Toxic shock syndrome is a rare complication of Staphylococcus aureus infection associated with small burn wounds of under 5 % total body surface area; it is predominantly observed in young children. Environmental factors that occur within a burn wound have been suggested to increase the risk of TSS developing, and wound dressings have been implicated to contribute to this risk. This study examined the effects of 11 wound dressings on the production of TSST-1 by two strains of S. aureus (strains T1 and T4). Initially, the effects of the wound dressings on growth and exotoxin production were assessed using a liquid culture medium, as this was used in other studies. The results indicated that growth was not markedly affected using this system, however there were a number of problems associated with the evaluation. TSST-l production was altered (increased or decreased) depending upon the dressing type, the gaseous environment or the strain of S. aureus used. Other exotoxins did not appear to be greatly affected by any of the dressings. When a semi-solid system was developed to minimise disintegration of the dressings and simulate a more appropriate wound model in terms of support and environment, similar results were observed as to those found in a liquid culture system. A l-layered semi-solid agarose system incubated in 6 % (v/v) carbon dioxide supported optimum TSST-1 production by both test strains in the presence and absence of most wound dressings. Actisorb Plus™ and crepe increased TSST-l production. Levels of TSST-l increased over time and Actisorb Plus™ continued to stimulate increased toxin production. Gamgee, and Biobrane ™previously implicated. in TSS, increased TSST-1 production t.. . I between 48-72 hours. Serine, thiol and metalloproteases were produced by both strains of S. aureus and proportions of each were altered by the presence of dressings. This study showed that some wound dressings may potentially increase the risk of a patient developing TSS, but further studies need to be done in vivo.

Summary Puerperal sepsis is a rare but serious and potentially lethal syndrome. It is imperative that severe postpartum malaise is taken seriously; early initiation of antibiotic therapy before sepsis becomes manifest can save lives.

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Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1985. / Bibliography: leaves 119-128. / Photocopy. / Microfilm. / ix, 128 leaves, bound ill. 29 cm

Toxic shock syndrome toxin-1 (TSST-1), an exoprotein produced by some strains of Staphylococcus aureus, is implicated in the pathogenesis of menstrual TSS. However, its role in nonmenstrual TSS is less certain. In order to study the pathogenetic role of TSST-1 in TSS, three approaches were taken: a) to develop an ELISA for detection of TSST-1 in biologic fluids in order to verify TSST-1 production in vivo in TSS patients, b) to quantitate TSST-1 specific antibodies in the serum of TSS patients and controls to determine whether such antibodies are protective, and c) to attempt to identify other staphylococcal products which may be implicated in some forms of TSS. A sensitive and specific noncompetitive enzyme-linked immunosorbent assay (ELISA) capable of detecting TSST-1 at concentrations from 0.5 to 16 ng/ml was developed. This assay did not detect other staphylococcal enterotoxins including A, B, C₁, C₂, C₃, D and E. Possible interference by protein A was readily eliminated by pretreatment of test samples with 10% nonimmune rabbit serum. The assay was adapted for rapid screening of TSST-1 production by S. aureus isolates in culture supernatants in vitro, and for the detection of TSST-1 in vaginal washings and urine of TSS patients and healthy controls in vivo. All 35 S. aureus isolates confirmed to be TSST-1 positive by Ouchterlony immunodiffusion, and 59 of 60 isolates confirmed to be TSST-1 negative, gave concordant results by ELISA. Interestingly, toxigenic S. aureus strains isolated from TSS patients quantitatively produced significantly more toxin in vitro compared to toxigenic control strains (p<0.05, Mann-Whitney rank sum test). TSST-1 could be detected by ELISA in 3 of 4 vaginal washings collected within 3 days of hospitalization from 3 women with acute menstrual TSS, compared to 0 of 17 washings from 9 TSS women collected greater than 3 days after hospitalization (p=0.003, Fisher's exact test) and 1 of 15 washings from 14 healthy control women (p=0.016). TSST-1 was not detected in the urine of 4 acute TSS patients, 2 convalescent TSS patients or in 3 control urine tested. A sensitive and reproducible ELISA was also developed for the quantitation of TSST-1 specific IgG in serum. Anti-TSST-1 was assessed in acute and convalescent sera from 16 nonmenstrual (9 female, 7 male) and 14 menstrual TSS patients, and from 87 healthy women and 66 healthy men as controls. Quantitative levels of anti-TSST-1 in the study groups were calculated as the percent of standard activity (POSA) relative to a medium titre reference serum standard. ELISA titers in acute sera from menstrual TSS (26.2 ± 5.2, mean POSA ± S.E.M.), but not nonmenstrual TSS women (71.8 ± 18.6), were significantly lower than in healthy controls (78.9 ± 7.3) (p<0.01, Mam-Whitney test). Titers from menstrual TSS patients remained low (25.2 ± 10.7) even during late convalescence (mean duration 20 months after illness onset), compared to healthy female controls (p<0.05). Acute titers in males with TSS (37.0 ± 15.6) were also significantly lower than those in control men (114.6 + 11.0) (p<0.05). An inverse relationship of recovery of toxigenic S. aureus and anti-TSST-1 titers in acute sera of TSS patients was observed. Interestingly, antibody titers in control men were significantly higher than in control women (p<0.001). No age-dependent effects or interactive effects of age and sex on ELISA titers were observed. To enable immunoblot analyses, TSST-1 was produced and partially purified using column chromatography techniques. Percent recovery of TSST-1 from culture supernatant through to the final procedure was approximately 15.5%. The relative purity of TSST-1 (TSST-l/total protein, w/w) was increased from 0.21% in culture supernatants to 94.4% in the final product. Ouchterlony immunoprecipitation against reference rabbit antitoxin demonstrated identity with reference TSST-1 as well as with TSST-1 prepared in other laboratories. Physical characterization demonstrated a molecular weight of 24 kd and a pi of 7.0. Using pooled normal human serum as a first antibody probe, several bands in addition to the 24 kd TSST-1 band were visualized by immunoblot against our partially purified toxin as well as similar preparations obtained from other investigators. To determine whether any of the additional bands might be implicated in TSS, acute and convalescent sera from TSS patients were used to probe for immunoreactive bands in our partially purified TSST-1 as well as a commercially obtained preparation. Seroconversion was demonstrated to the 24 kd TSST-1 protein in 7 of 10 TSS patients from whom toxigenic S. aureus was isolated. In addition, seroconversion was noted to a 49 kd band in 4 patients, to a 21 kd band in 3 patients, to a 28 kd band in 1 patient and to a 32 kd band in 2 patients. In conclusion: 1) the ability to measure TSST-1 in biologic fluids lends stronger support for the role of TSST-1 in menstrual TSS patients; 2) the serologic data support the etiologic role of TSST-1 in menstrual TSS and in nonmenstrual TSS patients from whom toxigenic S. aureus could be cultured, but not for nonmenstrual TSS women from whom toxigenic S. aureus was not isolated; 3) immunoblotting results with acute and convalescent sera from TSS and control patients, not only add further support to the role of TSST-1 in patients from whom toxigenic S. aureus could be isolated, but also indicate that there may be several other staphylococcal products implicated in TSS, particularly in whom antibody to TSST-1 pre-existed in acute sera. The nonresponsiveness or lack of seroconversion to TSST-1 in some patients could suggest either: a) TSST-1 was not the etiologic agent for such patients; b) TSST-1 was the etiologic agent, but the exposure was sufficient for an immune response (similar to tetanus), or; c) some immunologic defect may be present. Future studies are required to clarify these possibilities. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Compartment syndrome is a rare but a well-documented complication in patients with trauma-induced group A streptococcus infection. Here, we present a case of a male who developed compartment syndrome on the left lower extremity after an injury inoculated by group A streptococcus on the right lower extremity. The patient was resuscitated with antibiotics, urgent fasciotomy, and immunoglobulin. The patient was eventually transferred to a burn center for further care.

