Blood biomarkers differentiating viral versus bacterial pneumonia aetiology: a literature review

A total of eleven studies involved CRP [14, 16, 17, 19, 20, 22, 23, 25‐29]. In all the studies analysing CRP as a diagnostic marker, the average CRP level was higher in the bacterial group than viral group [14, 20, 22, 23, 25‐29]. In an investigation by Esposito et al. [20] the mean level of CRP was 32.2 mg/L in 74 bacterial CAP cases as compared to 9.4 mg/L from 16 viral CAP cases. Similarly, in a different study by Esposito et al. [22] with larger sample size, the average CRP was 21.3 mg/L in 235 bacterial CAP patients and 8.0 mg/L in 111 viral CAP patients. The study by Bhuiyan et al. [14] showed that CRP concentration was more than 6 times higher in definite bacterial cases than in viral cases. This study also showed that elevation of CRP with the presence or absence of clinical symptoms, such as fever (≥38 °C) or the absence of rhinorrhea differentiates bacterial pneumonia from viral pneumonia better than CRP alone [14]. Although the mean levels of CRP were higher in bacterial CAP, there was overlap between viral and bacterial cases leading to issues in fixing a suitable cut-off point that is both sensitive and specific to differentiate between the two. In the study by Elemraid et al. 25% of viral CAP cases had CRP over 80 mg/L, and nearly 23% of bacterial cases had CRP less than 20 mg/L [27]. It is evident that a low reference point for CRP will diagnose almost all cases of bacterial aetiology but will include a significant number of false-positive cases. For example, with a cut-off value of 10 mg/l, the sensitivity and specificity are 95 and 49% respectively. Doubling the cut-off point to 20 mg/L led to a lower sensitivity of 85% and increased the specificity of 67%. However, a lower threshold value does not guarantee higher sensitivity. In a study by Esposito et al. [20] a cut-off value of 7.4 mg/L only resulted in 64% sensitivity. In a study by Naydenova et al. [23], the diagnostic value of respiratory rate, heart rate, oxygen saturation together with auscultation findings (presence or absence of grunting or crackles) was additionally analysed in association with CRP. The combination of these clinical parameters and CRP slightly improved the predictive power with a sensitivity of 64% and specificity of 88%. In the same study, the author added Lipocalin-2 (Lcn2) to CRP and clinical data which dramatically increased sensitivity to 81.8% and specificity to 90.6%. A significant limitation of almost all the studies was the lack of inclusion of primary care patients. This meant that before hospital admission, many patients might have had exposure to antibiotics which may have altered the level of CRP [27]. Another drawback was that only one study investigated co-infection (viral-bacterial) and concluded that CRP level did not correlate with co-infection [29].

Four studies analysed the diagnostic value of procalcitonin (PCT) [17, 22, 24, 26]. PCT is a precursor to calcitonin produced in the parafollicular cells of the thyroid gland by the transcription of CALC-1 gene. During an infection, CALC-1 gene is activated and upregulated to increase the production of PCT in not only endocrine glands but also many parenchymal tissues [30]. The sudden and marked increase (over 2 ng/ml) in PCT within four to six hours is a key indicator of bacterial infection [31]. It is hypothesized that viruses are not able to increase PCT to such a concentration as certain cytokines expressed during viral infection leads to decreased induction of PCT. This was reflected in the study by Esposito et al. [22] as the mean PCT was 1.1 ng/ml in viral CAP compared to 6.1 ng/ml in bacterial CAP cases. This studyshowed that specificity to identify viral aetiology was higher of PCT compared to CRP. Authors reported that a PCT cut-off value of ≤0.07 ng/ml had the highest combined sensitivity (48.7%) and specificity (81.1%) for viral CAP. Hoshina et al. noted that PCT value higher than 0.2 ng/ml had a sensitivity of 86%, the specificity of 80% to diagnose bacterial pathogen [26].

Six papers focused on WBC and/or neutrophil count [14, 17, 20, 24, 26, 27]. The total WBC count fluctuates in the paediatric population, especially in the early period of life. Therefore, the reference values differ between the age groups [32]. In general, a value greater than 11 × 10^9/L is considered to be leucocytosis [32]. Research by Elemraid et al. [27] showed that almost 40% of viral pneumonia cases presented with WBC > 15 × 10^9/L. Esposito et al. [20] highlighted that WBC had the lowest positive predictive value compared to PCT and CRP. According to Zhu et al. [24], the percentage of neutrophils compared to a total WBC count was to some extent better at discriminating viral from bacterial infection. According to the literature, passing neutropenia (Neutrophils < 1.5 × 10^9/L) is likely to begin from day three and last until day eight in many viral infections, including RSV, IV and AV [33, 34]. The lack of rise in neutrophil count correlates well with viral causes. In the study by Elemraid et al., 80% of patients with viral pneumonia had neutrophils less than 10 × 10^9/L [27].

