NEW DIAGNOSTIC TESTS FOR SEPSIS

UDAWATTAGE GEEWAN KAMAL

GROUP 21

• SIRS (Systemic inflammatory response syndrome): The clinical syndrome that results from a deregulated inflammatory response or to a noninfectious insult.

Temp > 38 or < 36HR > 90RR > 20 or PaCO2 < 32WBC > 12 or < 4 or Bands > 10%

• Sepsis: SIRS that is secondary to infection that has been diagnosed clinically. Positive cultures add to the validity but are not required for the diagnosis.

• Severe Sepsis: Sepsis plus at least one sign of hypoperfusion or organ dysfunction that is new, and not explained by other known etiology of organ dysfunction.

• Septic Shock: Severe sepsis associated with refractory hypotension (BP<90/60) despite adequate fluid resuscitation and/or a serum lactate level >=4.0 mmol/L.

• Rapid Response Team: A team of qualified clinicians that respond to a perceived change in, or deterioration of, the condition of patients by bringing critical care expertise to the bedside.

Sepsis Pathogenesis

Unbalanced Immune Reaction

Tissue Factor

Procoagulant State

MicrovascularThrombosis

Mediators of Inflammation

ROS

Vasodilation CapillaryLeak

Newest diagnostic criteria according to cdc 2012General variables• Fever (> 38.3°C)• Hypothermia (core temperature < 36°C)• Heart rate > 90/min–1 or more than two sd above the normal value for age• Tachypnea• Altered mental status• Significant edema or positive fluid balance (> 20 mL/kg over 24 hr)• Hyperglycemia (plasma glucose > 140 mg/dL or 7.7 mmol/L) in the absence of

diabetes

Inflammatory variables• Leukocytosis (WBC count > 12,000 µL–1)• Leukopenia (WBC count < 4000 µL–1)• Normal WBC count with greater than 10% immature forms• Plasma C-reactive protein more than two sd above the normal value• Plasma procalcitonin more than two sd above the normal value

Hemodynamic variables• Arterial hypotension (SBP < 90 mm Hg, MAP < 70 mm Hg, or an SBP decrease >

40 mm Hg in adults or less than two sd• below normal for age)

Organ dysfunction variables• Arterial hypoxemia (Pao2/Fio2 < 300)• Acute oliguria (urine output < 0.5 mL/kg/hr for at least 2 hrs despite adequate fluid

resuscitation)• Creatinine increase > 0.5 mg/dL or 44.2 µmol/L• Coagulation abnormalities (INR > 1.5 or aPTT > 60 s)• Ileus (absent bowel sounds)• Thrombocytopenia (platelet count < 100,000 µL–1)• Hyperbilirubinemia (plasma total bilirubin > 4 mg/dL or 70 µmol/L)

Tissue perfusion variables• Hyperlactatemia (> 1 mmol/L)• Decreased capillary refill or mottling• WBC = white blood cell; SBP = systolic blood pressure; MAP = mean arterial pressure;

INR = international normalized ratio; aPTT = activated partial thromboplastin• time.• Diagnostic criteria for sepsis in the pediatric population are signs and symptoms of

inflammation plus infection with hyper- or hypothermia (rectal temperature • > 38.5° or < 35°C), tachycardia (may be absent in hypothermic patients), and at least

one of the following indications of altered organ function: altered mental • status, hypoxemia, increased serum lactate level, or bounding pulses

• By shortening the time to pathogen identification and allowing for detection of organisms missed by blood culture, new molecular methods may provide clinical benefits for the management of patients with sepsis.multiplex PCR, mass spectrometry and array techniques

• Inappropriate antimicrobial treatment is a major concern and is associated with increased mortality . Reasons for inappropriate treatment include the lack of coverage of the underlying pathogen and antimicrobial resistance of the causative pathogen in nosocomial infections and in infections by emerging multiresistant Gram-negative bacteria. Increased mortality may also be found following inappropriate fungal therapy since empiric antimycotic coverage is only recommended in high-risk patients (e.g., neutropenia, intra-abdominal infections).

• These findings have led to a growing interest in the development of diagnostic tests for the rapid diagnosis of pathogens causing bloodstream infections to allow early administration of adequate targeted antimicrobial therapy in critically ill patients.

• Blood culture (BC) is still considered the gold standard for diagnosis and identification of bloodstream pathogens by many . However, this conventional laboratory method lacks sensitivity, has a low pre-test probability in certain clinical settings, and is impaired by the delay in the time to result.

