PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (1335332)

Clipboard (0)
None

Related Articles

1.  Multiplex Identification of Gram-Positive Bacteria and Resistance Determinants Directly from Positive Blood Culture Broths: Evaluation of an Automated Microarray-Based Nucleic Acid Test 
PLoS Medicine  2013;10(7):e1001478.
Nathan Ledeboer and colleagues assess the performance of a diagnostic platform for detecting Gram-positive bacteria in blood cultures, an important step in the diagnosis and treatment of patients with sepsis.
Please see later in the article for the Editors' Summary
Background
A multicenter study was conducted to evaluate the diagnostic accuracy (sensitivity and specificity) of the Verigene Gram-Positive Blood Culture Test (BC-GP) test to identify 12 Gram-positive bacterial gene targets and three genetic resistance determinants directly from positive blood culture broths containing Gram-positive bacteria.
Methods and Findings
1,252 blood cultures containing Gram-positive bacteria were prospectively collected and tested at five clinical centers between April, 2011 and January, 2012. An additional 387 contrived blood cultures containing uncommon targets (e.g., Listeria spp., S. lugdunensis, vanB-positive Enterococci) were included to fully evaluate the performance of the BC-GP test. Sensitivity and specificity for the 12 specific genus or species targets identified by the BC-GP test ranged from 92.6%–100% and 95.4%–100%, respectively. Identification of the mecA gene in 599 cultures containing S. aureus or S. epidermidis was 98.6% sensitive and 94.3% specific compared to cefoxitin disk method. Identification of the vanA gene in 81 cultures containing Enterococcus faecium or E. faecalis was 100% sensitive and specific. Approximately 7.5% (87/1,157) of single-organism cultures contained Gram-positive bacteria not present on the BC-GP test panel. In 95 cultures containing multiple organisms the BC-GP test was in 71.6% (68/95) agreement with culture results. Retrospective analysis of 107 separate blood cultures demonstrated that identification of methicillin resistant S. aureus and vancomycin resistant Enterococcus spp. was completed an average of 41.8 to 42.4 h earlier using the BC-GP test compared to routine culture methods. The BC-GP test was unable to assign mecA to a specific organism in cultures containing more than one Staphylococcus isolate and does not identify common blood culture contaminants such as Micrococcus, Corynebacterium, and Bacillus.
Conclusions
The BC-GP test is a multiplex test capable of detecting most leading causes of Gram-positive bacterial blood stream infections as well as genetic markers of methicillin and vancomycin resistance directly from positive blood cultures.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Severe sepsis—a life-threatening condition that is usually is triggered by a bacterial infection of the bloodstream—is a major global cause of illness and death. In the US alone, sepsis causes up to 500,000 hospital admissions and more than 250,000 deaths a year. Normally, when microbes enter the human body, the immune system efficiently kills the invaders. In sepsis, the immune system goes into overdrive and the chemicals it releases into the blood to combat the infection trigger widespread inflammation, which leads to the formation of small blood clots and leaky blood vessels that impair the flow of blood to vital organs. In the most severe cases, multiple organs fail and the patient dies. Anyone can get sepsis but people with weakened immune systems, the elderly, and the very young are most vulnerable. Symptoms of sepsis include fever, chills, rapid breathing, and a fast heart rate. In its early stages, sepsis can be treated with antibiotics alone, but people with severe sepsis need to be admitted to an intensive care unit where their vital organs can be supported while the infection is treated.
Why Was This Study Done?
The outcome of sepsis is affected by many factors, but fast, accurate identification of the bacterial infection and determination of its antibiotic susceptibility is essential to ensure that patients receive appropriate antibiotics. Laboratory diagnosis of bloodstream infections currently requires incubation of blood samples in a liquid medium (broth) followed by growth on solid media to identify the bacteria and to test for antibiotic sensitivity. It takes about 3 days after a positive broth culture is obtained to complete this process during which time patients are treated with broad spectrum antimicrobials, which may be ineffective. In this study, the researchers evaluate the sensitivity (a test's ability to identify patients who are positive for a specific condition) and specificity (a test's ability to identify patients who do not have a specific condition) of the Verigene BC-GP test. This multiplex, automated microarray-based nucleic acid test has been developed to directly and simultaneously identify 12 species of Gram-positive bacteria (the commonest cause of bacterial bloodstream infections) and three antibiotic resistance determinants in blood culture broths that contain Gram-positive bacteria.
What Did the Researchers Do and Find?
The researchers used the Verigene BC-GP test and reference culture methods to identify the bacterial species and antibiotic resistance determinants present in 1,252 blood cultures containing Gram-positive bacteria collected at five US clinical centers and in 387 contrived blood cultures that contained bacterial species rarely found in the bloodstream. Compared to the reference culture method, the sensitivity of the Verigene BC-GP test for the bacterial species included in the test ranged from 92.6% to 100%; its specificity was 94.5%–100%. Identification of the mecA gene (a genetic marker for methicillin resistance) in cultures that contained Staphylococcus aureus or S. epidermis (Gram-positive bacteria that are often methicillin resistant) was 98.6% sensitive and 94.3% specific. Identification of the vanA gene (a genetic marker for vancomycin resistance) was 100% sensitive and specific in cultures containing Enterococcus species. Only 7.5% of single-organism cultures contained Gram-positive bacteria not present in the Verigene BC-GP test panel. Importantly, results obtained with the new test agreed those obtained with reference culture methods in three-quarters of cultures that contained more than one bacterial species. Finally, in a retrospective analysis of turn-around-time, identification of methicillin-resistant S. aureus and vancomycin-resistant Enterococcus species with the Verigene BC-GP test was about 42 hours faster than with reference methods.
What Do These Findings Mean?
These findings show that the Verigene BC-GP test is capable of accurately identifying most leading causes of bloodstream infection with Gram-positive bacteria. Moreover, they show that the test can detect genetic markers of methicillin and vancomycin resistance directly from positive blood cultures, although they also reveal that the test cannot assign mecA positivity to a specific organism in a mixed culture, a finding that may mean that some patients are treated with unnecessary antibiotics. Overall, the researchers conclude that the Verigene BC-GP test has the potential to markedly reduce the turn-around-time for reporting bacterial identification from positive blood cultures. Its use should, therefore, improve the care of patients with sepsis by allowing physicians to prescribe appropriate antibiotics much earlier than is currently possible.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001478.
The US National Institute of General Medical Sciences has a fact sheet on sepsis
The UK National Health Service Choices website has information about sepsis
The Sepsis Alliance, a US not-for-profit organization, provides information about sepsis for patients and their families, including personal stories about sepsis
The not-for profit UK Sepsis Trust is another useful source of information about sepsis that includes patient stories
Wikipedia has a page on Gram-positive bacteria (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Medlineplus provides links to additional resources about sepsis and about bacterial infections (in English and Spanish)
doi:10.1371/journal.pmed.1001478
PMCID: PMC3699453  PMID: 23843749
2.  Severe Maternal Sepsis in the UK, 2011–2012: A National Case-Control Study 
PLoS Medicine  2014;11(7):e1001672.
Marion Knight and colleagues conducted a national prospective case-control study in the UK from June 2011 through May 2012 to estimate the incidence, describe the causative organisms and sources of infection, and identify the risk factors for severe maternal sepsis.
Please see later in the article for the Editors' Summary
Background
In light of increasing rates and severity of sepsis worldwide, this study aimed to estimate the incidence of, and describe the causative organisms, sources of infection, and risk factors for, severe maternal sepsis in the UK.