Staphylococcus aureus is a gram positive bacterium asymptomatically colonizing 30-40% of the human population. S. aureus causes a variety of infections including superficial skin lesions, toxic shock syndrome, and infective endocarditis (IE). There are 100,000 cases of IE each year in the United States. IE is a life threatening infection of native/prosthetic valves and the lining of the heart. It is characterized by the formation of vegetations, “cauliflower-like” structures composed of bacteria and host factors. S. aureus is the most commonly identified pathogen (up to 40%) in patients with IE. USA200 (Clonal Complex 30) strains of S. aureus are significantly associated with IE, all of which produce toxic shock syndrome toxin-1 (TSST-1) and β-toxin. TSST-1 characterizes the staphylococcal Group I superantigens (SAgs). The major mechanism of activity of TSST-1 and other SAgs is the ability to activate T-cells and APCs by non-specifically cross-bridging Vβ-chains of T-cell receptors (TCRs) with α and/or β-chains of major histocompatibility complex II (MHCII) molecules on antigen presenting cells (APCs). In a rabbit model of IE and sepsis, TSST-1 is critical for the development of vegetations and the associated colony forming units (CFUs). β-toxin has a molecular mass of 35 kDa, a basic pI (>10.0), and is a member of the DNase I superfamily. This cytotoxin has two distinct mechanisms of action: sphingomyelinase (SMase) activity and DNA biofilm ligase activity. β-toxin is critical for causing IE in a rabbit model that strongly resembles human disease. This toxin association had been observed, but studies have not been completed to determine what role TSST-1 and β-toxin play independently and in cooperation with one another, and more specifically which mechanism each uses, during IE infections. While TSST-1 and β-toxin are both important for IE, they are very different toxins. My studies determined that the presence of TSST-1 and β-toxin in combination results in the highest levels of lethality in a rabbit model of IE. A strain expressing TSST-1 lacking superantigenic activity has decreased lethality compared to the same strain expressing wild type TSST-1. My study is the first to begin characterization of the DNA biofilm ligase active site by identifying important residues via a DNA binding and biofilm formation assays. Furthermore, my research shows that a β-toxin mutant lacking SMase activity is decreased in lethality and vegetation formation compared to wild type. β-toxin mutants disrupted in biofilm ligase activity do not decrease lethality but are deficient in vegetation formation compared to wild type. Utilizing in vitro assays to assess cellular events during IE, I established that β-toxin causes changes to morphology and is cytotoxic to human aortic endothelial cells (HAECs), inhibits production of IL-8, and modulates the expression levels of cluster of differentiation 40 (CD40) and vascular cell adhesion molecule 1 (VCAM-1). My work shows these two virulence factors (TSST-1 and β-toxin) produced by USA200 strains and other clonal groups play important roles in causing IE.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective was to compare the hemodynamic and metabolic effects of treatment with hypertonic saline and colloid (expanders) associated with different vasoconstrictors in dogs subjected to experimental hemorrhagic shock. Twenty-four healthy adult mongrel dogs were included in the study, with mean body weight of 10.84±3.3kg, males and females. Following anesthetic induction by isoflurane inhalation, the animals were intubated and connected to a partial rebreathing system, and subjected to general inhalational anesthesia with isoflurane using a calibrated vaporizer, which were then maintained at 1MAC (minimum alveolar concentration). Hypovolemia was induced by withdrawal of 5mL kg-1 min-1 of blood from the femoral artery, until the mean arterial pressure reached values between 45 and 50mmHg. After 60 minutes, basal parameters were measured and the treatments were initiated. At this moment, the treatment with colloid and hypertonic solution (4mL kg-1) was carried out. After 10 minutes, the animals were randomly allocated into four groups, according to the continuous infusion to be administered. In GD (dopamine group, n=06), a continuous infusion of dopamine (10μg kg-1 min-1) was administered. The animals in GDB (dobutamine group, n=06) received a continuous infusion of dobutamine (5μg kg-1 min-1) and GV (vasopressin group, n=06) vasopressin at the dose of 0.02IU-1 min-1 by continuous infusion, both diluted in 0.9% NaCl. The GC (n=06), control group, was only treated with the association of hypertonic saline and colloid . The animals were monitored with regards to heart rate, respiratory rate, rectal temperature, systolic, diastolic and mean arterial pressure, central venous pressure, concentration of expired isoflurane, concentration of expired carbon dioxide, fraction of inspired oxygen and