Biomarkers

PROCALCITONIN AND OTHER HOST-RESPONSE-BASED BIOMARKERS FOR EVALUATION OF INFECTION AND GUIDANCE OF ANTIMICROBIAL TREATMENT

Philipp Schuetz, Ramon Sager, Yannick Wirz, Beat Mueller

Host-Response Markers in Systemic Infections and Sepsis

In systemic bacterial infections, pathogens and their antigens stimulate pro- and anti-inflammatory immune and host-response mediators that constitute the host defences and coordinate leukocyte recruitment to the site of acute infection. These molecules have multiple adaptive and maladaptive functions including regulation of the osmotic and volume status, appetite, blood circulation, and food intake among others. Precursors, mature forms, and degradation products of these various mediators leave the original site of action and enter the circulation, where, theoretically, they can all be measured. As surrogate host-response biomarkers, these substances mirror the extent and severity of an infection, with their levels falling upon resolution of the infection. Significant attempts have been made to correlate the levels of different mediators with the presence of infection as potential diagnostic markers and to use these markers for risk stratification of patients.

Host-Response Markers and the Definition of Sepsis

The recognition over 25 years ago that the host response plays an integral role in sepsis led to the definition of sepsis that has recently been updated to also include evidence of organ dysfunction as this has been shown to drive mortality in this patient population. Unfortunately, the systemic inflammatory response syndrome (SIRS) variables (i.e. body temperature, heart rate, tachypnoea, and white blood count) used in the original definition from the early 1990s turned out to be less useful than anticipated, lacking sensitivity, specificity, and ease of clinical application. Had a newer host-response biomarker been available when the SIRS criteria were initially put forth, it would arguably have been preferable to use white blood cell counts as a laboratory-based SIRS criterion. Indeed, specific immune and host-response markers (e.g. procalcitonin (PCT), presepsin, and lactate) measured on admission and during follow-up have been suggested to improve early sepsis recognition, severity assessment, and therapeutic decision-making for individual patients and may thereby allow transformation of bundled sepsis care based on prespecified protocols to more individualized patient management. Of these, PCT has generated much interest as it has a high diagnostic ability, helps in risk stratification of patients, and has been successfully used as a guide to antibiotic treatment decisions. As for diagnostic accuracy, a meta-analysis from 2013 that included 3244 critically ill patients classified as experiencing sepsis or SIRS of noninfectious origin pooled the diagnostic power of PCT. Studies between 1996 and 2011 were included and showed a good high discriminatory ability of PCT (area under the curve [AUC] of 0.85), with pooled sensitivity and specificity of 0.77 and 0.79, respectively.

Regulation of Procalcitonin During Infections

Procalcitonin is released ubiquitously in response to endotoxin or mediators released in response to bacterial infections (e.g. interleukin [IL]-1β, tumour necrosis factor [TNF]-α, and IL-6) and correlates with the extent and severity of bacterial infections. Because upregulation of PCT is attenuated by interferon gamma (INFγ), a cytokine released in response to viral infections, PCT is more specific for bacterial infections and may help to distinguish bacterial infections from viral illnesses. It shows a favourable kinetic profile for use as a clinical marker, with its levels promptly increasing within 6–12  h following infection and falling by half daily when the infection is controlled by the host immune system or through antibiotic therapy. Its levels correlate well with bacterial load and severity of infection, and its course predicts fatal outcome in patients with systemic infections and critically ill patients with sepsis, thus suggesting prognostic implications.

Procalcitonin as a Diagnostic Guide in Patients with Infections

Procalcitonin has been investigated as a diagnostic guide for patients with infections in several observational studies focusing on different clinical situations and different types and sites of infections.

Blood Stream Infections

Procalcitonin has been demonstrated to be very useful in the diagnosis of blood stream infections and bacteraemia. It was superior to white blood counts and C-reactive protein (CRP) in its ability to distinguish blood contamination from true blood stream infection in patients with growth of coagulase-negative staphylococci in their blood cultures. At a cut-off of 0.1ug/L, PCT had a very high sensitivity to correctly exclude true bacterial infection. Two other studies that focused on the use of PCT to predict bacteraemia in patients with urinary tract infections (UTI) and pneumonia revealed a PCT cut-off of 0.25ug/L to be most helpful to exclude bacteraemic disease, with a high negative predictive value in both settings.

Urinary Tract Infections

Evidence for the utility of PCT in urinary tract infections comes primarily from the paediatric literature, where it has a similar sensitivity but superior specificity as compared to CRP for the prediction of pyelonephritis in children with febrile UTIs. It correlates well with both the extent of renal involvement and with renal scarring. The utility of PCT in this setting was also investigated in a randomized trial.The study showed an antibiotic usage reduction of 30% with PCT-guided treatment compared to the standard treatment. The elaborated algorithm combined serum PCT concentration and quantitative pyuria measurement. Patients were grouped on the basis of uncomplicated or complicated UTI and on the basis of the treatment setting (outpatient versus inpatient) resulting in differences in antibiotics administered and lengths of treatment or were subjected to a monitored approach with measurement of PCT and the degree of pyuria. There were no negative effects. The authors concluded that a PCT/pyuria-based approach is safe in terms of outcome and has the potential to reduce antibiotic consumption.