Methods and Findings
A prospective case-control study included 365 confirmed cases of severe maternal sepsis and 757 controls from all UK obstetrician-led maternity units from June 1, 2011, to May 31, 2012. Incidence of severe sepsis was 4.7 (95% CI 4.2–5.2) per 10,000 maternities; 71 (19.5%) women developed septic shock; and five (1.4%) women died. Genital tract infection (31.0%) and the organism Escherichia coli (21.1%) were most common. Women had significantly increased adjusted odds ratios (aORs) of severe sepsis if they were black or other ethnic minority (aOR = 1.82; 95% CI 1.82–2.51), were primiparous (aOR = 1.60; 95% CI 1.17–2.20), had a pre-existing medical problem (aOR = 1.40; 95% CI 1.01–1.94), had febrile illness or were taking antibiotics in the 2 wk prior to presentation (aOR = 12.07; 95% CI 8.11–17.97), or had an operative vaginal delivery (aOR = 2.49; 95% CI 1.32–4.70), pre-labour cesarean (aOR = 3.83; 95% CI 2.24–6.56), or cesarean after labour onset (aOR = 8.06; 95% CI 4.65–13.97). Median time between delivery and sepsis was 3 d (interquartile range = 1–7 d). Multiple pregnancy (aOR = 5.75; 95% CI 1.54–21.45) and infection with group A streptococcus (aOR = 4.84; 2.17–10.78) were associated with progression to septic shock; for 16 (50%) women with a group A streptococcal infection there was <2 h—and for 24 (75%) women, <9 h—between the first sign of systemic inflammatory response syndrome and a diagnosis of severe sepsis. A limitation of this study was the proportion of women with sepsis without an identified organism or infection source (16.4%).
Conclusions
For each maternal sepsis death, approximately 50 women have life-threatening morbidity from sepsis. Follow-up to ensure infection is eradicated is important. The rapid progression to severe sepsis highlights the importance of following the international Surviving Sepsis Campaign guideline of early administration of high-dose intravenous antibiotics within 1 h of admission to hospital for anyone with suspected sepsis. Signs of severe sepsis in peripartum women, particularly with confirmed or suspected group A streptococcal infection, should be regarded as an obstetric emergency.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Every year, nearly 300,000 women worldwide die during pregnancy or labour, or shortly after. According to a recent World Health Organization estimate, sepsis (blood poisoning) is responsible for 10.7% of these maternal deaths. Sepsis is caused by an inappropriate immune response to an infection. Normally, when bacteria or other microbes enter the human body, the immune system efficiently destroys the invaders. In sepsis, the immune system goes into overdrive, and the chemicals it releases into the blood to combat infection trigger widespread inflammation. This inflammation leads to the formation of small blood clots and leaky blood vessels that block the flow of blood to the vital organs. In the most severe cases (septic shock), blood pressure falls to dangerously low levels, multiple organs fail, and the patient can die. Symptoms of sepsis include fever, rapid breathing, and a fast heart rate. Sepsis, which often progresses rapidly, can be treated in its early stages with antibiotics alone. People with severe sepsis need to be admitted to an intensive care unit, where their vital organs can be supported while the infection is treated.
Why Was This Study Done?
Deaths from maternal sepsis mainly occur in low- and middle-income countries, but the rate of such deaths is increasing in countries with advanced healthcare systems. In the UK, for example, the incidence (the number of cases) of fatal maternal sepsis has increased markedly over the past two decades, and although the absolute risk of maternal death from sepsis is low, increasing numbers of women are experiencing severe maternal sepsis. To avoid preventable maternal illness and death in the UK, it is essential that clinical management and infection control strategies for maternal sepsis are improved. Here, to learn more about the incidence of maternal sepsis, the causative organisms and sources of infection, and the risk factors for maternal sepsis in the UK, the researchers undertake a national case-control study of severe maternal sepsis. A case-control study compares the characteristics of individuals with and without a given disease.
What Did the Researchers Do and Find?
For this study, clinicians in all the UK obstetrician-led maternity units (obstetricians care for women throughout pregnancy, labour, and the post-labour period) sent information about every woman who developed severe sepsis between June 2011 and May 2012 (365 cases) and about two unaffected (control) women per case to the United Kingdom Obstetric Surveillance System (UKOSS). Using this information and data on the number of maternities in the UK during this 12-month period, the researchers calculated that the incidence of severe sepsis was 4.7 per 10,000 maternities. Seventy-one women with severe sepsis (19.5% of cases) developed septic shock, and five women (1.4% of cases) died. The most common source of sepsis (implicated in about a third of cases) was a genital tract infection. Statistical analyses identified several risk factors for severe maternal sepsis, including having a fever or taking antibiotics in the two weeks preceding sepsis and all types of operative delivery (including cesarean delivery). Importantly, although Escherichia coli was the most common causative organism in severe maternal sepsis (present in a fifth of cases), infection with group A streptococcus was strongly associated with progression to septic shock. Moreover, in half the women with a group A streptococcal infection, severe sepsis was diagnosed within two hours of the first signs of a systemic inflammatory response.
What Do These Findings Mean?
These findings show that for every death from maternal sepsis in the UK, about 50 women develop life-threatening severe sepsis, that the onset of severe sepsis is very rapid, and that women who have recently had an infection are at particularly high risk of developing maternal sepsis. Although some pregnant women who developed severe sepsis during the study period may not have been included in the study, these findings have important clinical implications for the management of maternal sepsis in the UK and elsewhere. The findings suggest that pregnant or recently pregnant women with an infection need closer attention than women who are not pregnant, and adequate follow-up to ensure eradication of the infection. The findings also highlight the importance of giving high-dose intravenous antibiotics to anyone with suspected sepsis within an hour of admission to hospital as recommended by the international Surviving Sepsis Campaign, an initiative that was developed to improve the management, diagnosis, and treatment of sepsis. Finally, these findings suggest that signs of severe sepsis, particularly in women with a confirmed or suspected group A streptococcal infection, should be regarded as an obstetric emergency.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001672.
The UK National Health Service Choices website has information about sepsis
The international Surviving Sepsis Campaign guidelines for the treatment of sepsis are available through the campaign's website
The Sepsis Alliance, a US not-for-profit organization, also provides information about sepsis for patients and their families (in English and Spanish), including information about maternal sepsis and several personal stories about maternal sepsis (see the stories of Alanna Basinger, Alisa Proctor, Sandy C, and Natalie Banathy)
The not-for profit UK Sepsis Trust is another useful source of information about sepsis that includes patient stories
MedlinePlus provides links to additional resources about sepsis (in English and Spanish)
UKOSS provides more information about its national case-control study on severe maternal sepsis in the UK
doi:10.1371/journal.pmed.1001672
PMCID: PMC4086731  PMID: 25003759
3.  The clinical diagnostic accuracy of rapid detection of healthcare-associated bloodstream infection in intensive care using multipathogen real-time PCR technology 
BMJ Open  2011;1(1):e000181.
Background
There is growing interest in the potential utility of real-time PCR in diagnosing bloodstream infection by detecting pathogen DNA in blood samples within a few hours. SeptiFast is a multipathogen probe-based real-time PCR system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection and has European regulatory approval. The SeptiFast pathogen panel is suited to identifying healthcare-associated bloodstream infection acquired during complex healthcare, and the authors report here the protocol for the first detailed health-technology assessment of multiplex real-time PCR in this setting.
Methods/design
A Phase III multicentre double-blinded diagnostic study will determine the clinical validity of SeptiFast for the rapid detection of healthcare-associated bloodstream infection, against the current service standard of microbiological culture, in an adequately sized population of critically ill adult patients. Results from SeptiFast and standard microbiological culture procedures in each patient will be compared at study conclusion and the metrics of clinical diagnostic accuracy of SeptiFast determined in this population setting. In addition, this study aims to assess further the preliminary evidence that the detection of pathogen DNA in the bloodstream using SeptiFast may have value in identifying the presence of infection elsewhere in the body. Furthermore, differences in circulating immune-inflammatory markers in patient groups differentiated by the presence/absence of culturable pathogens and pathogen DNA will help elucidate further the patho-physiology of infection developing in the critically ill.
Ethics and dissemination
Ethical approval has been granted by the North West 6 Research Ethics Committee (09/H1003/109). Based on the results of this first non-commercial study, independent recommendations will be made to The Department of Health (open-access health technology assessment report) as to whether SeptiFast has sufficient clinical diagnostic accuracy to move forward to efficacy testing during the provision of routine clinical care.
Article summary
Article focus
To highlight the unmet need for accurate and rapid infection diagnostics in the setting of life-threatening infection.
