The efficacy of antivenom in loxoscelism treatment

ARTICLE IN PRESS Toxicon 48 (2006) 123–137 www.elsevier.com/locate/toxicon Review The efficacy of antivenom in loxoscelism treatment Isolete Paulia,b,, Juliana Pukac, Ida Cristina Gubertd, João Carlos Minozzoa a Production and Research Centre of Immunobiological Products, State Department of Health, Paraná, Brazil Department of Post-graduation in Biotechnological Processes, Federal University of Paraná, Paraná, Brazil c City Department of Health, Curitiba, Paraná, Brazil d Department of Pathology, Federal University of Paraná, Paraná, Brazil b Received 1 September 2005; received in revised form 26 April 2006; accepted 8 May 2006 Abstract Loxoscelism or brown spider envenomation is the most important form of araneism in some countries and constitutes the third cause of accidents by venomous animals in Brazil. The treatment of Loxosceles bites is still controversial, with a variety of interventions proposed and tried, such as antivenom. The majority of clinical studies demonstrate a significant delay between a spider’s bite and presentation for treatment, and this delay is thought to lead to an ineffective administration of a specific antivenom. Even in Brazil, where the antivenom therapy has been indicated more frequently than in other countries, there are still doubts about its real capacity to neutralize local and systemic effects of the envenomation and the ideal period for its administration. Thus, various studies in animal models have tried to correlate the time of envenomation with the application of the antivenom and the permanence of the venom in circulation or in dermonecrotic lesions. The purpose of this study was to evaluate the use of antivenom in loxoscelism treatment and to systematize the results of studies in animals and humans available in the last 30 years, making possible a more critical analysis of the efficacy of the antivenom or its therapeutic value in bites by spiders of the genus Loxosceles. r 2006 Elsevier Ltd. All rights reserved. Keywords: Loxosceles; Loxoscelism; Brown spiders; Necrotic arachnidism; Antivenom therapy Contents 1. 2. 3. 4. 5. 6. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Antivenom treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Studies in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Studies in experimental models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 125 126 127 131 134 135 135 Corresponding author. Production and Research Centre of Immunobiological Products, State Department of Health, Paraná, Brazil. Tel./fax: +55 41 3673 8811. E-mail address: isops@ibest.com.br (I. Pauli). 0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.05.005 ARTICLE IN PRESS 124 I. Pauli et al. / Toxicon 48 (2006) 123–137 1. Introduction Spiders of the genus Loxosceles have a worldwide distribution. There are more than 100 species described in many countries of Europe, Africa, Oceania, Asia, North America, Central America and mainly South America, where more than 30 species have been described (Gertsch, 1967; da Silva et al., 2004; Platnick, 2005). In Brazil, they have been recognized since 1891, but only in 1954 were implicated as agents capable of causing cutaneousnecrotic accidents (Cardoso and De Cillo, 1990; Barbaro and Cardoso, 2003), especially in south and southeast regions, where Loxosceles gaucho, Loxosceles intermedia and Loxosceles laeta are the prominent species (Eickstedt, 1994; Sezerino et al., 1998; Málaque et al., 2002; Gonc- alves de Andrade and Tambourgi, 2003). L. laeta is also endemic in Chile and Peru (Zavaleta, 1987; Schenone et al., 1989; da Silva et al., 2004). However, those three species, associated with Loxosceles reclusa from North America and Loxosceles deserta from USA and Mexico, are considered the most important Loxosceles spiders in the world based on their geographic distribution and the high number of notified bites with considerable morbidity and mortality (Gertsch and Ennik, 1983; Wong et al., 1987; Escalante-Galindo et al., 1999; Schenone et al., 2001). Loxoscelism, the term used to describe lesions and clinical manifestations caused by bites of Loxosceles, was first described in the USA (Caveness, 1872). Presently, it is the most severe form of necrotic araneism in several countries (Rees et al., 1987; Wong et al., 1987; Baldwin et al., 1988; Futrell, 1992; Sams et al., 2001; Wendell, 2003; da Silva et al., 2004; Hogan et al., 2004; Isbister and White, 2004; Pommier et al., 2005) and an important matter of public health in South America, with accidents reported in Brazil, Chile, Peru e Argentina (Schenone et al., 1989; Ribeiro et al., 1993; Sezerino et al., 1998; de Roodt et al., 2002; Málaque et al., 2002). Envenomation in humans can result in relatively mild features, such as cutaneous loxoscelism with edematous predominance (Futrell, 1992; Schenone, 1998; da Silva et al., 2004) or pruritic papular and vesicular lesions (Alario et al, 1987; Anderson, 1991), but presents two well-defined clinical variants: cutaneous loxoscelism and viscerocutaneous loxoscelism. The first situation, which is more common (67–100%), is characterized by the pre- sence of painful cutaneous lesion, of slow and gradual evolution, where signs as edema, induration, erythema, ischemia, ecchymosis and mixed area of erythema, ecchymosis and ischemia, known as red, white and blue sign, appear. These lesions may evolve to necrosis, eschar and necrotic ulcer of slow healing or may require surgical excision and skin grafting (Wong et al., 1987; Futrell, 1992; Sams et al., 2001; da Silva et al., 2004). The viscerocutaneous loxoscelism, also known as systemic or cutaneous-hemolytic loxoscelism, is less common (0–30%, according to region and specie studied), but constitutes the most serious form. It shows hematologic disturbances and/or renal injury associated to cutaneous picture, and may evolve, regardless of the cutaneous course (Málaque et al., 2002; Barbaro and Cardoso, 2003), to hemolysis, thrombocytopenia, jaundice, hematuria, hemoglobinuria, rhabdomyolysis, shock and, in some cases, disseminated intravascular coagulation and acute renal failure. These are the main causes of death in Loxosceles envenomation (Futrell, 1992; Sams et al., 2001; Hogan et al., 2004; da Silva et al., 2004). It is suggested that these manifestations may be due to the amount of injected venom, time of envenoming, spider factors—such as species, sex and ontogenetic variations—and features of the patients—such as age, bite site, genetic variations and the involvement of different endogenous mediators (Rees et al., 1981; Futrell, 1992; Sezerino et al., 1998; Gonc- alves de Andrade, 1998; Tambourgi et al., 1998; Greenfield et al., 2000; Málaque et al., 2002; Barretto et al., 2003; Barbaro and Cardoso, 2003; da Silva et al., 2004; de Oliveira et al., 2005). Various interventions have been proposed and tried for the treatment of loxoscelism (King Jr., 1985; Rees et al., 1985; Rees et