Endocarditis

In patients with infectious endocarditis, two independent studies revealed that circulating PCT levels were elevated compared to noninfected patients. Unfortunately, a reliable PCT threshold for diagnosing or excluding infective endocarditis has not been proposed or tested in interventional studies. Importantly, subacute forms of endocarditis or prosthetic valve endocarditis may show different characteristics compared to acute forms due to their low inflammatory nature and possible biofilm production.

Meningitis

Several older studies that evaluated PCT-guided therapy in meningitis revealed that PCT-guided therapy reduces antimicrobial consumption during viral outbreaks. Two recent meta-analyses confirmed its accuracy in differentiating viral from bacterial meningitis. The most recent meta-analysis from 2016 included 2058 subjects and showed a sensitivity of 0.95 (95% CI, 0.89–0.97), a specificity of 0.97 (95% CI, 0.89–0.99), a positive likelihood ratio of 31.7 (95% CI, 8.0–124.8), and a negative likelihood ratio of 0.06 (95% CI, 0.03–0.11). The diagnostic performance was even better when PCT levels were combined with those of cerebrospinal fluid lactate. Serum PCT was found to be more sensitive and specific compared to cerebrospinal fluid PCT. Furthermore, PCT was useful for prognostication of poor outcome, for follow-up of treatment, and for differentiating other bacterial meningitis from tuberculous meningitis.

Intra-abdominal Infections

Few studies have investigated the utility of PCT in intra-abdominal infections. While PCT as a marker promised to exclude perforation and ischaemia in obstructive bowel syndrome, its utility in acute appendicitis and pancreatitis was limited, and it was more helpful as a prognostic marker for severe disease and adverse outcome. While localized infections may not induce a massive upregulation of PCT, studies found PCT to be of diagnostic utility in patients with arthritis and osteomyelitis, particularly when subtle increases were considered and a low PCT cut-off (0.1ug/L) was employed.

Febrile Neutropenia

Multiple studies have evaluated the utility of PCT in patients with febrile neutropenia. A systematic review of 30 studies on the topic concluded that PCT has value as a diagnostic and prognostic tool in patients with febrile neutropenia but that due to differences in patient populations and study quality, further research is needed. Importantly, it is worth noting that the production of PCT seems not to be attenuated by corticosteroids and that it does not rely on white blood cells. A study that included 102 critically ill patients with systemic infections in a medical ICU found significantly lower CRP and IL-6 levels, but similar PCT levels, in patients treated with systemic corticosteroids (20–1500  mg per day of prednisone parenterally) compared to untreated patients. These observations were confirmed in healthy male volunteers who received different doses of prednisolone up to 30  mg before a sepsis-like syndrome was induced with Escherichia coli lipopolysaccharide (LPS) injections. While other biomarkers were significantly inhibited in a dosedependent manner, levels of PCT showed no inhibition during the study. Whether this is also true for other corticosteroid doses, however, remains unknown. The value of PCT in febrile neutropenia may be as part of a combination with other biomarkers of bacterial infection such as IL-6 and IL-8, as shown in a small study of paediatric febrile neutropenia.

Antibiotic Stewardship Aided by the Use of Procalcitonin

The clinical implications of the above-mentioned observational studies may be limited by differences in disease definitions and patient populations, the use of insensitive (semi-quantitative) PCT assays, methodological issues such as observer bias and selection bias, and issues with sample availability, co-infection, and colonization. To overcome these limitations, several randomized-controlled studies have investigated the utility of PCT in assisting with decisions regarding initiation and/or duration of antibiotic therapy (antibiotic stewardship). The benefit of PCT was measured through clinical outcomes, assuming that if the patient recovers without antibiotics, there was no relevant bacterial illness in need of chemotherapy.

Antibiotic Stewardship Algorithms

All published studies on antibiotic stewardship have used similar clinical algorithms with recommendations for or against antibiotic treatment based on PCT cut-off ranges. For moderate-risk patients with respiratory tract infections seen in the emergency department (Fig. 1), algorithms recommended initiation and discontinuation of antibiotic therapy based on four different cut-off ranges (<0.1, 0.1–0.25, 0.25– 0.5 > 0.5 ng/mL). Initial antibiotics were withheld mostly in patients with low risk for systemic infection with acute bronchitis or exacerbation of COPD [ECOPD]). Clinical re-evaluation and repeat measurement of PCT were recommended after 6–24 h if the clinical condition did not improve spontaneously. If PCT values were higher and antibiotic therapy was initiated, repeated PCT measurements every 1–2 days, depending on the clinical severity of disease, were recommended, and antibiotics were discontinued using the same cut-off ranges or if a marked drop in PCT levels by 80–90% was seen if initial levels had been high (e.g. >5 μg/L). To ensure safety, specific criteria where this algorithm could be overruled, such as lifethreatening disease or immediate need for ICU admission, were predefined. For high-risk patients in the ICU setting (Fig. 2), algorithms focused on discontinuation of antibiotic therapy if a patient showed a clinical recovery and PCT levels decreased to ‘normal’ levels, or by at least 80–90%.