To describe the systematic plans of a clinical diagnostic validity study of a new real-time PCR technology, designed to detect circulating pathogen DNA associated with bloodstream infection.
To describe the clinical standards for sepsis and healthcare-associated infection diagnosis and identify how these standards will be utilised to determine the clinical validity of the new real-time PCR test in critically ill patients.
Key messages
The study will provide the first independent, systematic, clinical validity study of real-time PCR technologies in the focused setting of suspected life-threatening healthcare-associated infections during the provision of routine emergency critical care.
Based on the results of this study, independent recommendations will be made to the UK's Department of Health as to whether the real-time PCR technology has sufficient clinical diagnostic accuracy to move forward to efficacy testing during the provision of routine clinical care.
Strengths and limitations of this study
The study is focused on a carefully delineated clinical cohort at significant risk of developing life-threatening infection.
The study is non-commercial and has been planned systematically by a multidisciplinary team of experts and patient representatives, working on behalf of the key stakeholders within a nationalised healthcare system.
Current clinical infection diagnosis standards may not have a high diagnostic accuracy in all settings and with all infections.
There is a documented high rate of broad-spectrum antimicrobial therapies delivered to critically ill patients empirically which could confound the comparison between culture methods and pathogen DNA-detection methods.
doi:10.1136/bmjopen-2011-000181
PMCID: PMC3191580  PMID: 22021785
Intensive &critical care; adult intensive & critical care; molecular diagnostics; adult intensive & critical care; adult thoracic medicine; adult surgery; Colorectal surgery; inflammatory bowel disease; Nutritional support; wound management
4.  Comparison of broad range 16S rDNA PCR and conventional blood culture for diagnosis of sepsis in the newborn: a case control study 
BMC Pediatrics  2009;9:5.
Background
Early onset bacterial sepsis is a feared complication of the newborn. A large proportion of infants admitted to the Neonatal Intensive Care Unit (NICU) for suspected sepsis receive treatment with potent systemic antibiotics while a diagnostic workup is in progress. The gold standard for detecting bacterial sepsis is blood culture. However, as pathogens in blood cultures are only detected in approximately 25% of patients, the sensitivity of blood culture is suspected to be low. Therefore, the diagnosis of sepsis is often based on the development of clinical signs, in combination with laboratory tests such as a rise in C – reactive protein (CRP). Molecular assays for the detection of bacterial DNA in the blood represent possible new diagnostic tools for early identification of a bacterial cause.
Methods
A broad range 16S rDNA polymerase chain reaction (PCR) without preincubation was compared to conventional diagnostic work up for clinical sepsis, including BACTEC blood culture, for early determination of bacterial sepsis in the newborn. In addition, the relationship between known risk factors, clinical signs, and laboratory parameters considered in clinical sepsis in the newborn were explored.
Results
Forty-eight infants with suspected sepsis were included in this study. Thirty-one patients were diagnosed with sepsis, only 6 of these had a positive blood culture. 16S rDNA PCR analysis of blinded blood samples from the 48 infants revealed 10 samples positive for the presence of bacterial DNA. PCR failed to be positive in 2 samples from blood culture positive infants, and was positive in 1 sample where a diagnosis of a non-septic condition was established. Compared to blood culture the diagnosis of bacterial proven sepsis by PCR revealed a 66.7% sensitivity, 87.5% specificity, 95.4% positive and 75% negative predictive value. PCR combined with blood culture revealed bacteria in 35.1% of the patients diagnosed with sepsis. Irritability and feeding difficulties were the clinical signs most often observed in sepsis. CRP increased in the presence of bacterial infection.
Conclusion
There is a need for PCR as a method to quickly point out the infants with sepsis. However, uncertainty about a bacterial cause of sepsis was not reduced by the PCR result, reflecting that methodological improvements are required in order for DNA detection to replace or supplement traditional blood culture in diagnosis of bacterial sepsis.
doi:10.1186/1471-2431-9-5
PMCID: PMC2635358  PMID: 19152691
5.  Multiplex PCR Allows Rapid and Accurate Diagnosis of Bloodstream Infections in Newborns and Children with Suspected Sepsis▿†§ 
Journal of Clinical Microbiology  2011;49(6):2252-2258.
Sepsis is a major health problem in newborns and children. Early detection of pathogens allows initiation of appropriate antimicrobial therapy that strongly correlates with positive outcomes. Multiplex PCR has the potential to rapidly identify bloodstream infections, compensating for the loss of blood culture sensitivity. In an Italian pediatric hospital, multiplex PCR (the LightCycler SeptiFast test) was compared to routine blood culture with 1,673 samples obtained from 803 children with suspected sepsis; clinical and laboratory information was used to determine the patient infection status. Excluding results attributable to contaminants, SeptiFast showed a sensitivity of 85.0% (95% confidence interval [CI] = 78.7 to 89.7%) and a specificity of 93.5% (95% CI = 92.1 to 94.7%) compared to blood culture. The rate of positive results was significantly higher with SeptiFast (14.6%) than blood culture (10.3%) (P < 0.0001), and the overall positivity rate was 16.1% when the results of both tests were combined. Staphylococcus aureus (11.6%), coagulase-negative staphylococci (CoNS) (29.6%), Pseudomonas aeruginosa (16.5%), and Klebsiella spp. (10.1%) were the most frequently detected. SeptiFast identified 97 additional isolates that blood culture failed to detect (24.7% P. aeruginosa, 23.7% CoNS, 14.4% Klebsiella spp., 14.4% Candida spp.). Among specimens taken from patients receiving antibiotic therapy, we also observed a significantly higher rate of positivity of SeptiFast than blood culture (14.1% versus 6.5%, respectively; P < 0.0001). On the contrary, contaminants were significantly more frequent among blood cultures than SeptiFast (n = 97 [5.8%] versus n = 26 [1.6%]), respectively; P < 0.0001). SeptiFast served as a highly valuable adjunct to conventional blood culture in children, adding diagnostic value and shortening the time to result (TTR) to 6 h.
doi:10.1128/JCM.02460-10
PMCID: PMC3122766  PMID: 21471340
6.  Accuracy of LightCycler® SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review protocol 
BMJ Open  2012;2(1):e000392.
Background
There is growing interest in the potential utility of molecular diagnostics in improving the detection of life-threatening infection (sepsis). LightCycler® SeptiFast is a multipathogen probe-based real-time PCR system targeting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here the protocol of the first systematic review of published clinical diagnostic accuracy studies of this technology when compared with blood culture in the setting of suspected sepsis.
Methods/design
Data sources: the Cochrane Database of Systematic Reviews, the Database of Abstracts of Reviews of Effects (DARE), the Health Technology Assessment Database (HTA), the NHS Economic Evaluation Database (NHSEED), The Cochrane Library, MEDLINE, EMBASE, ISI Web of Science, BIOSIS Previews, MEDION and the Aggressive Research Intelligence Facility Database (ARIF). Study selection: diagnostic accuracy studies that compare the real-time PCR technology with standard culture results performed on a patient's blood sample during the management of sepsis. Data extraction: three reviewers, working independently, will determine the level of evidence, methodological quality and a standard data set relating to demographics and diagnostic accuracy metrics for each study. Statistical analysis/data synthesis: heterogeneity of studies will be investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in Receiver Operator Characteristic (ROC) space. Bivariate model method will be used to estimate summary sensitivity and specificity. The authors will investigate reporting biases using funnel plots based on effective sample size and regression tests of asymmetry. Subgroup analyses are planned for adults, children and infection setting (hospital vs community) if sufficient data are uncovered.
Dissemination
Recommendations will be made to the Department of Health (as part of an open-access HTA report) as to whether the real-time PCR technology has sufficient clinical diagnostic accuracy potential to move forward to efficacy testing during the provision of routine clinical care.
Registration
PROSPERO—NIHR Prospective Register of Systematic Reviews (CRD42011001289).
Article summary
Article focus
To describe the plans of a systematic review aimed at determining the diagnostic accuracy of a new real-time PCR technology (LightCycler® SeptiFast), designed to detect bloodborne pathogens in the setting of life-threatening infection (sepsis).
