Adjunctive Rifampicin Increases Antibiotic Efficacy in Group A Streptococcal Tissue Infection Models.

Biofilm has recently been highlighted as a complicating feature of necrotizing soft tissue infections (NSTI) caused by Streptococcus pyogenes (i.e., group A Streptococcus [GAS]) contributing to a persistence of bacteria in tissue despite prolonged antibiotic therapy. Here, we assessed the standard treatment of benzylpenicillin and clindamycin with or without rifampin in a tissue-like setting. Antibiotic efficacy was evaluated by CFU determination in a human organotypic skin model infected for 24 or 48 h with GAS strains isolated from NSTI patients. Antibiotic effect was also evaluated by microcalorimetric metabolic assessment in in vitro infections of cellular monolayers providing continuous measurements over time. Adjunctive rifampin resulted in enhanced antibiotic efficacy of bacterial clearance in an organotypic skin tissue model, 97.5% versus 93.9% (P = 0.006). Through microcalorimetric measurements, adjunctive rifampin resulted in decreased metabolic activity and extended lag phase for all clinical GAS strains tested (P < 0.05). In addition, a case report is presented of adjunctive rifampin treatment in an NSTI case with persistent GAS tissue infection. The findings of this study demonstrate that adjunctive rifampin enhances clearance of GAS biofilm in an in vitro tissue infection model.

Diabetes and necrotizing soft tissue infections-A prospective observational cohort study: Statistical analysis plan.

BACKGROUND: Necrotizing soft tissue infections (NSTIs) are rare but carry a high morbidity and mortality. The multicenter INFECT project aims to improve the understanding of the pathogenesis, clinical characteristics, diagnosis, and prognosis of NSTIs. This article describes the study outline and statistical analyses that will be used. METHODS: Within the framework of INFECT project, patients with NSTI at 5 Scandinavian hospitals are enrolled in a prospective observational cohort study. The goal is to evaluate outcome and characteristics for patients with NSTI and diabetes compared to patients with NSTI without diabetes. The primary outcome is mortality at 90 days after inclusion. Secondary outcomes include days alive and out of ICU and hospital, SAPS II, SOFA score, infectious etiology, amputation, affected body area, and renal replacement therapy. Comparison in mortality between patients with diabetes type 1 and 2 as well as between insulin-treated and non-insulin-treated diabetes patients will be made. Clinical data for diabetic patients with NSTI will be reported. CONCLUSION: The study will provide important data on patients with NSTI and diabetes.

Necrotizing soft tissue infections - a multicentre, prospective observational study (INFECT): protocol and statistical analysis plan.

BACKGROUND: The INFECT project aims to advance our understanding of the pathophysiological mechanisms in necrotizing soft tissue infections (NSTIs). The INFECT observational study is part of the INFECT project with the aim of studying the clinical profile of patients with NSTIs and correlating these to patient-important outcomes. With this protocol and statistical analysis plan we describe the methods used to obtain data and the details of the planned analyses. METHODS: The INFECT study is a multicentre, prospective observational cohort study. Patients with NSTIs are enrolled in five Scandinavian hospitals, which are all referral centres for NSTIs. The primary outcomes are the descriptive variables of the patients. Secondary outcomes include identification of factors associated with 90-day mortality and amputation; associations between affected body part, maximum skin defect and Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) score and 90-day mortality; 90-day mortality in patients with and without acute kidney injury (AKI) and LRINEC score of six and above or below six; and association between affected body part at arrival and microbiological findings. Exploratory outcomes include univariate analyses of baseline characteristics associations with 90-day mortality. The statistical analyses will be conducted in accordance with the predefined statistical analysis plan. CONCLUSION: Necrotizing soft tissue infections result in severe morbidity and mortality. The INFECT study will be the largest prospective study in patients with NSTIs to date and will provide important data for clinicians, researchers and policy makers on the characteristics and outcomes of these patients.

Necrotizing skin and soft-tissue infections in the intensive care unit.

