagnosis. Herein, studying patients with cutaneous or cutaneous-hemolytic loxoscelism in a geographical area where most accidents are caused by L. gaucho, we observed that systemic manifestations were present in almost 90% of patients. In addition, we demonstrated that they were present not only in patients with clinical and/or laboratory evidence of mild or severe intravascular hemolysis, but also in those without intravascular hemolysis. Thus, we opted here to use the term cutaneous-hemolytic loxoscelism, instead of viscerocutaneous or systemic loxocelism, to refer to cases with intravascular hemolysis, since the latter terms do not seem appropriate to evidence the development of intravascular hemolysis. The most frequent alterations noticed in clinical chemistry tests were those regarding LDH and bilirubin assays, whose values were augmented suggesting the presence of

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hemolysis. The existence of hemolysis in loxoscelism is well reported, but its frequency is variable: 15.7% in Chile (Schenone et al., 1989), 13.1% in southern Brazil (Sezerino et al., 1998), and even no case of hemolysis in Israel (Ingber et al., 1991). In this study, in a region where the most frequent accidents are due to L. gaucho, although only 10% of patients manifested clinical jaundice, one-third of patients had elevated values of indirect bilirubin and LDH, suggesting that hemolysis is more frequent than expected and is usually mild. Hemoglobin levels were similar in groups with normal or high indirect bilirubin levels, and this nding may be ascribed to the fact that most patients with increased bilirubin levels showed a laboratory picture of mild hemolysis. Only two patients with massive hemolysis exhibited hemoblogin <10 g/dL on admission. Reticulocyte count increased twofold from week 1 to week 2. It is well known that in patients who had an acute hemolytic episode, signs of increased red cell production, like reticulocytosis, appear only after 36 days (Glader, 2003). Therefore, reticulocyte counts within the reference range obtained soon after Loxosceles bites may not be concordant with the clinical picture of patients with evidence of hemolysis. Later evaluations are necessary to evidence the increase in reticulocyte count in loxoscelism. Some patients also exhibited elevation of ALT and AST levels. Although the highest levels were found in patients with hemolysis, suggesting that this increase in serum might be associated with intravascular hemolysis (Lippi et al., 2006), few patients without evidence of hemolysis also had AST and ALT levels reaching up twofold the upper reference range. In addition, our ndings showed that 60% of patients who exhibited increased CK levels also showed other clinical chemistry tests suggestive of hemolysis. Therefore, such an increase in CK levels in those patients might also be attributed to the interference of hemolysis in the optical reaction for CK determination (Greenson et al., 1989). Changes in levels of urea, creatinine, sodium or potassium rarely occurred, only in those patients with massive hemolysis. A positive correlation was not observed herein between creatinine and IB levels, suggesting that AKI is associated with massive and not mild hemolysis in humans, even though direct toxicity of Loxosceles venom has been reported on renal structures in mice and renal cells in vitro (Luciano et al., 2004; Chaim et al., 2006). In experimental studies in rabbits, leukopenia was observed in the rst 24 h after Loxosceles envenomation, followed by leukocytosis, which peaked at 72 h (Tavares et al., 2004; Pauli et al., 2009). Neutrophilic leukocytosis was the most frequent alteration observed in complete blood count of patients, and it was noticed in both week 1 and 2. Leukocyte counts were higher in week 2 than in week 1, likely the result of corticosteroid administration to patients. Polymorphonuclears have been well established to have a critical role in the genesis of necrotic lesions induced by Loxosceles reclusa venom (Smith and Micks, 1970; Barbaro et al., 2010). Histopathological changes in loxoscelism are characterized by polymorphonuclear accumulation in and around blood vessels (Ospedal et al., 2002). Loxosceles venom is a potent endothelial cell agonist, stimulating the release of IL-8, granulocyte-macrophage colonystimulating factor and eliciting expression of E selectin

