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Poor efficacy of preemptive amoxicillin clavulanate for preventing secondary infection from Bothrops snakebites in the Brazilian Amazon: A randomized controlled clinical trial

  • Jacqueline A. G. Sachett,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Manaus, Brazil

  • Iran Mendonça da Silva,

    Roles Data curation, Investigation, Validation, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Manaus, Brazil

  • Eliane Campos Alves,

    Roles Data curation, Investigation, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Manaus, Brazil

  • Sâmella S. Oliveira,

    Roles Data curation, Investigation, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Manaus, Brazil

  • Vanderson S. Sampaio,

    Roles Formal analysis, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Manaus, Brazil, Núcleo de Sistemas de Informação, Fundação de Vigilância em Saúde do Amazonas, Manaus, Brazil

    ORCID http://orcid.org/0000-0001-7307-8851

  • Fábio Francesconi do Vale,

    Roles Data curation, Investigation, Validation, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Faculdade de Medicina, Universidade Federal do Amazonas, Manaus, Brazil

  • Gustavo Adolfo Sierra Romero,

    Roles Conceptualization, Formal analysis, Methodology, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Núcleo de Medicina Tropical, Faculdade de Medicina, Universidade de Brasília, Brasília, Brazil

    ORCID http://orcid.org/0000-0003-1425-926X

  • Marcelo Cordeiro dos Santos,

    Roles Investigation, Methodology, Supervision, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Manaus, Brazil

  • Hedylamar Oliveira Marques,

    Roles Investigation, Supervision, Validation, Writing – review & editing

    Affiliation Diretoria de Ensino e Pesquisa, Fundação Hospitalar de Hematologia e Hemoterapia do Amazonas, Manaus, Brazil

  • Mônica Colombini,

    Roles Conceptualization, Writing – review & editing

    Affiliation Divisão de Desenvolvimento Científico, Instituto Butantan, São Paulo, Brazil

  • Ana Maria Moura da Silva,

    Roles Investigation, Resources, Supervision, Validation, Writing – review & editing

    Affiliation Divisão de Desenvolvimento Científico, Instituto Butantan, São Paulo, Brazil

  • Fan Hui Wen,

    Roles Validation, Writing – original draft, Writing – review & editing

    Affiliation Divisão de Desenvolvimento Científico, Instituto Butantan, São Paulo, Brazil

  • Marcus V. G. Lacerda,

    Roles Conceptualization, Formal analysis, Methodology, Validation, Writing – original draft, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Manaus, Brazil, Instituto de Pesquisas Leônidas & Maria Deane, FIOCRUZ, Manaus, Brazil

  • Wuelton M. Monteiro ,

    Roles Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    wueltonmm@gmail.com

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Manaus, Brazil

  • Luiz C. L. Ferreira

    Roles Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliations Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil, Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Manaus, Brazil, Faculdade de Medicina, Universidade Federal do Amazonas, Manaus, Brazil

    ORCID http://orcid.org/0000-0002-9657-939X

Poor efficacy of preemptive amoxicillin clavulanate for preventing secondary infection from Bothrops snakebites in the Brazilian Amazon: A randomized controlled clinical trial

  • Jacqueline A. G. Sachett, 
  • Iran Mendonça da Silva, 
  • Eliane Campos Alves, 
  • Sâmella S. Oliveira, 
  • Vanderson S. Sampaio, 
  • Fábio Francesconi do Vale, 
  • Gustavo Adolfo Sierra Romero, 
  • Marcelo Cordeiro dos Santos, 
  • Hedylamar Oliveira Marques, 
  • Mônica Colombini
PLOS
x

Abstract

Background

Secondary bacterial infections from snakebites contribute to the high complication rates that can lead to permanent function loss and disabilities. Although common in endemic areas, routine empirical prophylactic use of antibiotics aiming to prevent secondary infection lacks a clearly defined policy. The aim of this work was to estimate the efficacy of amoxicillin clavulanate for reducing the secondary infection incidence in patients bitten by Bothrops snakes, and, secondarily, identify risk factors for secondary infections from snakebites in the Western Brazilian Amazon.

Methods and findings

This was an open-label, two-arm individually randomized superiority trial to prevent secondary infection from Bothrops snakebites. The antibiotic chosen for this clinical trial was oral amoxicillin clavulanate per seven days compared to no intervention. A total of 345 patients were assessed for eligibility in the study period. From this total, 187 accomplished the inclusion criteria and were randomized, 93 in the interventional group and 94 in the untreated control group. All randomized participants completed the 7 days follow-up period. Enzyme immunoassay confirmed Bothrops envenoming diagnosis in all participants. Primary outcome was defined as secondary infection (abscess and/or cellulitis) until day 7 after admission. Secondary infection incidence until 7 days after admission was 35.5% in the intervention group and 44.1% in the control group [RR = 0.80 (95%CI = 0.56 to 1.15; p = 0.235)]. Survival analysis demonstrated that the time from patient admission to the onset of secondary infection was not different between amoxicillin clavulanate treated and control group (Log-rank = 2.23; p = 0.789).Secondary infections incidence in 7 days of follow-up was independently associated to fibrinogen >400 mg/dL [AOR = 4.78 (95%CI = 2.17 to 10.55; p<0.001)], alanine transaminase >44 IU/L [AOR = 2.52 (95%CI = 1.06 to 5.98; p = 0.037)], C-reactive protein >6.5 mg/L [AOR = 2.98 (95%CI = 1.40 to 6.35; p = 0.005)], moderate pain [AOR = 24.30 (95%CI = 4.69 to 125.84; p<0.001)] and moderate snakebites [AOR = 2.43 (95%CI = 1.07 to 5.50; p = 0.034)].

Conclusions/Significance

Preemptive amoxicillin clavulanate was not effective for preventing secondary infections from Bothrops snakebites. Laboratorial markers, such as high fibrinogen, alanine transaminase and C-reactive protein levels, and severity clinical grading of snakebites, may help to accurately diagnose secondary infections.

Trial registration

Brazilian Clinical Trials Registry (ReBec): RBR-3h33wy; UTN Number: U1111-1169-1005.

Author summary

Bothrops genus is responsible by 80–90% of the snakebites in the Brazilian Amazon, resulting in a subcutaneous and muscular lesion at the site of bite, which many times evolve to local complications, mostly secondary bacterial infections. In this region, late medical assistance is common and probably contributes to the high complication rates related to local necrosis and secondary bacterial infections, which can lead to permanent function loss and disabilities. Even with this high frequency, routine empirical use of antibiotics aiming to prevent secondary infection lacks a clearly defined protocol. In this work, we estimated the efficacy of amoxicillin clavulanate for reducing the secondary infection incidence in patients bitten by Bothrops snakes, and, identified factors related to secondary infections from snakebites. Amoxicillin clavulanate was not effective for preventing secondary infections from Bothrops snakebites, probably because of the resistance to β-lactam antibiotics in bacteria species commonly found infecting the snakebite site. This finding highlights the need of previous knowledge of the secondary infections epidemiology as a cornerstone in the preemptive antibiotics trials in snakebites. Laboratorial markers, such as high fibrinogen, alanine transaminase and C-reactive protein levels, and severity clinical grading of snakebites, may help to accurately diagnose secondary infections.