BACKGROUND: Necrotizing skin and soft-tissue infections (NSTI) are rare but potentially life-threatening and disabling infections that often require intensive care unit admission. OBJECTIVES: To review all aspects of care for a critically ill individual with NSTI. SOURCES: Literature search using Medline and Cochrane library, multidisciplinary panel of experts. CONTENT: The initial presentation of a patient with NSTI can be misleading, as features of severe systemic toxicity can obscure sometimes less impressive skin findings. The infection can spread rapidly, and delayed surgery worsens prognosis, hence there is a limited role for additional imaging in the critically ill patient. Also, the utility of clinical scores is contested. Prompt surgery with aggressive debridement of necrotic tissue is required for source control and allows for microbiological sampling. Also, prompt administration of broad-spectrum antimicrobial therapy is warranted, with the addition of clindamycin for its effect on toxin production, both in empirical therapy, and in targeted therapy for monomicrobial group A streptococcal and clostridial NSTI. The role of immunoglobulins and hyperbaric oxygen therapy remains controversial. IMPLICATIONS: Close collaboration between intensive care, surgery, microbiology and infectious diseases, and centralization of care is fundamental in the approach to the severely ill patient with NSTI. As many aspects of management of these rare infections are supported by low-quality data only, multicentre trials are urgently needed.

Human polyspecific immunoglobulin attenuates group A streptococcal virulence factor activity and reduces disease severity in a murine necrotizing fasciitis model.

OBJECTIVES: Streptococcus pyogenes causes life-threatening invasive infections including necrotizing fasciitis (NF). Current treatment guidelines recommend the use of a cell-wall-active antibiotic combined with a protein synthesis inhibitor and surgical debridement in NF patients. Adjunctive therapy with intravenous immunoglobulin (IVIG) has been proposed for superantigen-associated streptococcal toxic shock syndrome. So far, benefits of IVIG treatment remain unclear and prospective clinical studies are scarce. Thus, we aimed to assess the effects of IVIG on virulence factor activity in vitro, ex vivo in patients and in vivo in a NF mouse model. METHODS: We investigated the effect of IVIG on the activity of the virulence factors streptolysin O (SLO), streptodornase 1 (Sda1), S. pyogenes cell envelope protease and streptococcal pyrogenic exotoxin B in vitro and ex vivo in patient sera. Additionally, we assessed the influence of IVIG on the clinical outcome in a murine NF model. RESULTS: In vitro, IVIG inhibited various streptococcal virulence factors. Further, IVIG treatment of group A Streptococcus-infected mice led to a reduced skin lesion size (median (interquartile range) day 3 intraperitoneal administration: 12 mm2 (9-14.5) vs. 4 mm2 (0.8-10.5), subcutaneous: 10.3 mm2 (6.9-18.6) vs. 0.5 mm2 (0.1-6.8)) and lower SLO activity. After treatment with IVIG, patient sera showed an elevated titre of specific SLO (7/9) and Sda1 (5/9) antibodies, reducing SLO and Sda1 activity. CONCLUSIONS: The clear reduction in disease severity in IVIG-treated mice and inhibition of virulence factor activity in mouse and human sera suggest that IVIG may be beneficial in invasive group A Streptococcus infections such as NF in addition to streptococcal toxic shock syndrome.

Viridans streptococcal septicaemia in neutropenic patients: role of proinflammatory cytokines.

The immunostimulatory activity of viridans streptococcal strains isolated from neutropenic patients with severe sepsis (n=9) or uncomplicated bacteraemia (n=10) was compared. Peripheral blood mononuclear cells from healthy individuals were stimulated with heat-killed bacteria or culture supernatants, and cytokine production assessed. All strains were potent inducers of IL1beta, IL8, and TNFalpha production. Heat-killed bacteria induced consistently higher IL1beta and TNFalpha production than did the cell-free bacterial supernatants (P<0.01). The strains did not induce any proliferative response, nor any significant TNFbeta or IFNgamma production. No difference in cytokine-inducing capacity could be detected between the cohorts of severe and nonsevere isolates. Comparison of strains causing severe and nonsevere episodes in the same patient (n=2) revealed a significantly higher induction of IL1beta by the severe episodes associated isolates as compared to the nonsevere (P<0.04). The study underscores the importance of the host-pathogen interplay in determining the level of inflammation, and hence the severity of disease.