al., 1987; Alario et al., 1987; Futrell, 1992; Barrett et al., 1994; Phillips et al., 1995; Maynor et al., 1997; Masters et al., 1998; Masters, 1998; Sams et al., 2001; Wendell, 2003; Isbister et al., 2003; da Silva et al., 2004; Swanson and Vetter, 2005). However, there are no unanimous criteria regarding the best therapeutic schedule and a definitive therapy has not yet been established, with a great deal of controversy about the effectiveness of drugs, antivenom or their combination (Wasserman and Anderson, 1983–84; de Roodt et al., 2002; Isbister et al., 2003; Barbaro and Cardoso, 2003; Isbister and White, 2004; da Silva et al., 2004; Hogan et al., 2004). Regarding all interventions evaluated, the specific antivenom was mentioned as having the greater potential when ARTICLE IN PRESS I. Pauli et al. / Toxicon 48 (2006) 123–137 administered in the first hours after envenomation, during the acute phase (Rees et al., 1981; Futrell, 1992; Gomez et al., 1999; Hogan et al., 2004). However, even in Brazil, where serum therapy has been indicated more frequently than in other countries, due to considerable morbidity and mortality of loxoscelism and where the local Ministry of Health has developed guidelines for identification, diagnosis and management in this kind of envenomation, there are divergences regarding the effectiveness of the serum in neutralizing local effects (dermonecrosis) and the ideal period for its administration (Guilherme et al., 2001; Ministry of Health, Brazil, 2001; City Department of Health, Curitiba, Brazil, 2002; Isbister et al., 2003; Barbaro and Cardoso, 2003; Isbister and White, 2004; da Silva et al., 2004; Hogan et al., 2004). Thus, the purpose of this study was to evaluate the use of antivenom for the treatment of loxoscelism and systematize the results of studies in humans and animals available in the last 30 years, in order to make possible a more critical analysis of the antivenom efficacy or its therapeutic value in bites by spiders of the genus Loxosceles. 2. Treatment Different interventions and medications were proposed for the treatment of loxoscelic envenomation, such as local care, analgesics, aspirin, heparin, phentolamine, vasodilators, dextran, topical nitroglycerine, electric shock, hyperbaric oxygen, antihistamines, antibiotics, corticosteroids (parenteral, oral, intralesional or topical), surgical excision, antivenom (parenteral or intralesional) and dapsone (King Jr., 1985; Rees et al., 1985,1987; Alario et al., 1987; Barrett et al., 1994; Maynor et al., 1997; Masters et al., 1998; Masters, 1998; Isbister et al., 2003; da Silva et al., 2004; Swanson and Vetter, 2005). Nevertheless, researches were not conclusive, with little evidence to support this variety of treatments (Futrell, 1992; Phillips et al., 1995; Sams et al., 2001; Wendell, 2003). Some of them are not recommended while others are expensive, painful or potentially toxic and much of what is known results from case studies, retrospective reviews and individual clinical experience, since no intervention was submitted to controlled randomized trials, which could prove its efficacy in humans (Jarvis et al., 2000; Sams et al., 2001; Swanson and Vetter, 2005). 125 In order to reduce inflammation and systemic effects and to prevent secondary infection, interventions may be used, such as cold or cool compresses, wound cleansing, sterile padding, relative rest, elevation and immobilization of the extremity, tetanus prophylaxis, analgesics and antihistamines and, in viscerocutaneous cases, supportive interventions such as hydration, blood transfusion, diuretics and dialysis (Futrell, 1992; Sams et al., 2001; Ministry of Health, Brazil, 2001; City Department of Health, Curitiba, Brazil, 2002; Wendell, 2003; da Silva et al., 2004). However, therapeutic interventions frequently used in different countries (according to regional experience and envenoming characteristics) such as antibiotics, surgical excision, corticoids, dapsone and antivenom, still remain controversial (Hogan et al., 2004). In countries such as the USA, necrotic lesions are usually treated with oral antibiotics to prevent infection (Hogan et al., 2004), since the presence of Clostridium perfringes in venom and fangs of L. intermedia was evidenced, associated to a greater degree of dermonecrosis in animal studies (Monteiro et al., 2002). Moreover, it is known that an infection markedly increases the temperature in bite site and, thus, the activity of the enzymes responsible for dermonecrosis and local inflammation, which can result in slow evolution and hard healing lesions (King Jr., 1985). However, in other countries, antibiotics administration is not a routine procedure, since the secondary infections are not common (Sezerino et al., 1998; Málaque et al., 2002; Schenone, 2003; Barbaro and Cardoso, 2003). Some authors consider their use inappropriate in the without evidences of infection, being reserved as prophylaxis when wounds begin to show signs of tissue breakdown, or to prevent cellulites, or for the treatment of established infections (King Jr., 1985; Rees et al., 1987; Escalante-Galindo et al., 1999; City Department of Health, Curitiba, Brazil, 2002; Wendell, 2003; Hogan et al., 2004). Immediate or early surgical intervention, especially in hands, is not recommended, since it can increase local inflammation and exacerbate the venom effects, prolonging tissue injury, increasing the lesion extension and consequently contributing to skin graft rejections, chronic ulcerations and poor functional and cosmetic results (King Jr., 1985; Rees et al., 1985; Futrell, 1992; Ship, 1998; Sams et al., 2001; Wendell, 2003; da Silva et al., 2004). Besides, one cannot know which bites may progress to a systemic disease or to a large ARTICLE IN PRESS 126 I. Pauli et al. / Toxicon 48 (2006) 123–137 cutaneous necrosis and the majority of lesions resolve without surgery or skin grafting and with better results (Anderson, 1991; Masters et al., 1998; da Silva et al., 2004). The necrotic eschar removal may be appropriate when the lesion shows complications or limited and prolonged healing (a stable lesion). In cases of permanent tissue loss, with unacceptable scarring, a reconstructive plastic surgery may be necessary (Sams et al., 2001; Wendell, 2003; Barbaro and Cardoso, 2003; Hogan et al., 2004; da Silva et al., 2004). Although there are no sufficient data about the use of systemic corticosteroids in cutaneous or viscerocutaneous loxoscelism, they were advocated for severe cases, especially in children, because they might be helpful in preventing hemolysis and renal injury, if applied earlier (Rees et al., 1981; Smith and Baldwin, 1988; Futrell, 1992; Gomez et al., 1999). However, corticosteroids do not inactivate the venom or stop its primary actions and, even if given very early, do not prevent the development of cutaneous necrosis or necrotic ulcers, being reserved for patients with systemic problems (Rees et al., 1981; Wasserman