PLoS One. 2015 Mar 18;10(3):e0120012.

A novel host-proteome signature for distinguishing between acute bacterial and viral infections

 

Kfir Oved1*, Asi Cohen1, Olga Boico1, Roy Navon1, Tom Friedman1,2, Liat Etshtein1,3, Or Kriger4, Ellen Bamberger1,3,5, Yura Fonar3, Renata Yacobov4, Ron Wolchinsky6, Galit Denkberg7, Yaniv Dotan3,8, Amit Hochberg4, Yoram Reiter6, Moti Grupper3,9, Isaac Srugo3,5, Paul Feigin10, Malka Gorfine10, Irina Chistyakov3,5, Ron Dagan11, Adi Klein4, Israel Potasman3,9, and Eran Eden1*

*E-mail: eraneden@me-med.com; kfiroved@me-med.com

1 MeMed Diagnostics, Tirat Carmel, Israel
2 Rambam Medical Center, Haifa, Israel
3 Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
4 Department of Pediatrics, Hillel Yaffe Medical Center, Hadera, Israel
5 Department of Pediatrics, Bnai-Zion Medical Center, Haifa, Israel
6 Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel
7 Applied Immune Technologies, Haifa, Israel
8 Department of Internal Medicine, Bnai-Zion Medical Center, Haifa, Israel
9 Infectious Diseases Unit, Bnai-Zion Medical Center, Haifa, Israel
10 Faculty of Industrial Engineering and Management, Technion-Israel Institute of Technology, Haifa, Israel
11 Pediatric Infectious Disease Unit and Clinical Microbiology Laboratory, Soroka Medical Center, Beer-Sheva, Israel

 

Abstract

Bacterial and viral infections are often clinically indistinguishable, leading to inappropriate patient management and antibiotic misuse. Bacterial-induced host proteins such as procalcitonin, C-reactive protein (CRP), and Interleukin-6, are routinely used to support diagnosis of infection. However, their performance is negatively affected by inter-patient variability, including time from symptom onset, clinical syndrome, and pathogens. Our aim was to identify novel viral-induced host proteins that can complement bacterial-induced proteins to increase diagnostic accuracy. Initially, we conducted a bioinformatic screen to identify putative circulating host immune response proteins. The resulting 600 candidates were then quantitatively screened for diagnostic potential using blood samples from 1002 prospectively recruited patients with suspected acute infectious disease and controls with no apparent infection. For each patient, three independent physicians assigned a diagnosis based on comprehensive clinical and laboratory investigation including PCR for 21 pathogens yielding 319 bacterial, 334 viral, 112 control and 98 indeterminate diagnoses; 139 patients were excluded based on predetermined criteria. The best performing host-protein was TNF-related apoptosis-inducing ligand (TRAIL) (area under the curve [AUC] of 0.89; 95% confidence interval [CI], 0.86 to 0.91), which was consistently up-regulated in viral infected patients. We further developed a multi-protein signature using logistic-regression on half of the patients and validated it on the remaining half. The signature with the highest precision included both viral- and bacterial-induced proteins: TRAIL, Interferon gamma-induced protein-10, and CRP (AUC of 0.94; 95% CI, 0.92 to 0.96). The signature was superior to any of the individual proteins (P<0.001), as well as routinely used clinical parameters and their combinations (P<0.001). It remained robust across different physiological systems, times from symptom onset, and pathogens (AUCs 0.87-1.0). The accurate differential diagnosis provided by this novel combination of viral- and bacterial-induced proteins has the potential to improve management of patients with acute infections and reduce antibiotic misuse.

PMID: 25785720

 

Supplement

One of the most alarming consequences of antibiotic overuse is the emergence and spread of multidrug resistant bacteria. Resistance of microbial pathogens to antibiotics is increasing world-wide at an accelerating rate. At least 2 million people are infected with antibiotic resistant bacteria each year in the US alone, and at least 23,000 people die as a direct result of these infections. [1] In the European Union, an estimated 400,000 patients present with resistant bacterial strains each year, of which 25,000 patients die. [2] Paradoxically, the inability to rapidly differentiate infections also results in the underuse of antibiotics, estimated to occur in 20-40% of all bacterial infections [3–6], putting patients at risk of complications and increasing healthcare costs.

 

fig11

Figure1. A blood sample was obtained for host-protein measurements from 1002 patients with suspected acute infection and non-infectious controls.