In recent years new biomarkers have been tested in children. Of this, MxA1 has shown promising results. Compared to other markers, MxA1 protein tends to rise significantly during viral rather than a bacterial infection. Type I or III Interferon (IFN) can activate MxA1 but not type II IFN signaling pathway or the direct interaction of bacteria or viruses [35]. IFN is classified into three groups depending on the similarities in their amino acid sequence. Type I IFN is called alpha, beta, tau, and -omega and are produced in all cells in the body [36]. IFN is also elevated in autoimmune conditions and some hematological cancers. Therefore, the value of IFN induced MxA1 in this population may not be relevant [37].

The study by Engelmann et al. was the largest prospective study analysing the role of MxA1. A cut off value of 200 ng/mL was 96.4% sensitive and 66.7% specific for identifying patients with viral CAP [25]. The author also hypothesized that if a bacterial infection is diagnosed and high levels of MxA1 are detected, this is an indication that bacterial organism preceded a viral cause [25]. This is because MxA1 stays elevated for approximately ten days after a viral insult in comparison to IFN which has a very short half-life [38]. The authors also made a correlation with CRP. Low CRP (< 40 mg/l) and MxA1 > 200 ng/ml is highly indicative of viral aetiology [26]. The study sample size was the biggest drawback of this investigation. Out of 553 children who were enrolled, only 41 had CAP. Moreover, not all cases of CAP were microbiologically confirmed. Therefore, more studies are needed to confirm the diagnostic value of MxA1.

Lcn2 has been studied in the above data. This protein is stored and released by neutrophils which distorts iron transportation within bacteria. This marker is of a high interest in diagnosing aetiological factor of CAP. Two studies involving Lcn2 were carried out in very different settings which may have contributed to the different results. The study by Esposito et al. [20] concluded that Lcn2 was a poor predictor compared to CRP or WBC [20]. While Naydenova et al. [23] found the use of Lcn2 to help discriminate bacterial and viral pneumonia. The latter study was set in a developing nation amongst children with malaria, which is known to affect the concentration of Lcn2 [39]. According to Huang et al. [39], a cut-off value of more than 130 ng/ml strongly correlates with bacterial aetiology (sensitivity 83.67% and specificity 85.71%). Also, Lcn2 of more than 160 ng/ml is highly indicative of a positive isolate from a blood culture [39].

HMGB1 is a protein which binds to DNA and causes the transcription of several inflammatory markers. Furthermore, it has some extracellular roles such as promoting migration and enhancing the production of pro-inflammatory markers and cytokines such as Interleukin 6 (IL-6), Tumour necrosis factor (TNF) or Interferon gamma (IFN-γ). This protein is elevated during CAP, sepsis and viral-bacterial co-infections, especially bacterial and Influenza virus co-infection [40]. A study by Zhou et al. [29] evaluated changes in the expression of HMGB1 gene in peripheral monocytes as opposed to measuring the concentration of HMGB1 in the serum. By using the PCR technique, gene expression was quantified by comparing HMGB1 proteins density to an 18S ribosomal ribonucleotide acid. It was found that co-infection (virus and bacteria) can be concluded when HMGB1 expression is greater than 1.0256. Furthermore, in this study HMGB1 expression < 1.0256 and a WBC > 13 × 10⁹/L had 92.3% positive predictive value for single bacterial pneumonia [29]. Therefore, HMGB1 seems to be a good marker. However, the need to isolate specific blood cells (monocytes) and to adopt PCR makes this method prolonged and expensive. Moreover, measurement of RNA may not necessarily correlate with functional serum HMGB1 protein.

Several new markers, such as Syndecan 4 (SYN4), were explored with poor reliability. Results from the study by Esposito et al. revealed that SYN4 had an AUC on ROC of only 0.54 (95% CI 0.40–0.69) compared to 0.67 (95% CI 0.53–0.80) for CRP [20]. Proteins that can affect the cardiovascular system: Midregional Proadrenomedullin and Midregional pro-atrial natriuretic peptide were found to be not useful in predicting aetiology; instead, these were indicative of CAP severity [22]. Few microRNAs have been recently reported as a biomarker for several diseases. Study by Huang et al. [15] found that miR-450a-5p/miR-103a-3p and miR-103b/miR-98-5p could be considered as potential diagnostic biomarkers for adenovirus infection-associated pneumonia.

It is willing that another recently discovered biomarker chitinase-like protein (YKL-40) could also be used to differentiate viral and bacterial pneumonia. This protein is involved in airway imflammation and potential of this marker is under observation. The recent study by Yang et al. [16] showed that levels of YKL-40 in the bronchoalveolar lavage fluids specimens compared with serum levels of patients with bacterial pneumonia were significantly higher than with viral pneumonia [16]. This study also showed that YKL-40 reductions in serum levels on day 5 after receiving therapy is a possible prognostic biomarker for children with viral pneumonia [16]. The results of this study let us believe that YKL-40 has potential value in the differentiating viral and bacterial pneumonia.