Clinical Trials in Respiratory Infections

The first interventional study testing PCT as a guide for antibiotic decisions included patients with different types and severities of respiratory infections. Clinical outcomes were similar regardless of whether therapy was guided by PCT levels or not, but patients receiving PCT-guided treatment, particularly those with ECOPD and acute bronchitis, had markedly lower rates of antibiotic prescriptions (44% vs 83%). Two subsequent trials that evaluated the impact of PCT guidance on antibiotic prescription patterns in patients with CAP and exacerbation of COPD revealed that PCT guidance reduced the duration of antibiotic therapy by 65% in CAP patients and the numbers of prescriptions for antibiotics from 72% to 40% in ECOPD patients. A subsequent multicentre trial confirmed the earlier results and documented a reduction in antibiotic prescriptions by 32% in patients with CAP, by 50% in patients with ECOPD, and by 65% in patients with acute bronchitis. Again, antibiotic exposure in patients with ECOPD and acute bronchitis decreased mainly by not initiating treatment at all, whereas in patients with CAP, it was principally due to reduction in the duration of therapy. Importantly, the overall rate of adverse events was similar in both study arms and excluded a risk of more than 0.4% for patients receiving PCT-guided therapy. Not surprisingly, patients with bacteraemic CAP had markedly increased PCT concentrations resulting in longer treatment durations as compared to culture-negative CAP patients with a lower infection-related risk. Similar results were also reported in trials from Denmark and China in patients with respiratory infections and recently from an observational ‘real-life’ quality control survey in Switzerland. Arguably, the most important (over-) use of antibiotics occurs in primary care. As many as 75% of patients with upper and lower respiratory infections receive antibiotics, despite the mostly viral origin of the condition. Two non-inferiority multicentre primary care trials recently investigated the safety and feasibility of PCT-guided algorithms in patients with upper and lower respiratory infections, essentially relying on an initial PCT measurement only. Both trials found substantial reductions in antibiotic exposure (by 75% and 42%), and similar clinical outcomes, particularly a similar time to recovery. A 2012 Cochrane meta-analysis that was based on individual patient data from 14 randomized-controlled trials (RCT) and focused on the impact of PCT-guided therapy for respiratory infections found an appreciable reduction in the initial use of antibiotics (by approximately 60–70%) in patients with low-severity respiratory infections (e.g. bronchitis, upper respiratory infections, and AECOPD). In patients with higher-severity respiratory infections (e.g. pneumonia), monitoring of PCT resulted in earlier cessation of antibiotic treatment with a relative reduction in the duration of antibiotic treatment by approximately 40% for pneumonia and approximately 25% in critical care patients with sepsis due to lung infections.

Clinical Trials in Sepsis

In more high-risk patients in the ICU setting, multiple trials have investigated the use of PCT, mainly for discontinuation of antibiotics. The first small proof of concept study found a 4-day reduction in the duration of antibiotic therapy in patients with severe sepsis but only in the per protocol analysis. A subsequent large multicentre trial in France recently validated this concept in more than 600 patients. Patients receiving PCT-guided therapy had similar 30-day mortality rates and similar rates of relapse but markedly more antibiotic-free days alive (14.3 vs. 11.6). A recent large RCT published in 2016 evaluated the effects of using PCT to deescalate and stop antibiotics in 1,575 critically ill patients in the Netherlands who received antibiotics for an assumed or proven infection within 24 h before inclusion in the study. The study found that the PCT-guided protocol significantly shortened the length of antibiotic treatment (5 days versus 7 days in the first 28 days upon admission) and significantly lowered 28-day mortality from 25% to 19.6%. Another multinational ICU study focused on ventilator-associated pneumonia (VAP) and found that PCT guidance resulted in a higher number of antibiotic freedays alive (13 versus 9.5 days). Two German studies that assessed the effect of PCT guidance in surgical ICU patients with suspected postoperative bacterial infections found PCT guidance to result in a significant reduction in the duration of antibiotic therapy, with no adverse effects on medical outcomes. In addition, the length of intensive care treatment in the PCT-guided group was significantly shorter than that in the control group (15.5 vs. 17.7 days), a finding similar to that in the first ICU study. Importantly, the use of PCT for discontinuation of antibiotics in ICU patients is still limited by a relatively small number of patients included in previous trials and awaits further large-scale validation. There are currently multiple ongoing trials focusing on this vulnerable patient population that should shed further light on the benefits and limitations of PCT-guided therapy in ICU patients.

Current Guideline Recommendations

Based on this body of evidence, recent guidelines on the treatment of lower respiratory tract infections (LRTI) emphasize the concept of using a biomarker to guide antibiotics and state that ‘.. biomarkers can guide treatment duration by the application of predefined stopping rules for antibiotics. It has been shown that such rules work even in most severe cases, including pneumonia with septic shock…’ In addition, the recently updated guidelines for the management of severe sepsis and septic shock put forth by the ‘Surviving Sepsis Campaign’ (SSC) now recommend the use of PCT as a marker to guide decisions pertaining to de-escalation of therapy in patients with clinical signs of recovery.