Introduction

Bothrops snakebites result in a subcutaneous and muscular lesion at the site of bite, which many times evolve to local complications [15]. In 2015, the Brazilian Ministry of Health recorded 18,741 snakebites across the country [6]. In this country, a higher incidence is observed in the Brazilian region (37 cases/100.000 inhabitants). These values could be higher in remote areas of the Amazon because of the considerable case underreporting [7]. Snakebites are recorded mostly from remote rural or riverine areas, from where patients’ rescue to the health units is made exclusively by boat transportation, lasting several hours or even days [810]. Thus, late medical assistance is common and probably contributes to the high complication rates related to local necrosis [1012] and secondary bacterial infections [1015], that can lead to permanent function loss and disabilities [3,10,1619]. It was suggested that secondary bacterial infections from snakebites are related to the oral and fangs microbiota of the perpetrating snake [2026]. Besides, traditional treatment also contributes for the emergence of secondary infections, such as tourniquet use, local alternative medicines, incision and suction of the bite site [8,18,27]. These factors increase the development of expressive forms of secondary infection, which has been primarily diagnosed with identification of cellulitis or abscesses [12,13,22].

In the Brazilian Amazon, Bothrops atrox is the most important venomous snake, causing 80–90% of the snake envenomings [28], Despite the wide geographic distribution in the Amazon, B. atrox venoms share the same family of toxins, as PIII and PI snake venom metalloproteinase, phospholipase A2, serine proteinase, cysteine-rich secretory protein, L-amino acid oxidase and C-type lectin-like [29,30] and are characterized by coagulant, hemorrhagic and proteolytic or acute inflammatory activities [3133]. Spontaneous systemic bleeding and acute renal failure are common systemic complications from Bothrops envenomings [13,34]. Local envenomation range from a painless reddened injury to intense pain and swelling at the site of bite, starting minutes after the event. Enlargement of the regional lymph nodes draining the site of bite and bruising can also be observed some hours after bite, especially if patient delayed in reaching a health service [33,34][34][33][33][33][33]. In the first 24 hours, blistering and tissue necrosis may be evident. Cellulitis or abscess occurs mostly in the moderate or severe cases, generally as a polymicrobial infection. Gram-negative bacteria have been implicated in secondary bacterial infection, which frequency may vary according to region [35]. In Manaus, secondary bacterial infections were observed in around 40% of the Bothrops snakebites [13].

Several antimicrobial schemes were suggested for the treatment of secondary infections, but in general these recommendations were not based on good evidences from clinical trials [36,37]. For example, ampicillin/cephalosporin/cloxacillin [14,27], ciprofloxacin [15,22] and clindamycin [36,38,39] were previously used for secondary bacterial infections resulted from snakebites, with variable effectiveness. In the Amazon, basic information about the bacterial agents responsible by the wound infection is still lacking since secondary infection diagnosis is mostly based only from clinical features without microbiological confirmation. Although common in endemic areas, routine empirical prophylactic use of antibiotics aiming to prevent secondary infection lacks a clearly defined policy, leading to wasteful inappropriate antibiotic use, which is costly and may promote bacterial antibiotic resistance [22,40]. Preemptive treatment efficacy of oral chloramphenicol monotherapy in Bothrops snakebites [41] and intravenous chloramphenicol plus gentamicin in Crotalus snakebites [42] showed no statistical difference between patients treated and untreated groups. Nowadays, however, the Infectious Diseases Society of America (IDSA) guidelines for diagnosis and management of skin and soft-tissue infections indicate amoxicillin clavulanate to reduce complications by prevention of secondary infection from animal bites [38,39]. Evidence supporting this recommendation came from a clinical trial carried out with patients bitten by dogs [43], but efficacy of this regimen is still not available in snakebites.

The aim of this work was to estimate the efficacy of amoxicillin clavulanate for reducing the secondary infection incidence in patients bitten by Bothrops snakes, and, secondarily, identify associated factors for secondary infections from snakebites in the Western Brazilian Amazon.

Methods and materials

Ethics statement

Ethical approval was obtained from the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) (approval number 492.892/2013). Written informed consent was obtained from all participants prior to randomization. This study was registered in the Brazilian Clinical Trials Registry (ReBec): RBR-3h33wy and UTN Number: U1111-1169-1005.

Study design and participants

This was an open-label, two-arm individually randomized superiority trial to estimate the efficacy of the preemptive amoxicillin clavulanate administration compared to no intervention for preventing secondary infection from Bothrops snakebites. Clinical trial was performed at the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD), in Manaus, Western Brazilian Amazon, from August 2014 to September 2016. This tertiary hospital is the reference in the Amazonas state for snakebites treatment. In Manaus, FMT-HVD is the only hospital unit that performs the distribution and administration of snakebite antivenom. At admission, Bothrops snakebites were diagnosed with basis in clinico-epidemiological characteristics of the patient and, when the patient brought the snake responsible by the envenomation, by its identification made by a trained biologist.

Sample size calculation was based on the mean of 240 snakebites/year attended at FMT-HVD, with an expected frequency of secondary infection of 40% [13], a 50% risk reduction of infection, at an 80% power and 5% of significance level and an 1:1 randomization ratio. Adding 10% of losses in the follow-up, a sample size of 186 participants was obtained, with 93 patients in the intervention group and 93 in the untreated group.

Eligibility, randomization and intervention

Patient was eligible if admitted to the hospital with less than 24 hours after the bite, without antivenom therapy in other hospital and without any sign of secondary infection at this time. Patients that used any antibiotic in the past 30 days, pregnant women or patients with previous history of allergic reactions to antibiotics were not included in this trial.

After application of eligibility criteria, the study pharmacist was contacted to obtain the allocation group to the patient. Randomization sequences with an allocation ratio of 1:1 were computer-generated by a random table, to the intervention group (preemptive amoxicillin clavulanate) or to the control group (no preemptive antibiotic prescription). All laboratory staff was blinded for treatment assignment. The antibiotic chosen for this trial was oral tablet amoxicillin clavulanate 875/125 mg to adults and 25 mg/kg/day to children twice per day for seven days, starting at the admission day.