arterial blood gas analysis to obtain values of PO2, PCO2, bicarbonate, pH, Na+, K+ and base deficit, hemoglobin and hematocrit. In addition, blood samples were collected to evaluate serum lactate, thromboplastin time, prothrombin time, platelets and complete blood count. The collected data were submitted to variance analysis, where the mean values between groups were analyzed using t-test, and between times within the same group using Tukey test, where the differences were considered statistically significant when p≤0.05. The differences between times within each group were not observed only in the variables of HR in GV and GC, PaO2, K+, MCV, MCHC, total leukocytes (except in GC), segmented neutrophils, monocytes, PT and APTT. The differences between groups were essentially regarding HR (with less proportion of alteration in GV and GC), MAP (GV with higher pressure), PaO2 (lower in GV), K+ (only one time higher in GV) and platelets, in which GC showed the lowest mean value. It can be concluded that the evaluated experimental model is efficient for hemorrhagic shock induction in dogs and requires a blood withdrawal of 42.75±9.2% of the circulating blood volume; the volemia expansion with hypertonic saline and colloid (4mL kg-1) associated or not with dopamine (10μg kg-1 min-1), dobutamine (5μg kg-1 min-1) or vasopressin (0.02IU-1 min-1) is efficient for hemodynamic recovery and metabolic stabilization; and the vasopressin group (GV), though not statistically significant, shows a more favorable clinical tendency in the recovery of hemodynamic and metabolic status in dogs submitted to experimental hemorrhagic shock. / Objetivou-se comparar os efeitos hemodinâmicos e metabólicos do tratamento com solução hipertônica e colóide (expansores) associada a diferentes vasopressores em cães submetidos a choque hemorrágico experimental. Foram utilizados 24 cães adultos, SRD, peso médio de 10,84+3,3kg, de ambos os sexos, hígidos. Os animais foram induzidos a anestesia geral por meio da vaporização de isofluorano, intubados e conectados a um sistema com reinalação parcial de gases, e anestesia geral inalatória com isofluorano em vaporizador calibrado, sendo após, mantidos em 1CAM (concentração alveolar mínima). Induziu-se a hipovolemia, por meio da retirada de 5mL kg-1 min-1 de sangue da artéria femoral, até que a pressão arterial média atingisse valores entre 45 e 50mmHg. Após 60 minutos mensurou-se os parâmetros basais e deu-se início aos tratamentos. Neste momento realizava-se o tratamento com os expansores (4ml kg-1). Após 10 minutos os animais foram alocados aleatoriamente em quatro grupos, conforme a infusão contínua que receberiam. No GD (grupo dopamina, n=06), a infusão contínua de dopamina (10μg kg-1 min-1). Os animais do GDB (grupo dobutamina, n=06), infusão contínua de dobutamina (5μg kg-1 min-1) e o GV (grupo vasopressina, n=6), vasopressina (0,02UI-1min-1) em infusão continua ambos em diluição com NaCl 0,9%. O GC (n=6), grupo controle, contou apenas com o tratamento dos expansores. Os animais foram monitorados quanto a freqüência cardíaca, freqüência respiratória, temperatura retal, pressão arterial sistólica, pressão arterial diastólica, pressão arterial média, pressão venosa central, concentração expirada de isofluorano, concentração expirada de dióxido de carbono, fração inspirada de oxigênio e, hemogasometria do sangue arterial, obtendo-se valores de PO2, PCO2, Bicarbonato, pH, Na+, K+, déficit de base, hemoglobina e hematócrito. Foram coletadas ainda amostras de sangue para avaliação de lactato sérico, tempo de tromboplastina, tempo de protrombina, plaquetas e hemograma completo. Os dados coletados foram submetidos à análise de variância, sendo que as médias entre grupos foram analisadas pelo Teste t e entre os tempos dentro do mesmo grupo pelo Teste de Tukey, sendo as diferenças consideradas estatisticamente significativas quando P0,05. Não ocorreram diferenças entre os tempos dentro de cada grupo quanto as variáveis de FC para GV e GC, PaO2, K+, VCM, CHCM, leucócitos totais (exceção de GC), bastonetes segmentados, monócitos, TP e TTPA. Ente grupos, as diferenças fixaram-se basicamente em FC (sendo que o GV e o GC, alterou com menor proporção), PAM (GV com pressão mais alta), PaO2 (GV menor), K+ (apenas um tempo com GV maior) e Plaquetas, tendo o GC o menor valor médio desta. Conclui-se que o modelo experimental, avaliado é eficiente para indução de choque hemorrágico em cães e requer uma expoliação de sangue de 42,75+9,2% do volume sanguíneo circulante; a expansão da volemia com a associação de hipertônica e colóide (4ml kg-1) associada ou não à dopamina (10μg kg-1 min-1), dobutamina (5μg kg-1 min-1) ou vasopressina (0,02UI-1min-1) é eficaz na restauração hemodinâmica e estabilização metabólica; e o grupo vasopressina (GV), embora não estatisticamente significativo, demonstra clinicamente ser mais eficaz na restauração dos padrões hemodinâmicos e metabólicos de cães induzidos a choque hemorrágico experimental.