and Anderson, 1983–84; Rees et al., 1985; Alario et al., 1987; Masters et al., 1998; Goddard, 1998; Sams et al., 2001; Wendell, 2003; Hogan et al., 2004). Dapsone, long used in leprosy treatment, has been recommended for the treatment of bites of Loxosceles spiders because it probably limits the migration and infiltration of neutrophils in the bite site, which is an essential factor for the development of dermonecrotic lesions. This could decrease the neutrophil-mediated tissue injury and promote important reduction of induration and necrosis (Rees et al., 1985; King Jr., 1985; Rees et al., 1987; Alario et al., 1987; Boba, 1988; Smith and Baldwin, 1988; Anderson, 1991; Barrett et al., 1994; Goddard, 1998; Sams et al., 2001). Yet, as dapsone’s side effects can be severe, its administration should be strictly supervised (Wille and Morrow, 1988; Wright et al., 1997; Masters et al., 1998). Additionally, dapsone is not indicated in systemic cases and some authors question its effectiveness, mentioning its lack of advantages in experimental studies or when administered many hours after the bite (Zavaleta, 1987; Futrell, 1992; Phillips et al., 1995; Wendell, 2003; Hogan et al., 2004). In South America, the considerable morbidity and mortality in loxoscelic accidents has led to the development and use of specific therapy with antivenom. However, there are divergences regard- ing its effectiveness or its therapeutic value (Ministry of Health, Brazil, 2001; Hogan et al., 2004; da Silva et al., 2004). 2.1. Antivenom treatment The Loxosceles antivenom, developed by Vellard in 1954 (Barbaro and Cardoso, 2003), has been produced in Brazil since the early 1960s (Furlanetto, 1961) and now two antivenoms are available from the Brazilian Ministry of Health: antiloxoscelic serum and anti-arachnidic serum. The antiloxoscelic antivenom is the polyspecific serum produced at Centro de Produc- ão e Pesquisa de Imunobiológicos of the State of Paraná, Brazil, containing antibodies against venoms of the three Loxosceles species medically most important in the country: L. gaucho, L. laeta and L. intermedia. The other serum, antiarachnidic antivenom from Instituto Butantan, São Paulo, Brazil, is produced from venoms of L. gaucho, Phoneutria nigriventer, Tityus serrulatus and Tityus bahiensis, containing Loxosceles venom in the immunization pool. Besides, there are two other commercial producers of Loxosceles antivenom: the Institutos Nacionales de Salud, in Lima, Peru, and the Instituto Bioclon, in Mexico (Hogan et al., 2004). These sera are heterologous immunoglobulins, fragment F(ab0 )2 of equine origin and, thus, it is not a treatment without risks, due to possibility of complications such as allergic reactions or delayed serum sickness (Hogan et al., 2004). In studies developed in the State of São Paulo and in the State of Santa Catarina, Brazil (Sezerino et al., 1998; Málaque et al., 2002), early reactions were observed, respectively, in 20% and 6.5% of the patients who received antivenom therapy, but manifestations were mild, such as urticaria and nausea, with no risks to life and promptly reversed by proper measures. The purpose of the antivenom therapy is to neutralize the greatest possible amount of circulating venom, as it is believed that this therapy decreases the risk of systemic envenomation and potentially fatal complications, such as hemolysis, renal failure and disseminated intravascular coagulation. In Brazil, serum therapy with anti-arachnidic or anti-loxoscelic sera associated to the corticoids constitute the most employed intervention (Guilherme et al., 2001) and the Brazilian Ministry of Health recommends its use in moderate and severe ARTICLE IN PRESS I. Pauli et al. / Toxicon 48 (2006) 123–137 cases, in the presence of systemic illness, and also to decrease the reaction’s severity and reduce the healing time, depending on how soon it is administered (Ministry of Health, Brazil, 2001; City Department of Health, Curitiba, Brazil, 2002; Barbaro and Cardoso, 2003; Hogan et al., 2004). However, there is little evidence to support this effectiveness, especially against local effects, one of the main controversies found in literature (Isbister et al., 2003). Furthermore, the administration frequency has varied according to regional experiences. Even in Brazil, where the antivenom has been indicated more frequently than in other countries, disagreements with respect to the serum’s efficacy in neutralizing local effects and the ideal period for its administration determine use percentages of 11.9%, 46.8%, 54.9% and 70% in different states of the country, such as Paraná, Santa Catarina, Rio Grande do Sul and São Paulo, respectively (Barbaro et al., 1996a; Ministry of Health, Brazil, 2001). Schenone et al. (2001) mention that Loxosceles venom acts quickly on the cellular structures for which it has greater affinity, thus stopping to circulate and bringing greater damage to other tissues. It is thought that the damaging effects, whether cutaneous or visceral, are established at the moment of bite and appear hours later and therefore there is no free venom that can be neutralized by an antivenom. Furthermore, many authors question the antivenom value in arachnidic bites, since it is a clinical condition with no immediate risk to life, with manifestations predominantly cutaneous (Schenone et al., 1989, 2001). On the other hand, Rees et al. (1981) observed the reduction of necrotic areas in animal models after the administration of the antivenom and, in countries where the antivenom has been introduced, an important reduction of the mortality in children and teenagers was observed (Isbister et al., 2003). Moreover, Guilherme et al. (2001) experimentally confirmed the importance of antibodies for the neutralization of dermonecrosis, when administered 6–12 h after envenoming. They also emphasized the differences in composition and toxicity of Loxosceles venoms, indicating that effective serumtherapy should contain antibodies against all medically important species in the region. Thus, even though some experimental studies show the reduction of antivenom efficacy in cutaneous form when administered from 12 to 24 h (Furlanetto, 1961; Guilherme et al., 2001), in Hospital Vital Brazil’s experience (Instituto Butan- 127 tan, São Paulo, Brazil), patients with acute cutaneous lesions, in which necrosis was not yet present, receive the antivenom until 72 h after the bite and, in the cutaneous-hemolytic form, the serumtherapy is indicated at any time (Málaque et al., 2002; Barbaro and Cardoso, 2003). Málaque et al. (2002) routinely use the antivenom, mentioning that even though the serum does not prevent the dermonecrotic lesions, this does not mean that it is totally inefficient in loxoscelism, since it prevents these lesions from extending and limits the occurrence of systemic effects. Thus, the recommendations for use of serumtherapy in loxoscelism depend on the seriousness classification, the time between bite and medical assistance, and the risks and benefits of each case. According to Hogan et al. (2004), all available studies, if taken together, suggest that the antivenom has a potential value to decrease lesion’s size or to limit systemic illness, even in delayed use. Nevertheless, more prospective clinical studies in humans are needed. 