Other Immune Biomarkers for Management of Sepsis Patients

Other promising host-response biomarkers from different pathways involved in the pathogenesis of sepsis have previously been evaluated observationally. For example, markers of endothelial cell activation have been found to be associated with sepsis severity and organ dysfunction and were predictors of mortality. Repeated measurements of endothelin-1 (ET-1) precursor peptides have been shown to be correlated with the disease course and to significantly improve risk assessment in pneumonia patients. In addition to providing predictive information, endothelial immune biomarkers may also help physicians in selecting specific sepsis drugs for patients. Because antagonism of the endothelial system by Bosentan has shown beneficial effects in experimental models of sepsis, it is tempting to hypothesize that if these antagonistic therapies find their way into routine clinical practice, the measurement of endothelial activation may help to identify patients who might benefit most from these therapies. Another interesting immune monitoring system is the endotoxin activity assay (EAA) which not only measures blood endotoxin levels but also helps to monitor immunological reactions against the endotoxemia.

Areas of Uncertainty and Limitations of Procalcitonin and Other

Host-Response Markers Importantly, PCT as well as other currently used host-response markers are far from being perfect and thus levels must be evaluated in the context of a careful clinical and microbiological assessment of the patient. Because the kinetics of these markers are of particular diagnostic and prognostic importance, repeated measurements should be performed. This is particularly true for persistently sick patients and in situations where antibiotics are withheld. Limitations of PCT include false-positive and false-negative results. Moreover, PCT levels may increase in the absence of bacterial infections in patients with severe trauma or surgery. Here, PCT usually shows a rapid decline in follow-up measurements when the patient recovers. In addition, patients with chronic renal failure may have a slower PCT decrease. Levels of PCT may also be low early in the course of an infection or when an infection is localized, with later measurements showing an increase in PCT levels. Here again, repeated PCT measurements are advised in case of uncertainty. Another important consideration is the cost of PCT measurement, especially given that multiple measurements may be required in specific instances, as discussed above. While some studies have found PCT to be cost-effective in respiratory infections when the use of antibiotics can be reduced by the measurement of this marker, this may not be true for other indications.

Conclusions

Protocol-based bundled sepsis care has been shown to improve outcomes in patients. Because all medical therapies can have potentially harmful effects if used indiscriminately, selection of patients most likely to benefit from such therapies is pivotal. Prognostic host-response biomarkers may facilitate selection of patients for whom early discharge is safe and may thereby help to divert the resources to patients most in need. Host-response biomarkers may also help physicians to identify patients who would, and would not, benefit from individually tailored therapies and thus may allow a transition from bundled sepsis care to more personalized approaches, which in turn could result in improved patient outcomes.

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VALIDATING DENGUE DUAL INFECTION SCORE IN ADULT DENGUE PATIENTS AT HOSPITAL CANSELOR TUANKU MUHRIZ

Mokhtar MF1, Tan TL2 

1 Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia
2 Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia

Introduction: Clinical features of dengue infection are rather non-specific and can be complicated by concurrent bacterial infection. Ability to perform risk stratification early in the patient’s course of illness may guide physicians to early bacterial cultures and empirical antibiotic. 

Methods: We aim to evaluate the usefulness of the Dengue Dual Infection Score (DDIS) as a diagnostic model for identifying bacterial coinfection in adult dengue patients who present to the Emergency Department. The study was conducted between August 2016 to March 2017 whereby adult dengue patients who presented to our department were assigned one point to each of five risk factors for bacterial coinfection; pulse rate at ED ≥90 beats/minute, total white cell count ≥6x10^9/L, hematocrit <40%, serum sodium <135 mmol/l and serum urea ≥5 mmol/L. We defined bacterial coinfection as any clinical diagnosis of bacterial infection or microbiological diagnosis of bacteremia from cultures; or pneumonia from clinical presentation and radiological examination, within 72 hours of admission. 

Results: Among the frequently treated coinfection in our cohort were leptospirosis and pneumonia. We found a DDIS of ≥4 had a specificity of 95.5%, sensitivity of 25.0% (PPV of 50.0%, NPV of 87.5%) for bacterial coinfection. Patients with bacterial coinfection had longer duration of fever (5 days; p value = 0.047), had higher white cell count (4.8 x 109; IQR Q75, Q25 = 4.8, 3.1; p value = 0.024), had lower sodium level (mean ± SD = 131 ± 5; p value =0.025), and lower hematocrit level (41.1%; IQR Q75, Q25 = 44.3, 40.2, p value = 0.009). 

Conclusion: the use of DDIS would be an advantage in situations where dengue is endemic as it will be a better method for resource allocation in antibiotic selection among dengue patients. 

Oral Presentation in Emergency Medical Annual Meeting (EMAS 2018)

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EVALUATION OF PERSISTENCE NS1 ANTIGEN POSITIVITY AFTER DAY 5 OF ILLNESS AND DENGUE SEVERITY IN EMERGENCY DEPARTMENT, UNIVERSITI KEBANGSAAN MALAYSIA MEDICAL CENTRE

Syed Farid Almufazal Syed Salim1, Shamsuriani Md Jamal1, Toh Leong Tan1

1 Department of Emergency Medicine, Hospital Canselor Tuanku Mukhriz, UKM Medical Centre, Kuala Lumpur, Malaysia

Introduction: Early diagnosis and risk prediction of severe dengue are necessary, especially in high case burden areas. In order for early detection of severe dengue, frequent clinical and laboratory parameters are measured in all patients admitted to the hospital with dengue infection, posing a huge cost to the healthcare. 