Infections due to group A streptococcus: new concepts and potential treatment strategies.

Important new information has been gained on the pathogenesis and treatment of life-threatening invasive infections caused by group A streptococci (GAS), i.e. the streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF). Both STSS and NF lead to superantigen reactions with activation of up to 10% of the CD4+ lymphocytes and release of large amounts of cytokines; mainly tumour necrosing factor beta, interferon gamma, interleukin 1 and interleukin 6. Streptococcal products known to trigger the superantigen reactions are the pyrogenic exotoxins, spe A, spe B and spe C and the M-proteins. Therapeutically clindamycin has been shown to reduce mortality in animal experiments in comparison to penicillin treatment. A possible mechanism is the effect of clindamycin on protein synthesis which might decrease the production of superantigens. In man, the use of intravenous immunoglobulin has been shown to significantly reduce mortality in STSS and NF. The most probable mechanism is neutralisation of superantigens by antibodies in the immunoglobulin preparations used.

Extracellular adherence protein (Eap) from Staphylococcus aureus does not function as a superantigen.

Extracellular adherence protein (Eap) from Staphylococcus aureus has been reported to have strong anti-inflammatory properties, which make Eap a potential anti-inflammatory agent. However, Eap has also been demonstrated to trigger T-cell activation and to share structural homology with superantigens. In this study, we focused on whether Eap fulfilled the definition criteria for a superantigen. We demonstrate that T-cell activation by Eap is dependent on both major histocompatibility complex class II and intercellular adhesion molecule type 1, that cellular processing is required for Eap to elicit T-cell proliferation, and that the kinetics of proliferation resemble the profile of a conventional antigen and not that of a superantigen.

Invasive group B Streptococcal disease in non-pregnant adults : a review with emphasis on skin and soft-tissue infections.

Streptococcus agalactiae, commonly referred as group B Streptococcus (GBS), is a major cause of neonatal sepsis and infections in pregnant women. However, the number of invasive infections in non-pregnant adults is growing. Elderly patients and those with chronic underlying conditions, such as diabetes mellitus or compromised immune defence, are at increased risk of invasion. The spectrum of clinical manifestations is broad and includes necrotizing fasciitis and toxic shock syndrome. Although, primary bacteremia and skin and soft-tissue infections are the most frequently reported diagnosis. This article reviews the epidemiology, pathogenesis and treatment of invasive GBS disease in non-pregnant adults, with an emphasis on skin and soft-tissue infections.

Intravenous polyclonal IgM-enriched immunoglobulin therapy in sepsis: a review of clinical efficacy in relation to microbiological aetiology and severity of sepsis.

The efficacy of intravenous polyclonal immunoglobulin (IVIG) as adjunct therapy in sepsis has long been debated. Clinical trials have yielded contradicting results, in part due to the varying study design and varying microbiological aetiologies. In most trials, the study drug has been IVIG containing polyclonal IgG. However, in recent reports, the efficacy of IgM-enriched IVIG as adjunct therapy in sepsis has been highlighted. Here we review studies on IgM-enriched IVIG therapy in sepsis and we discuss the clinical efficacy in relation to microbiological aetiology and severity of sepsis. The results suggest that patients most likely to benefit from IgM-enriched IVIG therapy are those with Gram-negative septic shock.

Novel therapies in streptococcal toxic shock syndrome: attenuation of virulence factor expression and modulation of the host response.

The systemic manifestations of severe invasive group A streptococcal infections, such as streptococcal toxic shock syndrome, are mediated by an overwhelming inflammatory response induced by streptococcal superantigens and other virulence factors. The high mortality rates associated with streptococcal toxic shock syndrome demonstrate a need for better therapy in these diseases. Novel strategies to attenuate or prevent streptococcal toxic shock syndrome at different stages of illness have been proposed. The most promising therapies include agents that by various mechanisms attenuate the inflammatory response or the action of streptococcal toxins/superantigens, or both.

Culture-negative severe septic shock: indications for streptococcal aetiology based on plasma antibodies and superantigenic activity.