3. Studies in humans Retrospective studies made in Brazil (Mello Guimarães et al., 1989; Mello da Silva et al., 1990; Ribeiro et al., 1993; Sezerino et al., 1998; Málaque et al., 2002), Chile (Schenone et al., 1989; Schenone et al., 2001; Schenone, 2003), Peru (Zavaleta, 1987), Mexico (Escalante-Galindo et al., 1999) and the USA (Wright et al., 1997), associated to prospective studies (Cacy and Mold, 1999; Mold and Thompson, 2004; Rees et al., 1987) and some reviewed case reports (Alario et al., 1987; Anderson, 1991; Baldwin et al., 1988; Masters, 1998; Zambrano et al., 2005), showed that loxoscelic bites usually occur in adults, from 20 to 50 years of age, with discrete predominance in women, in the hottest days or periods of the year, such as spring and summer months. The identification of which Loxosceles species caused the accident is an important factor to determine the clinical evolution and prognosis, and to direct the treatment. Important differences are observed with respect to venom’s proteic composition, experimentally associated to different degrees of toxicity, which may reflect differences in clinical state and in severity of envenoming (Barbaro et al., 1996a, b; Guilherme et al., 2001; Málaque et al., 2002; de Oliveira et al., 2005). However, since the brown spiders habits are ARTICLE IN PRESS 128 I. Pauli et al. / Toxicon 48 (2006) 123–137 predominantly nocturnal and the bite is initially relatively painless, few patients can identify the moment it happens and capture the offending agent. Zavaleta (1987) mentions that L. laeta and Loxosceles rufipes were frequently found in urban areas in the northern region of Lima, Peru, and along the coastal region of the country. However, the spider was not seen or brought for identification in more than 50% of the 279 reported cases between 1943 and 1981 in that country. This was also observed in Chile (Schenone et al., 1989; Schenone, 2003) where only 10% of the patients captured and brought the spider for identification, even though the moment of the bite was identified by 60% of these patients, who recall being bitten or seeing a spider by the time of the bite. However, Loxosceles species compatible with the reported predominant species in these regions was observed in more than 66% of the inspected houses of the patients with diagnosis of loxoscelism (Table 1). In studies carried out in Santa Catarina and in Paraná, Brazil, Sezerino et al. (1998) and Ribeiro et al. (1993) did not inform the number of spiders captured by patients or captured in the patients’ houses and surroundings, but also found Loxosceles species compatible with those reported in the literature: in Paraná, L. intermedia, predominantly, and L. laeta in restricted areas; in Santa Catarina, L. laeta in central and southern areas and L. intermedia throughout the state. It is known that the majority of fatal cases occur in children and elders and/or usually associated to species like L. laeta, possibly the most toxic of all (Futrell, 1992; Gonc- alves de Andrade and Tambourgi, 2003; de Oliveira et al., 2005), whereas relatively mild lesions are more often associated to the North American species L. deserta, Loxosceles arizonica and Loxosceles rufescens (Sams et al., 2001). In all reviewed studies, there was greater incidence of cutaneous loxoscelism, ranging from 67.9% to 100% of the cases (Table 2), as can be seen in Escalante-Galindo et al. (1999), Cacy and Mold (1999) and Mold and Thompson (2004) studies, in Mexico and in Oklahoma, USA, in which all patients were diagnosed with cutaneous loxoscelism. In these regions, L. reclusa was the predominant species and no death was registered, even though 1.8% of systemic cases by the same species have been reported by Wright et al. (1997) in Tennessee. The systemic or viscerocutaneous loxoscelism occurred more frequently in regions where L. laeta is the predominant species, such as in Chile, Peru Table 1 Identification of spiders involved in loxoscelic accidents Region (number of patients) Bite identification Spiders Predominant Species Captured Domiciliary inspection Identified species Paraná, Brazil (923)1 NR L. intermedia NR NR São Paulo, Brazil (359)2 93% L. gaucho 14% NR Santa Catarina, Brazil (267)3 Santiago, Chile (216)4 Santiago, Chile (56)5 Santiago, Chile (250)6 Limaa, Peru (279)7 NR L. laeta 2.6% NR 60.2% 61% 60.8% NR L. L. L. L. laeta laeta laeta laeta 10.6% NR 10.4% NR 66% 70% NR NR Ciudad de Mexico, Mexico (11)8 Oklahoma, USA (149)9 Oklahoma, USA (254)10 Tennessee, USA (111)11 72.7% L. laeta 63.6% NR L. intermedia and L. laeta L. gaucho, Loxosceles sp. and L. laeta L. laeta and L. intermedia L. laeta L. laeta L. laeta L. laeta and L. rufipes L. reclusa 14% 15% 20% L. reclusa L. reclusa L. reclusa 15% 13% 12% 54% 57% NR L. reclusa L. reclusa L. reclusa Key: NR—not reported. References: 1Ribeiro et al. (1993), 2Málaque et al. (2002), 3Sezerino et al. (1998), 4Schenone et al. (1989), 5Schenone et al. (2001), 6Schenone (2003), 7Zavaleta (1987), 8Escalante-Galindo et al. (1999), 9Cacy and Mold (1999), 10Mold and Thompson (2004), 11Wright et al. (1997). a Mainly in Lima, with some cases reported in Arequipa, Ica, Junin, Trujillo and Camaná. ARTICLE IN PRESS 129 I. Pauli et al. / Toxicon 48 (2006) 123–137 Table 2 Systemic loxoscelism and lethality occurrence according to age Region (Years) Predominant species n CL (%) SL (%) Death/ n (%) Death/SL (%) Age Paraná, Brazil (1989–90)1 Rio Grande do Sul, Brazil (1987–88)2 Rio Grande do Sul, Brazil (1989)3 São Paulo, Brazil (1985–96)4 Santa Catarina, Brazil (1985–95)5 Santiago, Chile (1955–88)6 Santiago, Chile (1955–2000)7 Santiago, Chile (1955–2000)8 Limaa, Peru (1943–81)9 Ciudad de Mexico, Mexico (1994–97)10 Oklahoma, USA (1996–98)11 Oklahoma, USA (1995–2000)12 Tennessee, USA (1993–95)13 L. intermedia Loxosceles sp. Loxosceles sp. L. gaucho L. laeta L. laeta L. laeta L. laeta L. laeta L. reclusa L. reclusa L. reclusa L. reclusa 923 64 116 359 267 216 56 250 279 11 149 254 111 NR 84.4 78 96.4 86.9 84.3 67.9 81.2 72.8 100 100 100 98.2 NR 15.6 22 3.6 13.1 15.7 32.1 18.8 27.2 0 0 0 1.8 0.2 1.5 0 0 1.5 3.7 7.1 3.6 NR 0 0 0 0 NR 10 0 0 11.4 23.5 22.2 19.1 NR 0 0 0 0 20.7%o20 NR 26.5%o20 7%o15 33%o15 26.8%o20 100%o15 NR NR 100%o15 17%o20 17%o20 NR Key: NR—Not Reported; n—Number of Patients; CL—Cutaneous Loxoscelism; SL—Systemic Loxoscelism. References: 1Ribeiro et al. (1993), 2Mello Guimarães et al. (1989), 3Mello da Silva et al. (1990), 4Málaque et al. (2002), 5Sezerino et al. (1998), 6Schenone et al. (1989), 7Schenone et al. (2001), 8Schenone (2003), 9Zavaleta (1987), 10Escalante-Galindo et al. (1999), 11Cacy and Mold (1999), 12Mold and Thompson (2004), 13Wright et al. (1997). a Mainly in Lima, with some cases reported in Arequipa, Ica, Junin, Trujillo and Camaná. and the State of Santa Catarina, Brazil. In these regions, a great number of patients under 15 years of age was observed, reaching 33% in Santa Catarina, Brazil (Sezerino et al., 1998) and 100% in Chile (Schenone et al., 2001). In both places, the greater lethality was also observed, 1.5% and 7.1%, respectively. This is more evident when more homogeneous groups with respect to species and age are compared (Sezerino et al., 1998; Schenone et al., 1989), with more similar incidences of cutaneous-hemolytic loxoscelism (Table 2). In the first group, 13.1% of the patients presented systemic loxoscelism, 62.9% of them less than 15 years old, which was similar to the 15.7% incidence in Chile, where 31% of the patients under 20 developed systemic loxoscelism, against only 10% of those over 20. Except in studies presented by Schenone et al. (1989, 2001), in which approximately 70% of the cases seek help earlier, few patients sought medical care until 24 h after the accident because there is usually a significant delay between the bite and the presentation for treatment. In the majority of clinical studies, symptoms started some hours after the bite and increased in 24–72 h, when patients looked for medical care, presenting pain, edema with induration, erythema and the red, white and blue sign (ecchymosis and ischemia) as the most frequent local manifestations. Unspecific signs and symptoms were observed in approximately 50% of patients with cutaneous loxoscelism and in almost all systemic loxoscelism cases, with reports of weakness, chills, myalgia, nausea, vomiting, diarrhea, turbid vision and petechiae and especially fever, generalized pruritic rash, headache and malaise in adults and, in children, fever, irritability and disturbances of consciousness. Regarding the treatment schedules, systemic corticoids were predominantly used, associated or not to analgesics and antihistamines especially in studies made in Chile, in which almost all patients were treated with corticoids associated with antihistamines in cutaneous cases and, in systemic cases, with supportive interventions (Schenone et al., 1989, 2001; Schenone, 2003). In Brazil, corticoids were in average administered in 30% of patients, similarly to what occurred in Oklahoma, USA, where 39% used systemic corticoids (Wright et al., 1997; Cacy and Mold, 1999; Mold and Thompson, 2004). The second mostly used medication was the antivenom, preferred in Brazilian states, where 60% of patients between 1984 and 1996 received intravenous serum, in contrast to 3.5% in Chile (Mello Guimarães et al., 1989; Mello da Silva et al., 1990; Ribeiro et al., 1993; Sezerino et al., 1998; Ministry of Health, Brazil, 2001; Málaque et al., 2002). Antibiotics were more often used in the USA. They were given as prophylaxis or treatment to 70% ARTICLE IN PRESS 130 I. Pauli et al. / Toxicon 48 (2006) 123–137 of patients, while in Mexico and in Santa Catarina, Brazil, they were administered to 63.6% and 38.5%, respectively, because of secondary infection, which had a small incidence in the other groups. Dapsone was prescribed to all patients in Mexico (EscalanteGalindo et al., 1999), but in other studies it was little used, in average 19.5% of patients, approximately 16% in the USA and 30% in São Paulo, Brazil (Málaque et al., 2002). In the follow-up period, the presence of necrosis was homogeneously observed in regions that have the same species, more evidently in Chile and in Santa Catarina, Brazil, where L. laeta is the predominant species (Table 3). Approximately, two-thirds of all patients with necrosis evolved to necrotic ulcer that healed without complications in the majority of cases, with healing sequels in 9.7% of them, in average, but more frequently in patients studied in Oklahoma, USA, who showed evident scars in 21% of the Mold and Thompson (2004) patients and in 13% of the Cacy and Mold (1999) ones, who were predominantly treated with antibiotics. Schenone et al. (1989) reported that hemolysis and skin ulcerations that evolve slowly leaving healing sequels were observed despite the specific treatment with antivenom. In studies carried out in Chile, the antiloxoscelic serum was administered by subcutaneous or intramuscular route in 4 of 385 patients with cutaneous loxoscelism diagnosis and in 12 of 81 patients with cutaneous-hemolytic loxoscelism. From the 12 patients that received serum for systemic loxoscelism, 5 died, associated to 12 that were treated only with corticoids and supportive interventions (Schenone et al., 1989; Schenone, 2003). In another study, with patients under 15 years of age (Schenone et al., 2001), the antiloxoscelic serum was applied by parenteral route in 2 of 18 patients with hemolytic manifestations, but these 2 patients died in addition to other 2, who were treated with corticoids and supportive interventions. There was no relation with site of bite or with the extension of cutaneous lesion; it was only mentioned that the treatment was initiated 24 h after the accident and that the deaths occurred in average 24–30 h after the bite, without more precise information about the time of presentation to the hospital and the beginning of treatment with antivenom, corticoids or other interventions. On the other hand, in a study performed in São Paulo, Brazil, Málaque et al. (2002) observed that the specific or polyvalent antivenom was administered intravenously to 237 of 359 patients available, in association with corticoids in 47% of cases and dapsone in 30%. All these patients arrived at hospital until 72 h after the bite and had no necrotic ulcer when admitted. Beyond the 237 patients, 2 other patients also sought medical care until 72 h, Table 3 Evolution of cutaneous and systemic loxoscelism, and antivenom use Region (n) Species Necrosis (%) Ulcer (%) Scar (%) LCH (%) Death/n Death/ (%) SL (%) AV (%) Paraná, Brazil (923)1 Rio Grande do Sul, Brazil (64)2 Rio Grande do Sul, Brazil (116)3 São Paulo, Brazil (359)4 Santa Catarina, Brazil (267)5 Santiago, Chile (216)6 Santiago, Chile (56)7 Santiago, Chile (250)8 Limaa, Peru (279)9 Ciudad de Mexico, Mexico (11)10 Oklahoma, USA (149)11 Oklahoma, USA (254)12 Tennessee, USA (111)13 L. intermedia Loxosceles sp. Loxosceles sp. L. gaucho L. laeta L. laeta L. laeta L. laeta L. laeta L. reclusa L. reclusa L. reclusa L. reclusa NR NR 85.0 29.1 56.9 50.9 66.0 55.2 NR 45.5 40.0 NR 37% NR NR NR 29.1 NR 35.1 45.0 38.0 NR NR NR NR NR NR 1.6 11.1 4.0 NR 8.3 10.8 8.8 NR 9.0 13.0 21.0 NR 0.2 1.5 0 0 1.5 3.7 7.1 3.6 NR 0 0 0 0 37.0 100.0 85.1 66.0 46.8 3.7 3.6 3.2 NR 0 0 0 0 NR 15.6 22.0 3.6 13.1 15.7 32.1 18.8 27.2 0 0 0 1.8 NR 10.0 0 0 11.4 23.5 22.2 19.1 NR 0 0 0 0 Key: NR—not reported; n—number of patients; CL—cutaneous loxoscelism; SL—systemic loxoscelism; AV—antivenom. References: 1Ribeiro et al. (1993), 2Mello Guimarães et al. (1989), 