• In order to increase the speed of diagnosis, to improve sensitivity and the clinical benefit of detection of pathogens in the blood, new methods have been developed.

• Molecular detection techniques for bacterial and fungal DNA have been implemented but are not in widespread clinical use.

• the use of molecular methods to identify pathogens following blood culture can be faster than the standard techniques involving phenotypic identification and antimicrobial susceptibility testing requiring up to 72 h after the blood culture became positive.

Hybridization

• Fluorescent in situ hybridization (FISH) is among the most studied commercial techniques suitable for the detection of pathogens in positive blood cultures.

• In only 2.5–3 h, FISH may identify more than 95% of bacteria and yeasts commonly found in blood.

• Slides of positive blood cultures are prepared, hybridized with fluorochrome-labeled oligonucleotide probes targeted to rRNA, and visualized microscopically

Broad-range amplification• Broad-range assays use primers that recognize

conserved sequences of bacterial/fungal chromosomal genes encoding ribosomal DNA. The clinical usefulness of these methods is, however, limited because after the PCR amplification of a target sequence, further identification procedures are necessary

• Hyplex Blood Screen (BAG, Germany) is a multiplex PCR assay with subsequent identification of bacterial species (methicillin-sensitive S. aureus, methicillin-resistant S. aureus, S. epidermidis, Streptococcus pyogenes,S. pneumoniae, E. faecalis, and Enterococcus faecium, E. coli, Enterobacter aerogenes, P. aeruginosa, andKlebsiella spp.) from positive blood cultures using hybridization in an ELISA-like format.

Multiplex amplification

• The StaphPlex system (Qiagen, USA) detects S. aureus using a unique target-enriched multiplex PCR method.

• This assay is designed for simultaneous detection and species-level identification of Panton–Valentine leukocidin (PVL) and several antimicrobial resistance determinants of staphylococci directly from blood culture in which Gram-positive cocci in clusters have been detected by Gram staining.

• The system amplifies and detects 18Staphylococcus-specific genes simultaneously in one reaction

• However, the use of multiplex PCR or broad-range amplification followed by sequence analysis of microorganisms after growth in conventional blood culture does provide the necessary clinical benefit within a few hours; the costs compared to conventional identification techniques are high.

• Studies using direct detection of pathogen DNA from blood have been confounded by the presence of pathogen DNA contamination introduced at the time of specimen collection and/or preparation

Diagnosis of sepsis independent of blood culture

• Molecular techniques applied directly on whole blood samples are the best choice for rapid identification of a microorganism in the blood.

• The main advantages of PCR detection directly from the blood are the increased sensitivity and the avoidance of time-consuming culture, resulting in substantial reduction in turnaround time even compared with PCR identification from positive blood cultures.

Broad-range nucleic acid amplification

• Broad-range PCR assays have been implemented for the detection of bacteria or fungi in blood based on the 16S or 23S rRNA gene of bacteria and the 18S rRNA gene of fungi.

• After amplification, the amplicons can be identified by different methods such as capillary sequencing analysis, pyrosequencing, or hybridization with specific probes .

Multiplex nucleic acid amplification• The multiplex real-time PCR assays allow the rapid identification of pathogens

directly from blood. Multiplex PCR involves amplifying multiple targets of DNA in the same sample at the same time using a mix of primers.

SepsiTest• SepsiTest (Molzym, Germany) is a PCR-based detection and sequence identification

system for organisms causing sepsis. Using 1 mL of blood, the presence of bacteremia or fungemia can be detected within 4 h via broad-range PCR for 16S and 18S rRNA genes. In positive cases, sequence analysis of the amplicon is performed for identification of more than 300 bacteria and fungi in 8–12 h. The diagnostic sensitivity and specificity of this test were 87.0% and 85.8% when compared to blood culture . Grif and collaborators have recently confirmed the diagnostic sensitivity and specificity (88.5% and 83.5%).

SeptiFast• The LightCycler® SeptiFast test (Roche Molecular Systems, Germany) is a multiplex

PCR assay designed to detect 25 microorganisms that cause approximately 90% of all bloodstream infections directly from blood. SeptiFast uses real-time PCR in a non-quantitative mode to identify ten bacteria at the species level, several more at the genus level, as well as five Candida spp. and Aspergillus fumigatus. These organisms are thought to be responsible for more than 90% of all the cases of bloodstream infection .

THANK YOU!!!

• cdc

• http://www.sccm.org/Documents/SSC-Guidelines.pdf