We present a severe septic shock syndrome patient with negative blood cultures. Acute and convalescent plasma samples from the patient were analysed for anti-streptolysin O titres, superantigen-neutralizing activity and presence of superantigenic activity. The plasma analyses implicated superantigen-producing Streptococcus pyogenes as the causative agent.

Differential induction of Th1 versus Th2 cytokines by group A streptococcal toxic shock syndrome isolates.

The majority of group A streptococcal (GAS) isolates from patients with streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) express numerous virulence factors, including several superantigens (SAgs). Purified SAgs are potent inducers of inflammatory (Th1) cytokines that contribute to the pathogenesis of severe infections. However, GAS-infected individuals are likely to be exposed to a mixture of GAS SAgs as well as other virulence factors produced by the bacteria, and therefore, our goal was to characterize the mitogenic and cytokine induction profiles of this mixture. All GAS isolates tested had brisk mitogenic activity and induced potent cytokine responses, with higher frequencies of Th1 than Th2 cytokine-producing cells. The mitogenic activity produced in culture supernatants of three selected clinical GAS isolates was significantly different, but no marked difference was found in their overall cytokine induction profiles. However, significant differences (P < 0.0062) were noted in the induction of Th2 cytokines between GAS supernatants and recombinant streptococcal pyrogenic exotoxin A (rSpeA), suggesting that the presence of other SAgs and/or the production of additional virulence factors may alter the overall cytokine induction profile of SAgs. A significant individual variation in the level of proliferative and cytokine responses to the same GAS culture supernatants or to rSpeA was noted. Individuals with higher frequencies of cells producing Th2 cytokines mounted lower levels of Th1 cytokine responses, and vice versa. Furthermore, quantification of the intensity and cell area of interleukin-1beta (IL-1beta)-producing cells by image analysis revealed that individuals with higher Th2 responses had significantly lower IL-1beta production (P < 0.0001) than the individual with a strong Th1 response. Differences in the ability to induce Th1 versus Th2 cytokines, as well as the individual variations in cytokine responses to streptococcal SAgs, may play a central role in determining the severity of invasive GAS infections.

Detection and nucleotide sequence analysis of the speC gene in Swedish clinical group A streptococcal isolates.

The production of pyrogenic exotoxins SpeA, SpeB, and SpeC by group A streptococci has been associated with streptococcal toxic shock syndrome. Several epidemiological studies using DNA hybridization and PCR analysis have been performed in attempts to correlate one or several of the toxins with streptococcal toxic shock syndrome. The results reveal great variation in the occurrence of the speA and speC genes among clinical isolates. In this study, we show that the speC gene could be detected by nested PCR in five Swedish T1M1 strains isolated from patients infected with group A streptococci as well as in three Norwegian T1M1 isolates, previously reported to lack speC as determined by dot blot hybridization. To verify the identities of the amplified products, the nucleotide sequences of the PCR fragments from one Swedish T1M1 strain and from the toxin reference strain NY5 were determined. The nucleotide sequences showed that the amplified products were speC and of allele type C2, on the basis of the nucleotides in positions 438 and 456. However, one additional base pair substitution was found in NY5 at position 147 and in the Swedish isolate at position 157, which resulted in nonsynonymous amino acid changes. Thus, these speC genes represent two new allelic variants.

Host variation in cytokine responses to superantigens determine the severity of invasive group A streptococcal infection.

Cytokines elicited by superantigens have been suggested to play a central role in severe systemic clinical manifestations of gram-positive sepsis. Here we provide evidence for a potent inflammatory cytokine response in acute invasive group A streptococcal infections, and show a direct correlation between the magnitude of this response and the severity of systemic manifestations of the disease. Severe invasive cases suffering from toxic shock and/or necrotizing fasciitis had significantly higher frequencies of IL-2-, IL-6-, and TNF-alpha-producing cells in their circulation as compared to non-severe invasive cases (p=0.05-0.01). This difference was even more accentuated when severe and non-severe cases infected with a clonal M1T1 strain were compared (p=0.03-0. 004). To determine whether host factors were responsible for this difference in magnitude of cytokine responses, paired age- and gender-matched severe and non-severe M1T1 cases (n=8) were tested in vitro during their convalescent phase for immune response to superantigens produced by their infecting isolate. The results showed persistent and inherent differences in the magnitude of proliferative and cytokine responses of severe and non-severe patients to the streptococcal superantigens to which they had been exposed during infection. Thus, the study provides evidence that patients with a propensity to produce higher levels of inflammatory cytokines in response to streptococcal superantigens develop significantly more severe systemic manifestations than patients who have a propensity to produce lower levels of inflammatory cytokines to the same superantigens. We therefore conclude that host factors influence the magnitude of cytokine responses to superantigens and consequently the clinical outcome of the infection.