(Patel et al., 1994; Desai et al., 1999; Gomez et al., 1999), and thus it may induce polymorphonuclear activation. High levels of CRP were noticed in most patients in week 1. In humans, plasma CRP levels rise rapidly and markedly after an acute inammatory stimulus. Induction of CRP in hepatocytes is mainly regulated at the transcriptional level by the cytokine interleukin-6 (IL-6), and this effect can be enhanced by interleukin-1b (IL-1b) (Kushner et al., 1995; Black et al., 2004). Barbaro et al. (2010) showed that L. gaucho venom stimulated local secretion of IL-6 at 2 h and 24 h after venom injection. Thus, cytokine release at the site of the bite might induce the increase in CRP levels noticed in plasma. Interestingly, CRP levels were signicantly higher in patients with high indirect bilirubin levels in week 1, suggesting an association with the hemolysis. de Sousa et al. (2008) observed that IL-6 and tumor necrosis factor-alpha were notably elevated in the serum of a patient with massive hemolysis due to Loxosceles bite. These data suggest an association between CRP levels and hemolysis, as well as with the cutaneous inammatory injury. Fibrinogen levels remained elevated in week 1 and 2 after the bite. Fibrinogen transcription is signicantly upregulated by IL-6 (Amrani, 1990). In rabbits, brinogen levels were raised as early as 12 h after experimental administration of Loxosceles venom, reaching the highest levels at 48 h. In fact, brinogen levels remained higher than control animals until day 10 after envenomation (Tavares et al., 2004; Pauli et al., 2009). In this investigation, D-dimer levels were also raised in 53.5% of patients in week 1, suggesting that endothelial activation, caused by either cutaneous lesion or hemolysis, likely result in increased expression of tissue factor on the luminal surface of endothelial cells (Berger et al., 1973; Veiga et al., 2001; Szotowski et al., 2005), and consequent activation of coagulation cascade and brinolysis (Rosser, 2009). This assumption is conrmed by experimental studies showing that factor VII was decreased in rabbits injected with L. gaucho venom (Tavares et al., 2004). However, most patients showed aPTT and INR values within the normal range, evidencing that either consumptive coagulopathy has not occurred in loxoscelism or that brinogen and factor VIII, which are acute phase reactants (Colman et al., 2005), might have balanced such coagulation disturbance. In effect, some patients even showed shortening instead of prolongation of clotting times. Moreover, no correlation was observed between bilirubin levels and platelet consumption, demonstrating that thrombocytopenia was not directly associated with hemolysis. The cause of thrombocytopenia in loxoscelism needs further investigation, but experimental studies also showed that soon after injection of L. gaucho venom in rabbits, platelet counts abruptly fell during the rst 48 h and thereafter reactive thrombocytosis was observed (Tavares et al., 2004). The diagnosis of overt DIC on admission could not be ascertained for any patient, including those two patients with massive hemolysis, given that few laboratory hemostasis tests were available to diagnose DIC according to criteria established elsewhere (Taylor et al., 2001). In addition to laboratory tests, we assessed whether symptoms other than clinical jaundice were related to hemolysis. Only the presence of fever showed a positive