Le syndrome de choc de dengue (DSS) est une complication potentiellement mortelle dela dengue, première arbovirose humaine et problème majeur de santé publiquemondial. Il survient chez une fraction des patients, et résulte d’une fuite plasmatiquemassive, non prédictible, dont la physiopathologie est mal connue. Le décryptage de laréponse de l’hôte est donc essentiel pour améliorer le pronostic et le traitement despatients. Ce travail de thèse a abordé les mécanismes de la fuite plasmatique du DSS dedeux façons : versant immunitaire et versant endothélial. D’une part, nous avons comparéen ex vivo les profils transcriptionnels sanguins de patients présentant différentes formescliniques de dengue, afin d’identifier des mécanismes contribuant à la survenue du DSS.Cette étude a révélé l’activation chez les patients en DSS, de signatures proinflammatoiresà l’interface entre immunité innée et métabolisme lipidique, représentantde nouveaux bio-marqueurs potentiels du DSS. D’autre part, les études in vitro desinteractions entre un virus de dengue et deux lignées de cellules endothélialesmicrovasculaires humaines (CEM), a révélé des différences d’intensité de réponseantivirale, ainsi que des différences dans l’expression de protéines impliquées dans laperméabilité, selon l’origine des territoires endothéliaux. Ces résultats suggèrent que levirus contribue directement au dysfonctionnement endothélial, au côté de mécanismesindirects médiés par des facteurs de l’hôte. Les deux types d’approches mises en oeuvreont ainsi établi de nouvelles données sur la physiopathologie du DSS, qui pourraient àterme trouver des applications dans la prise en charge des malades. / Dengue Shock Syndrome (DSS) is a life-threatening form of dengue infection, which is thefirst arboviral disease worldwide and a major public health problem. This severecomplication happens in a fraction of patients, and is the consequence of anunpredictable massive plasma leakage. The pathophysiology underlying DSS is stillunknown. Deciphering the host response to dengue infection is essential to improve boththe prognosis and the therapeutic management of dengue patients. This thesis workintended to study the mechanisms involved in DSS’ plasma leakage at both immunity (exvivo study) and endothelium (in vitro study) levels. First, in a ex vivo study, we comparedwhole blood cells’ transcriptional profiles of patients suffering from different clinicalpresentations of dengue disease, in order to identify mechanisms contributing to DSSoutcome. This study revealed the activation of pro-inflammatory signatures at theinterface of innate immunity and lipid metabolism, in DSS patients. Those signatures maybe new bio-markers of DSS. Second, in vitro studies of the consequences of a directinteraction between a dengue virus and human microvascular endothelial cells (MEC),revealed differences in antiviral response intensities and in the expression of proteinsinvolved in the endothelial permeability, depending on the endothelial origin of theMEC. Those results suggest that the virus directly contributes to the endotheliumdysfunction, together with indirect mechanisms triggered by soluble and cellular factors.Our investigations have produced new data on the pathophysiology of DSS that couldhave applications to the monitoring and treatment of the patients.