Admission and follow-up procedures

After patient inclusion, demographic and epidemiological information was collected using a standardized questionnaire, including gender, age (in years), area of occurrence (urban or rural), anatomical site of the bite, work-related bite (yes or no), time elapsed from bite to medical assistance (in hours), walking after bite (in minutes), previous history of snakebite and pre-admission conduits (use of topical or oral medicines, use of tourniquet and other procedures). A detailed clinical and laboratorial characterization was also made at this time. Pain assessment was made using the Numerical Rating Scale, with values rating from 1 to 10 [44]; pain was further classified as absent (rate 0), mild (rated from 1 to 3), moderate (rated from 4 to 7) and severe (rated from 8 to 10). Edema was classified as absent, mild (affecting 1–2 limb segments), moderate (affecting 3–4 limb segments) and severe (affecting more than 5 limb segments) [11]. Bite site temperature (oC) was measured using an infrared digital thermometer (Color Check AC322); the difference between the bite site temperature and the contralateral limb site was calculated. Presence of local bleeding, lymphadenitis and necrosis was also assessed. Systemic signs and symptoms, such as systemic bleeding, signs of acute renal failure, headache, dizziness and vomiting were recorded from patients. Vital signs (blood pressure, heart rate, respiratory rate and axillary temperature) were also assessed. All the clinical information was collected through a standardized clinical registration form. Immediately after clinical examination, a 15 mL blood sample was taken for laboratorial analysis. Tests included leukocyte count (cells/μL), fibrinogen (mg/dL), platelet count (number/μL), hemoglobin (mg/dL), creatine phosphokinase (IU/L), creatine phosphokinase-MB (ng/mL), erythrocyte sedimentation rate (mm/hour), lactate dehydrogenase (IU/L), creatinine (mg/dL), urea (mg/dL), aspartate transaminase (IU/L), alanine transaminase (IU/L), clotting time (in minutes), prothrombin time (in seconds) and C-reactive protein (mg/dL). An aliquot was submitted to an enzyme immunoassay to confirm Bothrops envenoming diagnosis and to determine circulating venom levels in all patients [45]. All the laboratory results were transferred to a standardized registration form.

According to clinical severity, patients were classified using to the Brazilian Health Ministry guidelines [7]: i) mild cases: local pain, local swelling and bruising for Bothrops; ii) moderate cases: local manifestations without necrosis and minor systemic signs (coagulopathy and bleeding, no shock); iii) severe cases: life- threatening snakebite, with severe bleeding, hypotension/shock and/or acute renal failure.

Both groups were submitted to the same local wound care with daily 0.9% saline cleaning. Thirty minutes before antivenom therapy, the intervention group of patients took amoxicillin clavulanate, supervised by a nurse. Twenty minutes after pre-medication with IV hydrocortisone (500 mg), IV cimetidine (300 mg) and oral dexchlorpheniramine (5 mg), antivenom therapy was given to all patients from both arms in a dosage corresponding to the severity grading, according the Brazilian official guidelines [7].

Intervention and control groups were hospitalized for 3 days and returned to the hospital 7 days after admission. A full clinical and laboratory examination were performed 24 hours, 48 hours, 72 hours and 7 days after admission.

Endpoints

The primary efficacy endpoint of this trial was the time free of secondary infection at snakebite site, defined as the presence of cellulitis and/or abscess [38,39], until 7 days after hospital admission. The onset of secondary infection until 48 hours at admission was considered a secondary outcome. Cellulitis was defined by the presence of local inflammation signs (erythema, edema, bruising and pain) with association to fever, leukocytosis, lymphangitis and/or lymphadenitis [46]. An abscess was characterized by individual injuries, floating, presenting purulent secretion or serous-purulent secretion [46,47]. Two independent observers evaluated all the patients and came to a final agreement. Patients clinically diagnosed with secondary infection were additionally evaluated by ultrasonography and a sample collection for microbiology was obtained only in cases evolving to abscess.

After secondary infection diagnosis, amoxicillin clavulanate was interrupted and patient was treated accordingly medical discretion.

Statistical analysis

Before statistical analysis, two independent typists entered information using Epi Info 3.5.1. A study researcher solved disagreements. The primary efficacy analysis was done on all randomized participants finishing the follow-up (per protocol population). The primary efficacy endpoint, secondary infection-free at 7 days, was analyzed using Kaplan-Meier estimates. A two-sided log-rank test was done over the period using a 5% significance level. Patients that were secondary infection-free at day 7 were censored at this point. The effect of drug use (amoxicillin clavulanate) was assessed as a single block. Relative risk, relative risk reduction, absolute risk reduction and number needed to treat were assessed for primary and secondary outcomes. For the secondary infection risk analysis, at 48 hours and 7 days of follow-up, explanatory variables were grouped in hierarchical blocks [48]. Proximal block was composed by laboratory findings at admission, intermediate block by clinical findings at admission and distal block by demographic and epidemiological variables. Univariate regression analysis was carried out for each block individually. Variables with a significance level of p<0.2 were included in the multivariate analysis by block. All variables with a significance level of p<0.05 in the multivariate analysis by block were thus included in the overall model (all blocks together). Crude odds ratios (OR), adjusted odds ratios (AOR) with their respective confidence intervals were calculated for each hierarchical level and for the overall model. Accuracy of the final model was evaluated by Hosmer-Lemeshow goodness-of-fit test. Where zeros caused problems with computation of the OR and 95% CI, 0.5 was added to all cells [49,50]. Mann-Whitney tests were carried out to assess differences between median of treated and untreated groups. Statistical analyses were performed using the STATA statistical package version 13 (Stata Corp. 2013).

Results

Patients’ characterization

A total of 345 patients were assessed for eligibility in the study period. From this total, 187 accomplished the inclusion criteria and were randomized, with 93 in the interventional group and 94 in the untreated control group. One patient of the control group was lost to follow up (Fig 1). Enzyme immunoassay confirmed Bothrops envenoming diagnosis in all included patients.

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Fig 1. Flow chart of inclusion.

Recruitment of the patients attended by snakebite at FMT-HVD, allocation and follow-up in the clinical trial.

https://doi.org/10.1371/journal.pntd.0005745.g001

Epidemiological characterization showed predominance of males (82.3%), mostly occurring in rural areas (87.1%). The most affected age group was the 21–30 years old (22.6%). The most affected anatomical site was the foot (66.1%). A total of 40.3% of cases were classified as work-related bites and 65.6% of the patients walked after the snakebite. Time elapsed from bite to medical assistance was higher than 3 hours in 42.4% of the cases. Use of topical medicines was informed in 34.4%, oral medicines in 28.5% and tourniquet in 24.7% of the cases (Table 1).