To highlight the unmet need for accurate and rapid infection diagnostics in the setting of life-threatening infection (sepsis).
Key messages
The study will provide the first independent systematic review of clinical validity studies of multiplex real-time PCR technology aimed at detecting circulating pathogen DNA straight from blood in the setting of suspected life-threatening infections (sepsis).
Based on the results of this study, independent recommendations will be made to the UK's Department of Health to help determine whether the real-time PCR technology has sufficient clinical diagnostic accuracy to move forward to efficacy testing during the provision of routine clinical care.
Strengths and limitations
The systematic review is focused on a single Conformité Européenne (CE)-marked real-time PCR technology designed for use in the setting of life-threatening infection (sepsis)
The systematic review is non-commercial and has been planned systematically by a multidisciplinary team of experts, working on behalf of the key stakeholders within a nationalised healthcare system.
Current clinical infection diagnostic reference standards may not have high diagnostic accuracy in all clinical settings and with all infections
doi:10.1136/bmjopen-2011-000392
PMCID: PMC3278490  PMID: 22240646
7.  Clinical impact of a commercially available multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis 
Background
Timely identification of pathogens is crucial to minimize mortality in patients with severe infections. Detection of bacterial and fungal pathogens in blood by nucleic acid amplification promises to yield results faster than blood cultures (BC). We analyzed the clinical impact of a commercially available multiplex PCR system in patients with suspected sepsis.
Methods
Blood samples from patients with presumed sepsis were cultured with the Bactec 9240™ system (Becton Dickinson, Heidelberg, Germany) and aliquots subjected to analysis with the LightCycler® SeptiFast® (SF) Test (Roche Diagnostics, Mannheim, Germany) at a tertiary care centre. For samples with PCR-detected pathogens, the actual impact on clinical management was determined by chart review. Furthermore a comparison between the time to a positive blood culture result and the SF result, based on a fictive assumption that it was done either on a once or twice daily basis, was made.
Results
Of 101 blood samples from 77 patients, 63 (62%) yielded concordant negative results, 14 (13%) concordant positive and 9 (9%) were BC positive only. In 14 (13%) samples pathogens were detected by SF only, resulting in adjustment of antibiotic therapy in 5 patients (7,7% of patients). In 3 samples a treatment adjustment would have been made earlier resulting in a total of 8 adjustments in all 101 samples (8%).
Conclusion
The addition of multiplex PCR to conventional blood cultures had a relevant impact on clinical management for a subset of patients with presumed sepsis.
doi:10.1186/1471-2334-9-126
PMCID: PMC2739209  PMID: 19671147
8.  Diagnostic Utility of Broad Range Bacterial 16S rRNA Gene PCR with Degradation of Human and Free Bacterial DNA in Bloodstream Infection Is More Sensitive Than an In-House Developed PCR without Degradation of Human and Free Bacterial DNA 
Mediators of Inflammation  2014;2014:108592.
We compared a commercial broad range 16S rRNA gene PCR assay (SepsiTest) to an in-house developed assay (IHP). We assessed whether CD64 index, a biomarker of bacterial infection, can be used to exclude patients with a low probability of systemic bacterial infection. From January to March 2010, 23 patients with suspected sepsis were enrolled. CD64 index, procalcitonin, and C-reactive protein were measured on admission. Broad range 16S rRNA gene PCR was performed from whole blood (SepsiTest) or blood plasma (IHP) and compared to blood culture results. Blood samples spiked with Staphylococcus aureus were used to assess sensitivity of the molecular assays in vitro. CD64 index was lower in patients where possible sepsis was excluded than in patients with microbiologically confirmed sepsis (P = 0.004). SepsiTest identified more relevant pathogens than blood cultures (P = 0.008); in three patients (13%) results from blood culture and SepsiTest were congruent, whereas in four cases (17.4%) relevant pathogens were detected by SepsiTest only. In vitro spiking experiments suggested equal sensitivity of SepsiTest and IHP. A diagnostic algorithm using CD64 index as a decision maker to perform SepsiTest shows improved detection of pathogens in patients with suspected blood stream infection and may enable earlier targeted antibiotic therapy.
doi:10.1155/2014/108592
PMCID: PMC4120914  PMID: 25120284
9.  Etiology of Illness in Patients with Severe Sepsis Admitted to the Hospital from the Emergency Department 
Background
Patients identified with sepsis in the emergency department often are treated on the basis of the presumption of infection; however, various noninfectious conditions that require specific treatments have clinical presentations very similar to that of sepsis. Our aim was to describe the etiology of illness in patients identified and treated for severe sepsis in the emergency department.
Methods
We conducted a prospective observational study of patients treated with goal-directed resuscitation for severe sepsis in the emergency department. Inclusion criteria were suspected infection, 2 or more criteria for systemic inflammation, and evidence of hypoperfusion. Exclusion criteria were age of <18 years and the need for immediate surgery. Clinical data on eligible patients were prospectively collected for 2 years. Blinded observers used a priori definitions to determine the final cause of hospitalization.
Results
In total, 211 patients were enrolled; 95 (45%) had positive culture results, and 116 (55%) had negative culture results. The overall mortality rate was 19%. Patients with positive culture results were more likely to have indwelling vascular lines (P = .03) be residents of nursing homes (P = .04), and have a shorter time to administration of antibiotics in the emergency department (83 vs 97 min; P = .03). Of patients with negative culture results, 44% had clinical infections, 8% had atypical infections, 32% had noninfectious mimics, and 16% had an illness of indeterminate etiology.
Conclusion
In this study, we found that >50% of patients identified and treated for severe sepsis in the emergency department had negative culture results. Of patients identified with a sepsis syndrome at presentation, 18% had a noninfectious diagnosis that mimicked sepsis, and the clinical characteristics of these patients were similar to those of patients with culture-positive sepsis.
doi:10.1086/650580
PMCID: PMC2921279  PMID: 20144044
10.  Quantitative data from the SeptiFast real-time PCR is associated with disease severity in patients with sepsis 
BMC Infectious Diseases  2014;14:155.
Background
The commercial test, SeptiFast, is designed to detect DNA from bacterial and fungal pathogens in whole blood. The method has been found to be specific with a high rule-in value for the early detection of septic patients. The software automatically provides information about the identified pathogen, without quantification of the pathogen. However, it is possible to manually derive Crossing point (Cp) values, i.e. the PCR cycle at which DNA is significantly amplified. The aim of this study was to find out whether Cp values correlate to disease severity.
Methods
We used a study cohort of patients with positive results from SeptiFast tests for bacteria from a recent study which included patients with suspected sepsis in the Emergency department. Cp values were compared with disease severity, classified as severe sepsis/septic shock or non-severe sepsis, according to the criteria of the American College of Chest Physicians/Society of Critical Care Medicine.
Results
Ninety-four patients were included. The prevalence of severe sepsis/septic shock in the study was 29%. SeptiFast positive tests from patients with severe sepsis/septic shock had significantly lower Cp values compared with those from patients with non-severe sepsis, median 16.9 (range: 7.3 - 24.3) versus 20.9 (range: 8.5 - 25.0), p < 0.001. Positive predictive values from the SeptiFast test for identifying severe sepsis/septic shock were 34% at Cp cut-off <25.0, 35% at Cp cut-off <22.5, 50% at Cp cut-off <20.0, and 73% at Cp cut-off <17.5. Patients with a positive Septifast test with a Cp value <17.5 had significantly more severe sepsis/septic shock (73% versus 15%, p < 0.001), were more often admitted to the Intensive Care Unit (23% versus 4%, p = 0.016), had positive blood culture (BC) more frequently (100% versus 32%, p < 0.001) and had longer hospital stays (median 19.5 [range: 4 - 78] days versus 5 [range: 0 - 75] days, p < 0.001) compared with those with a Cp value >17.5.