Methods: The purpose of the study is to look whether persistence NS1 antigen, using rapid bedside test, can be used to predict dengue severity among adult dengue patients. Methods: Adults with suspected dengue fever, presented to Emergency Department of Universiti Kebangsaan Malaysia Medical Centre were recruited in the study. Serial clinical and laboratory parameters were taken in patients, including rapid NS1 antigen. Clinical outcomes of each patient were determined, including severe dengue. 

Results: From the total of 66 patients recruited with positive NS1 antigen in earlier phase, 12% (n=8) developed severe dengue. There was no correlation noted between persistence NS1 antigen beyond day 5 of illness and severe dengue (P value 0.519). 

Conclusion: Our study did not show a statistically significant relationship between a persistent level of NS1 antigen beyond day 5 of illness and severe dengue. Further study, with a higher number of study populations will need to be performed to determine the relationship between persistence NS1 antigenaemia and dengue severity.

Oral Presentation in Emergency Medical Annual Meeting (EMAS 2018)

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EVALUATION OF SECRETORY PHOSPHOLIPASE A2 GROUP IIA AND SERUM PROCALCITONIN AS PREDICTORS OF SEPSIS IN THE EMERGENCY DEPARTMENT OF A TEACHING HOSPITAL

TAN TL1, Kang WL1, Nasuruddin DN2, Ithnin A2, Neoh H3*  

1 Department of Emergency Medicine,

2 Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia, 

3UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia                                                                                                                                                                                              

Background: Diagnosis of sepsis has proven to be a challenge due to its mimicry to other inflammatory response; while diagnosis of bacterial infection is dependent on the use of microbiological cultures. Early identification of sepsis and bacterial infection in the emergency department (ED) is important for initiation of antibiotic administration, as it is associated with good prognosis and reduced mortality. The aim of this study is to evaluate the usefulness of 2 different biomarkers in predicting sepsis and bacterial infection, namely secretory phospholipase A2 group IIa (sPLA2-IIa) and procalcitonin (PCT). 

Materials/Methods: Patients aged ≥ 18 years old with confirmed sepsis based on systematic inflammatory response syndrome and suspected infections were recruited from the ED of Universiti Kebangsaan Malaysia Medical Centre. The diagnosis of bacterial infection was confirmed either clinically or via microbiological culture. Serum levels of sPLA2-IIA and PCT level were measured using ELISA. 

Results: Two hundred and eleven adult patients with suspected sepsis on admission to the ED were included into the study. Relevant blood investigations, cultures and serology tests were performed. Serum levels for sPLA2-IIa and PCT were also analyzed. Sepsis was confirmed in 167 patients while 172 patients had bacterial infections from a total of 211 recruited subjects. Both biomarkers were able to distinguish between sepsis and non-sepsis groups and bacterial and non-bacterial infection groups. The area under receiver operating characteristic (AUROC) curves showed that sPLA2-IIa was better than PCT in the diagnosis of sepsis (AUROC = 0.81, 95% confidence interval (CI) = 0.71-0.86 and AUROC = 0.79, 95% CI = 0.72-0.89, respectively). SPLA2-IIa showed superiority over PCT (sPLA2-IIa, AUROC = 0.93, 95% CI = 0.88-0.96, and PCT, AUROC = 0.89, CI = 0.84-0.96, respectively) in diagnosing bacterial infections. 

Conclusion: sPLA2-IIa showed excellent performance in diagnosis of sepsis and bacterial infection in comparison to PCT; this may be useful for clinicians in decision making for early antibiotic administration. We advocate including both sPLA2-IIa and PCT into the diagnostic algorithm of sepsis in ED.

Poster presented in The Annual Scientific Meeting On Emerging Infection Treat 2017

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THE NEMATODE CHEMOTAXIS ASSAY: C. ELEGANS, A BIOSENSOR FOR IDENTIFICATION OF SEPSIS
Ling Fei Tee1, Toh Leong Tan2, Hui-min Neoh1, Ren Sen Lau3, Rahman Jamal1

1UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia

2Department of Emergency Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia

3Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia

Introduction: 

Sepsis is a serious medical condition which can lead to death. Its affirmative diagnosis requires at least 24 hours as it involves serological tests and bacterial cultures from patients’ blood. Recently, a Caenorhabditis elegans chemotaxis assay was used for cancer detection in urine samples. In this proof-of concept study, we modified parameters of the chemotaxis assay and determined its versatility for sepsis detection in patients admitted to the Department of Emergency Medicine, UKM Medical Centre (UKMMC). 

Methods: 

Eleven septic patients’ samples (whole blood, serum or urine) were used for this study. Control sample was from a healthy subject. The C. elegans chemotaxis assay was carried out using CESDA (C. elegans Sepsis Detection Assay). Nematodes were tested for their chemotaxis attraction towards the samples in terms of duration (time) needed for their migration towards the samples versus control. Chemotaxis index of “> 0” represents attraction of the nematodes towards tested samples; “≤ 0” represents non-attractant or repulsion.