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Table 1. Demographic end epidemiological characteristics of the randomized patients according to the experimental group.

https://doi.org/10.1371/journal.pntd.0005745.t001

The most frequent manifestations observed at admission were severe pain (46.2%), mild edema (48.4%) and local bleeding (46.8%). The difference of temperature between the bite site and the contralateral site was predominantly <1°C (51.9%). The most frequent systemic manifestations were headache (26.9%), dizziness (14.5%), gingival bleeding (8.6%), nausea (8.1%) and vomiting (7.0%). Mean axillary temperature was 36.1 oC, mean heart rate was 79.3 bpm, mean respiratory rate was 20 bpm, mean systolic pressure (mmHg) was 129.8 mmHg and mean diastolic pressure (mmHg) was 82.6 mmHg. Snakebites were classified as moderate in 48.9% of the cases (Table 2). No compartmental syndrome, sepsis, gangrene, amputation or death was seen.

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Table 2. Clinical characterization of the randomized participants according to the experimental group.

https://doi.org/10.1371/journal.pntd.0005745.t002

Laboratorial characterization revealed mild leukocytosis, hypofibrinogenemia, increased creatine phosphokinase and creatine phosphokinase-MB activities, increased erythrocyte sedimentation rate and mildly increased lactate dehydrogenase activity. Clotting time presented incoagulable in 57.5% of patients. Prothrombin time presented incoagulable in 40.9% of patients. C-reactive protein was >6.5 mg/dL in 18.8% of the cases. Mean blood venom concentration was 50.9 ng/mL (Table 3). No statistical differences were observed between treated (median = 12) and untreated (median = 6.5) groups when CRP medians were compared (Z = -0.161, p-value = 0.872).

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Table 3. Laboratorial characterization of the randomized participants according to the experimental group.

https://doi.org/10.1371/journal.pntd.0005745.t003

Outcome rates and efficacy estimates

Of the 74 patients with secondary infection, cellulitis was diagnosed in 64 and abscess in 29. Secondary infection rates observed in the intervention and control groups are shown in Table 4. Survival analysis demonstrated that the time from patient admission to the onset of secondary infection was not different between amoxicillin clavulanate treated and control group (Log-rank = 2.23; p = 0.789) (Fig 2). Secondary infection incidence until 7 days after admission was 35.5% in the intervention group and 44.1% in the control group [RR = 0.80 (95%CI = 0.56 to 1.15; p = 0.235)]. Cellulitis rate was 30.1% in the intervention group and 38.7% in the control group [RR = 0.78 (95%CI = 0.52 to 1.16; p = 0.279)]. The abscess rate was 15.1% in the intervention group and 16.1% in the control group [RR = 0.93 (95%CI = 0.48 to 1.82; p = 0.999)].

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Fig 2. Time free of secondary infection at snakebite site, until 7 days after hospital admission, for both groups.

Survival analysis demonstrating that the time from patient admission to the onset of secondary infection was not different between amoxicillin clavulanate treated and control group (Log-rank = 2.23; p = 0.789).

https://doi.org/10.1371/journal.pntd.0005745.g002

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Table 4. Secondary infection rates of the patients followed up in the clinical trial, according to the experimental group.

https://doi.org/10.1371/journal.pntd.0005745.t004

Secondary infection incidence until 48 hours after admission was 22.6% in the intervention group and 36.6% in the control group [RR = 0.62 (95%CI = 0.38 to 0.98; p = 0.038)]. Actually, survival analysis has shown a later onset of secondary infection in the treated group (Fig 2). No late secondary infection (after 7 days of follow-up) was observed.

From the total of 74 patients presenting secondary infections, 88.2% were males and 88.2% occurred in the rural area. The age groups more affected by secondary infections after snakebite were 31–40 and 51–60 years old, with 20.6% each. Secondary infections were recorded mostly from bites in the foot (61.8%). Infections were secondary to work-related snakebites in 41.2%. Time to medical assistance was less than 3 hours after snakebite in 61.8% of the secondary infections cases. Secondary infections were mostly observed in moderate snakebites (58.8%). Use of local products was made in 35.5% of the secondary infections cases and of tourniquets in 26.5%.

Samples from secondary infection injuries were collected for culture from 11 patients, with 6 positive cases. Microorganisms isolated were Morganella morganii (five cases) and Staphylococcus aureus (one case).

Factors associated to secondary infections

Considering proximal variables, secondary infections incidence in 7 days of follow-up was significantly associated to fibrinogen >400 mg/dL [AOR = 3.39 (95%CI = 1.72 to 6.66; p<0.001)], alanine transaminase >44 IU/L [AOR = 2.21 (95%CI = 1.03 to 4.75; p = 0.006)] and C-reactive protein >6.5 mg/L [AOR = 3.90 (95%CI = 1.98 to 7.69; p = <0.001)]. Regarding intermediate variables, moderate [AOR = 11.75 (95%CI = 2.47 to 55.86; p = 0.002)] and severe pain [AOR = 16.97 (95%CI = 2.05 to 340.80; p = 0.001)], moderate [AOR = 3.46 (95%CI = 1.63 to 7.35; p = 0.001)] and severe edema [AOR = 3.78 (95%CI = 1.20 to 11.90; p = 0.023)] and moderate [AOR = 2.52 (95%CI = 1.32 to 4.82; p = 0.005)] and severe snakebites [AOR = 2.80 (95%CI = 0.90 to 8.77; p = 0.076)]. No distal variable was associated to secondary infections incidence (Table 5).

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Table 5. Factors associated to secondary infection until 7 days of the snakebite patients attended in the hospital in Manaus, 2014 to 2016.

https://doi.org/10.1371/journal.pntd.0005745.t005

In the final multivariate analysis model, secondary infections incidence in 7 days of follow-up remained significantly associated to fibrinogen >400 mg/dL [AOR = 4.78 (95%CI = 2.17 to10.55; p<0.001)], alanine transaminase >44 IU/L [AOR = 2.52 (95%CI = 1.06 to 5.98; p = 0.037)], C-reactive protein >6.5 mg/L [AOR = 2.98 (95%CI = 1.40 to 6.35; p = 0.005)], moderate pain [AOR = 24.30 (95%CI = 4.69 to 125.84; p<0.001)] and moderate snakebites [AOR = 2.43 (95%CI = 1.07 to 5.50; p = 0.034)] (Table 6).

