Presymptomatic diagnosis of postoperative infection and sepsis using gene expression signatures.

PURPOSE: Early accurate diagnosis of infection ± organ dysfunction (sepsis) remains a major challenge in clinical practice. Utilizing effective biomarkers to identify infection and impending organ dysfunction before the onset of clinical signs and symptoms would enable earlier investigation and intervention. To our knowledge, no prior study has specifically examined the possibility of pre-symptomatic detection of sepsis. METHODS: Blood samples and clinical/laboratory data were collected daily from 4385 patients undergoing elective surgery. An adjudication panel identified 154 patients with definite postoperative infection, of whom 98 developed sepsis. Transcriptomic profiling and subsequent RT-qPCR were undertaken on sequential blood samples taken postoperatively from these patients in the three days prior to the onset of symptoms. Comparison was made against postoperative day-, age-, sex- and procedure- matched patients who had an uncomplicated recovery (n =151) or postoperative inflammation without infection (n =148). RESULTS: Specific gene signatures optimized to predict infection or sepsis in the three days prior to clinical presentation were identified in initial discovery cohorts. Subsequent classification using machine learning with cross-validation with separate patient cohorts and their matched controls gave high Area Under the Receiver Operator Curve (AUC) values. These allowed discrimination of infection from uncomplicated recovery (AUC 0.871), infectious from non-infectious systemic inflammation (0.897), sepsis from other postoperative presentations (0.843), and sepsis from uncomplicated infection (0.703). CONCLUSION: Host biomarker signatures may be able to identify postoperative infection or sepsis up to three days in advance of clinical recognition. If validated in future studies, these signatures offer potential diagnostic utility for postoperative management of deteriorating or high-risk surgical patients and, potentially, other patient populations.

Developing and understanding biofluid vibrational spectroscopy: a critical review.

Vibrational spectroscopy can provide rapid, label-free, and objective analysis for the clinical domain. Spectroscopic analysis of biofluids such as blood components (e.g. serum and plasma) and others in the proximity of the diseased tissue or cell (e.g. bile, urine, and sputum) offers non-invasive diagnostic/monitoring possibilities for future healthcare that are capable of rapid diagnosis of diseases via specific spectral markers or signatures. Biofluids offer an ideal diagnostic medium due to their ease and low cost of collection and daily use in clinical biology. Due to the low risk and invasiveness of their collection they are widely welcomed by patients as a diagnostic medium. This review underscores recent research within the field of biofluid spectroscopy and its use in myriad pathologies such as cancer and infectious diseases. It highlights current progresses, advents, and pitfalls within the field and discusses future spectroscopic clinical potentials for diagnostics. The requirements and issues surrounding clinical translation are also considered.

The ability of CpG oligonucleotides to protect mice against Francisella tularensis live vaccine strain but not fully virulent F. tularensis subspecies holarctica is reflected in cell-based assays.

CpG DNA is a potent activator of the innate immune system. Here the protective effects of CpG DNA are assessed against the facultative intracellular pathogen Francisella tularensis. Dosing of mice with CpG DNA provided protection against disease caused by F. tularensis subsp. holarctica live vaccine strain (LVS) but did not protect against the fully virulent F. tularensis subsp holarctica strain HN63. Similarly, in vitro studies in J774A murine macrophage-like cells demonstrated that stimulation with CpG DNA enables control of intracellular replication of LVS but not HN63. These data confirm findings that CpG DNA may have limited efficacy in providing protection against fully virulent strains of F. tularensis and also suggest that in vitro assays may be useful for the evaluation of novel treatments for virulent F. tularensis.

Protective cellular responses to Burkholderia mallei infection.

Burkholderia mallei is a Gram-negative bacillus causing the disease glanders in humans. During intraperitoneal infection, BALB/c mice develop a chronic disease characterised by abscess formation where mice normally die up to 70 days post-infection. Although cytokine responses have been investigated, cellular immune responses to B. mallei infection have not previously been characterised. Therefore, the influx and activation status of splenic neutrophils, macrophages and T cells was examined during infection. Gr-1+ neutrophils and F4/80+ macrophages infiltrated the spleen 5 h post-infection and an increase in activated macrophages, neutrophils and T cells occurred by 24 h post-infection. Mice depleted of Gr-1+ cells were acutely susceptible to B. mallei infection, succumbing to the infection 5 days post-infection. Mice depleted of both CD4 and CD8 T cells did not succumb to the infection until 14 days post-infection. Infected µMT (B cell) and CD28 knockout mice did not differ from wildtype mice whereas iNOS-2 knockout mice began to succumb to the infection 30 days post-infection. The data presented suggests that Gr-1+ cells, activated early in B. mallei infection, are essential for controlling the early, innate response to B. mallei infection and T cells or nitric oxide are important during the later stages of infection.

Host immunity in the protective response to vaccination with heat-killed Burkholderia mallei.

BACKGROUND: We performed initial cell, cytokine and complement depletion studies to investigate the possible role of these effectors in response to vaccination with heat-killed Burkholderia mallei in a susceptible BALB/c mouse model of infection. RESULTS: While protection with heat-killed bacilli did not result in sterilizing immunity, limited protection was afforded against an otherwise lethal infection and provided insight into potential host protective mechanisms. Our results demonstrated that mice depleted of either B cells, TNF-alpha or IFN-gamma exhibited decreased survival rates, indicating a role for these effectors in obtaining partial protection from a lethal challenge by the intraperitoneal route. Additionally, complement depletion had no effect on immunoglobulin production when compared to non-complement depleted controls infected intranasally. CONCLUSION: The data provide a basis for future studies of protection via vaccination using either subunit or whole-organism vaccine preparations from lethal infection in the experimental BALB/c mouse model. The results of this study demonstrate participation of B220+ cells and pro-inflammatory cytokines IFN-gamma and TNF-alpha in protection following HK vaccination.

Pathogenesis of Yersinia pestis infection in BALB/c mice: effects on host macrophages and neutrophils.

The pathogenesis of infection with Yersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+ macrophages and Ly-6G+ neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 10(3) to 10(4) CFU per spleen. Flow cytometric analysis revealed that there was even distribution of Y. pestis within both CD11b+ macrophage and Ly-6G+ neutrophil populations on day 2 postinfection. However, from day 3 postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+ macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages of Y. pestis infection, splenic neutrophils are responsible for limiting the growth of Y. pestis and that splenic macrophages provide safe intracellular shelters within which Y. pestis is able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma interferon and tumor necrosis factor alpha.

VP22 enhances antibody responses from DNA vaccines but not by intercellular spread.

In some species DNA vaccines elicit potent humoral and cellular immune responses. However, their performance in humans and non-human primates is less impressive. There are suggestions in the literature that an increase in the intercellular distribution of protein expressed from a DNA vaccine may enhance immunogenicity. We incorporated the Herpes Simplex Virus type 1 (HSV) VP22 gene, which encodes a protein that has been described as promoting intercellular spread, into a DNA vector in which it was fused to enhanced green fluorescent protein (EGFP). Following transfection of the plasmid DNA into mammalian cells, distribution of the fusion protein VP22-EGFP was not increased compared to EGFP alone. Furthermore, we found no evidence to suggest that VP22 was capable of mediating intercellular spread. However, when these constructs were used as DNA vaccines to immunise mice, antibody levels specific to EGFP were significantly enhanced when EGFP was fused to VP22. These data suggest that amplification of the immune response may occur via mechanisms other than VP22-mediated intercellular spread of antigen.