Conclusions
Our results suggest that introducing quantitative data to the SeptiFast test could be of value in assessing sepsis severity. Moreover, such data might also be useful in predicting a positive BC result.
doi:10.1186/1471-2334-14-155
PMCID: PMC3994454  PMID: 24656148
11.  Liver Dysfunction and Phosphatidylinositol-3-Kinase Signalling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis 
PLoS Medicine  2012;9(11):e1001338.
Experimental studies in a rat model of fecal peritonitis conducted by Michael Bauer and colleagues show that in this model, changes in liver function occur early in the development of sepsis, with potential implications for prognosis and development of new therapeutic approaches.
Background
Hepatic dysfunction and jaundice are traditionally viewed as late features of sepsis and portend poor outcomes. We hypothesized that changes in liver function occur early in the onset of sepsis, yet pass undetected by standard laboratory tests.
Methods and Findings
In a long-term rat model of faecal peritonitis, biotransformation and hepatobiliary transport were impaired, depending on subsequent disease severity, as early as 6 h after peritoneal contamination. Phosphatidylinositol-3-kinase (PI3K) signalling was simultaneously induced at this time point. At 15 h there was hepatocellular accumulation of bilirubin, bile acids, and xenobiotics, with disturbed bile acid conjugation and drug metabolism. Cholestasis was preceded by disruption of the bile acid and organic anion transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. This was partially confirmed by an increase in plasma bile acids (e.g., chenodeoxycholic acid [CDCA] and taurodeoxycholic acid [TDCA]) observed in 48 patients on the day severe sepsis was diagnosed; unlike bilirubin (area under the receiver-operating curve: 0.59), these bile acids predicted 28-d mortality with high sensitivity and specificity (area under the receiver-operating curve: CDCA: 0.77; TDCA: 0.72; CDCA+TDCA: 0.87).
Conclusions
Liver dysfunction is an early and commonplace event in the rat model of sepsis studied here; PI3K signalling seems to play a crucial role. All aspects of hepatic biotransformation are affected, with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reflected by early alterations in plasma bile acids. These observations carry important implications for the diagnosis of liver dysfunction and pharmacotherapy in the critically ill. Further clinical work is necessary to extend these concepts into clinical practice.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Sepsis (blood poisoning)—a life-threatening condition caused by an inappropriate immune response to an infection—is a major global cause of death. Normally, when bacteria or other microbes enter the human body, the immune system efficiently destroys the invaders. In sepsis the immune system goes into overdrive, and the chemicals it releases into the blood to combat the infection trigger widespread inflammation (swelling). This leads to the formation of small blood clots and leaky blood vessels that block the flow of blood to vital organs such as the kidneys and liver. In the most severe cases, multiple organs fail and the patient dies. Anyone can get sepsis, but people with weakened immune systems, the very young, and the elderly are most vulnerable. Symptoms of sepsis include fever, chills, rapid breathing, a fast heart rate, and confusion. In its early stages, sepsis can be treated with antibiotics alone, but people with severe sepsis need to be admitted to an intensive care unit where the vital organs can be supported while the infection is treated.
Why Was This Study Done?
Thirty to fifty percent of people who develop severe sepsis die. If sepsis could be diagnosed in its early stages, it might be possible to save more people. Unfortunately, the symptoms of sepsis mimic those of other conditions, and, because sepsis tends to develop very quickly, it is often not diagnosed until it is too late to save the patient's life. The development of liver (hepatic) dysfunction and jaundice are both regarded as late features of sepsis (jaundice is yellowing of the skin and eyes caused by a build-up of bilirubin in the blood). However, the researchers hypothesized that changes in liver function occur early in sepsis and could, therefore, be used to improve the diagnosis and management of sepsis.
What Did the Researchers Do and Find?
The researchers induced sepsis in rats by injecting bacteria into the peritoneal cavity (the gap between the abdominal wall and the abdominal organs), separated the infected animals into predicted survivors and non-survivors based on their heart stroke volume measured using cardiac ultrasound, and then examined their liver function. The expression of genes encoding proteins involved in “biotransformation” and “hepatobiliary transport” (the processes that convert waste products and toxic chemicals into substances that can be conjugated to increase solubility and then excreted) was down-regulated within six hours of sepsis induction in the predicted non-survivors compared to the predicted survivors. Functional changes such as bilirubin and bile acid accumulation in the liver (cholestasis), poor excretion of xenobiotics (molecules not usually found in the body such as antibiotics), and disturbed bile acid conjugation were also seen in predicted non-survivors but not in survivors. Moreover, phosphatidylinositol-3-kinase (PI3K) signaling (which is involved in several immune processes) increased soon after sepsis induction in non-survivor but not in survivor animals. Notably, mice lacking the PI3Kγ gene did not develop cholestasis or show impaired bile acid conjugation after induction of sepsis. Finally, in human patients, plasma bile acids were increased in 48 patients on the day that severe sepsis was diagnosed, and these increases accurately predicted death in these patients.
What Do These Findings Mean?
These findings show that liver dysfunction is an early event in animal models of sepsis and that PI3K signalling plays a crucial role in the development of liver dysfunction. They show that all aspects of liver biotransformation are affected during sepsis and suggest that outcomes are related to the severity of these changes. The limited clinical data included in this study also support the hypothesis that changes in liver function occur early in sepsis, although these data need confirming and extending. Taken together, these findings suggest that liver function tests might aid early diagnosis of sepsis and might also provide information about likely outcomes. They also have important implications for the use of drugs in patients who are critically ill with sepsis, in that some of the drugs routinely administered to such patients may not be adequately detoxified and may, therefore, contribute to organ injury. Finally, these findings suggest that inhibition of PI3Kγ may alleviate sepsis-associated cholestasis.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001338.
This study is further discussed in a PLOS Medicine Perspective by John Marshall
The US National Institute of General Medical Sciences has a fact sheet on sepsis
The UK National Health Service Choices website has information about sepsis and about jaundice
The Surviving Sepsis Campaign, which was developed to improve the management, diagnosis, and treatment of sepsis, provides basic information about sepsis
The Sepsis Alliance, a US not-for-profit organization, also provides information about sepsis for patients and their families, including personal stories about sepsis
The not-for profit UK Sepsis Trust is another useful source of information about sepsis that includes patient stories
MedlinePlus provides links to additional resources about sepsis and jaundice (in English and Spanish)
doi:10.1371/journal.pmed.1001338
PMCID: PMC3496669  PMID: 23152722
12.  Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit 
Critical Care  2010;14(4):R159.
Introduction
Sepsis is a serious medical condition that requires rapidly administered, appropriate antibiotic treatment. Conventional methods take three or more days for final pathogen identification and antimicrobial susceptibility testing. We organized a prospective observational multicenter study in three study sites to evaluate the diagnostic accuracy and potential clinical utility of the SeptiFast system, a multiplex pathogen detection system used in the clinical setting to support early diagnosis of bloodstream infections.
Methods
A total of 212 patients, suspected of having systemic inflammatory response syndrome (SIRS) caused by bacterial or fungal infection, were enrolled in the study. From these patients, 407 blood samples were taken and blood culture analysis was performed to identify pathogens. Whole blood was also collected for DNA Detection Kit analysis immediately after its collection for blood culture. The results of the DNA Detection Kit, blood culture and other culture tests were compared. The chosen antimicrobial treatment in patients whose samples tested positive in the DNA Detection Kit and/or blood culture analysis was examined to evaluate the effect of concomitant antibiotic exposure on the results of these analyses.
Results
SeptiFast analysis gave a positive result for 55 samples, while 43 samples were positive in blood culture analysis. The DNA Detection Kit identified a pathogen in 11.3% (45/400) of the samples, compared to 8.0% (32/400) by blood culture analysis. Twenty-three pathogens were detected by SeptiFast only; conversely, this system missed five episodes of clinically significant bacteremia (Methicillin-resistant Staphylococcus aureus (MRSA), 2; Pseudomonas aeruginosa, 1; Klebsiella spp, 1; Enterococcus faecium, 1). The number of samples that tested positive was significantly increased by combining the result of the blood culture analysis with those of the DNA Detection Kit analysis (P = 0.01). Among antibiotic pre-treated patients (prevalence, 72%), SeptiFast analysis detected more bacteria/fungi, and was less influenced by antibiotic exposure, compared with blood culture analysis (P = 0.02).