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Table 6. Final model of factors associated to secondary infection until 7 days of the snakebite patients attended in the hospital in Manaus.

https://doi.org/10.1371/journal.pntd.0005745.t006

Secondary infections incidence in 48 hours of follow-up was significantly associated to C-reactive protein >6.5 mg/L [AOR = 4.28 (95%CI = 1.81 to 10.14; p = 0.001)], moderate [AOR = 5.87 (95%CI = 2.06 to 16.74; p = 0.001)] and severe pain [AOR = 17.89 (95%CI = 1.71 to 186.97; p = 0.016)]. Preemptive amoxicillin clavulanate was protective for secondary infections in 48 hours of follow-up [AOR = 0.42 (95%CI = 0.19 to 0.94; p = 0.034)] (S1 File).

Discussion

Preemptive amoxicillin clavulanate efficacy interpretation

Antimicrobial schemes for prevention or treatment of secondary infections from snakebites are not based on good evidences from randomized clinical trials [36,37]. Although the Infectious Diseases Society of America (IDSA) guidelines for diagnosis and management of skin and soft-tissue infections indicates amoxicillin clavulanate to reduce complications by prevention of secondary infection from animal bites [38,39], to the best of our knowledge this is the first trial assessing the efficacy of this regimen for snakebites. In this trial, although lower rates of secondary infection was observed in the intervention group such differences between amoxicillin clavulanate treated and control groups over a follow-up of 7 days did not achieve statistical significance. Consistently, analysis of the subgroups of patients who had abscesses or cellulitis presented also not significantly difference in terms of intervention efficacy. A previous study with oral chloramphenicol showed a poor efficacy in preventing secondary infection from Bothrops snakebites [41], although this drug was suggested as a good alternative for the treatment of local infections which may complicate bites by this snake genus [26,51,52]. Accordingly, intravenous chloramphenicol plus gentamicin showed no statistical difference between patients treated and untreated groups in Crotalus snakebites [42]. In a previous study amoxacillin was also used preemptively without any clinical benefit [53]. In general, these trials were not guided by the investigation of the bacterial agents responsible by the snakebite site infection in that area. Indeed, even for routine treatment purposes, secondary infection diagnosis is mostly based only from clinical features without microbiological confirmation followed of antimicrobial resistance profile.

Oral microbiota of snakes comprises a wide range aerobial and anaerobial microorganisms, including Enterobacteriaceae (namelly Morganella spp. and Escherichia coli), Streptococcus, Aeromonas spp., Staphylococcus aureus and Clostridium spp. [2022]. Few reports of microbiological confirmation of bacteria responsible for snakebites abscesses demonstrated a predominance of aerobics Enterobacteriaceae, mainly Morganella morganii [20,23,24]. The infection may not be necessarily associated to snake's mouth flora but also to local disorders induced by venom. The significant difference present at 48 hours but not at 7th day could be explained by the contamination between 3rd day and 7th day with origin other than oral microbiota of the snake, such as patient microbiota or iatrogeny. A limitation of this study was the absence of definitive identification of bacteria responsible by the infections for most of the participants. From six bacterial isolations, however, Morganella morganii was present in five snakebite site infections, however no antibiogram was routinely performed. Resistance to β-lactam antibiotics in Morganella species is very common and usually mediated by the presence of chromosomally encoded β-lactamases belonging to the AmpC β-lactamase family. These β-lactamases are typically inducible in the presence of β-lactam antibiotics [54]. As a result, agents such as ampicillin, amoxicillin, and first-generation and some second-generation cephalosporins may be ineffective [55]. This finding highlights the need of previous knowledge of the secondary infections epidemiology as a cornerstone in the preemptive antibiotics trials in snakebites.

Secondary infection incidence until 48 hours after admission had a relative risk reduction of 38.3% for the group using preemptive amoxicillin clavulanate compared to control, showing that antibiotic regimen delayed the onset of secondary infection among treated patients. As the preventive effect did not extend until the end of the follow-up, this delay may represent a severe risk for patients using ineffective preventive antibiotics regimens, because in the absence of signs of local complications patients are usually discharged after 48 hours of hospitalization, and may develop secondary infection without proper medical attention. In the difficulty for riverine and indigenous populations living in remote areas returning to health centers for treatment of this complication, severe clinical conditions such as functional loss, amputation, sepsis and even deaths are possible [28]. Although common in endemic areas, our results point that routine empirical prophylactic use of antibiotics aiming to prevent secondary infection lacks a clearly defined policy, leading to a costly and wasteful inappropriate antibiotic use, and even a risk for the patient [22,40]. Unfortunately due to the limited funding, a double-blinded study was not performed, a possible limitation of the study. Another limitation was the enrollment of patients with less than 24 hours after the bite, what may have selected those less prone to develop secondary infection.

Factors associated to secondary bacterial infection from Bothrops snakebites

In Bothrops snakebites, studies mostly describe factors associated to systemic complications, such as coagulopathy [11,20], acute renal failure [11,5659] and death [11,60,61], with extreme age groups and time to medical assistance associated with these poor outcomes [61]. There is scarce information in relation to local complications, especially necrosis [59,62] and amputation [63], associated to anatomical region bitten, systemic bleeding, renal failure, older age and use of tourniquet. Although secondary bacterial infections were observed in around 40% of the B. atrox snakebites in the Amazon [13], as confirmed in this work, no epidemiological or clinical predictive marker is known for this complication.

In this work, secondary infections incidence was significantly associated to higher levels fibrinogen, alanine transaminase and C-reactive protein, suggesting these laboratorial markers as auxiliary tools for the diagnosis of secondary infections allied to clinical signs of cellulitis and abscesses. Fibrinogen is an acute-phase protein and its serum concentration may be elevated in inflammatory and infectious conditions associated with vascular damage [64,65]. Even that B. atrox metalloproteinases cleave fibrinogen and induces a drop of fibrinogen levels with an increase in fibrin/fibrinogen degradation products (FDP) levels in vivo, with a foremost role in the pathogenesis of coagulopathy and intravascular hemolysis in the acute envenoming [66,67], the intense inflamatory reaction linked to secondary infection persisting after the antivenom therapy may trigger an acute phase reactant response in affected patients. High C-reactive protein paralleling with high fibrinogen in patients with soft-tissue secondary infections [39]. Our study suggests that the proinflammatory profile present in secondary infections from snakebites may be responsible for hepatocellular dysfunction and further elevated alanine transaminases, as previously reported in patients with sepsis and urinary tract infections [6872]. Cellulitis or abscesses occur mostly in the moderate or severe snakebite cases, as previously reported in southern Brazil [52].

Conclusions and perspectives

The Infectious Diseases Society of America (IDSA) guidelines for diagnosis and management of skin and soft-tissue infections indicate amoxicillin clavulanate to prevent secondary infections from animal bites [38,39]. However, in this study, patients did not benefit from preemptive amoxicillin clavulanate in preventing secondary infection from Bothrops snakebites. As a perspective, antimicrobial selection to be used in future clinical trials should be pursued diligently in comprehensive snakebites infection management. Secondary infections incidence was significantly associated to higher levels fibrinogen, alanine transaminase and C-reactive protein, suggesting these laboratorial markers as auxiliary tools for a more accurate diagnosis of secondary infections allied to clinical evaluation.