Results: 

Using CESDA, whole blood septic samples (n=3, 27.3%) showed negative chemotaxis index, while serum samples (n=11, 100%) did not show conclusive results (positive index: n=6, 54.5%; negative index: n= 4, 36.4%, 0 index: n=1, 9.1%). Urine samples, although few, were all positive for chemotaxis index (n= 2, 18.2%). Interestingly, the attraction of the worms towards septic urine samples were as immediate as 20 minutes. 

Summary: 

The versatility of using urine samples for sepsis detection and C. elegans’s rapid attraction towards septic urine samples makes CESDA an interesting protocol to be further explored for sepsis diagnosis.

Poster presented in Minggu Penyelidikan Perubatan dan Kecemasan ke18 (2016)

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SERUM LACTATE AS A BIOMARKER FOR RISK STRATIFICATION OF SEPSIS IN THE EMERGENCY DEPARTMENT OF A TEACHING HOSPITAL

Tan TL^a, Neoh HM^b, Nurul Saadah A^c

^aDepartment of Emergency Medicine, Faculty of Medicine, Hospital Canselor Tuanku Muhriz,Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur.

^b UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur.

^c Department of Biochemistry, Faculty of Medicine, Hospital Canselor Tuanku Muhriz,

Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur.

Abstract

Introduction:

Serum lactate is reported to be useful in assisting early risk stratification for sepsis, improving its management outcome.

Objective:

This study was carried out to determine whether initial serum lactate level is associated with 28-days all cause mortality of septic patients in the emergency department of a teaching hospital in Malaysia.

Method:

This was a single center prospective cross-sectional study. Seventy-three patients were recruited from December 2010 to September 2011. Initial blood lactate levels were measured and patients were followed-up via phone call or case records to determine for 28-days mortality outcome.

Results:

Serum lactate level proved to be a good predictor for 28-days mortality in sepsis, severe sepsis and septic shock (ROC-AUC=0.812). Results suggested a threshold lactate level of 2.85mmol/L for the three groups with 75% sensitivity (95% CI, 0.22-0.99) and 77% specificity (95% CI, 0.65-0.85). After adjustment for the sepsis group, the subset for severe sepsis and septic shock group (n=53) showed the same lactate threshold of 2.85mmol/L (ROC-AUC=0.735) with 75% sensitivity (95% CI, 0.02-0.19) and 67% specificity (95% CI, 0.52-0.80). Although the association between serum lactate levels and 28-day mortality for the 3 groups was not significant (p=0.052), the trend indicated a possible clinical value which may be seen with a larger sample size. Patients with higher initial lactate levels appeared to have a higher risk of 28-days mortality.

Conclusion

Serum lactate of more than 2.85mmol/L is fairly sensitive and specific to predict 28-days mortality in septic patients.
Poster Presentation Minggu Penyelidikan Perubatan Dan Kesihatan ke-17, 2015 (Best poster presentation award)

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ELEVATION OF SECRETORY PHOSPHOLIPASE A2 GROUP IIA (SPLA2-IIA) AND CHANGES OF INFLAMMATION MARKERS IN BACTERIAL SEPSIS SYNDROME PATIENTS OF AN EMERGENCY DEPARTMENT

NURUL SAADAH AHMAD1, TOH LEONG TAN2, KHAIZURIN TAJUL ARIFIN1, WAN ZURINAH WAN NGAH1, YASMIN ANUM MOHD YUSOF1

1Department of Biochemistry, 2Department of Emergency Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur.

Corresponding author: sebastianttl@yahoo.co.uk 

Introduction: Bacterial sepsis involves complex sequence of inflammatory events towards infection.  Elevation of secretory phospholipase A2 Group IIA (sPLA2-IIA) level and changes in cyclooxygenases and prostaglandins in blood have been implicated in bacterial sepsis. 

Methods: This study aimed to determine changes in sPLA2-IIA and Prostaglandin D Synthase (PGDS) levels, and cyclooxygenase 1 & 2 (COX-1 & COX-2) enzyme activities in bacterial sepsis (bs) and severe bacterial sepsis/septic shock (bss/ss) patients.  

Results: A total of 107 subjects (healthy subjects = 45, bs = 45 and bss/ss = 17; age ≥18 years old) were recruited from the Emergency Department, Universiti Kebangsaan Malaysia Medical Centre.  Levels of sPLA2-IIA, PGDS, COX-1 & COX-2 activities were measured with ELISA kits and tested with One-Way ANOVA. Mean serum concentration of all markers in healthy subjects, bs and bss/ss, respectively, were as follows: sPLA2-IIA (1.04±0.13, 18.33±1.73 and 19.33±3.18 ng/ml); PGDS (4.11±0.30, 8.73±0.65 and 8.54±1.29 ng/ml); COX-1 (61.66±4.6, 35.88±4.14 and 28.86±8.02 nmol/unit/ml); COX-2 (24.81±4.77, 64.06±4.89, 40.84±7.04 nmol/unit/ml). Both sPLA2-IIA and PGDS levels were elevated significantly in bs & bss/ss compared to healthy subjects (p<0.05). There was a significant decrease of COX-1 activity in bs & bss/ss compared to healthy subjects (p<0.05). No significant differences of sPLA2-IIA, PGDS and COX-1 activity among the patients were found. In contrast to COX-1, COX-2 activity was raised significantly in bs compared to healthy subjects (p<0.05). 