Conclusions
This rapid multiplex pathogen detection system complemented traditional culture-based methods and offered some added diagnostic value for the timely detection of causative pathogens, particularly in antibiotic pre-treated patients. Adequately designed intervention studies are needed to prove its clinical effectiveness in improving appropriate antibiotic selection and patient outcomes.
doi:10.1186/cc9234
PMCID: PMC2945143  PMID: 20731880
13.  Development and validation of a novel molecular biomarker diagnostic test for the early detection of sepsis 
Critical Care  2011;15(3):R149.
Introduction
Sepsis is a complex immunological response to infection characterized by early hyper-inflammation followed by severe and protracted immunosuppression, suggesting that a multi-marker approach has the greatest clinical utility for early detection, within a clinical environment focused on Systemic Inflammatory Response Syndrome (SIRS) differentiation. Pre-clinical research using an equine sepsis model identified a panel of gene expression biomarkers that define the early aberrant immune activation. Thus, the primary objective was to apply these gene expression biomarkers to distinguish patients with sepsis from those who had undergone major open surgery and had clinical outcomes consistent with systemic inflammation due to physical trauma and wound healing.
Methods
This was a multi-centre, prospective clinical trial conducted across four tertiary critical care settings in Australia. Sepsis patients were recruited if they met the 1992 Consensus Statement criteria and had clinical evidence of systemic infection based on microbiology diagnoses (n = 27). Participants in the post-surgical (PS) group were recruited pre-operatively and blood samples collected within 24 hours following surgery (n = 38). Healthy controls (HC) included hospital staff with no known concurrent illnesses (n = 20). Each participant had minimally 5 ml of PAXgene blood collected for leucocyte RNA isolation and gene expression analyses. Affymetrix array and multiplex tandem (MT)-PCR studies were conducted to evaluate transcriptional profiles in circulating white blood cells applying a set of 42 molecular markers that had been identified a priori. A LogitBoost algorithm was used to create a machine learning diagnostic rule to predict sepsis outcomes.
Results
Based on preliminary microarray analyses comparing HC and sepsis groups, a panel of 42-gene expression markers were identified that represented key innate and adaptive immune function, cell cycling, WBC differentiation, extracellular remodelling and immune modulation pathways. Comparisons against GEO data confirmed the definitive separation of the sepsis cohort. Quantitative PCR results suggest the capacity for this test to differentiate severe systemic inflammation from HC is 92%. The area under the curve (AUC) receiver operator characteristics (ROC) curve findings demonstrated sepsis prediction within a mixed inflammatory population, was between 86 and 92%.
Conclusions
This novel molecular biomarker test has a clinically relevant sensitivity and specificity profile, and has the capacity for early detection of sepsis via the monitoring of critical care patients.
doi:10.1186/cc10274
PMCID: PMC3219023  PMID: 21682927
14.  DNAemia Detection by Multiplex PCR and Biomarkers for Infection in Systemic Inflammatory Response Syndrome Patients 
PLoS ONE  2012;7(6):e38916.
Fast and reliable assays to precisely define the nature of the infectious agents causing sepsis are eagerly anticipated. New molecular biology techniques are now available to define the presence of bacterial or fungal DNA within the bloodstream of sepsis patients. We have used a new technique (VYOO®) that allows the enrichment of microbial DNA before a multiplex polymerase chain reaction (PCR) for pathogen detection provided by SIRS-Lab (Jena, Germany). We analyzed 72 sepsis patients and 14 non-infectious systemic inflammatory response syndrome (SIRS) patients. Among the sepsis patients, 20 had a positive blood culture and 35 had a positive microbiology in other biological samples. Of these, 51.4% were positive using the VYOO® test. Among the sepsis patients with a negative microbiology and the non-infectious SIRS, 29.4% and 14.2% were positive with the VYOO® test, respectively. The concordance in bacterial identification between microbiology and the VYOO® test was 46.2%. This study demonstrates that these new technologies offer great hopes, but improvements are still needed.
doi:10.1371/journal.pone.0038916
PMCID: PMC3376137  PMID: 22719987
15.  Efficacy of bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR for detecting neonatal sepsis: a case control study 
BMC Pediatrics  2010;10:53.
Background
Neonatal sepsis is difficult to diagnose and pathogens cannot be detected from blood cultures in many cases. Development of a rapid and accurate method for detecting pathogens is thus essential. The main purpose of this study was to identify etiological agents in clinically diagnosed neonatal sepsis using bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR (BrRNA-RT-qPCR) and to conduct comparisons with the results of conventional blood culture. Since BrRNA-RT-qPCR targets bacterial ribosomal RNA, detection rates using this approach may exceed those using conventional PCR.
Methods
Subjects comprised 36 patients with 39 episodes of suspected neonatal sepsis who underwent BrRNA-RT-qPCR and conventional blood culture to diagnose sepsis. Blood samples were collected aseptically for BrRNA-RT-qPCR and blood culture at the time of initial sepsis evaluation by arterial puncture. BrRNA-RT-qPCR and blood culture were undertaken using identical blood samples, and BrRNA-RT-qPCR was performed using 12 primer sets.
Results
Positive rate was significantly higher for BrRNA-RT-qPCR (15/39, 38.5%) than for blood culture (6/39, 15.4%; p = 0.0039). BrRNA-RT-qPCR was able to identify all pathogens detected by blood culture. Furthermore, this method detected pathogens from neonates with clinical sepsis in whom pathogens was not detected by culture methods.
Conclusions
This RT-PCR technique is useful for sensitive detection of pathogens causing neonatal sepsis, even in cases with negative results by blood culture.
doi:10.1186/1471-2431-10-53
PMCID: PMC2922101  PMID: 20667142
16.  Multiplex Polymerase Chain Reaction Pathogen Detection in Trauma, Emergency, and Burn Surgery Patients with Suspected Septicemia 
Surgery  2011;151(3):456-463.
Background
The goal of this study is to determine the clinical value of multiplex PCR for enhancing pathogen detection in trauma, emergency, and burn surgery patients with suspected septicemia. PCR-based pathogen detection quickly reveals occult bloodstream infections in these high-risk patients and may accelerate initiation of targeted antimicrobial therapy.
Methods
We conducted a prospective observational study comparing results for 30 trauma and emergency surgery patients to 20 burn patients. Whole-blood samples collected with routine blood cultures (BC) were tested using a new multiplex PCR-based pathogen detection system. PCR results were compared to culture data.
Results
PCR rapidly detected more pathogens than culture methods. Acute Physiology and Chronic Health Evaluation II (APACHE, P = 0.019), Sequential Organ Failure Assessment (SOFA, P = 0.011), and Multiple Organ Dysfunction (MODS, P = 0.033) scores were significantly higher in PCR positive versus PCR negative trauma and emergency surgery patients. Negative PCR results (OR 0.194; 95% CI, 0.045 – 0.840, P = 0.028) acted as an independent predictor of survival for the combined surgical patient population.