3Mello da Silva et al. (1990), 4Málaque et al. (2002), 5Sezerino et al. (1998), 6Schenone et al. (1989), 7Schenone et al. (2001), 8Schenone (2003), 9Zavaleta (1987), 10Escalante-Galindo et al. (1999), 11Cacy and Mold (1999), 12Mold and Thompson (2004), 13Wright et al. (1997). a Mainly in Lima, with some cases reported in Arequipa, Ica, Junin, Trujillo and Camaná. ARTICLE IN PRESS I. Pauli et al. / Toxicon 48 (2006) 123–137 but they did not receive the antivenom, possibly by the presence of established necrotic lesion. Necrosis was more frequently observed when the bite occurred in proximal anatomic areas, such as trunk, thigh and upper arm (p ¼ 0:0002) and in those patients who arrived at the hospital 72 h or more after the bite (po0:0001). Necrotic lesions that dropped off leaving ulcers were observed in 191 patients (53%), being 122 on admission who did not receive serum, and 69 after antivenom therapy. Of the 237 patients that received antivenom, 69 (29.1%) developed necrosis, in contrast to the 168 (70.9%) patients who had no necrosis, necrotic ulcers or healing sequels. Moreover, no acute renal failure, disseminated intravascular coagulation or death were registered, even though cutaneoushemolytic loxoscelism has developed in 13 (3.6%) patients, some of these with hemoglobin level next to 5.0 g/dl and packed cell volume next to 10%. In Sezerino et al. (1998) study, which is more similar to those performed in Chile (Schenone et al., 1989, 2001; Schenone, 2003) due to L. laeta predominance in the region, similar incidences of systemic loxoscelism and necrosis were observed, but with differences in the number of patients that received antivenom and the number of deaths, which were approximately twice as those observed in the Chilean studies mentioned. However, it is not known if these differences are due to different therapeutic schedules, to variations among the studied populations or other factors, because there are no detailed information about the time between the bite and the beginning of medication in this specific group. Besides, there is no information that allows the correlation between the type of treatment received with the evolution of the cutaneoushemolytic loxoscelism cases, such as with the evolution of the cutaneous cases in other studies (Mello Guimarães et al., 1989; Mello da Silva et al., 1990; Ribeiro et al., 1993; Ministry of Health, Brazil, 2001; Málaque et al., 2002; Zavaleta, 1987; Escalante-Galindo et al., 1999; Wright et al., 1997; Cacy and Mold, 1999; Mold and Thompson, 2004). 4. Studies in experimental models The lesions induced by the brown spiders toxins can be experimentally reproduced. In mice, the venom toxins promote damage in liver, kidneys, heart and central nervous system with death, but without the occurrence of the dermonecrotic lesions 131 that appear in rabbits and in humans (Babcock et al., 1981). Furlanetto (1961) was recognized by the production of the first antiloxoscelic serum in industrial scale and a standardization that is adequate to therapeutic use, instituting the methodology for the titration of this serum. This is based on the antinecrotizing unit (AU), which is the smallest amount of antiloxoscelic serum that, intravenously inoculated, is capable of totally neutralizing the effect of Loxosceles venom minimal necrotizing dose (MND) injected by intradermal route into a rabbit’s ear. One MND is the smallest amount of venom intradermally injected into a rabbit’s ear able to induce a necrotic lesion of about one centimeter of diameter in 48–72 h. This author also demonstrated that rabbits are the best animals for reproduction of loxoscelic envenomation signs in humans and are also the more suitable animal model for serum neutralization trials. Studies in animal models have demonstrated the potential value of the antivenom in loxoscelism treatment (Furlanetto, 1961; Rees et al., 1981; Bravo et al., 1993; Braz et al., 1999; Gomez et al., 1999; Guilherme et al., 2001; Barbaro et al., 2005), but one of the most important questions in loxoscelism is the ideal time for the antivenom administration. In this direction, several experimental studies have tried to correlate the time of envenoming with the antivenom administration (Table 4). In experiments with in vivo neutralization, performed with independent injections of venom and antibody into a rabbit’s ear, Furlanetto (1961) observed complete inhibition of dermonecrosis when the antivenom was intravenously administered until 4 h after the venom inoculation; almost complete inhibition until 8 h, and reduction of the necrotic lesion to half its size when antivenom was administered at 16–24 h (Table 4). In this study, the author cited the use of L. rufipes venom and L. rufescens antivenom, but cytogenetic studies carried out at that time, showed that these spiders were actually variations of the same species. Rees et al. (1981), when comparing surgical excision, heparin, steroids and specific antivenom administered by intradermal route at the lesion site, demonstrated that the antivenom is the most effective method to prevent the toxic effects of L. reclusa venom. Microscopic findings of vessel occlusion, polymorphonuclear neutrophils infiltration and necrosis were present when the antivenom ARTICLE IN PRESS 132 I. Pauli et al. / Toxicon 48 (2006) 123–137 Table 4 Dermonecrotic activity neutralization by antivenoms in rabbits Venom Antivenom Results Study Loxosceles Sp. Route Type Route L. rufipes ID Equine polyclonal anti L. rufescens antibody; F(ab’)2 fragments IV Antivenom given until 4 h promoted total neutralization of necrosis; until 8 h, almost total neutralization and, from 16 to 24 h, reduction to half the dermonecrosis Furlanetto (1961) L. reclusa ID Rabbit polyclonal anti L. reclusa antibody; not reported if complete IgG or fragments ID, at the lesion site Antivenom given until 6–12 h did not totally inhibit the microscopic signs of envenoming; given until 24 h, weakly attenuated the toxic effects of the venom Rees et al. (1981) L. deserta ID Rabbit polyclonal anti L. deserta antibody; Fab fragments ID, at the lesion site Antivenom given until 4 h inhibited the inflammation and the dermonecrosis Gomez et al. (1999) L. gaucho ID Rabbit monoclonal anti L. gaucho antibody IV Antivenom given until 6 h reduced dermonecrotic area by around 97% Guilherme et al. (2001) L. gaucho ID Equine polyclonal anti L. gaucho antibody; F(ab0 )2 fragments IV Antivenom given until 12 h reduced dermonecrotic area by around 76% Guilherme et al. (2001) L. laeta and intermedia ID Rabbit monoclonal anti L. gaucho antibody IV Antivenom was not effective in the neutralization of the toxic effects of L. laeta and L. intermedia Guilherme et al. (2001) Key: ID—intradermal; IV—intravenous. was administered 6–12 h post-envenomation, but were decreased when compared to control rabbits (Table 4). In addition, they observed that larger doses of the antivenom inhibited the initial amount of erythema. Gomez et