Acknowledgments

Initially, we thank all patients that participated of this study. We thank the participation of medical and nursing staff of the FMT-HVD hospital, mainly Antônio Magela and Silvio Fragoso. We also thank all health professionals of the Dermatology ward, medical residents and nursing staff. We had the important contribution of the FMT-HVD clinical laboratory, namely Geraldo Majela, Yonne Francis and Rossiclea Monte. We thank also Diego Britto, Sanmile Holanda, Fernanda Oliveira, Ramisés Santos, Josué Brutus, Ana Paula Damião and Elizandra Nascimento for their technical assistance.

References

  1. 1. White J (2000) Bites and stings from venomous animals: a global overview. Ther Drug Monit 22: 65–8. pmid:10688262
  2. 2. Chippaux JP (1998) Snake-bites: Appraisal of the global situation. Bull World Health Organ 76: 515–524. pmid:9868843
  3. 3. Gutiérrez JM, Theakston RDG, Warrell D (2006) Confronting the neglected problem of snake bite envenoming: the need for a global partnership. PLoS Med 3: e150. pmid:16729843
  4. 4. Kasturiratne A, Wickremasinghe AR, Silva N, Gunawardena NK, Pathmeswaran A, Premaratna R, et al. (2008) The Global Burden of Snakebite: A Literature Analysis and Modelling Based on Regional Estimates of Envenoming and Deaths. PLoS Med 5: 1591–1604. pmid:18986210
  5. 5. Warrell DA (2010). Snake bite. Lancet 375: 77–88. pmid:20109866
  6. 6. Brazilian Ministry of Health. Sistema de Informação de Agravos de Notificação—SINAN. In: Ministério da Saúde [Internet]. 2016 [cited 12 Nov 2016]. Available: http://portalsaude.saude.gov.br/images/pdf/2016/janeiro/20/1-Casos-Ofidismo-2000-2015.pdf
  7. 7. Brazilian Ministry of Health. Secretaria de Vigilância em Saúde. Guia de vigilância em saúde [Internet]. Ministério da Saúde, editor. Guia de vigilância em saúde. Brasília; 2014. Available: http://portalsaude.saude.gov.br
  8. 8. Moreno E, Queiroz-Andrade M, Lira-da-silva RM, Tavares-Neto J (2005) Clinical and epidemiological characteristics of snakebites in Rio Branco, Acre. Rev Soc Bras Med Trop 2005;38: 15–21. pmid:15717089
  9. 9. Saraiva MG, Oliveira DDS, Filho GMCF, Coutinho LASDA, Guerreiro JV (2012) Epidemiological profile of snake bites in the State of Paraiba, Brazil, 2005 to 2010. Epidemiol Serviços Saúde 21: 449–456.
  10. 10. Borges CC, Sadahiro M, Dos-Santos MC (1999) Epidemiological and clinical aspects of snake accidentes in the municipalities of the State of Amazonas, Brazil. Rev Soc Bras Med Trop 32: 637–646. Available: http://www.scielo.br/pdf/rsbmt/v32n6/0860.pdf pmid:10881100
  11. 11. Otero R, Gutiérrez J, Beatriz Mesa M, Duque E, Rodríguez O, Luis Arango J, et al. (2002) Complications of Bothrops, Porthidium, and Bothriechis snakebites in Colombia. A clinical and epidemiological study of 39 cases attended in a university hospital. Toxicon 40: 1107–1114. Available: http://www.ncbi.nlm.nih.gov/pubmed/12165312 pmid:12165312
  12. 12. Saboriâo P, Gonza M, Cambronero M (1998) Accidente Ofídico en Niños en Costa Rica: Epidemiología y Deteccíon de Factores de Riesgo en el Desarrollo de Absceso y Necrosis. Toxicon 1998;36: 359–366. pmid:9620583
  13. 13. Souza ARB (2002) Snakebite by Bothrops atrox (Lin. 1758) in the State of Amazonas—Brazil: Study of 212 cases with identified snake. Rev Patol Trop 31: 267–268. Available: http://www.revistas.ufg.br/index.php/iptsp/article/viewFile/14573/9140
  14. 14. Alkaabi JM, Al Neyadi M, Al Darei F, Al Mazrooei M, Al Yazedi J, Abdulle AM (2011) Terrestrial snakebites in the South East of the Arabian Peninsula: patient characteristics, clinical presentations, and management. PLoS One 6: e24637. pmid:21931788
  15. 15. Chen C-M, Wu K-G, Chen C-J, Wang C-M (2011) Bacterial infection in association with snakebite: a 10-year experience in a northern Taiwan medical center. J Microbiol Immunol Infect 44: 456–460. pmid:21700517
  16. 16. Ozay G, Bosnak M, Ece A, Davutoglu M, Dikici B, Gurkan F, et al. (2005) Clinical characteristics of children with snakebite poisoning and management of complications in the pediatric intensive care unit. Pediatr Int 2005;47: 669–675. pmid:16354222
  17. 17. David S, Matathia S, Christopher S (2012) Mortality predictors of snake bite envenomation in southern India: A ten-year retrospective audit of 533 patients. J Med Toxicol 8: 118–123. pmid:22234395
  18. 18. Pierini S V, Warrell DA, Paulo A, Theakston RD (1996) High incidence of bites and stings by snakes and other animals among rubber tappers and Amazonian Indians of the Juruá Valley, Acre State, Brazil. Toxicon 34: 225–236. pmid:8711756
  19. 19. Ribeiro LA, Jorge MT (1997) Acidente por serpentes do gênero Bothrops: série de 3.139 casos. Rev Soc Bras Med Trop 30: 475–480. pmid:9463193
  20. 20. Jorge MT, Mendonça JS, Ribeiro LA, Silva MLR, Kusano EJU, Cordeiro CLS (1990) Flora bacteriana da cavidade oral, presas de Bothrops jararaca: possível fonte de infecção no local da picada. Rev Inst Med Trop São Paulo 32: 6–10.
  21. 21. Goldstein EJC (1992) Bite wounds and infection. Clin Infect Dis 14: 633–640. Available: http://www.ncbi.nlm.nih.gov/pubmed/1562653 pmid:1562653
  22. 22. Garg A, Sujatha S, Garg J, Acharya NS, Parija SC (2009) Wound infections secondary to snakebite. J Infect Dev Ctries 3: 221–223. pmid:19759478