Conclusions
PCR detected the presence of pathogens more frequently than blood culture. These PCR results were reported faster than blood culture results. Severity scores were significantly higher in PCR-positive trauma and emergency surgery patients. The lack of pathogen DNA as determined by PCR served as a significant predictor of survival in the combined patient population. PCR testing independent of traditional prompts for culturing may have clinical value in burn patients. These results warrant further investigation through interventional trials.
doi:10.1016/j.surg.2011.07.030
PMCID: PMC3304499  PMID: 21975287
Antimicrobial therapy; blood culture; nucleic acid recognition; sepsis; serial testing; turnaround time
17.  Diagnostic performance and therapeutic impact of LightCycler SeptiFast assay in patients with suspected sepsis 
Rapid and reliable identification of pathogens is very important in the management of septic patients. We retrospectively evaluated the diagnostic accuracy and clinical utility of a multiplex real-time polymerase chain reaction (PCR) assay (SeptiFast (SF)) in patients with suspected sepsis in a tertiary care hospital in Tallinn, Estonia. A total of 160 blood samples from 144 patients were included in the study. SF results were compared with corresponding blood culture (BC) results. The concordance between SF and BC was 78.8%. The rate of positive results was significantly higher in SF than in BC (33.7% vs. 21.2%, respectively; p < 0.001). A total of 27 samples were found positive by both SF and BC, 27 by SF only, and seven by BC only. Of a total of 83 microorganisms detected SF identified 71, and BC 42 (p < 0.001). SF detected markedly more patients with candidemia: 11 patients were detected by SF compared to four patients by BC. Antimicrobial treatment was changed in 21 (38.9%) of 54 SF positive cases. In conclusion, our results demonstrated the high diagnostic accuracy of SF in detection of sepsis pathogens. In conjunction with its impact on therapeutic decisions, SF proved to be a useful adjunct to conventional blood culture in the diagnosis of sepsis etiology.
doi:10.1556/EuJMI.3.2013.1.10
PMCID: PMC3832085  PMID: 24265921
bacteremia, bloodstream infection, fungemia, real-time PCR, sepsis, SeptiFast
18.  Survival Analysis of 314 Episodes of Sepsis in Medical Intensive Care Unit in University Hospital: Impact of Intensive Care Unit Performance and Antimicrobial Therapy 
Croatian medical journal  2006;47(3):385-397.
Aim
To evaluate epidemiology of sepsis in medical intensive care unit (ICU) in an university hospital, and the impact of ICU performance and appropriate empirical antibiotic therapy on survival of septic patients.
Methods
Observational, partly prospective study conducted over 6 years assessed all patients meeting the criteria for sepsis at ICU admission at the Sisters of Mercy Hospital in Zagreb. Clinical presentation of sepsis was defined according to 2001 International Sepsis Definitions Conference. Demographic data, admission category, source of infection, severity of sepsis, ICU or hospital stay and outcome, ICU performance, and appropriateness of empirical antibiotic therapy were analyzed.
Results
The analysis included 314 of 5022 (6.3%) patients admitted to ICU during the study period. There were 176 (56.1%) ICU survivors. At the ICU admission, sepsis was present in 100 (31.8%), severe sepsis in 89 (28.6%), and septic shock in 125 (39.8%) patients with mortality rates 17%, 33.7%, 72.1%, respectively. During ICU treatment, 244 (77.7%) patients developed at least one organ dysfunction syndrome. Of 138 (43.9%) patients who met the criteria for septic shock, 107 (75.4) were non-survivors (P<0.001). Factors associated with in-ICU mortality were acquisition of sepsis at another department (odds ratio [OR] 0.06; 95% confidence interval [CI], 0.02-0.19), winter season (OR 0.42; 0.20-0.89), limited mobility (OR 0.28; 0.14-0.59), ICU length of stay (OR 0.82; 0.75-0.91), sepsis-related organ failure assessment (SOFA) score on day 1 (OR 0.80; 0.72-0.89), history of global heart failure (OR 0.33; 0.16-0.67), chronic obstructive pulmonary disease (COPD)-connected respiratory failure (OR 0.50; 0.27-0.93), septic shock present during ICU treatment (OR 0.03; 0.01-0.10), and negative blood culture at admission (OR 2.60; 0.81-6.23). Microbiological documentation of sepsis was obtained in 235 (74.8%) patients. Urinary tract infections were present in 168 (53.5%) patients, followed by skin or soft tissue infections in 58 (18.5%) and lower respiratory tract infections in 44 (14.0%) patients. Lower respiratory tract as focus of sepsis was connected with worse outcome (P<0.001). Empirical antibiotic treatment was considered adequate in 107 (60.8%) survivors and 42 (30.4%) non-survivors. Patients treated with adequate empirical antibiotic therapy had significantly higher survival time in hospital (log-rank, P = 0.001).
Conclusion
The mortality rate of sepsis was unacceptably high. The odds for poor outcome increased with acquisition of sepsis at another department, winter season, limited mobility, higher SOFA score on day 1, history of chronic global heart failure, COPD-connected respiratory failure, and septic shock present during ICU treatment, whereas longer ICU length of stay, positive blood culture, and adequate empirical antibiotic therapy were protective factors.
PMCID: PMC2080418  PMID: 16758516
19.  Diagnostic Performance of a Multiple Real-Time PCR Assay in Patients with Suspected Sepsis Hospitalized in an Internal Medicine Ward 
Journal of Clinical Microbiology  2012;50(4):1285-1288.
Early identification of causative pathogen in sepsis patients is pivotal to improve clinical outcome. SeptiFast (SF), a commercially available system for molecular diagnosis of sepsis based on PCR, has been mostly used in patients hospitalized in hematology and intensive care units. We evaluated the diagnostic accuracy and clinical usefulness of SF, compared to blood culture (BC), in 391 patients with suspected sepsis, hospitalized in a department of internal medicine. A causative pathogen was identified in 85 patients (22%). Sixty pathogens were detected by SF and 57 by BC. No significant differences were found between the two methods in the rates of pathogen detection (P = 0.74), even after excluding 9 pathogens which were isolated by BC and were not included in the SF master list (P = 0.096). The combination of SF and BC significantly improved the diagnostic yield in comparison to BC alone (P < 0.001). Compared to BC, SF showed a significantly lower contamination rate (0 versus 19 cases; P < 0.001) with a higher specificity for pathogen identification (1.00, 95% confidence interval [CI] of 0.99 to 1.00, versus 0.94, 95% CI of 0.90 to 0.96; P = 0.005) and a higher positive predictive value (1.00, 95% CI of 1.00 to 0.92%, versus 0.75, 95% CI of 0.63 to 0.83; P = 0.005). In the subgroup of patients (n = 191) who had been receiving antibiotic treatment for ≥24 h, SF identified more pathogens (16 versus 6; P = 0.049) compared to BC. These results suggest that, in patients with suspected sepsis, hospitalized in an internal medicine ward, SF could be a highly valuable adjunct to conventional BC, particularly in patients under antibiotic treatment.
doi:10.1128/JCM.06793-11
PMCID: PMC3318564  PMID: 22322348
20.  Bench-to-bedside review: The promise of rapid infection diagnosis during sepsis using polymerase chain reaction-based pathogen detection 
Critical Care  2009;13(4):217.
Early infection diagnosis as the cause of a patient's systemic inflammatory syndrome is an important facet of sepsis care bundles aimed at saving lives. Microbiological culture provides the main route for infection diagnosis but by its nature cannot provide time-critical results that can impact on early management. Consequently, broad-spectrum and high-potency antibiotics are essential during the immediate management of suspected sepsis in critical care but are associated with the development of drug-resistant organisms and superinfections. Established molecular laboratory techniques based on polymerase chain reaction (PCR) technology can detect pathogen DNA rapidly and have been developed for translation into a clinical diagnostic setting. In the setting of sepsis in critical care, emerging commercial systems are now available for the analysis of whole blood within hours, with the presumed aim of adoption into the current care bundles. In this review, we consider the importance of early infection diagnosis in sepsis, how this is limited by culture approaches and how the emerging PCR methods are showing promise in early clinical observational studies. The strengths and weaknesses of culture and PCR pathogen detection in whole-blood samples will be highlighted and recommendations made for urgent appropriately powered diagnostic validation studies in advance of clinical effectiveness trials before these emerging PCR pathogen detection techniques can be considered for adoption in clinical practice.
doi:10.1186/cc7886
PMCID: PMC2750130  PMID: 19664174
21.  Performance Evaluation of Hematologic Scoring System in Early Diagnosis of Neonatal Sepsis 
Objectives:
The present study was undertaken to evaluate and highlight the importance of hematological scoring system (HSS) in the early detection of neonatal sepsis.