Conclusion: sPLA2-IIA level was shown to have a positive relationship with changes in PGDS and COX-2 and may be used as a combine markers for early diagnosis of sepsis. 

Keywords

Sepsis, Bacterial Infection, sPLA2-IIA, PGDS, COX

Oral Presentation 

41st Annual Conference Of The Malaysia Society For Biochemistry And Molecular Biology

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A PILOT STUDY FOR THE USAGE OF SECRETORY PHOSPHOLIPASE A2 GROUP IIA AND CD64 LEVELS IN DISTINGUISHING BACTERIAL INFECTIONS AND SEPSIS IN ADULTS PRESENTED TO THE EMERGENCY DEPARTMENT OF A TEACHING HOSPITAL  

TL TAN1* , DN Nasuruddin2, A Ithnin2, H Neoh3 

1 Department of Emergency Medicine,

2 Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia, 

3 UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia Medical Centre                                                                                                                              

Purpose Early diagnosis of bacterial infections is important because they can evolve rapidly; in addition, effective treatment depends on antibiotic administration. Clinicians are in need of good diagnostic biomarkers to identify infected patients who would benefit from prompt antibiotic therapy, thus improving the survival rate. 

Methods This was a single-center prospective observational study. Patients with suspected bacterial sepsis were included into the study upon admission to the emergency department (ED) of a teaching hospital from March to August 2014. From 69 patients with probable infection, 51 adults were recruited into this study. Relevant cultures and serology tests were performed for all study subjects. Serum secretory phospholipase A2 Groupd IIA (sPLA2-IIA) and CD64 levels for each subject were subsequently analyzed. 

Results Forty-four patients presented with sepsis and 33 patients were confirmed to have bacterial infections. Both biomarkers were shown to be good in distinguishing between sepsis and non-sepsis groups. For the ROC curves, the area under the curve (AUC) for  sPLA2-IIa was better than CD64 (AUC=0.92, 95% confidence interval: 0.85-0.99, p=0.001 and AUC=0.91, 95% confidence interval: 0.81-1.00, p=0.001 respectively). In diagnosing bacterial infection, both sPLA2-IIa and CD64 levels were found to be fair tools to differentiate both bacterial and non-bacterial infections (sPLA2-IIA AUC=0.97, 95% confidence interval: 0.93-1.00, p=0.001, and CD64 AUC = 0.95, 95% confidence interval: 0.90-1.00 and p= 0.001 respectively.)  Interestingly,there was a 2-fold increase in sPLA2-IIa levels in bacterial infection compared to sepsis. 

Conclusion sPLA2-IIa had the best overall performance compared to CD64 and appears to be a good biomarker to diagnose both sepsis and bacterial infection, either alone or in combination with other biomarkers, thereby enabling early antimicrobial administration. We recommend incorporating sPLA2-IIa and CD64 level investigations into the diagnostic algorithm of sepsis in ED.

Presented in Regional Congress of Molecular Medicine, Malaysia (RCMM 2015)

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ELEVATED SECRETORY PHOSPHOLIPASE A2 GROUP IIA (SPLA2-IIA) LEVEL AND NEUTROPHIL COUNT AMONG SUSPECTED SEPSIS SYNDROME PATIENTS

NS Ahmad1, TL Tan2, K Tajul Arifin1, WZ Wan Ngah1

1Department of Biochemistry, 2Department of Emergency Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur.

Background

Sepsis is defined as systemic inflammatory response towards infection. Secretory phospholipase A2 Group IIA (sPLA2-IIa) and neutrophil count are documented as potential biomarkers for sepsis specifically caused by bacteria. sPLA2-IIa has been demonstrated to play an important role in generating bioactive lysophopholids implicated in inflammation caused by bacteria while neutrophil activation at certain level will indicate the infection is caused by bacteria or not. The aim of this study is to compare the level of secretory phospholipase A2 Group IIA (sPLA2-IIa) and neutrophil count among Systematic Inflammatory Response Syndrome (SIRS), suspected sepsis, severe sepsis and septic shock patients.  

Method

A total of 103 suspected sepsis patients aged above 18 years old were recruited from Department of Emergency Medicine, Universiti Kebangsaan Malaysia Medical Centre. Blood samples were collected upon consent. The level of sPLA2-IIa were measured by using commercial available ELISA kit and neutrophil count by computerized blood count. Comparison of the sPLA2-IIa level and the neutrophil count among sepsis groups were analyzed using Kruskal-Wallis test (non-parametric test).

Results

The median of sPLA2-IIa level in SIRS, suspected sepsis, severe sepsis and septic shock are 1953.43, 6161.67, 7119.47 and 15634.68 respectively. The median of neutrophil count among groups are 71.10, 74.50, 83.25 and 92.95 respectively. There are significant increases of sPLA2-IIa level and neutrophil count among SIRS, suspected sepsis, severe sepsis and septic shock patients (p<0.05).

Conclusion

The significant elevation of sPLA2-IIa level and neutrophil count among suspected sepsis syndrome patients suggest that these potential sepsis biomarkers have major role as indicator of bacterial infection sepsis in clinical setting. 