Relation between low capacity of human sera to inhibit streptococcal mitogens and serious manifestation of disease.

The proliferative response of lymphocytes induced by a new streptococcal mitogenic factor (MF) and the streptococcal pyrogenic exotoxins (Spe) A and B was determined in sera from 6 healthy persons. Responses were compared to those obtained from reference serum that lacked mitogen-specific ELISA antibodies. The sera showed individual variations in the levels of MF- and Spe-specific antibodies, as determined by ELISA. The experiments showed that most human sera, which contained mitogen-specific antibodies, could neutralize the mitogenicity of the proteins. However, there were sera that contained mitogen-specific antibodies but that did not inhibit the toxin-induced proliferation. Thus, the ELISA antibody titer did not always equal the neutralizing capacity. Sera from 27 patients with group A streptococcal bacteremia had significantly lower neutralizing ability against MF and SpeB than did sera from 25 uncomplicated tonsillitis cases.

Similar cytokine induction profiles of a novel streptococcal exotoxin, MF, and pyrogenic exotoxins A and B.

The cytokine production induced by a newly discovered streptococcal exotoxin, MF, and the pyrogenic exotoxins SpeA and SpeB was determined by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) obtained from healthy blood donors. The induction and kinetics of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-1 receptor antagonist, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, gamma interferon, tumor necrosis factor alpha (TNF-alpha), TNF-beta, and granulocyte-macrophage colony-stimulating factor were studied at the single-cell level by use of cytokine-specific monoclonal antibodies and intracellular immunofluorescent juxtanuclear staining. The cytokine-producing cells, with the exception of IL-1-expressing cells, had a characteristic morphology generated by the accumulation of cytokines in the Golgi organelle. MF, SpeA, and SpeB induced a massive gamma interferon and TNF-beta response in 10 to 16% of the PBMCs after 48 to 96 h of cell stimulation. In contrast, IL-2 and TNF-alpha production was detected in only 1 to 3% of the PBMCs. The induction of a lymphocyte TH2 phenotype response, including production of IL-3, IL-4, IL-5, and IL-10, was weak. However, the monokines, IL-1 alpha, IL-1 beta, IL-1 receptor antagonist, and IL-8, were consistently found and gradually produced, peaking at 24 h in approximately 5 to 8% of the PBMCs. MF showed extensive cytokine- and proliferation-inducing capacities equal to those of SpeA and SpeB, which suggests that MF is also a superantigen. A marked interindividual variation could be noted both in the proliferative response and in the cytokine induction of lymphocytes isolated from different individuals, which may be one explanation for the varying clinical severity noticed during group A streptococcal infections.

Association of human leukocyte antigen with outcomes of infectious diseases: the streptococcal experience.

The role of host genetic factors in determining susceptibility to infections has become more evident. Certain individuals appear to be predisposed to certain infections, whereas others are protected. By studying the immune response and the genetic makeup of susceptible and resistant individuals a better understanding of the disease process can be achieved. Infections caused by group A streptococci offer an excellent model to study host-pathogen interactions and how the host genetic variation can influence the infection outcome. These studies showed that the same clone of these bacteria can cause severe or non-severe invasive disease. This difference was largely related to the human leukocyte antigen class 11 type of the patient. Certain class II haplotypes present the streptococcal superantigens in a way that results in responses, whereas others present the same superantigens in a way that elicits very potent inflammatory responses that can lead to organ failure and shock. These findings underscore the role of host genetic factors in determining the outcome of serious infections and warrants further investigations into how the same or different genetic factors affect susceptibility to other emerging and re-emerging pathogens.