 

For the past four years, our team has been collaborating with leading clinicians and scientists from around the world to develop and validate our novel approach for distinguishing between bacterial and viral infections. Unlike most traditional diagnostics, this approach builds on an exquisitely informative system crafted by nature – the human immune system. Bacteria and viruses are recognized by distinct molecular entities and trigger an immune cascade that is partly common (e.g., inflammatory cytokines) and partly infection specific (e.g., induction of IL-6 by bacterial infections and Type I interferons by viral infections; Figure 5 )[7,8]. We conducted extensive screening of over 600 immune system proteins in patients with acute infections. A few of the proteins showed distinctly different patterns in bacterial and viral infected patients. In particular, the most informative protein we found, called TRAIL, dramatically increased in the blood of patients infected with a wide range of viruses, but surprisingly, decreased in bacterial infections. We then developed an algorithm that computationally integrates TRAIL with other immune proteins to produce an immune signature that diagnose the cause of the infection with high accuracy.

In the PLOS ONE study, the immune signature was developed and independently validated on a cohort of 1002 patients with acute infections and yielded highly accurate results, with sensitivity and specificity greater than 90%. The assay was validated in a diverse group of pediatric and adult patients at different time points after the onset of symptoms (from the first day up to 12 days) and across 56 different pathogen species. The signature remained robust over all sub-groups studied. The predictive power of the immune signature outperformed routine biomarker and laboratory tests, as well as combinations of these tests. The signature is amenable to rapid measurement using a blood test run on standard hospital and laboratory-deployed automated platforms, or using a point-of-care device now in development.

 

fig22

Figure 2. We conducted extensive screening of over 600 blood-borne proteins to generate an immune signature that diagnose the cause of the infection with high accuracy.

 

Why is the new signature superior to known host proteins and their combinations? Single host-proteins, such as procalcitonin (PCT), CRP and IL-6, are routinely used to assist in the diagnosis of bacterial infections because they convey additional information over clinical symptoms, blood counts and microbiology.[9] However, these markers are sensitive to inter-patient variability, including time from symptom onset, co-morbidities, site of infection, and pathogen species. For example, PCT is useful to diagnose bacterial sepsis [10], yet its utility in less severe infections, remains unclear.[11,12] Elevated CRP levels are highly suggestive of a bacterial infection [13], but similar levels may be observed in patients with some viral strains (e.g. adenovirus and influenza)[14], and inflammatory diseases. Finally, while IL-6 rises quickly in response to a bacterial trigger, its utility is dampened by a limited window of opportunity for measurement due to its rapid decay within roughly 36 hours.[15,16] To improve the performance of individual host-proteins, combinations of several proteins into a single predictive score have been proposed.[9,17–19] Different combinations of host-proteins tested by other groups have thus far yielded insignificant-to-moderate diagnostic improvement over individual proteins.[9,17–19] This modest improvement may be due to the reliance on combinations of bacterial-induced proteins, which share biological pathways, and are thus inherently sensitive to the same factors.

 

fig33

Figure 3. TRAIL and IP-10 were measured using commercial ELISA kits (MeMed Diagnostics)

 

The robustness of the new signature across a wide range of patient characteristics stems from the convergence of novel viral-induced proteins that complement routinely used bacterial-induced proteins. The three proteins are not strongly correlated and thus each of them has its own unique differential expression pattern in bacterial and viral patients: TRAIL is induced in response to viruses and suppressed by bacteria; IP-10 is more elevated in viral than bacterial infections; CRP is more elevated in bacterial than viral infections, which is well-established. [13,20] Combining proteins with complementary behaviors conveys valuable information that provides accurate and stable results, superior to current practice and previous findings.

 

fig44

Figure 4. Using the patient own immune system to diagnose the type of infection.

 

The immune-based approach overcomes inherent limitations of many traditional diagnostics tools. It is accurate and rapid, it can diagnose infections that are not readily accessible such as pneumonia (because immune system components circulate throughout the entire body), and it prevents false alarms due to the benign presence (“carriage”) of potentially pathogenic bacteria and viruses that are not causing active disease. Thus, the new immune signature has the potential to assist physicians make better informed antibiotic treatment decisions. This should result in more bacterial infected patients receiving timely therapy that is actually useful for treating their illness. Additionally, it can lead to fewer prescriptions to viral patients for whom antibiotics do nothing to speed recovery, while causing potential harm to the larger community.

 

fig55

Figure 5. TRAIL, IP-10 and CRP participate in different signaling pathways and exhibit complementary dynamics in response to bacterial (B) and viral (V) infections. PAMPs – pathogen-associated molecular patterns; PGN – peptidoglycan; LPS – lipopolysaccharide.

copyright: © 2015 Oved et al. PLoS One. 2015 Mar 18;10(3):e0120012. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 

Contact

Eran Eden, PhD, CEO – eraneden@me-med.com

Kfir Oved, MD/PhD, CTO – kfiroved@me-med.com

MeMed Diagnostics Ltd.   5 Nahum Heth St., Haifa Park High-Tech North, 39120, Israel Website: www.me-med.com

 

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