Keywords

sPLA2-IIa, Neutrophil count, , Sepsis, Infection

Presented in International Conference on Antioxidants and Degenerative Diease (ICADD 2015)

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DISTINGUISHING BACTERIAL AND NON-BACTERIAL SEPSIS BY USING SECRETORY PHOSPHOLIPASE A2 GROUP IIA LEVEL AND C - REACTIVE PROTEIN IN EMERGENCY DEPARTMENT – A PROSPECTIVE STUDY

Tan Toh Leong¹, Ida Zarina¹, Neoh Hui-min², Nidzwani Mahdi¹, Fadzlon Yatim¹, Dian Nasriana ³, Azlin Ithnin³, Ramliza Ramli⁴

¹Department of Emergency Medicine, ²UKM Medical Molecular Biology Institute, ³Department of Pathology, ⁴Department of Medical Microbiology and Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre,

Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia.

Corresponding author: sebastianttl@yahoo.co.uk

Introduction: Confirmation of bacterial sepsis is time consuming and challenging in emergency department (ED). Secretory phospholipase A2 group IIA (sPLA2-IIA) and C-reactive protein (CRP) are helpful laboratory investigations in diagnosing bacteria sepsis in ED. Early diagnosis would enable prompt antibiotic therapy soonest possible, thus improve survival rates 
Objective: To compare the performance of SPLA2-IIA and CRP with positive blood culture assays in differentiating bacterial sepsis from non-bacterial sepsis. 
Methodology: sPLA2-IIA and CRP levels were compared in septic patients admitted to ED of UKM Medical Centre from March to August 2014. A total of 84 patients were identified. After exclusion, 62 paired samples were analyzed. The median of SPLA2-IIA and CRP was compared using Mann-Whitney test. The accuracy of the tests were analysed with Receiver Operative Characteristic (ROC) Curve.  
Results: Twenty-six samples were confirmed to have bacterial infections, while 9 and 2 samples were confirmed to have viral and fungal infections, respectively based on cultures or serology assays. The remaining samples were blood culture-negative. There was a significant difference in sPLA2-IIA (P<0.001) and CRP (P=0.004) levels between bacterial and non-bacterial sepsis samples. Both SPLA2-IIA and CRP assay are able to differentiate bacterial, viral and fungal sepsis (P<0.05). The area under the ROC curve for sPLA2-IIA and CRP were 0.77 and 0.73 respectively. 
Conclusions: Both SPLA2-IIA and CRP assays show significant association with bacterial sepsis. These assays have a role in facilitating ED physicians to initiate antibiotic therapy in a timely manner. 

Presented at International Congress of The Malaysian Society for Microbiology (ICMSM 2015)

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28-DAYS MORTALITY PREDICTION IN SEPSIS PATIENTS USING STATIC LACTATE CONCENTRATION AND EARLY LACTATE CLEARANCE:

AN OBSERVATIONAL STUDY

Ahmad Fuad Fahmi bin Mohd Noor1,2 , Noor Asmidar binti Anuar1,2, Ong Wei Jie1,2, Chieng Zhin Liang1,2, Noor Akmal Shareela Ismail2, Tan Toh Leong1

1. Department of Emergency Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, 56000, Kuala Lumpur, Malaysia.

2. Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

Abstract

Background: Sepsis cause significant mortality. Static lactate concentration and lactate clearance are cite to be a predictor for sepsis survival. This study examines the clinical utility of the lactate clearance within the first 6 hours of admission in Emergency Department (ED) to predict 28-day mortality rate among sepsis. This study was designed towards sepsis patients in Emergency Department by focusing on the early lactate clearance within the first 6 hours of admission and its association with 28-day of survival rate.

Method: PPatients who presented with sepsis, severe sepsis or septic shock admitted to  Emergency DepartmentED of National University of MalaysiaUniversiti Kebangsaan Malaysia Medical Centre (ED UKMMC) were recruited. Blood lactate concentrations were measured upon admission (H 0), at 1st hour (H 1) and 6th hour (H 6) respectively. Either sStandard treatment of sepsis either septic bundle or eEarly gGoal dDirected tTherapy (EGDT) was initiated according to sepsis severity. once the sepsis was identified. A follow-up report was conducted at 28 days via telephone call, e-mail or case notes. and. Ppatients were later classified into survivor and non-survivor as final outcome. 

Results: Among Total of 19 patients were recruited for 2 months duration., Tthe mortality rate was 25% in lactate non clearance group versus lactate clearance group. The trend reflects no relationship between lactate clearance and 28-day mortality. The static lactic concentration  appeared to be significantly higher for non-survivor compare with the survival group at H0(p=0.03), H1(p=0.01)  and H6 (p=0.01). However, there were significant differences for static lactate concentration in both survivor and non- survivor groups of H0, H1 and H6 (p<0.05).

Conclusions: Static lactate concentration showed a superior predictor for sepsis as compared to early lactate clearance. Although early lactate clearance was unable to prove its ability to predict 28-day mortality, it iswe suggested it as a useful tool to monitor gauge the resuscitation efforts.outcome. 

Keywords: Sepsis, Static Lactate Concentration, Early Lactate Clearance, Prognostic indicator, Mortality rate

Type of presentation: poster 

6th Medical  Undergraduates' Annual Scientific Research Meeting 2014