  23. 23. Brazilian MInistry of Health. Fundação Nacional de Saúde. Manual de Diagnóstico e Tratamento de Acidentes por Animais Peçonhentos. Brasília: Fundação Nacional de Saúde; 2001.
  24. 24. Jorge MT, Mendonça JS, Ribeiro LA, Cardoso JLC, Silva ML (1987) Bacilos Gram-negativos aeróbios em abscessos por acidente botrópico. Rev Soc Bras Med Trop 20: 55.
  25. 25. Bastos HM, Lopes LFL, Gattamorta MA, Matushima ER (2008) Prevalence of enterobacteria in Bothrops jararaca in São Paulo State: microbiological survey and antimicrobial resistance standards. Acta Sci Biol Sci 30: 321–326.
  26. 26. Jorge MT, Ribeiro LA, Da Silva MLR, Kusano EJU, Mendonça JS (1994) Microbiological studies of abscesses complicating Bothrops snakebite in humans: A prospective study. Toxicon 32: 743–748. pmid:7940580
  27. 27. Michael GC, Thacher TD, Shehu MIL (2011) The effect of pre-hospital care for venomous snake bite on outcome in Nigeria. Trans R Soc Trop Med Hyg 105: 95–101. pmid:21035155
  28. 28. Wen FH, Monteiro WM, Silva AMM, Tambourgi D V., Silva IM, Sampaio VS, et al. (2015) Snakebites and Scorpion Stings in the Brazilian Amazon: Identifying Research Priorities for a Largely Neglected Problem. PLoS Negl Trop Dis 9: e0003701. pmid:25996940
  29. 29. Calvete JJ, Sanz L, Pérez A, Borges A, Vargas AM, Lomonte B, et al. (2011) Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management. J Proteomics 74: 510–527. pmid:21278006
  30. 30. López-Lozano JL, Sousa MV, Ricart CAO, Chávez-Olortegui C, Sanchez EF, Muniz EG, et al. (2002) Ontogenetic variation of metalloproteinases and plasma coagulant activity in venoms of wild Bothrops atrox specimens from Amazonian rain forest. Toxicon 40: 997–1006. pmid:12076654
  31. 31. Assakura MT, Furtado MF, Mandelbaum FR (1992) Biochemical and biological differentiation of the venoms of the lancehead vipers (Bothrops atrox, Bothrops asper, Bothrops marajoensis and Bothrops moojeni). Comp Biochem Physiol Part B Comp Biochem 102: 727–732.
  32. 32. Moreira V, Dos-Santos MC, Nascimento NG, Silva HB, Fernandes CM, D’Império Lima MR, et al. (2012) Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators. Toxicon 60: 12–20. pmid:22465491
  33. 33. Otero R, Gutiérrez JM, Núñez V, Robles A, Estrada R, Segura E, et al. (1996) A randomized double-blind clinical trial of two antivenoms in patients bitten by Bothrops atrox in Colombia. Trans R Soc Trop Med Hyg 90: 696–700. pmid:9015522
  34. 34. Pardal PPO, Souza SM, Monteiro MRCC, Fan HW, Cardoso JLC, França FOS, et al. (2004) Clinical trial of two antivenoms for the treatment of Bothrops and Lachesis bites in the north eastern Amazon region of Brazil. Trans R Soc Trop Med Hyg 98: 28–42. Available: http://www.ncbi.nlm.nih.gov/pubmed/14702836 pmid:14702836
  35. 35. Oliveira SS, Sampaio VS, Sachett JAG, Alves EC, Silva VC, Lima JAA, et al. Snakebites in the Brazilian Amazon: Current Knowledge and Perspectives. In: Gopalakrishnakone P., Faiz S.M.A., Gnanathasan Christeine Ariaranee, Habib Abdulrazaq Garba, Fernando Ravindra, Yang Chen-Chang, Vogel Carl-Wilhelm, Denise V. Tambourgi SAS, editor. Clinical Toxinology. 1st ed. Holanda: Springer Netherlands; 2016. 22 p.
  36. 36. Albuquerque PLMM Jacinto CN, Silva Junior GB Lima JB, Veras MSB Daher EF (2013) Acute kidney injury caused by Crotalus and Bothrops snake venom: a review of epidemiology, clinical manifestations and treatment. Rev Inst Med Trop Sao Paulo 55: 295–301. pmid:24037282
  37. 37. Cheng AC, Currie BJ (2004) Venomous Snakebites Worldwide with a Focus on the Australia-Pacific Region: Current Management and Controversies. J Intensive Care Med 19: 259–269. pmid:15358944
  38. 38. Stevens DL, Bisno AL, Chambers HF, Dellinger EP, Goldstein EJC, Gorbach SL, et al. (2014) Practice guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the infectious diseases society of America. Clin Infect Dis 59: 1–43.
  39. 39. Stevens DL, Bisno AL, Chambers HF, Everett ED, Dellinger P, Goldstein EJC, et al. (2005) Practice Guidelines for the Diagnosis and Management of Skin and Soft-Tissue Infections. Clin Infect Dis 41: 1373–1406. Available: http://cid.oxfordjournals.org/ pmid:16231249
  40. 40. Tagwireyi DD, Ball DE, Nhachi CFB (2001) Routine prophylactic antibiotic use in the management of snakebite. BMC Clin Pharmacol 1: 1–7.
  41. 41. Jorge MT, Malaque C, Ribeiro LA, Fan HW, Cardoso JLC, Nishioka SA, et al. (2004) Failure of chloramphenicol prophylaxis to reduce the frequency of abscess formation as a complication of envenoming by Bothrops snakes in Brazil: a double-blind randomized controlled trial. Trans R Soc Trop Med Hyg 98: 529–534. pmid:15251401
  42. 42. Kerrigan K, Mertz B, Nelson S, Dye J (1997) Antibiotic prophylaxis for pit viper envenomation: prospective, controlled trial. World J Surg 21: 369–373. Available: papers2://publication/uuid/2CEFE28B-F057-4003-94EB-4C292221FF19 pmid:9143566
  43. 43. Brakenbury PH, Muwanga C (1989) A comparative double blind study of amoxycillin/clavulanate vs placebo in the prevention of infection after animal bites. Arch Emerg Med 6: 251–256. pmid:2692580
  44. 44. Huskisson EC (1982) Measurement of pain. J Rheumatol 9: 768–769. pmid:6184474
  45. 45. Colombini M. Reatividade antigênica cruzada entre os venenos de Bothrops atrox e Lachesis muta muta e desenvolvimento de um teste imunoenzimático diferencial para acidentes causados por essas serpentes. Universidade de São Paulo. 2003.