al. (1999) verified that anti L. reclusa Fab fragments intradermally injected at the lesion site inhibited the inflammation and necrosis associated to Loxosceles envenomation in rabbits until 4 h after the venom inoculation, but did not inhibit them when administered 8 h after venom inoculation (Table 4). However, the authors call attention to several limitations and questionings that must be observed in this study: the unblinded design in lesion area determination, which does not permit control for investigator bias; the injured area and the mieloperoxidade activity levels had been followed only up to 48 h after the beginning of treatment, and it is not known which would be the evolution if the accompaniment was made by a longer period. In addition, the use of the same animals for both treatment and control lesions could have affected the outcome. On the other hand, if the venom spreads from the initial envenomation site, one antivenom injection in the bite site could possibly not neutralize the venom in double lesions. Thus, it is questioned whether larger groups and optimized study conditions would demonstrate efficacy by longer periods of time post-envenomation and whether the intralesional antivenom administration would be a useful therapy in systemic loxoscelism. Guilherme et al. (2001) studied the ability of the specific antibodies to neutralize dermonecrosis, lethal activity and differences of the main toxic proteins of medically important Loxosceles venom in Brazil (L. gaucho, L. laeta and L. intermedia) using monoclonal antibodies (MAbs) produced ARTICLE IN PRESS 133 I. Pauli et al. / Toxicon 48 (2006) 123–137 against the 35 kDa dermonecrotic component of L. gaucho venom, the MoALgs. It was observed that MoALg1 efficiently reduced L. gaucho venominduced dermonecrosis, but other MAbs did not show any effect even on homologous venom and none of the MAbs was able to significantly neutralize local reactions induced by L. laeta and L. intermedia venom. Furthermore, only MoALg1 delayed the lethality induced by L. gaucho venom, protecting 91% of the mice studied, but these animals died 96 h after venom injection. The other MAbs were unable to neutralize or delay this lethal effect of homologous venom and all MAbs failed to neutralize the activity of the heterologous venom (L. laeta and L. intermedia). On the other hand, MoALg1 reduced the dermonecrotic activity in approximately 97%, even when injected (intravenously) 6 h after the envenoming. However, it failed again to neutralize the dermonecrotic activity of L. laeta and L. intermedia venoms, suggesting the existence of important differences among the epitopes present in the dermonecrotic and lethal components of the three Loxosceles venoms. In this study, an equine polyclonal antiloxoscelic serum was also utilized, which initially neutralized 90–100% of the dermonecrotic activity and, when given 12 h post-envenomation, neutralized 76% of the dermonecrosis (Table 4). Barbaro et al. (2005) characterized and compared aspects of the five major medically important Loxosceles venoms in the Americas (L. gaucho, L. laeta, L. intermedia, L. reclusa and L. deserta), particularly considering their neutralization by the Brazilian commercial antivenoms: the anti-arachnidic serum and the antiloxoscelic serum. These antivenoms were able to completely neutralize the dermonecrotic activity as well as the local reaction of all venoms. The ability of the antivenoms was demonstrated by incubating venoms of the Loxosceles species with each antivenom, for one hour at 37 1C, and the supernatant material was intradermally injected into a rabbit’s dorsum. This study showed strong cross-reactivity among all venoms and the Brazilian antivenoms, determined by enzyme-linked immunosorbent assay (ELISA). However, against L. laeta venom, the anti-arachnidic serum showed titers significantly lower than the antiloxoscelic serum (Table 5). Braz et al. (1999) also observed lower neutralizing dermonecrotic capacity of the anti-arachnidic serum than the antiloxoscelic serum against L. intermedia venom in trials in vivo, suggesting that the homologous antivenoms would be the most efficient. In another study, de Oliveira et al. (2005) analyzed biochemical and toxicity variations in L. laeta and L. intermedia venoms, focused in finding a correlation with the severity of the bite. Differences in protein and glycoprotein expression and in sphingomyelinase activity between venoms of these two species were observed, which were reflected in venoms toxicity, including the capacity to induce complement-dependent hemolysis, dermonecrosis and lethality. In addition, the experimental antivenom raised against the L. laeta female venom showed the highest efficacy in neutralizing venoms of males and females of both L. laeta and L. intermedia. Based on this, it is suggested that for accidents involving L. laeta, a specific serum therapy is necessary (de Oliveira et al., 2005). With the purpose of determining the venom permanence time in the lesion site, Cardoso et al. (1990) also showed the presence of L. gaucho venom Table 5 Antigenic cross reactivity between venoms of the different Loxosceles species Antivenom Venom L. deserta (ELISA titer) L. gaucho (ELISA titer) L. intermedia (ELISA titer) L. laeta (ELISA titer) L. reclusa (ELISA titer) Anti-arachnidic Serum: anti L. gaucho antibodies; F(ab’)2 fragmentsa 128,000 256,000 128,000 64,000 128,000 Antiloxoscelic Serum: anti L. laeta, L. intermedia and L. gaucho antibodies; F(ab’)2 fragmentsb 256,000 512,000 512,000 512,000 512,000 Reference: Barbaro et al. (2005). a Batch 0211124, produced by Instituto Butantan, São Paulo, Brazil. b Batch S 01/00, produced by Centro de Produc- ão e Pesquisa de Imunobiológicos (CPPI) of the State of Paraná, Brazil. ARTICLE IN PRESS 134 I. Pauli et al. / Toxicon 48 (2006) 123–137 in dermonecrotic lesion in patients by ELISA 49 days after the bite. In animal models, Krywko and Gomez (2002) verified the presence of L. reclusa venom in hair and tissue aspirate and biopsy until 7 days, but the venom is not detectable with 24 h when searched in serum. However, Gubert (2005), using rabbits inoculated by subcutaneous route, showed the presence of L. intermedia circulating venom 4 h after venom inoculation, and ChávezOlórtegui et al. (1998), in a study with 112 patients, found circulating venom in serum of the 30 patients victim of L. intermedia that arrived at the hospital until 24 h after the bite. 5. Discussion The ability of the specific antibodies to neutralize dermonecrotic activity induced by Loxosceles venoms was already argued by several authors, being presently unquestioned (Furlanetto, 1961; Rees et al., 1981; Bravo et al., 1993; Braz et al., 1999; Gomez et al., 1999; Guilherme et al., 2001; Barbaro et al., 2005; Swanson and Vetter, 2005). However, the antivenom efficacy is time- and dosedependent, as demonstrated in studies in animals, with variable neutralization of inflammation and dermonecrosis