Materials and Methods:
The cross-sectional study enrolled 110 neonates who were clinically suspected of infection (study group) and normal neonates for comparison (controls), during the 1st week of life. All peripheral blood smears were analyzed using HSS of Rodwell et al., by pathologists blinded to the infection status of the newborns. HSS assigns a score of 1 for each of seven findings significantly associated with sepsis: Abnormal total leukocyte count, abnormal total polymorphonuclear neutrophils (PMN) count, elevated immature PMN count, elevated immature: Total (I:T) PMN ratio, immature: Mature (I:M) PMN ratio ≥0.3, platelet count ≤150,000/mm3, and pronounced degenerative or toxic changes in PMNs. Score of ≤2 was interpreted as sepsis unlikely; score 3-4: Sepsis is possible and ≥5 sepsis or infection is very likely. Blood culture was taken as a standard indicator for septicemia. The perinatal history, clinical profile and laboratory data were recorded and correlated in each case. Each hematological parameter was assessed for its individual performance and also with the culture-proven sepsis. Sensitivity, specificity, positive and negative predictive values (NPVs) were calculated for each parameter and for different gestational ages. P value was also calculated for different parameters.
Results:
Out of the 110 infants, based on clinical findings and laboratory data were classified into three categories: Sepsis (n=42), probable infection (n=22) and normal (n=46). Out of these, 42 (38.2%) newborns had positive blood culture. 63 (57%) neonates were preterm and 47 (43%) term. Male: female ratio was 0.96:1. The P value was significant for the different gestational ages (0.0002) and sex ratio (0.003). Immature polymorphonuclear neutrophils (PMN) count was the most sensitive and I:M PMN ratio, the most specific indicator of sepsis. For sepsis and probable sepsis, I:T PMN count and immature PMN count have highest sensitivity whereas I:T and I:M PMN ratio have highest specificity. HSS has much higher sensitivity and specificity in preterms than in term neonates. Positive predictive value and NPV is also higher in preterm than term for HSS. It was also seen that with increasing scores, the likelihood of sepsis also increased.
Conclusion:
The sensitivities of the various screening parameters were found to be satisfactory in identifying early onset neonatal sepsis. It is a simple and feasible diagnostic tool to guide towards the decision-making for a rationale treatment.
doi:10.4103/2249-4847.109243
PMCID: PMC3761960  PMID: 24027741
Culture; hematological scoring system; neonatal sepsis; polymorph; preterm
22.  Use of PCR Coupled with Electrospray Ionization Mass Spectrometry for Rapid Identification of Bacterial and Yeast Bloodstream Pathogens from Blood Culture Bottles ▿  
Journal of Clinical Microbiology  2010;49(1):345-353.
Sepsis is among the top 10 causes of mortality in the United States. Rapid administration of antibiotics is one of the most important contributors to patient survival, yet only a limited number of methods exist for rapid identification of microbes cultivated from bloodstream infections, which can lead to sepsis. While traditional single-target molecular methods have been shown to greatly improve survival for septic patients by enabling rapid deescalation of broad-spectrum antibiotics, multiplex methods offer even greater possibilities. A novel multiplex method, PCR coupled to electrospray ionization mass spectrometry (PCR/ESI-MS), was used to identify the genus and species of microorganisms found to cause human bloodstream infections. DNA was directly extracted from 234 BacT-Alert blood culture bottles, and results were compared to those obtained by clinical reference standard methods. The study results demonstrated 98.7% and 96.6% concordance at the genus and species levels, respectively. Mixtures of microbes were identified in 29 blood culture bottles, including mixed species of the same genus, as well as mixtures containing Gram-positive and Gram-negative organisms, exemplifying the PCR/ESI-MS capability to identify multiple organisms simultaneously without the need for cultivation. This study demonstrates high analytical accuracy in comparison to routine subculture of blood culture bottles and phenotypic identification of microbes. Without foreknowledge of the microorganisms potentially present, the PCR/ESI-MS methods can deliver accurate results in as little as 5 to 6 h after a positive alarm from the automated blood culture system; however, current batch mode testing limits the method's clinical utility at this time.
doi:10.1128/JCM.00936-10
PMCID: PMC3020459  PMID: 21048006
23.  Discriminative Value of Inflammatory Biomarkers for Suspected Sepsis 
Background
Circulating biomarkers can facilitate sepsis diagnosis enabling early management and improved outcomes. Procalcitonin (PCT) has been suggested to have superior diagnostic utility compared to other biomarkers.
Methods
Adults with suspected sepsis in the Emergency Department were enrolled. PCT, CRP, and IL-6 were correlated with infection likelihood, sepsis severity, and septicemia. Multivariable models were constructed for length-of-stay and discharge to a higher level of care.
Results
Of 336 enrolled subjects, 60% had definite infection, 13% possible infection and 27% no infection. Of those with infection, 202 presented with sepsis, 28 with severe sepsis, and 17 with septic shock. Overall, 21% of subjects were septicemic. PCT, IL6, and CRP levels were significantly higher in septicemia (median PCT 2.3 vs. 0.2ng/mL; IL-6 178 vs. 72pg/mL; CRP 106 vs. 62mg/dL, p<0.001). Biomarker concentrations increased with greater likelihood of infection and sepsis severity. Using ROC analysis, PCT best predicted septicemia (0.78 vs. IL-6 0.70 and CRP 0.67) but CRP better identified clinical infection (0.75 vs. PCT 0.71 and IL-6 0.69). A PCT cut-off of 0.5ng/mL had 72.6% sensitivity and 69.5% specificity for bacteremia as well as 40.7% sensitivity and 87.2% specificity for diagnosing infection. A combined clinical-biomarker model revealed that CRP was marginally associated with length-of-stay (p=0.015), but no biomarker independently predicted discharge to a higher level of care.
Conclusions
In adult Emergency Department patients with suspected sepsis, PCT, IL-6, and CRP highly correlate with several infection parameters, but do not meaningfully predict length-of-stay or need for discharge to a higher level of care.
doi:10.1016/j.jemermed.2011.05.072
PMCID: PMC3740117  PMID: 22056545
Sepsis; Procalcitonin; Interleukin-6; C-Reactive Protein; Sensitivity and Specificity
24.  A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients 
BMC Infectious Diseases  2014;14:406.
Background
Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients.
Methods
DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures.
Results
The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients.
Conclusions
Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection.
doi:10.1186/1471-2334-14-406
PMCID: PMC4223582  PMID: 25047415
Candidaemia; Candida spp; Multiplex PCR; ICU; Paediatric; Diagnosis
25.  Genomic transcriptional profiling identifies a candidate blood biomarker signature for the diagnosis of septicemic melioidosis 
Genome Biology  2009;10(11):R127.
A diagnostic signature for sepsis caused by Burkholderia pseudomallei infection was identified from transcriptional profiling of the blood of septicemia patients.
Background
Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei, a Gram-negative bacillus classified by the National Institute of Allergy and Infectious Diseases (NIAID) as a category B priority agent. Septicemia is the most common presentation of the disease with a 40% mortality rate even with appropriate treatments. Better diagnostic tests are therefore needed to improve therapeutic efficacy and survival rates.
Results
We have used microarray technology to generate genome-wide transcriptional profiles (>48,000 transcripts) from the whole blood of patients with septicemic melioidosis (n = 32), patients with sepsis caused by other pathogens (n = 31), and uninfected controls (n = 29). Unsupervised analyses demonstrated the existence of a whole blood transcriptional signature distinguishing patients with sepsis from control subjects. The majority of changes observed were common to both septicemic melioidosis and sepsis caused by other infections, including genes related to inflammation, interferon-related genes, neutrophils, cytotoxic cells, and T-cells. Finally, class prediction analysis identified a 37 transcript candidate diagnostic signature that distinguished melioidosis from sepsis caused by other organisms with 100% accuracy in a training set. This finding was confirmed in 2 independent validation sets, which gave high prediction accuracies of 78% and 80%, respectively. This signature was significantly enriched in genes coding for products involved in the MHC class II antigen processing and presentation pathway.
Conclusions
Blood transcriptional patterns distinguish patients with septicemic melioidosis from patients with sepsis caused by other pathogens. Once confirmed in a large scale trial this diagnostic signature might constitute the basis of a differential diagnostic assay.
doi:10.1186/gb-2009-10-11-r127
PMCID: PMC3091321  PMID: 19903332

Results 1-25 (1335332)