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C.M.S. Malaque et al. / Toxicon 58 (2011) 664671 Glader, B., 2003. Anemia: general considerations. In: Greer, J.P., Foerster, J., Lukens, J.N., Rodgers, G.M., Paraskevas, F., Glader, B. (Eds.), Wintrobes Clinical Hematology. Lippincot Williams & Wilkins, Philadelphia, pp. 779809. Gomez, H.F., Miller, M.J., Desai, A., Warren, J.S., 1999. Loxosceles spider venom induces the production of alpha and beta chemokines: implications for the pathogenesis of dermonecrotic arachnidism. Inammation 23, 207215. Gonalves de Andrade, R.M., De Oliveira, K.C., Giusti, A.L., Dias da Silva, W., Tambourgi, D.V., 1999. Ontogenetic development of Loxosceles intermedia spider venom. Toxicon 37, 627632. Gonzalez, C., Safe, A., Fardella, C., Contreras, A., 1986. Insuciencia renal aguda en loxocelismo cutaneo-visceral: 11 casos. Rev. Med. Chil 114, 11551159. Greenson, J.K., Farber, S.J., Dubin, S.B., 1989. The effect of hemolysis on creatine kinase determination. Arch. Pathol. Lab. Med. 113, 184185. Hendrickson, J.E., Hillyer, C.D., 2009. Noninfectious serious hazards of transfusion. Anesth. Analg. 108, 759769. Hogan, C.J., Barbaro, K.C., Winkel, K., 2004. Loxoscelism: old obstacles, new directions. Ann. Emerg. Med. 44, 608624. Hostetler, M.A., Dribben, W., Wilson, D.B., Grossman, W.J., 2003. Sudden unexplained hemolysis occurring in an infant due to presumed Loxosceles envenomation. J. Emerg. Med. 25, 277282. Ingber, A., Trattner, A., Cleper, R., Sandbank, M., 1991. Morbidity of brown recluse spider bites. Clinical picture, treatment and prognosis. Acta Derm. Venereol. 71, 337340. Isbister, G.K., Fan, H.W., 2011. Spider bite. Lancet. Kushner, I., Jiang, S.L., Zhang, D., Lozanski, G., Samols, D., 1995. Do posttranscriptional mechanisms participate in induction of C-reactive protein and serum amyloid A by IL-6 and IL-1? Ann. N. Y. Acad. Sci. 762, 102107. Lippi, G., Salvagno, G.L., Montagnana, M., Brocco, G., Guidi, G.C., 2006. Inuence of hemolysis on routine clinical chemistry testing. Clin. Chem. Lab. Med. 44, 311316. Luciano, M.N., da Silva, P.H., Chaim, O.M., dos Santos, V.L., Franco, C.R., Soares, M.F., Zanata, S.M., Mangili, O.C., Gremski, W., Veiga, S.S., 2004. Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue. J. Histochem. Cytochem. 52, 455467. Malaque, C.M., Castro-Valencia, J.E., Cardoso, J.L., Frana, F.O., Barbaro, K.C., Fan, H.W., 2002. Clinical and epidemiological features of denitive and presumed loxoscelism in Sao Paulo, Brazil. Rev. Inst. Med. Trop. Sao Paulo 44, 139143. McDade, J., Aygun, B., Ware, R.E., 2010. Brown recluse spider (Loxosceles reclusa) envenomation leading to acute hemolytic anemia in six adolescents. J. Pediatr. 156, 155157. McGlasson, D.L., Harroff, H.H., Sutton, J., Dick, E., Elston, D.M., 2007. Cutaneous and systemic effects of varying doses of brown recluse spider venom in a rabbit model. Clin. Lab. Sci. 20, 99105. Murray, L.M., Seger, D.L., 1994. Hemolytic anemia following a presumptive brown recluse spider bite. J. Toxicol. Clin. Toxicol. 32, 451456. Nance, W.E., 1961. Hemolytic anemia of necrotic arachnidism. Am. J. Med. 31, 801807. Ospedal, K.Z., Appel, M.H., Fillus Neto, J., Mangili, O.C., Sanches Veiga, S., Gremski, W., 2002. Histopathological ndings in rabbits after experimental acute exposure to the Loxosceles intermedia (brown spider) venom. Int. J. Exp. Pathol. 83, 287294. Paixo-Cavalcante, D., van den Berg, C.W., de Freitas Fernandes-Pedrosa, M., Gonalves de Andrade, R.M., Tambourgi, D.V., 2006. Role of matrix metalloproteinases in HaCaT keratinocytes apoptosis induced by Loxosceles venom sphingomyelinase D. J. Invest. Dermatol. 126, 6168. Patel, K.D., Modur, V., Zimmerman, G.A., Prescott, S.M., McIntyre, T.M., 1994. The necrotic venom of the brown recluse spider induces dysregulated endothelial cell-dependent neutrophil activation. Differential induction of GM-CSF, IL-8, and E-selectin expression. J. Clin. Invest. 94, 631642. Pauli, I., Minozzo, J.C., da Silva, P.H., Chaim, O.M., Veiga, S.S., 2009. Analysis of therapeutic benets of antivenin at different time intervals after experimental envenomation in rabbits by venom of the brown spider (Loxosceles intermedia). Toxicon 53, 660671. Platnick, N.I., 2010. The World Spider Catalog, Version 11.0. <http:// research.amnh.org/entomology/spiders/catalog/index.html>. Rosser Jr., E.J., 2009. Use of the D-dimer assay for diagnosing thrombosis in cases of canine cutaneous vasculitis. Vet. Dermatol. 20, 586590. Schenone, H., Saavedra, T., Rojas, A., Villarroel, F., 1989. Loxoscelismo en Chile. Estudios epidemiologicos, clinicos y experimentales. Rev. Inst. Med. Trop. Sao Paulo 31, 403415. Sezerino, U.M., Zannin, M., Coelho, L.K., Gonalves Junior, J., Grando, M., Mattosinho, S.G., Cardoso, J.L., von Eickstedt, V.R., Frana, F.O.,

correlation with the increase in IB. In fact, fever has been reported to occur in hemolytic transfusion reaction (Hendrickson and Hillyer, 2009). In the patient group with high IB levels, males were more frequent than females (p 0.002), in accordance with results reported elsewhere (Al-Sweedan et al., 2009). As previously reported (Malaque et al., 2002), no association was noticed between hemolysis and patient age in this study, conversely to another study showing a higher frequency of Loxosceles accidents in children (Sezerino et al., 1998). In conclusion, this investigation reports that hemolysis is more frequent in loxoscelismin an area where L. gaucho is more abundantthan previously reported, and that most patients manifest a clinical and laboratory picture of mild hemolysis. AKI was infrequent in this study, and only occurred in cases manifesting massive hemolysis. Conict of interest statement The authors declare that there are no conicts of interest.

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