after administration of antivenom in different periods, which was complete 4 h postenvenomation (Furlanetto, 1961; Gomez et al., 1999), of about 76% in 12 h (Guilherme et al., 2001) and 50% in 16–24 h (Furlanetto, 1961), and more evident with gradually larger doses of antivenom (Rees et al., 1981) or with species-specific serumtherapy (Guilherme et al., 2001; de Oliveira et al., 2005). In these experiments, different protocols to correlate the envenomation time with the administration of antivenom were applied, but better results were observed with the use of standardized equine serum, administered intravenously. It is known that the majority of venom’s damaging effects in studies in animals occur in 3–6 h after the brown recluse spider bite (Rees et al., 1981; Wasserman and Anderson, 1983–84; Patel et al., 1994; Ospedal et al., 2002) and since the majority of clinical studies usually demonstrated a significant delay between bite and presentation for treatment, it is thought that this delay leads to an ineffective administration of antivenom. However, an important reduction of mortality associated to loxoscelism in children and teenagers was observed in countries where antivenom was introduced (Wendell, 2003), as well as less healing complications in some institutions that were using serum in patients who searched for medical help until 72 h after the loxoscelic accident without necrosis (Málaque et al., 2002). In fact, the majority of patients who looked for medical care until 24 h after the bite did not present dermonecrosis (Barbaro et al., 1992), as evidenced in Málaque et al. (2002) study. This work demonstrated that the majority of patients seen before or until 72 h had no necrosis, suggesting that establishment of dermonecrosis in humans is a slower process than the one verified in rabbits (Guilherme et al., 2001; Barbaro et al., 2005). In this direction, it is believed that even a delayed antivenom therapy can be beneficial to decrease the lesion size and cure time or to limit systemic effects (Rees et al., 1981; Guilherme et al., 2001; Hogan et al., 2004; Barbaro et al., 2005). Furthermore, some care is necessary when extrapolating results of experimental studies for clinical management of Loxosceles bites in patients, because studies in rabbits are not truly comparable to studies in humans, since chronic ulcerations do not develop in rabbits and venom-induced lesions heal much faster in those animals than in humans (Masters et al., 1998; Guilherme et al., 2001). Also, the genetic and immunological differences between human and animal models must be considered. The differences in protocols used in animal trials should also be considered, such as: different amounts of venoms from different Loxosceles species; different antivenoms obtainment and standardization of methods, administered by different routes and with different doses; and the choice of antibody type (complete IgG or Fab and F(ab0 )2 fragments). These differences reflect on the optimization of toxin neutralization by specific antibodies. On the other hand, for the majority of studies in humans there is no detailed information about the time between bite and beginning of medication, nor information that may allow a correlation between the received treatments and the evolution of the loxoscelic pictures. In addition, it is not known if the differences observed are due to different therapeutic schedules, to variations between the studied populations or to other factors. Thus, the systematization of the observed data and their statistical analysis, which would make possible a more critical analysis of the antivenom’s efficacy in bites of Loxosceles, were not possible in this study. ARTICLE IN PRESS I. Pauli et al. / Toxicon 48 (2006) 123–137 Even presently, the early and adequate treatment is not possible because of diagnostic difficulties, since few patients can identify the moment of bite and/or capture the spider for identification. There is disinformation of doctors and patients and there are no specific laboratory tests for diagnosis or prognosis tests (Barbaro and Cardoso, 2003; da Silva et al., 2004). Some immunoassays to detect the presence of venom in blood circulation (Chávez-Olórtegui et al., 1998), hair, skin biopsies or aspirate tissue (Cardoso et al., 1990; Miller et al., 2000; Gomez et al., 2002; Krywko and Gomez, 2002) were studied, with potential for future application, as diagnosis tests. However, the diagnosis of loxoscelic accidents is still fundamentally clinical-epidemiological, based on clinical history, signs and symptoms associated to the spider identification or possible contact and other etiology exclusions (Futrell, 1992; Schenone, 1996; Wright et al., 1997; Sams et al., 2001; Wendell, 2003; da Silva et al., 2004; Isbister and White, 2004; Swanson and Vetter, 2005). Moreover, the diagnosis depends on medical experience and medical care infrastructure. Thus, the development of a sensitive, fast and specific kit of laboratorial diagnosis would allow the early diagnosis but, despite existing studies, there are no laboratory tests commercially available and with enough clinical correlation (da Silva et al., 2004). Additionally, medical care of suspected cases must be prioritized in different health centers, adequate information for patients must be encouraged and technical subsidies to identify, diagnose and treat this type of injury must be supplied to doctors, especially in endemic regions. This correct diagnosis leads to an immediate treatment, thus contributing to the decrease in morbidity, suffering and mortality. 6. Conclusion Of all the evaluated interventions, antivenom showed the greatest therapeutic potential, but there are no adequately performed clinical experiments that may assure the efficacy of the different treatments in loxoscelic bites or lead to a greater consensus of which would be the ideal therapy for the treatment of loxoscelism. We believe that even delayed administrations of antivenom can contribute to the reduction of dermonecrosis and healing time, as well as to limit systemic damages. 135 Issues about serum’s capacity to neutralize local effects and about the correlation between envenomation time and serumtherapy efficacy still have no answers. Thus, we reaffirm the need for controlled prospective clinical studies for its elucidation— using patients with early presumptive lesions, that is, anticipated lesions—which constitutes a crucial step in the treatment of loxoscelism. References Alario, A., Price, G., Stahl, R., Bancroft, P., 1987. Cutaneous necrosis following a spider bite: a case report and review. Pediatrics 79 (4), 618–621. Anderson, P.C., 1991. Loxoscelism threatening pregnancy: five cases. Am. J. Obstet. Gynecol. 165, 1454–1456. Babcock, J.L., Civello, D.J., Geren, C.R., 1981. Purification and characterization of a toxin from brown recluse spider (Loxosceles reclusa) venom gland extracts. 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