  46. 46. Bisno AL, Stevens DL (1996) Streptococcal Infections of Skin and Soft Tissues. N Engl J Med 1996;334: 240–245. pmid:8532002
  47. 47. Andrade JG, Pinto RNL, Andrade ALSS, Martelli CMT, Zicker F (1989) Estudo bacteriológico de abscessos causados por picada de serpentes do gênero Bothrops. Rev Inst Med Trop São Paulo 31: 363–367.
  48. 48. Victora CG, Huttly SR, Fuchs SC, Olinto MT (1997) The role of conceptual frameworks in epidemiological analysis: A Hierarchical Approach. Int J Epidemiol 26: 224–227. pmid:9126524
  49. 49. Pagano M, Gauvreau K. Principles of Biostatistics. 2nd ed. Brooks/Cole, editor. Belmont: Brooks/Cole; 2000.
  50. 50. Deeks J, Higgings J. Statistical algorithms in Review Manager 5 [Internet]. 2010. Available: http://ims.cochrane.org/revman/documentation/Statistical-methods-in-RevMan-5.pdf
  51. 51. Jorge MT, Nishioka A, Oliveira RB, Ribeiro LA, Silveira PVP (1998) Aeromonas hydrophila soft-tissue infection as a complication of snake bite: report of three cases. Ann Trop Med Parasitol 92: 213–217. pmid:9625918
  52. 52. Bucaretchi F, Herrera SRF, Hyslop S, Bacarat ECE, Vieira RJ (2001) Snakebites by Bothrops spp in children in Campinas, São Paulo, Brazil. Rev Inst Med Trop São Paulo 43: 329–333. pmid:11781603
  53. 53. Boels D, Hamel JF, Deguigne MB, Harry P (2012) European viper envenomings: Assessment of ViperfavTM and other symptomatic treatments. Clin Toxicol 50: 189–196. pmid:22372786
  54. 54. Bush K, Jacoby GA, Medeiros AA (1995) A Functional Classification Scheme for β-Lactamases and Its Correlation with Molecular Structure. Antimicrob Agents Chemother 39: 1211–1233. pmid:7574506
  55. 55. Biedenbach DJ, Jones RN, Erwin ME (1993) Interpretive Accuracy of the Disk Diffusion Method for Testing Newer Orally Administered Cephalosporins against Morganella morganii. J Clin Microbiol 31: 2828–2830. pmid:8253998
  56. 56. Albuquerque PLMM Silva GB, Jacinto CN Lima JB, Lima CB Amaral YS, et al. (2014) Acute kidney injury after snakebite accident treated in a Brazilian tertiary care centre. Nephrology 19: 764–70. pmid:25123203
  57. 57. Amaral CFS, Rezende NA de, Silva OA da, Ribeiro MMF, Magalhães RA, Reis RJ dos, et al. (1986) Insuficiência renal aguda secundária a acidentes ofídicos botrópico e crotálico. Análise de 63 casos. Rev Inst Med Trop Sao Paulo 28: 220–227. pmid:3563305
  58. 58. Pinho FMO, Yu L, Burdmann EA (2008) Snakebite-Induced Acute Kidney Injury in Latin America. Semin Nephrol 28: 354–362. pmid:18620958
  59. 59. Ribeiro LA, Gadia R, Jorge MT (2008) Comparison between the epidemiology of accidents and the clinical features of envenoming by snakes of the genus Bot. Rev Soc Bras Med Trop 41: 46–49. pmid:18368270
  60. 60. Ribeiro LA, Albuquerque MJ, Pires de Campos VAF, Katz G, Takaoka NY, Lebrão ML, et al. (1998) Óbitos por serpentes peçonhentas no Estado de São Paulo: avaliação de 43 casos, 1988/93. Rev Assoc Med Bras 44: 312–318.
  61. 61. Feitosa EL, Sampaio VS, Salinas JL, Queiroz AM, Silva IM, Gomes AA, et al. (2015) Older Age and Time to Medical Assistance Are Associated with Severity and Mortality of Snakebites in the Brazilian Amazon: A Case-Control Study. PLoS One 10: e0132237. pmid:26168155
  62. 62. Ribeiro LA, Jorge MT, Lebrão ML (2001) Prognostic factors for local necrosis in Bothrops jararaca (Brazilian pit viper) bites. Trans R Soc Trop Med Hyg 95: 630–634. pmid:11816436
  63. 63. Jorge MT, Ribeiro LA, O’Connell JL (1999) Prognostic factors for amputation in the case of envenoming by snakes of the Bothrops genus (Viperidae). Ann Trop Med Parasitol 93: 401–408. pmid:10656041
  64. 64. Adams R, Passino M, Sachs B, Nuriel T, Akassoglou K (2004) Fibrin mechanisms and functions in nervous system pathology. Mol Interv 4: 163–176. pmid:15210870
  65. 65. Davalos D, Akassoglou K (2012) Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol 34: 43–62. pmid:22037947
  66. 66. Jacob-Ferreira AL, Menaldo DL, Sartim MA, Riul TB, Dias-Baruffi M, Sampaio S V (2017) Antithrombotic activity of Batroxase, a metalloprotease from Bothrops atrox venom, in a model of venous thrombosis. Int J Biol Macromol 95: 263–267. pmid:27876598
  67. 67. Jacob-Ferreira AL, Menaldo DL, Bernardes CP, Sartim MA, Angelis CD, Tanus-Santos JE, et al. (2016) Evaluation of the in vivo thrombolytic activity of a metalloprotease from Bothrops atrox venom using a model of venous thrombosis. Toxicon 109: 18–25. pmid:26556655
  68. 68. Szabo G, Romics L, Frendl G (2002). Liver in sepsis and systemic inflammatory response syndrome. Clin Liver Dis 6: 1045–1066. pmid:12516206
  69. 69. Koo DJ, Chaudry IH, Wang P (1999) Kupffer cells are responsible for producing inflammatory cytokines and hepatocellular dysfunction during early sepsis. J Surg Res 83: 151–157. pmid:10329110
  70. 70. Dhainaut JF, Marin N, Mignon A, Vinsonneau C (2001) Hepatic response to sepsis: interaction between coagulation and inflammatory processes. Crit Care Med 29: S42–47. pmid:11445733
  71. 71. Campos J, Alende R, Gonzalez-Quintela A (2009) Abnormalities in aminotransferase levels during acute pyelonephritis. Eur J Intern Med 20: e53–e56. pmid:19393479
  72. 72. Park JY, Ko KO, Lim JW, Cheon EJ, Yoon JM (2013) Increase in Aminotransferase Levels during Urinary Tract Infections in Children. Pediatr Gastroenterol Hepatol Nutr 16: 89–94. pmid:24010112